Scenesse/Msh will with high likelihood cure/alleviate Multiple Sclerosis but with the regulatory system and distractions, this drug will not get to these patients for over 20 years.

[Uhohinc]

https://groups.google.com/forum/#!topic/clinuvel/P74TIqCRYvk  Being that "the regulators read what is posted on the investor websites" perhaps they might take note of the evidence leading to this supposition.

https://groups.google.com/forum/#!topic/clinuvel/P74TIqCRYvk

[Uhohinc]

http://ici2016.org/cms/wp-content/uploads/2016/05/ICI-Program-6-May-20161.pdf One of the authors of the above study from the U of Muenster is Nadine Sucker (Note M. Bohm also whom worked for Clinuvel on some trials) will be giving an lecture in August in Melbourne  on neurodegeneration and autoimmunity thru melanocortin receptor 1.

435: Stimulation of the melanocortin-1 receptor leads to direct neuroprotection via orphan nuclear 4 receptor in inflammatory neurodegeneration Nadine Sucker

(not much to find on any orphan receptors nor orphan nuclear receptor 4, they appear to be important, but big unknowns)

[MrPoonz1]

That's a big call... I don't dispute your research and analysis and I hope it to be true but I'm not readily convinced yet..

[Uhohinc]

I did carefully state "high likelihood" not to game it, but I think there is a preponderance of compelling studys and reasonable conclusions.

[Vallaurix]

Did anyone manage to attend the conference?

Stimulation of the melanocortin-1 receptor leads to direct neuroprotection via orphan nuclear 4 receptor in inflammatory neurodegeneration

Sucker. N.', Herrmann, A.M.2, Faber, C.3, Wiendl, H.2, Luger, T.A. ',Meuth, S.G.l, Loser, K. '

'University of Munster, Department of Dermatology, Munster, Germany, 2University of Munster, Department of Neurology, Munster, Germany, University of Munster, Institute for Clinical Radiology, Munster, Germany

In inflammation-associated progressive neurodegenerative disorders, e.g. multiple sclerosis (MS), inflammatory infiltrates, including Th 1 and Th 17 cells, cause emyelination and axonal/neuronal damage. Regulatory T cells (Treg) control the activation and infiltration of autoreactive T cells into the central nervous system (CNS). However, in experimental autoimmune encephalomyelitis (EAE) and MS, Treg function is severely impaired. Here we demonstrated that Nle4-D-Phe7-a-melanocyte-stimulating hormone (NDP-MSH), by binding to the melanocortin-1 receptor (MC-1 R), induced functional Treg, which efficiently inhibited EAE progression. Strikingly, NDPMSH also prevented immune cell infiltration into the CNS by restoring the integrity of the blood-brain barrier and exerted strong, long-lasting direct neuroprotective effects in an active (MOG-immunization) and spontaneous EAE model (Devic mice). Moreover, NDP-MSH prevented excitotoxic neuronal cell death in isolated mouse as well as human neuronal cells and reestablished action potential firing in embryonic hippocampal neurons. Gene expression studies performed in brain and spinal cord tissue from MOG-immunized and NDP-MSH-treated mice revealed that neuroprotection by NDP-MSH was mediated via signaling through MC-1 R, phosphorylation of the cAMP response element-binding protein (CREB) and subsequent activation of the orphan nuclear 4 receptor NR4A 1 in mouse and human neurons. NDP-MSH has recently received European Medicines Agency (EMA) approval for the treatment of erythropoietic porphyria and thus, our novel data might support its use in treating neuroinflammatory and neurodegenerative diseases, e.g. relapsing-remitting MS.

http://react-profile.org/ebook/ICI2016/AbstractBook/#48/z

http://ici2016.org/

[Uhohinc]

Note that Luger is one of the authors, whom has done several trials with Clinuvel. Inclusive of the acne trial.
New here is one item I suspected as it is logical. That is the blood brain barrier is apparently reset to homeostasis with Afamelanotide. I think based on the chemistry here it was Afamelanotide and not just generic research grade melanocytes stimulating hormone.

In homeostasis here, the mcr1 containing epithelial cells that make up the blood brain barrier epithelium should be tightly together and only allow one blood cell in a line to flow thru. And complete junctions to not let any microbe or foreign to get thru to brain cells. But if a neuron is signaling for death or a microbe has gotten thru, the immune cells cascade will come in and signal for the epithelial cells to spread openings so immune cells can get thru.

For unknown reason, the immune cells think the protein named mylein which I think is made by ------which insulates the electro micro current that flows thru axioms is not a host human protein. And they attack and remove the mylein. This is coalesced to preponderantly thought to be the cause of multiple sclerosis.

I think a recent observation in cancer patients with lymphoma who also had multiple sclerosis that then had radiation therapy which entirely destroyed their bone marrow, where immune cells stem from. As with a vaccination, certain immune cells test infectious microbes that enter the host human. If and when just one sentinel immune cell figures out how to spear, or lock into like a puzzle pieces, or attach to the side, or inject the microbe with a peroxide. A vaccination is giving a human the shell of a dead microbe or a very weak living version of a microbe which the immune system can shuffle up to millions of immune cells till it find one that has figured out how to kill the shell or weakly alive microbe.......then this one successful immune cell goes to the spleen and signals to create millions more just like it to combat.

These will go to the infection thru the blood whereby the epithelial cells spread apart to let these immune cells thru.

I think it very serendipitous that when the immune system in blood cancers or lymphoma was destroyed and replaced with donor marrow that the multiple sclerosis went away. This would in my thinking indicate and narrow down that multiple sclerosis is not originating in the brain neurons nor bad signals nor the mylein protein. As nothing is destroyed by chemo or radiation treatment here.

This would indicate multiple sclerosis is eminating from an immune cell, which keeps propagating that is errant or a miscreant cell modified in error to think the mylein is a foreign invader.

Afamelanotide is indicated to be lowering the white blood cell counts to a more homeostasis but must in someway interfere in this chain.

[Uhohinc]

In the below excerpt from the post by Vallaurix summarization of the presentation is a key word that to recall is what I posted in links of recent as to the basis of Lou Gherigs disease or aka Amyolateral sclerosis........"excitotoxicity" in which Astrocytes attack neurons abherant glutamatic acid levels. I wonder if Afamelanotide brings the excitotoxicity level to homeostasis or if it just stops the immune cells from "neuronal cell death"

The bottom line here is the information in just this summary of the Melbourne seminar presentation above by Vallaurix is that Afamelanotide with specificity to melanocortin 1 into the brain cells and immune cells is therapeutic to nuerodengenerative diseases. That of which I have posted about other pharma companies like Biogen which became tens of billions of $ in market cap for much less therapeutic value with multiple sclerosis.

Coupling this with the recent post referencing Dr Wolgen patent of Afamelanotide to therapy of four of the major nuerodengenerative disorders in multiple sclerosis, Huntingtons, Amyolateral sclerosis and Parkinson's and when these move from concepts on papers to phase II trials and so forth this is like having the knowledge to the biggest mega lottery numbers before they are drawn for at least ten billion in Stocks market cap.

Someday.......eventually.

[Uhohinc]

https://clinicaltrials.gov/ct2/show/NCT02446886?term=Melanocyte+stimulating+hormone&rank=4

[Uhohinc]

Multiple sclerosis: Progression-reduction drug in the pipeline

Written by Hannah Nichols
Published: Today
email
4.5
Multiple sclerosis affects around 400,000 people in the United States and 2.5 million people worldwide. While there is no cure, scientists have found a potential treatment to stop multiple sclerosis progression in its tracks in the form of the experimental drug laquinimod.
[woman taking tablet with water]
A potential oral drug may help prevent and reduce the progression of MS.
Multiple sclerosis (MS) is a disease of the central nervous system, whereby the immune system attacks tissue in the brain and spinal cord. The damage to the tissue called the myelin sheath - an insulated, fatty covering that protects the nerve fibers - affects how nerves carry electrical signals from the brain and the spinal cord.

Currently, MS treatment involves the use of prescription drugs and rehabilitation. MS is a lifelong disease with no known cure. However, the disease can be controlled using both disease-modifying and symptomatic treatments.

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Disease-modifying therapies reduce the number of relapses and may help slow disease progression; symptomatic therapies contribute to relieving some of the symptoms.

The research - published online in an American Academy of Neurology medical journal Neurology: Neuroimmunology & Neuroinflammation - discovered that laquinimod might prevent the development of MS or slow down the progression of MS in mice.

"These results are promising because they provide hope for people with progressive MS, an advanced version of the disease for which there is currently no treatment," says study author Scott Zamvil, M.D., Ph.D., of the University of California-San Francisco and a Fellow of the American Academy of Neurology.

Laquinimod is a drug in development for relapsing-remitting MS (RRMS) and primary progressive MS. RRMS is characterized by unexpected, recurring relapses. While in around 80 percent of all patients the disease begins as RRMS, most go on to develop secondary progressive MS after 10 years, which describes gradually increasing disability, without recovery periods.

The precise way in which laquinimod works is unclear. It is suggested that the drug alters the behavior of immune cells and prevents them from entering the brain and spinal cord, thus reducing damage to myelin.

Studies have indicated that laquinimod may have both an anti-inflammatory action and the capacity to protect the nerve structure and function.

For this research, Zamvil and colleagues gave mice that develop a spontaneous type of MS oral laquinimod or a placebo (water) daily. The number of T and B cells in the mice were then analyzed.

Laquinimod reduces clusters of myelin-destroying cells
T and B cells usually assist with the body developing immunity to infection. However, in MS, T and B cells help create antibodies that attack and destroy myelin.

In the first study, which included 50 mice, results showed that of the mice that were given oral laquinimod, 29 percent developed MS, compared with 58 percent of mice that received the placebo. Also noted was a 96 percent reduction in harmful clusters of B cells, which are only found in people with progressive MS. The researchers say that this evidence indicates that the drug may prevent MS.

In the second study, which included 22 mice, laquinimod was administered after mice had developed paralysis. The team observed a reduction in progression of the disease. Compared with the mice receiving the placebo, the mice that were given laquinimod presented a 49 percent decrease of dendritic cells that help create specialized T cells, a 46 percent reduction in T cells and a 60 percent drop in harmful antibodies.

"This study has given us more insight into how laquinimod works. But because this was an animal study, more research needs to be done before we know if it could have similar results in people."

Scott Zamvil, M.D., Ph.D.

People with MS have to receive medication throughout their lifetime. Active Biotech, the manufacturer of laquinimod, points out that, a once-daily oral treatment creates a substantial advantage for patients compared with existing products on the market, all of which need to be injected.

[Uhohinc]

RESEARCH ARTICLEMULTIPLE SCLEROSIS
Melanocortin-1 receptor activation is neuroprotective in mouse models of neuroinflammatory disease

Nadine Mykicki1,2, Alexander M. Herrmann2,3, Nicholas Schwab3, René Deenen4, Tim Sparwasser5, Andreas Limmer6, Lydia Wachsmuth7, Luisa Klotz3, Karl Köhrer4, Cornelius Faber2,7,8, Heinz Wiendl2,3,8, Thomas A. Luger1,2, Sven G. Meuth2,3,8,* and Karin Loser1,2,8,*,†
+ Author Affiliations
↵†Corresponding author. Email: los...@uni-muenster.de
↵* These authors contributed equally to this work.
Science Translational Medicine 26 Oct 2016:
Vol. 8, Issue 362, pp. 362ra146
DOI: 10.1126/scitranslmed.aaf8732

[Uhohinc]

If one can connect the above dots, Scenesse stimulates mcr1 and mcr1 will prevent or stop or alleviate multiple sclerosis.
There are about 400,000 diagnosed multiple sclerosis patients in USA. This link indicates the drug costs to Be about $60,000 annual to "treat" multiple sclerosis which would be $24 billion. Many patients are not on the latest expensive drugs yet, such as marketed by Biogen's Tecfedera, Avonex, Copaxan, and one called Beta something.http://www.everydayhealth.com/multiple-sclerosis/treatment/costs-of-ms-treatment/

Additionally, as such patient ages the become reliant on assisted living. I estimate 100,000 of the 400,000 are in care facilities at a cost of $50,000 per year each. That's another $5 billion. These costs are all embedded in US healthcare and Medicare and Obamacare. If the governments and insurance companies would let Clinuvel have a free market share price now, the societal reverberation and compounding exponential benefits are humongous. Yet shortsighted thinking politicians want to blame pharmaceutical management and are numb to reality of stock investors.

And numerous other nueroinflammatory disorders are potential therapies for Clinuvel Scenesse.
My suspicion is that Scenesse will not cure these reactive immune responses on the different nerve cells in different proteins in the brain and chord. I think Scenesse would need a intermittent long term therapy of perhaps a minimum of two to be determined dosages per year. Even if the one million people worldwide were successfully treated just one Scenesse implant and the T cells immune response reset that is $20 billion in sales.

[Uhohinc]

To note, Thomas Luger at U . of Muenster is a coauthor on the Mcr1 study in multiple sclerosis. He has been involved in May published Melanocyte stimulating hormone and melanocortins and I think I recall he was in on the Clinuvel Acne trial also

[Uhohinc]

http://www.sciencedirect.com/science/article/pii/S0197018615300656

Latest on major neurodegenerative disorders and the main pathways affected by all effectuating drugs
And plant polyphenols and flavonoids that appear to help and their pathways

[Uhohinc]

I do not think we will have to wait 20 years with the Thomas Luger study with Scenesse in the recent post. In this links of posts that I was just collecting bits and pieces and abstracts inferences are now in one abstract where Scenesse is strongly purported to be therapeutic in Multiple sclerosis as well as similar immune cell attacks in all neuro degenerative disorders including Alzheimer's and Parkinson's.

[Farma Zutical]

You called it, Uho. Very exciting.

[Uhohinc]

For these researchers to have finished this study I would think they conceptulized it a year ago based on recent observations and logical assertions of other study's over the last two to three years with melanocyte stimulating hormone other than Scenesse.

I would think Luger and the others at Muenster took away a lot of ideas and thinking after participating when Clinuvel sponsored the Afamelanotide trial thru them in acne.

The white blood cells, which encompass a general name now for about ten cell types inter related to each other in immune response is more being learned. But this is breaking news for drug research where the incremental advances have been measured in .........decades.
The TH17 cells did not even have a name as they were so recently discovered and most pathways to these and in other subsets big unknowns and are New within the last ten years.
I imagine Clinuvel contemplating a proof of concept study in a specific population of patients with advancing multiple sclerosis. Such study would require neurological backgrounds of physicians and a lot of magnetic resonance imaging appointments to measure white lesions. And probably need two years but if substantial patients are in the study maybe it could be one year. Maybe five years to see a difference as the disease advances in not a gradual way.
If Scenesse reversed the lesions as is indicated in the study, then such result would be visual in the MRI and probably speed up the trial. About five drugs which have mostly been bought by Biogen are approved to slow down or hinder multiple sclerosis are on market. Though they have the data to back it up, I do not think much about the efficacy of Avonex, Copaxine or one called Beta ?.
But the recent one called Tecfedera appears to have a partial pathway to genes actioned thru nrf2, which the Hailey Hailey researchers in Italy ascribed to Scenesse helping in that disorder. And which they stated their intent to give Tecfedera a try in a new Hailey Hailey trial

As a side note, I am picking up in searches brief mentions on resumes and curriculae vitals of researchers and grad students whom participated in the Hailey Hailey aka phemigus giving scant info that ten patients completed the study in 2015. No results published.

[Uhohinc]

http://www.nature.com/articles/srep41513
In this link in the second paragraph of Introduction it indicates Sox10 is a critical regulator of mylein. Msh strongly upregulates Sox10 which is a transcription factor.

Mylein is a protein made by olig? Cells as the insulation for nerve impulses, and its destruction by immune cells is the heart of multiple sclerosis.

[Uhohinc]

http://www.medicalnewstoday.com/articles/316382.php
Reversal of multiple sclerosis damage to neurons.

Regulatory T cells play a part in progenitors cells to become the Oligiodentridyte cells, the cells which create the mylein protein, which is insulative to the micro electric impulses.

[Uhohinc]

(From the above study, this a very telling study as in this excerpt)

Regulatory T cells also have a regenerative role
The researchers found that regulatory T cells release a protein that stimulates the differentiation of the progenitor cells into mature oligodendrocytes.

They showed that mice deficient in regulatory T cells showed substantially impaired myelin regeneration, which was restored when they transferred regulatory T cells into the mice.

Using cultured brain tissue, the researchers also showed that regulatory T cells "accelerated developmental myelination and remyelination, even in the absence of overt inflammation."

They conclude that their findings reveal a regenerative function for regulatory T cells that is separate from their role in regulating the immune response.

"This exciting study gives us an important understanding of how myelin repair can be promoted, which could open up new areas for treatment development."

Dr. Sorrel Bickley, Head of Biomedical Research at the UK MS Society

[dan.mur...@gmail.com]

What do you think Scenesse can do for motor neurone disease Uho? I was watching "The Theory of Everything", the movie about Stephen Hawking and how the doctor was saying something about the myelin sheath slowly being destroyed. I wonder if Scenesse could be used as a preventative therapy for those with possible predisposition to it OR it could delay the onset of motor neurone disease somewhat...

[Uhohinc]

Dr Wolgen does not say much, but in the Nov-Jan AGM and newsletters, he indicated Neurogenisis as a therapeutic area of focus (along with photophysics and inflammation) Neurogenisis is just a way of stating create nerve cells.

Stephen Hawking has Amnio lateral sclerosis which is a little different then Multiple Sclerosis in that it is ones own immune cells, T cells most thought to be (Cd4 and ? Precommitted in transformation) and these T cells are "trained" or whereby they had one "trainer" t cell which is supposed to recognize and identify a foreign cell or protein as a threat to the real host human cells, and then go to thymus and make more transformed to specific attack the same protein.
For some unknown reason the originating T cell thinks mylein proteins, which are created by oligiodendrites cells to insulate an electrochemical impulse from neuron to other neurons are proteins to be killed and removed.

A study which followed up antecdotal reports of patients with multiple sclerosis AND lymph or luekimia cancers in which all ones bone marrow producing T cells and all T cells were destroyed with radiation and chemotherapeutics incidentally ended their multiple sclerosis as well as cancer, upon reintroduced from donor blood marrow.

It appears msh or Scenesse is increasing the number and activity of the mylein producing Oligiodendrite cells (all cells mentioned have mcr) and causing the overacting conversioned cd4 cells to not only calm down but self destroy their over numbers.

As for ALS I sent a email to Hawkins sister's manager and attempted other initial dialogues but no responses, including the Italian drug price authority whom has ALS and has publicly trashed Clinuvel over the costs of Scenesse.. I have collected the referenced studies which I think pinpoint how msh and Sceness will be productive responses to the Astrocytes cells which are being signaled to attack area specific neurons and chord which is hallmark of ALS.

Dr Wolgen pointed to the Cd4 cells so many years back I do not recall now how long. I do see that he is years ahead of the research community in the pathways and genes of anything and where Scenesse msh is involving and may become important.

[Uhohinc]

https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm549325.htm This drug under brand name Ocrevus mechanic of action is on CD20 cells and it is affecting axon damages. And it is only show to work on a certain aggressive form of ms. It got every fast waiver fast track.

I know nothing about cd20 cells. I have never read them to be a cause or in the pathway of the disorder to whereby the immune cells attack mylein proteins sheathing axons. The trial compared the drug to beta interferon. Which I think is a drug along with Copaxine and Avonex I now think this drug as well as these others are manipulated patients for manipulated trial results. I think there are a lot of ms patients whom give up on these drugs because they are not working.

MS takes 15 to 40 years to progress and progresses so differently and with unusual stagnations different in each patient. And pregnancy halts it in females until birth.All patients are anomalies and the trial in my opinion would have to be decades. Roche Genetech did not do a comparator to the best Ms drug with I think best results. Tecfedera.
I think Scenesse really will be effective on MS. And note a few days ago the abstract on Axons being repaired by msh.

[Uhohinc]

https://www.uni-muenster.de/imperia/md/content/gebin-2017/gebin_2017_meeting_booklet.pdf

Top of its page 17. I think they used Scenesse in this observation.

[Uhohinc]

On Wednesday, April 12, 2017 at 11:27:59 PM UTC-7, Uhohinc wrote:
> https://www.uni-muenster.de/imperia/md/content/gebin-2017/gebin_2017_meeting_booklet.pdf
>
> Top of its page 17. I think they used Scenesse in this observation.

STIMULATION OF THE MELANOCORTIN-1 RECEPTOR LEADS TO DIRECT NEUROPROTECTION IN INFLAMMATORY NEURODEGENERATION
N. Mykicki1, A. Herrmann2, C. Faber3, H. Wiendl2, T. A. Luger1, S. G. Meuth2, K. Loser1 1Department of Dermatology, 2Department of Neurology and 3Institute for Clinical Radiology, University of Münster, 48149 Münster, Germany
In inflammation-associated progressive neurodegenerative disorders, e.g. multiple sclerosis (MS), inflammatory infiltrates, including Th1 and Th17 cells, cause demyelination and axonal/neuronal damage. Regulatory T cells (Treg) control the activation and infiltration of autoreactive T cells into the central nervous system (CNS). However, in experimental autoimmune encephalomyelitis (EAE) and MS, Treg function is severely impaired. Here we demonstrated that Nle4-D-Phe7-α- melanocyte-stimulating hormone (NDP-MSH), by binding to the melanocortin-1 receptor (MC-1R), induced functional Treg, which efficiently inhibited EAE progression. Strikingly, NDP-MSH also prevented immune cell infiltration into the CNS by restoring the integrity of the blood-brain barrier and exerted strong, long-lasting direct neuroprotective effects in an active (MOGimmunization) and spontaneous EAE model (Devic mice). Moreover, NDP-MSH prevented excitotoxic neuronal cell death in isolated mouse as well as human neuronal cells and reestablished action potential firing in embryonic hippocampal neurons. Gene expression studies performed in brain and spinal cord tissue from MOG- immunized and NDP-MSH-treated mice revealed that neuroprotection by NDP-MSH was mediated via signaling through MC-1R, phosphorylation of the cAMP response element-binding protein (CREB) and subsequent activation of the orphan nuclear 4 receptor NR4A1 in mouse and human neurons. NDP-MSH has recently received European Medicines Agency (EMA) approval for the treatment of erythropoietic porphyria and thus, our novel data might support its use in treating neuroinflammatory and neurodegenerative diseases, e.g.

[Uhohinc]

In the just above link, Abbivie is at the conference

[Uhohinc]

https://www.google.com/search?client=safari&channel=ipad_bm&ei=CRmRWauHNZSajwPgt7nQCA&q=vitamin+d+multiple+sclerosis&oq=vitamin+d+multipl&gs_l=mobile-gws-serp.1.0.0l5.4933.16163.0.17771.19.18.1.8.8.0.263.2913.0j15j2.17.0....0...1.1.64.mobile-gws-serp..1.18.1768.3..35i39k1j0i131k1j0i20k1.6heAYhPpHe4

Msh and vitamin d are connected to sunlight, and multiple sclerosis.

[Uhohinc]

http://www.mdpi.com/2076-3425/7/8/104/pdf Coincidentally this comes out today.

[Uhohinc]

http://www.sciencemag.org/news/2017/09/gut-microbes-could-help-trigger-multiple-sclerosis
Inference here very strong that gut microbiome is contributing to immune cells attacking insulating myelin proteins in neurons. Same inferences in other neurodegenerative disorders in other neurons in other nervous system cells.

[Uhohinc]

https://endpts.com/megablockbuster-day-for-roche-too-as-ms-game-changer-ocrevus-gets-a-green-light-at-the-fda/ Tecfedera sales down, and Ocrevus looks to have what's future market

[Uhohinc]

http://www.sigmaaldrich.com/catalog/product/aldrich/d95654?lang=en&region=US&gclid=Cj0KCQjw9afOBRDWARIsAJW4nvyiSIqGqa0QKvN5HgDh1AE1K-KFrjyQyp3aVjmJdzwFZTL1-KlU3A0aAjGjEALw_wcB

Was shopping online at a major USA pet store chain Petsmart and recognized the close in name Diethylfumerate which recalled here as a antifungal for furniture and now a very similar to Dimethylfumerate for multiple sclerosis.

Though close in name, chemistry and one for dogs and one for humans.......not close in price. About $50,000 difference in price.

[Uhohinc]

A29 Real-time biosensor technology reveals the temporal dynamic changes in BBB endothelial barrier strength following exposure to inflammatory cytokines present in the serum of MS patients

Rebecca H. Johnson1,2, Sarah Gough1, Dan T. Kho1,2, Simon J. O’Carroll1,3, Catherine E. Angel4, Jennifer Pereira5, E. Scott Graham1,2

1Centre for Brain Research, University of Auckland, Auckland, New Zealand, 2 Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand, 3 Department of Anatomy and Medical Imaging, School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand, 4 School of Biological Sciences, Faculty of Science, University of Auckland, Auckland, New Zealand, 5 Neurology Department, Auckland District Health Board, Auckland, New Zealand

Correspondence: E. Scott Graham (s.gr...@auckland.ac.nz)

Fluids and Barriers of the CNS 2017, 14(Supp 2):A29

In this research we have investigated the temporal regulation of brain endothelial barrier function following exposure to a variety of inflammatory mediators elevated in the serum of MS patients. It is well known that Relapsing Remitting MS is a chronic inflammatory disease with evidence of autoimmune dysfunction, therefore our serum cytokine panel was biased towards Th1, Th17 and leukocyte recruitment/activation mediators. In the MS sera, we detected elevated levels of (i) IL-1β, IL2, IL-4, IL6, IL-12, IL17, IFNs, (ii) the chemokines fractalkine, MCP-1, IP-10, RANTES, IP-10, MIP1α, and (iii) various growth factors including GCSF and GMCSF. Each of these were then applied to an immortalised human brain endothelial cell culture model system with barrier function measured using ECIS technology. Of all of the cytokines assessed, IL-1β and TNFα had the greatest effect on barrier strength. We observed a small transient reduction in barrier strength (in the first 10-14 hours), which was followed by a sustained strengthening by both IL-1β and TNFα (50-500pg/mL). IFNγ mediated a similar change in barrier strength. These responses were concentration dependent with IL-1β being the most potent. In contrast, IFNα caused the immediate strengthening of the barrier, whereas both IL6 and IL4 caused progressive weakening. Even though these cytokines influenced barrier function in different ways as demonstrated by the temporal power of ECIS technology, they each stimulated the brain endothelial cells causing increased secretion of various pro-inflammatory cytokines and chemokines. Most of the other factors measured in the sera from the MS patients did not have any influence on the barrier function or viability of the brain endothelial cells used in this study. We conclude that human brain endothelial cells are sensitive to a range of pleiotropic cytokines that are elevated in the sera of patients with RRMS. It is quite plausible that some or a combination of these may influence the BBB integrity and contribute to the leakiness occurring in regions of the brain susceptible to the formation of lesions.

Research Support: Neurological Foundation of New Zealand, and the University of Auckland Faculty Research Development Fund.

[Uhohinc]

The just above study is significant in relation to other study’s relative to msh causation of homeostasis in endothelial cells of the blood brain barrrier mostly thru mcr1 and with the inference of open or inflamed junctions letting foreign fats, microbes or particulates into brain to cause a immune reaction that infers may be cause of multiple sclerosis.

[Uhohinc]

A35 Molecular control of the brain barriers in multiple sclerosis

Melissa A. Lopes-Pinheiro2, Jamie Lim2, Alwin Kamermans2, Jack van Horssen2, Wendy W.J. Unger1, Ruud Fontijn2, Helga E. de Vries2

1Department of Pediatrics, ErasmusMC, Rotterdam, The Netherlands, 2Department of Molecular Cell Biology and Immunology, VUmc MS Center Amsterdam, VU medical center, Amsterdam, The Netherlands

Correspondence: Helga E. de Vries (he.de...@vumc.nl)

Fluids and Barriers of the CNS 2017, 14(Supp 2):A35

Dysfunction of the neuroprotective blood–brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) and inflammation thereof are key events in multiple sclerosis (MS). In early stages of MS, inflammation of the brain barriers actively aids in immune cell influx, inducing neuro-inflammation and demyelination. Upon MS progression, chronic BBB dysfunction is apparent, which contributes to gliosis and neurodegeneration. Moreover, barrier dysfunction goes hand in hand with a profound inflammation of the endothelial cells, thereby promoting infiltration of auto-reactive T-cells and monocytes, which further induces irreversible tissue damage. In turn, inflamed brain endothelium contributes to disease progression by its release of pro-inflammatory factors which activates the surrounding glia cells, together reinforcing astrogliosis, microglia activation and consequently neurodegeneration.

Our recent work suggests that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin which was routed to endo-lysosomal compartment for processing in a time-dependent manner and subsequently induce T-cell activation and migration. In the transmigration of leukocytes across inflamed BECs, the enzyme acid sphingomyelinase (ASM), involved in the production of bio-active lipids such as ceramide, is essential for the coordination of the function of the endothelial adhesion molecule ICAM-1. These results demonstrate that BECs are capable of enhancing antigen-specific T cell recruitment. Moreover, current treatment regimens for MS, such as dimethylfumarate, are capable of reducing immune cell trafficking through the induction of endogenous anti-oxidant enzyme systems.

Together this illustrates that identification of the pathways that counteract BBB inflammation and reinstate proper BBB function will provide new and selective means to fight MS in different stages of disease.

Work was funded through grants of the Dutch MS research foundation

1.
Lim JL, van der Pol SM, Di Dio F, van Het Hof B, Kooij G, de Vries HE, van Horssen J. Protective effects of monomethyl fumarate at the inflamed blood–brain barrier. Microvasc Res. 2016;105: 61–9.

2.
Lopes Pinheiro MA, Kamermans A, Garcia-Vallejo JJ, et al. Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration. Westbrook GL, ed. eLife. 2016;5: e13149.

3.
Lopes Pinheiro MA, Kroon J, Hoogenboezem M, Geerts D, van het Hof B, van der Pol SMA, van Buul JD and de Vries HE. Acid Sphingomyelinase–Derived Ceramide Regulates ICAM-1 Function during T Cell Transmigration across Brain Endothelial Cells. J Immunol. 2016;196: 72–9.

[Uhohinc]

http://www.mdpi.com/2076-3425/7/8/104/pdf Very important implications of msh thru melanocortins including mcr1 in bbb therapeutic.

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PEER-REVIEWED Brain and Cognition section
Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone
Research articleCell BiologyNeurosciencePharmacology
András Harazin​1,2, Alexandra Bocsik​1, Lilla Barna1,2, András Kincses1, Judit Váradi3, Ferenc Fenyvesi3, Vilmos Tubak4, Maria A. Deli​1, Miklós Vecsernyés​3
May 15, 2018
Author and article information
Abstract

The blood–brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which α-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-α and IL-1β, we hypothesized a protective effect of α-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide α-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-α and IL-1β induced cell damage, measured by impedance and MTT assay, which was attenuated by α-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and β-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-κB were also reduced by α-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of α-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB.

Cite this as

Harazin A, Bocsik A, Barna L, Kincses A, Váradi J, Fenyvesi F, Tubak V, Deli MA, Vecsernyés M. (2018) Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone. PeerJ 6:e4774 https://doi.org/10.7717/peerj.4774
Main article text

Introduction
The neuropeptide α-melanocyte stimulating hormone (α-MSH) belongs to the family of melanocortins, which are created from pro-opiomelanocortin (Catania et al., 2010). The α-MSH, a 13 amino acid long peptide hormone, is mainly produced in the hypothalamic region (Brzoska et al., 2008). Induction of melanogenesis in pigment cells and immunomodulation are among its main physiological functions (Catania, 2008). There are five melanocortin receptors (MCRs), from which four, MC1R, MC3R, MC4R, and MC5R, bind α-MSH (Brzoska et al., 2008). MCRs are G-protein-coupled and exert their effects via cyclic 3′,5′-adenosine monophosphate (cAMP)-dependent signaling pathways. The predominant receptor of α-MSH is MC1R, which binds the neuropeptide with high affinity, and is expressed in both the brain and periphery. MC1R was demonstrated in several tissues, such as brain, skin, immune system, and gut (Brzoska et al., 2008).

Melanocortins are evolutionary conserved defense molecules against tissue injury and bacterial invasion (Catania, 2008). The immunomodulatory and anti-inflammatory effects of α-MSH are well known and widely investigated. The anti-inflammatory action of α-MSH was proven in animal models of systemic or local inflammation, like sepsis, arthritis, uveitis, dermatitis, pancreatitis, and colitis (Brzoska et al., 2008). The protective effect of α-MSH was also shown in ischemic conditions in the heart (Vecsernyés et al., 2003, 2017), retina (Varga et al., 2013), kidney and intestine (Brzoska et al., 2008). In addition to animal models of gut inflammation (Rajora et al., 1997a), the effects of α-MSH were examined on cell cultures. Our groups have recently described that pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) disrupted the barrier integrity of Caco-2 human intestinal epithelial cells, which was attenuated by α-MSH via the inhibition of the NF-κB pathway (Váradi et al., 2017).

In addition to systemic inflammatory conditions, the beneficial effect of α-MSH was also investigated in models of acute and chronic injuries of the central nervous system (CNS; Catania, 2008). Treatment with α-MSH was neuroprotective in cerebral ischemia, traumatic spinal cord injury, kainic acid induced excitotoxic brain damage, and lipopolysaccharide (LPS)-induced cerebral inflammation in animal studies. One of the main mechanisms of action of α-MSH is the down-regulation of pro-inflammatory cytokines TNF-α and/or IL-1β in blood and brain tissue, as it was described in LPS-induced brain inflammation (Rajora et al., 1997b) and unilateral middle cerebral artery occlusion (Huang & Tatro, 2002) in mice, and in kainic acid-induced brain damage in rats (Forslin Aronsson et al., 2007). Studies on cultured astrocytes, neurons, and microglia revealed that, similarly to the periphery, inhibition of the NF-κB pathway and downstream blockade of pro-inflammatory cytokine release and nitric oxide overproduction contribute to the protective effects of α-MSH (Catania, 2008).

Dysfunction of the blood–brain barrier (BBB) has been described in many systemic and CNS inflammatory diseases (Erickson & Banks, 2018) and plays a central role in the pathomechanism of many neurological diseases (Zhao et al., 2015; Liebner et al., 2018). TNF-α and IL-1β are the two most studied major pro-inflammatory cytokines in neuroinflammation observed in CNS pathologies (Sochocka, Diniz & Leszek, 2017; Liebner et al., 2018), and induced by systemic inflammation (Hoogland et al., 2015). The elevated levels of these two cytokines, confirmed in chronic neurodegenerative diseases (Sochocka, Diniz & Leszek, 2017), open the BBB (Liebner et al., 2018). TNF-α directly increases the permeability of the BBB for marker molecules in animal studies (Megyeri et al., 1992; Ábrahám et al., 1996), as well as in culture models (Deli et al., 1995) and a similar effect was described for IL-1β in BBB in vitro experiments (for review see Deli et al., 2005). Since these two pro-inflammatory cytokines are major players in both systemic and CNS inflammation and induce BBB dysfunction, TNF-α and IL-1β were selected for our experiments.

A binding site was found on murine brain endothelial cells by radiolabeled α-MSH (de Angelis et al., 1995), suggesting the presence of MCR(s) at the BBB, but they were not identified. MCRs expressed on cells of the neurovascular unit could mediate the neuroprotective actions of melanocortins (Catania, 2008), however, the effects of α-MSH on the BBB, especially on brain endothelial cells have not been investigated yet.

Therefore, our aim was to investigate the effects of α-MSH peptide on a culture model of the BBB, its possible protective action against barrier disruption induced by TNF-α and IL-1β cytokines by measuring cellular viability, permeability for marker molecules, structure of interendothelial tight junctions, production of reactive oxygen species (ROS) and the nuclear translocation of NF-κB p65 subunit.

Materials and Methods
Materials

All reagents were purchased from Sigma-Aldrich Corporation (subsidiary of Merck KGaA, Darmstadt, Germany) unless otherwise indicated.

Cell culture

Primary rat brain endothelial cells were isolated from three week-old outbred Wistar rats (Harlan Laboratories, Indianapolis, IN, USA) as described in our previous paper (Veszelka et al., 2013). Forebrains were collected in ice-cold sterile phosphate buffered saline (PBS). After meninges were removed the tissue was cut into 1 mm3 pieces by scalpel and digested with enzymes (1 mg/ml collagenase type II, and 15 μg/ml deoxyribonuclease type I; Roche, Basel, Switzerland) in Dulbecco’s modified Eagle medium (DMEM/F12, Gibco; Life Technologies, Carlsbad, CA, USA) at 37 °C for 55 min. Microvessels were separated from myelin rich fraction by centrifugation in 20% BSA–DMEM gradient (1,000×g, 20 min, three times). The collected vessels were further digested with enzymes (1 mg/ml collagenase–dispase, and 15 μg/ml deoxyribonuclease type I; Roche, Basel, Switzerland) in DMEM/F12 at 37 °C for 35 min. Brain microvascular endothelial cell clusters were separated on a 33% continuous Percoll gradient (1,000×g, 10 min), collected, and washed twice in DMEM/F12. Cells were seeded onto Petri dishes (100 mm; Orange Scientific, Braine-l’Alleud, Belgium) coated with collagen type IV and fibronectin (100 μg/ml each). Cultures were maintained in DMEM/F12 supplemented with 15% plasma-derived bovine serum (First Link, Wolverhampton, UK), 5 μg/ml insulin-transferrin-sodium selenite (Pan Biotech, Aidenbach, Germany), 1 ng/ml basic fibroblast growth factor (Roche, Basel, Switzerland), 100 μg/ml heparin, and 5 μg/ml gentamycin. For the first three days of culture cells were grown in medium with 3 μg/ml puromycin to eliminate P-glycoprotein negative contaminating cell types (Perrière et al., 2005). When endothelial cells reached 90% confluency, they were subcultivated for different experiments.

Primary rat brain pericytes were isolated by the same method, except that the second digestion lasted only for 15 min. After isolation cells were plated onto uncoated dishes and they were cultured in low-glucose DMEM medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Pan Biotech, Aidenbach, Germany) and gentamycin. No puromycin was applied. Pericytes were used at third passage for experiments.

Primary rat glial cells were isolated from one-day-old Wistar rats. After meninges were removed brain tissue was mechanically dissociated by a syringe equipped with a long needle. The tissue homogenate was filtered through a nylon mesh (40 μm; Millipore, Billerica, MA, USA) to remove large tissue pieces and vessels. Cell clusters in the filtrate were plated onto uncoated 75 cm2 flasks (TPP, Trasadingen, Switzerland), cultured until 90% confluency in DMEM containing 10% FBS (Lonza, Basel, Switzerland) and gentamycin. For the BBB co-culture model glial cells were passaged at a cell number of 5 × 104 cells/well for 12-well plates (Corning Costar, Corning, NY, USA) and cultured for two weeks before use. Confluent glia cultures contained 90% of GFAP immunopositive astroglia, and 10 % CD11b immunopositive microglia.

To induce BBB characteristics brain endothelial cells were co-cultured with brain pericytes and glial cells (Nakagawa et al., 2009) using 12-well format tissue culture inserts (Transwell clear, polyester membrane, 0.4 μm pore size, 1.12 cm2 surface; Corning Costar, Corning, NY, USA). Rat pericytes (1.5 × 104 cells/cm2) were subcultivated to the bottom side of the insert membranes and brain endothelial cells (7.5 × 104 cells/cm2) were passaged to the upper side of the collagen type IV and fibronectin coated inserts. Both compartments received endothelial culture medium. After two days of co-culture, brain endothelial cells reached confluency and 550 nM hydrocortisone was added to the culture medium to tighten junctions (Walter et al., 2015). Before experiments 250 μM 8-(4-chlorophenylthio)-cAMP and 17.5 μM Ro 20-1724 (Roche, Basel, Switzerland) were added to the endothelial cells for 24 h to tighten junctions and elevate resistance (Deli et al., 2005; Perrière et al., 2005).

Treatments

The neuropeptide α-MSH (Mw. 1664.8 Da) was tested at 1 pM to 1 μM concentrations. The use of cytokine cocktails is common in CNS culture studies (Sochocka, Diniz & Leszek, 2017). We have also used a combination of TNF-α (50 ng/ml) and IL-1β (25 ng/ml) on intestinal epithelial cells in our previous study (Váradi et al., 2017). Based on this study, we have tested a combination of the pro-inflammatory cytokines TNF-α (10–50 ng/ml) and IL-1β (10–25 ng/ml) for 24 h to induce damage in brain endothelial cells. The control group received culture medium only.

Total RNA isolation and RT-PCR

Brain microvessels were isolated from adult rat brains as published earlier (Veszelka et al., 2007). For receptor expression analysis total RNA was isolated from isolated brain microvessels and brain endothelial cells by TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). One microgram of total RNA was transcribed to cDNA by Maxima First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA). Specific oligonucleotide primers were designed for the Mc1r gene (XM_006222790). Primer sets were MC1R_fwd 5′-TGCACCTCTTGCTCATCGTT-3′ and MC1R_rvs 5′-ACCTCCTTGAGTGTCATGCG-3′. The predicted length of PCR product was 160 bps. Primers for β-actin were used as internal controls (NM_031144). Primer sets were ACT_fwd 5′-TACTCTGTGTGGATTGGTGGC-3′ and ACT_rvs 5′-GGTGTAAAACGCAG CTCAGTAA-3′. The predicted length of PCR product was 150 bps. PCR was performed with FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia) in T100 thermal cycler (BioRad, Hercules, CA, USA). After initial heat inactivation (95 °C for 3 min) the following cycling conditions were applied: melting 94 °C for 15 s, annealing 50 °C for 15 s, polimerization 72 °C for 20 s (35 cycles). After a final 5 min extension at 72 °C PCR products were analyzed on 3% MetaPhor agarose gel (Cambrex BioScience, Rockland, ME, USA) and fragments were verified by capillary DNA sequencing.

Measurement of cell viability: real-time cell electronic sensing and MTT assay

Real-time cell electronic sensing is an impedance-based, label-free technique for dynamic monitoring of living adherent cells, including barrier forming epithelial and brain endothelial cells (Kiss et al., 2014; Lénárt et al., 2015). The RTCA-SP instrument (ACEA Biosciences, San Diego, CA, USA) registers the impedance of cells in every 10 min and for each time point cell index is calculated as (Rn − Rb)/15, where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the medium alone. E-plates, special 96-well plates with built in gold electrodes, were coated with collagen type IV and fibronectin for brain endothelial cells at room temperature and dried for 20 min under UV and air-flow. For background measurements culture medium (60 μl) was added to each well, then 50 μl of rat brain endothelial cell suspension was distributed at a cell density of 5 × 103 cells/well. After cells reached a steady growth phase they were treated with α-MSH and cytokines.

In parallel we also used an endpoint colorimetric cell viability assay (Kiss et al., 2014). The yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye is converted by viable cells to purple formazan crystals. Brain endothelial cells were grown in 96-well plates coated with collagen type IV and fibronectin. After treatment of confluent monolayers, cells were incubated with 0.5 mg/ml MTT solution in cell culture medium for 3 h in CO2 incubator. Formazan crystals were dissolved in dimethyl sulfoxide, and dye concentration was determined by absorbance measurement at 570 nm with a multiwell plate reader (Fluostar Optima; BMG Labtechnologies, Offenburg, Germany).

Measurement of permeability for marker molecules

Penetration of fluorescein isothiocyanate-labeled dextran (FITC-dextran, MW: 4.4 kDa) and Evans blue-labeled albumin (EBA, MW: 67 kDa) across the BBB model was determined in permeability studies (Veszelka et al., 2007). Rat brain endothelial cells co-cultured with pericytes and glia on cell culture inserts were treated with cytokines and/or α-MSH for 1 h. After treatment inserts were transferred to 12-well plates containing 1.5 ml Ringer-HEPES buffer (150 mM NaCl, 2.2 mM CaCl2, 0.2 mM MgCl2, 5.2 mM KCl, 6 mM NaHCO3, 5 mM glucose, and 10 mM HEPES, pH 7.4) in the lower compartments. In the upper compartments culture medium was replaced by 500 μl Ringer-HEPES buffer containing EBA complex (1 mg/ml bovine serum albumin and 167.5 μg/ml Evans blue) and FITC-dextran (100 μg/ml). The plates were kept for 30 min on a rocking platform (100 rpm) at 37 °C in an incubator with 5% CO2. After the incubation the concentrations of the marker molecules in samples from the upper and lower compartments were determined by a fluorescence multiwell plate reader (Fluostar Optima; BMG Labtechnologies, Offenburg, Germany; excitation wavelength: 485 nm, emission wavelength: 520 nm for FITC-dextran; excitation wavelength: 584 nm, emission wavelength: 680 nm for EBA). The apparent permeability coefficients (Papp) were calculated by the following equation (Váradi et al., 2017):
(1)
Papp=[C]B×VBA×[C]A×t
P
app
 
=
[
C
]
B
 
×
  
V
B
A
×
[
C
]
A
×
t
where [C]B is the concentration of the tracer in the lower (basal) compartment after 1 h, [C]A is the concentration of the tracer in the upper (apical) compartment at 0 h, VB is the volume of the basal compartment (1.5 ml) and A is the surface area available for permeability (1.12 cm2).

Immunohistochemistry

Melanocortin-1 receptor

Isolated rat brain microvessels and brain endothelial cells, co-cultured with brain pericytes and glial cells, were immunostained for MC1R as we described in our previous paper for epithelial cells (Váradi et al., 2017). Cells were washed in PBS, fixed with ice cold acetone/methanol solution (1:1) for 5 min, and blocked with 3% BSA–PBS for 1 h. A rabbit anti-human MC1R antibody (M9193; 1 mg/ml) was used as primary antibody for overnight at 4 °C. Incubation with secondary antibody, Alexa Fluor 488 conjugated goat-anti-rabbit IgG (A11029; ThermoFisher Scientific, Waltham, MA, USA; 2 mg/ml) and nucleus stain ethidium homodimer-1 (1 μM) lasted for 1 h. The samples were thoroughly washed with PBS after each step and mounted in Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Stainings were visualized by a Leica SP5 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Junctional proteins and NF-κB p65 subunit

Rat brain endothelial cells treated with cytokines and/or α-MSH for 1 h were fixed and permeabilized with ice cold acetone/methanol solution (1:1) and blocked in 3% BSA–PBS for 1 h. Cell were incubated with rabbit anti-rat β-catenin polyclonal (C2206; 1 mg/ml) or rabbit anti-rat claudin-5 polyclonal (SAB4502981; 1 mg/ml) primary antibodies overnight, followed by secondary labeling with Cy3-conjugated goat-anti-rabbit IgG (C2306; 2 mg/ml) and a nucleus stain (H33342; 1 μg/ml) for 1 h. For NF-κB p65 subunit immunostaining a rabbit anti-human p65 polyclonal antibody (sc-372; Santa Cruz Biotechnology, Dallas, TX, USA; 100 μg/ml, overnight incubation) was used, followed by incubation with Alexa Fluor 488-conjugated goat-anti-rabbit IgG (A11029; ThermoFisher Scientific, Waltham, MA, USA; 2 mg/ml) and H33342 for 1 h. Cells were washed and mounted as described above. For both TJ protein and NF-κB stainings samples were observed by a Leica SP5 confocal laser scanning microscope. Images showing TJ staining were analyzed using MATLAB software (MathWorks, Natick, MA, USA). The backgrounds of each image were determined and subtracted from the corresponding image to compensate the occasional non-uniform background. Then, the grayscale images were converted to binary. Objects with size less than 6 pixels were eliminated to reduce any false structure. The areas of the structures were determined by the pixel number of the structures on the binary images. The object number defines the number of the separated, non-contact structure elements of the images. Images of NF-κB immunostaining were analyzed using ImageJ software (National Institutes of Health, Rockville, MD, USA) as described in our previous paper (Sántha et al., 2016).

Measurement of ROS production

Reactive oxygen species production was detected by chloromethyl-dichloro-dihydro-fluorecein diacetate (DCFDA; Life Technologies, Carlsbad, CA, USA) as we described previously (Veszelka et al., 2013; Lénárt et al., 2015). The indicator penetrates into the cells and interacts with intracellular esterases. After its oxidation fluorescent molecules are produced, and the fluorescent signal is proportional to the produced ROS. Confluent brain endothelial cells were cultured in black 96-well plates with glass bottoms (Corning Costar, Corning, NY, USA). After 1 h treatment cells were incubated with Ringer-HEPES buffer containing 2 μM DCFDA for 1 h at 37 °C. Hydrogen peroxide (100 μM) was used as a reference compound (positive control). Fluorescence was measured at 485 nm excitation and 520 nm emission wavelengths by a Fluostar Optima multiwell plate reader (BMG Labtechnologies, Offenburg, Germany) every 5 min for 1 h. Fluorescence intensities are shown in arbitrary units.

Statistical analysis

Statistical analysis was done by GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). Data are presented as mean ± SEM. Comparison of groups was performed using ANOVA and Dunnett or Bonferroni tests. Differences were considered significant at P < 0.05.

Results
Expression of melanocortin-1 receptor on rat brain microvessels and cultured brain endothelial cells

MC1R gene expression was detected on both freshly isolated rat brain microvessels and confluent rat brain endothelial cell cultures with reverse transcription followed by PCR amplification using the specifically designed rat primers (Fig. 1A). This result was strengthened by immunohistochemistry for MC1R. The brain endothelial staining (Fig. 1B) was similar to the apical surface staining of MC1R on Caco-2 cells in our previous work (Váradi et al., 2017). Positive MC1R immunostaining was found on freshly isolated brain microvessels, too (Fig. 1C).

[Uhohinc]

https://res.mdpi.com/def50200c1bba91ac63fd90442840104d55e52b0c2a9cdf9d45b5edda3e5b778aa428b7b0f5033d30c890ffa8e416cceb5be87e4a045db2196cc3bbe581bdd93a7d63ff19b57db99a0c683346417cc8d6608bdf21a479d55ea3f2915163d22477b03e317c4309c93784ee1df67cbaf78d4a4d27257f3ba31ca1d92199d62b6fc18338cb154d2ca?filename=&attachment=1

Scenesse is a strong Ppar agonist.

[Vallaurix_CUV]

Tracking the Patent Applications. Note the same examiner is reviewing both Luger’s and Clinuvel’s application, John D Ulm.
https://examiner.ninja/examiners/ULM-JOHN-D

I hope Clinuvel schedule an interview with Ulm, seems the biggest variable in success and better than the written back and forth over Office Actions.

1) US20160158322A1
Ndp-msh for treatment of inflammatory and/or neurodegenerative disorders of the central nervous system
Inventor Thomas A. LUGER, Karin Loser
Current Assignee Westfaelische Wilhelms - Universitaet Muenster
Priority date 2013-08-05
https://patentimages.storage.googleapis.com/a2/66/bc/3ffa2e949ded11/US20160158322A1.pdf
https://patentscope.wipo.int/search/en/detail.jsf?docId=US174018836&recNum=1&tab=NatCollDocuments&maxRec=&office=&prevFilter=&sortOption=&queryString=

2) US20170304406A1
New indication for alpha-msh analogues
Inventor Philippe Wolgen
Current Assignee CLINUVEL AG
Priority date 2014-10-28
https://patentimages.storage.googleapis.com/6f/c5/0c/3e5bd8f1a0a9d2/US20170304406A1.pdf
https://patentscope.wipo.int/search/en/detail.jsf?docId=US205400485&redirectedID=true

[Uhohinc]

From search on google groups :

Autoimmune Encephalomyelitis msh prevents paralysis and promoted early motor function recovery. A more than ten fold increase in LAP expression on Msh induced Treg cells
11:06 AMme
https://www.researchgate.net/publication/51665072_The_Alpha-Melanocyte_Stimulating_Hormone_Induces_Conversion_of_Effector_T_Cells_into_Treg_
11:11 AMme
https://en.wikipedia.org/wiki/Experimental_autoimmune_encephalomyelitis Related to multiple sclerosis with the https://en.m.wikipedia.org/wiki/Demyelinating_disease

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Posts: 287
Thanks: 1067


Very impressive lecture from Mr. Luger from the University of Münster about the anti-inflammatory and neuroprotective effects of afamelanotide. They've found many surprising effects that haven't been published yet. I really hope, that CUV is going after these indications! Unfortunately, it's only in german language.

http://www.medroom.at/p.php?action=vortrag...2061&PREV=0

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575624/

Melanocortins, Athcar, theraputic inferences in Central Nervous System, specifically Multiple Sclerosis.

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https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008034 Two types of stem cells in hair bulb/skin

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> IL015. Invited Lecture
Symposium MED-3 Photoimmunology and photocarcinogenesis (Scott Byrne)
MECHANISMS BY WHICH NARROWBAND UVB PHOTOTHERAPY MAY REDUCE CONVERSION TO MULTIPLE SCLEROSIS BY PEOPLE WITH CLINICALLY ISOLATED SYNDROME, A PRE-FORM OF MS
Authors: Prue Hart1, Stephanie Trend1, Anderson Jones1, Robyn Lucas2, David Booth3, Scott Byrne3, Allan Kermode4 Presenting Author: Prue Hart
1) Telethon Kids Institute, University of Western Australia, Perth, Australia 2) National Centre for Epidemiology & Population Health, Australian National University, Canberra, Australia 3) University of Sydney, Faculty of Medicine and Health, Westmead Institute for Medical Research, Westmead, Australia 4) Centre for Neuromuscular and Neurological Disorders, Perron Institute for Neurological and Translational Science, University of Western Australia, Perth, Australia
Introduction
Clinically isolated syndrome (CIS) is the earliest clinical episode in multiple sclerosis (MS). Low environmental exposure to UV radiation is implicated in risk of developing MS, and therefore, narrowband UVB phototherapy may delay progression to MS in people with CIS. Twenty individuals with CIS were recruited, and half were randomised to receive 24 sessions of narrowband UVB phototherapy over a period of 8 weeks. After 12 months, 7/10 of those receiving narrowband UVB phototherapy had progressed to MS whilst 10/10 in the no-phototherapy group had progressed (ref 1).
Methods
Peripheral blood samples for all participants were collected at baseline, and 1, 2, 3, 6 and 12 months after recruitment. An extensive panel of leukocyte populations, including subsets of T cells, B cells, monocytes, dendritic cells, and natural killer cells were examined in phototherapy-treated and control participants, and immunoglobulin as well as 25(OH)vitamin D levels measured in serum.
Results and Discussion
Conflicting with our initial hypothesis, there were no significant changes in Tregulatory cells in phototherapy-treated participants. There were significant short-term increases in the frequency of naïve B cells and intermediate monocytes, as well as reduced concentrations of switched memory B cells and classical monocytes in phototherapy-treated individuals. There was reduced arginase mRNA expression after 3 months by blood cells from phototherapy-treated participants. Although phototherapy increased serum 25(OH) vitamin D levels, the changes in B cells and monocytes have not been associated previously with vitamin D supplementation.
Conclusions
Several changes in blood cell subsets have been detected in CIS people receiving narrowband UVB phototherapy. More functional analyses are required to link cell changes with a UVB-induced reduction in CIS to MS conversion. UVB-induced pathways independent of vitamin D have been implicated.
Reference
1. Hart et al., A randomised, controlled clinical trial of narrowband UVB phototherapy for clinically isolated syndrome: The PhoCIS study. Mult. Scler. J. Exp. Transl. Clin. 4, 2055217318 d12, doi:10.1177/2055217318773112 (

[Uhohinc]

OCTOBER 4, 2019
Targeting a rogue T cell prevents and reverses multiple sclerosis in mice
by Nancy Fliesler, Children's Hospital Boston

Targeting certain rogue T cells prevents and reverses multiple sclerosis in mice
T helper cells bearing the CXCR6 surface marker drive multiple sclerosis by producing a host of proteins that damage nerve fibers, by attacking their protective myelin sheath. The cells are shown here releasing GM-CSF, which stimulates other immune cells, macrophages, to release damaging inflammatory compounds. Credit: Lifei Hou/Boston Children's Hospital
Multiple sclerosis is an autoimmune disease affecting both adults and children. It's driven by "helper" T cells, white blood cells that mount an inflammatory attack on the brain and spinal cord, degrading the protective myelin sheath that covers nerve fibers. But there are many different kinds of T helper cells, and up until now, no one knew which ones were the bad actors.


Researchers at Boston Children's Hospital have now pinpointed the specific helper T cells that cause MS, as well as a protein on their surface that marks them. As reported this week in PNAS, an antibody targeting this protein, CXCR6, both prevented and reversed MS in a mouse model.

If human studies bear out the findings, targeting these rogue T cells could potentially ameliorate MS, says senior investigator Eileen Remold-O'Donnell, PhD, of the hospital's Program in Cellular and Molecular Medicine. She believes the findings could also apply to other forms of autoimmune encephalomyelitis (inflammation of the brain and spinal cord), as well as inflammatory arthritis.

"We've demonstrated in mice you can target these cells and get rid of them," she says. "We don't know if this approach would be appropriate for all cases of MS, but it could be effective in the early inflammatory stages of the disease, and in targeting newly formed cells during disease exacerbations."

Profiling MS-inducing cells

Recent efforts to pinpoint the T helper cells causing MS have pointed to TH17 cells, but some TH17 cells appear not to be involved. Remold-O'Donnell and her former postdoctoral fellow Lifei Hou, PhD, zeroed in on a subset of fast-proliferating TH17-derived cells, all bearing the CXCR6 marker. These cells, they showed, are highly damaging to nerve fibers, producing one set of proteins that directly damage cells and others, including GM-CSF, that stimulate an inflammatory attack by other immune cells known as macrophages.

Targeting a rogue T cell prevents and reverses multiple sclerosis in mice
Credit: Micheal Goderre/Boston Children’s Hospital
The CXCR6-positive cells also produce increased amounts of a protein called SerpinB1 (Sb1), the researchers showed. When they deleted the Sb1 gene in T cells in their mouse model, fewer immune cells survived to infiltrate the spinal cord. The mice also displayed fewer symptoms of disease than control mice. Moreover, these Sb1-containing cells could be readily identified with antibodies targeting the CXCR6 surface protein.



Human counterparts

But are CXCR6+ cells relevant in human disease? To investigate, Remold-O'Donnell and Hou worked with rheumatologists, immunologists, and neuro-immunologists at Boston Children's and Brigham and Women's Hospital.

They obtained and tested samples of blood and synovial fluid (from the cavities of joints) from patients with inflammatory autoimmune arthritis. Levels of CXCR6+ cells were indeed elevated in the inflamed joints, but not in circulating blood from the arthritis patients, patients with MS, or healthy controls.

Targeting a rogue T cell prevents and reverses multiple sclerosis in mice
T helper cells bearing the CXCR6 marker drive multiple sclerosis by producing proteins that damage nerve fibers by attacking their protective myelin sheath. The cells are shown here releasing GM-CSF, which stimulates other immune cells, macrophages, to release inflammatory compounds. Credit: Lifei Hou/Boston Children’s Hospital
Targeting CXCR6 in multiple sclerosis

Remold-O'Donnell and Hou, first author on the paper, believe treatments to deplete CXCR6+ cells could mitigate MS and possibly other autoimmune disorders while largely leaving other T cell immune defenses intact. When they used monoclonal antibodies to target CXCR6, the harmful cells largely disappeared, and mice, which were primed to get MS, did not develop the disease.

The two have filed a patent covering the work and have formed a company, Edelweiss Immune, Inc., in which they have equity ownership together with Boston Children's Hospital. The new company will be carrying the research forward.

"Many drugs have been developed to treat autoimmune diseases, such as glucocorticoids and cytotoxic reagents," says Hou. "However, none selectively target pathogenic T cells, and long-term use of immunosuppressive agents results in broad immunosuppression and compromised immune defenses. Therapeutics with better selectivity, safety, and efficacy are needed."

[Uhohinc]

On Friday, May 6, 2016 at 6:57:45 AM UTC-7, Uhohinc wrote:
> https://groups.google.com/forum/#!topic/clinuvel/P74TIqCRYvk  Being that "the regulators read what is posted on the investor websites" perhaps they might take note of the evidence leading to this supposition.
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> https://groups.google.com/forum/#!topic/clinuvel/P74TIqCRYvk



This implication of multiple sclerosis to immune B cell Lymphocytes may have tremendous insights as to the mcr1 on these cells and the pathways in these B cells to proper cell death (Apoptosis) thru Scenesse.

https://montrealgazette.com/news/local-news/udem-researchers-find-molecule-to-block-multiple-sclerosis-progression?utm_medium=Social&utm_source=Facebook&fbclid=IwAR150YwcsKdj1Qz1-WWeK-CDxUQQHR0J6JKV9JAngRyMvROTL9qDXZSyBFc#Echobox=1573823570

[Uhohinc]

ons

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-Melanocyte-Stimulating Hormone Suppresses Antigen-Induced Lymphocyte Proliferation in Humans Independently of Melanocortin 1 Receptor Gene Status

Article (PDF Available) in The Journal of Immunology 175(7):4806-13 · November 2005 with 33 Reads 
DOI: 10.4049/jimmunol.175.7.4806 · Source: PubMed
Cite this publication
Ashley Cooper
Ashley Cooper
Samantha J Robinson
Samantha J Robinson
+ 2
Chris Pickard
Chris Pickard
Claire Jackson at University of Southampton
Claire Jackson
33.47University of Southampton
Show more authors
Abstract
Studies in mice indicate that α-melanocyte-stimulating hormone (αMSH) is immunosuppressive, but it is not known whether αMSH suppresses human immune responses to exogenous Ags. Human PBMCs, including monocytes, express the melanocortin 1 receptor (MC1R), and it is thought that the ability of αMSH to alter monocyte-costimulatory molecule expression and IL-10 release is mediated by this receptor. However, the MC1R gene Is polymorphic, and certain MC1R variants compromise receptor signaling via cAMP, resulting in red hair and fair skin. Here, we have investigated whether αMSH can suppress Ag-induced lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. αMSH suppressed streptokinase-streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10 -13-10 -11 M αMSH. Anti-IL-10 Abs failed to prevent suppression by αMSH, indicating that it was not due to MC1R-mediated IL-10 release by monocytes. Despite variability in the degree of suppression between subjects, similar degrees of αMSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R alleles. RT-PCR of streptokinase-streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macrophages, MC1R and MC3R expression by B lymphocytes, but no melanocortin receptor expression by T lymphocytes. In addition, αMSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas αMSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that αMSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lymphocytes, has immunosoppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by αMSH is independent of MC1R gene status.




Evidence is there about B lymphocytes affected by msh, but not strong or clear if value by Msh.

[Uhohinc]

https://jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-019-1593-2

The B cells and their subsets overly proliferated and or overly activated appear to be the immune cells destroying the protein known as Mylan (spelling ?) which insulates the electrical travel thru neuron to nueron......which is multiple sclerosis.

Not much known yet of B cells. But tis study compared several drugs effect on patients on the various drugs and the study indicate B Cell counts.

The DMF is branded as Tecfedera. The drug was a Chemical used in furniture cushions to abate fungal. It took 65 years to take these observations to FDA approval. Tecfedera cost about $1,000. Dmf costs about a one cent.

To my pont. Tecfedera closely somewhat expresses gene pathway profile a little similar to
Scenesse.

Studys whereby all B cells were removed from rats caused to many infections or death.

Wil Scenesse lessen or regulate down B cells or the subset most ivolved........?

[Uhohinc]

https://www.technologynetworks.com/neuroscience/news/vitamin-d-deficiency-linked-to-loss-in-brain-plasticity-315688?fbclid=IwAR3Zo1DJ2JGoOMOkB_QMaof91-XUKBzPijsvuRc7AjZRDE6MMFHMPLZSIt8

Somewhere I posted a study but can not locate, about 6 years ago, whereby massive amounts of supplements of vitamin d alleviated multiple sclerosis v control.

[Uhohinc]

Because of nrf2 expression by Tecfedera, aka i have a post in gg not under the above header, whereby Dimethyl fumerate is an already on market drug, so the next sixth teaser to be announced indication i do not think is multiple sclerosis and 400,000 USA patients , does not meet Dr Wolgen criteria.

Incidental, I think Afamelanotide will do more positive oaths than just nrf2, in the treg cells attackiking insulative proteins of neurons.

[Uhohinc]

We have surprisingly found that the invention allows for effective yet safe and convenient treatment of neurodegenerative disorders using alpha-MSH analogues. 

Details description of the invention 

For the purpose of this invention, treatment is defined as encompassing prevention of a disorder. 

According to the invention, we have surprisingly found that alpha-MSH analogues are effective in treatment of CNS disorder of human subjects, preferably neurodegenerative disorders. For the purpose of this invention, neurodegenerative disorders are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. According to the invention, neurodegenerative disorders preferably include the diseases of Multiple Sclerosis, Alzheimer's Disease and other dementias, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS) and which is also called Lou Gehrig's Disease, Huntington's Disease, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, and Prion Diseases. 

Accordingly, in one aspect, the present invention is directed to the above-mentioned neurodegenerative disorders, preferably the juvenile form thereof, each separately and as a group. 

In another aspect, the present invention is directed to Multiple Sclerosis, preferably the juvenile form which is defined according to the invention as occurring before the age of 18 years. In another aspect, the present invention is directed to dementia, preferably the juvenile form. In another aspect, the present invention is directed to Alzheimer's Disease, preferably the juvenile form which is defined according to the invention as occurring before the age of 65 years. In another aspect, the present invention is directed to Parkinson's Disease, preferably the juvenile form which is defined according to the invention as occurring before the age of 20 years. In another aspect, the present invention is directed to Amyotrophic Lateral Sclerosis (ALS) which is often called Lou Gehrig's Disease, and preferably the juvenile form which is defined according to the invention as occurring before the age of 25 years. In another aspect, the present invention is directed to Huntington's Disease, preferably the juvenile form which is defined according to the invention as occurring before the age of 20 years. 

[Uhohinc]

https://news.clinuvel.com/2021/01/15/publication-brief-sunlight-exposure-exerts-immunomodulatory-effects-to-reduce-multiple-sclerosis-severity/

[Uhohinc]

After reading Clinuvel post today on Vitamin D and Multiple Sclerosis, I reviewed the posts above here and refreshed in my mind and can now look back with more knowledge, that Clinuvel thru mcr1 can cure multiple sclerosis. I am even more convinced this is the sixth indication......Also interesting one can follow the U of Muenster researchers Clinuvel hired to collaborate first on an acne study with a msh and see how the HUGE financial potential and the observations of Bohm and Luger led to them involved in jumping out of line and filing patents which Clinuvel had to litigate away from them, alledgedly imo.

An incredible luck, to find a person whom had leuikemia AND multiple sclerosis gave info that MS was gone when that patients entire immune cell originating from their marrow was destroyed by radiation proved that the cause of MS was in cells originating in the marrow, not the brain or cells in it giving signaling yet those cells accessing the brain and destructing the myelin proteins.

And everything (every single gene expression, interlukine,  oligodentrite, etc......etc......too many to re mention......of Scenesse effects those pathways in the T reg cells to block the attacking immune cells on the myelin.

AND Scenesse on the mcr1 on the cells which repair the myelin, there is corrobative info that it will instigate repair to these proteins.......The negative here for Clinuvel is all the drugs such as Tecfedera, which i have alot of research on gg, Copaxan, Avonex, Beta something and a couple of newer ones which are FDA EMA appproved for ms but i do not think really work well. These are billions in revenue and these companys will not like Clinuvel disrupting.........

Uhohinc<michaelba...@gmail.com>

Dec 4, 2020, 11:33:04 AM

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to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9

Imo.......Alzheimer’s is LOGICALLY NOT Clinuvel’s next prospective therapy......going for it would be great for stock hype publicity and grandiose but not Clinuvel managment style..Clinuvel has been great at managing rabbit holes and not just laborious at what they have done at the time they done it but in what they did not do at the time..(..That is melanocortin  maniPalatinuation 6 months ago working their stockholders with illusions/delusions of announcing for Covid therapy and trials timeline with years with easy topoint then of vaccines by December.)

Despite this, Imo melanocortins will be very therapeutic in Alzheimer and Covid type virus. Separating Alzheimers from complexing just the part related to melanocortins first. Decades and billions of research dollars into AD has generally consensoused  thinking AD was the buildup of beta amyloid plaques in neurons in brain......and that removing or preventing this will stop prevent AD.....This made sense as AD brains had these plaques that appeared to be cause/symptomatic/affect/effect.

In the last few years the researchers started getting contradictory results, and some started to look away from the beta amyloids to tau protien entanglements........i recall posting one study about a year ago which summarized the dumbfoundeness I read into Pfizer closing a trial with huge investment into AD based beta amyloid reduction....

AD has so much more complexity whereby just not as much is known ......to many open spaces between dots and connecting info.  A lot more than Multiple Sclerosis and Parkinson. Huntington’s is more directly gentic. Dementia in general is too generally just encompassing of various symptoms. More theory to AD still needing some fact.

There are five plus at least, real positive prospective  pathways an msh Afamelanotide will affect in varioius neurons and area brain cells.......autophagy for clean out of mitochondria, and incredibly neurogenisis apparently involving Sirt1 (phenominal implication) etc.....but which melanocortin and receptor, with Clinuvel mostly mcr1 oriented and as the patent states surprise.

Noted, AD is probably an inflammatory orgined and responsed disorder......which suits msh.

Of the major neurodegenerative disorders. Imo AD is a very slow progression comparatively. And less predictive of the graduation of that progression in individual to individual. Parkinsons is similar in general. Multiple Scerosis is more sporadic with starts and stops......(symptoms stop or progression stops when a women gets pregnant)

....other than mice, gerbils, and rats,  it is very difficult to attest or observe stages of Alzheimers in rodents.....and its longer term.......for Clinuvel to “ surprise”  (patent term used) then we may assume the observation was or has been in human Afamelanotide trial or now EPP patients.

With all the neurodegenatives, we are talking years in most Multiple Sclerosis, ALS, PD, ( Huntingtons, to rare and genitic) but with Alzheimers we are looking at even longer progression.

We can assume no Afamelanotide patient whom was diagnosed with Alzheimers has died and an brain autopsy confirmed a positive affect in that patients observered or self reporting......just another aspect of the complexity of and attempting a trial by Clinuvel in AD. 

Of many. With what is not known about AD just adds to many more impracticalities in making it a target when there are targets still big but easier in sites...

The odds of a on Afamelanotide patient with EPP also having AD, PD, or MS are there now with these disorder prevalences.....but MS is much more (in compare) in the youth starting at age 18 and on.

Despite Michael Fox, PD is rare until age 50 but more so in older than that. And AD is in aged.

Clinuvel is not prognosed in 75 and older.

Logically I think there are more informational links in knowledge to me to make me think PD would make a more sure target.........but collectively with all info to all taken into account I think Multiple Sclerosis is the Clinuvel next target indication......

And what is the number one reason, but not only, that I think points to Multiple Sclerosis is really not taking into account any logicals ir data, or abstracts, or scientific based studies...........(number 2 reason is the German Homm with MS and his history to Clinuvel)

.............its because Multiple Sclerosis was the first of mention in the patent by Clinuvel, which secondarily to sequentially  mentioned the other neurodegenerative disorders. Sometime human behavior is simple.



[Uhohinc]

sharelookerWell-known member

21 minutes ago  

• #4,812

seeva222 said:

@macgyver All signs point to MS

Not really! PW stated very clearly "...for which there is absolutely no effective treatment"

The MS space is very crowded. There are a lot of highly effective treatments available, manufactured by the big sharks like NVS, RHHBY, BIBB etc. And after reading the impressive preclinical results of the BNTX MS vaccine, i think it's not a good idea to go after this indication.

I agree with the incongruity and worth pointing at again in "for which there is absolutely no effective treatment" and have explained away in that essentially MS, PD, AD, Huntigtons and essentially every degenerative neuron disorder has some "treatment"  (not a cure, and effective ?)

But Dr. Wolgen qualified it with "effective"........so it is hard to come to terms with the semantic of and subjective of this adjective and then find any disorder that qualifies.

Aside from this rationalizing of the words Dr Wolgen used, the revolution taking place with RNA is a huge door that already has been shown in innovation and speed of the covid vaccines.

The BNTX MS vaccine in its press release, looks like a game changer and pivotal. Need more detail, as typically a vaccine (as described) creates an customized anti-body to specifically go after the culpritting protein. In MS, it is one own proteins and immune cells.....

The RNA descriptive would be more suitable as my understanding in generalization is that RNA is mostly but not conclusively a messenger protein that interferes with or  a blocking of an DNA actions.

I still think Clinuvel has in mind MS as the 6th indication that has been progressing, but this BNTX therapy in mice may be a Clinuvel disruptive technology. To all present drugs. RNA therapy proteins may be faster in getting here than Crispr Cas 9 gene editing, or in conjunction.

[Uhohinc]

the below, I think is a very interesting correlation to multiple sclerosis, note the study above  in which super super high dose of vitamin D stopped the progression of MS

Synergy of melanin and vitamin-D may play a fundamental role in preventing SARS-CoV-2 infections and halt COVID-19 by inactivating furin protease

• Kishalay Paria, 
• Debarati Paul, 
• Trinath Chowdhury, 
• Smritikana Pyne, 
• Ranadhir Chakraborty & 
• Santi M. Mandal 

Translational Medicine Communications volume 5, Article number: 21 (2020) Cite this article

• 6733 Accesses

• 34 Altmetric

• Metricsdetails

Abstract

Since the birth of Christ, in these 2019 years, the man on earth has never experienced a survival challenge from any acellular protist compared to SARS-CoV-2. No specific drugs yet been approved. The host immunity is the only alternative to prevent and or reduce the infection and mortality rate as well. Here, a novel mechanism of melanin mediated host immunity is proposed having potent biotechnological prospects in health care management of COVID-19. Vitamin D is known to enhance the rate of melanin synthesis; and this may concurrently regulate the expression of furin expression. In silico analyses have revealed that the intermediates of melanin are capable of binding strongly with the active site of furin protease. On the other hand, furin expression is negatively regulated via 1-α-hydroxylase (CYP27B1), that belongs to vitamin-D pathway and controls cellular calcium levels. Here, we have envisaged the availability of biological melanin and elucidated the bio-medical potential. Thus, we propose a possible synergistic application of melanin and the enzyme CYP27B1 (regulates vitamin D biosynthesis) as a novel strategy to prevent viral entry through the inactivation of furin protease and aid in boosting our immunity at the cellular and humoral levels.

Introduction

SARS-CoV-2, is the root cause for the late pandemic, novel coronavirus disease 2019 (COVID-19). A sick individual experiences mild to serious respiratory problems. Among infected populations, barely any symptomatic patients recoup without hospitalization and the vast majority of them are hospitalized for extraordinary treatment. This is a remarkable worldwide war, where hospitals are in the front line and doctors being commanding officers along with the medical support team, are constantly battling against COVID-19 [1, 2]. By and large, COVID-19 manifests symptoms like other SARS-CoV- infected diseases, advances rapidly towards developing ARDS (acute respiratory distress syndrome) with septic stun, in worst circumstances failure of multiple organs take place because of viral-infection-instigated cytokine storm in the body [3]. The novel corona virus or SARS-CoV-2 is commonly spread via little miniscule droplets liberated into the surrounding environment when the infected persons unguardedly sneeze, cough or even talk with people in close contact [4].

There is no affirmed anti-COVID-19 medication in the existing shelf [5]. A few clinical trials are currently in progress and a few drugs, for example, chloroquine, remdesivir, arbidol, and favipiravir have been tried yet none of them is fruitful altogether to improve the survivability rate [6]. As of now, there are no particular guidelines and treatment regime for COVID-19. Most treatment methodologies are symptomatic and based on supportive therapy. Scarcely any medications have demonstrated great adequacy at the cell level which need further trial and approval. A few antimicrobials including antiviral drugs were utilized to treat COVID-19 patients, for example, blend of remdesivir or lopinavir or ritonavir and chloroquine [7,8,9] also several drugs are in pipeline [10]. Following application of drugs hostile to viral and other microbes, the shattered natural parity of the gut microbiome further contributes to the progression of morbidity in patients. Corona patients predominantly experience the ill effects of decreasing white-blood corpuscles (WBC) and lymphocytes when there is an urgency of maintaining a threshold level of cytokine level including IL-6 and IL-10 [11]. In this situation, coordinated host-immune based treatments remain decisive to get by against COVID-19.

Nutrient treatment, especially Vit-C and Vitamin D, is a long known practice against the coronavirus affected patients [12]. As of late, phase-3 trials of Vitamin D treatment with different dose management for COVID patients are in progress [13]. Vitamin-D might be a possibly intriguing steady treatment against SARS-CoV-2 infection. Anyway, no logical proof or scientific evidence has been perceived up to this point. Here, a cross-talk between Vitamin D and melanin synthesis pathway has been reported with a fascinating observation where by-products of melanin synthesis unequivocally tie to the dynamic site of human protease furin which is vital for the SARS-CoV-2- mediated disease progression [14].

Vitamin D: contribution to human health

Vitamin D is commonly obtained from food sources or synthesized within human skin [15]. It is widely documented that this vitamin modulates both the adaptive or innate immuity (Fig. 1). Vitamin- D receptors (VDRs) that are displayed on B-cells/ T-cells or on the APCs (antigen presenting cells) can synthesize an active metabolite from vitamin-D. An essential activity of Vitamin D is the maintainance of calcium homeostasis and skeletal health. In the liver, hydroxylated Vitamin D enters a dynamic form, i.e. 25 OH vitamin D3 (also called 25D). Inside the kidney, 25D gets transformed to another dynamic form i.e. -1,25,dihydroxy vitamin D (1, 25 D), also called calcidiol via the action of an enzyme, 1-α-hydroxylase (CYP27B1). Subsequently, 24-hydroxylase (CYP24) converts 1,25 D to an inactive compound i.e. 1,24,25 vitamin D (Fig. 2). 1, 25 D in active form impacts the intestine and bones after binding to the VDR [16].

Fig. 1

Schematic representation of the cross road between sunlight, vitamin D synthesis, activation of melanocytes and their regulation of immune molecules

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Fig. 2

Consequences of Vitmain D in human health and their metabolism in liver. The hydroxylated form, 25 OH vitamin D3 (25 D) in synthesized in liver and converted to most active form, 1,25,dihydroxyvitamin D (1, 25 D) by 1-α-hydroxylase (CYP27B1). CYP27B1, is the key factor to vitamin D biosynthesis

Full size image

A few cross-sectional investigations uncovered that Vitamin D content in the body are directly connected to the progression of diseases such as flu [17], similar to other infections caused by virus for example, HIV etc. [18, 19]. There is an immediate relationship between low levels of vitamin D with diminished resistance and higher rates of viral diseases (Table 1). It is well documented that macrophages use toll like receptors (TLR) to detect lipopolysaccharide (LPS), LPS being a preffered target for bacterial infection. TLR on the macrophages prompts enhancement in expression of both, 1-α-hydroxylase and the VDR. People are increasingly inclined to viral infections of the upper part of respiratory tract, owing to lower vitamin D content, as compared to people who have adequate levels. The level of vitamin D fluctuates with the age, season, sex, race and body mass which is like wise connected with viral infections [32]. The expression of VDR is seen in brain, breast, bone marrow, colon, malignant cells and immunogenic cells other than skeletal and intestine, propose ingthat vitamin D have major protective role by maintaing the homeostasis of calcium in bone tissue as well as increasing the immunity to battle against human pathogens [33].

Table 1 List of viral pathogens evidenced for anti-viral feat by the consequence of Vitamin D

Full size table

Vitamin D mediated innate immunity

During respiratory infections, the pathogens go into the respiratory tract and begin to colonize in epithelial cells, also immediately the innate immune and inflammatory signalling network start to respond [34]. Firstly, neutrophils enter the parenchyma cells of the lung and colonization begins within an hour of infection. Subsequently within few days of infection, it effects the natural killer cells, monocytes/macrophages and T cells. RIG- like receptor and TLRs recognize viral pathogen and participate in anti-viral defence system by producing cytokines and type I interferon (IFN). The IFN and proinflammatory cytokines hinder viral replication and translation thereby controlling the infection [25]. These immune molecules additionally actuate viral cleavage, repress viral fusion by the activation of cytolytic cells and stimulate humoral factors, for example, acute-phase proteins, collectins, defensins, including complement proteins [35, 36].

In the presence of viral antigens, macrophages and dendritic cells start processing the CD 4+ and CD 8+ T cells inpresent in the affected lymphatic nodes. After that T cells move into the infected tissue to intervene pro-inflammatory and cytolytic impacts [37]. Then again, T-helper cell promotes B cell proliferation as well as differentiation into plasma and memory cell, they undergo antibody class switching to synthesize both IgA and IgG [35]. B cell delivering antibody forestalls the entry of virus into cells and prompts phagocytosis by the innate immunogenic cells. Subsequently, the innate and adaptive immune responses can assume cooperative role for protection of individuals from respiratory viral infections [37].

Among all vitamins, only vitamin D is synthesized by the exposure of sunlight (280–315 nm) on the skin. During exposure of sunlight to the keratin rich cutaneous membrane, 7-dehydrocholesterol is finally converted to vitamin D3, calcitriol [38]. Majorly systemic vitamin D is skin- based while a small fraction likewise originates from the dietary supplementation [39]. The intermediate, 1,25(OH)2D3 regulates over 1000 genes after interaction with VDR. Epithelial cells express a critical level of VDR with SNP, related to risk posed by of RSV infection [40, 41]. 1,25(OH)2D3 invigorates maturation of NK cells, neutrophils and macrophages inside the respiratory tract, and furthermore a few antimicrobial peptides (AMPs), for example, cathelicidins and defensins [42]. Such AMPs have shown anti-viral (mainly anti-influenza) impact by associating with hCAP18/LL-37 [43]. Then again, the expression of CD14 and TLR are also likewise affected by 1,25(OH)2D intermediate [44]. The increased activity of macrophages decreases the activity of autophagy during the infection [45]. Autophagy is a cytokine induced cellular homeostasis process. Autophagy is coupled to IFN-α /CXCL10 release to stall viral replication, during infection by Influenza-A [46]. Therefore, vitamin D interceded autophagy restraint can control the respiratory viral disease of lungs. The innate immune response by the action vitamin D become more extensive to energize the movement of myeloid dendritic cells to lymph organs to stimulate specific TH cells/ and B cells [47, 48]. Pro- inflammatory cytokine is likewise hindered by Vitamin D. At the time of influenza-A infection, 1,25 (OH)2D can reduce tumour necrosis factor (TNF)-α and interleukins e.g. IL-8, IL-6, IFN-β, and RANTES in epithelial layer of lungs [21]. Especially viral replication rate, level of cytokine is high in more pathogenic viral strain than less pathogenic strain [49]. Respiratory viral infection rate are lower in summer than winter [47]. Vitamin D can control respiratory viral infection by modulation of adaptive immunity through down regulation of cytokine level of TH1 and TH2 [50] but upregulation of T regulatory cell [51]. Strikingly, it was seen that Infants who are not exposed to sunlight are found to suffer from lower respiratory tract infection and a low level of 25(OH) D was detected in blood sera [52]. Report showed that sunlight-exposed mother give a high measure of vitamin D to a kid than mother without exposure to sunlight. Sunlight exposure as well as dilatory vitamin D is necessary to maintain foetal growth and development of immunity, along these lines sunlight is an important stimulator of vitamin D synthesis [53].

Cross-talk between vitamin D and melanin biosynthesis

Human skin spontaneously produces vitamin D during exposure to sunlight. This procedure is a photochemical reaction initiated after 7-dehydrocholesterol present in our epidermis absorbs UV-B and therefore the synthesis depends on the few factors such as UV-B dose, temperature, and lipid environment [54]. Melanin pigmentary system affect vitamin D signalling via the linkage between melanogenic machinery in skin and circulating 25(OH) D of Caucasian individuals [55]. Melanin absorbs UV-B (290–320 nm) and participates in the filtration of light which determines the amount of the UV-B radiation to be penetrated in the skin epithelium [56]. It is confirmed that the world’s racial distribution by latitude is regulated by vitamin D production in individuals [57]. When people migrated from lower latitude to higher latitudes; their skin colour faded due to decreased sunlight. Thus, skin pigmentation is a dominant variable for regulating vitamin D3 synthesis in competition to melanin with 7-dehydrocholesterol [58]. In these conditions, a famous hypothesis “vitamin D-folate hypothesis”, portrays the explanation behind an apparent adaption of human skin shading in UV radiation situations. Vitamin D and folate have diverse sensitivity level against UV radiation, strangely when vitamin D is blended utilizing UVR exposure and afterward folate is degraded. The proposed supposition of “vitamin D-folate hypothesis” is that pigmentation of skin keeps up the cell homeostasis of vitamin D [59]. Several alternative theories restrict this hypothesis. Experts in the area of vitamins do accept theories on converse relationship between a cutaneous pigmentary framework and vitamin D creation [60, 61].

In normal daylight, UVB is the dynamic radiation for vitamin D synthesis over UVA. In an investigation, it is reported that fair complexion individuals need an exposure of 20–30 min and a few times in 7 days to create 20,000 IU of vitamin D3, while a brown complexion individual with high melanin level requires 2–10 fold more exposure time for equivalent level of vitamin D3 [62]. A synergistic action of Vitamin D and melanin in the skin, is fundamentally imperative to screen the levels of 25-(OH) D in youngsters, the pregnant lady just as young and old. Both, melanin and vitamin D have protective role against viral infection as well as bacterial or fungal diseases [63]. Several factors mediate melanin and vitamin D syntheses in the skin. However, information is not adequate on the synthesis of various categories of pigment and their direct correlation with vitamin D. At the point when a patient is being hospitalized with limitation of UVB exposure, the individual must have constraint of vitamin D synthesis. As a matter of fact, the regulation of vitamin D synthesis and involvement of melanocytes with their regulatory activities have not been studied in details under diseased condition.

Melanin

Indole polymer containing melanin pigments were found in five kingdoms from Monera to Animalia. Melanin goes about as biomarker for evolutionary study. It is considered as most ancient pigments of nature which have been recognizedin fossils of dinosaurs and feathered creatures. From the Jurassic time frame these are found in cephalopod ink sacs [64]. Melanins are three sorts, for example, eumelanins, pheomelanins and allomelanins [65]. Alongside natural conditions, for the most part, two shades are liable for the hue of human skin, for example, eumelanin and pheomelanin. In melanin synthesis pathway, principally the catecholamine precursor 3,4-dihydroxyphenyl alanine (DOPA) is produced from tyrosine, after oxidation which is converted to 3,4-dioxyphenylalanine (dopaquinone) which cyclisize to 5,6-indole quinones and polymerize to melanin (Fig. 3). Dissolvable melanins are orchestrated after L-ß-3,4-dihydroxyphenylalanine (L-DOPA) is oxidized or after L-tyrosine is chemically oxidized [66].

Fig. 3

Schematic representation of the melanin synthesis including pheomelanin and eumelanin in human

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Melanin not just secures photo-induced damage during absorption of high range of the electromagnetic spectrum, but also ensure protection against both chemical and thermal stresses. In this way, melanins are widely utilised for manufacturing photo protective creams/ cosmetics, and also for designing eye glasses or for treating radioactive wastes [67]. Melanin can not only shield our skin from UV radiation-damage and keep up thermoregulation, it also aids in managing stress response, metabolism, and immunity [68]. Prior reports confirm that both, alfa -melanocyte stimulating hormone and melanin-concentrating hormone synthesizing gene of vertebrate are highly conserved, that bind to MRC (melanocortin receptor) of tissue and liable for different physiological activities including defence against parasitic infection [69]. Then again, melanocortin ligand (alfa MSH and ACTH) helps in the expression of MHCI [70].

Along these lines, melanin is every now and again utilized as healthcare material as it have cancer prevention properties, antiviral, antimicrobial, antiinflammatory, antitumor, immuno-stimulating, and radioprotective activities [71]. Synthetic melanin under in-vitro condition was found to inhibit replication of HIV-1or HIV-2 and impeded HIV-1 envelope surface glycoprotein but did not interfere with the activity of reverse transcriptase enzyme [66] and is additionally used for treating metastatic melanoma in human [67]. Therefore, it is accepted that melanin pigment has a significant preventive role against both malignant growth and infectious diseases.

Furin mediates the pathogenesis of cancer and viral infections

In excess of 500 proteases are accounted for in human genome having significant job as molecular scissors in all physiological process. Proteolytic cleavage regulates several physiological and pathogenesis pathways leading to either health and disease. One of the generally significant and ubiquitously expressed proteases is the serine protease furin [72]. Furin is responsible for the activation of several virus particles. Viruses of enveloped proteins, just as non-enveloped, are processed by this protease along with other proteases for the entry for producing mature virions that are ready for infection. Hormones, growth factors, cytokines, and receptors are the mammalian substrates of furin and unusual activity of furin is directly associated with a multiplicity of dieases, including cancer and viral or bacterial infections [73].

Furin has a place with the group of exceptionally explicit, calcium-subordinate proprotein/prohormone convertases (PCs) [74]. This endoproteinases highlighted a synergist area of homology to subtilisin and initiate an enormous number of emitted proteins by constrained proteolysis. Furin is a sort I transmembrane serine-protease that is universally communicated and cycles from trans-Golgi systems to cells, via the endosomal framework. In warm-blooded animals, the PC family grasps seven individuals that divide after different fundamental deposits at the site of cleavage (R/K)Xn(R/K)↓ (here “↓” denotes scissile peptide security), with furin especially perceiving the sequence R-X-K/R-R↓ [75]. This exceptional succession explicit cleavage is basic for the initiation of various PC substrates.

Although, furin is normally expressed by various cells, its mRNA and protein levels fluctuate depending on the type of cells/ tissues; significant quantities being present in bone marrow, salivary glands, and liver while, in muscle cells furin production is comparatively lower [76]. The pro-peptide of furin is transferred to the trans-Golgi network (TGN) from endoplasmic reticulum, during which autoproteolytic process occurs in two steps such that furin becomes enzymatically active [77]. Simultaneously, N-linked oligosaccharides are incorporated and the peptide is trimmed. Since furin levels are elevated in the TGN, it can be transferred to the surface of cells and back to the TGN, via the endosomal pathway [78]. At last, furin is shed and discharged as extracellular protein after the proteolytic cleavage of its catalytic membrane-bound domains [79]. The capability to act upon a range of cellumar substrates within the cell or in the extracellular spaces is related to the ubiquitous presence of furin, not only within the TGN and endosomal- compartments, but also on cellular surfaces.

Mostly glycoproteins present on the viral envelope are proteolytically severed before entering host cells. May a times, viruses utilise cellular enzymes e.g. trypsin or subtilisin-like endo-proteases for such actions. Furin which is a subtilisin-like protease recognise and cleave at polybasic locii; a trypsin-like protease however, perceives mono-basic sites to cut next to any single Arg or Lys residue [80]. Various reports have shown that glycoproteins belonging to the coat of several viruses (Borna, Pneumo, Orthomyxo, Herpes, Flavi, Toga, Bunya, Filo, Paramyxo, Corona, and Retroviridae) are cleaved by Furin, although these viruese are evolutionarily divergent.

In silico analysis among furin and melanin intermediates

Albeit viral furin substrates by and large contain polybasic canonical cleavage site, its active site binding pocket is conserved in many species. Viral glycoproteins and furin protein, both enter the secretory pathway, allowing proteolytic cleavage at various times during replication of viral genome. The proteins coat of few viral strains are produced separately and not along with the genome in producer cells, while in others the protein envelop is extracellularly processed before the virus attacks another target host cell. A good number of viruses utilise furin and other proprotein convertases (PCs) in order to regulate their entry into host cell and develop high pathogenicity [72]. It is well documented that several growth factors, receptors, matrix metalloproteinases and viral envelope glycoproteins are involved in the conversion to their bioactive forms [81, 82]. Recently, in silico to in vitro strategies are undertaken to hinder the furin activity for SARS-CoV-2 spike glycoprotein cleavage repression [83]. Therefore, furin is a taget molecules to halt the entry a number of viruses. There is a direct correlation was also observed between furin and melanosome biogenesis. It was evidenced that the intralumenal fibrils are required to cleave the Pmel17 by a furin-like proprotein convertase (PC). The cleavage of Pmel17 liberates a lumenal domain fragment that helps to regulate the melanosome biogenesis by controlling the fibrillogenic activity [84].

The catalytic domain of furin binds to the target site of catalytic triad (ASP153, HIS194, SER368) with a distinguished oxyanion hole (ASN295). Apart from that, the residue from SER253 to PRO256 likewise demonstrates a strong affinity to small molecule to bind to furin. The accompanying sections feature the interaction of furin with few important intermediates (available structures in PubChem) of melanin biosynthesis pathway.

Energy minimization and molecular docking

Protein Data Bank file for Human Furin (PDB ID: 4RYD) was utilizedas receptor molecule and Melanine (PubChem CID:6325610), Eumelanine (PubChemCID:102582077), L-DOPA (l− 3,4-dihydroxyphenylalanine) (PubChem CID:6047), L-Dopaquinone (PubChemCID:44229226),was taken as ligand molecule for docking. Each molecule was subjected to energy minimization using ChemBio3DUltra 13.0 software, a high quality workstation where MM2 energy minimization of each molecule was identified with stable molecular conformation. Least RMS gradient taken was 0.010. Studies on docking of Melanin, Eumelanine, L-DOPA and L-Dopaquinone with Human Furin was performed using iGEMDOCK v2.1 software by using a basic algorithm to perform automated dockings. The software called AutoDockVina was additionally utilized analysis of results obtained after molecular docking. This software used Pyrex tools or Auto-Dock Tools (ADT) [85]. Gasteiger charges were determined after removing water residues from macromolecules. The ligands and macromolecules were fed into the Pyrex tool [86]. Finally, ligand and receptor files were exported as “pdbqt” format files.

Molecular docking analysis and self-protective benefit

From the results of molecular docking, it was observed that melanin, eumelanine, L-dopaquinone and L-DOPA emphatically bind with the active site of furin protein and therefore, forestalling the viral entry unwaveringly. In silico docking investigation of melanin with furin protein clearly delineates a binding affinity of − 95.25 kcal/mol (Table 2). Melanin interacts with the residues-HIS194, ASP258, ALA292, SER253, TRP254, GLY255, SER293, GLY294, ASN295, THR367 of furin protein. From Fig. 4A and A’, the interaction of melanin with the residues SER253, TRP254 and GLY255, the inhibitor binding site, where melanin binds alongside the single residue of catalytic triad (HIS194) with the oxyanion opening (ASN295) of furin protein. Another docking was performed with eumelanine with furin protein which shows strong binding affinity of − 119.51 kcal/mol (Table 2). Eumelanine binds with the residues-ARG197, ASP153, ASP191, ARG193, ARG197, GLU257, HIS364, THR365, HIS194, LEU227 of furin protein (Fig. 4B and B’). In this interaction study, the interacting residues ASP153 and HIS194 are part of catalytic triad where eumelanine emphatically binds strongly. In the docking study of L-Dopaquinone with furin protein, the binding free energy was determined to be − 77 kcal/mol (Table 2). L-dopaquinone interact with the six residues, ARG197, ARG193, HIS194, ARG197, HIS364, andTHR365, of furin protein (Fig. 5A and A’). Here, additionally one of the residues of catalytic triad (HIS194) was associated with the interaction study. Docking study of L-DOPA with furin protein additionally uncovers a binding affinity of − 77.15 kcal/mol (Table 2). The interaction of L-DOPA with furin obviously shows an involvement of PRO256, ASP258, SER293, GLY294, ASN295, ASP306, SER368, TRP254, GLY255 residues of furin (Fig. 5b and b’). The residues TRP254, GLY255, PRO256 are the inhibitor binding site of furin protein where L-DOPA binds alongside the association of one residue of catalytic triad (SER368).

Table 2 Determination of binding free energy of docking interactions between melanine, eumelanine, l-dopaquinone and l-dopa with human furin (4RYD) using iGEMDOCK

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Fig. 4

Docked image ofHuman Furin (PDB ID: 4RYD) with Melanine viewed in in PyMO (A). Zoomed image of ligand binding site of Furin-Melanine complex (A’).Docked image of Human Furin (PDB ID: 4RYD) with Eumelanine viewed in in PyMO (B). Zoomed image of ligand binding site of Furin-Eumelanine complex (B’)

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Fig. 5

Docked image ofHuman Furin (PDB ID: 4RYD) with L-Dopaquinone viewed in in PyMO (A). Zoomed image of ligand binding site of Furin-L-Dopaquinone complex (A’).Docked image of human Furin (PDB ID: 4RYD) with L-DOPA viewed in in PyMO (B). Zoomed image of ligand binding site of Furin-L-DOPA complex (B’)

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Availability of melanin and perceived opportunity for pharmaceutical/ biotech-industry

As the tremendous preventive role of melanin in curbing the spread of several diseases could be perceived (Table 3), a real opportune-market of pharmaceuticals will be ensuing very shortly. Melanin synthesized in skin with the aid of sunlight, is impracticable for a patient to replenish during hospitalization. In case of long run, the genetic level of expression varies among individuals, races and seasonal. Therefore, the availability of melanin is an significant issue about its availability in healthcare application as ‘preventive medicine’. Other than mammals, melanin from microbial sources is available in the market [92]. Next to chemical-synthesis, it can be resourced from many living entities including plant, animal, fungi and bacteria. As of late, eco-friendly microbe-derived melanin has stood out as a biotechnology option in contrast to synthetic/chemical production [93]. By and the large microbial melanin- synthesizing enzyme belongs to laccase and tyrosinase group. The tyrosinases are prevalently associated with melanogenesis. In view of amino acid sequences and catalytic activity, microbial tyrosinases belong to five main classes [94]. Although, tyrosinases (mono-oxygenases) contain dinuclear copper catalytic centre, which catalyse ortho-hydroxylation of mono-phenols (cresolase activity), it can also oxidize catechols (catecholase activity) for synthesis of ortho-quinone. Microbial biosynthetic route of melanin production is summarized in Fig. 6. Be that as it may, tyrosinase of a few microorganisms, for example, Rhizobium etli, Bacillus megaterium, and Bacillus thuringiensis don’t require copper for activation of chaperone protein. Then again, another melanogenic enzyme laccase (copper-dependent oxidoreductases) is principally found in plant, fungi and bacteria [95].

Table 3 Definite action of melanin against bacterial and viral pathogens

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Fig. 6

Biosynthesis pathway of melanin in microbes

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Melanin from bacteria

Like mammals, bacteria are also capable of synthesizing melanin [67]. Several bacterial genera, such as Aeromonas, Bacillus, Azotobacter, Legionella, Proteus, Micrococcus, Mycobacterium, Azospirillum, Pseudomonas, Rhizobium, Shewanella, Streptomyces, Escherichia, Bacillus, Klebsiella and Vibrio. have been reported to produce endogenous melanin [64]. Streptomyces spp. has melanin operon (melC) constituted of melC1 and melC2 genes that codes for apotyrosinase and tyrosinase enzyme repectively [93]. A group of bacteria is known to produce melanin from L-tyrosine using the key enzyme 4-hydroxyphenylacetic acid hydroxylase [96]. Furthermore, bacterial genes were used in the form of reporter genes to screen recombinant bacterial strains, for synthesis of melanin [97, 98]. A list of selected bacterial and fungal origin of melanin production is provided in Table 4.

Table 4 Enhanced melanin production from microbial in origin at a glance

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Melanin from fungi

Several fungi can produce appreciable amount of melanin from dihydroxynaphthalene (DHN), γ-glutaminyl-4-hydroxybenzene, HGA, tyrosine and catechol. The habitat of about 80% of melanin producing endophytic fungi, in Antractica, is a herbaceous plant, Deschampsia Antarctica Desv (Poaceae). Melanized fungi, for example, Trimmatostroma salinum, Hortaea werneckii, Aureo basidium pullulans, Phaeotheca triangularis, and Cladosporium sp. live in salterns [105]. Some melanin producing fungi are found in habitats polluted with heavy metals and unsaturated hydrocarbons from industries and urban wastes [92]. A. fumigatus by the virtue of its ability to synthesize dihydroxynaphthalene (DHN) melanin and mobilize the same in the gray-green colour conidia, can inhibit many pathogens. DHN melanin,the product of pksP gene, has been shown to protect A. fumigatus from reactive oxygen intermediates (ROI). PksP inhibit the lysosome - phagosome fusion to destroy the conidia of A. fumigatus [106]. Cryptococcus neoformans, Aspergillus fumigatus and Pneumocystis cariniietc are the major fungal strains reported to produce melanin [107]. Besidesphotoprotection and antioxidant activities, fungal melanins arecapable of rendering resistance against several biotic, abiotic, and radiation stresses, such as antifungal agents, oxidizing agents, salinity, draught, heavy metals, thermal, UV & electromagnetic radiation [106,107,108]. Human melanin has structural similarity with fungal melanin, more so, melanin extracted from the fungus, Cryptococcus neoformans, has been used for the production of monoclonal antibodies (mAb) for treating patients suffering from metastatic melanoma; this monoclonal antibody is capable of binding tohuman melanin [98].

Recombinant melanin production

Alongside wild type bacteria, many recombinant bacterial strains were constructed for production of melanin. E. coli was utilized as a host for producing the first recombinant melanin by cloning and expression of specific genes of actinomycete Streptomyces antibioticus. Primarily mel, and ORF438S genes are involved in melanin production [67]. The modified strain of E. coli produced eumelanin at 30 °C in the culture medium following consumption of L-tyrosine [108]. Synthetic amino acids, e.g., L- tyrosine ethyl ester and N-acetyl-L-tyrosine have been propsed for use melanin synthesis by S. antibioticus tyrosinase [109]. E. coli strain JM109 was used to clone the mel gene of S. antibioticus and phage T5 promoter and lac operators were used for transcription [67]. In laboratory culture condition, recombinant strain of Streptomyces kathirae SC1 was also used to produce considerableamount of melanin by tuning the expression of melC gene [93].

Cross-talk between vitamin-D and furin protease

The function of our inherint immune system depends on vitamin D content that in turn leads to the protection of our health and well being. As discussed previously macrophages attach to LPS, during bacterial infections, viaTLRs, leading to increase in expression of CYP27B1 (1-α-hydroxylase) and the VDR (Fig. 2). Individuals showing low levels of vitamin D (< 30 ng/ml) were bound to complain about constant infections in the upper respiratory tract than individuals possesing adequate Vitamin D levels, depending on their, age, gender, body mass, race and season [32].

VDRs are expressed by the brain cells, bone marrow, breast cells and skeletal and intestininal cells. This suggestes that vitamin D may have a role to play, other than the established function of maintaining calcium and bone homeostasis [110]. Furin (ubiquitous endoprotease present within constitutive secretory pathways) activity in other pathways (parathyroid hormone processing, pro-factor IX, etc) is controlled negatively by both, calcium levels and vitamin D in the secretory pathway [111,112,113]. Increased Furin activity enhances the role of TMPRSS 2 in viral-entry intothe host cells. Negative regulation by Vitamin D may bring down the furin activity and associated TMPRSS2 action thereby decreasing the chances of coronavirus infection (Fig. 7).

Fig. 7

The diagram shows the probable associations between various proteins (analysed using STRING software). The main nodes are formed by FGF23, CYP27B1, CYP24A1, PTH, FURIN, etc. The diagram indicates the importance of vitamin metabolism involving FGF23, CYP27B1, CYP24A1 and the regulation of Furin ultimately, which in turn is responsible for entry of coronavirus into the cell by interaction with TMPRSS2

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Conclusion and future perspective

Melanin is an useful polymer classified as a natural product having numerous potential applications in the industry based on bioresources including plants, microbes, animal cells and from semisynthetic blends. Skin pigmentation is, in fact, a predominant variable regulating the production of vitamin D3 under conditions of low degrees of solar-illumination since melanin retains UV photons in rivalry with 7-dehydrocholesterol. A few examinations have indicated that lower amounts of vitamin D is correlates with not only an increased susceptibility to acute infections, but also with chronic infections (such as HIV infection) in some cases. Taking vitamin D supplements may improve ones response to treatment for diseases caused by virus or bacteria, such as chronic hepatitis C or pulmonar tuberculosis. No benefit could be derived from solo treatment using vitamin D in reducing pulmonary infections. The dose of vitamin D supplemention along with antiviral or antibacterial drug is not well defined. Unmistakably, 1,25(OH)2D has numerous immunomodulating properties which might decrease risks of respiratory infections caused by viruses. As discussed in this review, one toward this path can be founded on the applications of natural melanin bioextracted from novel melanogenic organisms. We have reviewed the applicability of vitamin D and D3 for modulating immunity in human beings, which is suggests using vitamin D/D3 to treat Covid-19. We have inferred three main issues- First, vitamin D induces immunity. Second, vitamin D production may be influenced by melanin. Third, both vitamin D and melanin may have significant impact in management of COVID-19. Determining the right category of melanin pigment (eumelanine or pheomelanin) to be used in specified amounts for vitamin D production, its mobility in the body, and initial hydroxylation, should be studied in future. We trust that this short review article will be useful in shaping the future course of treating COVID-19.

Abbreviations

[Uhohinc]

K-dt147Well-known member

Yesterday at 7:41 AM  

• #5,939

This forum is also growing independently of CUV. Have you registered all the side threads yet? There is a community growing here and I'm glad to be a part of it. And even though my thoughts were with ironbark today, also something about CUV.

Waterman said:

He still talks about MS as a possible indication

The reason why Homm keeps talking about MS in connection with CUV could also be hope. As far as I know, he suffers from the disease himself. It would be interesting to know if he still has any relationships to CUV. And one other thing, and I'm not sure if I have mentioned it before. The first quarter is almost over. We have been waiting since december. I want to know what the 6th indication is. NOW.

• 
  

[Uhohinc]

In reference to just above commentary by K-dt147 speculation about Florian Homm, he has publicly acknowledged having Multiple Sclerosis. 

[Uhohinc]

I was intrigued by reading a post on Sharetease which I can not find to add here, of an observation that Florian Homm no longer is of use of a cane in more recent videos. 

[Uhohinc]

Luger patentPublication number: 20200360484 Type: Application Filed: Feb 26, 2020 Publication Date: Nov 19, 2020 has substantial changes over previous patent

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Uhohinc<michaelba...@gmail.com>

Apr 25, 2021, 8:37:01 PM (4 hours ago) 

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to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9

NDP-MSH FOR TREATMENT OF INFLAMMATORY AND/OR NEURODEGENERATIVE DISORDERS OF THE CNS

Feb 26, 2020

The present invention is related to NDP-MSH or pharmaceutically acceptable salts thereof for therapeutic and/or prophylactic therapeutic treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis. The present invention is further related to pharmaceutical compositions and a kit comprising NDP-MSH or pharmaceutically acceptable salts thereof.

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Description

FIELD OF THE INVENTION

The present invention is related to NDP-MSH or pharmaceutically acceptable salts thereof for therapeutic and/or prophylactic therapeutic treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis. The present invention is further related to pharmaceutical compositions and a kit comprising NDP-MSH or pharmaceutically acceptable salts thereof.

BACKGROUND

Disorders of the central nervous system (CNS) are highly prevalent and can affect the brain and/or the spinal cord, resulting in neurological or psychiatric disorders, and occasionally a severe impairment of quality of life. The development of new methods of treatment has addressed a multitude of disorders; but, however, still lags behind other therapeutic areas. This is due to several factors including the complexity of the diseases and the problem of delivering drugs through the blood-brain barrier (BBB). The development of new therapies for

CNS disorders could provide patients with significant improvements in quality of life, and reduce the economic burden on health-care systems.

CNS disorders involving inflammation and/or neurodegeneration account for a large proportion of disorders affecting the CNS. They include widely known diseases such as Alzheimer's Disease, Parkinson's Disease and Multiple Sclerosis.

Multiple sclerosis (MS) is a disease of the central nervous system (CNS). It is estimated that number of people affected by MS amounts to 2-2.5 million (approximately 30 per 100,000) worldwide. Pathological manifestations of MS can include multiple inflammatory foci, plaques of demyelination, neuronal injury or loss within the brain or spinal cord, and neuronal dysfunction. MS is typically accompanied by neurological symptoms of variable degrees, including motor, sensory and cognitive deficits, ataxia and visual impairment.

Although the events triggering the onset of MS are still not fully understood, most evidence points toward an autoimmune etiology, possibly together with environmental factors or genetic predisposition. Many elements of the cascade of events leading to MS have been studied in experimental autoimmune encephalomyelitis (EAE), an animal model of autoimmune inflammatory diseases of the CNS which resembles MS in many respects (Constantinescu et al., 2011). Active EAE is induced by immunization of susceptible animals with CNS tissue or myelin peptides, for example myelin basic protein (MBP), proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), or their encephalitogenic fragments such as PLP139-151 or MOG35-55, and appropriate adjuvants. Passive or adoptive-transfer EAE can be induced by transferring pathogenic, myelin-specific T cells to recipient animals. In 2006, Krishnamoorthy et al. further developed transgenic mouse with MOG specific T and B cell receptors that spontaneously develops an inflammatory demyelinating disease resembling Devic's disease, which is often considered a variant of MS.

α-melanocyte-stimulating hormone (α-MSH) is a 13 amino acid peptide derived from a large precursor hormone called pro-opiomelanocortin (POMC). Post-translational cleavage of POMC gives rise to α-MSH in a tissue-specific manner. It has been detected in various regions of the brain and peripheral organs including the skin. Cells producing α-MSH include keratinocytes, melanocytes, Langerhans cells, monocytes, macrophages, endothelial cells, fibroblasts and mast cells. It has been established that α-MSH is not only involved in melanogenesis, but also plays a role in immunity and inflammation (see Luger et al. (2003) for review).

α-MSH exerts its effects through activation of cell-surface bound melanocortin receptors. Five melanocortin receptors (MC-1R to MC-5R) are known. They belong to the G-protein coupled receptors with seven transmembrane domains and are expressed in a cell- and tissue specific manner (see Brzoska et al. for review). The majority of anti-inflammatory effects of α-MSH are associated with to the detection of MC-1R, however, several in vivo studies have linked α-MSH activity to MC-4R (Carniglia et al. 2013).

The anti-inflammatory potential of α-MSH and its role in immunological cascades has been elucidated by several studies. It has been shown to down-regulate the production of pro-inflammatory cytokines (IL-1, IL-6, TNF-α, IL-2, IFN-γ, IL-4, IL-13) and the expression of co-stimulatory molecules (CD86, CD40) and adhesion molecules (ICAM-1, VCAM-1, E-selectin) on antigen-presenting cells. Furthermore, the production of the cytokine synthesis inhibitor IL-10 is up-regulated by α-MSH (Brzoska et al. (2008), Luger et al. (2003)).

The large majority of studies concerned with the investigation of the neuroprotective effect of melanocortins assess the effects of α-MSH, as reviewed in Catania (2008), but fail to recognized the therapeutic potential of NDP-MSH in MS treatment. Brod and Hood (2008) reported that orally administered α-MSH delayed disease onset and decreased disease severity in EAE. Mice were fed with 1, 10 or 100 μg α-MSH starting one week prior to EAE induction by active immunization and continuing through day 14 post immunization. α-MSH prevented or delayed disease onset and was able to reduce the clinical score of affected animals (patented in U.S. Pat. No. 7,807,143). However, the fact that preventive administration of relatively high dosages was necessary on a daily basis renders the approach impracticable for treatment of MS in humans.

Two groups pursued a gene therapy approach in order to deliver sufficient amounts of α-MSH: Yin et al. (2003) generated expression constructs encoding peptides with α-MSH activity and assessed their potential for treatment of EAE in mice. Intramuscular injection of 100 μg of DNA constructs was accomplished concurrently with EAE induction and repeated weekly for a total period of 4 weeks. Treatment with the DNA constructs resulted in delayed disease onset (about 2 days) and a decreased mortality, accounting for the slight reduction of the mean clinical score that was observed.

Han et al. (2008) employed activated transduced T cells specific for the CNS proteolipid (PLP) 139-151 as α-MSH “shuttles”. α-MSH producing T cells exhibited an altered cytokine secretion profile and, when transferred to animals with induced or established EAE, could reduce disease incidence delay disease onset. However, although the idea of using auto-reactive T cells as targeted α-MSH shuttles may seem intriguing, the fact that 12.5% of healthy recipient animals developed EAE renders this approach untenable with regard to safety and acceptance as a potential MS therapy.

Therapeutic treatment using α-MSH is hampered because of its inherent instability and short plasma half-life, and its weak receptor interaction (Rudman et al., 1983; Sawyer et al., 1980), resulting in the need of repeated high-dose administration.

However, in 1980 Sawyer et al. succeeded in synthesizing the synthetic α-MSH analog NDP-MSH which exhibited superior biological properties including prolonged biological activity, enhanced potency and resistance to enzymatic degradation (EP0292291). Today, NDP-MSH is marketed as SCENESSE® as a photoprotective drug and has been authorized by the European Medicine's Agency for treatment of erythropoietic protoporphyria. The role of NDP-MSH in inflammatory processes has been assessed, i.a., by Carniglia et al. (2013) who reported that NDP-MSH stimulates the release of IL-10 and TGF-β via MC-4R signaling in rat primary astrocytes and microglia in vitro. The mere observation that rat primary cells—obtained from healthy rat pups—release anti-inflammatory cytokines upon addition of NDP-MSH in vitro can however not suffice to foresee the surprising effects of NDP-MSH on the complex events contributing to disease onset and progression in adult MS model animals. Further, the observations presented herein clearly indicate involvement of MC-1R signaling whereas the effects observed by Carniglia et al. were linked to the detection of MC-4R expression, thereby indicating that the present inventors have revealed a novel mechanism of action of NDP-MSH in inflammatory and/or neurodegenerative processes within the CNS. Ter Laak et al. (2003) discovered that the α-MSH analog melanotan-II is effective in nerve regeneration and neuroprotection, but did not investigate the effect of NDP-MSH, let alone in MS treatment.

There is currently no cure for MS. Therapeutic treatment of MS includes disease-modifying and symptomatic treatments. FDA-approved disease-modifying agents for treatment of relapsing-remitting MS include immunosuppressive agents (mitoxantrone and teriflunomide), immunomodulatory agents such as glatiramer acetate (GA) and the cytokine inhibitor IFN-β, cell-migration modifying therapies including natalizumab and finglomod and neuroprotective agents such as dimethyl-fumarate. While treatment of relapsing-remitting MS is still hampered by adverse side effects or limited clinical efficacy, therapeutic options for secondary progressive MS or primary progressive MS are severely limited (for review see Chen et al. (2012)). There still exists a need in the art to develop alternative drugs for multiple sclerosis treatment.

The technical problem can thus be seen in the provision of an alternative treatment for inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis.

SUMMARY

The present inventors have surprisingly discovered that NDP-MSH is able to significantly ameliorate clinical and pathological manifestations in different EAE models, and even prevented recurrence of the disease after the treatment was discontinued. Thus, the present invention provides NDP-MSH or pharmaceutically acceptable salts thereof for use in treatment of multiple sclerosis or inflammatory and/or neurodegenerative disorders of the CNS in a subject. Further, NDP-MSH or pharmaceutically acceptable salts thereof can be used for therapeutic and/or therapeutic prophylactic treatment of inflammatory and neurodegenerative disorders of the CNS or multiple sclerosis in a subject. The subject is preferably a mammal, and in a particularly preferred embodiment the subject is a human.

Preferably, the treatment of inflammatory and/or neurodegenerative disorders of the CNS or MS with NDP-MSH or pharmaceutically acceptable salts thereof thus has an anti-inflammatory and/or neuroprotective effect.

NDP-MSH can be chemically modified, including, e.g., modifications of the C terminus and/or the N-terminus of the peptide. Thus, in some embodiments, treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis in a subject can be accomplished with NDP-MSH or a pharmaceutically acceptable salt thereof that is chemically modified.

Some inflammatory and/or neurodegenerative disorders of the CNS, for example MS, can establish various clinical courses. NDP-MSH or a pharmaceutically acceptable salt thereof can be administered during any phase of the disease, e.g. before onset of the disease, during relapse, remission and/or progression of the disease. It can be administered in any suitable form, however, in one preferred embodiment NDP-MSH or a pharmaceutically acceptable salt thereof is administered intravenously. Alternatively, NDP-MSH or pharmaceutically acceptable salts thereof can be administered as subcutaneous dissolving implants.

A suitable dosage range for NDP-MSH or its pharmaceutically acceptable salt is 0.,01 μg-1000 μg/kg of body weight. Preferably, the dosage is about 1-1000 μg/kg, about 1-500 μg/kg or about 1-250 μg/kg of body weight.

For multiple sclerosis treatment or treatment of inflammatory and/or neurodegenerative disorders of the CNS in a patient, NDP-MSH can be administered once, or it can be administered repeatedly, for example in intervals, e.g. every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours or every 72 hours. In other embodiments, NDP-MSH can be administered every week or every month.

The invention further relates to a pharmaceutical composition comprising NDP-MSH for treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis.

In another aspect, the invention is related to kit for use in the treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis comprising NDP-MSH or pharmaceutically acceptable salts thereof and a carrier. The kit may further comprise one or more agents selected from the group consisting of immunosuppressive agents and anti-inflammatory agents together with a pharmaceutically acceptable carrier or diluent.

DESCRIPTION OF THE FIGURES

FIG. 1 Effect of systemic NDP-MSH treatment on ongoing Experimental Autoimmune Encephalomyelitis (EAE). C57BL/6 mice were actively immunized by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG35-55) emulsified in Complete Freund's Adjuvant and systemically treated with 5 μg NDP-MSH or PBS every 48 hours beginning at a clinical score of 2-3. Mice were monitored for clinical score illustrated in FIG. 1(A) and body weight illustrated in FIG. 1(B) and sacrificed at day 17. Data from n=17 mice in each group is depicted, *, p<0.05 versus PBS-treated controls.

FIG. 2 Histological analyses of brain tissue obtained from mice treated as described in FIG. 1. FIG. 2(A) shows H&E staining of a representative overview (I) and a section enlargement (II) and myelin staining with luxol fast blue (III). FIG. 2(B) shows fluorescence marker staining DAPI/RILP2 (I) and DAPI/APP (II). One representative image for each group (+NDP-MSH, +PBS) is shown.

FIG. 3 Effect of NDP-MSH treatment of numbers of pathogenic Th1 and Th17 cells in the CNS. At day 17 post immunization brain and spinal cord from EAE mice treated with PBS and NDP-MSH as described for FIG. 1 were isolated. Cells were analyzed by multi-color flow cytometry using antibodies against CD4, IL-17, ROR-γt, IFN-γ and T-bet. One representative image illustrated in FIG. 3(A) as well as the statistical evaluation from n=8 mice in each group illustrated in FIG. 3(B) is shown. Cells are gated for CD4 and IL-17, ROR-γt, IFN-γ as well as T-bet staining was performed after cell permeabilization.

FIG. 4 Induction of functional regulatory T cells in the CNS by NDP-MSH treatment. Numbers, phenotype, and function of Foxp3+ regulatory T cells isolated from brain tissue of NDP-MSH and PBS treated mice were analyzed. One representative dotplot illustrated in FIG. 4(A) as well as the statistical evaluation from n=6 mice illustrated in FIG. 4(B) is depicted. Cells are gated for CD4 and Foxp3 as well as Helios staining was performed after cell permeabilization. *, p<0.05 versus PBS treated mice.

FIG. 5 Generation of tolerogenic dendritic cells by NDP-MSH treatment. The DC phenotype in regional lymph nodes from immunized NDP-MSH and PBS treated mice was analyzed. One representative dot plot illustrated in FIG. 5(A) as well as the statistical evaluation from n=2-6 mice illustrated in FIG. 5(B) is depicted. Cells are gated for MHCII and IL-10 as well as IFN-y staining was performed after cell permeabilization. *, p<0.05 versus PBS treated mice.

FIG. 6 Involvement of the melanocortin-1 receptor in effects of NDP-MSH on EAE progression. EAE was induced in MC-1R deficient mice as described in FIG. 1. Subsequently, disease development was monitored over time.

FIG. 7 Devic mice at the age of 38 days and a clinical score of 7 (severe hind limb paralysis) were injected intravenously with 5 μg NDP-MSH every other day for 3 weeks. At day 60, NDP-MSH treatment was interrupted and mice were observed for the onset of clinical symptoms. Disease progression was monitored over time.

FIG. 8 Effect of NDP-MSH in DEREG mice with induced EAE. Treg were depleted in DEREG mice as described by Lahl et al. (2007) by systemic treatment with diphtheria toxin.

Subsequently, EAE was induced as described in FIG. 1. 5 μg NDP-MSH were injected intravenously every 48 hours and disease progression was monitored over time.

FIG. 9 Effect of NDP-MSH in C11c-DTR mice with induced EAE. DC were depleted in C11c-DTR mice as described by Hochweller et al. (2008) by systemic treatment with diphtheria toxin. Subsequently, EAE was induced as described in FIG. 1. 5 μg NDP-MSH were injected intravenously every 48 hours and disease progression was monitored over time.

FIG. 10 Expression of pro-apoptotic (caspase-8) and anti-apoptotic (Bcl-2) genes in primary mouse neurons after treatment with 50 μm Glutamat or 50 μM Glutamat +1 nM NDP-MSH. Primary murine neurons were stimulated with glutamate which results in apoptosis (increased expression of pro-apoptotic caspase-8 and a reduced expression of anti-apoptotic Bcl-2 relative to PBS-treated controls). Addition of NDP-MSH to the glutamate-stimulated neuron cultures prevents induction of cell death (reduced expression of pro-apoptotic caspase-8, increased expression of anti-apoptotic Bcl-2).

FIG. 11 Long lasting direct neuroprotective effects of NDP-MSH in mice. FIG. 11(A) illustrates NDP-MSH treatment from days 36 to 64 of age prevented TCRMOG×IgHMOG mice from relapse for >8 weeks after cessation of therapy. Clinical scores from n=8 mice are depicted (individual mice are marked by different symbols). FIG. 11(B) illustrates flow cytometry of CD4+T cells in spinal cord tissue from NDP-MSH-treated TCRMOG×IgHMOG mice at days 124 and 194 after birth. Representative histograms are shown. FIG. 11(C) illustrates H&E, Luxol Fast Blue (LFB), and immunofluorescence staining using antibodies against CD4 (red), IL-17 (green), and Lama5 (gray) in lumbar spinal cord from NDP-MSH-treated TCRMOG×IgHMOG mice and PBS-treated controls at days 124 and 194 after birth. One representative image is shown. Areas of demyelination (LFB) and reduced Lama5 expression in the basement membrane are indicated by arrows.

FIG. 12 NDP-MSH modulates action potential generation in TCRMOG×IgHMOG mice. FIG. 12(A) illustrates numbers of action potentials (AP) in hippocampal neurons from TCRMOG×IgHMOG mice before disease development (black, day 30 after birth), autoimmune-prone, PBS-treated TCRMOG×IgHMOG mice (dark grey, day 60 after birth) and NDP-MSH-treated TCRMOG×IgHMOG mice (light grey, day 60 after birth), n=3 mice in each group. FIG. 12(B) illustrates firing behavior of PBS- or NDP-MSH-treated hippocampal neurons from TCRMOG×IgHMOG mice after Glutamate-provoked by depolarization. One representative image per group is show.

FIG. 13 A single subcutaneous injection of NDP-MSH-loaded microparticles is sufficient to attenuate CNS inflammation. FIG. 13(A) illustrates C57BL/6 mice were immunized with MOG-peptide to induce EAE and injected with NDP-MSH peptide (i.v., white arrows), placebo particles (s.c., black arrow) or NDP-MSH-loaded microparticles (s.c., red arrow) when clinical symptoms were detectable in the first mouse. Mean EAN scores from n=7 mice per group are shown; *, p<0.05 vs. mice treated with placebo particles. FIG. 13(B) illustrates representative images of CNS tissue after H&E as well as Luxol Fast Blue (LFB) staining. Inflammatory foci and demyelinated areas are marked with arrows. FIG. 13(C) illustrates flow cytometry of regulatory T cells in the CNS at disease maximum. Representative FACS plots are shown, cells are gated for CD4 and Foxp3 as well as Helios staining was performed after cell permeabilization.

FIG. 14 NDP-MSH down-regulated the expression of potassium channels associated with CNS inflammation and/or neurodegeneration in the CNS from MOG-immunized mice. FIG. 14(A) illustrates using the STRING 10 database, a network of potential protein interactions focusing on potassium channels has been generated. Kcnc3 (Kv3.3) is known to cause cerebellar neurodegeneration (Zhang et al. Cell 2016; 165:434-48) whereas Kcnc1 (Kv3.1) was suggested as a therapeutic target tor neuroprotection in Alzheimer's disease (Francosi et al. J Neurosci 2006; 26:11652-64). FIG. 14(B) illustrates representative immunofluorescence staining of brain tissue using antibodies to Kcnc3 (Kv3.3), Kcnc1 (Kv3.1) and NeuN. Nuclei are counterstained with DAPI, original magnification 200 X.

FIG. 15 NDP-MSH impacts on cognitive effects. FIG. 15(A) illustrates the object recognition (NOR) test is a commonly used behavioral test in mice. A mouse is presented with two similar objects during the first session (familiarization, 1 h per day for 4 consecutive days). Thereafter, one of the two objects is replaced by a new object (test session, 1 h at day 5). The amount of time taken to explore the new object provides an index of recognition memory (NOR-index). FIG. 15(B) illustrates C57BL/6mice (WT) were systemically treated with Scopolamin (Scm) daily from day 1-4, which is known to impair cognitive effects and memory. 30 or 60 minutes after Scopolamin treatment mice received an intravenous injection of 5 μg NDP-MSH in 100 μl PBS or an equal amount or PBS and the NOR-index was assessed. n =10 mice in each group; *;p<0.05vs.WT+Scopolamin.

DETAILED DESCRIPTION

To their surprise, the present inventors have discovered that NDP-MSH, a synthetic α-MSH analog that had initially been developed as a potent and stable stimulator of melanogenesis, ameliorates clinical and pathological manifestations in experimental autoimmune encephalomyelitis (EAE) models in mice. Interestingly, NDP-MSH was able to reduce inflammation in the CNS and promote re-myelination of neurons, resulting in attenuation of EAE progression and even complete recovery from EAE symptoms. Notably, even several weeks after NDP-MSH treatment was stopped, no disease recurrence was observed. Thus, NDP-MSH holds considerable potential as a drug for treatment of inflammatory and/or neurodegenerative disorders of the CNS, multiple sclerosis and other inflammatory demyelinating diseases in humans.

The neuropeptide α-MSH is a potent immunomodulator capable of inducing immunosuppression and tolerance. Using the mouse model of experimental autoimmune encephalomyelitis (EAE) the present inventors systemically treated MOG-immunized mice with NDP-MSH before and after the onset of hind limb paralysis. Whereas control mice showed a significant weight loss and developed severe ascending paralysis, mice preemptively injected with NDP-MSH were resistant to EAE development. Notably, therapeutic treatment attenuated EAE progression and prevented mice from weight loss. Flow cytometry, immunofluorescence staining and gene expression analyses revealed the absence of pathogenic Th17 and Th1 cells from brain tissue of NDP-MSH-treated animals. This effect was mediated by up-regulated numbers of Foxp3+ regulatory T cells (Treg) in α-MSH-injected mice versus controls. Since α-MSH has been shown to expand Treg by the induction of tolerogenic dendritic cells (DC) the DC phenotype at different stages of disease was analyzed. DC from NDP-MSH-treated mice expressed increased levels of PD-L1 or IL-10 and down-regulated maturation markers pointing to the induction of a tolerogenic DC phenotype. Since signaling via melanocortin-1-receptor (MC-1R) mediates the immunomodulatory effects of α-MSH, EAE was induced in MC-1R-deficient mice. Interestingly, upon α-MSH injection these mice developed hind limb paralysis similar to PBS treated controls, demonstrating that binding to MC-1R is essential for the NDP-MSH-mediated prevention of EAE. Together, these data indicate that NDP-MSH induces tolerogenic DC and expands functional Treg in vivo. These Treg suppress pathogenic Th1 and Th17 cells during EAE development, suggesting NDP-MSH as a potential therapeutic option for the treatment of patients with moderate multiple sclerosis. Moreover, NDP-MSH was shown to have a strong neuroprotective effect, which was further elucidated by NDP-MSH treatment of EAE in Treg- or DC-depleted mice. Notably, while PBS-treated controls developed severely progressing symptoms from day 10 after immunization, disease development and progression was significantly reduced in NDP-MSH treated animals even in the absence of Treg or DC indicating that NDP-MSH elicits its effects not only by induction of Treg and tolerogenic DC, but also plays a considerable neuroprotective role.

The neuroprotective role of NDP-MSH was further confirmed analyzing neurons from NDP-MSH-treated animals and vehicle-treated controls (isolated before and after MOG immunization) using histological staining tests for the detection of myelin, NeuN, act. Caspase 3 and TUNEL. Further, in vitro stimulation of neurons from embryonic mice with glutamate—which causes cell damage—in the presence or absence of NDP-MSH and subsequent histological staining tests for the detection of myelin, NeuN, act. Caspase 3 and TUNEL indicated that NDP-MSH was able to reduce the glutamate-induced neuronal damage or MOG significantly. Thus, a neuroprotective effect of NDP-MSH is a very likely explanation for the observed effect in the EAE model.

To further examine the long term effect of NDP-MSH, and to strengthen the data obtained in MOG-induced EAE, the effects of NDP-MSH was further investigated in a second, independent, spontaneous model of inflammatory/demyelinating diseases of the CNS, TCRMOG×IgHMOG mice (Bettelli et al., 2006), as described in Examples 8 and 9. In Example 8, TCRMOG×IgHMOG mice that had been treated with NDP-MSH were examined for relapses more than 8 weeks after treatment cessation, as shown in FIG. 11. Here it was found that NDP-MSH treatment from days 36 to 64 of age prevented TCRMOG×IgHMOG mice from relapse for >8 weeks after cessation of therapy. This shows that the effect of NDP-MSH lasts beyond the time of treatment and is independent of the MS mouse model used.

In a further experiment, Example 9, the TCRMOG×IgHMOG mice were examined with the results shown in FIG. 12. Specifically, the numbers of action potentials (AP) in hippocampal neurons were measured in TCRMOG×IgHMOG mice before disease development, in control mice and NDP-MSH-treated TCRMOG×IgHMOG mice, and it was found that NDP-MSH reduced the number of action potentials significantly, which indicates a direct effect on the hippocampal neurons.

Further, because NDP-MSH is proteolytically cleaved in serum and the half-life of the peptide after intravenous injection has been estimated to 90 min, which might complicate the further development towards a potential clinical application, a slow-release microparticular formulation of NDP-MSH was tested as described in Example 10. It was found that the slow release formulation released NDP-MSH over a period of >30 days and release of NDP-MSH reached ˜90% after 50 days. As shown in FIG. 13, a single subcutaneous injection of NDP-MSH-loaded microparticles into MOG-immunized C57BL/6 mice after the first clinical symptoms appeared is sufficient to attenuate CNS inflammation. It was found that after injection, the neuropeptide release from the particles lasts for more than 30 days.

In a further experiment shown as Example 11, it was found that NDP-MSH down-regulated the expression of potassium channels associated with CNS inflammation and/or neurodegeneration in the CNS from MOG-immunized mice. As shown in FIG. 14 (A), using the STRING 10 database, a network of potential protein interactions focusing on potassium channels has been generated. Kcnc3 (Kv3.3) is known to cause cerebellar neurodegeneration (Zhang et al. Cell 2016; 165:434-48) whereas Kcnc1 (Kv3.1) was suggested as a therapeutic target tor neuroprotection in Alzheimer's disease (Francosi et al. J Neurosci 2006; 26:11652-64). Further, FIG. 14 (B) shows representative immunofluorescence staining of brain tissue using antibodies to Kcnc3 (Kv3.3), Kcnc1 (Kv3.1) and NeuN.

Finally, to show the impact of NDP-MSH on cognitive effects, the object recognition (NOR) test, which is a commonly used behavioral test in mice, was used as outlined in Example 12 and FIG. 15 (A). In the NOR test, a mouse is presented with two similar objects during the first session (familiarization, 1 h per day for 4 consecutive days). Thereafter, one of the two objects is replaced by a new object (test session, 1 h at day 5). The amount of time taken to explore the new object provides an index of recognition memory (NOR-index). In order to study the impacts of NDP-MSH, C57BL/6mice (WT) were systemically treated with Scopolamin (Scm) daily from day 1-4, which is known to impair cognitive effects and memory. 30 or 60 minutes after Scopolamin treatment mice received an intravenous injection of 5 μg NDP-MSH in 100 μl PBS or an equal amount or PBS and the NOR-index was assessed. n=10 mice in each group; *;p<0.05vs.WT+Scopolamin. As can be seen from FIG. 15 (B), the NOR score oft eh NDP-MSH mice was higher than that of the mice treated with Scolopamin alone, indicating that NDP-MSH has a neuroprotective effect on WT mice.

Without wishing to be bound by theory, it is speculated that NDP-MSH exerts its neuroprotective effect by inducing Nur77 expression, a receptor that is normally associated with the T-cell activation. As early as 2008 it was shown that Nur77 can be activated by MC-1R mediated signals and in 2010 Volakakis et al. noted that this receptor, in addition to the activation of T cells, also controls induction of neuroprotective genes in response to oxidative stress (Smith, et al. (2008) Volakilis, et al.(2010)). The present inventors observed an induction of the neuroprotective Nur77 receptor in NDP-MSH stimulated neurons as compared to vehicle-treated controls. To show whether the effect of NDP-MSH on the progression of EAE in vivo was caused by induction of Nur77, Nur77 deficient mouse mutants were subjected to a MOG-induced EAE ±NDP-MSH treatment. Without wishing to be bound by theory, it is expected that NDP-MSH has no effect on the progression of EAE in Nur77-deficient mice.

“NDP-MSH” also referred to as Afamelanotide or Melanotan-1 or [Nle4, D-Phe7]-α-MSH is a synthetic analog of α-MSH. The term “synthetic analog” is used herein to describe a non-naturally occurring or artificially synthesized compound that is structurally related to a parent compound. “alpha-MSH ” or “α-MSH” as used herein means alpha-melanocyte stimulating hormone, a peptide hormone of the melanocortin family. Typically, α-MSH consists of thirteen amino acids having the sequence reflected in SEQ ID NO: 1. Compared to SEQ ID NO: 1, in NDP-MSH, the amino acid corresponding to the amino acid at position 4 is norleucine (abbreviated Nle), and the amino acid corresponding to the amino acid at position 7 is D-phenylalanine (i.e. phenylalanine configurated as D-enantiomer, abbreviated D-Phe). The amino acid sequence of NDP-MSH is shown in SEQ ID NO: 2.

The term NDP-MSH″ also includes the alpha-MSH analogues described in U.S. Pat. Nos. 4,457,864; 4,485,039; 4,866,038; 4,918,055; 5,049,547; 5,674,839 and 5,714,576 and Australian Patent Nos. 597630 and 618733 which are herein incorporated by reference for their teachings with respect to alpha-MSH analogues and their synthesis thereof. An alpha-MSH analogue is sometimes also referred to herein as alpha-MSH derivative and, thus, these terms can mutually replace each other.

In one aspect, the alpha-MSH analogue may be a compound as disclosed in AU-Patent No. 597630, selected from compounds of the formula:

R1-W-X—Y—Z—R2

wherein

• R1 is absent; n-Pentadecanoyl, Ac, 4-phenylbutyryl; Ac-Gly-, Ac-Met-Glu, Ac-Nle-Glu-, or Ac-Tyr-Glu-;
• W is -His- or -D-His-;.
• X is -Phe-, -D-Phe-, -Tyr-, -D-Tyr-, or -(pNO)D-Phe7-;
• Y is -Arg- or -D-Arg-;
• Z is -Trp- or -D-Trp-; and
• R2 is —NH2; or -Gly-Lys-NH2.

In another aspect, the alpha-MSH analogue may be selected from cyclic analogues which are disclosed in Australian Patent No. 618733 where an intramolecular interaction (such as a disulfide or other covalent bond) exists (1) between the amino acid residue at position 4 and an amino acid residue at position 10 or 11, and/or (2) between the amino acid residue at position 5 and the amino acid residue at position 10 or 11.

The alpha-MSH analogue may be a linear analogue as disclosed in U.S. Pat. No. 5,674,839, selected from the group consisting of:

Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Lys-Gly- Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly- Pro-Val-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH2 Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-Gly-Pro-Val-NH2 Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-NH2 Ac-Nle-Asp-HiS-D-Phe-Arg-Trp-Lys-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Orn-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Lys-NH2 Ac-Nle-Asp-His-D-Phe-Arg-Trp-Lys-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Orn-NH2 Ac-Nle-Asp-His-D-Phe-Arg-Trp-Orn-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Dab-NH2 Ac-Nle-Asp-His-D-Phe-Arg-Trp-Dab-NH2 Ac-Nle-Glu-His-D-Phe-Arg-Trp-Dpr-NH2 Ac-Nle-Glu-His-L-Phe-Arg-Trp-Lys-NH2 Ac-Nle-Asp-His-L-Phe-Arg-Trp-Lys-NH2

The alpha-NISH analogue may also be a cyclic analogue as disclosed in U.S. Pat. No. 5,674,839, selected from the group consisting of:

wherein Ala=alanine, Arg=arginine, Dab=2,4-diaminobutyric acid, Dpr=2,3-diaminopropionic acid, Glu=glutamic acid, Gly=glycine, His=histidine, Lys=Met=methionine, Nle=norleucine, Om=ornithine, Phe=phenylalanine, (pNO2)Phe=paranitrophenylalanine, Plg=phenylglycine, Pro=proline, Ser=serine, Trp=tryptophan, TrpFor=N1- formyl-tryptophan, Tyr=tyrosine, Val=valine.

All peptides are written with the acyl-terminal end at the left and the amino terminal end to the right; the prefix before an amino acid designates the D-Isomer configuration, and unless specifically designated otherwise, all amino acids are in the L-isomer configuration.

In one aspect of the present invention, the alpha-MSH analogue can be

• [D-Phe7]-alpha-MSH,
• [Nle4, D-Phe7]-alpha-MSH,
• [D-Ser1, D-Phe7]-alpha-MSH,
• [D-Tyr2, D-Phe7]-alpha-MSH,
• [D-Ser3, D-Phe7]-alpha-MSH,
• [D-Met4, D-Phe7]-alpha-MSH,
• [D-GLu5, D-Phe7]-alpha-MSH,
• [D-His6, D-Phe7]-alpha-MSH,
• [D-Phe7, D-Arg8]-alpha-MSH,
• [D-Phe7, D-Trp9]-alpha-MSH,
• [D-Phe7, D-Lys11]-alpha-MSH
• [D-Phe-7, D-Pro12]-alpha-MSH,
• [D-Phe7, D-Val13]-alpha-MSH,
• [D-Ser1, Nle4, D-Phe7]-alpha-MSH,
• [D-Tyr2, Nle4, D-Phe7]-alpha-MSH,
• [D-Ser3, Nle4, D-Phe7]-alpha-MSH,
• [Nle4, D-Glu5, D-Phe7]-alpha-MSH,
• [Nle4, D-His6, D-Phe7]-alpha-MSH,
• [Nle4, D-Phe7, D-Arg8]-alpha-MSH,
• [Nle4, D-Phe7; D-Trp9]-alpha-MSH,
• [Nle4, D-Phe7, D-Lys11]-alpha-MSH,
• [Nle4, D-Phe7, D-Pro12]-alpha-MSH,
• [Nle4, D-Phe7, D-Val13]-alpha-MSH,

• [Nle4, D-Phe7]-alpha-MSH4-10,
• [Nle4, D-Phe7]-alpha-MSH4-11,
• [D-Phe7]-alpha-MSH5-11,
• [Nle4, D-Tyr7]-alpha-MSH4-11,
• [(pNO2)D-Phe7]-alpha-MSH4-11,
• [Tyr4, D-Phe7]-alpha-MSH4-10,
• [Tyr4, D-Phe7]-alpha-MSH4-11,
• [Nle4]-alpha-MSH4-11,
• [Nle4, (pNO2)D-Phe7]-alpha-MSH4-11,
• [Nle4, D-His6]-alpha-MSH4-11,
• [Nle4, D-His6, D-Phe7]-alpha-MSH4-11,
• [Nle4, D-Arg8]-alpha-MSH4-11,
• [Nle4, D-Trp9]-alpha-MSH4-11,
• [Nle4, D-Phe7, D-Trp9]alpha-MSH4-11,
• [Nle4, D-Phe7]-alpha-MSH4-9, or
• [Nle4, D-Phe7, D-Trp9]-alpha-MSH4-9.
  In a further aspect, the alpha-MSH analogue is:
• [Nle4, D-Phe7]-alpha-MSH4-10,
• [Nle4, D-Phe7]-alpha-MSH4-11,
• [Nle4, D-Phe7, D-Trp9]-alpha-MSH4-11, or
• [Nle4, D-Phe7]-alpha-MSH4-9.
  In a particularly preferred aspect, the alpha-MSH analogue is [Nle4, D-Phe7]-alpha-MSH.

For the purpose of the invention the active compound as defined above also includes the pharmaceutically acceptable salt(s) thereof. The phrase “pharmaceutically or cosmetically acceptable salt(s)”, as used herein, means those salts of compounds of the invention that are safe and effective for the desired administration form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The use of salt formation as a means of varying the properties of pharmaceutical compounds is well known and well documented. Salt formation can be used to increase or decrease solubility, to improve stability or toxicity and to reduce hygroscopicity of a drug product. There are a wide range of chemically diverse acids and bases, with a range of pKa values, molecular weights, solubilities and other properties, used for this purpose. Of course, any counterions used in pharmaceuticals must be considered safe, and several lists of pharmaceutically approved counterions exist, which vary depending on the source. Approved salt formers can e.g. be found in the Handbook of Pharmaceutical Salts (Stahl PH, Wermuth CG, editors. 2002. Handbook of pharmaceutical salts: Properties, selection and use. Weinheim/Zurich: Wiley-VCH/VHCA.). Thus, the present invention also comprises the use of pharmaceutically acceptable salts of NDP-MSH for the treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis.

“Inflammatory and/or neurodegenerative disorders of the CNS” are disorders associated with inflammation and/or neurodegeneration that affect the CNS. However, some disorders may also affect the peripheral nervous system (PNS). “Inflammatory and/or neurodegenerative disorders” means that some of the disorders are associated with neurodegeneration in the CNS, while others are associated with inflammation in the CNS, and some are associated with both neurodegeneration and inflammation in the CNS. Inflammatory and/or neurodegenerative disorders of the CNS include, but are not limited to, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA), Multiple Sclerosis (MS), Marburg variant of MS, Baló's concentric sclerosis, Schilder's disease, acute disseminated encephalomyelitis (ADEM), and Devic's disease, also referred to as neuromyelitis optica (NMO), Optic-spinal MS, Acute hemorrhagic leukoencephalitis, Solitary sclerosis, Optic neuritis, Transverse myelitis. It is to be understood that treatment of any other disorder involving inflammation and/or neurodegeneration in the CNS is also envisaged.

“Multiple sclerosis” or “MS”, also sometimes referred to as disseminated sclerosis or encephalomyelitis disseminata, is a disease that affects the central nervous system (CNS). The CNS is a part of the nervous system comprising the brain and the spinal cord. MS can be associated with a wide range of neurological symptoms, including paralysis, sensory and cognitive defects, spasticity, tremor, lack of coordination and visual impairment.

Typically, MS is categorized in the following subtypes: a) relapsing-remitting MS (RRMS), which affects about 85% of MS patients and is characterized by relapses (acute attacks) of disease followed by periods of partial or full recovery (remission); b) secondary progressive MS (SPMS) which begins with an initial relapsing-remitting disease course, followed by ongoing disease progression that may include occasional relapses and minor remissions and plateaus, i.e. periods without a change in health condition, c) primary progressive MS (PPMS), which affects approximately 10% of MS patients and is characterized by disease progression from the onset, most frequently in the absence of relapses, and d) progressive-relapsing MS, which is the least common disease course showing disease progression from onset but with clear acute relapses.

It is envisaged that NDP-MSH can be used for treatment of MS in any of the forms and/or phases described herein. For example, NDP-MSH can be used for treatment of relapsing-remitting MS during relapse and/or during remission. It is further envisaged that NDP-MSH can be used for prophylactic therapeutic treatment of subjects that are at risk of developing MS or another inflammatory and/or neurodegenerative disease, for example individuals that have developed clinically isolated syndrome (CIS), the first clinical episode of symptoms and signs suggestive of an inflammatory demyelinating disorder of the central nervous system.

Preferably, NDP-MSH treatment results in amelioration and/or remission of clinical and/or pathological manifestations and/or symptoms associated with MS or the inflammatory and/or neurodegenerative disorder.

Typical pathological manifestations of MS or inflammatory and/or neurodegenerative disorders include, but are not limited to, inflammation, de-myelination and neurodegenerationin the CNS. Thus, treatment with NDP-MSH preferably results in an anti-inflammatory and/or neuroprotective effect.

Without wishing to be bound by a specific theory, it is thought that MS is triggered by CNS-autoreactive T cells that become activated in the periphery and differentiate into Th1 (producing, e.g., IFN-γ) or Th17 cells (producing, e.g., IL-17, IL-22, IL-21). Activated T-cells can up-regulate integrins such as VLA-4 and cross the blood brain barrier (BBB), the interface that separates the brain from the circulatory system and protects the CNS. On encountering their cognate antigen in the CNS, the T cells proliferate and secrete pro-inflammatory cytokines which in turn stimulate microglia, macrophages and astrocytes, and recruit B cells, ultimately resulting in demyelination and axonal loss.

Having an “anti-inflammatory” effect in general means controlling and/or reducing any step of the inflammation cascade triggering and/or contributing to MS pathology or pathology of the inflammatory and/or neurodegenerative disorder. The person skilled in the art readily knows how to assess the anti-inflammatory effect of NDP-MSH, e.g. by measuring the expression of certain marker proteins associated with CNS inflammation, such as, e.g., Rab-interacting lysosomal protein (RILP) 2. This can, for example, be accomplished by immunofluorescence staining with antibodies recognizing the marker protein and linked to (labeled with) a fluorophore. Other methods include monitoring populations of pro-inflammatory cells in the CNS that are associated with disease onset and/or progression. For example, in MS, Th1 and Th17 cell populations are thought to be involved in inflammatory processes in the CNS contributing to disease onset/progression. The person skilled in the art knows how to assess specific cell populations, e.g., by fluorescence-activated cell sorting (FACS). The method has been extensively described in the prior art. Another method to survey inflammatory processes is to assess levels of pro-inflammatory cytokines, e.g. by ELISA (enzyme-linked immunosorbent assay).

Having a “neuroprotective” effect as used herein means having the effect of preventing neurodegeneration. “Neurodegeneration” is used herein to describe neuronal and/or axonal injury and/or loss. The events leading to neurodegeneration have not fully been elucidated, however, without wishing to be bound by a specific theory, it is presumed that in some inflammatory and/or neurodegenerative disorders of the CNS or MS, inflammation and/or de-myelination may be involved. “Demyelination” means damage and/or loss of the myelin sheath. Myelin is composed of water, lipids and proteins and is typically deposited in layers around axons. The myelin sheath functions as an electrical insulation and thereby increases the speed of impulses propagating along the myelinated axons. When myelin is damaged or degenerated, conduction of signals along the nerve can be impaired or lost. It is assumed that loss of the myelin sheath may result in neurodegeneration. Demyelination can, for example, be visualized with a suitable dye, such as, e.g. luxol, in a sample. Further, magnetic resonance imaging (MRI) can be used for visualizing plaques of demyelination in the brain.

NDP-MSH or pharmaceutically acceptable salts thereof used according to the invention may be chemically modified. Generally, all kind of modifications of NDP-MSH or pharmaceutically acceptable salts thereof are comprised by the present invention as long as they do not inhibit the therapeutic effect of the peptide or salt respectively. E.g. modifications at the N terminus and/or at the C terminus of the peptide might be performed, for example by an acyl group, preferably an acetyl group at the N terminus and/or an amidation or esterification of the C terminus.

Other chemical modifications of the compounds of the invention such as alkylation (e. g., methylation, propylation, butylation), arylation, etherification and esterification may be possible and are also envisaged. It is preferred that the mentioned modifications do not significantly alter the advantageous capabilities of the compounds of the invention as described herein, i.e. the chemically modified compounds of the invention have capabilities which are comparable with the capabilities of the compounds which were evaluated in the appended examples. “Comparable” is explained herein below.

It may be necessary, for reasons of resistance to degradation, to employ a protected form of the compounds of the invention. The nature of the protecting group must obviously be a biologically compatible form. Many biologically compatible protective groups are suitable, such as, for example, those provided by acylation or acetylation of the amino-terminal end or amidation of the carboxy-terminal end.

Thus, the invention also features the compounds of the invention in a protected or unprotected form. Protective groups based either on acylation or acetylation of the amino-terminal end or on amidation of the carboxy-terminal end or, alternatively, on both, are the preferred.

Further protective groups known per se are likewise possible. The modifications may also affect the amino group in the side chains of the amino acids. As stated above, it is preferred that these modifications do not significantly alter the advantageous capabilities of the compounds of the invention as described herein.

In a more preferred embodiment of the invention the above mentioned tripeptides are amidated at the C-terminus.

Thus, a further embodiment of the present invention is the use of the NDP-MSH or pharmaceutically acceptable salts thereof which are chemically modified.

In the context with the present invention the term “treatment” and all its grammatical forms thereof includes therapeutic or prophylactic treatment. A “therapeutic or prophylactic treatment” comprises prophylactic treatments such as complete prevention of occurrence of symptoms or therapeutic treatment for improvement or amelioration of already occurred symptoms or in order to prevent further aggravation of disease (activity). As for effectiveness of the prophylactic and/therapeutic treatment, the term should be construed in its broadest sense including improvement of findings diagnosed by a doctor and improvement of rational symptoms.

NDP-MSH or a pharmaceutically acceptable salt thereof as described above is preferably applied in the treatment of mammals, particularly of humans.

According to one embodiment of the present invention the inventive use of NDP-MSH or a pharmaceutically acceptable salt thereof leads to a direct or indirect interaction with the melanocortin receptor 1 (MC-R1).

NDP-MSH or the pharmaceutically acceptable salts thereof might also be used as part of a composition. Thus, a further embodiment of the invention is the use of NDP-MSH or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical composition for treatment of multiple sclerosis or inflammatory and/or neurodegenerative disorders of the CNS. NDP-MSH or the pharmaceutically acceptable salts thereof can also be used to produce a medicament for the treatment and/or prevention of multiple sclerosis or inflammatory and/or neurodegenerative disorders of the CNS. The embodiments indicated above are encompassed analogously by this use. NDP-MSH or the pharmaceutically acceptable salts thereof are normally mixed with a pharmaceutically acceptable carrier or diluent. Processes known per se for producing medicaments are indicated in Forth, Henschler, Rummel (1996) Allgemeine und spezielle Pharmakologie und Toxikologie, Urban & Fischer.

Pharmaceutical compositions of the invention comprise a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof and can be formulated in various forms, e.g. in solid, liquid, powder, aqueous, lyophilized form.

The pharmaceutical composition may be administered with a pharmaceutically acceptable carrier to a patient, as described herein. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. Accordingly, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers, which may be used in formulating the composition according the invention, comprise those described below for the composition. Other suitable pharmaceutically acceptable carriers and excipients are inter alia described in Remington's Pharmaceutical Sciences, 151h Ed., Mack Publishing Co., New Jersey (1991) and Bauer at al, Pharmazeutische Technologie, 5th Ed., Govi-Verlag Frankfurt (1997).

It is also envisaged that the pharmaceutical composition may optionally further comprise one or more of the group selected from immunosuppressive agents and anti-inflammatory agents. The person skilled in the art knows how to select suitable agents for treatment of the specific inflammatory and/or neurodegenerative disorder of the CNS or MS. Exemplary suitable agents include, but are not limited to, corticosteroids.

The present invention relates also to a kit for the treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis comprising NDP-MSH or pharmaceutically acceptable salt thereof and a carrier. It is also envisaged that the kit may optionally further comprise one or more of the group selected from immunosuppressive agents and anti-inflammatory agents. The person skilled in the art knows how to select suitable agents for treatment of the specific inflammatory and/or neurodegenerative disorder of the CNS or MS. Exemplary suitable agents include, but are not limited to, corticosteroids.

Generally all carriers are suitable that are pharmaceutically acceptable. Generally all types of carriers are suitable for the use according to the present invention that enable a release at the desired sit of action. The person skilled in the art knows which type of carrier is suitable depending on the correspondent application form.

Carriers might be biodegrade such as Liposomes; Microspheres made of the biodegradable polymer poly(lactic-co-glycolic) acid, albumin microspheres; synthetic polymers (soluble); nanofibers, protein-DNA complexes; protein conjugates; erythrocytes virosomes. Various carrier based dosage forms comprise solid lipid nanoparticles (SLNs), polymeric nanoparticles, ceramic nanoparticles, hydrogel nanoparticles, copolymerized peptide nanoparticles, nanocrystals and nanosuspensions, nanocrystals, nanotubes and nanowires, functionalized nanocarriers , nanospheres, nanocapsules, liposomes, lipid emulsions, lipid microtubules/microcylinders, lipid microbubbles, lipospheres, lipopolyplexes, ethosomes, multicomposite ultrathin capsules, aquasomes, pharmacosomes, colloidosomes, niosomes, discomes, proniosomes, microspheres, microemulsions and polymeric micelles.

Polymers are the backbone of the typical transdermal drug delivery systems. Systems for transdermal delivery are fabricated as multi-layered polymeric laminates in which a drug reservoir or a drug-polymer matrix is sandwiched between two polymeric layers: an outer impervious backing layer that prevents the loss of drug through the backing surface and an inner polymeric layer that functions as an adhesive and/or rate-controlling membrane. Transdermal drug delivery systems comprise different systems such as the reservoir systems, microreservoir systems, and the combination of reservoir and matrix-dispersion systems.

In the reservoir system, the drug reservoir is embedded between an impervious backing layer and a rate-controlling membrane. The drug releases only through the rate-controlling membrane, which can be microporous or non-porous. In the drug reservoir compartment, the drug can be in the form of a solution, suspension, or gel or dispersed in a solid polymer matrix. On the outer surface of the polymeric membrane a thin layer of drug-compatible, hypoallergenic adhesive polymer can be applied. In the Matrix systems and Drug-in-adhesive system the drug reservoir is formed by dispersing the drug in an adhesive polymer and then spreading the medicated polymer adhesive by solvent casting or by melting the adhesive (in the case of hot-melt adhesives) onto an impervious backing layer. On top of the reservoir, layers of unmedicated adhesive polymer are applied. In the Matrix-dispersion system the drug is dispersed homogeneously in a hydrophilic or lipophilic polymer matrix. This drug-containing polymer disk then is fixed onto an occlusive baseplate in a compartment fabricated from a drug-impermeable backing layer. Instead of applying the adhesive on the face of the drug reservoir, it is spread along the circumference to form a strip of adhesive rim. The drug delivery system is a combination of reservoir and matrix-dispersion systems. The drug reservoir is formed by first suspending the drug in an aqueous solution of water-soluble polymer and then dispersing the solution homogeneously in a lipophilic polymer to form thousands of unleachable, microscopic spheres of drug reservoirs. The thermodynamically unstable dispersion is stabilized quickly by immediately cross-linking the polymer in situ. Transdermal drug delivery technology represents one of the most rapidly advancing areas of novel drug delivery. This growth is catalyzed by developments in the field of polymer science. This article focuses on the polymeric materials used in transdermal delivery systems, with emphasis on the materials' physicochemical and mechanical properties, and it seeks to guide formulators in the selection of polymers. Polymers are used in transdermal delivery systems in various ways, including as matrix formers, rate-controlling membranes, pressure-sensitive adhesives (PSAs), backing layers or release liners.

Polymers used in transdermal delivery systems should have biocompatibility and chemical compatibility with the drug and other components of the system such as penetration enhancers and PSAs. They also should provide consistent, effective delivery of a drug throughout the product's intended shelf life or delivery period and have generally-recognized-as-safe status.

Depending on the correspondent need the skilled person will choose the suitable carrier in order to apply NDP-MSH or pharmaceutically acceptable salt according to the present invention. E.g. carriers in the context with e.g. a rectal application are e.g. multi matrix systems using methacrylic acid copolymers.

If e.g. the desired site of action is the colon and NDP-MSH or a pharmaceutically acceptable salt thereof is applied orally the carrier has to be resistant to gastric acid in order to enable a release of NDP-MSH or the pharmaceutically acceptable salt thereof in the colon.

The administration of or the pharmaceutical composition comprising NDP-MSH or pharmaceutically acceptable salts thereof can be done in a variety of ways, including, but not limited to, topically, transdermally, subcutaneously, intravenously, intraperitoneally, intramuscularly or intraocularly. Subcutaneous administration can be accomplished by providing a subcutaneous implant comprising a suitable amount of NDP-MSH or the pharmaceutically acceptable salt thereof, for example about 16-20 mg. However, any other NDP-MSH dosage may be applied if necessary. In one preferred embodiment, NDP-MSH or pharmaceutically acceptable salts thereof or the pharmaceutical composition according to the present invention is administered intravenously.

The exact dose will depend on the purpose of the treatment (e.g. remission maintenance vs. acute flare of disease), and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. A typical dose can be, for example, in the range of 0.01 to 1000 μg/kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

A suitable dose for administration lies e.g. in the range of 0,1-1000 μg/kg, for example about 1-1000 μg/kg, about 1-500 μg/kg, or about 1-250 μg/kg of body weight.

NDP-MSH can be administered once, or it can be administered repeatedly, for example in intervals, e.g. every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 60 hours or every 72 hours. In other embodiments, NDP-MSH can be administered every week or every month.

The pharmaceutical composition according to the invention may be in solid, liquid or gaseous form and may be, inter alia, in the form of an ointment, a cream, transdermal patches, a gel, powder, a tablet, solution, an aerosol, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tincture or fluid extracts or in a form which is particularly suitable for the desired method of administration, in particular systemic administration.

Rectal applications can be compounded in many forms. Liquid rectal medicine solutions are given by enema. Creams, lotions and ointments are applied externally or inserted internally using an applicator. Suppositories might be prepared by mixing medicine with a wax-like substance to form a semi-solid, bullet-shaped form that will melt after insertion into the rectum.

Intraperitoneal injection or IP injection is the injection of a substance into the peritoneum (body cavity). In humans, the method is used to administer chemotherapy drugs to treat some cancers. A further form of administration of an inventive composition is the topic administration, for instance in form of an ointment or cream. Such an ointment or cream may additionally comprise conventional ingredients, like carriers or excipients as described above.

NDP-MSH or the pharmaceutically acceptable salts thereof can also be used in sprays, for example for inhalation. NDP-MSH or the pharmaceutically acceptable salts thereof may also be added to foods.

The present invention is also related to a kit for treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis comprising NDP-MSH or pharmaceutically acceptable salts thereof and a carrier. The inventive kit might be a kit of two or more parts and might be prepared for use in order to apply the kit in in order to treat inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis.

It is to be understood that all embodiments, definition, etc. disclosed in the context of treatment are fully applicable to methods of treatment as well. The present invention relates to a method of treatment of inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis in a subject in need thereof, comprising administering a pharmaceutically effective amount of NDP-MSH or a pharmaceutically acceptable salt thereof. By “therapeutically effective amount” or “therapeutically active” is meant a dose of a NDP-MSH or a pharmaceutically acceptable salt thereof that produces the therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for age, body weight, general health, sex, diet, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. The therapeutic effect of the respective methods or method steps of the present invention is additionally detectable by all established methods and approaches which will indicate a therapeutic effect. It is, for example, envisaged that the therapeutic effect is detected by way of an improvement or amelioration of the neurological symptoms known in the art for inflammatory and/or neurodegenerative disorders of the CNS or multiple sclerosis, e.g., those described herein. Additionally or alternatively it is also possible to evaluate the general appearance of the respective patient (e.g., fitness, well-being) which will also aid the skilled practitioner to evaluate whether a therapeutic effect is already there. The skilled person is aware of numerous other ways which will enable him or her to observe a therapeutic effect of the compounds of the present invention.

A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

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EXAMPLESExample 1Systemic NDP-MSH Treatment of Ongoing Experimental Autoimmune Encephalomyelitis (EAE)

To investigate whether NDP-MSH is able to inhibit autoimmunity and inflammation in organs different from the skin, such as the central nervous system (CNS), the mouse model of experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory autoimmune disease resembling human multiple sclerosis, was used.

Therefore, C57BL/6 mice were actively immunized by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG35-55) emulsified in Complete Freund's Adjuvant at the back skin (day 0). At day 0 and day 2 mice received intraperitoneal injections of 400 ng pertussis toxin and disease development was monitored daily. When mice reached a clinical score of 2-3 (beginning hind limb paralysis, day 12) they were injected intravenously with 5 μg NDP-MSH or an equal amount of PBS every 48 h. Mice treated with NDP-MSH started gaining weight after the first injection of the hormone (right) and recovered from paralysis whereas control animals exhibited a significant weight loss and continued developing severe ascending paralysis (left). All mice were sacrificed at day 17 and analyzed on a cellular and molecular level. Data were obtained from n=12 mice in each group is depicted, *, p<0.05 versus PBS-treated controls.

Example 2Effect of NDP-MSH on Inflammation in the CNS Myelination Status of Neurons

Brain tissue from mice treated with NDP-MSH or PBS at day 17 post immunization obtained from Ex. 1 was analyzed. H&E staining showed substantial numbers of mononuclear, pro-inflammatory cells infiltrating the brain of PBS-treated controls whereas almost no cell infiltrates were detectable in brain tissue from NDP-MSH treated animals. One representative overview (A) as well as a sectional enlargement (B) is depicted for each group. (C) Myelin staining using luxol fast blue showed complete demyelination of the brain in PBS-treated mice as well as tremendous re-myelination in NDP-MSH treated animals. (representative D) Dramatically reduced expression of markers associated with tissue inflammation (RILPL2) and tissue destruction (APP) in the brain of NDP-MSH-treated mice compared to PBS-treated controls was observed. One representative image for each group is shown.

Immunofluorescence staining and histology of the brain tissue revealed reduced numbers of pro-inflammatory mononuclear cells infiltrating the brain of NDP-MSH treated mice compared to PBS treated controls. Moreover, the expression of markers characteristic for CNS inflammation or neurodegeneration, like RILPL2 or APP, respectively were significantly reduced in NDP-MSH treated mice versus controls. Besides reducing the CNS inflammation NDP-MSH also induced the re-myelination of neurons as evidenced by luxol fast blue staining, which detects myelin.

Example 3Numbers of Pathogenic Th1 and Th17 Cells in the CNS after NDP-MSH Treatment

In support of the beneficial effects of NDP-MSH on the progression of ongoing EAE, flow cytometry of CNS revealed decreased levels of pathogenic Th1 as well as Th17 effector cells in NDP-MSH treated mice versus PBS treated controls.

Reduced numbers of pathogenic Th1 and Th17 cells in the CNS from NDP-MSH treated mice compared to PBS-treated controls. At day 17 post immunization brain and spinal cord were isolated from PBS- and NDP-MSH-treated mice and single cell suspensions were prepared using density gradient centrifugation. Subsequently, cells were analyzed by multi-color flow cytometry using antibodies against CD4, IL-17, ROR-gt, IFN-g and T-bet. One representative image (left) as well as the statistical evaluation from n=8 mice in each group (right) is depicted, showing a significantly reduced infiltration of Th17 cells (factor 5) and Th1 cells (factor 2) in the CNS from NDP-MSH-treated mice compared to controls. Cells are gated for CD4 and IL-17, ROR-gt, IFN-g as well as T-bet staining was performed after cell permeabilization.

Example 4NDP-MSH Induces Functional Regulatory T Cells in the CNS by Generating Tolerogenic Dendritic Cells

Numbers, phenotype and function of Foxp3+ regulatory T cells (Treg) isolated from brain tissue of NDP-MSH and PBS treated mice as described in Ex. 1 was analyzed at day 17 post immunization by flow cytometry analysis.

Notably, up-regulated levels of Foxp3+ Treg expressing characteristic markers, such as Helios or CTLA-4, were present at higher numbers in brain tissue from NDP-MSH treated mice compared to controls. Of note, these Treg were functional as they efficiently inhibited the proliferation of effector T cells in vitro. Further, the DC phenotype in regional lymph nodes from immunized NDP-MSH and PBS treated mice was analyzed. Interestingly, DC from NDP-MSH injected mice expressed increased levels of PD-L1 or IL-10 and down-regulated typical maturation markers like CD80 and IFN-y pointing to the induction of tolerogenic DC in MOG35-55 immunized and NDP-MSH treated animals.

Example 5Effects of NDP-MSH on EAE Progression by Signaling via Melanocortin-1 Receptor

To investigate whether the NDP-MSH induced effects on the progression of EAE were mediated via binding to a functional MC-1R, EAE was induced in MC-1R deficient mice with a point mutation in the MC-1R gene resulting in a truncated protein (Roberts et al., 1993). Mice were immunized with MOG3555, injected with pertussis toxin and treated with NDP-MSH or PBS as described in Ex. 1. Subsequently, disease development was monitored over time.

Notably, NDP-MSH treated MC-1R deficient mice developed hind limb paralysis similar to PBS treated controls demonstrating that signaling via a functional MC-1R is essential for the NDP-MSH mediated amelioration of disease. Together, these data indicate that NDP-MSH by binding to MC-1R induces tolerogenic DC and expands functional Treg in vivo. These Treg suppress pathogenic Th1 and Th17 effector cells during EAE progression. The extensive re-myelination of neurons from NDP-MSH treated mice compared to PBS injected controls furthermore suggests a neuroprotective effect of NDP-MSH.

Example 6Effects of NDP-MSH in a Spontaneous EAE Model (Devic Mice)

To characterize the effects of NDP-MSH in a spontaneous EAE model Devic mice were used. Devic mouse mutants express transgenic T- and B-cell receptors specific for MOG and spontaneously develop EAE at the age of 4-5 weeks (Bettelli et al., 2006).

Starting at the age of 38 days when mice reached a clinical score of 7 (severe hind limb paralysis) animals were injected intravenously with 5 μg NDP-MSH every other day. In total, treatment of mice with NDP-MSH for 3 weeks resulted in a significant amelioration of disease in all animals. Whereas PBS treated control mice showed a considerable weight loss and continued developing severe ascending paralysis, mice injected with NDP-MSH gained weight and recovered from clinical symptoms of EAE. Part of the mice almost completely recovered from disease. At day 60, NDP-MSH treatment was interrupted and mice were monitored for the onset of clinical symptoms. Notably, even after several weeks without NDP-MSH injection EAE pathology was stable in all animals and no disease recurrence in any of the NDP-MSH treated Devic mice was observed. Together, these data point to a long-lasting neuroprotective effect of NDP-MSH.

Example 7Effect of NDP-MSH on EAE in Mice After Depletion of Regulatory T Cells or Dendritic Cells

To further elucidate the mode of action of NDP-MSH on EAE, Treg and DC were depleted in DEREG (Lahl et al., 2007) or CD11c-DTR mice (Hochweller et al. 2008), respectively by systemic treatment with diphtheria toxin. Subsequently, EAE was induced as described in Ex. 1. Intravenous injection of 5 μg NDP-MSH every 48 hours resulted in reduced disease severity in NDP-MSH treated DEREG mice (FIG. 8) and prevented disease onset in CD11c-DTR mice (FIG. 9) even in the absence of Treg or DC. These data demonstrate that not only Treg and tolerogenic DC, which are induced by NDP-MSH, account for the observed effects in EAE but in contrast indicate a strong neuroprotective role of NDP-MSH in inflammatory as well as neurodegenerative disorders of the CNS.

Example 8Long lasting Direct Neuroprotective Effects of NDP-MSH in Mice

To further examine the long term effect of NDP-MSH, and to overcome the limitations of MOG-induced EAE we investigated the effects of NDP-MSH in a second, independent, spontaneous model of inflammatory/demyelinating diseases of the CNS. Hence, TCRMOG×IgHMOG mice (Bettelli et al., 2006), that had been treated with NDP-MSH were examined for relapses more than 8 weeks after treatment cessation, as shown in FIG. 11. In FIG. 11 (A), it is shown that NDP-MSH treatment from days 36 to 64 of age prevented TCRMOG×IgHMOG mice from relapse for >8 weeks after cessation of therapy. Clinical scores from n=8 mice are depicted (individual mice are marked by different symbols). FIG. 11 (B) shows Flow cytometry of CD4+ T cells in spinal cord tissue from NDP-MSH-treated TCRMOG×IgHMOG mice at days 124 and 194 after birth. Representative histograms are shown. Furthermore, as shown in FIG. 11 (C), H&E, Luxol Fast Blue (LFB), and immunofluorescence staining using antibodies against CD4 (red), IL-17 (green), and Lama5 (gray) in lumbar spinal cord from NDP-MSH-treated TCRMOG×IgHMOG mice and PBS-treated controls at days 124 and 194 after birth was analysed and one representative image is shown. Areas of demyelination (LFB) and reduced Lama5 expression in the basement membrane are indicated by arrows. This shows that the effect of NDP-MSH lasts beyond the time of treatment.

Example 9NDP-MSH Modulates Action Potential Generation in TCRMOG×IgHMOG Mice

In a further experiment, the TCRMOG×IgHMOG mice were examined with the results shown in FIG. 12. Specifically, hippocampal neuronal cell cultures were obtained from TCRMOG×IgHMOG embryos (E18) and incubated at 37° C. and 5% CO2 for 5 to 7 days, stimulated with 1 nM NDP-MSH or an equal amount of PBS two times per day for the last 3 days of the culture. Electrophysiologic analyses of neuronal function (action potential generation, firing behavior) were performed by treating neuronal cells for 6 hours in standard artificial cerebrospinal fluid medium (ACSF; 120 mM NaCl, 2.5 mM KCI, 1.25 mM NaH2PO4, 22 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2, and 20 mM dextrose; pH 7.35 adjusted by bubbling with a mixture of 95% O2 and 5% CO2). In FIG. 12 (A) numbers of action potentials (AP) in hippocampal neurons from TCRMOG ×IgHMOG mice before disease development (black, day 30 after birth), autoimmune-prone, PBS-treated TCRMOG×IgHMOG mice (dark grey, day 60 after birth) and NDP-MSH-treated TCRMOG×IgHMOG mice (light grey, day 60 after birth), n=3 mice in each group were measured. In FIG. 12 (B), the firing behavior of PBS- or NDP-MSH-treated hippocampal neurons from TCRMOG×IgHMOG mice after Glutamate-provoked by depolarization is shown. One representative image per group is shown in the corresponding Figure.

Example 10A Single Subcutaneous Injection Of NDP-MSH-Loaded Microparticles is Sufficient to Attenuate CNS Inflammation

Because NDP-MSH is proteolytically cleaved in serum and the half-life of the peptide after intravenous injection has been estimated to 90 min, which might complicate the further development towards a potential clinical application, we generated a slow-release formulation by encapsulating the peptide into. These microparticles released NDP-MSH over a period of >30 days. NDP-MSH loaded microparticles increased release of NDP-MSH which reached ˜90% after 50 days. In a further experiment, as shown in FIG. 13, it was shown that a single subcutaneous injection of NDP-MSH-loaded microparticles into MOG-immunized C57BL/6 mice after the first clinical symptoms appeared is sufficient to attenuate CNS inflammation. After injection, the neuropeptide release from the particles lasts for more than 30 days. FIG. 13 (A) shows data from the C57BL/6 mice that were immunized with MOG-peptide to induce EAE and injected with NDP-MSH peptide (i.v., white arrows), placebo particles (s.c., black arrow) or NDP-MSH-loaded microparticles (s.c., red arrow) when clinical symptoms were detectable in the first mouse. Mean EAN scores from n=7 mice per group are shown; *, p<0.05 vs. mice treated with placebo particles. Further, FIG. 13 (B) shows representative images of CNS tissue after H&E as well as Luxol Fast Blue (LFB) staining. Inflammatory foci and demyelinated areas are marked with arrows. Finally, Flow cytometry of regulatory T cells in the CNS at disease maximum was performed. In FIG. 13 (C) representative FACS plots are shown, cells are gated for CD4 and Foxp3 as well as Helios staining was performed after cell permeabilization.

Example 11NDP-MSH Down-Regulated The Expression Of Potassium Channels Associated with CNS Inflammation and/or Neurodegeneration in the CNS from MOG-Immunized Mice

As shown in FIG. 14 (A), using the STRING 10 database, a network of potential protein interactions focusing on potassium channels has been generated. Kcnc3 (Kv3.3) is known to cause cerebellar neurodegeneration (Zhang et al. Cell 2016; 165:434-48) whereas Kcnc1 (Kv3.1) was suggested as a therapeutic target tor neuroprotection in Alzheimer's disease

(Francosi et al. J Neurosci 2006; 26:11652-64). FIG. 14 (B) shows representative immunofluorescence staining of brain tissue using antibodies to Kcnc3 (Kv3.3), Kcnc1 (Kv3.1) and NeuN. Nuclei are counterstained with DAPI, original magnification 200 X. To assess the gene expression of voltage-gated potassium channels in brain and spinal cord from MOG-immunized and NDP-MSH treated mice as well as controls total RNA was extracted from 5 to 10 mg of tissues at disease maximum. Afterwards, total RNA preparations were analyzed for integrity using Agilent 2100 Bioanalyzer (Agilent Technologies). All samples showed high quality (mean RNA Integrity Number 9.3). RNA was further analyzed by photometric NanoDrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies). Synthesis of biotin-labeled cDNA was performed by converting 100 ng of total RNA to cDNA. After amplification by in vitro transcription and second cycle synthesis, cDNA was fragmented and biotin-labeled by terminal transferase. Finally, end-labeled cDNA was hybridized to Affymetrix Mouse Gene 2.0 ST Gene Expression Microarrays for 16 hours at 45° C., stained by streptavidin/phycoerythrin conjugate, and scanned. Data analyses on Affymetrix CEL files were conducted using GeneSpring GX software (version 12.5; Agilent Technologies). Probes within each probe set were summarized by GeneSpring' s ExonRMA16 algorithm after quantile normalization of probe level signal intensities across all samples to reduce inter-array variability. Input data preprocessing was concluded by baseline transformation to the median of all samples. Differential gene expression was statistically determined by moderated t-tests. The significance threshold was set to P=0.05. Differentially expressed genes passing a fold change cutoff of >1.5 and a P value of <0.05 in all replicates of one experimental group were further characterized and known as well as predicted interactions of proteins encoded by the differentially expressed genes (focus on voltage-gated potassium channels) were calculated using STRING 10 software (http://string-db.org).

Example 12NDP-MSH Impacts on Cognitive Effects

Further, to show the impact of NDP-MSH on cognitive effects, the object recognition (NOR) test, which is a commonly used behavioral test in mice, was used as outlined in FIG. 15 (A). In the NOR test, a mouse is presented with two similar objects during the first session (familiarization, 1 h per day for 4 consecutive days). Thereafter, one of the two objects is replaced by a new object (test session, 1 h at day 5). The amount of time taken to explore the new object provides an index of recognition memory (NOR-index). In order to study the impacts of NDP-MSH, C57BL/6mice (WT) were systemically treated with Scopolamin (Scm) daily from day 1-4, which is known to impair cognitive effects and memory. 30 or 60 minutes after

Scopolamin treatment mice received an intravenous injection of 5 μg NDP-MSH in 100 μl PBS or an equal amount or PBS and the NOR-index was assessed. n=10 mice in each group; *;p<0.05vs.WT+Scopolamin. As can be seen from FIG. 15 (B), the NOR score oft eh NDP-MSH mice was higher than that of the mice treated with Scolopamin alone, indicating that NDP-MSH has a neuroprotective effect on WT mice.

Claims

1-9. (canceled)

10. A method of ameliorating the symptoms of multiple sclerosis (MS) in a subject in need thereof comprising administering NDP-MSH or pharmaceutically acceptable salts thereof

11. The method of claim 10, wherein the method comprises therapeutic and/or a therapeutic prophylactic treatment.

12. The method of claim 10, wherein the method has an anti-inflammatory and/or neuroprotective effect.

13. The method of claim 10, wherein the subject is a mammal.

14. The method of claim 10, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is chemically modified.

15. The method of claim 10, wherein NDP-MSH is administered during relapse, progression and/or remission.

16. The method of claim 10, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is administered intravenously.

17. The method of claim 10, wherein 1-500 μg/kg of body weight of NDP-MSH or the pharmaceutically acceptable salt is administered.

18. The method of claim 10, wherein NDP-MSH is administered repeatedly in intervals of 12-72 hours.

Patent History

Publication number: 20200360484
Type: Application
Filed: Feb 26, 2020
Publication Date: Nov 19, 2020
Inventors: THOMAS LUGER (MUENSTER), KARIN LOSER (LTENBERGE), SVEN MEUTH (Münster)
Application Number: 16/802,016

Classifications

International Classification: A61K 38/22 (20060101); A61P 25/00 (20060101); A61P 29/00 (20060101);

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[Uhohinc]

I reference here, I am going to compare the Luger 2014 USA patent, to the Luger 2020 patent for the same what I will call the Multiple Sclerosis patent. I think from this we can see where Clinuvel defended its rights and see how the team of Drs. with law degrees at Munster (i am misspelling intentionally so it does not come up in searches) fought, mostly had to back off somewhat, and where Clinuvel patents IP stands. It appears imo, That Clinuvel's lawyers beat up Munster and Luger lawyers!!!!!!!

Claims in Luger 2014 Patent

1. A method of ameliorating the symptoms of inflammatory and/or neurodegenerative disorders of the Central Nervous System (CNS) in a subject in need thereof comprising administering NDP-MSH or pharmaceutically acceptable salts thereof, wherein the inflammatory and/or neurodegenerative disorder of the CNS is acute disseminated encephalomyelitis (ADEM).   

2. The method of claim 1, wherein the method comprises therapeutic and/or a therapeutic prophylactic treatment.

3. The method of claim 1, wherein the method has an anti-inflammatory and/or neuroprotective effect.

4. The method of claim 1, wherein the subject is a mammal.

5. The method of claim 1, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is chemically modified.

6. The method of claim 1, wherein NDP-MSH is administered during relapse, progression and/or remission.

7. The method of claim 1, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is administered intravenously.

8. The method of claim 1, wherein 1-500 μg/kg of body weight of NDP-MSH or the pharmaceutically acceptable salt is administered.

9. The method of claim 1, wherein NDP-MSH is administered in repeatedly in intervals of 12-72 hours.

10. A method of ameliorating the symptoms of inflammatory and/or neurodegenerative disorders of the Central Nervous System (CNS) in a subject in need thereof comprising administering NDP-MSH or pharmaceutically acceptable salts thereof, wherein the inflammatory and/or neurodegenerative disorder of the CNS is acute hemorrhagic leukoencephalitis (AHLE).

11. The method of claim 10, wherein the method comprises therapeutic and/or a therapeutic prophylactic treatment.

12. The method of claim 10, wherein the method has an anti-inflammatory and/or neuroprotective effect.

13. The method of claim 10, wherein the subject is a mammal.

14. The method of claim 10, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is chemically modified.

15. The method of claim 10, wherein NDP-MSH is administered during relapse, progression and/or remission.

16. The method of claim 10, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is administered intravenously.

17. The method of claim 10, wherein 1-500 μg/kg of body weight of NDP-MSH or the pharmaceutically acceptable salt is administered.

18. The method of claim 10, wherein NDP-MSH is administered in repeatedly in intervals of 12-72 hours.

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

Claims in Luger 2020 Patent

Claims

1-9. (canceled)                              (alll wiped out!!!!!!)

10. A method of ameliorating the symptoms of multiple sclerosis (MS) in a subject in need thereof comprising administering NDP-MSH or pharmaceutically acceptable salts thereof

11. The method of claim 10, wherein the method comprises therapeutic and/or a therapeutic prophylactic treatment.                                        (same)

12. The method of claim 10, wherein the method has an anti-inflammatory and/or neuroprotective effect.                                                              (same)

13. The method of claim 10, wherein the subject is a mammal.                                                                                                                                         (same)

14. The method of claim 10, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is chemically modified.                                       (same)

15. The method of claim 10, wherein NDP-MSH is administered during relapse, progression and/or remission.                                                   (same)

16. The method of claim 10, wherein NDP-MSH or a pharmaceutically acceptable salt thereof is administered intravenously.                          (same)

17. The method of claim 10, wherein 1-500 μg/kg of body weight of NDP-MSH or the pharmaceutically acceptable salt is administered.       (same)

18. The method of claim 10, wherein NDP-MSH is administered repeatedly in intervals of 12-72 hours.                                                                 (same)

[Uhohinc]

CITED LITERATURE                   (from 2014 Luger patent)

• Brod, S. A., & Hood, Z. M. (2008). Ingested (oral) alpha-MSH inhibits acute EAE. Journal of Neuroimmunology, 193, pp. 106-112.

• Brzoska, T., Luger, T. A., Maaser, C., Abels, C., & Bohm, M. (2007). alpha-Melanocyte Stimulating Hormone and Related Tripeptides: Biochemistry, Antiinflammatory and Protective Effects in Vitro and in Vivo, and Future Perspectives for the Treatment of Immune-mediated Inflammatory Diseases. Endocrine Reviews, 29(5), pp. 581-602.

• Carniglia, L., Durand, D., & Lasaga, M. (2013). Effect of NDP-alpha-MSH on PPAR-gamma and -beta Expression and Anti-Inflammatory Cytokine Release in Rat Astrocytes and Microglia. PLoS ONE, 8(2).
• Catania, A. (2008). Neuroprotective actions of melanocortins: a therapeutic opportunity. Trends Neurosci., 31(7), pp. 353-360.
• Chen, S.-J., Wang, Y.-L., Fan, H.-C., Lo, W.-T., Wang, C.-C., & Sytwu, H.-K. (2012). Current Status of Immunomodulation and Immunimediated Therapeutic Strategies for Multiple Sclerosis. Clinical and Developmental Immunology.

• Constantinescu, C. S., Farooqi, N., O'Brien, K., & Gran, B. (2011). Experimental autoimmune ecephalomyelitis (EAE) as a model for multiple sclerosis (MS). British Journal of Pharmacology, 164, pp. 1079-1106.

• Han, D., Tian, Y., Zhang, M., Zhou, Z., & Lu, J. (2007). Prevention and treatment of experimental autoimmune encephalomyelitis with recombinant adeno-associated virus-mediated alpha-melanocyte stimulating hormone-transduced PLP-139-151-specific T cell. Gene Therapy, 14, pp. 383-395.

• Hochweller, K., Striegler, J., Hammerling, G. J., & Garbi, N. (2008). A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. European Journal of Immunology, 38(10), pp. 2776-83.

• Krishnamoorthy, G., Lassmann, H., Wekerle, H., & Holz, A. (2006). Spontaneous opticospinal encephalomyelitis in a double-transgenic mouse model of autoimmune T cell/B cell cooperation. The Journal of Clinical Investigation, 116(9), pp. 2385-2392.
• Kurtzke, J. F. (1983). Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology, 33(11), pp. 1444-1452.
• Lahl, K., Loddenkemper, C., Drouin, C., Freyer, J., Arnason, J., Eberl, G., . . . Sparwasser, T. (2007). Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. The Journal of Experimental Medicine, 204(1), pp. 57-63.

• Luger, T. A., Scholzen, T. E., Brzoska, T., & Bohm, M. (2003). New Insights into the Fuctions alpha-MSH and Related Peptides in the Immune System. Annals New York Academy of Sciences, pp. 133-140.

• Rudman, D., Hollins, B. M., Kutner, M. H., Moffitt, S. D., & Lynn, M. J. (1983). Three types of alpha-melanocyte-stimulating hormone: bioactivities and half-lives. American Journal of Physiology, 245(1), pp. E47-54.
• Sawyer, T. K., Sanfilippo, P., Hruby, V. J., Engel, M. H., Heward, C. B., Burnett, J., & Hadley, M. E. (1980). 4-Norleucine, 7-D-phenylalanine-alpha-melanocyte-stimulating hormone: A highly potent alpha-melanotropin with ultralong biological activity. (5758, Ed.) Proceedings of the National Academy of Sciences of the United States of Americs, 77(10), pp. 5754-5758.

• Smith, A. G., Luk, N., Newton, R. A., Roberts, D. W., Sturm, R. A., & Muscat, G. E. (2008). Melanocortin-1 receptor signaling markedly induces the expression of the NR4A nuclear receptor subgroup in melanocytic cells. 283(18), pp. 12564-12570.
• Ter Laak, M., Brakkee, J., Adan, R., Hamers, F., & Gispen, W. (2003). The potent melanocortin receptor agonist melanotan-II promotes peripheral nerve regeneration and has neuroprotective properties in the rat. Eur J Pharmacol., 462(1-3), pp. 179-183.

• Volakilis, N., Kadkhodaei, B., Joodmardi, E., Wallis, K., Panman, L., Silvaggi, J., . . . Perlmann, T. (2010). NR4A orphan nuclear receptors as mediators of CREB-dependent neuroprotection. 107(27), pp. 12317-12322.
• Yin, P., Luby, T. M., Chen, H., Etemad-Moghadam, B., Lee, D., Aziz, N., . . . Hedley, M. L. (2003). Generation of expression constructs that secrete bioactive alphaMSH and their use in the treatment of experimental autoimmune encephalitis. Gene Therapy, 10, pp. 349-355.

(cited literature from Luger 2020 patent

CITED LITERATURE

• Bettelli, et al. (2006). Myelin oligodendrocyte glycoprotein-specific T and B cells cooperate to induce a Devic-like disease in mice. Journal of Clinical Investigation, 116(9). pp. 2393-2402       (new not in 2014 Luger patent)

Perlmann, T. (2010). NR4A orphan nuclear receptors as mediators of CREB-dependent neuroprotection. 107(27), pp. 12317-12322.           (new, not in 2014 luger patent)

[Uhohinc]

the below here, was added to the 2020 luger patent that was not in the 2014 Luger patent

Example 8Long lasting Direct Neuroprotective Effects of NDP-MSH in Mice

To further examine the long term effect of NDP-MSH, and to overcome the limitations of MOG-induced EAE we investigated the effects of NDP-MSH in a second, independent, spontaneous model of inflammatory/demyelinating diseases of the CNS. Hence, TCRMOG×IgHMOG mice (Bettelli et al., 2006), that had been treated with NDP-MSH were examined for relapses more than 8 weeks after treatment cessation, as shown in FIG. 11. In FIG. 11 (A), it is shown that NDP-MSH treatment from days 36 to 64 of age prevented TCRMOG×IgHMOG mice from relapse for >8 weeks after cessation of therapy. Clinical scores from n=8 mice are depicted (individual mice are marked by different symbols). FIG. 11 (B) shows Flow cytometry of CD4+ T cells in spinal cord tissue from NDP-MSH-treated TCRMOG×IgHMOG mice at days 124 and 194 after birth. Representative histograms are shown. Furthermore, as shown in FIG. 11 (C), H&E, Luxol Fast Blue (LFB), and immunofluorescence staining using antibodies against CD4 (red), IL-17 (green), and Lama5 (gray) in lumbar spinal cord from NDP-MSH-treated TCRMOG×IgHMOG mice and PBS-treated controls at days 124 and 194 after birth was analysed and one representative image is shown. Areas of demyelination (LFB) and reduced Lama5 expression in the basement membrane are indicated by arrows. This shows that the effect of NDP-MSH lasts beyond the time of treatment.

Example 9NDP-MSH Modulates Action Potential Generation in TCRMOG×IgHMOG Mice

In a further experiment, the TCRMOG×IgHMOG mice were examined with the results shown in FIG. 12. Specifically, hippocampal neuronal cell cultures were obtained from TCRMOG×IgHMOG embryos (E18) and incubated at 37° C. and 5% CO2 for 5 to 7 days, stimulated with 1 nM NDP-MSH or an equal amount of PBS two times per day for the last 3 days of the culture. Electrophysiologic analyses of neuronal function (action potential generation, firing behavior) were performed by treating neuronal cells for 6 hours in standard artificial cerebrospinal fluid medium (ACSF; 120 mM NaCl, 2.5 mM KCI, 1.25 mM NaH2PO4, 22 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2, and 20 mM dextrose; pH 7.35 adjusted by bubbling with a mixture of 95% O2 and 5% CO2). In FIG. 12 (A) numbers of action potentials (AP) in hippocampal neurons from TCRMOG ×IgHMOG mice before disease development (black, day 30 after birth), autoimmune-prone, PBS-treated TCRMOG×IgHMOG mice (dark grey, day 60 after birth) and NDP-MSH-treated TCRMOG×IgHMOG mice (light grey, day 60 after birth), n=3 mice in each group were measured. In FIG. 12 (B), the firing behavior of PBS- or NDP-MSH-treated hippocampal neurons from TCRMOG×IgHMOG mice after Glutamate-provoked by depolarization is shown. One representative image per group is shown in the corresponding Figure.

Example 10A Single Subcutaneous Injection Of NDP-MSH-Loaded Microparticles is Sufficient to Attenuate CNS Inflammation

Because NDP-MSH is proteolytically cleaved in serum and the half-life of the peptide after intravenous injection has been estimated to 90 min, which might complicate the further development towards a potential clinical application, we generated a slow-release formulation by encapsulating the peptide into. These microparticles released NDP-MSH over a period of >30 days. NDP-MSH loaded microparticles increased release of NDP-MSH which reached ˜90% after 50 days. In a further experiment, as shown in FIG. 13, it was shown that a single subcutaneous injection of NDP-MSH-loaded microparticles into MOG-immunized C57BL/6 mice after the first clinical symptoms appeared is sufficient to attenuate CNS inflammation. After injection, the neuropeptide release from the particles lasts for more than 30 days. FIG. 13 (A) shows data from the C57BL/6 mice that were immunized with MOG-peptide to induce EAE and injected with NDP-MSH peptide (i.v., white arrows), placebo particles (s.c., black arrow) or NDP-MSH-loaded microparticles (s.c., red arrow) when clinical symptoms were detectable in the first mouse. Mean EAN scores from n=7 mice per group are shown; *, p<0.05 vs. mice treated with placebo particles. Further, FIG. 13 (B) shows representative images of CNS tissue after H&E as well as Luxol Fast Blue (LFB) staining. Inflammatory foci and demyelinated areas are marked with arrows. Finally, Flow cytometry of regulatory T cells in the CNS at disease maximum was performed. In FIG. 13 (C) representative FACS plots are shown, cells are gated for CD4 and Foxp3 as well as Helios staining was performed after cell permeabilization.

Example 11NDP-MSH Down-Regulated The Expression Of Potassium Channels Associated with CNS Inflammation and/or Neurodegeneration in the CNS from MOG-Immunized Mice

As shown in FIG. 14 (A), using the STRING 10 database, a network of potential protein interactions focusing on potassium channels has been generated. Kcnc3 (Kv3.3) is known to cause cerebellar neurodegeneration (Zhang et al. Cell 2016; 165:434-48) whereas Kcnc1 (Kv3.1) was suggested as a therapeutic target tor neuroprotection in Alzheimer's disease

(Francosi et al. J Neurosci 2006; 26:11652-64). FIG. 14 (B) shows representative immunofluorescence staining of brain tissue using antibodies to Kcnc3 (Kv3.3), Kcnc1 (Kv3.1) and NeuN. Nuclei are counterstained with DAPI, original magnification 200 X. To assess the gene expression of voltage-gated potassium channels in brain and spinal cord from MOG-immunized and NDP-MSH treated mice as well as controls total RNA was extracted from 5 to 10 mg of tissues at disease maximum. Afterwards, total RNA preparations were analyzed for integrity using Agilent 2100 Bioanalyzer (Agilent Technologies). All samples showed high quality (mean RNA Integrity Number 9.3). RNA was further analyzed by photometric NanoDrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies). Synthesis of biotin-labeled cDNA was performed by converting 100 ng of total RNA to cDNA. After amplification by in vitro transcription and second cycle synthesis, cDNA was fragmented and biotin-labeled by terminal transferase. Finally, end-labeled cDNA was hybridized to Affymetrix Mouse Gene 2.0 ST Gene Expression Microarrays for 16 hours at 45° C., stained by streptavidin/phycoerythrin conjugate, and scanned. Data analyses on Affymetrix CEL files were conducted using GeneSpring GX software (version 12.5; Agilent Technologies). Probes within each probe set were summarized by GeneSpring' s ExonRMA16 algorithm after quantile normalization of probe level signal intensities across all samples to reduce inter-array variability. Input data preprocessing was concluded by baseline transformation to the median of all samples. Differential gene expression was statistically determined by moderated t-tests. The significance threshold was set to P=0.05. Differentially expressed genes passing a fold change cutoff of >1.5 and a P value of <0.05 in all replicates of one experimental group were further characterized and known as well as predicted interactions of proteins encoded by the differentially expressed genes (focus on voltage-gated potassium channels) were calculated using STRING 10 software (http://string-db.org).

Example 12NDP-MSH Impacts on Cognitive Effects

Further, to show the impact of NDP-MSH on cognitive effects, the object recognition (NOR) test, which is a commonly used behavioral test in mice, was used as outlined in FIG. 15 (A). In the NOR test, a mouse is presented with two similar objects during the first session (familiarization, 1 h per day for 4 consecutive days). Thereafter, one of the two objects is replaced by a new object (test session, 1 h at day 5). The amount of time taken to explore the new object provides an index of recognition memory (NOR-index). In order to study the impacts of NDP-MSH, C57BL/6mice (WT) were systemically treated with Scopolamin (Scm) daily from day 1-4, which is known to impair cognitive effects and memory. 30 or 60 minutes after

Scopolamin treatment mice received an intravenous injection of 5 μg NDP-MSH in 100 μl PBS or an equal amount or PBS and the NOR-index was assessed. n=10 mice in each group; *;p<0.05vs.WT+Scopolamin. As can be seen from FIG. 15 (B), the NOR score oft eh NDP-MSH mice was higher than that of the mice treated with Scolopamin alone, indicating that NDP-MSH has a neuroprotective effect on WT mice.

[Uhohinc]

• Review Article
• Open Access
• Published: 06 May 2021

Paving the way towards an effective treatment for multiple sclerosis: advances in cell therapy

• M. J. Mansilla, 
• S. Presas-Rodríguez, 
• A. Teniente-Serra, 
• I. González-Larreategui, 
• B. Quirant-Sánchez, 
• F. Fondelli, 
• N. Djedovic, 
• D. Iwaszkiewicz-Grześ, 
• K. Chwojnicki, 
• Đ. Miljković, 
• P. Trzonkowski, 
• C. Ramo-Tello & 
• E. M. Martínez-Cáceres 

Cellular & Molecular Immunology (2021)Cite this article

• Metricsdetails

Abstract

Multiple sclerosis (MS) is a leading cause of chronic neurological disability in young to middle-aged adults, affecting ~2.5 million people worldwide. Currently, most therapeutics for MS are systemic immunosuppressive or immunomodulatory drugs, but these drugs are unable to halt or reverse the disease and have the potential to cause serious adverse events. Hence, there is an urgent need for the development of next-generation treatments that, alone or in combination, stop the undesired autoimmune response and contribute to the restoration of homeostasis. This review analyzes current MS treatments as well as different cell-based therapies that have been proposed to restore homeostasis in MS patients (tolerogenic dendritic cells, regulatory T cells, mesenchymal stem cells, and vaccination with T cells). Data collected from preclinical studies performed in the experimental autoimmune encephalomyelitis (EAE) model of MS in animals, in vitro cultures of cells from MS patients and the initial results of phase I/II clinical trials are analyzed to better understand which parameters are relevant for obtaining an efficient cell-based therapy for MS.

Multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease that affects the central nervous system (CNS) and is characterized by inflammation, multifocal demyelination, axonal loss, and gliosis in both the white and gray matter. MS is a complex disease with considerable clinical and radiological heterogeneity. It was initially classified into four different phenotypes: relapsing-remitting MS (RRMS), secondary-progressive MS (SPMS), primary-progressive MS (PPMS), and relapsing-progressive MS (RPMS).1 RRMS (85%) is characterized by acute relapses (acute or subacute episodes of new or increasing neurologic dysfunction in the absence of fever or infection) followed by remission with full or partial recovery. SPMS is defined as progressive clinical worsening over time after an initial relapsing course, with or without acute exacerbations during the progressive course. PPMS, accounting for ~15% of MS, is characterized by clinical progression without relapse from disease onset. The term RPMS is used to describe the progressive accumulation of disability from onset with occasional relapses. This subtype is rarely diagnosed since it overlaps with other phenotypes in terms of its features.

In addition, since 2014, these phenotypes have also included the concept of “disease activity” based on clinical and MRI criteria in an effort to achieve better patient classification.2 For MS diagnosis, a combination of clinical, radiological, and laboratory criteria (presence of oligoclonal bands in the cerebrospinal fluid (CSF)) is used. The most recent diagnostic criteria are the 2017 McDonald criteria.3

MS pathogenesis

Currently, the cause of MS remains unknown. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, myelin-specific T cells are believed to play a crucial role in its pathogenesis.4 In fact, the presence of circulating myelin-reactive T cells in MS patients has been extensively reported. However, the specific mechanisms that cause the activation and entrance of these cells into the CNS are still unknown. It has been postulated that a complex interaction between multiple genetic and environmental factors contributes to the dysregulation of peripheral immune homeostasis and the activation of autoreactive T cells.5

Several environmental factors, including infectious agents (mainly viruses), tobacco, diet (long-chain fatty acids, salt), gut microbiota, stress, sex hormones, and vitamin D deficiency, have been shown to be related to the triggering and development of disease.5 The incidence of MS is higher in women than in men. MS symptoms often improve during late pregnancy, coinciding with high levels of estriol.6 In contrast, men are more prone to develop PPMS later in life, correlating with the physiological decline in testosterone with age.7

Extensive studies have been performed to understand the genetic contribution to MS, and more than 200 loci that promote a predisposition to MS have been identified, suggesting a complex disease etiology. Nearly all the gene regions identified so far contain genes involved in immune mechanisms. The major HLA-DRB1∗1501 locus accounts for 30% of the overall risk.8,9 Moreover, emerging evidence indicates that the DNA methylome actively participates in gene × environment interactions, and several studies have shown an aberrant DNA methylome profile develops in MS.10

Factors such as the presence of dysfunctional regulatory T cells (Tregs)11 or dendritic cells (DCs)12 and alterations in cytokine production may facilitate the entry of proinflammatory myelin-specific autoreactive T cells into the CNS (reviewed in13,14). In this context, IFN-γ-producing Th17.1 (CCR6+CXCR3+CCR4–) cells have been identified as relevant in disrupting the permeability of the BBB in MS.15 Memory B cell precursors and IFN-γ-producing CD8+ T cells also express high levels of CCR6, as they can enter the CNS. In addition to chemokine receptors and proinflammatory cytokines, adhesion molecules such as integrin α4β1 (VLA-4), which induces firm adhesion to vascular cell adhesion protein 1 (VCAM-1) on brain endothelial cells, and activated leukocyte cell adhesion molecule enhance the transmigration of pathogenic B and T cell subsets.16,17

As an alternative to the immune-mediated cause of MS, it has been postulated that CNS-intrinsic events (for example, CNS viral infection or processes leading to primary neurodegeneration) may trigger disease development, with the infiltration of autoreactive lymphocytes occurring as a secondary phenomenon.18,19

The brain and spinal cord are surrounded by the meninges, which provide first-line protection to the CNS. They are constituted by three layers: the dura mater, located directly under the skull or vertebral column; arachnoid mater; and pia mater, in close contact with the CNS parenchyma. The CSF drains through the subarachnoid space, an anatomical gap between the arachnoid mater and pia mater (both known as the leptomeninges). Interestingly, the existence of a rudiment of lymphatic vessels within the meninges was recently described, supporting the existence of a physical connection between the fluids, immune cells, and macromolecules of the CNS and the deep cervical lymph nodes, establishing physical contact between the CNS and the immune system.19,20

Most knowledge of MS pathogenesis has been obtained in the EAE model, the animal model of MS. This model is typically induced by either active immunization with myelin-derived proteins or peptides in adjuvant or by passive transfer of activated myelin-specific CD4+ T lymphocytes and reproduces most of the main clinical and histopathological characteristics of MS. Although this model has some limitations, as it is an induced model, it is a powerful tool to investigate MS pathogenesis and potential therapeutic strategies.21,22 In EAE, several days before inflammatory cells are detected in the CNS, an influx of peripherally derived immune cells within the meninges occurs.23 Myelin antigens that drain from the CNS to the meninges via the CSF are presented by infiltrating or resident antigen-presenting cells (APCs) (perivascular macrophages or resident APCs, e.g., microglial cells).24,25,26 This causes myelin-specific T cell reactivation. Following myelin recognition by autoreactive pathogenic T cells, a complex immune response is produced, facilitated by the entrance of other cell types (B cells, NK cells, macrophages, and innate immune cells) and the production of proinflammatory cytokines and reactive oxygen and nitrogen species (ROS and RNS, respectively); this results in disruption of the blood–brain barrier (BBB) and the entrance of cells into the CNS parenchyma, leading to perivascular inflammation, demyelination, and neuronal damage.27

Interestingly, in contrast to EAE, CD8+ T cells are found more frequently than CD4+ cells in acute and chronic MS lesions. CD8+ T cells directly attack oligodendrocytes (via the secretion of granzymes and perforin), causing apoptosis and damaging neurons via the release of cytolytic granules, leading to axonal dissection.28 Up to a quarter of CD8+ T cells in the active lesions of MS patients can produce IL-17 and are thought to be mucosa-associated invariant T (MAIT) cells. It has been suggested that these CD8+ cells, characterized by the expression of a semi-invariant T cell receptor (a dimer of Vα7.2 with Jα12, Jα20, or Jα33), play an important role in disease pathogenesis.29

In recent years, the pathogenic role of B cells in MS has been highlighted.30 Clonally expanded B cells can be found in the meninges, parenchyma and CSF. B cells produce antibodies intrathecally with an oligoclonal pattern (which can be observed by comparison with serum samples from the same patient). Ectopic lymphoid follicles, which sequester antigens and facilitate B and T cell activation, have been observed proximal to cortical demyelinating lesions in the meninges of MS patients, and their frequency correlates with disease severity.31,32,33 The presence of tertiary lymph follicles suggests that B cell maturation is sustained locally, contributing to the intrathecal synthesis of immunoglobulins.31,32 In addition to their roles in antigen presentation to Th1 and Th17 cells and antibody production, B cells secrete proinflammatory cytokines (e.g., IL-6, TNF, granulocyte-macrophage colony-stimulating factor (GM-CSF)), promoting CNS inflammation and demyelination.34

There is increasing evidence that innate immune cells (NK, DCs, macrophages, mast cells, and innate lymphoid cells (ILCs)), normal residents of the meninges, are involved in the pathogenesis of MS, affecting both its initiation and progression (reviewed by Brown et al.33). Mast cell transcripts that encode mast cell-associated molecules, such as tryptase histamine or FcεR1, have been observed in the demyelinating lesions of MS patients.35 They can increase BBB permeability, contributing to the initiation of chronic inflammation.36 Moreover, interactions between resident mast cells and autoreactive T cells in the meninges induce caspase-1-dependent IL-1β production by mast cells, activating GM-CSF production by T cells.37,38 GM-CSF is an essential growth factor for T cell encephalitogenicity, inducing the recruitment of CCR2+ inflammatory monocytes into the CNS.39,40,41,42 Additionally, TNF expression by mast cells is essential for the early recruitment of neutrophils to the meninges and CNS.23,36,43 Neutrophil products, i.e., proteolytic enzymes, including matrix metalloproteinase 9, ROS and structures composed of DNA and proteins called neutrophil extracellular traps, damage the BBB, thus supporting a role for neutrophils in MS pathogenesis.43,44,45

CD45+Lin−IL-7Rα+ ILCs constitute a heterogeneous group of innate immune cells that have more recently been related to MS pathogenesis: group 1 (ILC1), group 2 (ILC2), and group 3 (ILC3) cells, analogous to Th1, Th2, and Th17 cells, respectively. These cells mostly remain in tissues and exert their effects locally.46 Similar to Th cells, ILCs can also exhibit considerable phenotypic plasticity.46 Each group of ILCs plays a different role in EAE-related inflammatory responses. ILC2s express ST2, the heterodimeric IL-33 receptor. Upon activation, these cells produce the Th2 cytokines IL4, IL-5, IL-9, and IL-13.47 IL-33 induces ST2+ ILC2s to produce IL-13 and other Th2-polarizing cytokines, which in turn promote a nonpathogenic Th2-dominated response. In contrast, ILC1s and ILC3s produce IFN-γ, IL-17, GM-CSF, and other cytokines that have been linked to EAE pathogenesis. Interestingly, the lymphoid tissue inducer subset of ILC3s drives ectopic lymphoid follicle formation.48

Natural killer (NK) cells have been widely studied in the context of MS and EAE and assigned both pathogenic and protective roles (reviewed in49,50). CD56dim cells, the major cytotoxic NK population, can kill oligodendrocytes, astrocytes, and microglia in vitro. In contrast, regulatory CD56hi NK cells produce neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3, consistent with a role in neural repair.

Additionally, CNS-resident cells (mainly microglia) are highly sensitive to homeostatic disturbances and can produce neurotoxic inflammatory mediators (cytokines, chemokines, and ROS), promoting and sustaining neurodegeneration (reviewed in13). Importantly, astrocytes are key players recruiting lymphocytes and inducing the innate response during the early stage of white matter lesion formation. In contrast, astrocytes can also restrict inflammation through scar formation and trigger neuroprotection and tissue repair.51 Regulatory B cells (Bregs), T cells (CD4+FoxP3+ (Tregs), CD4+Tr1+ and CD8 Tregs), tolerogenic DCs (tolDCs) and regulatory CD56hi NK cells can regulate effector T cells in the periphery or CNS through different mechanisms. It has been postulated that either a defect in the regulatory function of these cells or the increased resistance of effector T cells to suppressive mechanisms contributes to the pathogenic function of autoreactive T and B cells in MS. Alternatively, the dysfunction of peripheral regulatory cells could be indirectly driven by the dysregulation of tolerogenic APCs (reviewed in13).

Neuropathology of MS

In contrast to the increased inflammation of relapsing forms of MS, neurodegenerative events are more severe in progressive MS. Nevertheless, there is no clear distinction between progressive and relapsing MS; both pathobiologies can occur during the disease.

Comparison of PPMS and SPMS reveals some quantitative differences in the presence of focal and active classical white matter lesions and in the global degree of inflammation, which is lower in PPMS than in SPMS.52 These differences can be explained by the two types of inflammation in MS patients (Fig. 1). During disease progression, patients can exhibit both types of inflammation, although the composition can be altered during relapses and progressive periods.

Fig. 1

Multiple sclerosis pathogenesis in both relapsing and progressive disease. Scheme representing the major cells and molecules that play a role in the two different stages of MS. The dashed line allows comparison of the differences between relapsing and progressive MS. Arrows indicate release, while inhibitors indicate inhibition. The black arrowhead on a dotted line indicates transmigration. CSF cerebrospinal fluid, DC dendritic cells, GM-CSF granulocyte-macrophage colony-stimulating factor, IFN interferon, IL interleukin, ILCs innate lymphoid cells, MAIT cells mucosal-associated invariant T cells, MS multiple sclerosis, RNS reactive nitrogen species, ROS reactive oxygen species, SAS subarachnoid space, Th T helper

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During the acute and relapsing phases of the disease, in which leakage of the BBB occurs, the major players are T (CD4+ and CD8+) and B lymphocytes that attack myelin, leading to demyelination. CD8+ cells have the phenotype of tissue-resident memory cells. CD8+ T cells proliferate focally and show signs of activation or clonal expansion, indicating local antigen recognition. The pathogenic role of B cells is inferred by the beneficial effects observed after anti-CD20 therapies. However, B cells are thought to play different roles depending on their stage of differentiation or the activity stage of the lesions. Plasmablasts and plasma cells within MS lesions express high levels of IL-10,53 suggesting that they may ameliorate inflammation.

Inflammatory infiltrates may lead to focal areas of primary demyelination with variable axonal injury, which is mainly carried out by activated microglia and macrophages. Antibodies that recognize oligodendrocytes or astroglia may contribute to MS pathogenesis at this stage. After the initial autoimmune attack, lymphoid cells in the parenchyma undergo apoptosis, and macrophages and microglia can switch to an anti-inflammatory/reparative phenotype.

In the progressive phases of MS, the second pattern of inflammation predominates. Leakage of the BBB is less pronounced, and T and B lymphocytes slowly accumulate in the connective tissue spaces of the brain and spinal cord, affecting the meninges and periventricular spaces. Infiltrating cells form focal aggregates resembling tertiary lymph follicles. CD8+ cells have the phenotype of tissue-resident memory cells with focal activation. Most cells of the B cell lineage in chronic lesions are plasmablasts and plasma cells. Tissue injury may at least be partly mediated by microglia and macrophage activation, oxidative injury and mitochondrial damage. This inflammation is associated with the expansion of pre-existing lesions, as well as diffuse neurodegeneration in normal-appearing white or gray matter. Interestingly, it has been found that this second type of inflammation is already present in the early stages of MS, after which it increases gradually with disease duration and patient age.52 The inflammation induced by peripheral immune cell infiltration and CNS-resident innate immune cells may contribute to acceleration of the aging processes in the CNS, followed by pronounced progressive neurodegenerative decline (reviewed in13).

Cell-based tolerogenic therapies

Targeting the fundamental cause of autoimmunity, i.e., the loss of tolerance to self-antigens, will provide the next steps forward to avoid the general immunosuppression induced by current treatments. Accumulating knowledge on the mechanisms of immune tolerance and activation has led to the development of tolerance-inducing cellular therapies with the specific objective of limited unwanted immune reactions over the long term (reviewed in54). Phase I clinical trials using Tregs, mesenchymal stem cells (MSCs), or tolerogenic antigen-presenting cells, such as tolDCs and regulatory macrophages, for the treatment of autoimmune diseases (ADs) and prevention of transplant rejection have expanded in recent years. The results have confirmed so far that these cellular therapies are safe, with no relevant side effects, and well tolerated by patients.54 In this context, several cellular products have been developed for MS (Fig. 2), and phase I clinical trials are currently ongoing or finished. To advance the use of these therapies in the clinic, we should analyze the results and address remaining challenges, such as the optimal dose, administration route, frequency of administration, antigen specificity, and biomarkers of clinical and biological response, to design the next generation of clinical trials.

Fig. 2

Proposed mechanisms of action of approved treatments for multiple sclerosis and cell-based therapies. Representation of the mechanisms of action of current treatments (black boxes) and cell-based therapies (gray boxes). Arrows indicate induction, while inhibition symbols indicate inhibition. Breg regulatory B cells, CNS central nervous system, DC dendritic cells, MAIT cell mucosal-associated invariant T cells, NK natural killer cells, Th T helper, Treg regulatory T cells

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Tolerogenic dendritic cells

DCs are key players in controlling the immune response. They are highly efficient APCs that are able to activate the immunogenic T cell response and suppress it by inducing regulatory mechanisms.55 TolDCs are defined as semimature DCs with an intermediate phenotype between the phenotypes of immature (iDCs) and mature DCs (mDCs). It is not clear whether tolDCs constitute a different DC subset by themselves. TolDCs are characterized by one or more of these features: the expression of low levels of costimulatory molecules (i.e., CD80, CD86, and CD40) and MHC class II, a reduced capacity to produce proinflammatory cytokines, upregulated expression of inhibitory molecules such as PDL1, ILT3 and ILT4 and secretion of immunoregulatory cytokines and mediators (IL-10, TGF-β, IDO, heme oxygenase-1 or FasL).56,57 Because of the semimature or mature resistance phenotype of tolDCs, the proper activation of T cells by costimulatory molecules and proinflammatory cytokines following antigen recognition is limited. Under these conditions, instead of inducing the activation and clonal expansion of T cells, tolDCs promote T cell hyporesponsiveness that is mainly mediated by the induction of T cell anergy, T cell depletion triggered by apoptosis induction or the induction of Treg differentiation (reviewed in58,59).

Importantly, tolDCs can be generated ex vivo from autologous human peripheral blood monocytes. Indeed, over the last 20 years, a wide variety of protocols describing the induction of tolDCs with several stimuli, such as anti-inflammatory cytokines (IL-10 and TGF-β), pharmacological agents and immunosuppressant compounds (rapamycin, different corticosteroids, vitamin D3, aspirin, mitomycin C, and the NF-κB inhibitor BAY11-7082), and other strategies, such as genetic engineering for the selective repression or induction of key molecules and pathways, among many others, have emerged (reviewed in54,58). Most of these protocols share several features, such as the differentiation of monocytes in the presence of GM-CSF and IL-4, as well as the addition of a maturation stimulus (which usually consists of different combinations of LPS, monophosphoryl lipid A, TNF, IL-1β, prostaglandin E2 (PGE2) and IL-6), to maintain tolDCs in an activation-resistant state, as this is an absolute requirement for tolDC therapy.

Analysis of preclinical data for the evaluation of tolDC treatment in EAE, as well as in vitro studies on peripheral blood leukocytes from MS patients (Table 1), is crucial to better understand the requirements and characteristics for the design of efficient tolDC products for MS patients.

Table 1 Tolerogenic DCs therapies in EAE and in vitro studies using MS patients tolDCs

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Antigen specificity

Several studies in the EAE model of MS have demonstrated that clinical efficacy of tolDCs is achieved only when the cells are loaded with disease-related autoantigens60,61,62,63,64,65,66,67,68,69,70,71 (Table 1). Although the etiology of MS is unknown, it is widely accepted that it is an autoimmune-mediated disease directed against several myelin proteins, such as MBP, proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). Consequently, when thinking about the translation of tolDC therapy to humans, the loading of a single antigen on tolDCs is unlikely to be sufficiently effective. Moreover, due to the epitope spreading phenomenon, the same patient may also exhibit extended reactivity to additional myelin epitopes following disease progression. Therefore, in tolDC phase I clinical trials for MS patients, a cocktail of myelin peptides containing the most relevant autoreactive MOG, PLP, and MBP peptides seems to be a better strategy.72,73 In fact, the three phase I clinical trials of peptide-loaded tolDCs in MS have employed a similar approach using a pool of myelin peptides (NCT02283671, NCT02903537, and NCT02618902) (Table 2).

Table 2 Clinical trials using tolDCs treatment in MS patients

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Dose, timing, and frequency

Although studies in animal models are extremely useful, extrapolation of the dose, timing, and frequency of administration to patients is not straightforward. From publications using different tolDC types to treat EAE, i.v. administration of a million antigen-specific tolDCs has demonstrated promising results in reducing EAE severity (Table 1). However, extrapolation of the dose from these studies is not feasible. The monocyte-derived tolDCs recovered after the in vitro differentiation process number a maximum of several million, depending on the patient. Consequently, thus far, direct extrapolation of tolDC doses from EAE studies is not feasible. In fact, in a phase I trial of i.v. dexamethasone-tolDC (dexa-tolDC) administration, a technical limitation was reported related to reaching 300 × 106 cells.74 Since cells administered intravenously exhibit a wide biodistribution of cells (meaning that a large number of tolDCs would be necessary to reach the secondary lymphoid organs), other routes of administration are currently being tested by our group and others (Table 2). This issue will be discussed below.

As shown in Table 1, many of the EAE studies analyzed the efficacy of tolDCs before the onset of clinical symptoms (prophylactic or late prophylactic administration). In contrast, other authors have attempted to reproduce the real-world situation of MS patients for therapeutic administration.61,66,67,70 From those studies, antigen-specific VitD3-tolDCs demonstrated a remarkable therapeutic effect.61,66,67

Focusing on therapeutic studies in EAE, the administration of at least 3 doses of antigen-specific tolDCs treated with VitD3 or BD750 (a JAK3/STAT5 inhibitor) in the early stage of disease abrogated disease progression.61,66,67,70 Considering translation to MS, the use of multiple tolDC injections seems optimal. However, this would imply repeated production of tolDC batches (which would require several rounds of leukapheresis and in vitro differentiation under good manufacturing practice conditions). Hence, the use of cryopreserved cells is a feasible option.66,75 In fact, at least for VitD3-tolDCs, both human and murine cryopreserved cells retain phenotypical and functional tolerogenic characteristics. Importantly, therapeutic administration of cryopreserved MOG40–55-VitD3-tolDCs in EAE mice showed clinical efficacy, reducing T cell autoreactivity and triggering the generation of FoxP3+ Tregs in vivo.66 All these data were comparable with those obtained using fresh cells.67 To go one step further, we explored the effect of long-term treatment with cryopreserved MOG40–55-VitD3-tolDCs in EAE. Following 3 therapeutic administrations of MOG40–55-tolDCs every 4 days, additional doses were administered when the mean clinical score of the group increased. Strikingly, we observed that periods of long-lasting clinical stability increased progressively. Immunological examination of mice revealed increased proportions of Bregs and activated NKT cells as well as a reduction in immunogenic NK cells in the spleens of treated mice, which might explain (at least in part) the beneficial effect of MOG40–55-tolDCs.66

The effect of tolDCs in the chronic phase of EAE has been analyzed using MOG35–55-BD750-tolDCs. Unfortunately, no clinical benefits were observed even though 3 doses of MOG35–55-tolDCs were administered every 4 days.70 Although more studies are needed, these results suggest that tolDC therapy should be used in the first stages of the disease.

Route of administration

The safety of i.v., intraperitoneal (i.p.), intradermal (i.d.), intranodal (i.n.) and even intra-articular routes of human tolDC administration has been demonstrated in different phase I clinical trials.74,76,77,78,79 The route of administration is crucial due to two important issues. On the one hand, the treatment must promote tolerogenic in vivo effects. In this context, i.v. administration is considered the most tolerogenic route of administration, showing tolerogenic effects superior to those of i.d. administration in nonhuman primates.80 On the other hand, the selected route of administration must allow tolDCs to reach the draining lymph nodes or inflamed tissues. In this regard, either i.d. administration near the draining lymph nodes or direct i.n. tolDC injection could be an alternative to i.v. administration. Interestingly, two coordinated phase I clinical trials using VitD3-tolDCs will compare these two routes of administration81 (Table 2).

Mechanism of action

The mechanism of action of several types of tolDCs has been analyzed.54 Most tolDCs impair T cell alloproliferation in in vitro cultures. Moreover, they also act through different pathways. Dexa-tolDCs produce IL-10, tolDCs generated in the presence of exogenous IL-10 (DC-10) induce IL-10-producing Tregs (Tr1 cells), and VitD3-tolDCs induce T cell hyporesponsiveness in an antigen-specific manner without affecting the ability of other T cells to respond to unrelated antigens.75,82 In addition, a transcriptomic analysis of autologous CD4+ T-cells primed with antigen-specific VitD3-tolDCs revealed profound genetic downregulation, mainly affecting factors related to the cell cycle and proinflammatory immune response processes.83

Dexa+VitD3-tolDCs regulate CD4+ T cell cytokine production in RA patients in a TGF‐β1‐dependent manner.84 Additionally, TGF-β secreted by tolDCs is an important immunoregulatory mediator involved in the induction of Tregs. Interestingly, tolDCs differentiated in the presence of low doses of GM-CSF and in the absence of IL-4, referred to as autologous tolerogenic dendritic cells (ATDCs), have the capacity to reduce T cell proliferation via a novel mechanism involving lactate secretion.85 Lactate secreted by ATDCs exerts its immunosuppressive effect by downregulating T cell glycolysis. Currently, the specific mechanisms triggered by different tolDCs in vivo remain elusive. Inhibition of antigen-specific T cell proliferation, increases in FoxP3+ Treg numbers, decreases in proinflammatory Th17 and Th1 cell numbers in both the periphery (spleen and lymph nodes) and CNS, and increased levels of IL-10 have been described60,61,62,63,64,65,67,68,69,70,75,82,84,86,87 (Table 1). Interestingly, an increased frequency of Bregs was found after MOG-VitD3-tolDC therapy in EAE,64,66 similar to the results of the first phase I clinical trial with genetically modified tolDCs conducted by Giannoukakis et al. in patients with type I diabetes.77 In MS, an increase in IL-10 production by PBMCs isolated from treated patients was described.74 In addition, tolDCs can induce the secretion of indoleamine 2,3-dioxygenase (IDO), a potent regulatory enzyme that catalyzes the degradation of tryptophan required for T cell proliferation.88 Altogether, these data indicate that tolDC therapy can trigger a complex tolerogenic immune cascade, with anergy or elimination of pathogenic Th1/Th17 cells and induction of regulatory cells (FoxP3+ Tregs, Tr1 cells, and Bregs), that can reduce EAE severity, even in mice with established clinical signs of paralysis.

Phenotype, function, and stability

Currently, no common biomarkers of tolerogenic function in different types of monocyte-derived tolDCs have been identified.89 Thus, their phenotypic, as well as functional, characterization requires comparison with mDCs generated in parallel to certify their correct production before administration to patients. In addition, the stability of tolDCs is crucial, and stability can be analyzed in vitro in tolDCs exposed to a proinflammatory milieu to ensure that there is no conversion of tolerogenicity to immunogenicity.90 No data regarding the stability of these cells in vivo after their administration to patients have been obtained so far. In this context, it is expected that the use of cell trackers will provide relevant information to better understand the in vivo mechanism of action of tolDCs.91

Clinical trials in MS

To the best of our knowledge, a total of three phase I clinical trials using tolDCs for the treatment of MS patients are ongoing or have recently finished (Table 2). In one dose-escalation phase Ib clinical trial, patients with MS (n = 8) and neuromyelitis optica (n = 4) received 3 i.v. injections (50, 100, 150, or 300 × 106 tolDCs in total) as three independent doses administered every 2 weeks. However, the last group did not receive the planned doses due to a technical limitation in obtaining the required number of cells. Clinically, the treatment was safe and well tolerated.74

Two coordinated phase I clinical trials in MS patients treated with autologous VitD3-tolDCs loaded with myelin peptides are currently ongoing simultaneously in Belgium and Spain in the context of the European H2020 framework.81 Both studies tested the safety and tolerability of autologous peptide-loaded VitD3-tolDCs in a dose-escalation study using 5, 10, and 15 × 106 VitD3-tolDCs/administration, with the first four of six independent doses administered every 2 weeks and the last two administered every 4 weeks. In addition, exhaustive immunomonitoring is being performed (Table 2).

Regulatory T cells

Since Treg induction is one of the most relevant and consistent mechanisms to achieve immunoregulation, cell therapy administering Treg cells is one of the most promising strategies that has been extensively investigated worldwide.92

Mechanism of action

Tregs in the body comprise the naturally occurring/thymic (tTreg) and induced/peripheral (pTreg) compartments. The latter compartment is further divided into several subsets, with Tr1 cells and Th3 cells being the main representatives. While tTregs are specifically designed to regulate the immune response from the progenitor stage in the thymus, pTregs are generated via the conversion of conventional CD4+ T cells in the periphery during the immune response.93 Both subsets are efficient regulators of the immune response, but their origins suggest somewhat different activities. Thymic Tregs are anergized towards self-antigens in the thymus. These cells migrate to a site of inflammation and the local lymphoid tissue surrounding it and limit the immune response when self-antigens are sensed. In this way, tTregs protect the body from possible autoreactivity. This suppression is very precise, occurring mainly locally via cell-to-cell interactions with other cells that take part in the immune response. The main receptor of tTregs is CTLA-4 (cytotoxic T lymphocyte antigen 4, CD152), which links to receptors from the B7 family on APCs and limits the presentation of autoantigens. Surface TGF-β and LAG-3 are involved in the suppression of NK cells, and PD-1/PD-L coupling is involved in the suppression of T cells and B cells. High expression of CD25 (IL2Rα subunit) allows tTregs to preferentially take up the majority of available IL-2, which triggers apoptosis of overactivated effector T cells (conventional T cells, Tconvs) in the surrounding microenvironment. This suppression is not limited to self-antigens, as the interaction with APCs can result in so-called “linked suppression”. Specifically, when APCs present tolerized autoantigens with some alloantigens, tTregs interacting with APCs can also impose tolerance towards the alloantigen. This is an important advantage of the polyclonal preparation of tTregs used in cell therapy. The fact that pTregs arise in the periphery during immune responses, mainly from naïve CD4+ T cells, implies that they are specific to the antigen that triggers the immune response. The main regulatory mechanism of pTregs involves the secretion of suppressive factors such as IL-10, produced mainly by Tr1 cells, or TGF-β, produced by Th3 cells.92,94 Currently, tTregs are the main subset used in the clinic, but a limited number of trials using Tr1 cells have also been conducted.

Phenotype, function, and stability

Expression of the transcription factor FoxP3 is currently the main marker of Tregs.93 CD127 (the IL7 receptor) is negatively correlated with FoxP3.11,95 Importantly, mutations that render FoxP3 inactive in humans are responsible for immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX). Nevertheless, the phenotype should always be confirmed with the suppressive function. Throughout their development from the progenitor stage to the immune response, tTregs undergo many changes that affect their activity. For example, there is a developmental link between Th17 cells and tTregs, which implies the plasticity of cells of these phenotypes.96 This is important for the stability of Tregs not only during in vitro manufacturing but also when tTregs are expected to function in the body. The stability of Tregs is also substantially affected by epigenetic changes in mature cells. For example, methylation of the Treg-specific demethylated region within the foxp3 gene significantly impairs suppressive function.97 Additionally, the inflammatory cytokine milieu can counteract the suppressive effects of tTregs, which should be considered when therapies with tTregs are designed. For example, TNF secreted by inflammatory cells can abrogate the suppressive effects of tTregs.98 This fragile phenotype should be taken into account during the manufacturing of Tregs for clinical applications, since trivial factors such as the time or temperature of expansion might affect the final activity of the cellular product.99,100

Therapy with Tregs in EAE

While animal studies in many cases assume very early intervention, when there is little or no damage to the CNS, therapy in humans usually starts when the pathology is very advanced, as the first symptoms often occur only then. Likewise, the burden of inflammation and the location of the inflammatory process may be different between an animal model and humans in a trial, and this needs to be considered when translating the results into the clinic. One variable to consider is the time when the treatment is administered. In this context, analysis of different studies on Tregs shows that their administration in EAE was performed at different times: prophylactically (before or with immunization), late prophylactically (before the first clinical signs appeared), therapeutically (with the first clinical signs), and late therapeutically (5–8 days after the initial clinical signs). Details of the studies are given in Table 3. Here, we will focus on studies in which Tregs are administered therapeutically, as they more closely resemble the potential application of Tregs in MS.

Table 3 CD4+CD25+FoxP3+ Tregs in EAE treatment

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Stephens et al. administered Tg4 CD25+CD62Lhi MBP(Ac1-9)-reactive Tregs to Ac1-9 peptide-immunized B10.PL or B10.PL x SJL mice at 18 days post immunization (d.p.i.).101 This was the time of remission from the first EAE relapse. While mice that were not treated with Tregs developed more relapses and chronicity of EAE in the subsequent days, the severity of EAE relapse was markedly reduced in Treg-treated mice. The effect was more pronounced in B10.PL x SJL mice, as Treg-treated mice were nearly disease-free at the end of the observation period (100 d.p.i.). These results imply that Treg application could be efficient in MS, even at the later stages of the disease when the vicious cycle of autoimmune reactivity is well established. In another study, Fransson et al. used Tregs derived from CD4+ T cells that had been modified with a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting MOG in trans with the FoxP3 gene. They were able to produce Tregs with a strong affinity for MOG and persistent FoxP3 expression.102 Tregs were applied to C57BL/6 mice with MOG35–55-induced EAE on day 15, i.e., at the time of the EAE peak. The treatment reduced the severity of EAE in the chronic phase, and the treated mice were symptom-free at the end of the observation period (30 d.p.i.). Importantly, Treg-treated mice were reimmunized with MOG35–55 at 30 d.p.i., and only one mouse developed any clinical manifestations of EAE. This effect on the clinical score occurred in parallel with inhibition of IL-12 and IFN-γ expression in the CNS. Interestingly, Tregs were applied to mice intranasally (i.n.), and the authors were able to demonstrate their migratory capacity (presumably via olfactory pathways), as they detected the Tregs within the CNS.

Niedbala et al. used nitric oxide (NO)-induced Tregs in EAE.103 They applied Tregs in C57BL/6 mice with MOG35–55-induced EAE at day 10, i.e., when the initial clinical signs appeared. The treatment led to a reduction in the severity of chronic EAE throughout the observation period, which ended at 30 d.p.i. This effect occurred in parallel with the limitation of immune cell infiltration into the CNS and a specific reduction in Th17 cell numbers within the CNS.

Malviya et al. generated transgenic T cells expressing a TCR specific for MOG and neurofilament medium (NF-MT) and used these T cells to treat C57BL/6 mice with EAE induced by MOG35–55 or PLP178–191.104 These engineered Tregs reduced the severity of EAE when applied at 9 d.p.i. (when clinical symptoms were evident), and their effect increased towards the peak of EAE. Importantly, these Tregs containing TCRs specific for MOG were equally efficient in EAE induced by MOG35–55 and EAE induced by an unrelated CNS antigen, PLP178–191. Such efficacy in restricting autoimmune reactivity against unrelated CNS antigens, if extrapolated to humans, would be a beneficial therapeutic property in MS. The engineered Tregs were detected in the CNS of the treated mice, and the authors suggested that the therapeutic effect of the Tregs was achieved within the CNS.

To summarize, at least in the EAE model of MS, a single application of Tregs to mice when the disease is well established is successful and has persistent effects. The cells can be applied systemically (i.v. or i.p.)101,103,104,105,106,107,108,109,110,111 or locally (i.n.).102 Nevertheless, the number of cells used for i.n. application was 10 times lower than that used for systemic application, which could be of interest when thinking about translation to humans. However, this difference in the number of Tregs required for efficient application could also be a consequence of different backgrounds and preparations. In conclusion, the results obtained in EAE clearly imply that the application of Tregs is a promising approach for the treatment of ongoing CNS autoimmunity, as observed in MS; therefore, clinical trials with Tregs in MS are expected to yield promising results.

Clinical trials in MS patients

Tregs, mainly tTregs, have been extensively tested in clinical trials for the treatment of not only AD and graft-versus-host disease after bone marrow transplantation as a prophylaxis for solid organ rejection but also for unexpected indications such as thalassemia, muscle dystrophies or amyotrophic lateral sclerosis.92 We recently accomplished a phase I/IIa trial of autologous CD4+CD25hiCD127-FoxP3+ Tregs administered to RRMS patients, either i.v. or intrathecally (i.t.) (trial registration: EudraCT: 2014-004320-22.94 The therapy proved to be safe. Although very preliminary, the results also suggested that intrathecal administration was more effective than intravenous administration. Experiments with adoptive transfer of Tregs suggested the good safety profile of Tregs administered via both tested routes.94

Dose, timing, and route of administration

To translate a treatment from early animal use into late human pathology, the dose and route of administration of the cells should be adjusted to address the level of inflammation and follow the progression of the disease. We tried to address these issues with the use of two routes of Treg administration. Patients treated i.v. received 40 × 106 Tregs/kg b.w., which in our experience is a relatively high dose. Within this arm of the trial, we tried to address the hypothesis that systemic dysregulation between Tconvs and Tregs triggers the disease and relapse.112 The results suggest that we were, at least partially, ‘too late’, as half of the treated patients experienced relapse and progression of disease, as confirmed with MRI. This somewhat confirms that the clinical onset of disease may occur very late in the pathogenesis of MS, when the core of the process has already moved from the periphery to the CNS. The initiation of RRMS occurs somewhere in the peripheral lymphoid system with the presentation of myelin peptides and the generation of autoaggressive Tconvs, as in EAE, which is not adequately controlled by Tregs.113 However, Tconvs very quickly traffic to the CNS, destroy the BBB, attack myelin sheaths, and cause the development of lesions. Hence, the systemic administration of drugs is of limited value when symptoms have already occurred. The results of immunophenotyping in our trial seem to confirm such overactivity of Tconvs, which were mainly of an ‘experienced’ memory phenotype in all patients. At the same time, the majority of Tregs were naïve, confirming their relative inactivity. Surprisingly, MS patients exhibited an extraordinarily high percentage of peripheral Helios−FoxP3+ Tregs (20–30% of all FoxP3+ Tregs). These cells arise during the immune response, which suggests a history of long/massive immune activation in the periphery, with ineffective regulation of this process in MS. There have been reports that Tregs in RRMS patients follow autoreactive Tconvs and, attracted by inflammation, move quickly to the CNS, accumulating in the CSF. Moreover, remission occurs only when Tregs have accumulated in the CSF.114

This finding justifies the second arm of our trial, in which patients received Tregs i.t. The dose could be lower (1.0 × 106 Tregs) in these patients, as 100% of the cells were delivered behind the BBB. The patients did not experience relapse, and MRI confirmed stable nonprogressing lesions in the CNS of these patients, which proved that this approach should be further tested in future trials.94

Novel Treg therapies

The approach based on engineered Tregs came from cancer studies in which the receptor specific for a particular molecule expressed on cancer cells was inserted into Tconvs using a vector (chimeric antigen receptor T (CAR-T) cells). This approach allows CAR-T cells to identify and kill cancer cells in a very specific and efficient way. Several drugs based on this therapy, i.e., axicabtagene ciloleucel (Yescarta, Gilead) and tisagenlecleucel (Kymriah, Novartis), are already routinely used. The first CAR-Tregs were constructed with specificity towards allo-HLA to quench the possible rejection of an allotransplant.112,115,116 The challenge is much higher in ADs such as MS, in which the target antigens are not as obvious. In the majority of such diseases, the complete list of autoantigens is not known.117 Moreover, it is possible that the target antigens evolve with the progression of the disease due to epitope spread.114 The complexity of the response is also caused by the fact that the same epitopes can trigger responses in both Tregs and Tconvs, and the final outcome depends on which subset prevails.118 Nevertheless, there have been attempts to create Tregs with engineered TCRs to direct them to particular sites and protect particular organs from autoimmune attack.119 These attempts for MS are at the EAE stage. There have been reports on CAR-T cells with specificity towards MOG manufactured from Tconvs and directed toward a regulatory function through foxP3 gene delivery. This cellular product trafficked to the brain and exerted suppressive activity.102 More recently, human Tregs that had a transgenic TCR specific for MBP and had proven immunosuppressive activity were described.111

Mesenchymal stem cells

MSCs are nonhematopoietic multipotent and self-renewing progenitor cells with the potential to differentiate into different lineages under specific conditions. They were described for the first time in 1968 by Friedenstein et al. as an adherent fibroblast-like population in the bone marrow that was able to differentiate into adipocytes, chondrocytes and osteocytes.120 In 1991, Caplan et al. named these cells “mesenchymal stem cells”.121 The authors demonstrated that MSCs are involved in bone and cartilage turnover and examined how surrounding conditions play a crucial role in their differentiation. Therefore, MSCs were postulated to be a novel therapeutic strategy for self-cell repair.121

MSCs can be isolated from various tissues, including the bone marrow, adipose tissue, placenta, umbilical cord, fetal liver, muscle, and lung. Among these tissues, bone marrow and adipose tissue are the most widely used sources of MSCs for therapeutic purposes. According to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapies,122 the cells must be plastic adherent and able to differentiate into osteoblasts, adipocytes and chondroblasts, and their phenotype must be characterized by flow cytometry as CD105+ CD73+ CD90+ CD45− CD34− CD14−/CD11b− CD79a−/CD19− HLA-class II−. MSCs can be easily cultured and expanded ex vivo and have several properties, such as functions in tissue repair and homeostasis maintenance, immunomodulatory properties and low immunogenicity.

Due to their potential as an immunomodulatory and regenerative therapy, MSCs have been considered an optimal candidate cellular therapy for inflammatory and neurodegenerative diseases of the CNS, such as MS.

Preclinical studies

MSC-based cell therapy is the most investigated and has been examined in numerous in vitro and in vivo studies (EAE). In vivo treatment of EAE mice with bone marrow-derived MSCs (BM-MSCs) using several routes of administration (i.v., intraventricular (i.v.t.), and i.p.) has shown clinical amelioration of EAE severity, with reduced inflammatory infiltration, demyelination, and axonal damage. Of note, this beneficial effect was not found when BM-MSCs were infused during the chronic phase of the disease.123

It has been widely reported that MSC therapy in EAE exerts an important immunomodulatory effect and, to a lesser extent, a neuroprotective effect that results in axonal and neuronal protection through the release of antiapoptotic, antioxidant, and neurotrophic factors (systematically reviewed in124). Induction of Tregs, TGF-β1, and IL-10 mRNA in the spleen and lymph nodes of treated mice was the main immunological mechanism involved in the induction of peripheral tolerance following MSC transplantation in EAE mice.

Mechanisms of action of MSCs

The specific mechanisms that mediate the clinical benefits of BM-MSCs likely involve a combination of peripheral autoimmune modulation and the induction of CNS tissue protection. MSCs have four main properties: (1) migration capacity, (2) immunomodulation, (3) differentiation and neuroregeneration, and (4) secretion of soluble factors.

After their systemic administration, MSCs can migrate to and engraft in inflamed locations, exerting a local effect. Injured cells and immune cells involved in the immune response regulate MSC migration through the secretion of a broad range of signals, such as growth factors and chemokines. In vitro studies proved that MSC migration is regulated by receptors such as platelet-derived growth factor and insulin-like growth factor 1 and chemokine receptors such as CCR2, CCR3, CCR4, and CCL5.125 Studies in animal models have shown that MSCs can roll and tether to the endothelium, crossing the BBB through the VLA-4/VCAM-1 interaction. Moreover, MMPs play an important role in the transit of MSCs through the endothelial membrane. Interestingly, it is important to keep in mind that culture conditions during MSC ex vivo expansion can affect the expression of some receptors, such as VLA-4 and MMP, thus altering the migration capacity of MSCs.125,126,127

The immunomodulatory properties of MSCs can be exerted via cell-to-cell interactions or paracrine effects. On T cells, MSCs inhibit T cell proliferation via a mechanism independent of apoptosis induction.128 After coculture with T cells, MSCs decreased the Th1 response and induced a switch towards the Th2 response (a decrease in IFN-γ secretion and an increase in IL-4 secretion).129 In addition, MSCs induce the expansion of Treg subsets, increasing Foxp3 and CD25 expression. These Treg cells express TGF-β1 and PGE2.129,130

Regarding B cells, it has been reported that PDL1 inhibits their proliferation in murine cells and arrests the cell cycle in human cells.131,132 Chemotactic properties are also affected by soluble factors secreted by MSCs. A decrease in the expression of some chemokine receptors, such as CXCR4, CXCR5, and CCR7, was observed in B cells, together with a decrease in CXCL12 (a CXCR4 ligand) and CXCL13 (a CXCR5 ligand) expression in MSCs. In contrast, B cell costimulatory molecule expression and cytokine production were unaltered.132 Furthermore, suppression of B cell terminal differentiation by soluble factors secreted by MSCs, such as MCP-1 or IL-6, was also reported in C57BL/6 mice.133

MSCs induced the inhibition of NK cell proliferation when cocultured in IL-15-supplemented medium via both cell-to-cell contact and soluble factors, such as TGF-β1 and PGE2. Moreover, under these conditions, a decrease in the production of IFN-γ and IL-10, a decrease in the surface expression of CD56 in NK cells (although no changes were observed in the ratio of the CD56dim and CD56bright subsets), and lower cytotoxicity against HLA class I targets were found.129,134,135

MSCs can inhibit the maturation of DCs, resulting in a decrease in their capacity to activate alloreactive T cells. Furthermore, MSCs can inhibit TFN-α release by DCs, resulting in a tolerogenic state. Moreover, it has been postulated that PGE2 secreted by MSCs plays an important role in promoting the Th2 cell response, acting against Th1 cells, during the DC-induced Th cell response.129,136

As previously mentioned, MSCs release multiple soluble and encapsulated molecules, such as growth factors, cytokines, and chemokines (secretome), which can exert a biological effect in tissues. The conditioned medium of MSCs was shown to have a beneficial effect in damaged tissue in the liver and myocardium. Among these molecules are PGE2, which has anti-inflammatory and antiproliferative effects; IL-10 and IL-1 receptor antagonist (which have anti-inflammatory effects); and TGF-β1 and hepatocyte growth factor (which suppress T cell proliferation).126,127 Moreover, it was recently reported that exosomes released by MSCs can cross the BBB due to their small size and transfer bioactive molecules. In this regard, MSC-derived exosomes polarized microglial cells mainly into the M2 anti-inflammatory phenotype, promoting a shift to an anti-inflammatory profile and, consequently, reducing the clinical symptoms of EAE in rats.137

One important characteristic of MSCs is their ability to differentiate towards both mesenchymal and ectodermal cells, such as neurons, astrocytes, and oligodendrocytes. When analyzing the neuroprotective and regenerative functions of MSCs in MS, different experiments demonstrated that after i.v. administration of MSCs, they spread and homed preferentially to inflamed tissues, to the CNS in the case of EAE, to induce neuronal axon protection and the regeneration of damaged areas.138,139 Unfortunately, MSCs have not been demonstrated to transdifferentiate into neuronal cells. Their protective function is likely related to the secretion of antiapoptotic, anti-inflammatory, and neurotropic factors (through the activation of astroglial cells to secrete neurotrophins such as BDNF, glial cell-derived neurotrophic factor, and nerve growth factor),139 as well as the probable recruitment of local progenitor cells for subsequent differentiation into neurons and oligodendrocytes.139,140,141 These promising results encourage the use of MSCs in MS.

Clinical trials in MS

The number of clinical trials investigating MSC treatment for RRMS and progressive MS patients has increased quickly over the past two decades. Currently, more than twenty clinical trials are registered at ClinicalTrials.gov (see Table 4). Thus far, the results have shown that MSC therapy in MS patients is safe, with no relevant side effects.142,143,144,145,146,147,148,149,150,151 Due to variability in the protocols used (different types of MSCs, routes of administration, and doses) and the limited number of patients enrolled in each trial, most studies have been unable to draw conclusions about the efficacy of these treatments. Nevertheless, some trials have reported beneficial effects of MSCs through a decrease in relapse rate or disability.143,146,147

Table 4 Clinical trials using mesenchymal stem cells in MS patients

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To establish a consensus protocol for the use of MSCs for treating MS patients, in 2010, a group of experts created the International Mesenchymal Stem Cell Transplantation Study Group (IMSCTSG). As a result, a large multicenter randomized, double-blind, crossover phase I/II clinical trial was initiated to analyze the safety and efficacy of a single i.v. dose of autologous BM-derived MSCs (Mesenchymal StEm cells for Multiple Sclerosis: MESEMS trial; NCT02403947).142 The results of this trial have not yet been published.

Recently, Sarkar et al. reported relevant data about the MSC secretome that must be considered in the context of treatment with MSCs. These authors found that chronic inflammatory stress in MS patients limits MSC functionality by altering the MSC secretome.152 Moreover, other factors, such as aging and the in vitro expansion of MSCs, also exert detrimental effects on their functionality.152 Therefore, an in-depth analysis of the autologous MSC secretome should be considered as a quality control measure before administrating MSC therapy.

Regarding the effect of MSC treatment on the immune response, only a few clinical trials have performed immune monitoring to elucidate the mechanism of action of this therapy. Llufriu et al. reported a decrease (although it was not statistically significant) in Th1 and Th17 cells and an increase in Breg cells, as analyzed by the expression of IFN-γ, IL-17, and IL-10, respectively, using intracellular cytokine staining.143 Karussis et al. described an increase in Treg cells (identified as CD4+CD25+), together with a decrease in the expression of CD83 and CD86 in DCs and CD40 in activated cells. Moreover, they performed functional analysis of the T cell response, which showed a decrease in proliferation in response to phytohemagglutinin (PHA).147 Finally, a clinical trial of the administration of autologous BM-MSCs is currently ongoing in Jordan and will analyze the levels of IgG, IgA, and IgM and complement factors C3 and C4 in treated patients (NCT03069170).

Hematopoietic stem-cell transplantation

Hematopoietic stem-cell transplantation (HSCT) was established for the treatment of hematological malignancies such as multiple myeloma and leukemias due to the capacity of hematopoietic stem cells to differentiate into all hematopoietic cell types.153 Surprisingly, treated patients who had concomitant ADs experienced amelioration of their clinical symptoms following HSCT.154 As a result, high-dose immunosuppression followed by autologous HSCT (aHSCT) has been investigated for patients with severe MS, with the rationale of this therapy based on a “reset” of the immune repertoire to eliminate autoreactive T and B cells; thus, subsequent aHSCT would allow reconstitution with the hope that a new and more self-tolerant immune system is developed.155

HSCT is carried out through different steps: mobilization, harvesting, ablative conditioning, and transplantation of aHSCs.156 First, HSCs are collected from the peripheral blood of patients after the mobilization of HSCs from bone marrow using treatment with granulocyte colony-stimulating factor (G-CSF) or GM-CSF with cyclophosphamide to prevent possible MS relapse or worsening of clinical symptoms as a result of G-CSF or GM-CSF administration.156 After 4 or 5 days, cells are harvested by leukapheresis and cryopreserved. Additionally, an HSC purification step is performed by CD34 positive selection to eliminate possible autoreactive lymphocytes. Then, the patient receives ablative conditioning to eradicate autoreactive cells. Different regimens of immune ablative conditioning are used based on the intensity of ablation using different chemotherapeutics and immunosuppressive drugs. High-intensity ablative regimens involve a high i.v. dose of immunosuppressive therapy and are associated with high toxicity and, in a small percentage of patients, mortality.156,157 In contrast, low-intensity regimens are nonmyeloablative and produce fewer adverse effects but may be associated with the early reappearance of MS disease activity post infusion. Hence, intermediate-intensity ablative regimens, referred to as BEAM (BCNU (or carmustine), etoposide, cytosine arabinoside, melphalan) or modified BEAM, are becoming more accepted.157 Following ablative conditioning, cryopreserved HSCs are thawed and reinfused into the patient.

Despite the promising positive results obtained using aHSCT (see below), some aHSCT-related risks must be considered: as stated above, the risk of transplant-related mortality, which is most severe in the first 100 days after transplantation, and the increased susceptibility to infection as a result of the accompanying chemotherapeutic immunosuppressive regimen. In addition, long-term side effects include the development of secondary autoimmune problems and/or fertility issues.158 Interestingly, aHSCT has dramatically improved over the years, showing a 0.3% treatment-related mortality rate since 2005.157

Clinical trials in MS

Since the end of the 1990s, several clinical trials evaluating the safety and efficacy of aHSCT using different conditioning regimens have been performed. Published results have shown that aHSCT can inhibit MS disease activity for 4–5 years in 70–80% of patients. Interestingly, this rate is higher than that achieved with any other therapy for MS. The results were better in young patients with inflammatory-active RRMS (reviewed in157).

To go one step further in examining aHSCT, currently, a phase III randomized clinical trial in RRMS patients with significant inflammatory disease activity is being conducted to compare the efficacy of aHSCT using a nonablative conditioning regimen with that of alemtuzumab (anti-CD52), which is considered the most effective available drug for RRMS (NCT03477500). Importantly, if the results indicate improved efficacy of aHSCT over alemtuzumab, aHSCT will likely be approved as a part of the current standard treatment recommendations for a significant proportion of RRMS patients (except in the case of Sweden, where aHSCT has been already approved). In the same way, another phase III clinical trial to analyze the efficacy of aHSCT (using high-dose myeloablative conditioning) in comparison with the best available therapy (BAT) in treatment-resistant RRMS is currently ongoing (BEAT-MS trial, NCT04047628). This multicenter, randomized, blinded study was conducted in a total of 156 RRMS patients distributed to each treatment arm at a ratio of 1:1. Treatments included in the BAT arm are natalizumab (anti-CD49d), alemtuzumab (anti-CD52), ocrelizumab (anti-CD20) and rituximab (anti-CD20). Relapse-free survival up to 3 years will be determined and used to compare the efficacy of aHSCT with that of the other treatments.

T cell vaccination

Autologous T cell vaccination (TCV) involves collecting and expanding myelin-reactive T cells from MS patients and reinfusing them after their attenuation by irradiation. The rationale is that, as a result of this process, the immune system will attack pathogenic myelin-specific T cells, causing their deletion or inactivation while maintaining protective immunity.

The first study using attenuated MBP-reactive T lymphocytes was conducted in Lewis rats in 1981.159 The adoptive transfer of MBP-reactive T cells in rats induced EAE disease onset. Interestingly, administration of the same MBP-T cells attenuated by irradiation before adoptive induction resulted in disease prevention. Increased interest in TCV led to the initiation of multiple trials in MS patients in the late 1990s. The results from phase I pilot studies demonstrated that TCV treatment was safe and well tolerated160,161,162,163,164,165 (Table 5) and depleted MBP-reactive T cells after only 2 administrations.160 Interestingly, a correlation between MBP-reactive T cell depletion and a 40% reduction in the relapse rate was found in RRMS patients. Nevertheless, no relevant reduction in EDSS score was observed in the RRMS patients. In contrast, a slight increase in EDSS score after 2 years was reported in SPMS patients166 (Table 5).

Table 5 Clinical trials using T cell vaccination in MS patients

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To improve TCV outcomes in MS patients, T lymphocytes isolated from CSF were used to develop CSF-derived T cell lines against myelin peptides, since CSF was thought to contain infiltrating pathogenic lymphocytes relevant to the disease process due to the proximity of these lymphocytes to the CNS. Data from two pilot studies indicated that the administration of attenuated autoreactive CSF-derived T cell clones was feasible and safe, and no adverse effects were observed. Phase II studies with a large number of MS patients are required to evaluate the clinical and radiological efficacy of CSF-derived TCV.167,168

To improve clinical remission, a new strategy using multivalent TCV was developed using immune-dominant epitope sequences of MBP, MOG and PLP. A phase I clinical trial was conducted by Achiron et al. using attenuated T cell lines specific for different MBP and/or MOG peptides.169 The results of the trial showed clinical as well as radiological benefits with no adverse effects in RRMS patients who did not respond to disease-modifying treatments.169 In addition, multivalent TCV allows personalized therapy following prescreening for myelin reactivity. In this regard, Tcelna (Imilecleucel-T, previously known as Tovaxin), a TCV composed of autologous preselected T cells reactive against up to six immunodominant peptides derived from MBP, MOG and PLP, was shown to be safe and well tolerated in a phase II clinical trial; however, its clinical and radiological efficacy was not demonstrated (TERMS trial: Tovaxin for Early Relapsing Multiple Sclerosis, NCT00245622,170). An extension study (OLTERMS trial: Open label extension of TERMS study, NCT00595920) was initiated to further evaluate the clinical efficacy of Tcelna/Tovaxin. However, the study was terminated because it did not accomplish the predefined primary endpoint of reducing brain atrophy or the secondary endpoint of decreasing disability progression (NCT00595920).

Combined therapies

Evidence from phase I clinical trials conducted in type 1 diabetes, rheumatoid arthritis, Crohn’s disease and MS patients has demonstrated that tolDCs, Tregs, TCV, MSCs, and HSCT are safe and well tolerated. However, due to the different cell types and mechanisms involved in the maintenance of immune tolerance and the difficulty of establishing an optimal dose, route, and frequency of administration, it has been postulated that a combined therapy of antigen-specific cells with conventional immunomodulatory drugs is most likely necessary to potentiate their beneficial effects and restore immune homeostasis.54

TolDCs with immunomodulatory/immunosuppressive drugs

A single infusion of donor-derived VitD3+IL-10-tolDCs before transplantation in combination with CD28 costimulatory signal blockade using the fusion protein CTLA-4 Ig (abatacept) and rapamycin significantly prolonged allograft survival in nonhuman primates by attenuating donor‐reactive memory T cells.171

More recently, the results of the ONE Study, an analysis of seven non-randomized, single-arm, phase I/IIa trials in living donor kidney transplantation in which different types of regulatory cells, including tolDCs, were combined with conventional immunosuppressive drugs reducing the proliferative response of lymphocytes by inhibiting IL-2 signaling (basiliximab and tacrolimus), blocking de novo synthesis of guanosine nucleotides (mycophenolate mofetil) or using a feedback mechanism to control inflammation and the immune response to steroids. Data from the trial showed the safety and feasibility of combined therapy. Interestingly, a decrease in infectious complications was reported in the group administered combined therapy.100 In this context and to move one step forward in the clinical application of tolDCs in MS, our group investigated the effect of tolDCs combined with IFN-β. We found that the anti-inflammatory and immunomodulatory properties of IFN-β combined with VitD3-tolDCs induced a reduction in Th1 and Th17 cells, favoring a more potent antigen-specific regulatory effect of VitD3-tolDCs both in vivo (EAE model) and in vitro in cultures of peripheral blood cells from MS patients (Quirant-Sánchez et al. unpublished data).

Tregs with immunomodulatory/immunosuppressive drugs

The relationship between Tregs and IL-2 is probably the most explored area in MS research, with the longest history of clinical trials.172 Tregs have high expression of IL-2R and are highly dependent on IL-2; therefore, this cytokine or its muteins have been used alone or in combination with Tregs in many conditions, such as GvHD, transplantation or T1D.96,97 Other approaches include the administration of rapamycin as an immunosuppressive agent able to induce Tregs and tolDCs98,99 or adjuvant therapy with Tregs added to standard immunosuppression in organ transplantation.100 Our team has been testing Tregs combined with anti-CD20 antibody in T1D.173

TolDCs in combination with Tregs

The combination of tolDC and Treg cell therapy has been proposed for the treatment of ADs since these cells can interact to stabilize, maintain and potentiate their tolerogenic effects.174 Although coadministration or serial administration of antigen-specific tolDCs and Tregs a priori does not seem to be feasible, a single leukapheresis could provide enough monocytes to generate tolDCs and lymphocytes for autologous Treg expansion.174 However, to date, no studies investigating the efficacy of this combined therapy have been reported.

TolDCs/Tregs in combination with MSCs

A different combination therapy approach for ADs is the administration of antigen-specific cells, such as tolDCs or Tregs, with MSCs. In this context, synergistic suppression of autoimmune arthritis was reported when collagen-induced arthritis (CIA) mice were treated with RelB-silenced tolDCs and MSCs. Combined therapy was able to inhibit disease progression, decrease the clinical symptoms of CIA and reduce joint damage. Immunological studies revealed inhibition of the collagen T cell response and a shift towards an anti-inflammatory profile, although the most potent synergistic effect elicited by RelB-silenced tolDC and MSC therapy was a strong reduction in Th17 cells.175

Another example was published by Lee et al., who used a murine model of acute GVD (aGVHD) to determine the efficacy of combined Treg and MSC therapy.176 Researchers have shown that the adoptive transfer of donor-derived MSCs and Tregs reduces the severity of aGVHD by controlling Th1 and Th17 responses (related to the function of Tregs), accompanied by increased long-term survival of transferred Tregs and induction of endogenous Treg repopulation in target organs (related to MSC function).176

In conclusion, all these data suggest that combination therapies have the advantage of increasing the possible clinical effectiveness of antigen-specific cell-based tolerogenic therapies and will contribute to their optimal application in the future.

Legal rules for cellular products

Cellular therapies are classified as either transplants, in which unmodified cells are immediately administered, or drugs, in which substantial laboratory modification of the cells and/or nonhomologous use of the cells occurs. In Europe, the use of these two kinds of products is regulated by directive 2004/23/EC, which sets standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells, and directive 1394/2007, which is focused on advanced therapy medicinal products (ATMPs). Clinical trials to test new investigational medicines are also centrally regulated by regulation 536/2014. In general, these directives implement the guidelines established by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), a common worldwide effort of national regulatory authorities and the pharmaceutical industry to discuss scientific and technical aspects of pharmaceuticals. Similar acts based on ICH rules are also in place in other parts of the world and describe the path of new medicines from discovery to routine use. This path for cellular drugs is similar to that for other drugs. It starts from preclinical assessment, which establishes the most important toxic, pharmacokinetic and pharmacokinetic features of the investigational drug. This step in humans consists of three trials: a first-in-man phase I trial to establish safety and dose, a phase II trial to test efficacy and a phase III trial to confirm safety and efficacy in a larger population. The last study is usually pivotal and the basis for marketing authorization. Drugs are also continuously surveyed post authorization, which is phase IV. In Europe, the authorization of cellular drugs is centralized, as it occurs in one step for all EU countries and is granted by the EMA. Some possibilities for early access to cellular products, such as hospital exemption rules, exist, but these are very limited, and there is pressure to provide standardized, equal treatment to all citizens of the EU. The importance of cellular drugs and high expectations that these cellular drugs will be a ‘game changer’ for many unmet medical needs are highlighted by the fact that there is a dedicated legal board within the EMA, the Committee for Advanced Therapies (CAT), which addresses only regulations on cellular therapy. There are also academic initiatives to regulate the way cellular drugs are manufactured. For example, academic guidelines for tolerance-inducing cellular products based on the minimal information model exist.177,178

Concluding remarks

Over the past 20 years, an extraordinary effort has been made to develop treatments that can halt the natural evolution of MS. Thanks to this effort, we have a variety of drugs that, due to their powerful anti-inflammatory/immunosuppressive effects, decrease the rate of relapse and radiological activity, thus slowing the onset of disability. However, by acting in an immunologically nonspecific manner and suppressing one or more branches of the immune response, these treatments have the potential to cause serious adverse effects. In this context, cell therapy appears to be a promising strategy.

Phase I clinical trials with tolDCs, MSCs and Tregs in MS patients have shown these therapies to be safe and well tolerated, with no relevant adverse effects. Among cell-based strategies, MSCs have a potent immunomodulatory effect. TolDCs loaded with self-antigens against which tolerance is induced, Tregs, and more recently CAR-Tregs have the potential to specifically act against the cause of the disease, i.e., the autoimmune response to CNS myelin, while maintaining protective immunity. In addition, although less explored, some cell therapies show neuroprotective and neurorepair potential. These cells have shown promising results in experimental models, and some, such as hfNSCs, MSC-NPs and hESCs, are already being tested in patients.

All these developments paint a very hopeful picture for the next few years. The personalized combination of treatments will allow us to approach this disease from various fronts and, without a doubt, increase the possibility of obtaining a suitable therapy for each patient at the right time to ensure the highest quality of life possible because each patient is unique, as is their disease.

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2. 2.

[Uhohinc]

Interesting, no one under about age 16 ever gets MS

[Uhohinc]

https://www.healthline.com/health/multiple-sclerosis/in-children-and-teens 

very rare.

[Uhohinc]

Neurodegeneration and cholesterol: What is the link?

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MNT’s Sunday Supplement: Neurodegeneration and cholesterol
Is there a link between brain health and how brain cells are able to process cholesterol? We know that cholesterol is an essential molecule for healthy cell membranes and hormone production throughout the body, but 23–25% of a person’s cholesterol is in the brain.

There, it helps build protective myelin sheaths around nerve cells, insulating them and allowing them to transmit nerve signals precisely and efficiently. Damage to these myelin sheaths is linked to neurological conditions, such as Alzheimer’s and Parkinson’s disease, so researchers are very keen to learn more about the mechanism involved.

This week, Medical News Today reported on the discovery that a specific protein, TDP-43, is implicated in the loss of myelin sheathing. Cells that lack this protein are unable to absorb or process cholesterol properly, so targeting TDP-43 offers hope of new treatments for neurodegenerative diseases.

Cholesterol has had a bad press over the years, with high levels often claimed as a risk factor for heart disease and stroke. However, this new research reminds us that a healthy level of cholesterol metabolism is essential for proper brain functioning.

To learn more about this discovery and how it may help scientists develop treatments for neurodegenerative diseases — including Alzheimer’s and Parkinson’s — jump to “Neurodegeneration and cholesterol: What is the link?”

We also have a selection of articles from the past week that you may have missed. We reported on a new rapid saliva test for the Delta variant that costs as little as $2 a time, how insights about how the brain processes silence may lead to new tests for dementia, and the arguments for and against compulsory vaccination for people working in healthcare. You’ll find these and five more new stories below.

We’ll return with our regular daily newsletter tomorrow. Until then, please share your thoughts on this weekend’s stories with us by email.

Robin Hough
Editor-in-Chief, Medical News Today

[Uhohinc]

SCIENCEVOL. 374, NO. 6569MYELIN: A GATEKEEPER OF ACTIVITY-DEPENDENT CIRCUIT PLASTICITY?

REVIEWNEURODEVELOPMENT

Myelin: A gatekeeper of activity-dependent circuit plasticity?

GIULIA BONETTO HTTPS://ORCID.ORG/0000-0003-1469-2004DAVID BELIN HTTPS://ORCID.ORG/0000-0002-7383-372X AND RAGNHILDUR THÓRA KÁRADÓTTIR HTTPS://ORCID.ORG/0000-0001-9675-2722 Authors Info & Affiliations

SCIENCE•7 Oct 2021•Vol 374, Issue 6569•DOI: 10.1126/science.aba6905

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• Insulation for circuit regulation
• Structured Abstract
• Abstract
• References and Notes
• 0eLetters

Insulation for circuit regulation

Lipid-rich myelin, which wraps around and insulates neuronal axons, improves the speed and efficiency of signal propagation. Myelination is not universally applied in the central nervous system, and even axons within the same circuit can vary in the amount of myelination they carry. 

Bonetto et al. reviewed what is known about how such variability can affect computational functions in brain circuits. Myelin plasticity may be one mechanism linking experiential learning to modified brain connections. Whether you are learning to juggle or learning to read, changes in myelination may tune the circuits underlying those skills. —PJH

Structured AbstractBACKGROUND

Myelin, produced by oligodendrocytes, supports the rapid and synchronized transfer of information across the central nervous system (CNS). Earlier views of myelin as inert and immutable have been reformed with the discovery that myelin changes throughout the life span with experience or the acquisition of new skills, adapting the function of neuronal circuits like those underlying learning and memory. We discuss the functional implications of myelin changes, capitalizing on previous reviews of mechanisms of myelin plasticity to assess how such changes might be linked to circuit function underlying learning and memory.

In the CNS, there remains a substantial proportion of unmyelinated or partially myelinated axons, which can potentially become myelinated during the life span. This offers a dynamic range for activity-dependent structural plasticity, whereby newly generated oligodendrocytes myelinate these axons and axonal segments, or existing oligodendrocytes alter myelin internodes in response to neuronal activity. Neuronal activity seems to promote oligodendrocyte precursor cells (OPCs), evenly distributed throughout the adult brain, to differentiate into new myelinating oligodendrocytes and to alter the internodal length and thickness of existing myelin, as well as increasing lactate release from oligodendrocytes via myelin to fuel conduction. Computational models indicate that neuronal activity–dependent myelin alterations can promote neural phase synchronization, which is needed for learning and memory. Myelin plasticity therefore represents a mechanism by which experience and associated learning may modify brain connections, presumably by shaping the computation of neuronal circuits via alterations in the timing of neuronal signal transmission.

ADVANCES

The few studies that have directly investigated the role of myelin plasticity in learning and memory have provided evidence that brain plasticity in the form of de novo myelin formation may play a role in the encoding (learning) and storage (memory) of information. Collectively these studies reveal that OPC proliferation is fast, almost immediate, upon initiation of training in a behavioral task, driving change at a speed similar to that of structural synaptic plasticity. However, new oligodendrocytes and new myelin do not appear until days or weeks later. These dynamic changes do not seem to be universal nor to follow the ongoing constitutive myelination program. Instead, they differ in brain regions and timing depending on the task, the performance of which relies on distinct and/or interacting memory processes, including prefrontal cortex–dependent short-term/working memory, hippocampus-dependent spatial memory, dorsal striatum–dependent procedural memory, or amygdala-dependent Pavlovian and emotional memory. As a result, it appears that although the proliferation of oligodendrocytes seems nonspecific to the function and underlying neural substrate that is being recruited to the task, the pattern of myelination is indeed memory system–dependent.

The functional role of OPC differentiation, as these cells form new oligodendrocytes at different times during learning and memory formation, can be causally interrogated in transgenic mouse models that use inducible OPC-specific Cre lines to genetically delete transcription factors needed for this process. Preventing OPC differentiation into new myelinating oligodendrocytes affects memory across an array of behavioral tasks, including prefrontal cortex– and hippocampus-dependent spatial navigation (in the Morris water maze), contextual fear conditioning, and motor cortex/dorsal striatum–dependent skill learning. Despite differences in study designs that preclude direct comparison, collectively the results of these studies converge on one overarching picture: The time at which the deficit emerges after blockade of OPC differentiation seems to coincide with the time when multiple brain regions coordinate functionally to mediate long-term storage of a memory.

OUTLOOK

Various studies demonstrate that suppression of adult oligodendrogenesis differentially impairs memory across memory/neural systems. Collectively, they support the conclusion that myelin plasticity could support systems-wide memory consolidation, a mechanism underlying long-term memory. Together with the evidence that dysfunctional myelin or altered myelination during development impairs learning and adaptive behavior, this body of data suggests that even small changes in myelin across the life span can have an impact on circuit- and systems-level mechanisms involved in cognition and behavior. Myelin might have a broader function than previously assumed: Dysfunctional or maladaptive myelination may contribute to neurodegenerative and neuropsychiatric disorders, which have hitherto been considered to have a neuronal basis. A deeper knowledge of the functional role of myelin plasticity may unlock alternative therapeutic approaches. Collectively, this calls for further investigation of the functional role of myelin plasticity.

Myelin plasticity underpins learning and memory.

Stylized diagram of myelinating oligodendrocytes connecting different types of long-term memory, including spatial memory, Pavlovian fear memory, and motor skills, assessed in specific behavioral paradigms (clockwise from top left: single-pellet forelimb reach, complex wheel, contextual fear, Morris water maze) and underpinned by distinct neural systems, representing how oligodendrocyte and myelin changes in the brain are involved in the modification of circuit functions underlying learning and behavior.

OPEN IN VIEWER

Abstract

The brain is responsive to an ever-changing environment, enabling the organism to learn and change behavior accordingly. Efforts to understand the underpinnings of this plasticity have almost exclusively focused on the functional and underlying structural changes that neurons undergo at neurochemical synapses. What has received comparatively little attention is the involvement of activity-dependent myelination in such plasticity and the functional output of circuits controlling behavior. The traditionally held view of myelin as a passive insulator of axons is changing to one of lifelong changes in myelin, modulated by neuronal activity and experience. We review the nascent evidence of the functional role of myelin plasticity in strengthening circuit functions that underlie learning and behavior.

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[Uhohinc]

Regarding the Luger patent: Is the general assumption that there is a transfer process going on in the background? That PW and Luger have reached an agreement that Luger proceeds with obtaining the patent and that it will subsequently be transferred to Clinuvel?

 Reactions:Justinian, Alexius and Toktok

mrdaxModerator

Staff member

Friday at 7:37 PM  

• #13,070

Interesting study trial design... sunburn thresholds comparing untreated vs. afamelanotide implant injected

 Reactions:johnnytech, jack2, Justinian and 7 others

johnnytechModerator

Staff member

Yesterday at 3:10 AM  

• #13,071

@Jalu06

Patent

The Luger patent was indirectly but officially rescinded by the applicant himself at the European patent office. Further, an apology from Luger to PW is documented stating the intellectual property belongs to PW. However, the American version of the patent was awarded, but no doubt will be overturned if pursued.

I think the indications above will absolutely be pursued, but is not critical to stock performance in the next 2 years.

The 18 month path seems clear now...

1. Good momentum with the 3 trials recently announced, and more with previous stroke trial. I expect 2 of 4 trials to go to market by 2023 end, I'll settle for 1. But, since all use same approved formulation, 3 is not out of the question. 4 is a stretch (DNA).
2. The OTC's (2022-2023).
3. A timely 2023 NASDAQ level 2 upgrade.
4. A little noise in the ACTH field (2023?).

This is all that's needed to make this company pop by the end of 2023 enough to meet performance condition #1, or sell the company for the same valuation.

Timing
All three new trials were announced in the last 60 days. Any one of those trials could have started at any time in the last 5 years. There's a reason they're all starting right now. They all use the same approved drug with a solid distribution network and appear to meet summer 2023 marketability targets. Only need 1 or 2 trials (of the 4) to hit market, and the two other listed above (nasdaq & ACTH) for PC1 to be seriously viable.

They know exactly what they are doing and have been planning it for a decade. CFO got what he deserved (maybe tiny high), but others deserve their payday too prorated to their length of service (Lachlan / Dr. P / etc...).

Target is November 2023. Take a sleeping pill and don't sell.

 Reactions:Kalaz, TheNormalOne, Sergio Marchionne and 31 others

macgyverWell-known member

Yesterday at 10:14 AM  

• #13,072

@johnnytech This is my interpretation of the Luger patent situation (please correct me anyone if I’m missing something here):

US20160158322: NDP-MSH FOR TREATMENT OF INFLAMMATORY AND/OR NEURODEGENERATIVE DISORDERS OF THE CENTRAL NERVOUS SYSTEM

- This patent has been awarded. But it is expressly for acute disseminated encephalomyelitis (ADEM). Luger has mouse model data for this disease as well as AHLE, of which he has used in the following US patent application for CNS disorders which is currently suspended at the request of the applicant:

US20200360484: NDP-MSH FOR TREATMENT OF INFLAMMATORY AND/OR NEURODEGENERATIVE DISORDERS OF THE CNS:

It is this US patent application which specifically mentions treating multiple sclerosis that is having trouble, the Examiner refuses to accept the mouse model data and states for the patent to be awarded, data from multiple sclerosis trials in humans (or mice if that’s possible) can only be used. It should be noted this patent application continued with its progress despite Clinuvel launching legal action in Europe. No information yet as to why the applicant initiated suspension, I don’t think it’s Clinuvel-related as they would only launch legal action at the last minute if it looked like the patent was about to be awarded. No point to pay a lawyer to take premature action when the patent application could fail on its own (they waited till the last minute with the European patent). Reasons for why the applicant suspended this patent application? Your guess is as good as mine but there’s only three reasons I can think of: Clinuvel launching legal action, Luger is performing MS trials to gather data (perhaps with funding from Clinuvel?), or Luger is going to withdraw the application outright.

EP3030256:

NDP-MSH FOR TREATMENT OF INFLAMMATORY AND/OR NEURODEGENERATIVE DISORDERS OF THE CNS

This is the contested European patent application which is due to be granted but currently there is a stay on proceedings due to the legal action launched by Clinuvel. Curiously this application shows up as being removed in Wipo Patentscope, however it still exists in the European Patent Office database and the renewal fee was paid last August 2021.

There was some confusion here previously about overlapping Euro patent claims, the serial numbers were the same on two patent applications except the most recent one had ‘A1’ added to it. I’m assuming this is the numbering system the European authority uses when there has been an application amended/updated (A1-A9, B1-B9 and so on for each amended application).

Regarding Luger and Clinuvel, I’d suggest for this CNS/multiple sclerosis IP dilemma/dispute to resolve itself they need each other. Clinuvel has the only relevant (and approved) peptide to engage in CNS trials, Luger has the patent application that has advanced the most, and with no apparent competitor peptide on the horizon it’s in his best interest to partner with Clinuvel. Or as a point of last resort Luger fails in his claims and gets moved out of the way so Clinuvel can lodge an application with relevant data and proceed on their own. But that’s highly unlikely, if that were to happen and the 6th indication is related to CNS, it will be years before the 6th sees the light of day.

Last edited: Yesterday at 10:56 AM

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johnnytechModerator

Staff member

Yesterday at 11:17 AM  

• #13,073

This is a lot of patent minutiae. I don't want others to get confused with my post above.

In my opinion, this post about patents has nothing to do with viability of PC1 by November 2023.

macgyver said:

No information yet as to why the applicant initiated suspension, I don’t think it’s Clinuvel-related as they would only launch legal action at the last minute

Keep in mind this is the American patent system, not the European one. They are government office and possibly like the FDA is very tight-lipped with correspondence until the case is closed. I doubt we're ever going to see lawyer letters either like we do with the European office.

In the European challenge, again I think Clinuvel waited until the last second to keep their name on the downlow, not for lawyer costs. They could have started that challenge a whole year earlier like the Italian scientists did.

But now that their desire to claim that intellectual property is known, I don't think they're going to wait until the last moment anymore. And if it's important to them for November 2023, they're definitely not going to wait (hint... it's not).

With the 2008 NDA and documentation that Luger agrees PW owns all intellectual property for everything covered in the European patent, this absolutely undermines Luger credibility and forward patents that backwards reference on anything related to MS and/or CNS. (Keep in mind, the patent even names the drug Scenesse and the company Clinuvel).

The very best Luger can accomplish is a payoff from Clinuvel to stop being a patent bully, otherwise it's going to be a long legal fight which he'll lose in the end.

macgyver said:

and the renewal fee was paid last August 2021

Of course the Renewal fee was paid. Probably by Clinuvel. Remember, Clinuvel's request to the patent office wasn't to stop the award, but to award it to Clinuvel. I'm sure their lawyers know what they're doing.

This is 2024 stuff and beyond. By then you'll be have two junior Macgyver researchers working and posting about patents for you.

 Reactions:Sherlock, gerritbakker57, Alexius and 1 other person

macgyverWell-known member

Yesterday at 11:32 AM  

• #13,074

@johnnytech

I wonder if the difference between NDP-MSH and afamelanotide matters.

Regarding the US patent you claimed Luger had, it appeared to be misrepresented as the crucial CNS patent Clinuvel was seeking. But instead it’s for an obscure disease, in that regard Luger and Clinuvel haven’t secured anything. I might add that where IP is concerned, transparency is of the utmost importance. Someone else may wish to make a counterclaim so it seems appropriate to have all available documentation out in the open.

To be clear, I’m just making sure people aren’t confused about which patents are secured and still in play. As for your 2023 predictions, I hope you’re right.

Last edited: Yesterday at 3:44 PM

[Uhohinc]

STUDY: MULTIPLE SCLEROSIS PATIENTS HAVE A DIFFERENT GUT MICROBIOME

The condition has confounded scientists. Do bacteria in the gut hold any answers?

Shutterstock

NICK KEPPLER

11 HOURS AGO

Multiple sclerosis still baffles scientists. Theories exist, but researchers still don’t understand what triggers the immune system to attack healthy nerves.

In a new study, researchers found that the microbiomes — the ecosystem of viruses, bacteria, and fungi that reside in our guts — of people with MS are distinct from those without the disease. Understanding why this difference exists could help researchers better understand and treat the condition.

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A PAIR OF GUT ENZYMES COULD BE KEY TO UNIVERSAL ORGAN DONATION — A HOLY GRAIL OF MEDICINE

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By NICK KEPPLER

EARN REWARDS & LEARN SOMETHING NEW EVERY DAY.

SCIENTISTS HAVE BEEN STIFLED BY THE ENDURING MYSTERY OF WHY THE IMMUNE SYSTEM MOUNTS THIS ATTACK ON HEALTHY NERVES.

WHAT’S NEW — In a study published last month in the journal EBioMedicine, researchers found that a group of people with MS had markedly different bacterial ecosystems in their guts compared to a similar cohort without MS.

In multiple sclerosis, the immune system attacks the protective sheaths of nerve fibers, disrupting the communication between those nerves and our brains. Researchers do not know why people develop the condition and have been left with a baffling list of risk factors: Caucasians are more likely to develop MS, and women are more likely to develop one type. MS is more prevalent in colder climates, and a history of certain infections might play a role in triggering the condition.

To examine the disease from another angle, a team of researchers, led by Washington University School of Medicine and the University of Connecticut’s clinical health unit, looked at the eating habits and digestive and metabolic characteristics of 24 people newly diagnosed with the condition. MS patients enrolled in the study had markedly different blood metabolites and microbiome contents than people without MS. They also, on average, ate more than twice as much meat.

Further research is needed to determine the significance (if any) of these correlations and how they might contribute to an understanding of MS.

WHY IT MATTERS — Multiple sclerosis afflicts 2.3 million people globally and can be debilitating, sometimes causing severe handicaps.

Many treatments are available, but there is no cure, and scientists have been stifled by the enduring mystery of why the immune system mounts this attack on healthy nerves. So every new angle could help.

The gut microbiome helps train and develop the immune system. Researchers know that diet plays a crucial role in shaping what species reside in the gut and in what amounts, so perhaps there are insights to be taken from looking at the digestive and metabolic health of people with MS.

Yanjiao Zhou, one of the coauthors, tells Inverse that this small study is a first crack at examining MS from a new perspective — with the technology that allows scientists to sequence gut bacteria from a stool sample and identify the telling aftereffects of metabolism as seen in the bloodstream, plus the knowledge that all these facets are connected.

“This paper is really just to try to leverage all this technology together, and try [to] review the association between all these components,” she says.

Zhou says that some of the bacteria lacking in MS patients have anti-inflammation properties. Inflammation is correlated with a host of chronic conditions, particularly autoimmune ones.

Also, there were differences in the gut microbiomes between those with mild and severe symptoms, which “suggest[s] that specific gut microbes may be associated with the degree of disability in MS patients,” according to the paper.

The most significant dietary difference between the 24 MS patients and 25 healthy people recruited as a control group was meat consumption. The MS patients ate 2.7 servings of meat for every serving eaten by the control group.

Previous research into MS and meat consumption have had inconclusive results. One study showed a higher prevalence of MS among people with high animal fat intake and a lower prevalence in vegetarians. But another study found that people with increased consumption of non-processed red meat had a reduced risk for neurological activity associated with MS. The authors noted that red meat is high in vitamin D and other components that have a neuroprotective effect.

Investigating this further, the researchers behind the new paper used the microbiome data to determine that, in their pool of participants, high meat consumption was correlated with a lack of B. thetaiotaomicron, a gut bacteria that helps the stomach digest carbohydrates.

Interestingly, the presence of that same bacteria “was strongly negatively correlated” with high circulation of an immune system component called T helper 17 cells. Scientists have thought T helper 17 cells play a role in the immune system’s haywire behavior in MS.

But all of that is inconclusive. As for what this means for one of medicine’s most frustrating conditions, only time and further research will tell.

Study participants with MS ate twice as much red meat as those who did not have MS. Research on MS and meat consumption has largely been inconclusive.Shutterstock

HOW THEY DID IT — The researchers recruited 24 newly diagnosed patients with as-yet untreated MS.

They also recruited 25 healthy people and populated that group to match approximately the age, gender, race, and body mass index of the MS group. They used this control group to tabulate what was standard.

The researchers had both groups complete food diaries and submit blood and stool samples.

WHAT’S NEXT — Zhou and her collaborates would like to do another study with more people to test the consistency of these findings. They would also like to include people with more severe MS to investigate the finding further that the microbiome deviates even further from the norm depending on the severity of the disease.

RELATED TAGS

• HEALTH
• MEDICINE
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GUT WEEK

WHY THE GUT MAY BE HUMANS’ “FIRST BRAIN”

This is not science fiction.

timandtim/Photodisc/Getty Images

JOCELYN SOLIS-MOREIRA

11.26.2021 12:00 PM

WHETHER IT’S DIVING OFF A PIER into the salty waves below or stepping up to the podium to address a room full of people, you likely know what “butterflies in your stomach” feel like when they start fluttering away inside. In truth, of course, you are not full of literal butterflies — it’s just your brain talking. But this brain isn’t the one in your head: This brain is the one that runs all the way through your body from your mouth to your butt.

Humans have two brains (stay with us): Sometimes nicknamed the “mini-brain,” your gut has its own nervous system made up of more than 100 million neurons working autonomously from the brain in your head to regulate the digestive system.

INVERSE is counting down the ten most-surprising discoveries about your wondrous gut in 2021. This is #2. Read the original story here.

But like the brain in your skull, the enteric system isn’t a one-trick pony: It regulates the immune system, blood flow, and secretes hormones. It’s also in constant communication with the central nervous system, helping to regulate your emotions and mood. But for all we’ve learned about the enteric nervous system, a dozen more questions remain unanswered.

One question stems from the classic “chicken or the egg” problem.

WHICH CAME FIRST: THE CEREBRAL BRAIN OR THE GUT BRAIN?

That the enteric nervous system evolved to function independently from the brain causes some researchers to argue that the so-called mini-brain came first — Nick Spencer, a professor at Flinders University in Australia, is one of them.

WHAT’S NEW — Earlier this year, Spencer and his colleagues published a study in support of the “first brain” theory. They showed how gut neurons evolved independently to communicate and control gut muscle movement — a key piece of evidence for that the gut’s nervous system may be our “first brain.”

In the study, Spencer and his colleagues find that neurons from the enteric nervous system send and receive messages to coordinate gut muscle movement. The firing of messages is synchronized to other neurons. Doing so is necessary to move items down for digestion, absorb nutrients from food, and expel waste products.

HERE’S THE BACKGROUND — Support for the ‘first brain’ theory ironically comes from animals without brains.

Freshwater creatures belonging to the genus Hydra have roamed the Earth for over 600 million years and yet, their tube-shaped bodies function without a brain.

How did Hydra survive for so long? According to Spencer, the hydra evolved a nervous system similar to today’s “gut brain.”

Hydra may be key to understanding how our own enteric nervous system evolved.CHOKSAWATDIKORN / SCIENCE PHOTO LIBRARY/Science Photo Library/Getty Images

“Despite the lack of a formal brain, as we know it, their intrinsic nervous system allows them to swim and feed and ingest food,” he says.

The enteric nervous system plays multiple roles but its main one involves moving food and other items in and out of the gut. The neurons cause muscles in the GI tract to contract and relax, allowing for digestion to take place.

WHY IT MATTERS — The study adds to the mountain of evidence for the gut’s starring role in your metabolism, brain function, and longevity. One recent study has even found that a diet feeding healthy gut bacteria could reduce multiple sclerosis symptoms.

And for better or for worse, increasing evidence has demonstrated the gut’s influence on your personality — it all depends on the diversity of your microbiome.

But like any other organ in the body, the enteric nervous system isn’t perfect. Studies like Spencer’s can help shift the needle on developing treatments that take this overlooked nervous system into account.

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GUT SCIENCE

SQUIRREL GUT STUDY COULD HELP TREAT A PAINFUL HUMAN CONDITION

Behold the wonders of the microbiome.

Robert Streiffer

TARA YARLAGADDA

1.27.2022 1:17 PM

THE THIRTEEN-LINED SQUIRREL, aptly named for the thirteen stripes on its back, spends much of its time above ground dodging angry humans. But for several months of the year, it gets a reprieve: Like many mammals, this furry rodent spends the winter months hibernating away in its burrows. But rather unlike other hibernating creatures, it reemerges from its long slumber just as brawny as before. Now, scientists may have figured out how it maintains its muscular build without working out — and these findings could help doctors treat muscle wasting diseases in humans, too.

[Uhohinc]

MULTIPLE SCLEROSIS MSJ JOURNAL http://msj.sagepub.com 1 Introduction Although the cause of multiple sclerosis (MS) is not known, it is generally believed that dynamic interactions of genetic and environmental factors, infections in particular, trigger the activation of autoreactive T and B cells present in the normal immune repertoire. 

A plethora of studies in animal models, the experimental autoimmune encephalomyelitis (EAE) model in particular, shows that autoreactive T and B cells activated in peripheral lymphoid organs can infiltrate the central nervous system (CNS) and trigger cascades of pathophysiological reactions, causing inflammation and tissue injury.1 However, despite years of intensive research, a single infectious trigger of MS has not been found. This implies that MS may either be induced by a variety of different triggers or, as proposed by us2 and others,3 that the cause of MS may lie inside the CNS. While considering potential triggers of MS, I asked why MS affects only the human primate (HP) and not closely related non-human primates (NHP). To my knowledge, spontaneous development of MS in NHP has not been reported, not even in chimpanzees, despite the >98% genetic similarity with humans. One might argue that in their natural habitat NHP might not be exposed to the environmental triggers of MS. However, even NHP held in captive colonies in moderate climate areas, where they are likely exposed to the same environmental cues as humans, do not develop MS spontaneously. 

A documented exception may be the spontaneous inflammatory demyelinating disease in a Japanese macaque colony (Macaca fuscata) at the Oregon Primate Center.4 A novel simian γ2-herpesvirus, Japanese macaque rhadinovirus (JMRV), was identified as the potential trigger of the disease. However, definitive proof in deliberately infected monkeys is still lacking. Are non-human primates resistant against multiple sclerosis? One could argue that NHP are (genetically) resistant to MS. Our work in the EAE models in marmosets (Callithrix jacchus) and rhesus monkeys (Macaca Why does multiple sclerosis only affect human primates? Bert A ‘t Hart Abstract Background: Multiple sclerosis (MS) develops exclusively in humans. Non-human primates are resistant against MS, although they are highly susceptible to the MS animal model, experimental autoimmune encephalomyelitis (EAE). Unravelling of the cause(s) underlying this discrepancy is highly relevant as insights might be gained into the elusive event(s) that trigger(s) MS. 

A well-established difference between the human primate (Homo sapiens) and non-human primates is that humans are unable to synthesize the sialic acid N-glycolylneuraminic acid (Neu5Gc). Viewpoint: We propose the concept that long-term ingestion by human primates of the foreign Neu5Gc, via red meat consumption, is an ignored environmental risk factor for MS. Conceptually, incorporation of dietary Neu5Gc into vital regions of the central nervous system, such as the blood–brain barrier (BBB) and the axon–myelin unit, creates targets for binding of de novo synthesized heterophilic anti-NeuGc antibodies. Binding of the antibodies can cause BBB leakage and destabilization of the axon–myelin coupling. The ensuing cytodegeneration and release of self-antigens could be a start of the characteristic pathological features of MS. 

Keywords: Axonal loss, demyelination, immunology, progressive, multiple sclerosis Date received: 5 February 2015; revised: 12 May 2015; accepted: 26 May 2015 Correspondence to: Prof. Bert A. ‘t Hart, PhD Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands. ha...@bprc.nl Prof. Bert A ‘t Hart, PhD University of Groningen, University Medical Center, Department of Neuroscience, Groningen, The Netherlands 591862 MSJ0010.1177/1352458515591862Multiple Sclerosis JournalBA ‘t Hart research-article2015 Personal Viewpoint Downloaded from msj.sagepub.com at Monash University on November 6, 2015 Multiple Sclerosis Journal 2 http://msj.sagepub.com mulatta) over the past 16 years shows no evidence for a low sensitivity of NHP to myelin sensitization; NHP rather seem remarkably sensitive. Marmosets sensitized against the CNS myelin-specific antigen myelin oligodendrocyte glycoprotein (MOG), an immunodominant CNS myelin component, formulated with the relatively weak incomplete Freund’s adjuvant, developed an autoimmune neurological disease that shares remarkable clinical and pathological features with MS.5 

MS risk factors segregate into genetic and environmental factors. To date more than 100 genes with a variable influence on MS have been identified. The vast majority of MS risk genes encode an immunological function, with HLA-DR on top of the hierarchy followed by polymorphisms in the promoter regions of cytokines and chemokines or their receptors.6 The genetic similarity between HP and NHP is very high, even in highly polymorphic genes, such as those encoding the major histocompatibility complex (MHC) and the antigen receptors of T and B cells. Our studies in common marmosets showed in addition that autoimmunity against myelin involves evolutionarily conserved epitopes. The T-cell epitopes that activate the two dominant pathogenic pathways are MOG24-36, activating MHC class II/ Caja-DRB1*W1201-restricted CD4+ T helper 1 cells, which initiate EAE, and MOG40-48, activating MHC class I/Caja-E restricted cytotoxic T lymphocytes (CTL), which drive EAE progression to irreversible neurological disease.5 The two epitopes are juxta-positioned in a highly conserved region (residues 20–50) of the MOG extracellular domain. While the pathological relevance of the Th1 pathway in MS is under debate, I noticed that the newly discovered CTL pathway may be implicated in irreversible demyelination by the killing of oligodendrocytes.7 Environmental factors enhancing the genetic risk to MS are Epstein Barr Virus (EBV) infection, smoking and possibly vitamin D deficiency.8 NHP obviously do not smoke, but they express chronic latent infection with EBV-like lymphocryptoviruses (LCV; including EBV in human, CalHV3 in marmosets, macaque LCV in rhesus monkeys). The hairy and pigmented skin of NHP likely limits the natural production of vitamin D from sunlight exposure, even more so in captive colonies housed indoors. Considering these data we regard it rather unlikely that the absence of MS in NHP can be explained by natural resistance. 

Are human primates uniquely susceptible to multiple sclerosis? The NHP EAE model supports the inside-out paradigm, that is, that autoimmunity results from a dysregulated immune reaction against CNS injury; chronic latent infection with cytomegalovirus (CMV) and EBV contribute to the hyperresponsive state.9 

Chronic infection of HP with CMV creates a (progressively expanding) repertoire of highly reactive cytotoxic T lymphocytes with NK cell markers (NK-CTL), which protect against reactivation of the virus.10 The same holds true for the rhesus monkey. Chronic CMV infection contributes also to changes in the immune system of very elderly people that likely underlie the increasing prevalence of chronic inflammatory diseases.1

0 We noticed that the autoreactive CTL identified in the NHP EAE model cross-react with the UL86 ORF-encoded major capsid protein of CMV and that they have a comparable effector memory phenotype and MHC restriction (MHC-E) as the NK-CTL of humans. Marmoset B cells infected with EBV have the capacity to recruit and activate the CTL.9 These findings indicate that humans may not be uniquely susceptible to MS, as the pathogeneducated immune system of HP and NHP may be similarly responsive to antigen released from a hypothesized primary CNS injury.3,11 A human-specific cytodegeneration paradigm

 The inside-out paradigm and the notion that MS affects only humans may imply a specifically human degenerative event as etiopathogenic trigger. However, myelin release per se, such as in stroke, usually does not elicit (chronic) autoimmune disease. Recent work shows that elicitation of autoimmunity may be prevented by glycoproteins expressed on the myelin surface (e.g. MOG-Lex) that bind C-type lectin receptors (e.g. DC-SIGN) of antigen presenting cells (APC) and inhibit APC maturation. 

The maturation block is removed when the glycan moiety of MOG is changed by inflammatory factors present in early MS lesions.12 The vast majority of glycoproteins that decorate body cells terminate by the structurally almost identical sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolyneuraminic acid (Neu5Gc)13 (Figure 1). Neu5Ac is the precursor of Neu5Gc and conversion is catalysed by CMP-N-acetylneuraminic acid hydroxylase (CMAH). Neu5Gc is absent in the CNS as expression of CMAH is suppressed, indicating that expression of Neu5Gc within the CNS may be detrimental.

14 Indeed, overexpression of CMAH in the mouse brain may have detrimental consequences.15 This warrants the question whether Neu5Gc might be implicated in the destabilization of the axon–myelin unit. Compact myelination of CNS axons is mediated by the interaction of myelin-associated glycoprotein (MAG) with Downloaded from msj.sagepub.com at Monash University on November 6, 2015 BA ‘t Hart http://msj.sagepub.com 3 gangliosides (GM1, GD1a, GD1b, GT1b) on the axon (Figure 1). MAG is a sialic acid-binding lectin (Siglec4) with high specificity for the Neu5Ac residue that caps the major axonal gangliosides. Humans are unique among hominoid primates (bonobos, chimpanzees, gorillas, orangutans, gibbons) because of their inability to synthesize Neu5Gc in any of their body tissues due to an irreversible exon deletion in the CMAH gene, which occurred after the most recent branching of hominoid NHP (around 3 million years ago)14. Nevertheless, detailed analysis of human necropsy tissues showed presence of Neu5Gc in a variety of healthy and malignant tissues.16 The incorporated Neu5Gc was shown to come from the consumption of high Neu5Gc-containing food, red meat from livestock (beef, pork and lamb) in particular. A second important finding has been that due to this defect humans generate Figure 1. Neu5Gc-induced degeneration of the axon–myelin unit. Depicted in panel A is a graphic representation of the effect that Neu5Gc incorporation may have on the axon–myelin unit; the figure is inspired by Simons et al.23 B. The axon–myelin unit is stabilized by myelin-associated glycoprotein (MAG) binding of gangliosides. MAG is a sialic acid-binding lectin (Siglec-4), with high specificity for N-acetylneuraminic acid (Neu5Ac). Panel C depicts the most important gangliosides of CNS axons, which all terminate with Neu5Gc. D. The conversion of Neu5Ac into N-glycolylneuraminic acid (Neu5Gc) is catalysed by CMP-N-acetylneuraminic acid hydroxylase (CMAH) and involves substitution of a -H for an -OH group (arrow). Panel A depicts the putative effect of Neu5Gc on the axon–myelin unit. The healthy unit (upper half) is stabilized by MAG molecules binding gangliosides expressed on the axon surface. Trophic support is provided from the oligodendrocyte to the axon via transport of monocarboxylates (pyruvate, lactate) through transporters (MCT) in the inner myelin lamellae and the axon. The nutrients are fed into the mitochondria to generate sufficient ATP for satisfying the high energy need of the axon, i.e. electron pulse conduction and vesicle transport to the synapses. The lower half shows the situation when MAG–ganglioside binding is blocked by Neu5Gc incorporation and heterophilic antibody binding. The ensuing dissociation of myelin from the axon antibody impairs trophic support, which causes metabolic problems to the axon. The incapacity to produce sufficient ATP causes a pulse-conduction block, mitochondrial degeneration and problems with Ca2+ homeostasis, which inevitably leads to the degeneration of the neuron/axon complex. Downloaded from msj.sagepub.com at Monash University on November 6, 2015 

Multiple Sclerosis Journal 4 http://msj.sagepub.com antibodies against the xeno-sialic acid (e.g. HanganutziuDeicher antibodies in cancer or Paul-Bunnell antibodies in infectious mononucleosis), which are likely induced by gut flora elements incorporating dietary Neu5Gc.17,18 These heterophilic antibodies were found to bind tissues with incorporated Neu5Gc and elicit low-burning inflammation.19 What is the relevance of this paradigm for MS? There is a remarkable overlap of global areas where the MS risk is high with areas where meat from livestock is part of the daily diet. Note also that the dramatic increase of MS incidence in Japan after World War 2 has been attributed to the introduction of the Western diet.20 While the Neu5Gc content of traditional Japanese food, based on fish, poultry and plants is low, it has been calculated that consumption of 250 g red meat per day equals to daily intake of 4.5–7.5 grams Neu5Gc.16 One pathogenically relevant site of Neu5Gc incorporation in the CNS is the blood–brain barrier, where binding anti-Neu5Gc antibody binding may induce endothelial leakage and antibody diffusion into the CNS parenchyma.21 The second relevant site of Neu5Gc incorporation is the interaction of MAG with axonal gangliosides (GM1, GD1a, GD1b, GT1b). It has been shown in neuroblastoma cell cultures that metabolic substitution of Neu5Ac by Neu5Gc abrogates MAG binding.22 It remains to be established whether a sufficient proportion of dietary Neu5Gc will be incorporated in the CNS, but heterophilic antibodies binding low numbers of Neu5Gc epitopes will most likely hinder MAG–ganglioside interaction already at low Neu5Gc incorporation. There is a relatively large body of literature about the consequences when the axon–myelin unit is disturbed, reviewed recently by Simons et al.23 The organization of the unit as bands of compactly wound myelin interspaced by (non-myelinated) nodes of Ranvier allows the fast saltatory conduction of action potentials, but the price paid is that axons depend on the oligodendrocyte for trophic support. Indeed, astrocytes and oligodendrocytes satisfy the high energy need of axons via the supply of monocarboxylate nutrients (pyruvate, lactate) through MCT transporters in the internodes and axons. The strong dependence of axons on glia for the functioning axon–myelin unit implies that disturbed couplings within the unit may result in its degeneration (Figure 1). Perspective for validation of the concept I propose that daily consumption of meat from livestock should be regarded as an environmental risk factor for MS. A great uncertainty in the concept, however, is whether the proportion of orally fed Neu5Gc that incorporates in the brain is sufficient for destabilizing the axon–myelin unit, for the induction of heterophilic antibodies and for activation of an autoimmune process. The common marmoset offers an attractive experimental animal model for answering these questions, as it shares the genetic CMAH defect with humans24 as well as MS-like autoimmune reactions against human myelin.25 Acknowledgements The author thanks Mr Henk van Westbroek (BPRC) for the artwork and Prof Dr Jeroen J Geurts (VUmc, Amsterdam) for valuable discussions and critical reading of the manuscript. Conflict of interest None declared. Funding This research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. References 1. Gold R, Linington C and Lassmann H. Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 2006; 129: 1953–1971. 2. ‘t Hart BA, Hintzen RQ and Laman JD. Multiple sclerosis – a response-to-damage model. Trends Mol Med 2009; 15: 235–244. 3. Stys PK, Zamponi GW, van Minnen J, et al. Will the real multiple sclerosis please stand up? Nat Rev 

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Vegetarians and rate of multiple sclerosis relative to the above inference of a protein in red meat facilitating a t cell response

multiple sclerosis rate in vegetarian

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12 more years plus. for Multiple Sclerosis patients.....Dr Wolgen and company have been putting up the good fight and endurance since FDA told Epitan in a letter that no tanning drug will be approved. Another reason the Mitsubishi Tanabe is wasting its resources thinking the FDA will let one take a pill or keep taking abusively to get tanner quicker.....There is no one that can understand or replace this FDA position, til some more retire.