AP1189 a Erk1/2 pathway without Mcr1 or Mcr3 activation, I do not see as a commercial threat to afamelanotide. Hruby patent for Mcr3 selectivity has gone nowhere.

[Uhohinc]

Biased Agonism as a Novel Strategy To Cooray, Thomas E. N. Jonassen and Mauro Perretti Trinidad Montero-Melendez, Thomas Gobbetti, Sadani N. http://www.jimmunol.org/content/194/7/3381 doi: 10.4049/jimmunol.1402645 February 2015; J Immunol 2015; 194:3381-3388; Prepublished online 27 Material Supplementary 5.DCSupplemental http://www.jimmunol.org/content/suppl/2015/02/27/jimmunol.140264 References http://www.jimmunol.org/content/194/7/3381.full#ref-list-1 This article cites 44 articles, 7 of which you can access for free at: *average • Fast Publication! 4 weeks from acceptance to publication • No Triage! Every submission reviewed by practicing scientists • Rapid Reviews! 30 days* from submission to initial decision Why The JI? Submit online. Subscription http://jimmunol.org/subscription Information about subscribing to The Journal of Immunology is online at: Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright © 2015 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., 

The Journal of Immunology is published twice each month by by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from The Journal of Immunology Biased Agonism as a Novel Strategy To Harness the Proresolving Properties of Melanocortin Receptors without Eliciting Melanogenic Effects Trinidad Montero-Melendez,* Thomas Gobbetti,* Sadani N. Cooray,* Thomas E. N. Jonassen,† and Mauro Perretti* There is a need for novel approaches to control pathologies with overexuberant inflammatory reactions. Targeting melanocortin (MC) receptors represents a promising therapy for obesity and chronic inflammation, but lack of selectivity and safety concerns limit development. A new way to increase selectivity of biological effects entails the identification of biased agonists. In this study, we characterize the small molecule AP1189 as a biased agonist at receptors MC1 and MC3. Although not provoking canonical cAMP generation, AP1189 addition to MC1 or MC3, but not empty vector, transfected HEK293 cells caused ERK1/2 phosphorylation, a signaling responsible for the proefferocytic effect evoked in mouse primary macrophages. Added to macrophage cultures, AP1189 reduced cytokine release, an effect reliant on both MC1 and MC3 as evident from the use of Mc1r2/2 and Mc3r2/2 macrophages. No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 elicited antiinflammatory actions in peritonitis and, upon administration at the peak of inflammation, accelerated the resolution phase by ∼3-fold. Finally, given the clinical efficacy of adrenocorticotropin in joint diseases, AP1189 was tested in experimental inflammatory arthritis, where this biased agonist afforded significant reduction of macroscopic and histological parameters of joint disruption. These proof-of-concept analyses with AP1189, an active oral anti-inflammatory and resolution-promoting compound, indicate that biased agonism at MC receptors is an innovative, viable approach to yield novel anti-inflammatory molecules endowed with a more favorable safety profile. The Journal of Immunology, 2015, 194: 3381–3388. Melanocortin (MC) receptors (MC1–MC5), a family of class A druggable G protein–coupled receptors (GPCRs), are attractive therapeutic targets for a number of conditions due to their wide distribution and diversity of physiological processes they regulate (1). MC1 regulates UV light–induced skin tanning and other immune responses because of its expression on leukocytes. MC2 regulates cortisol production on the adrenal glands, whereas MC5 plays a role on exocrine glands secretions. MC3 and MC4 exert nonredundant functions on energy homeostasis in addition to specific anti-inflammatory roles: whereas MC3 activation is particularly protective for arthritides (2), MC4 provides neuroprotection in brain inflammation (3). Accordingly, the array of pathological situations that could be targeted with MC drugs includes skin conditions (vitiligo, melanoma, erythropoietic protoporphyria), cardiovascular pathologies, joint inflammation (gout, rheumatoid arthritis [RA]), obesity, cachexia, or sebaceous glands disorders such as acne vulgaris (4). Peripheral MC1 and MC3 can be pharmacologically activated to induce anti-inflammation. The endogenous agonist a-melanocyte– stimulating hormone (aMSH), like other protective mediators, is released by immune cells to counterbalance proinflammatory signals, thus preventing excessive tissue damage (5, 6). In line with the resolution of inflammation concept, therapeutics targeting MC1 and MC3 will then be acting by mimicking the body’s own protective resources (7, 8) and might be characterized by a lighter burden of side effects. Shown to be effective in RA rheumatic diseases since the early 1950s (9), the use of corticotropin or adrenocorticotropin hormone (ACTH) declined when synthetic glucocorticoids became available. However, the discovery of an alternative anti-inflammatory mechanism for ACTH involving activation of peripheral MC receptors on immune cells (10) has revived the interest in developing novel ACTH-like molecules with no steroidogenic effects for the treatment of joint diseases such as gout or RA (11, 12). However, the limitation in the translational delivery of novel MC drugs besides the marketed ACTH formulations (H.P. Acthar Gel, Synacthen Depot, or Cortrosyn) is imposed by the lack of receptor selectivity achieved so far. Innovative approaches in G protein–coupled receptor drug discovery might help to overcome this limitation. Allosteric modulation consists in the ability of a molecule to enhance (positive modulation) or reduce (negative modulation) the effect of the endogenous ligand by binding to a distinct site of the receptor protein, termed allosteric site (13). A higher degree of selectivity is expected as allosteric regions are less conserved among the five MCRs, and indeed, allosteric modulators at MC4 are currently under development for the treatment of obesity (14). *William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and † Department of Biomedical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark Received for publication October 20, 2014. Accepted for publication January 28, 2015. This work was supported by a collaborative project between SynAct Pharma APS and William Harvey Research Limited. Address correspondence and reprint requests to Prof. Mauro Perretti and Dr. Trinidad Montero-Melendez, William Harvey Research Institute, Barts and The London School of Medicine, Charterhouse Square, London EC1M 6BQ, U.K. E-mail addresses: m.per...@qmul.ac.uk (M.P.) and t.monter...@qmul.ac.uk (T.M.-M.) The online version of this article contains supplemental material. Abbreviations used in this article: ACTH, adrenocorticotropin hormone; AGRP, agouti-related protein; EIA, enzyme immunoassay; MC, melanocortin; aMSH, a-melanocyte–stimulating hormone; NDP-aMSH, [Nle4,D-Phe7]-aMSH; RA, rheumatoid arthritis; WT, wild-type. Copyright 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402645 by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from Another emerging concept of significant therapeutic interest is the one of biased agonism. The obsolete notion that receptors could exist in two unique conformations, the active one and the inactive one, has been replaced with the conception that multiple active conformations can exist, each one creating a distinct signal yielding to multiple functional outcomes (15, 16). Receptor activation, rather than linear and static, is emerging as a highly dynamic and multidimensional process in which a diversity of active conformations may be induced by different molecules leading to distinct effects. We reasoned that this new pharmacological approach would have a positive impact on MC-based drug development because it sets the focus on “pathway selectivity” rather than “receptor selectivity,” given the proven difficulty of the latter strategy. In addition, finetuned molecules could be designed to engage only the therapeutically relevant pathway and not those leading to side effects. In this article, we present the first, to our knowledge, biased dual agonist at MC1 and MC3, and describe its anti-inflammatory properties together with its lack of effect on melanogenesis. Materials and Methods MC drugs and inhibitors aMSH and [Nle4,D-Phe7]-aMSH (NDP-aMSH) were purchased from Tocris Bioscience (Abingdon, U.K.). (E)-N-[trans-3-{1-(2-nitrophenyl)-1H-pyrrol2-yl} allylidenamino] guanidinium acetate (AP1189) was synthesized at Clauson-Kaas A/S (Copenhagen, Denmark). The ERK1/2 inhibitor FR180204 was obtained from Merck Millipore (Darmstadt, Germany). Cell culture and transfections HEK293A cells were maintained in DMEM containing 10% FCS and 1% penicillin/streptomycin and kept at 37˚C with 5% CO2. Clones for MC1 and MC3 and empty vector pCMV6 were purchased from Origene (Rockville, MD). Serum-starved cells were transfected with Lipofectamine 2000 (Invitrogen, Paisley, U.K.) according to manufacturer’s instructions and used 24 h after transfection. Radioligand binding assay Determination of binding affinities was performed by [125I]-NDP-aMSH radioligand binding. Cells were suspended in HEPES buffer and incubated with 0.05 nM [125I]-NDP-aMSH. AP1189 dissolved in DMSO was added, and cells were incubated for 90 min at 22˚C (MC1 cells -B16-F10-) or for 60 min at 37˚C (MC3 cells -CHO stable transfected cells-). After incubation, the amount of bound radioligand was determined and expressed in percentage of total binding in the absence of AP1189. ELISA and enzyme immunoassay IL-1b, IL-6, and TNF-a were measured in cell supernatants by ELISA using Ready-SET-Go ELISA kits from eBioscience (Hatfield, U.K.). cAMP was measured using the cAMP enzyme immunoassay (EIA) kit from Cayman Chemical (Cambridge, U.K.). Western blotting HEK293A cells were lysed using radioimmunoprecipitation assay buffer (Thermo Scientific, Waltham, MA) containing protease inhibitors mixture (Calbiochem, Hertfordshire, U.K.). Samples were subjected to standard SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore). These were incubated with rabbit anti–phospho-ERK1/2 (42/44 kDa, 1:1000; Cell Signaling, Leiden, the Netherlands) overnight and HRP-conjugated anti-rabbit IgG for 1.5 h (1:2000; Dako, Stockport, U.K.). Membranes were reblotted with anti–a-Tubulin (55 kDa, 1:5000; Sigma-Aldrich, Dorset, U.K.) for 1 h and HRP-conjugated anti-mouse IgG for 1 h (1:2000; Dako, Stockport, U.K.). Proteins were detected with Luminata Forte Western HRP substrate (Merck Millipore, Hertfordshire, U.K.) and visualized on the FluorChem E Digital Room (ProteinSimple, East Sussex, U.K.). Ca2+ mobilization assay Twenty-four hours after transient transfection, HEK293 cells were incubated with 2 mM Fura 2-AM (Molecular Probes, Paisley, U.K.) in HBSS without Ca2+ (Sigma-Aldrich) at 37˚C for 45 min in the dark. Subsequently, cells were washed three times in HBSS. HBSS containing 0.185g/l CaCl2 was then added before stimulation with agonists at the indicated concentrations. Ionomycin (1 mM) was used as a positive control. Mobilization of intracellular calcium was measured by recording the ratio of fluorescence emission at 510 nm after sequential excitation at 340 and 380 nm using the NOVOstar microplate reader (BMG LABTECH, Aylesbury, U.K.) during 70 s after drug addition. Then data corresponding to time 25 s after drug stimulation were selected to generate concentration–response curves. Determination of melanin accumulation B16-F10 cells were cultured in aMEM containing 10% FCS and 1% penicillin/streptomycin, and kept at 37˚C with 5% CO2. For melanin determination, cells were plated in 96-well plates using phenol red free media. Melanin content was determined spectrophotometrically at 405 nM in supernatants 72 h after drug stimulation (17). Isolation of primary peritoneal macrophages Mice were injected i.p. with 1 ml of 2% Biogel (Bio-Rad, Hemel Hempstead, U.K.). Four days later, peritoneal cells were collected by lavage using 4 ml of 3 mM EDTA in PBS and plated in 24-well plates at a density of 0.5 3 106 cells/well, in RPMI 1640 containing 10% FCS and 50 mg/ml gentamicin. After 2 h of incubation, nonadherent cells were removed. Compounds or vehicle were added 30 min before stimulation with 25 mg/ml zymosan A (Sigma-Aldrich). Supernatants were collected after 5 h. Isolation of human primary neutrophils Experiments using healthy volunteers were approved by the local research ethics committee (P/00/029 East London and The City Local Research Ethics Committee 1). Informed written consent was provided, according to the Declaration of Helsinki. Blood was collected into 3.2% sodium citrate and diluted 1:1 in RPMI 1640 before separation through a double-density gradient using Histopaque 10771 and 11191 (Sigma-Aldrich). Contaminating erythrocytes were removed by hypotonic lysis. Polymorphonuclear cells were incubated in 10% FCS overnight at 37˚C, 5% CO2 to let neutrophils undergo spontaneous apoptosis. Phagocytosis and efferocytosis Primary peritoneal macrophages were stimulated with compounds/vehicle for 30 min before the addition of zymosan A at 1:10 (macrophage/zymosan) ratio for 15 min, or apoptotic neutrophils (1:2, macrophage/neutrophil) for 1 h. Cells were fixed and neutrophils stained using the myeloperoxidase assay by adding 0.1 mg/ml dimethoxybenzidine (Sigma-Aldrich) and 0.03% (v/v) hydrogen peroxide for 1 h (18). Cells were analyzed by light microscopy with three random fields being acquired per well (n = 3 wells/ treatment). More than 400 cells were blindly counted per treatment point. Animals All animal studies were approved by and performed under the guidelines of the Ethical Committee for the Use of Animals, Barts and The London School of Medicine and Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986). Male (7–8 wk old) C57BL/6J wild-type (WT) and BALB/c mice were purchased from Charles River Laboratories. Breeding pairs of the Mc1r mutant (Mc1r2/2; recessive yellow e/e) and Mc3r2/2 colonies were originally obtained from Jackson Laboratories or donated by Dr. H.Y. Chen (Merck), respectively. Zymosan-induced peritonitis model Peritonitis was induced by the injection of 1 mg zymosan A (SigmaAldrich) i.p. in 0.5 ml sterile PBS. At the indicated times, mice were sacrificed by CO2 exposure, and peritoneal cavities were washed with 4 ml ice-cold PBS containing 3 mM EDTA. Cells were stained with Turk’s solution (0.01% crystal violet in 3% acetic acid) and counted using a Neubauer hemocytometer or were stained with FITC-conjugated mAb for Ly-6G/Gr1, F4/80, and corresponding isotype controls (eBioscience, Hatfield, U.K.), and subjected to flow-cytometry analysis using a BD FACSCalibur platform (BD Biosciences, Oxford, U.K.). K/BxN serum-induced arthritis model Arthritis was induced with two i.p. injections of 100 ml K/BxN serum on days 0 and 2. Disease was monitored daily until day 8 by assessing the paw volume using a plethysmometer (Ugo Basile, Comerio, Italy), disease incidence, and clinical score (score per paw: 0 = no signs of inflammation, 1 = subtle inflammation, localized, 2 = easily identified inflammation but localized, 3 = evident inflammation, not localized; maximum score = 12 3382 BIASED AGONISM AT MC1 AND MC3 REDUCES INFLAMMATION by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from per mouse). Pharmacological treatments were administered orally once daily from day 2 until the end of the experiment. Histological analysis Tissues (ankles) were fixed with 4% neutral-buffered formalin, decalcified with 10% formic acid, and paraffin embedded. Sections (4 mm) were stained with H&E (Sigma-Aldrich) or fast green and safranin O (SigmaAldrich). Sections were graded from 0 (no disease) to 3 (severe) based on the degree of synovitis and cartilage degradation. Statistical analysis Experiments were repeated from two to five independent times. Data were analyzed by Student t test, one- or two-way ANOVA, followed by Dunnett’s or Bonferroni’s multiple comparison test when appropriate. Nonlinear regression models were used to generate dose–response curves for cAMP and ERK phosphorylation. In all cases, data are presented as mean 6 SEM of n independent observations and were considered statistically significant when p , 0.05.

 Results AP1189 is a biased agonist at the MC receptors MC1 and MC3 AP1189 structure was designed on phenyl-pyrrole-aminoguanidine derivatives, shown to activate MC receptors (patent no. WO2007141343A1). However, when AP1189 was tested on HEK293A cells transfected with mouse receptors for activation of the canonical cAMP pathway, no signal was observed with MC3 cells and a response on MC1 cells only at high concentrations ($10 mM; Fig. 1A). In addition, no response was obtained in B16-F10 murine melanocytes (Supplemental Fig. 1A). cAMP accumulation for controls (empty vector pCMV6 transfected cells) are shown in Supplemental Fig. 2. These results are in agreement with binding assays where AP1189 displayed the tracer NDP-aMSH at high concentrations for MC1 with no effect at MC3 (Supplemental Fig. 1B). 

In contrast, AP1189 addition to mouse MC1 and MC3 transfected HEK293 cells afforded consistent and robust activation of ERK1/2 phosphorylation (Fig. 1B). This response was not restricted to the murine receptors and was confirmed in human MC1 and MC3 transfected cells (Supplemental Fig. 3). In addition, AP1189 induced Ca2+ mobilization, another melanocortin signaling response (Fig. 1C). To further explore this ligand bias observed with AP1189, we generated “biased plots” (15) using equimolar concentration– response curves for two signaling pathways (cAMP versus ERK1/2, as well as cAMP versus Ca2+) plotted as a function of each other (Fig. 2). These plots were constructed using the concentration–response curves for the three pathways shown in Fig. 1, and expressed as percentage maximal response of the reference aMSH. Considering the natural ligand aMSH as the reference standard, AP1189 showed preferential response to ERK1/2 phosphorylation and Ca2+ mobilization over cAMP, an effect observed for both MC1 and MC3 receptors (Fig. 2). 

Collectively, these results demonstrate that AP1189 is a biased agonist. Next, we questioned whether such a selective activation of MC receptors yielded biological activity. AP1189 exerts anti-inflammatory and proresolving actions in vitro Because mouse macrophages express both MC1 and MC3 receptors (18), we began the investigation of the functional properties of AP1189, using peritoneal macrophages from WT, Mc1r2/2, and Mc3r2/2 mice. Added to cells at nanomolar concentrations, AP1189 significantly reduced IL-1b, IL-6, and TNF-a in WT macrophages, whereas no effect was observed in Mc3r2/2 cells; the inhibitory effect was retained for IL-1b and IL-6, but not TNF-a, on Mc1r2/2 cells (Fig. 3A–C). Whereas cytokine inhibition is a typical anti-inflammatory effect, proresolving molecules must activate processes like phagocytosis and efferocytosis (19), effects we have reported for MC agonists (18). AP1189 promoted phagocytosis of zymosan particles by increasing both the proportion of phagocytic macrophages and the number of particles internalized per single cell (Fig. 3D). In addition, AP1189 promoted phagocytosis of apoptotic neutrophils (efferocytosis), a crucial event in resolution and for restoration of tissue homeostasis after an inflammatory event. AP1189 (optimal concentration of 1 nM) augmented efferocytosis of apoptotic neutrophils by ∼60% in WT macrophages (Fig. 3E). This effect was not evident in cells lacking either MC1 or MC3, suggesting a contribution of both receptors in the prophagocytic abilities of AP1189. To functionally associate biased agonism to biological outcome, we established the relevance of ERK1/2 phosphorylation in efferocytosis: addition of the ERK1/2 inhibitor FR180204 (1 mM) abrogated the proefferocytic actions of AP1189 (Fig. 3F). Melanogenesis is not induced by AP1189 Active in vitro concentrations of 0.1–1 nM AP1189 were tested on B16-F10 cells for potential promelanogenic properties. Incubated with this mouse melanocyte cell line, AP1189 did not induce formation of dendrites (at 24 h; Fig. 4A) or production of eumelanin, a response evident at day 3 on pelleted cells treated with the pan-agonists aMSH and NDP-aMSH (Fig. 4A, insets), as well as by the quantification of melanin in supernatants (Fig. 4B). These data, not unexpected because melanogenesis depends on the cAMP pathway, provide further functional confirmation to the biased properties of AP1189. AP1189 promotes resolution of acute inflammation in vivo The efficacy of AP1189 on cytokine release and phagocytosis justified follow-up studies to establish its ability to affect leukocyte migration in acute peritonitis using either a prophylactic (administration 30 min before zymosan) or a therapeutic (administration 2 h after the inflammatory insult) design. In addition, distinct administration routes were evaluated including i.p., i.v., and oral. Results, presented in Fig. 5A–D, report significant protective effects of AP1189 in vivo with ∼30–70% inhibition of neutrophil infiltration alongside both administration protocols and regardless of the administration route. Monocyte infiltration was also reduced (Supplemental Fig. 4A). In vivo proresolving effects were investigated by treating animals with AP1189 at the peak of inflammation (12 h postzymosan), where neutrophil numbers are the highest, and measuring the resolution indices as described previously (20). AP1189 accelerated resolution by reducing the time when peak values are reduced by 50% from 38 to 21 h (Fig. 5E). The resolution index (time interval from maximal response to time when peak values are reduced by 50%) was almost three times faster in the group of mice treated with AP1189 as compared with vehicle-treated mice (9 and 26 h, respectively). In addition, total cell (Fig. 5F) and monocyte/macrophage (Fig. 5G) infiltration were significantly reduced by AP1189. We also observed an increase in efferocytosis at 22 h afforded by AP1189 (Supplemental Fig. 4B), from 1.8 to 3. 1% phagocytic cells, although this difference did not reach statistical significance (p = 0.34). AP1189 did not alter the F4/80low/ Cd11blow population (12.1 and 15.12% for vehicle and AP1189 treated, respectively, p = 0.14, t test). These data indicate that tissues were more effectively cleared from recruited immune cells when treated with AP1189, effects conducive to resolution of inflammation and restoration of a physiological environment. The Journal of Immunology 3383 by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from Orally active AP1189 reduces arthritis in mice MC compounds are well-known for their antiarthritic actions in both preclinical (11, 21) and clinical settings, with ACTH reported to be clinically effective for the treatment of RA and gout (22, 23). We used the K/BxN serum transfer model, characterized by rapidonset severe inflammatory arthritis. Mice were challenged with two doses of arthritogenic serum (days 0 and 2), and AP1189 was administered once daily, 25 or 50 mg/kg orally, from day 2, when the arthritis begins to be macroscopically detectable. At 50 mg/kg, AP1189 reduced all signs of arthritis measured: clinical score (242%), paw swelling (287%), proportion of animals with all four paws affected (250%), and the severity of the inflammation (270%; Fig. 6A–E). Histological analyses (shown in Fig. 6F, 6G) revealed a significant reduction of synovitis, evident by the lower extent of leukocyte infiltration (monitored after H&E staining), whereas no evident effects were observed on cartilage protection (safranin O staining). FIGURE 1. Signaling profile of AP1189 at MC receptors. cAMP production, ERK1/2 phosphorylation, and intracellular Ca2+ mobilization were studied in HEK293A cells transiently transfected with mouse MC1 and MC3. (A) For cAMP assay, cells were stimulated with compounds for 15 min and samples analyzed by EIA. Forskolin (3 mM) was used as positive control. (B) For ERK1/2 phosphorylation analyses, cells were stimulated with AP1189 for 8 min and with aMSH for 5 min. ERK1/2 phosphorylation was analyzed by Western blot using a-Tubulin as loading control. Representative blots of three to four experiments are shown. Bands were quantified using ImageJ64 and the ratio of phospho-ERK1/2 and a-Tubulin calculated. Data were then expressed as the percentage of aMSH response. (C) Intracellular Ca2+ mobilization was measured using Fura-2 AM–labeled cells in the NOVOstar microplate reader. Ionomycin (1 mM) was used as positive control. The ratio of fluorescence emission at 510 nm after sequential excitation at 340 and 380 nm was recorded. Concentration–response curves were generated using the Ca2+ response 25 s after drug stimulation. Experiments were repeated two to five times and analyzed by nonlinear regression. FIGURE 2. Biased agonism at MC receptors by AP1189. Biased plots were generated using the cAMP concentration–response curves (EIA assays) and those generated from ERK1/2 Western blots densitometric analyses (A) or Ca2+ flux assays (B); see Fig. 1 for original data. All data for cAMP, ERK1/2, and Ca2+ are expressed as percentage maximal response of aMSH. 3384 BIASED AGONISM AT MC1 AND MC3 REDUCES INFLAMMATION by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from Discussion We report in this article that biased agonism at MC receptors can be a viable, and innovative, approach to exploit a large wealth of MC biology for therapeutic treatment of pathological inflammation. We performed an integrated series of experiments weaved on a rationale that started from the discovery of unorthodox agonism to the assessment of in vivo and in vitro biological properties of AP1189. Our results provide proof-of-concept data to effect therapeutic innovation beyond classical orthosteric ligands to deliver translational MC drugs. Biased agonists at MC receptors offer new opportunities for the development of therapeutics with improved profile by activating pathways that are therapeutically relevant and evading those associated with side effects (15, 16).

 The appreciation that therapeutic outcomes might derive from distinct downstream signaling events indicates that not all signaling pathways ascribed to a specific receptor need to be activated by a candidate drug molecule. Indeed, biased opiate analgesics devoid of respiratory depressive effects are under development (24). The concept of ligand bias, relatively new for therapeutic approaches, calls for: 1) a better dissection of signaling pathways required for therapeutic efficacy, 2) the identification of pathways associated to side effects, and 3) a careful design of drug screening and lead candidates optimization according to the desired pathways. In our attempts to establish new biology that supports approaches for novel anti-inflammatory/proresolving therapeutics, we focus on the MC receptor subtypes MC1 and MC3, both expressed and playing tonic and nonredundant protective roles in immune cells and synovial tissue, among others (2, 18, 21, 25). The MC peptide ACTH has long been known to be effective for the treatment of gout and RA (26). Subsequently, efficacy in inflammatory bowel disease, multiple sclerosis exacerbations, or nephrotic syndrome has been reported, but overall the use of ACTH is by and large limited for side effects associated with increased cortisol production through MC2 activation (23, 27, 28). MC1 represents an appealing anti-inflammatory target: its selective activation inhibits leukocyte adhesion and emigration in a model of ischemia-reperfusion injury (29) and evokes protection against lung injury as well as in a model of delayed-type hypersensitivity (30). However, its promelanogenic actions impose a considerable limitation to MC1 as a safe target candidate when chronic treatment is predicted. Selective targeting of MC3 may result in improved safety profile and particular efficacy for joint inflammation. This receptor mediates the antiarthritic actions of the synthetic polypeptide DTrp8 -gMSH in the K/BxN model, as well as the negative regulation of osteoclast generation and activation (21, 31), as well as crystal-induced inflammation (25). The FIGURE 3. In vitro anti-inflammatory actions of AP1189. Biogel-elicited peritoneal macrophages were collected from WT, Mc1r2/2, or Mc3r2/2 mice and stimulated for 30 min with AP1189 before the addition of zymosan A (25 mg/ml) for 5 h. IL-1b (A), IL-6 (B), and TNF-a (C) were measured in supernatants. (D) Phagocytosis was assayed in WT cells, pretreated with 1 nM AP1189 for 30 min, after addition of zymosan particles at 1:10 ratio (macrophages/zymosan) for 1 h. The proportion of phagocytic cells and the number of internalized particles per 100 cells (phagocytic index) were analyzed. (E and F) Efferocytosis of apoptotic neutrophils was assayed in WT, Mc1r2/2, and Mc3r2/2 macrophages (E) and in WT in the presence of the ERK1/2 inhibitor FR180204 at 1 mM (F). Apoptotic neutrophils were added to the macrophages at a ratio of 1:2 on AP1189-pretreated macrophages (as in D), and 1 h later, myeloperoxidase staining was performed to visualize ingested neutrophils. Data are the mean 6 SEM of three independent experiments. *p , 0.05 versus control (WT [A–C] or vehicle [D–F]). FIGURE 4. Effects of MC compounds on melanogenesis. (A) B16-F10 cells were treated with AP1189 or the pan-agonists aMSH and NDP-aMSH for 1 d (monolayer cells are shown; original magnification 340). At day 3, cells were collected by centrifugation and pellets photographed. (B) Melanin production was quantified spectrophotometrically by measuring absorbance at 405 nM, 72 h after drug stimulation. aMSH was used at 10 mM and NDPaMSH at 100 nM. Data are the mean 6 SEM of n = 8, representative of two independent experiments. ***p , 0.01 versus vehicle (Veh). The Journal of Immunology 3385 by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from major obstacle in this study derives from the absence of selective MC3 agonists devoid of off-target effects associated particularly with MC1 and MC4 activation. In this article, we characterize the orally active small molecule AP1189 as a biased agonist with the additional advantage of targeting both tissue-protective MC1 and MC3 receptors. AP1189 activation of MC receptors did not induce cAMP accumulation, the canonical pathway ascribed to MC agonists and used for drug screening programs (e.g., see Pantel et al. [14]). AP1189 interaction with MC1 and MC3 activated a second pathway centered on ERK1/2 phosphorylation, as well as intracellular Ca2+ mobilization. Although somehow neglected, these two signaling pathways have been described by downstream activation of most MC receptors (32). The immediate aspect we addressed was the analysis of the consequences of this unusual bent toward the currently considered noncanonical pathways. Eumelanogenesis is unquestionably dependent on cAMP induction by MC1 (33) whereas ERK1/2 phosphorylation seems to meFIGURE 5. In vivo anti-inflammatory actions of AP1189. Acute peritonitis was induced with one single injection of 1 mg zymosan, and leukocyte infiltration was analyzed by flow cytometry. (A–C) Prophylactic experimental design: AP1189 was administered 30 min before zymosan injection and cells were analyzed at the 4-h time point. (D) Therapeutic design: AP1189 was administered 2 h after zymosan, and cells were analyzed at the 6-h time point. (E) AP1189 promotes resolution. AP1189 (1 mg/kg) or vehicle were injected i.p. at 12 h postzymosan, and cells were analyzed at the 22- or 44-h time point. Neutrophils infiltration and resolution indices are shown: Cmax = maximal neutrophil infiltration; Tmax = time when maximal neutrophil infiltration is achieved; T50 = time when maximal neutrophil infiltration is reduced by 50%; Ri = time from maximal response to T50. (F and G) Total cells numbers and monocytes/macrophages values from the experiment in (E). AP1189 was administered i.p. (A and E–G), i.v. (B and C), or orally [p.o., (D)]. C57BL/6 mice were used in (A) and (E)–(G). BALB/c mice were used in (B)–(D). Data are the mean 6 SEM of n = 5–6 per group. *p , 0.05 versus control (vehicle). FIGURE 6. Antiarthritic actions of AP1189 in the K/BxN serum transfer model. Arthritis was induced on C57BL/6 mice by two i.p. injections of arthritogenic serum on days 0 and 2. From day 2, AP1189 or vehicle (PBS) were administered orally once daily. Clinical score (A), paw swelling (B), disease incidence (C), number of mice with all paws affected (D), and the number of paws per mouse severely affected (E) were recorded over 8 d. (F and G) Paraffin tissue sections were stained with H&E and fast green and safranin O. Sections were graded from 0 (no disease) to 3 (severe) based on the degree of synovitis (purple staining in the H&E sections) and cartilage degradation (loss of red coloration in the safranin O sections, arrows). Representative images are shown (original magnification 34). Data are the mean 6 SEM of n = 6 per group. *p , 0.05 versus control (vehicle). B, bone; S, synovitis. 3386 BIASED AGONISM AT MC1 AND MC3 REDUCES INFLAMMATION by guest on May 23, 2021 http://www.jimmunol.org/ Downloaded from diate the anorexigenic effects of MC4 (34); but whether ERK1/2 phosphorylation occurs downstream of cAMP is controversial. Inhibition of NF-kB activation upon MC activation also has been linked to the cAMP pathway (35). However, anti-inflammation via ERK1/2 activation, in conjunction or not with cAMP induction, has also been suggested (36, 37). In this respect, we show that AP1189 reduced cytokines release by zymosan-stimulated macrophages and promoted both phagocytosis and efferocytosis by this cell type. These effects were produced with distinct profiles when using Mc1r2/2 and Mc3r2/2 cells, suggesting nonredundant engagement for both receptors by AP1189. The protective actions of AP1189 were absent in Mc3r2/2 cells for IL-1b, IL-6, and TNF-a release and efferocytosis. With Mc1r2/2 cells, only the effects on TNF-a release and efferocytosis were prevented. In addition, promotion of efferocytosis by AP1189 was entirely dependent on ERK1/2 activation, as observed by using a selective inhibitor. As predicted, the biased signaling dissociated from cAMP also prevented the unwanted pigmentary effects on melanocytes, including eumelanin production and formation of dendrites. These findings, together with the fact that the in vivo experiments were performed in BALB/c mice (Fig. 5B–D), exclude any involvement of melanogenesis and melanin, a molecule with protective antioxidant properties (38), in the actions of AP1189. We have pioneered the concept that therapeutic innovation for inflammatory diseases could derive from appreciation and exploitation of the mechanisms operative during the resolution phase of inflammation (39, 40). Such an approach, we propose, would yield therapeutics that would not be resolution toxic by repressing or delaying tissue repair processes and the ensuing restoration of homeostasis (40). To this end, we took advantage of the resolution indices, introduced by Serhan’s group, to quantify the impact of novel drugs in the resolution phase of inflammation (20). When administered at the peak of neutrophil infiltration, that is, right before the beginning of resolution, AP1189 promoted resolution. Analysis of the resolution indices indicated that in AP1189-treated mice, recovery was achieved three times faster than in vehicletreated animals. Monocyte/macrophage counts were also reduced by AP1189. This effect could be because of reduced infiltration, because we observed that AP1189 at 10 mg/kg afforded a 37% reduction of peritoneal infiltration of this cell type after zymosan (Supplemental Fig. 4A). However, increased efflux of macrophages after increased efferocytosis could also contribute to the lower counts quantified at a given time point (Supplemental Fig. 4B). Exciting results were also obtained with the oral treatment with AP1189 in the serum transfer model of arthritis. This arthritogenic serum contains anti–glucose-6-phosphate-isomerase Abs that, when passively transferred to naive mice, form immunocomplexes on the cartilage and synovial surface, which attract and activate neutrophils and mast cells initiating joint inflammation (41). In line with data produced with classical MC agonists such as AP214 (18) or DTrp8 -gMSH (21, 31), oral administration of AP1189 reduced clinical score, paw swelling, incidence of severe disease incidence, and the overall disease severity. Microscopically, AP1189 significantly decreased leukocyte infiltration and synovitis, although no significant changes were observed on cartilage degradation. These findings, together with the wellknown protective effects of ACTH in human joint diseases, present the MC AP1189 as a promising oral candidate for pathologies of the joint including RA. MC receptors are products of genes that are very polymorphic, thus making it necessary to determine whether gene variants could impact on the pharmacological profile. In this vein, Herraiz et al. (42) found that red hair variants of MC1 presented reduced cAMP response to NDP-aMSH, but normal ERK1/2 response. Moreover, Doyle et al. (37) showed that red hair variants do not impact on the anti-inflammatory properties, measured as inhibition of NF-kB activation, of the MC1 selective small molecule BMS-470539. These studies indicate to us that although MC receptor polymorphism must be considered and tested for each MC candidate agonist, fully active molecules on MC1 variants are plausible, hence the low activity elicited by the endogenous ligand does not necessarily predict low activity for synthetic molecules. Indeed, a similar situation occurs for MC4: Haslach et al. (43) recently identified a number of peptides with full agonistic activity at lossof-function obesity-related MC4 variants. Another matter of interest derived from the work presented in this article will be the identification of molecular or chemical determinants that lead to biased agonism. AP1189 was not able to displace NDP-aMSH from binding to MC3 but was able to induce ERK1/2 phosphorylation and Ca2+ mobilization. The same was essentially true also for MC1, where apparent competition occurred at concentrations .1,000-fold higher than those active for ERK1/2 phosphorylation. 

Altogether these studies suggest that AP1189 binds to an allosteric site, hence it acts as a biased allosteric agonist, explaining the differential activation profile observed when compared with classical “orthosteric” agonists. Elucidation of these determinants might guide a rational design of a new class of drugs combining allosterism and ligand bias. We note how Ca2+ mobilization, albeit not novel for MC receptor agonists, is yet to be functionally linked to the anti-inflammatory actions of MC-based compounds. Our data on the biased agonist AP1189 also suggest the urgency for a redesign of current MC-based drug discovery approaches (by including noncanonical pathways) and possibly even reassessment of known compounds. In fact, AP1189 could have been mistakenly described as a weak antagonist at MC1, active at micromolar concentrations, if cAMP signaling and binding properties were solely studied. Similar conclusions have been suggested for the endogenous antagonist, or more correctly inverse agonist, agoutirelated protein (AGRP). AGRP can actually initiate Gi/o protein– induced signaling (44), as well as internalization of MC3 and MC4, a receptor turn-off mechanism attributed to agonistic activity (45). More recently, AGRP has been identified as a biased agonist at MC4 causing, indeed, activation of the ERK1/2 pathway (46). 

In summary, we report in this article a novel strategy to harness the tissue-protective properties of MC receptors in inflammatory settings. Our data indicate that, in addition to a redefinition of efficacy (quality/quantity), drug selectivity needs to be reconsidered in terms of “receptor” or “pathway” selectivity, given the impact this may have on the development and characterization of novel therapeutics and subsequent medicinal chemistry programs. We also show that ERK1/2 phosphorylation downstream MC1 and MC3 activation leads to proresolving and anti-inflammatory actions, despite absence of cAMP signaling. New knowledge from basic research needs to be better translated into industry and incorporated into the drug-discovery process: despite that the existence of ligand bias has been known for .20 y, it is only now that its relevance is being appreciated. We propose that in the MC receptor discovery arena, biased agonism is a novel approach that can lead to improved therapeutics, perhaps like AP1189 acting on two nonredundant anti-inflammatory targets at once, yet likely devoid of side effects associated with cAMP activation. Disclosures T.E.N.J. is shareholder i

[Uhohinc]

https://news.cision.com/synact/r/synact-pharma-initiates-phase-ii-study-with-ap1189-for-the-treatment-of-ards-in-covid-19-patients,c3201113.............rabbit hole covid...reminds me  how I just saw a truck with those spinner wheel hubs that turn, that was one of the fastest here and gone fads......I was trying to find how AP1189 gets to the actual point of expressing the Erk1/2 pathway.................with the "mechanics of action" given in here the link , I do not think they have a idea how .......

[Claes Svensson]

Wait and see! Melanocortin receptors 1 and 3 are the most important when regulating the inflammatory response. The key is to switch macrophages into M2-phenotype, Mc3r helps alot!

SynAct has good preclinical data in ARDS already, dating 10 years back. The covid19-situation gave a great opportunity to do human trials - SynAct are front runners in inflammation resolution.

Lots of data supporting melanocortins in the treatment of ARDS - central downregulation of the inflammatory response includes the lungs :)

One would think that achieving activation of Mc1R without cAMP and side effects, was a matter of company secrets. They are the only ones that know how. Thomas J is in the top5 on theese receptors!

[Uhohinc]

https://patents.google.com/patent/WO2003013509A1............the patent referenced in above post.

[Uhohinc]

N-phenylpyrrole guanidine derivatives as melanocortin reception ligands

Abstract

The present invention provides novel compounds of the general Formula (I) as ligands to the melanocortin receptors and/or treatment of disorders in the melanocortin system: wherein the variables are as defined in the claims; and the pharmacologically active salts thereof.

Classifications

 C07D207/335 Radicals substituted by nitrogen atoms not forming part of a nitro radical

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WO2003013509A1

WIPO (PCT)

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Other languagesFrenchInventorTorbjörn LundstedtAnna SkottnerArne BomanPer AnderssonElisabeth SeifertVictor Andrianov

Worldwide applications

2001  GB 2002  IL DE EP DK JP BR US WO ES AU CA KR SI NZ MX PT 2004  NO 2006  US 2008  US 2009  JP

Application PCT/GB2002/003620 events 

2001-08-06

Priority to GB0119172.5

2001-08-06

Priority to GBGB0119172.5A

2002-08-06

Application filed by Melacure Therapeutics Ab

2003-02-20

Publication of WO2003013509A1

InfoPatent citations (8) Cited by (44) Legal events Similar documents Priority and Related ApplicationsExternal linksEspacenetGlobal DossierPatentScopeDiscuss

Description

N- HENYL PYRROLE GUANIDINE DERIVATIVES AS MELANOCORTIN RECEPTOR LIGANDS

The present invention relates to phenyl pyrrole aminoguanidines. It further relates to the use of these guanidines for the treatment of obesity, anorexia, inflammation, mental disorders and other diseases associated with the melanocortin receptors or related systems, e.g. the melanocyte stimulating hormones.

A number of large linear and cyclic peptides are known in the art which show high specific binding to melanocortin (MC) receptors. The agonistic and/or antagonistic properties of these peptides are also known. See for example "Melanocortin Receptor ligands and methods of using same" by Dooley, Girten and Houghten (WO99/21571).

A number of low molecular weight compounds are known, e.g. isoquinolines, spiropyridines and benzimidazoles, which show activity on the MC- receptors. See "Isoquinoline compound melanocortin receptor ligands and methods of using same" by Basu et al, Trega Biosciences Inc. (WO 99/55679), "Spiropiperidine derivatives as melanocortin receptor agonists" by Nargung, Ye, Palucki, Bakshi, Patchett and van der Ploeg (WO 99/64002) and "Melanocortin receptor-3 ligands to treat sexual dysfunction" by Dines et al. (WO0105401). See also WO0074679, WO0058361, WO0218327, WO0212166, WO0155106, WO0155107, WO0155109, WO0211715 and WO0212178 for additional compounds acting on the melanocortin receptors.

However, there is still a large need to provide low molecular weight compounds showing agonistic or antagonistic properties to the melanocortin receptors. The compounds of the present invention are structurally different from the above-mentioned compounds and, consequently, constitute a new class of compounds that show activity to the MC-receptors.

A compound previously known in the art, which is similar to the presented compounds, is given below (see e.g. WO98/23267);

This hydroxyguanidine has proven activity against xanthine oxidase/xanthine dehydrogenase enzymes. Therefore it is very surprising that the phenyl pyrrole benzylideneamino guanidine compounds in the present invention show affinity to the melanocortin receptors as agonists and/or antagonists.

One aspect of the present invention is therefore to provide low molecular weight compounds showing activity on melanocortin receptors and which may be taken up after peroral administration and which may penetrate well through the blood brain barrier.

The present invention provides novel compounds of the general formula (I) and their tautomeric forms:

(I)

wherein X is (CH2)„ where n is 0, 1 or 2;

Ri, R2, R3, R4 and R5 may be the same or different and are selected from hydrogen, halogen, alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, hydroxy, alkylsulphonyloxy, cyano, nitro, trihaloalkyl, sulpho or one of the structures given in Scheme 1; and/ or two of R1? R2, R3, R and R5 may together form a methylenedioxy or ethylenedioxy moiety (preferably such methylenedioxy or ethylenedioxy moieties are formed using j and R5 and/ or two of R\, R2 and R3);

Scheme 1

Re, R7, R8 and R are the same or different and are selected from hydrogen, halogen, alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, hydroxy, amines (primary, secondary or tertiary) having 0, 1 or 2 carbon atoms, cyano, nitro, trihaloalkyl, amide or sulpho, and z where shown represents the point of attachment of the residue to the phenyl or pyrrole ring;

and the pharmacologically active salts thereof.

The term halogen includes fluoro, chloro, bromo and iodo.

The term "alkyl" includes straight or branched hydrocarbon chains. The term "alkoxy" includes straight and branched chain alkoxy groups.

Preferably, the "alkyl having 1 to 5 carbon atoms" is a lower alkyl such as methyl, ethyl, propyl, isopropyl or tert-butyl.

Preferably, the "alkoxy having 1 to 5 carbon atoms" is a lower alkoxy such as methoxy, ethoxy, propoxy, iso-propoxy or tert-butoxy. The term trihaloalkyl includes straight or branched hydrocarbon chains, preferably having 1 to 5 carbon atoms, and includes trichloroalkyl and trifluoroalkyl.

Preferably, the trihaloalkyl is trihalomethyl, trihaloethyl, trihalopropyl or trihaloiso-propyl.

Furthermore, it should be noted that the Scheme 1 residues may be attached to the carbon backbone of the compound of general formula (I) at any suitable point within the compound of Scheme 1, preferably at the 1, 2 or 3 position.

The compounds of formula (I) have basic properties and, consequently, they may be converted to their therapeutically active acid addition salts by treatment with appropriate physiologically acceptable acids, e.g. inorganic acids such as hydrochloric, hydrobromic, hydriodic, sulphuric, nitric and phosphoric acid, or organic acids such as acetic, propanoic, glycolic, lactic, malonic, succinic, fumaric, tartaric, citric, pamoic, oxalic and /?αra-toluene- sulphonic acid.

Conversely, the salt form may be converted into the free base form by treatment with alkali.

The present invention relates to novel aromatic amines. Compounds of the present invention have been biologically tested in the melanocortin system and have suφrisingly been shown to be capable of binding to melanocortin receptors as well as showing activity in functional assays.

Compounds of the present invention are either agonists or antagonists of a specific MC- receptor or of a number of MC-receptors, e.g. MCI, MC3, MC4 or/and MC5 receptors.

The MC-receptors belong to the class of G-protein coupled receptors which are all built from a single polypeptide forming 7 transmembrane domains. Five such receptors types, termed MCI, MC2, MC3, MC4 and MC5, have been described. The MC receptor's signaling is mainly mediated via cAMP but also other signal transduction pathways are known. They are distinctly distributed in the body. MC-receptors are linked to a variety of physiological actions that are thought to be mediated by distinct subtypes of the MC-receptors. In many cases, however, it is not entirely clear which of the subtypes is responsible for the effect.

It has long been known that MSH-peptides may affect many different processes such as motivation, learning, memory, behaviour (including feeding and sexual), inflammation (including immunostimulatory and immunosuppressive), body temperature, pain perception, blood pressure, heart rate, vascular tone, brain blood flow, trophic effects in different organs, nerve growth, placental development, endocrine and exocrine functions, aldosterone synthesis and release, thyroxin release, spermatogenesis, ovarian weight, prolactin and FSH secretion, effects or other hormones, uterine bleeding in women, sebum and pheromone secretion, blood glucose levels, natriuresis, intrauterine foetal growth, as well as other events surrounding parturition (Eberle, AN: The melanotropins: Chemistry, physiology and mechanisms of action. Basel: Karger, Switzerland. 1988, ISBN 3-8055-4678-5; Gruber, and Callahan, Am. J. Physiol. 1989, 257, R681-R694; De Wildt et al, J. Cardiovascular Pharmacology. 1995, 25, 898-905), as well as inducing natriuresis (Lin et al, Hypertension. 1987, 10, 619-627).

It is also well-known that the immunomodulatory action of α-MSH includes both immunostimulatory and immunosuppressive effects. Several studies have shown that α-MSH antagonizes the effects of pro-inflammatory cytokines such as IL-lα, IL-lβ, IL-6 and TNFα, and induces the production of the anti-inflammatory cytokine, IL-10 (for review see Catania & Lipton, 1993).

Eating behaviour is regulated by a complex network of physiological regulatory pathways that involve both the central nervous system and peripheral sites. Factors such as leptin, insulin, NPY (neuropeptide Y), orexins, CRF (Corticotropin-Releasing Factor, release hormone) and melanocortic peptides (Schwartz; Nature Medicine 1998, 4, 385-386) are known to control the amount of food intake both during short and long term, which may affect body weight, body fat mass and growth rate. Recent studies have shown a role of MC-receptors, especially the MC4 receptor, for control of food intake, and there is evidence indicating that the melanocortins and the MC4 receptor are important factors downstream of leptin. Intracerebro ventricular injections of the melanocortic peptides α-MSH and ACTH(l-24) have been shown to markedly inhibit feeding (Poggioli et al, Peptides, 1986, 7, 843-848; Nergoni et α/., Νeuropeptides, 1986, 7, 153-158).

The MC5-receptor has recently been attributed a role in control of exocrine gland function (van der Kraan, et al, Endocrinol. 1998, 139, 2348-2355; Chen et al, Cell. 1997, 91, 789- 798).

In addition, the melanocortic peptides have distinct effects on sexual functions in that they cause erection in males (Donovan, Psychol. Med. 1978, 8, 305-316), presumably mediated by a central agonistic effect of the peptide on MC-receptors. It has also been shown that a MC- receptor blocker could inhibit the erectogenic effect of melanocortic peptides (Vergoni et al, Eur. J. Pharmacol, 1998, 362; 95-101).

Compounds of formula (I) and/or their pharmaceutically acceptable salts have valuable pharmacological properties, making them useful for the treatment of mental disorders such as psychoses, depression, anxiety, senile dementia, Alzheimer's disease, drug abuse disorders and eating disorders such as anorexia and bulimia.

Compounds of formula (I) and/or their pharmaceutically acceptable salts have valuable pharmacological properties, making them useful for the treatment of dysfunctions of the endocrine system and other hormonal systems such as excessive menstruations, endometriosis, events related to parturition, dysfunctions related to prolactin, dysfunctions related to growth hormone, dysfunctions related to testosterone, dysfunctions related to estrogen, dysfunctions related to glucocorticoids, dysfunctions related to luteinizing hormone and follicle stimulating hormone, inducing abortion, for prevention of abortion and/or for treatment of events related to parturition.

Compounds of formula (I) and/or their pharmaceutically acceptable salts have valuable pharmacological properties, making them useful for the treatment of sexual functions / dysfunctions such as inducing erection in man, to induce erection in animal breeding, to stimulate intercourse in animals which are difficult to mate, in particular rare species or valuable strains, pets, cats, dogs, horses or to reduce sexual behaviour in animals, e.g. for pets, cats etc., to treat impotence and disorders related to sexual drive, including lack of sexual drive or abnormal sexual drive in both men and women.

Compounds of formula (I) and/or their pharmaceutically acceptable salts have valuable pharmacological properties, making them useful for the treatment of inflammation such as inflammations related to the production of nitric oxide, inflammation related to increased amounts (upregulated amounts) of inducible nitric oxide synthase, inflammation related to activation of transcriptional activators, inflammation related to nuclear factor kappa beta, inflammation related to macrophages, neutrophils, monocytes, keratinocytes, fibroblasts, melanocytes, pigment cells and endothelial cells, inflammation related to increased production and/or release of inflammatory cytokines, such as e.g. interleukins, in particular interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor a (TNF-α).

In the present specification, "increased production" refers to increased formation, increased release, or increased amount of an endogenous compound locally, regionally or systemically in a patient compared to the amount of said endogenous compound in a healthy individual. In the present specification, "upregulated" refers to an increased activity or amount of the compound compared with that in a healthy individual.

In the present specification, "decreased production" refers to decreased formation, decreased release, or decreased amount of an endogenous compound in a patient compared to the amount of said endogenous compound in a healthy individual. In the present specification, "downregulated" refers to a decreased activity or amount of the compound compared with that in a healthy individual.

In particular, positive treatment effects or preventive effects may be seen in conditions where inflammation or an inflammatory-like condition is caused by or being associated with one or more of the following: allergy, hypersensitivity, bacterial infection, viral infection, inflammation caused by toxic agent, fever, autoimmune disease, radiation damage by any source including UN-radiation, X-ray radiation, γ-radiation, α- or β-particles, sun burns, elevated temperature or mechanical injury. Moreover, inflammation due to hypoxia, which is optionally followed by reoxygenation of the hypoxic area, is typically followed by severe inflammation, which condition may be positively affected by treatment with a compound of the invention.

In very specific embodiments of the invention, a compound of the invention may be administered for the prevention or therapeutic treatment of inflammatory diseases of the skin (including the dermis and epidermis) of any origin, including skin diseases having an inflammatory component. Specific examples of this embodiment of the invention include treatment of contact dermatitis of the skin, sunburns of the skin, burns of any cause, and inflammation of the skin caused by chemical agents, psoriasis, vasculitis, pyoderma gangrenosum, discoid lupus erythematosus, eczema, pustulosis palmo-plantaris, and phemphigus vulgaris.

Also comprised by the invention is the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of an inflammatory disease in the abdomen, including an abdominal disease having an inflammatory component. Specific examples of the treatment of such a disease with a compound of the invention are gastritis, including one of unknown origin, gastritis perniciosa (atrophic gastritis), ulcerous colitis (colitis ulcerosa), morbus Crohn, systemic sclerosis, ulcus duodeni, coeliac disease, oesophagitis and ulcus ventriculi.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of systemic or general and/or local immunological diseases, including those of an autoimmune nature, and other inflammatory diseases of a general nature. Specific examples include treatment of rheumatoid arthritis, psoriatic arthritis, systemic sclerosis, polymyalgia rheumatica, Wegener' s granulomatosis, sarcoidosis, eosinophilic fasceitis, reactive arthritis, Bechterew's disease, systemic lupus erythematosus, arteritis temporalis, Behcet's disease, morbus Burger, Good Pastures' syndrome, eosinophilic granuloma, fibromyalgia, myositis, and mixed connective tissue disease. Included therein is also arthritis, including arthritis of unknown origin.

Further included in the invention is administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of a disease of the peripheral and/or central nervous system related to inflammation. Included in this aspect of the invention is the treatment of cerebral vasculitis, multiple sclerosis, autoimmune ophthalmitis and polyneuropathia. Comprised by the invention is also the administration of a compound of the invention for the treatment of an inflammation of the central nervous system to prevent apoptotic cell death. Moreover, as some of the compounds of the invention show a distinct ability to induce nerve regeneration, positive treatment effects are often seen in central nervous system diseases involving damage of cells in this region. This aspect of the invention also includes treatment of traumatic injuries to the central nervous system, brain edema, multiple sclerosis, Alzheimer's disease, bacterial and viral infections in the central nervous system, stroke, and haemorrhagia in the central nervous system.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases of the eye and tear glands related to inflammation. Specific examples of such diseases comprise anterior and posterior uveitis, retinal vasculitis, optic neuritis, optic neuromyelitis, Wegener's granulomatosis, Sjδgren's syndrome, episcleritis, scleritis, sarcoidosis affecting the eye and polychondritis affecting the eye.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases of the ear related to inflammation, specific examples of which include polychondritis affecting the ear and external otitis.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases of the nose related to inflammation, specific examples of which are sarcoidosis, polychondritis and mid-line granuloma of the nose.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to inflammation of the mouth, pharynx and salivary glands. Specific examples include Wegener's granulomatosis, mid-line granuloma, Sjogren's syndrome and polychondritis in these areas. Included in the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to inflammation in the lung. Specific examples include treatment of idiopathic alveolitis, primary pulmonary hypertension, bronchitis, chronic bronchitis, sarcoidosis, alveolitis in inflammatory systemic disease, pulmonary hypertension in inflammatory systemic disease, Wegener's granulomatosis and Good Pastures' syndrome.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to the inflammation of the heart. Specific examples include treatment of pericarditis, idiopathic pericarditis, myocarditis, Takayasus' arteritis, Kawasaki's disease, coronary artery vasculitis, pericarditis in inflammatory systemic disease, myocarditis in inflammatory systemic disease, endocarditis and endocarditis in inflammatory systemic disease.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to inflammation of the liver. Specific examples include treatment of hepatitis, chronic active hepatitis, biliary cirrhosis, hepatic damage by toxic agents, interferon induced hepatitis, hepatitis induced by viral infection, liver damage induced by anoxia and liver damage caused by mechanical trauma.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to inflammation of the pancreas. Specific examples include treatment (and prevention) of diabetes mellitus, acute pancreatitis and chronic pancreatitis.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to the inflammation of the thyroidea. Specific examples of these embodiments of the invention include treatment of thyreoiditis, autoimmune thyreoiditis and Hashimoto's thyreoiditis.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to inflammation of the kidney. Specific examples include treatment of glomerulonephritis, glomerulonephritis in systemic lupus erythematosus, periarteritis nodosa, Wegener's granulomatosis, Good- Pastures' syndrome, HLAb27 associated diseases, IgA nephritis (IgA = Immunoglobulin A), pyelonephritis, chronic pyelonephritis and interstitial nephritis.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to the inflammation of the joints. Specific examples include treatment of Bechterew's disease, psoriatic arthritis, rheumatoid arthritis, arthritis in colitis ulcerosa, arthritis in morbus Crohn, affection of joints in systemic lupus erythematosus, systemic sclerosis, mixed connective tissue disease, reactive arthritis, Reiter's syndrome. Moreover, included in this embodiment of the invention is treatment of arthrosis of any joint, in particular arthrosis of finger joints, the knee and the hip.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of diseases related to the inflammation of blood vessels. Specific examples include treatment of arteritis temporalis, periarteritis nodosa, arteriosclerosis, Takayasus' arteritis and Kawasaki's disease. Particularly advantageous is the capacity of some compounds of the invention to afford protection against and prevention of arteriosclerosis. This is in part due to the capacity of some compounds of Formula (I) or the pharmacologically acceptable salts thereof to prevent the induction of inducible nitric oxide synthesis (iNOS) caused by the action of oxidized Low Density Lipoprotein on endothelial cells and blood vessel walls.

Comprised by the invention is also the administration of a compound of the invention for the treatment of drug-induced disorders of the blood and lymphoid system, including the treatment of drug-induced hypersensitivity (including drug hypersensitivity) affecting blood cells and blood cell forming organs (e.g. bone marrow and lymphoid tissue). Specific embodiments of this aspect of the invention include the treatment of anemia, granulocytopenia, thrombocytopenia, leukopenia, aplastic anemia, autoimmune hemolytic anemia, autoimmune thrombocytopenia and autoimmune granulocytopenia. The compounds of the invention may also be administered for the treatment of fast allergic disorders (Type I allergy). Included in this embodiment of the invention is the treatment of anaphylactic reactions, anaphylactoid reactions, asthma, asthma of allergic type, asthma of unknown origin, rhinitis, hay fever and pollen allergy.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of inflammation related to infections of any origin. Specific examples include treatment of inflammation secondary to infection caused by virus, bacteria, helminths and protozoae.

Comprised by the invention is also the administration of a compound of formula (I) or a pharmacologically acceptable salt thereof for the treatment of inflammations related to trauma and/or tissue injury of any origin.

Compounds of formula (I) or pharmaceutically acceptable salts thereof have valuable pharmacological properties, making them useful for the treatment of disorders of the cardiovascular system such as disorders related to blood pressure, heart rate, vascular tone, natriuresis, bleeding, shock, disorders related to ischemia, infarction, repercussion injuries, arrhythmias of the heart, in particular during ischemia, or for the treatment of arrhythmias associated with reoxygenation of a previously ischemic period of the heart.

Compounds of formula (I) or the pharmaceutically acceptable salts thereof have valuable pharmacological properties, making them useful for the treatment of pain such as pain of central origin, pain seen after damage to the CNS, stroke, infarction, pain of peripheral origin, chronic pain, neuropathies and disorders where a treatment effect is achieved by stimulation of receptors in the periaqueductal grey area.

Because of the capacity of compounds of the invention to stimulate pigment formation in epidermal cells, some of the compounds of the invention may be also useful for inducing skin tanning for cosmetic reasons, for treatment of vitiligo, or any other condition where darkening of skin color is desired. Moreover, because of the ability of some of the compounds of the invention to inhibit pigment formation in cells of the skin, they may also be useful for inducing lighter skin color for cosmetic reasons, or during any condition where a lighter color of skin is desired.

Compounds of formula (I) or the pharmaceutically acceptable salts thereof have valuable pharmacological properties, making them useful to cause skin tanning, darkening the colour of the skin, to induce melanin synthesis in the skin, to reduce skin tanning, lightening the colour of the skin, to reduce or block melanin synthesis in the skin, to cause anti- inflammatory actions in the skin, to modulate epidermal growth, to improve wound healing, to treat acne, seborrhoea, acne roseacea, conditions related to malfunctions of the glands of the skin, e.g. sebacous glands and over or underproduction of sebum.

Compounds of the invention are useful for inhibiting or stimulating the in vivo formation of second messenger elements such as cAMP. Such inhibition stimulation may be used in cells or crushed cell systems in vitro, e.g. for analytical or diagnostic purposes.

For analytical and diagnostic purposes the compounds of the invention may be used in radioactive form where they comprise one or more radioactive labels or gamma or positron emitting isotopes, to be used in radioligand binding for the quantification as well as tissue localisation of MC-receptors, for analysis of dissociation/association constants, and for imaging of in vivo binding by the use of scintigraphy, positron emission tomography (PET) or single photon emission computed tomography (SPECT), or for the diagnosis of disease and treatment of any malignancy where the malignant cells contain MC receptors.

Alternatively the compounds of the invention can be labelled with any other type of label that allows detection of the respective compound, e.g. fluorescence, biotin, NMR, MRI, or labels activated by gamma-irradiation, light photons or biochemical processes, or by light or UN- light (the latter in order to obtain a compound useful for covalent labelling of MC receptors by a photoaffmity technique).

Compounds of formula (I) or the pharmacologically acceptable salts thereof may also be tagged with a toxic agent (i.e. doxorubicin, ricin, diphtheria toxin or other) and used for targeted delivery to malignant cells bearing MC receptors, or tagged with a compound capable of activating the endogenous immune system for triggering the immune system (for example a compound, monoclonal antibody or other, capable of binding to a T-cell antigen, e.g. CD3 or other) for treatment of malignancies and other MC receptor expressing diseases. The thus formed hybrid compound will direct cytotoxic cells to the malignant melanoma cells or the MCI -receptor bearing malignant cells and inhibit the tumor growth.

Compounds of formula (I) or a pharmacologically acceptable salt thereof may be attached to the antibody chemically by covalent or non-covalent bond(s).

Compounds of the invention may be used for the treatment and diagnosis of diseases, disorders and/or pathological conditions in an animal, in particular in man.

The present invention also relates to a pro-drug which, upon administration to an animal or a human, is converted to a compound of the invention. Pro-drugs of the compounds of Formula (I) and their pharmacologically acceptable salts may be used for the same purposes as described in this specification for the compounds of the invention, as well as is disclosed in the Examples given below.

The compounds of the present invention may be bound covalently or non-covalently to one or several of other molecule(s) of any desired structure(s); the thus formed modified compound or complex may be used for the same purposes as described in this specification for the compounds of the invention, as well as is disclosed in the Examples given below. In a particularly important embodiment of the invention, a radioactively-labelled molecule is covalently bound to a compound of Formula (I) or a pharmacologically acceptable salt thereof so as to make a compound of Formula (I) or a pharmacologically acceptable salt thereof radioactively labelled.

Some of the compounds of the invention have an effect on xanthine oxidase in mammals, including humans.

The invention also relates to processes for the manufacture of and pharmaceutical preparations comprising one or more of the compounds of the invention, as well as to their uses for various medical and veterinary practices related to melanocyte stimulating hormone receptors. Compounds of the general formula II are either commercially available or can be synthesised by methods well known in the art, see for example "Advanced Organic Chemistry", by Jerry March or "Organic Synthesis " by Michael B. Smith.

METHODS OF PREPARATION

We further provide a process for the preparation of a compound of formula (I) as defined in above, in which a compound of formula (II) wherein X, Rl5 R2, R3, R4 and R5 are as previously defined, is reacted with an aminoguanidine (III), or a salt or protected form thereof, using procedures known per se in the art, and, following deprotection if necessary or desired, a compound of formula (I) is obtained.

(II) (III)

The following examples are intended to illustrate but not to limit the scope of the invention, although the compounds named are of particular interest for the intended purposes. The preparation of the compounds of general formula (I) is presented schematically in Example 1 below. Specific synthetic procedures are given in methods 1-3. The compounds are numbered and listed with their complete name below.

EXAMPLES

Example 1

The compounds of the invention may be prepared by the following general method. IR, NMR, MS and elementary analysis have confirmed the structures of the compounds. When melting points (m.p.) are given, these are uncorrected.

Example 1

N-[l-((4-Chlorophenyl)-lH-Pyrrol-2-yl)methyleneamino]guanidine

5 g (24.3 mmol) l-(4-chlorophenyl)-pyrrol-2-carboxaldehyde and 4.96 g (36.5 mmol) aminoguanidine bicarbonate was suspended in 500 ml acetonitrile. 120 ml acetic acid was added and the reaction mixture was refluxed for 2h. The solution was cooled and the solvent was evaporated. The resulting crude product was dissolved in ether and crystallised after four hours in the freezer. The slightly yellow solid was recrystallised from acetonitrile/ethanol (5:1) to give the pure product (1) as white crystals. Yield 3 g (45%).

The novel compounds 2-50 were prepared in an analogous manner:

Compounds 1-50

No. Name 1 N- { 1 - [(4-Chlorophenyl)- 1 H-Pyrrol-2-yl]methyleneamino } guanidine

_ N- [ 1 -(5-propylamino- 1 -(2-butoxy-pheny 1)- 1 H-pyrrol-2-yl)- methyleneamino] -guanidine

_ N- { 3 - [ 1 -(2-Isobutyryl-phenyl)-5 -nitro- 1 H-pyrrol-2-yl] - propy lideneamino } -guanidine

. N-{ l-[l-(3-Cyanophenyl)-5-trifluoromethyl-lH-pyrrol-2-yl]- methyleneamino } -guanidine

N-{3-[l-(3-Fluorophenyl)-5-methyl-lH-pyrrol-3-yl]- propy lideneamino } -guanidine

, N- [ 1 -(4- Aminophenyl)-5 -hydroxy- 1 H-pyrrol-2- ylmethylideneaminoj-guanidine

N-{3-[l-(4-Chlorophenyl)-5-phenethyl-lH-pyrrol-3-yl]- propylideneamino } -guard dine

N- {3 -[ 1 -(4-Propylaminophenyl)-5-trichloromethyl- 1 H-pyrrol-3 -yl]- propy lideneamino } -guanidine

N-(5-tert-Butyl- 1 -phenyl- 1 H-pyrrol-2-ylmethylideneamino)- guanidine

N-[3-(5-Methoxy-l-phenyl-lH-pyrrol-3-yl)-ρropylideneamino]- guanidine

. N- [4-Pentyl- 1 -(2-trichloromethylphenyl)- 1 H-pyrrol-3 - ylmethylideneaminoj-guanidine 12 N-(4-Cyano- 1 -o-tolyl- 1 H-pyrrol-3-ylmethylideneamino)-guanidine

1 _ N- { 3 - [4-Hydroxy- 1 -(3 -trichloromethylphenyl)- 1 H-pyrrol-2-yl] - propy lideneamino } -guanidine

. N-{3-[l-(3-tert-Butylphenyl)-5-isobutyryl-lH-pyrrol-2-yl]- propy lideneamino} -guanidine

N-(l-Biphenyl-4-yl-4-chloro-lH-pyrrol-2-ylmethylideneamino)-

15 guanidine

16 N- { 3 - [ 1 -(4-Bromophenyl)-4-tert-butyl- 1 H-pyrrol-3 -yl]- propy lideneamino } -guanidine

17 N-(4-Butoxy- 1 -phenyl- 1 H-pyrrol-3 -ylmethylideneamino)-guanidine ι o N- [3 -(4-Methoxy- 1 -phenyl- 1 H-pyrrol-2-y l)-propy lideneamino] - guanidine

1 Q N- { 3 - [ 1 -(2-Nitropheny 1)- 1 H-pyrrol-2-yl] -propylideneamino } - guanidine „ N-{3-[l -(2-Hydroxypheny 1)- 1 H-pyrrol-2-yl] -propylideneamino } - guanidine

N- [ 1 -(3 -Methoxypheny 1)- 1 H-pyrrol-2-ylmethy lideneamino] -

21 guanidine

N- [ 1 -(3 -Butylaminopheny 1)- 1 H-pyrrol-3 -y lmethy lideneamino] -

22 guanidine

? ~ N- [ 1 -(4-tert-Butylphenyl)- 1 H-pyrrol-3 -ylmefhy lideneamino] - guanidine

9 . N- { 3 - [ 1 -(4-Trifluoromethy lphenyl)- 1 H-pyrrol-2-yl]- propylideneamino} -guanidine

25 N-( 1 -Phenyl- 1 H-pyrrol-3 -ylmethylideneamino)-guanidine

26 N- [3 -( 1 -Phenyl- 1 H-pyrrol-2-yl)-propy lideneamino] -guanidine

N- [3 -(4-Methy 1- 1 -phenyl- 1 H-pyrrol-3 -yl)-propy lideneamino] -

29 guanidine

N- [ 1 -(3 -Nitro-4-propylaminophenyl)- 1 H-pyrrol-3 - 30 ylmethylideneaminoj-guanidine

N-{3-[l-(3-Butylamino-4-methylphenyl)-4-methyl-lH-pyrrol-2-yl]- 31 propylidene} -guanidine

N- { 3 -[5-Bromo- 1 -(4-bromophenyl)- 1 H-pyrrol-3 -yl]-

32 propylideneamino } -guanidine

N-[(5-Chloro- 1 -(4-isobutyrylphenyl)-4-pentyl- 1 H-pyrrol-2- 33 yl)methyleneamino] -guanidine

N-[3-(5-Propoxy-l-m-tolyl-lH-pyrrol-2-yl)-propylideneamino]- 34 guanidine

N-[4-Methyl-5-propylamino- 1 -(3 -trichloromethylphenyl)- 1 H-pyrrol- 35 3 -y lmethy lideneamino] -guanidine

„ , N-{3-[l-(3-Nitrobiphenyl-4-yl)-lH-pyrrol-2-yl]-propylideneamino}- guanidine

_ „ N- {2- [4-tert-Butyl- 1 -(4-methoxy-2-propy laminophenyl)- 1 H-pyrrol- 3 -y l]-ethy lideneamino } -guanidine N- { 3 - [ 1 -(2-Bromo-3 -chloropheny 1)- 1 H-pyrrol-3 -yl] -

38 propylideneamino } -guanidine

N- [4-Bromo- 1 -(2-butoxy-3 -propoxypheny 1)- 1 H-pyrrol-2- 39 ylmethylideneamino]-guanidine

N- [5 -Hydroxy- 1 -(2-hydroxypheny 1)- 1 H-pyrrol-3 - 40 ylmethylideneaminoj-guanidine

N- { 3 - [ 1 -(2-tert-Butylphenyl)-4-chloro-5 -methyl- 1 H-pyrrol-2-yl] - 41 propylideneamino } -guanidine

N- [5 -Isobutyryl- 1 -(2,3 ,4-trimethoxypheny 1)- 1 H-pyrrol-2- 42 y lmethy lideneamino] -guanidine

N-{3-[5-tert-Butyl-l-(2-tert-butyl-3,4-bis-trichloromethylphenyl)-4- 43 trichloromethyl- 1 H-pyrrol-2-yl] -propylideneamino } -guanidine

N- { 2- [ 1 -(4-Pyrrol- 1 -y lphenyl)- 1 H-pyrrol-2-y 1] -ethy lideneamino } - 44 guanidine

. . N- [ 1 -(4-Morpholin-4-yl-phenyI)- 1 H-pyrrol-2-ylmethy lideneamino] - guanidine

N- [1 -(4-Pyrrolidin- 1 -yl-phenyl)- 1 H-pyrrol-3 -ylmethylidenamino]-

46 guanidine

47 N-((l-Phenyl-lH-pyrrol-2-yl)methyleneamino)guanidine

48 N-[(l-(4-Trifluoromethylphenyl)-lH-pyrrol-2- yl)methyleneamino]guanidine

49 N- [( 1 -(3 -cyanophenyl)- 1 H-pyrrol-2-y l)methyleneamino] guanidine

„ N- [( 1 -(3 ,5 -dichloropheny 1)- 1 H-pyrrol-2- yl)methyleneamino] guanidine

Test 1. Affinity for the MCl-receptor

The binding assay was carried out essentially as described by Lunec et al, Melanoma Res 1992; 2; 5-12 using I125-NDP-αMSH as ligand.

Test 2. Affinity for the MC3-receptors, the MC4-receptors and the MC5-receptors

The binding assays were carried out essentially as described by Szardenings et al, J Biol Chem 1997; 272; 27943-27948 and Schioth et al, FEBS Lett 1997; 410; 223-228 using 1125- NDP-αMSH as ligand.

Test 3. cAMP The stimulation of cAMP was carried out essentially as described by Schioth et al, Br J Pharmacol 1998; 124; 75-82, however, the response is given relative to α-MSH. Table la. Affinity for MC-receptors

Table lb. Influence on cAMP

Example 2

In vivo effects on food intake

Compounds have been tested for their effects on food intake and body weight in rats. In order to investigate the agonistic effect, ie decrease in food intake, of compounds, the nocturnal protocol was used.

Sprague-Dawley, male rats were used, which were cannulated intracerebroventricularly. Stainless steel guide cannulae were placed in the lateral ventricle and fixed in the skull. Animals were acclimatized for a week before the experiments took place. After the experiments were done, the rats were killed and placement of the cannulae were checked.

Nocturnal protocol:

Rats were cannulated as described above. They were used without prior starvation, and compounds were administered at 5 pm in a total volume of 5ml. Doses of compound 2:4 used were 1, 4 and lOnmoles. Food intake was measured at 3, 15 and 24 hours after dosing, and body weight was recorded at 24 hours. For comparison, the well known MC4 receptor agonist, Melanotan II (MTU) was used, at a dose of 1 nmole.

Example 3

Anti inflammatory effects

Control

Female BALB/c mice (weight 20-22 g) were sensitized by treatment of the shaved abdomen with 30 μl of 0.5% 2,4-dinitrofluorobenzene (DNFB). After 4 days they were challenged with 10 μl of 0.3 % DNFB to the paw. The unchallenged mice paws served as a control. Twenty- four hours after the last challenge, the difference in paws weight were determined as an indicator of the inflammation (paw edema).

alpha-MSH and prednisolone controls

Mice were treated as the control but were additionally injected i.p. with α-MSH (0.5 mg/kg) or prednisolone (20 mg/kg) two hours before sensitization (day 0) and the same dose was administered repeatedly after sensitization during four consecutive days. The paw edema inhibition was measured as described above.

Study of new compounds

Mice were treated as the control but were additionally injected i.p. with various doses (0.05, 0.15 or 0.25, 0.375, 0.5 and 0.75 mg/kg) of each compounds two hours before sensitization (day 0) and the same dose was administered repeatedly after sensitization during four consecutive days. The paw edema inhibition as described above.

Groups containing at least 10 mice each were used for all experiments.

Example 4

The following formulations are representative for all of the pharmacologically active compounds of the invention Example of a preparation comprising a capsule

Per capsule

Active ingredient, as salt 5 mg

Lactose 250 mg

Starch 120 mg

Magnesium stearate 5 mg

Total 380 mg

In case of higher amounts of active ingredient, the amount of lactose used may be reduced.

Example of a suitable tablet formulation.

Per tablet

Active ingredient, as salt 5 mg

Potato starch 90 mg

Colloidal Silica 10 mg

Talc 20 mg

Magnesium stearate 2 mg

5 % aqueous solution of gelatine 25 mg

Total 152 mg

Claims

Hide Dependent 

Claims

A compound of general formula (I), and tautomeric forms thereof

(I)

wherein X is (CH2)„ where n is 0, 1 or 2;

R\, R2, R3, R and R5 may be the same or different and are selected from hydrogen, halogen, alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, hydroxy, alkylsulphonyloxy, cyano, nitro, trihaloalkyl, sulpho and any of the following structures:

and/ or two of Ri, R2, R3, R4 and R5 may together form a methylenedioxy or ethylenedioxy moiety;

R6, R7, R8 and R9 are the same or different and are selected from hydrogen, halogen, alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, hydroxy, amines (primary, secondary or tertiary) having 0, 1 or 2 carbon atoms, cyano, nitro, trihaloalkyl, amide or sulpho, and z where shown represents the point of attachment of the residue to the phenyl or pyrrole ring; or a pharmacologically active salt thereof.

2. A compound as claimed in claim 1 wherein at least two of the substituents Ri, 5 R2, R3, R4 and R5 are hydrogen.

3. A compound as claimed in any of the previous claims wherein alkyl is selected from methyl, ethyl, n-butyl, n-pentyl or tert-butyl.

10 4. A compound as claimed in any of the previous claims wherein alkoxy is selected from methoxy, ethoxy, propyloxy, butoxy or tert-butoxy.

5. A compound as claimed in any of the previous claims wherein R6, R and R8 are hydrogen.

15

6. A compound as claimed in any of the previous claims wherein halogen is selected from fluoro, chloro or bromo.

7. A compound as claimed in any of the previous claims wherein n=0. 20

8. A compound as claimed in any of the previous claims wherein R5 is hydrogen.

9. A compound as claimed in any of the previous claims wherein R3 is hydrogen.

25 10. A compound as claimed in any of the previous claims wherein Rj is hydrogen.

11. A compound as in claimed any of the previous claims wherein the -X-CH=N-

NH-C(=NH)-NH2 moiety is in the 2 position of the pyrrole ring.

30 12. A compound as claimed in any of the previous claims wherein R] and R2 are positioned in the 3, 4 or 5 position in the phenyl ring.

13. A compound as claimed in any of the previous claims wherein R2 is hydrogen.

14. A compound as claimed in any of the previous claims wherein R\ is in the 4 position in the phenyl ring.

15. A compound as claimed in any of the previous claims wherein R is selected from alkyl, alkoxy, halogen, hydrogen, amino or trihaloalkyl.

16. A compound as claimed in claim 15 wherein R] is selected from halogen, hydrogen, trihaloalkyl, or moφholine.

17. A compound as claimed in claim 16 wherein halogen preferably is selected from chloro or bromo.

18. A compound as claimed in claim 16 wherein halogen most preferably is chloro.

19. A compound as claimed in claim 16 wherein trihaloalkyl is selected from trifluoromethyl or trichloromethyl.

20. A compound as claimed in claim 16 wherein trihaloalkyl most preferably is trifluoromethyl.

21. A compound as claimed in claim 1 having one of the following formulae:

No. Name

1 N-{ l-[(4-Chlorophenyl)-lH-Pyrrol-2-yl]methyleneamino}guanidine

N- [ 1 -(5 -propylamino- 1 -(2-butoxyphenyl)- 1 H-pyrrol-2-yl)-

2 methy leneamino] -guanidine

N- { 3 - [ 1 -(2-Isobutyrylpheny l)-5 -nitro- 1 H-pyrrol-2-yl] - 3 propylideneamino } -guanidine

N-{l-[l-(3-Cyanophenyl)-5-trifluoromefhyl-lH-pyrrol-2-yl]- 4 methy leneamino } -guanidine

N-{3-[l-(3-Fluorophenyl)-5-methyl-lH-pyrrol-3-yl]- 5 propy lideneamino } -guanidine . N-[l-(4-Aminophenyl)-5-hydroxy-lH-pyrrol-2- ylmethy lideneamino] -guanidine

N-{3-[l-(4-Chlorophenyl)-5-phenethyl-lH-pyrrol-3-yl]-

7 propy lideneamino } -guanidine

0 N- { 3 - [ 1 -(4-Propylaminophenyl)-5 -trichloromethyl- 1 H-pyrrol-3 -y 1]- propy lideneamino } -guanidine

„ N-(5 -tert-Butyl- 1 -phenyl- 1 H-pyrrol-2-ylmethylideneamino)- guanidine

1 π N-[3-(5-Methoxy-l-phenyl-lH-pyrrol-3-yl)-propylideneamino]- guanidine N- [4-Pentyl- 1 -(2-trichloromethylphenyl)- 1 H-pyrrol-3 -

11 y lmethy lideneamino] -guanidine

12 N-(4-Cyano- 1 -o-tolyl- 1 H-pyrrol-3 -ylmethylideneamino)-guanidine

N- { 3 -[4-Hy droxy- 1 -(3 -trichloromethylphenyl)- 1 H-pyrrol-2-yl]-

13 propy lideneamino } -guanidine

1 . N-{3-[l-(3-tert-Butylphenyl)-5-isobutyryl-lH-pyrrol-2-yl]- propy lideneamino } -guanidine

N-(l-Biphenyl-4-yl-4-chloro-lH-pyrrol-2-ylmethylideneamino)-

15 guanidine

N-{3-[l-(4-Bromophenyl)-4-tert-butyl-lH-pyrrol-3-yl]-

16 propy lideneamino } -guanidine

1177 N-(4-Butoxy- 1 -phenyl- 1 H-pyrrol-3 -ylmethylideneamino)-guanidine N- [3 -(4-Methoxy- 1 -phenyl- 1 H-pyrrol-2-yl)-propy lideneamino] - 18 guanidine

1 q N-{3-[l-(2-Nitrophenyl)-lH-pyrrol-2-yl]-propylideneamino}- guanidine

N- { 3 - [ 1 -(2-Hydroxyphenyl)- 1 H-pyrrol-2-yl] -propylideneamino } - 0 guanidine

N- [ 1 -(3 -Methoxyphenyl)- 1 H-pyrrol-2-y lmethy lideneamino] - 1 guanidine

N- [ 1 -(3 -Butylaminophenyl)-l H-pyrrol-3 -ylmethylideneamino]- 2 guanidine

N- [ 1 -(4-tert-Butylphenyl)- 1 H-pyrrol-3 -ylmethylideneamino] - 3 guanidine

N- { 3 - [ 1 -(4-Trifluoromethy lphenyl)- 1 H-pyrrol-2-yl] - 4 propylideneamino } -guanidine 5 N-( 1 -Phenyl- 1 H-pyrrol-3 -y lmethy lideneamino)-guanidine 6 N- [3 -( 1 -Phenyl- 1 H-pyrrol-2-yl)-propy lideneamino] -guanidine N- [3 -(4-Methyl- 1 -phenyl- 1 H-pyrrol-3 -yl)-propy lideneamino] - 9 guanidine

N-[l -(3-Nitro-4-propylaminophenyl)- 1 H-pyrrol-3- 30 ylmethy lideneamino] -guanidine

N- { 3 - [ 1 -(3 -Butylamino-4-methyl-phenyl)-4-methyl- 1 H-pyrrol-2-yl]- 31 propy lidene } -guanidine

N- { 3 - [5 -Bromo- 1 -(4-bromophenyl)- 1 H-pyrrol-3 -yl] -

32 propylideneamino } -guanidine

N- [(5 -Chloro- 1 -(4-isobutyrylphenyl)-4-penty 1- 1 H-pyrrol-2- 33 yl)methy leneamino] -guanidine

N- [3 -(5 -Propoxy- 1 -m-tolyl- 1 H-pyrrol-2-yl)-propylideneamino] - 34 guanidine

N-[4-Methyl-5-propylamino-l -(3-trichloromethyl-phenyl)- 1 H- 35 pyrrol-3 -ylmethylideneamino] -guanidine

N-{3-[l-(3-Nitro-biphenyl-4-yl)-lH-pyrrol-2-yl]- 36 propylideneamino } -guanidine N- { 2- [4-tert-Butyl- 1 -(4-methoxy-2-propylaminopheny 1)- 1 H-pyrrol-

37 3 -yl]-ethylideneamino } -guanidine

N- { 3 - [ 1 -(2-Bromo-3 -chlorophenyl)- 1 H-pyrrol-3 -yl]-

38 propy lideneamino } -guanidine o N- [4-Bromo- 1 -(2-butoxy-3 -propoxyphenyl)- 1 H-pyrrol-2- ylmethy lideneamino] -guanidine

. _ N- [5 -Hydroxy- 1 -(2-hy droxyphenyl)- 1 H-pyrrol-3 - ylmethylideneamino] -guanidine

. N-{3-[l -(2-tert-Butylphenyl)-4-chloro-5-methyl-lH-pyrrol-2-yl]- propy lideneamino } -guanidine

N- [5-Isobutyryl- 1 -(2,3 ,4-trimethoxyphenyl)- lH-pyrrol-2-

42 y lmethy lideneamino] -guanidine

N-{3-[5-tert-Butyl-l-(2-tert-butyl-3,4-bis-trichloromethylρhenyl)-4- 43 trichloromethyl- 1 H-pyrrol-2-yl] -propylideneamino } -guanidine

N- { 2- [ 1 -(4-Pyrrol- 1 -y lphenyl)- 1 H-pyrrol-2-yl] -ethy lideneamino } - 44 guanidine

N- [ 1 -(4-Moφholin-4-y lphenyl)- 1 H-pyrrol-2-ylmethylideneamino] - 45 guanidine

N- [ 1 -(4-Pyrrolidin- 1 -y lphenyl)- 1 H-pyrrol-3 -ylmethylidenamino] - 46 guanidine

47 N-(( 1 -Phenyl- 1 H-pyrrol-2-yl)methyleneamino)guanidine

N- [( 1 -(4-Trifluoromethylphenyl)- 1 H-pyrrol-2-

48 yl)methyleneamino] guanidine

49 N- [( 1 -(3 -cy anophenyl)- 1 H-pyrrol-2-yl)methy leneamino] guanidine

N-[(l-(3,5-dichloroρhenyl)-lH-ρyrrol-2-

50 yl)methyleneamino] guanidine

or a pharmacologically acceptable salt thereof.

22. A compound as claimed in any one of the previous claims which additionally 5 comprises a label, preferably a radioactive label, or a toxic agent.

23. A prodrug from which a compound as claimed in any one of claims 1 to 22 is formed in vivo.

10 24. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23, together with one or more adjuvants, carriers or excipients.

25. A compound as claimed in any one of claims 1 to 23 for use in therapy.

26. A process for the production of a compound as claimed in claim 1 which 5 comprises reacting a guanidine of formula (III), or a salt or protected form thereof, with an compound of formula (II)

(II) (III)

wherein X and Rj, R2, R3, R4 and R5 are as defined in claim 1, followed by deprotection if 10 necessary or desired.

27. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of inflammation.

15 28. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of mental disorders.

29. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of dysfunctions of the 0 endocrine system or a hormonal system.

30. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of sexual functions and/or sexual dysfunctions.

25

31. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of drug-induced or other disorders of the blood and/or lymphoid system.

5 32. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of allergic disorders.

33. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of disorders of the

10 cardiovascular system.

34. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of pain.

15 35. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for inducing skin tanning or for inducing lighter skin colour.

36. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as 20 claimed in claim 23 in the production of a medicament for the treatment of diabetes type II.

37. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of obesity.

25 38. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of anorexic conditions such as those caused by cancer, cachexia, geriatric conditions, HIN, trauma and psychological conditions.

30 39. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for inducing peripheral nerve regeneration.

40. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for inducing central nerve regeneration.

5 41. A method of treating inflammation comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

42. A method of treating mental disorders comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

10

43. A method of treating dysfunctions of the endocrine system or an hormonal system comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

15 44. A method of treating sexual functions and/or sexual dysfunctions comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

45. A method of treating drug-induced disorders of the blood and/or lymphoid 20 system comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

46. A method of treating disorders of the cardiovascular system comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as

25 claimed in claim 23.

47. A method of treating pain comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

30 48. A method of inducing skin tanning or for inducing lighter skin colour comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

49. A method of treating diabetes type II comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

50. A method of treating obesity comprising the use or administration of a 5 compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

51. A method of treating anorexic conditions such as those caused by cancer, cachexia, geriatric conditions, HIN, trauma and psychological conditions comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as

10 claimed in claim 23.

52. A method of inducing peripheral nerve regeneration comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

15

53. A method of inducing central nerve regeneration comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

20 54. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of skin disorders, including for the treatment of melanoma.

55. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as 25 claimed in claim 23 in the production of a medicament for the treatment and/or diagnosis of malignancies, such as melanoma and metastases.

56. A method of treating a skin disorder, including the treatment of melanoma, comprising the use or administration of a compound as claimed in any one of claims 1 to 22

30 or a prodrug as claimed in claim 23.

57. A method of treating and/or diagnosing malignancies, such as melanoma and metastases, comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

58. Use of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23 in the production of a medicament for the treatment of ischemia and/or ischemia/reperfusion.

59. A method of treating ischemia and/or ischemia/reperfusion comprising the use or administration of a compound as claimed in any one of claims 1 to 22 or a prodrug as claimed in claim 23.

[Uhohinc]

https://www.clinicaltrialsregister.eu/ctr-search/search

[Uhohinc]

https://www.clinicaltrialsregister.eu/ctr-search/search?query=ap1189

Usually I look into things to understand what is going on, with this drug AP1189 and the company, stock, and manager I now look and see so many contradictions, discrepancies, conceptual conflicts, that I am now trying to keep an open mind and not see what appears to be a scam.

[Claes Svensson]

And all I see is that you've mastered the art of copy/paste and done alot of highlighting on stuff that has nothing to do with anythin I said... Starting to question your abilities on reading articles.....
These guys running SynAct Pharma has taken quite a few substances through clinical phases and done some deals with Big Pharma such as Abbvie, Pfizer, Questcor etc. I think they know what they are doing.

[Claes Svensson]

Like, what is this? "AP1189 a Erk1/2 pathway without Mcr1 or Mcr3 activation" - this is proof enough that you havent understood anything.

AP1189 is a BIASED agonist. A so called melanocortin-receptor-agonist. It activates MC1R and MC3R. They've achieved in activating MC1R through ERK1/2 instead of the usual "cAMP" as all the other known melanocortins such as a-msh and Acthar Gel do.

Its called a BIASED agonist because it DOES NOT stimulate the part on MC1R that induces melanogenesis.

Its a selective melanocortin agonist in the form of a "small molecule". Selective in that way that it does not activate all 5 mc-receptors. Just the two that matters when you want to inhibit inflammation and trigger the resolution phase.

onsdag 26 maj 2021 kl. 18:21:57 UTC+2 skrev Uhohinc:

[Uhohinc]

I do not deny I steal everything. I bring it together and then I formulate my own opinion. I have seen the research of Perretti for years, he is stellar. But you deflect away from the one thing here in subject is the drug AP1189 and what is it about.....you have legitimatized it in your mind by associative to "Big Pharma" and Abbievie, Pfizer, Questcor," and they are legitimate. Then in place of argue of facts or the cut paste highlights I give, you "question (my) abilities on reading articles...."    YOU are the one stating Thomas J. is the one, (kind of like Charlie Manson type of coronation in that we should not observe, research, think, postulate but trust in the wizard behind the green curtain. 

Right back at you.

For those reading here the patents and the company press release and what I think is a prestigious publication, none of it is matching up.  I said I am keeping an open mind, trying real hard to, but incredible statements require incredible facts, and documentations.  

[Claes Svensson]

Peretti and Thomas J are friends and colleagues since many years. They did the preclinical trials together (the one you cited recently) on AP1189. Mauro Martins Teixeira, NO1 on inflammation in South America is the one doing the ARDS/covid19-study in Brazil at the moment. But naah.. They probably know less than you hahaha!

Just recently this was announced:
“The project is a natural next step in our long-standing collaboration with Dr Trini-Montero Melendez and Professor Mauro Perretti. Our investment of ~£400,000 is to enable an important task of the project, i.e. to link clinical outcome of treatment with our AP1189 compound to MCR1 polymorphism as it would give the possibility to apply a rational approach for patient selection for future clinical studies. In addition, new insight into pharmacology of specific MCR1 variants can be used for identification of next generation compounds”, says Thomas Jonassen, CSO SynAct Pharma."

Not matching up? Your brain isnt matching up. Get a clue. The patent cover all kind of formulations, one of them making up AP1189, another one AP1030 towards diabetes etc. They have peptides, small molecules in their pipeline.

Peretti is on the scientific board of SynAct. They have Thomas Boesen on board. The team behind SynAct is top notch. James Knight who sold Acthar Gel to Questcor is on board as portfolio manager etc.

But dont worry, they aint after to steal the market from your Scenesse. They are focused on RA, PSA, IBD and now also Covid19 induced ARDS.

[Uhohinc]

I have carefully read your post twice now, since you claim I do not read. And yes i cut past and now highlight. And no, Abbievie and Pfizer and Big Pharma are not with me here. Maybe you are tooo close to the research and tooooo emotionally, financially, and intellectually committed to your pet project here and can not see a perspective that might not be in reality to most. There is something here, but I read into it Thomas J. and the authors do not really know what is going on, as they state several times in the publication they are indicating they do not know even some basics. 

The patent they reference, which I cut and paste and highlight AND READ, states the compound can cause a tan, and in the same sentence it states it can prevent a tan. Forest Gump was a movie, he could not exist in the story in real life. These contradictions are all over in the patent, written maybe by a multiple personality psycho lawyer. You state " SynAct has good preclinical data in ARDS already, dating 10 years back" yet you cite none of it. Maybe try some cut and paste if it is not just a concept that you believe, or really really want to believe. Starting your comment with a "wait and see" on a stock investment research board is the old con man trick of "trust me"..........well I like to use my eyes and hopefully open unbiased thinking  and read into a commentary of such salacious claim with nothing to "read" with it. Why do we have to wait and see............if you have some thing why keep it secret. Oh, you did state "They are the only ones that know." And use of "company secrets, as an excuse to hide basic research and not explain what is the mechanics of action is not going to make me a believer in the religion you have bought and I suppose put your money into.  You claim the drug is miraculous in regulating the inflammatory response and to trust Thomas J. (esus) because he is in the top five........top five of what.  And just because YOU can cut and paste Phfizer, and Abbievie, is not making me drink that kool aide.  What is your affiliation to SynAct, Thomas J. and this drug, are you involved as an investor, a researcher, a employee of the company ?   

"Wait and see! Melanocortin receptors 1 and 3 are the most important when regulating the inflammatory response. The key is to switch macrophages into M2-phenotype, Mc3r helps alot!

SynAct has good preclinical data in ARDS already, dating 10 years back. The covid19-situation gave a great opportunity to do human trials - SynAct are front runners in inflammation resolution.

Lots of data supporting melanocortins in the treatment of ARDS - central downregulation of the inflammatory response includes the lungs :)

One would think that achieving activation of Mc1R without cAMP and side effects, was a matter of company secrets. They are the only ones that know how. Thomas J is in the top5 on theese receptors!"

[Uhohinc]

Whooooa, you come on here, put your post up, attack me personally for questioning your SynAct, and I respond, then you deleted it your posts.......Tell me what it is or what you wrote that made you have a second thought as to delete rather than explain what ....................?

[Claes Svensson]

"some of the compounds of the invention". The patent covers ALL the different structures of the molecule and their different actions. Maybe contradictory for some with reading disabilities....
All through that text - COMPOUNDS. Nothing is secret, but you probably suck at Google. The melanocortin-system is not fully understood, and therefore all of the action of AP1189 can not be understood.

But anyways, AP1189 shows great efficacy, and the team has delivered many times before. The preclinical studys on ARDS where performed in their previous company Action Pharma, that sold their lead candidate to AbbVie.

[Uhohinc]

https://covid19.elsevierpure.com/en/persons/mauro-martins-teixeira-2/publications/ You cited this researcher above, here I read and quote you; please explain how in my link he does not include collaboration, in his collaberations area, and how is it that this AP1189 is all about not stimulating cAMP thru Mcr1 and here you cite a "treatment of AP1189 compound to MCR1"

Note that this so called collaboration, the fine Brazillian is doing sars research, but it is into sewage effluent in river water containing Covid 19....

And if you know what it really means in the SynAct press release when it states "in our long-standing collaboration with Dr. ...Mlendez.........what it really means is they are embellishing a relation in type, that really is not longstanding...............

from you, above cut and paste......

"Just recently this was announced:

“The project is a natural next step in our long-standing collaboration with Dr Trini-Montero Melendez and Professor Mauro Perretti. Our investment of ~£400,000 is to enable an important task of the project, i.e. to link clinical outcome of treatment with our AP1189 compound to MCR1 polymorphism as it would give the possibility to apply a rational approach for patient selection for future clinical studies."

[Uhohinc]

Read very carefully what you wrote here, I have copied and pasted for your convenience.....read it......you just made all my points and you may read, and you may write, but do you comprehend........

Claes Svensson<classesve...@gmail.com>

10:33 AM (18 minutes ago) 







to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9

"some of the compounds of the invention". The patent covers ALL the different structures of the molecule and their different actions. Maybe contradictory for some with reading disabilities....
All through that text - COMPOUNDS. Nothing is secret, but you probably suck at Google. The melanocortin-system is not fully understood, and therefore all of the action of AP1189 can not be understood.

But anyways, AP1189 shows great efficacy, and the team has delivered many times before. The preclinical studys on ARDS where performed in their previous company Action Pharma, that sold their lead candidate to AbbVie.



[Uhohinc]

https://groups.google.com/g/clinuvel/search?q=synthactin    now it can not be a coincidence that the drug Synthactin is not anything to do with your company SynAct Pharma  which has a MAJOR problem with MCR2 stimulation but do not listen or read what i cut paste write,,,,,,lets listen TO YOU>>>............................"But anyways" ............................."The melanocortin-system is not fully understood, and therefore all of the action of AP1189 can not be understood."

[Uhohinc]

You got defensive when I pointed out just some of the incongruities with this AP1189 and SynAct Pharma................but your last post is indicative to meeeeee. That with the patent  and its absolute rediculous claims, you had a epiphony exemplified in your last above post. Your cognitive thinking portion of brain usurped the emotional limbic brain and recognized there is somethings here not making sense.  Now they could eventually be figured out and make more sense with more known. But you showed in your reply that you yourself are in conflict to try and explain these incongruities of mcr1 is or is not the pathway, magical erk stimulation, and a tan and no tan.........presto investo.....

Your feel good Dr. Phil explanation..........is to just say.............."But anyways"    

As to how or what is AP1189,......................you do not understand it as you now admit, I do not understand it, nor does your svengali Thomas J understand it.

Why would you invest in something you do not understand nor do the guys running the company......................and if it may fruition and the incongruities are explained some time in the long time to go with many years of research........................We got some old old old old guys invested in Clinuvel whom if not seen often I almost call for a welfare check, as seen on Sharetease and you may have time to chase your windmills. 

[Claes Svensson]

Wow, I think we have an ASPIE here, big time. Try keeping two thoughts in mind at the same time.
https://synactpharma.com/en/synact-pharma-initiates-resovir-scientific-and-clinical-collaboration-to-explore-ap1189-in-viral-infections/

A bit much asking me to explain why SynAct as of yet isnt mentioned on his site, still, a trial is being conducted.
The brazilian trial is named "EFEITOS DA ADMINISTRAÇÃO DO AP1189, UM ATIVADOR DE VIAS PRO-RESOLUTIVAS, NAS ALTERAÇÕES PULMONARES DE PACIENTES COM COVID-19 E OXIGENAÇÃO COMPROMETIDA (RESOLVIR)"

No, its not all about not stimulating cAMP, that is still a big break through though, as melanogenesis is an unwanted side effect coming from cAMP-activation. Having a melanocortin-agonist in the form of a stayble "small molecule", having an oral formula, selectivity on only MC1 and MC3 is the big thing. Being biased against MC1R is a big bonus.

THE PROJECT, is a new collaboration announced 2 weeks ago, and is about personalized medicine. Every person has a different set of genes making up their MC-receptors. AP1189 will bind better to some patients, and less to others. This is a study to find the different gene-types, and then by a simple blood sample be able to tell beforehand if the patient will be a responder or not. Not everyone responds to melanocortin treatment.

Peretti and Melendez did the preclinical studies on AP1189 many years ago, this is another step taking it to the next level.

Trinidad on twitter:

Trinidad Montero

@TriniMonteroM

·

May 13

We are delighted to continue our long-term collaboration with #SynAct Pharma. A long journey from early pre-clinical to clinical development of promising drug candidate #AP1189 for rheumatoid arthritis and more!

@QMULWHRI

@QMUL

@QMULBartsTheLon

@CiTI_QMUL

If you google "the melanocortin system" you will find that almost every scientific article about it states that the melanocortin system is not fully understood. That does not mean that SynAct doesnt know how to formulate the substance to avoid cAMP.

Damn.. Dont know if your stupid or just trolling. Done wasting time on you.
Like, why even highlight "sold their lead candidate to AbbVie"?

"some of the incongruities" the only ones can be found in your head. Not my fault that you cant comprehend.

Good luck to me, I hope the stellar guy Peretti knows what he's up to, even though you cant understand that MC1R-activation is possible in two ways.

Bye bye aspie <3

[Uhohinc]

 I have made enough of my points and refuse to engage you over and over, you know and showed you are not able of intelligently and backing up your points, statements and argueing and revert to not answering my points of arguemnet, but pubescently revert to what worked when you were not fully formed............namecalling..  You do not have an investment, you have a personality disorder in need of a target......You showed me your true ulterior state of mind as to your personal or financial investment with a positive  outcome to AP1189, that it does not matter when you state "but anyways"..

"But anyways" is another way in my English interpretation of saying "those facts do not matter to me" pertinent to what you stated as to the patent. 

Look up the meaning, the use and the synomyns for "But anyways"  ...............................these are your incongruities tooo.......The difference is You do not care to deal with it as it does not fit your (not reality) construct. Why you do not need to come to terms or explain or as you say "understand" it. Just pretend play it is not there. A basic defense mechanism in human psyche. That goes from seeing something that does not make sense, but you senselessly ignore it ..................The next time I pass by a church or synagogue or mosque, i might pray for you and your drug. But I will not put any money into the SynAct stock of a garbled patent into a obfuscated ACTH remake with the need for blind thinking foolowlers............In fact I got wealthy off people like you.......................and hope to research, read, cut paste and steal others work right here on GG clinuvel.  In America there was for over a hundred years a saying about selling swamp land in Florida, as well as a sucker born every minute...............well that saying is almost gone now, because Disney drained the swamp, and the water was channeled into millions of homes with boat docks..........................so maybe your Charlatan you blindly follow into maybe, something, kinda, might, be some kinda drug that is antiinflammatory in some kinda way, that he admitted in the first post above is not UNDERSTOOD, yet you have a narrative in your mind that you understand it will........do something.....or as you like to throw caution and fact to the wind as you say, "just wait and see"......................but anyways hope your dream come true and your swamp gets a Disney park.  But anways, when someone comes on here and says there is Unicorns and rainbows I want to see some Unicorns urine and shit and hear the noise it makes and I want pictures.  You are chasing an elusive rainbow..........for now.  I will not close my mind like you.

You do know, that cAMP is not even close to THE instigation of melaningenisis, ?  

 Can you elaborate how  AP1189  upon encountering an melanocyte, or any cell get thru to the cell nucleus for expressions of ( lets pretend here because you and SynAct claim you do not understand, but it is not thru mcr1, but at different times or in the patent it is thru mcr1, or in the press release you cite  a secret study by a Doctor in the Amazon wading thru sewage who is but is not in collaboration with SynAct ) and what are just say 4 pathways Ap1189 expresses and how does the drug do this ?.............................oh wait, I just realized you stated you do not understand.................Your in a Aphasia I and II and III clinical trial in your head..........

[Claes Svensson]

Many words were written. Not alot was said.
"by a Doctor in the Amazon wading thru sewage" - yup... https://scholar.google.com/citations?user=njJyXhsAAAAJ&hl=en

Mauro Teixeira holds a medical degree from the UFMG Medical School (1990) and a PhD in Immunopharmacology from the University of London (1994). He is a full professor of the Department of Biochemistry and Immunology of the Federal University of Minas Gerais, researcher 1A of CNPq, Member of the Brazilian Academy of Sciences, of the National Order of Scientific and Technological Merit and of the World Academy of Sciences (TWAS). Member of the editorial board of the journals "Pharmacology & Therapeutics (Oxford)", "Inflammation Research" and "Medical Microbiology and Immunology". He is the coordinator of INCT in dengue and the Pronex Network in Dengue of CNPq. It acts in the area of ​​inflammation, especially trying to understand the relevance and molecular mechanisms of the inflammatory response during infectious and autoimmune diseases. He is currently vice-president of the Brazilian Academy of Sciences (Regional CO-MG). He is a member of the Scientific Committee of Anvisa (CCVISA).

Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis:
https://pubmed.ncbi.nlm.nih.gov/8612494/

Another ignorant american at its best... By NOT activating cAMP on MC1r-receptors, which are found on melanocytes, ap1189 does not cause skin pigmentation. Why? Who the fuck cares.

Melanocortins, such as Acthar Gel sell for billions of dollars each year. AP1189 will be able to do pretty much the same as Acthar Gel without any side effects. None.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763663/ only one of many independent articles supporting my claims.

[Uhohinc]

Ignorant me here with a preface......This is the only SynAct Pharma 12 patient trial. It FAILED and that is why they are going to try and increase the dose 100%  and disguise it to gullible investors as a success and call the changes a part 2 of the trial. This public release is paid for by SynActin......This press release states the drug Ap1189 is a first in  class melanocortin receptor agonist.....................read that again......................There is no such word or terminology that an endogenous hormone is an melanocortin...............perhaps just bad translation and not ignorance............. "but recently the Danish biotech added AP1189 as an adjunctive therapy in hospitalised patients with Covid-19 infection in order to prevent ARDS,"    

This is what all the desperate small underfunded failing drug pharma throw out as the shinny sparkle to glitter to catch gullibles that I call a rabbit hole............Any one with sense or reads here has known for over a year that yes melanocortins will likely be of therapy in Covid................Covid is over to the foools out there. Where is the monetization of any Covid therapy here if or wheeeeeeeeen this company gets a drug to approval.  A trial in Brazil is worthless, just ask the Brazilians, if the drug works in Covid that why not try it in FDA or EMA jurisdictions where SynActin can actually the persue a commercial oppurtunity in the markets of USA and Europe........this Covid distraction is a scam..........no intent to seek monetary for it.................

 

Yesterday, preliminary data from SynAct Pharma’s Phase II study with the drug candidate AP1189 in patients with rheumatoid arthritis was released. The data indicate that AP1189, administered in doses of 50 mg, is safe and well-tolerated.     The blinded review of the data also indicates a substantial reduction in disease severity among two thirds of the patients, which may be attributed to AP1189. BioStock reached out to the company’s CEO Jeppe Øvlesen for a comment on the positive indicative results.

When you read about Ap1189 about the goal is it is really only an "add on treatment" to be used with other drugs.......................Aspirin is an anti inflammatory and will get better efficacy  results than this drug did in its own trial. Numerous anti inflammatory on market are already getting better results with no adverse effects........If AP1189 works, lets see it without the need to pair it to other known to work in extents drugs for RA.

the below excerpt, is gobbleydeegook double speak for 50mg did not show efficacy, so they intend to use 100mg and make you think it is intentional.....with an oppurtunity to show efficacy..............why not do that from the start..........you do not need another safety profile, that was phase I

Excerpted from below fake interview from SynActin.........................." In the short-term this means that we can focus on getting a safety profile on the next dose level of 100 mg and, if this dose, which was very well-tolerated in our Phase I study, turns out to be safe in the patients as well, we can continue in the second part of the study with two doses of active compound vs placebo. That gives us a great opportunity to show efficacy and thereby confirm the potential of AP1189 as new treatment option in rheumatoid arthritis."

Make sense of this, all I can figure is they are preparing to hide the patients that do not report the way SynActin finds positive......................................wthf.................a rigged trial result.....

"you state that it is unlikely that all patients in the planned 100 mg cohort in part 1 of the study will be registered, dosed and reported "

SynAct comments on positive Phase II data

 6 maj, 2020

 SynActs pipeline. Click to enlarge

SynAct Pharma´s leading drug candidate AP1189 is a First-in-Class melanocortin receptor agonist targeted at active inflammatory and autoimmune diseases. The primary indications for AP1189 are active rheumatoid arthritis (RA) and Nephrotic Syndrome (NS), but recently the Danish biotech added AP1189 as an adjunctive therapy in hospitalised patients with Covid-19 infection in order to prevent ARDS, to it’s pipeline, given the common denominator in all three indications; an uncontrollable inflammatory response.

Based on the endogenous hormone melanocortin

SynAct Pharma’s research platform is based on the endogenous hormone melanocortin, which is activated in inflammatory conditions and contributes with anti-inflammatory effects that are crucial for the healing process and recovery to normal tissue function.

The treatment concept is based on strengthening the immune system’s healing mechanisms by activating the body’s own immune cells, a so-called resolution therapy. By activating the body’s immune cells, SynAct Pharma´s concept differs from biological and immunosuppressive drugs, which act by inhibiting the activity of the immune system.

Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a chronic inflammatory disorder, which means that the immune system mistakenly attacks healthy cells in the body, causing inflammation and painful swelling in the affected parts of the body. RA affects not only the joints in hands, wrists, and knees, for but also organs like the skin, eyes, lungs, heart, as well as blood vessels. The tissue damage can cause long-lasting or chronic pain, unsteadiness and deformity. Thus, the disease is associated with progressive disability, premature death, and hence, also socioeconomic burdens.

While new types of medications have improved treatment options dramatically, severe RA can still cause physical disabilities. It is in this space where SynAct Pharma wants to position its leading candidate AP1189, with its potential to induce disease remission without concomitant immune-suppression and thereby increased risk for infections, both in RA and other inflammatory and auto-immune diseases.

An early add-on treatment in RA

In the ongoing study, AP1189 or placebo is dosed once daily in previously methotrexate (MTX)-naïve patients. Methotrexate, a chemotherapy agent and immune system suppressant, is one of the most effective medications to treat RA on the market.

Still, approximately 40% of the patients have an inadequate response, while next-line treatments are more efficacious, but often have severe side effects. Even with expensive injectable biologics, up to 30% still suffer from inadequate treatment.

SynAct Pharma´s strategy is to position AP1189 in this context as an early “add-on” treatment that could reduce the need for second-line treatment and/or reduce the dose levels of MTX.

New phase II data in RA

Yesterday, SynAct Pharma announced that the first of two tested doses (50 mg) with AP1189 in early RA patients with active joint disease, was safe and well-tolerated, based on a blinded review of data from the company’s ongoing placebo-controlled Phase II study, which, to date, has enrolled 12 dosed patients from sites in Denmark and Sweden.

The results also show a strong 50% reduction in disease severity in 8 of the 12 patients tested. However, since the review of the data was blinded in order to avoid inserting bias into the analysis, it remains inconclusive whether the reduction in disease severity can be attributed to AP1189.

Based on these results from the double-blind study of the safety, tolerability and efficacy of AP1189, the company has decided to proceed with dosing at the next dose level (100 mg) in part 1 of the study, and to include the 50 mg dose level in part 2 of the study. It will later determine whether 100 mg is safe and tolerable and whether the dose should be included in part 2.

BioStock reached out to SynAct Pharmas CEO Jeppe Øvlesen for a comment on the released data in RA.

Jeppe Øvlesen, vd SynAct Pharma

Jeppe Øvlesen, to begin with, the preliminary results based on the blinded data, show that administration of AP1189 seems to be safe and well-tolerated. Thus, you have achieved an important milestone in your RA project. What significance does this have for the future development of the candidate?

– For us, it is extremely encouraging to see that, according to the data, the treatment setup and the dose of the compound given to patients is safe. The interpretation on the blinded data on efficacy further support that we should continue dosing at this level in the second part of study.

– In the short-term this means that we can focus on getting a safety profile on the next dose level of 100 mg and, if this dose, which was very well-tolerated in our Phase I study, turns out to be safe in the patients as well, we can continue in the second part of the study with two doses of active compound vs placebo. That gives us a great opportunity to show efficacy and thereby confirm the potential of AP1189 as new treatment option in rheumatoid arthritis.

Going into the numbers, after a 4 week treatment period, a group of 8 patients showed a reduced clinical disease activity index (CDAI) score compared to pre-treatment levels with a median reduction of 50%, and a group of 4 patients with a worsening in their clinical status, where the median CDAI score was increased by 10%. The numbers – considering that it is the common understanding that it takes several weeks before MTX induces improvement in symptoms – sound indicative of a substantial clinical improvement, is that how you would interpret them?

– I have to emphasize that the data is blinded, the sample size is small and there could placebo treated among the subjects where we have identified a fall in clinical disease score. Saying that there is as we highlight in the PR patients that goes from high disease activity to low disease activity within the study period. Such reductions in disease activity are very seldom seen following 4 weeks treatment with MTX as monotherapy. However, we cannot exclude an extraordinary placebo effect. We have to wait to make any conclusions until we have the planned patients enrolled and unblind the data.

Considering the review of the data was blinded, it cannot be ruled out that there are placebo-treated patients among those showing clinical improvement. How does this caveat play into choosing the next dose level?

– The decision to go to the next dose level is based on the safety review not on blinded review of potential efficacy. We could have had a setting where most of the patients still had high disease activity at the end of the treatment period – which the data shows us we are not.

You plan to position AP1189 as an additional treatment given early to patients with RA, in combination with methotrexate (MTX). What reactions and support have you received from major pharmaceutical companies and researchers when presenting this new combination strategy?

– The setup is in many ways ambitious as we go in early  start dosing in parallel with methotrexate in patients with high disease activity. In these patients the physician often turns to intraarticular injections of glucocorticoids to induce pain relief in parallel with up-titration of methotrexate as the onset of clinical effect of methotrexate is slow. If the treatment profile of AP1189 in combination with Methotrexate turns out to be effective it could reduce the need for glucocorticoid injections and potentially reduce the need for second-line treatments like biologics or JAK inhibitors.

– If that turns out to be the case, the physicians would find AP1189 as a very attractive opportunity, also because the compound seems not to induce immunosuppression. The pharma industry would likewise see a big opportunity in such a scenario – where it would also serve as part of proof of our “resolution therapy treatment”.

In the press release yesterday, you state that it is unlikely that all patients in the planned 100 mg cohort in part 1 of the study will be registered, dosed and reported during the second quarter of 2020. This seems to have made the market somewhat ambivalent when looking at the relatively small increase in the share price. Would you like to comment on how your chances of success are affected by the Covid-19 outbreak?

– The Covid-19 outbreak has, until further put the recruitment in Sweden and Norway on hold. In Denmark recruitment continued but at a lower recruitment rate.  However, we now slowly see that the countries get better control with the corona situation and we hope we will get back to a more normal situation. This will help us in getting the recruitment rate up again and making it possible to get the patients for both the first and the second part of the study enrolled.

Finally, what milestones can we look forward to during the rest of the year?

– We still have the ambition for significant progress in the RA study. We expect to get the study in nephrotic syndrome up and running and then we have the opportunity in Covid-19 where we are working on the protocol and where we have had very good progress with the investigators.

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Biased Agonism as a Novel Strategy To Cooray, Thomas E. N. Jonassen and Mauro Perretti Trinidad Montero-Melendez, Thomas Gobbetti, Sadani N. http://www.jimmunol.org/content/194/7/3381 doi: 10.4049/jimmunol.1402645 February 2015; J Immunol 2015; 194:3381-3388; 

Prepublished online 27 Material Supplementary 5.DCSupplemental http://www.jimmunol.org/content/suppl/2015/02/27/jimmunol.140264 References http://www.jimmunol.org/content/194/7/3381.full#ref-list-1 This article cites 44 articles, 7 of which you can access for free at: *average • Fast Publication! 4 weeks from acceptance to publication • No Triage! Every submission reviewed by practicing scientists • Rapid Reviews! 30 days* from submission to initial decision Why The JI? Submit online. Subscription http://jimmunol.org/subscription Information about subscribing to The Journal of Immunology is online at: Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright © 2015 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., 

The Journal of Immunology is published twice each month by by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from The Journal of Immunology Biased Agonism as a Novel Strategy To Harness the Proresolving Properties of Melanocortin Receptors without Eliciting Melanogenic Effects Trinidad Montero-Melendez,* Thomas Gobbetti,* Sadani N. Cooray,* Thomas E. N. Jonassen,† and Mauro Perretti* 

There is a need for novel approaches to control pathologies with overexuberant inflammatory reactions. Targeting melanocortin (MC) receptors represents a promising therapy for obesity and chronic inflammation, but lack of selectivity and safety concerns limit development. A new way to increase selectivity of biological effects entails the identification of biased agonists.

 In this study, we characterize the small molecule AP1189 as a biased agonist at receptors MC1 and MC3. Although not provoking canonical cAMP generation, AP1189 addition to MC1 or MC3, but not empty vector, transfected HEK293 cells caused ERK1/2 phosphorylation, a signaling responsible for the proefferocytic effect evoked in mouse primary macrophages. Added to macrophage cultures, AP1189 reduced cytokine release, an effect reliant on both MC1 and MC3 as evident from the use of Mc1r2/2 and Mc3r2/2 macrophages.

 No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 elicited antiinflammatory actions in peritonitis and, upon administration at the peak of inflammation, accelerated the resolution phase by ∼3-fold. 

Finally, given the clinical efficacy of adrenocorticotropin in joint diseases, AP1189 was tested in experimental inflammatory arthritis, where this biased agonist afforded significant reduction of macroscopic and histological parameters of joint disruption. These proof-of-concept analyses with AP1189, an active oral anti-inflammatory and resolution-promoting compound, indicate that biased agonism at MC receptors is an innovative, viable approach to yield novel anti-inflammatory molecules endowed with a more favorable safety profile. 

The Journal of Immunology, 2015, 194: 3381–3388. Melanocortin (MC) receptors (MC1–MC5), a family of class A druggable G protein–coupled receptors (GPCRs), are attractive therapeutic targets for a number of conditions due to their wide distribution and diversity of physiological processes they regulate (1). MC1 regulates UV light–induced skin tanning and other immune responses because of its expression on leukocytes. MC2 regulates cortisol production on the adrenal glands, whereas MC5 plays a role on exocrine glands secretions. MC3 and MC4 exert nonredundant functions on energy homeostasis in addition to specific anti-inflammatory roles: whereas MC3 activation is particularly protective for arthritides (2), MC4 provides neuroprotection in brain inflammation (3). Accordingly, the array of pathological situations that could be targeted with MC drugs includes skin conditions (vitiligo, melanoma, erythropoietic protoporphyria), cardiovascular pathologies, joint inflammation (gout, rheumatoid arthritis [RA]), obesity, cachexia, or sebaceous glands disorders such as acne vulgaris (4). 

Peripheral MC1 and MC3 can be pharmacologically activated to induce anti-inflammation. The endogenous agonist a-melanocyte– stimulating hormone (aMSH), like other protective mediators, is released by immune cells to counterbalance proinflammatory signals, thus preventing excessive tissue damage (5, 6). In line with the resolution of inflammation concept, therapeutics targeting MC1 and MC3 will then be acting by mimicking the body’s own protective resources (7, 8) and might be characterized by a lighter burden of side effects. 

Shown to be effective in RA rheumatic diseases since the early 1950s (9), the use of corticotropin or adrenocorticotropin hormone (ACTH) declined when synthetic glucocorticoids became available. However, the discovery of an alternative anti-inflammatory mechanism for ACTH involving activation of peripheral MC receptors on immune cells (10) has revived the interest in developing novel ACTH-like molecules with no steroidogenic effects for the treatment of joint diseases such as gout or RA (11, 12). However, the limitation in the translational delivery of novel MC drugs besides the marketed ACTH formulations (H.P. Acthar Gel, Synacthen Depot, or Cortrosyn) is imposed by the lack of receptor selectivity achieved so far. 

Innovative approaches in G protein–coupled receptor drug discovery might help to overcome this limitation. Allosteric modulation consists in the ability of a molecule to enhance (positive modulation) or reduce (negative modulation) the effect of the endogenous ligand by binding to a distinct site of the receptor protein, termed allosteric site (13). A higher degree of selectivity is expected as allosteric regions are less conserved among the five MCRs, and indeed, allosteric modulators at MC4 are currently under development for the treatment of obesity (14). *William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and † Department of Biomedical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark Received for publication October 20, 2014. 

Accepted for publication January 28, 2015. 

This work was supported by a collaborative project between SynAct Pharma APS and William Harvey Research Limited. Address correspondence and reprint requests to Prof. Mauro Perretti and Dr. Trinidad Montero-Melendez, William Harvey Research Institute, Barts and The London School of Medicine, Charterhouse Square, London EC1M 6BQ, U.K. E-mail addresses: m.per...@qmul.ac.uk (M.P.) and t.monter...@qmul.ac.uk (T.M.-M.) The online version of this article contains supplemental material. Abbreviations used in this article: ACTH, adrenocorticotropin hormone; AGRP, agouti-related protein; EIA, enzyme immunoassay; MC, melanocortin; aMSH, a-melanocyte–stimulating hormone; NDP-aMSH, [Nle4,D-Phe7]-aMSH; RA, rheumatoid arthritis; WT, wild-type. Copyright 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402645 by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from Another emerging concept of significant therapeutic interest is the one of biased agonism. 

The obsolete notion that receptors could exist in two unique conformations, the active one and the inactive one, has been replaced with the conception that multiple active conformations can exist, each one creating a distinct signal yielding to multiple functional outcomes (15, 16). Receptor activation, rather than linear and static, is emerging as a highly dynamic and multidimensional process in which a diversity of active conformations may be induced by different molecules leading to distinct effects. 

We reasoned that this new pharmacological approach would have a positive impact on MC-based drug development because it sets the focus on “pathway selectivity” rather than “receptor selectivity,” given the proven difficulty of the latter strategy. 

In addition, finetuned molecules could be designed to engage only the therapeutically relevant pathway and not those leading to side effects. In this article, we present the first, to our knowledge, biased dual agonist at MC1 and MC3, and describe its anti-inflammatory properties together with its lack of effect on melanogenesis.

 Materials and Methods MC drugs and inhibitors aMSH and [Nle4,D-Phe7]-aMSH (NDP-aMSH) were purchased from Tocris Bioscience (Abingdon, U.K.). (E)-N-[trans-3-{1-(2-nitrophenyl)-1H-pyrrol2-yl} allylidenamino] guanidinium acetate (AP1189) was synthesized at Clauson-Kaas A/S (Copenhagen, Denmark). The ERK1/2 inhibitor FR180204 was obtained from Merck Millipore (Darmstadt, Germany).

 Cell culture and transfections HEK293A cells were maintained in DMEM containing 10% FCS and 1% penicillin/streptomycin and kept at 37˚C with 5% CO2. Clones for MC1 and MC3 and empty vector pCMV6 were purchased from Origene (Rockville, MD). Serum-starved cells were transfected with Lipofectamine 2000 (Invitrogen, Paisley, U.K.) according to manufacturer’s instructions and used 24 h after transfection. 

Radioligand binding assay Determination of binding affinities was performed by [125I]-NDP-aMSH radioligand binding. Cells were suspended in HEPES buffer and incubated with 0.05 nM [125I]-NDP-aMSH. AP1189 dissolved in DMSO was added, and cells were incubated for 90 min at 22˚C (MC1 cells -B16-F10-) or for 60 min at 37˚C (MC3 cells -CHO stable transfected cells-). After incubation, the amount of bound radioligand was determined and expressed in percentage of total binding in the absence of AP1189. ELISA and enzyme immunoassay IL-1b, IL-6, and TNF-a were measured in cell supernatants by ELISA using Ready-SET-Go ELISA kits from eBioscience (Hatfield, U.K.). cAMP was measured using the cAMP enzyme immunoassay (EIA) kit from Cayman Chemical (Cambridge, U.K.). Western blotting HEK293A cells were lysed using radioimmunoprecipitation assay buffer (Thermo Scientific, Waltham, MA) containing protease inhibitors mixture (Calbiochem, Hertfordshire, U.K.). Samples were subjected to standard SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore). These were incubated with rabbit anti–phospho-ERK1/2 (42/44 kDa, 1:1000; Cell Signaling, Leiden, the Netherlands) overnight and HRP-conjugated anti-rabbit IgG for 1.5 h (1:2000; Dako, Stockport, U.K.). Membranes were reblotted with anti–a-Tubulin (55 kDa, 1:5000; Sigma-Aldrich, Dorset, U.K.) for 1 h and HRP-conjugated anti-mouse IgG for 1 h (1:2000; Dako, Stockport, U.K.). Proteins were detected with Luminata Forte Western HRP substrate (Merck Millipore, Hertfordshire, U.K.) and visualized on the FluorChem E Digital Room (ProteinSimple, East Sussex, U.K.). Ca2+ mobilization assay Twenty-four hours after transient transfection, HEK293 cells were incubated with 2 mM Fura 2-AM (Molecular Probes, Paisley, U.K.) in HBSS without Ca2+ (Sigma-Aldrich) at 37˚C for 45 min in the dark. Subsequently, cells were washed three times in HBSS. HBSS containing 0.185g/l CaCl2 was then added before stimulation with agonists at the indicated concentrations. Ionomycin (1 mM) was used as a positive control. Mobilization of intracellular calcium was measured by recording the ratio of fluorescence emission at 510 nm after sequential excitation at 340 and 380 nm using the NOVOstar microplate reader (BMG LABTECH, Aylesbury, U.K.) during 70 s after drug addition. Then data corresponding to time 25 s after drug stimulation were selected to generate concentration–response curves. Determination of melanin accumulation B16-F10 cells were cultured in aMEM containing 10% FCS and 1% penicillin/streptomycin, and kept at 37˚C with 5% CO2. For melanin determination, cells were plated in 96-well plates using phenol red free media. Melanin content was determined spectrophotometrically at 405 nM in supernatants 72 h after drug stimulation (17). Isolation of primary peritoneal macrophages Mice were injected i.p. with 1 ml of 2% Biogel (Bio-Rad, Hemel Hempstead, U.K.). Four days later, peritoneal cells were collected by lavage using 4 ml of 3 mM EDTA in PBS and plated in 24-well plates at a density of 0.5 3 106 cells/well, in RPMI 1640 containing 10% FCS and 50 mg/ml gentamicin. After 2 h of incubation, nonadherent cells were removed. Compounds or vehicle were added 30 min before stimulation with 25 mg/ml zymosan A (Sigma-Aldrich). Supernatants were collected after 5 h. 

Isolation of human primary neutrophils Experiments using healthy volunteers were approved by the local research ethics committee (P/00/029 East London and The City Local Research Ethics Committee 1). Informed written consent was provided, according to the Declaration of Helsinki. Blood was collected into 3.2% sodium citrate and diluted 1:1 in RPMI 1640 before separation through a double-density gradient using Histopaque 10771 and 11191 (Sigma-Aldrich). Contaminating erythrocytes were removed by hypotonic lysis. Polymorphonuclear cells were incubated in 10% FCS overnight at 37˚C, 5% CO2 to let neutrophils undergo spontaneous apoptosis. 

Phagocytosis and efferocytosis Primary peritoneal macrophages were stimulated with compounds/vehicle for 30 min before the addition of zymosan A at 1:10 (macrophage/zymosan) ratio for 15 min, or apoptotic neutrophils (1:2, macrophage/neutrophil) for 1 h. Cells were fixed and neutrophils stained using the myeloperoxidase assay by adding 0.1 mg/ml dimethoxybenzidine (Sigma-Aldrich) and 0.03% (v/v) hydrogen peroxide for 1 h (18). Cells were analyzed by light microscopy with three random fields being acquired per well (n = 3 wells/ treatment). More than 400 cells were blindly counted per treatment point. Animals All animal studies were approved by and performed under the guidelines of the Ethical Committee for the Use of Animals, Barts and The London School of Medicine and Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986). Male (7–8 wk old) C57BL/6J wild-type (WT) and BALB/c mice were purchased from Charles River Laboratories. 

Breeding pairs of the Mc1r mutant (Mc1r2/2; recessive yellow e/e) and Mc3r2/2 colonies were originally obtained from Jackson Laboratories or donated by Dr. H.Y. Chen (Merck), respectively. Zymosan-induced peritonitis model Peritonitis was induced by the injection of 1 mg zymosan A (SigmaAldrich) i.p. in 0.5 ml sterile PBS. At the indicated times, mice were sacrificed by CO2 exposure, and peritoneal cavities were washed with 4 ml ice-cold PBS containing 3 mM EDTA. Cells were stained with Turk’s solution (0.01% crystal violet in 3% acetic acid) and counted using a Neubauer hemocytometer or were stained with FITC-conjugated mAb for Ly-6G/Gr1, F4/80, and corresponding isotype controls (eBioscience, Hatfield, U.K.), and subjected to flow-cytometry analysis using a BD FACSCalibur platform (BD Biosciences, Oxford, U.K.). K/BxN serum-induced arthritis model Arthritis was induced with two i.p. injections of 100 ml K/BxN serum on days 0 and 2.

 Disease was monitored daily until day 8 by assessing the paw volume using a plethysmometer (Ugo Basile, Comerio, Italy), disease incidence, and clinical score (score per paw: 0 = no signs of inflammation, 1 = subtle inflammation, localized, 2 = easily identified inflammation but localized, 3 = evident inflammation, not localized; maximum score = 12 3382 BIASED AGONISM AT MC1 AND MC3 REDUCES INFLAMMATION by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from per mouse). 

Pharmacological treatments were administered orally once daily from day 2 until the end of the experiment. Histological analysis Tissues (ankles) were fixed with 4% neutral-buffered formalin, decalcified with 10% formic acid, and paraffin embedded. Sections (4 mm) were stained with H&E (Sigma-Aldrich) or fast green and safranin O (SigmaAldrich). 

Sections were graded from 0 (no disease) to 3 (severe) based on the degree of synovitis and cartilage degradation. Statistical analysis Experiments were repeated from two to five independent times. Data were analyzed by Student t test, one- or two-way ANOVA, followed by Dunnett’s or Bonferroni’s multiple comparison test when appropriate. Nonlinear regression models were used to generate dose–response curves for cAMP and ERK phosphorylation. In all cases, data are presented as mean 6 SEM of n independent observations and were considered statistically significant when p , 0.05. 

Results AP1189 is a biased agonist at the MC receptors MC1 and MC3 AP1189 structure was designed on phenyl-pyrrole-aminoguanidine derivatives, shown to activate MC receptors (patent no. WO2007141343A1). However, when AP1189 was tested on HEK293A cells transfected with mouse receptors for activation of the canonical cAMP pathway, no signal was observed with MC3 cells and a response on MC1 cells only at high concentrations ($10 mM; Fig. 1A).

 In addition, no response was obtained in B16-F10 murine melanocytes (Supplemental Fig. 1A). cAMP accumulation for controls (empty vector pCMV6 transfected cells) are shown in Supplemental Fig. 2. These results are in agreement with binding assays where AP1189 displayed the tracer NDP-aMSH at high concentrations for MC1 with no effect at MC3 (Supplemental Fig. 1B). In contrast, AP1189 addition to mouse MC1 and MC3 transfected HEK293 cells afforded consistent and robust activation of ERK1/2 phosphorylation (Fig. 1B). 

This response was not restricted to the murine receptors and was confirmed in human MC1 and MC3 transfected cells (Supplemental Fig. 3). In addition, AP1189 induced Ca2+ mobilization, another melanocortin signaling response (Fig. 1C). 

To further explore this ligand bias observed with AP1189, we generated “biased plots” (15) using equimolar concentration– response curves for two signaling pathways (cAMP versus ERK1/2, as well as cAMP versus Ca2+) plotted as a function of each other (Fig. 2). 

These plots were constructed using the concentration–response curves for the three pathways shown in Fig. 1, and expressed as percentage maximal response of the reference aMSH. 

Considering the natural ligand aMSH as the reference standard, AP1189 showed preferential response to ERK1/2 phosphorylation and Ca2+ mobilization over cAMP, an effect observed for both MC1 and MC3 receptors (Fig. 2). 

Collectively, these results demonstrate that AP1189 is a biased agonist. Next, we questioned whether such a selective activation of MC receptors yielded biological activity. AP1189 exerts anti-inflammatory and proresolving actions in vitro Because mouse macrophages express both MC1 and MC3 receptors (18), we began the investigation of the functional properties of AP1189, using peritoneal macrophages from WT, Mc1r2/2, and Mc3r2/2 mice. Added to cells at nanomolar concentrations, AP1189 significantly reduced IL-1b, IL-6, and TNF-a in WT macrophages, whereas no effect was observed in Mc3r2/2 cells; the inhibitory effect was retained for IL-1b and IL-6, but not TNF-a, on Mc1r2/2 cells (Fig. 3A–C). Whereas cytokine inhibition is a typical anti-inflammatory effect, proresolving molecules must activate processes like phagocytosis and efferocytosis (19), effects we have reported for MC agonists (18). 

AP1189 promoted phagocytosis of zymosan particles by increasing both the proportion of phagocytic macrophages and the number of particles internalized per single cell (Fig. 3D). In addition, AP1189 promoted phagocytosis of apoptotic neutrophils (efferocytosis), a crucial event in resolution and for restoration of tissue homeostasis after an inflammatory event. AP1189 (optimal concentration of 1 nM) augmented efferocytosis of apoptotic neutrophils by ∼60% in WT macrophages (Fig. 3E). This effect was not evident in cells lacking either MC1 or MC3, suggesting a contribution of both receptors in the prophagocytic abilities of AP1189. To functionally associate biased agonism to biological outcome, we established the relevance of ERK1/2 phosphorylation in efferocytosis: addition of the ERK1/2 inhibitor FR180204 (1 mM) abrogated the proefferocytic actions of AP1189 (Fig. 3F). Melanogenesis is not induced by AP1189 Active in vitro concentrations of 0.1–1 nM AP1189 were tested on B16-F10 cells for potential promelanogenic properties. Incubated with this mouse melanocyte cell line, AP1189 did not induce formation of dendrites (at 24 h; Fig. 4A) or production of eumelanin, a response evident at day 3 on pelleted cells treated with the pan-agonists aMSH and NDP-aMSH (Fig. 4A, insets), as well as by the quantification of melanin in supernatants (Fig. 4B). These data, not unexpected because melanogenesis depends on the cAMP pathway, provide further functional confirmation to the biased properties of AP1189. AP1189 promotes resolution of acute inflammation in vivo The efficacy of AP1189 on cytokine release and phagocytosis justified follow-up studies to establish its ability to affect leukocyte migration in acute peritonitis using either a prophylactic (administration 30 min before zymosan) or a therapeutic (administration 2 h after the inflammatory insult) design. In addition, distinct administration routes were evaluated including i.p., i.v., and oral. Results, presented in Fig. 5A–D, report significant protective effects of AP1189 in vivo with ∼30–70% inhibition of neutrophil infiltration alongside both administration protocols and regardless of the administration route. Monocyte infiltration was also reduced (Supplemental Fig. 4A). In vivo proresolving effects were investigated by treating animals with AP1189 at the peak of inflammation (12 h postzymosan), where neutrophil numbers are the highest, and measuring the resolution indices as described previously (20). AP1189 accelerated resolution by reducing the time when peak values are reduced by 50% from 38 to 21 h (Fig. 5E). The resolution index (time interval from maximal response to time when peak values are reduced by 50%) was almost three times faster in the group of mice treated with AP1189 as compared with vehicle-treated mice (9 and 26 h, respectively). In addition, total cell (Fig. 5F) and monocyte/macrophage (Fig. 5G) infiltration were significantly reduced by AP1189. We also observed an increase in efferocytosis at 22 h afforded by AP1189 (Supplemental Fig. 4B), from 1.8 to 3. 1% phagocytic cells, although this difference did not reach statistical significance (p = 0.34). AP1189 did not alter the F4/80low/ Cd11blow population (12.1 and 15.12% for vehicle and AP1189 treated, respectively, p = 0.14, t test). These data indicate that tissues were more effectively cleared from recruited immune cells when treated with AP1189, effects conducive to resolution of inflammation and restoration of a physiological environment. The Journal of Immunology 3383 by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from Orally active AP1189 reduces arthritis in mice MC compounds are well-known for their antiarthritic actions in both preclinical (11, 21) and clinical settings, with ACTH reported to be clinically effective for the treatment of RA and gout (22, 23). We used the K/BxN serum transfer model, characterized by rapidonset severe inflammatory arthritis. Mice were challenged with two doses of arthritogenic serum (days 0 and 2), and AP1189 was administered once daily, 25 or 50 mg/kg orally, from day 2, when the arthritis begins to be macroscopically detectable. At 50 mg/kg, AP1189 reduced all signs of arthritis measured: clinical score (242%), paw swelling (287%), proportion of animals with all four paws affected (250%), and the severity of the inflammation (270%; Fig. 6A–E). Histological analyses (shown in Fig. 6F, 6G) revealed a significant reduction of synovitis, evident by the lower extent of leukocyte infiltration (monitored after H&E staining), whereas no evident effects were observed on cartilage protection (safranin O staining). FIGURE 1. Signaling profile of AP1189 at MC receptors. cAMP production, ERK1/2 phosphorylation, and intracellular Ca2+ mobilization were studied in HEK293A cells transiently transfected with mouse MC1 and MC3. (A) For cAMP assay, cells were stimulated with compounds for 15 min and samples analyzed by EIA. Forskolin (3 mM) was used as positive control. (B) For ERK1/2 phosphorylation analyses, cells were stimulated with AP1189 for 8 min and with aMSH for 5 min. ERK1/2 phosphorylation was analyzed by Western blot using a-Tubulin as loading control. Representative blots of three to four experiments are shown. Bands were quantified using ImageJ64 and the ratio of phospho-ERK1/2 and a-Tubulin calculated. Data were then expressed as the percentage of aMSH response. (C) Intracellular Ca2+ mobilization was measured using Fura-2 AM–labeled cells in the NOVOstar microplate reader. Ionomycin (1 mM) was used as positive control. The ratio of fluorescence emission at 510 nm after sequential excitation at 340 and 380 nm was recorded. Concentration–response curves were generated using the Ca2+ response 25 s after drug stimulation. Experiments were repeated two to five times and analyzed by nonlinear regression. FIGURE 2. Biased agonism at MC receptors by AP1189. Biased plots were generated using the cAMP concentration–response curves (EIA assays) and those generated from ERK1/2 Western blots densitometric analyses (A) or Ca2+ flux assays (B); see Fig. 1 for original data. All data for cAMP, ERK1/2, and Ca2+ are expressed as percentage maximal response of aMSH. 3384 BIASED AGONISM AT MC1 AND MC3 REDUCES INFLAMMATION by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from Discussion We report in this article that biased agonism at MC receptors can be a viable, and innovative, approach to exploit a large wealth of MC biology for therapeutic treatment of pathological inflammation. We performed an integrated series of experiments weaved on a rationale that started from the discovery of unorthodox agonism to the assessment of in vivo and in vitro biological properties of AP1189. Our results provide proof-of-concept data to effect therapeutic innovation beyond classical orthosteric ligands to deliver translational MC drugs. Biased agonists at MC receptors offer new opportunities for the development of therapeutics with improved profile by activating pathways that are therapeutically relevant and evading those associated with side effects (15, 16). The appreciation that therapeutic outcomes might derive from distinct downstream signaling events indicates that not all signaling pathways ascribed to a specific receptor need to be activated by a candidate drug molecule. Indeed, biased opiate analgesics devoid of respiratory depressive effects are under development (24). The concept of ligand bias, relatively new for therapeutic approaches, calls for: 1) a better dissection of signaling pathways required for therapeutic efficacy, 2) the identification of pathways associated to side effects, and 3) a careful design of drug screening and lead candidates optimization according to the desired pathways.

 In our attempts to establish new biology that supports approaches for novel anti-inflammatory/proresolving therapeutics, we focus on the MC receptor subtypes MC1 and MC3, both expressed and playing tonic and nonredundant protective roles in immune cells and synovial tissue, among others (2, 18, 21, 25). 

The MC peptide ACTH has long been known to be effective for the treatment of gout and RA (26). Subsequently, efficacy in inflammatory bowel disease, multiple sclerosis exacerbations, or nephrotic syndrome has been reported, but overall the use of ACTH is by and large limited for side effects associated with increased cortisol production through MC2 activation (23, 27, 28). 

MC1 represents an appealing anti-inflammatory target: its selective activation inhibits leukocyte adhesion and emigration in a model of ischemia-reperfusion injury (29) and evokes protection against lung injury as well as in a model of delayed-type hypersensitivity (30). However, its promelanogenic actions impose a considerable limitation to MC1 as a safe target candidate when chronic treatment is predicted. Selective targeting of MC3 may result in improved safety profile and particular efficacy for joint inflammation. This receptor mediates the antiarthritic actions of the synthetic polypeptide DTrp8 -gMSH in the K/BxN model, as well as the negative regulation of osteoclast generation and activation (21, 31), as well as crystal-induced inflammation (25). The FIGURE 3. In vitro anti-inflammatory actions of AP1189. 

Biogel-elicited peritoneal macrophages were collected from WT, Mc1r2/2, or Mc3r2/2 mice and stimulated for 30 min with AP1189 before the addition of zymosan A (25 mg/ml) for 5 h. IL-1b (A), IL-6 (B), and TNF-a (C) were measured in supernatants. (D) Phagocytosis was assayed in WT cells, pretreated with 1 nM AP1189 for 30 min, after addition of zymosan particles at 1:10 ratio (macrophages/zymosan) for 1 h. The proportion of phagocytic cells and the number of internalized particles per 100 cells (phagocytic index) were analyzed. (E and F) Efferocytosis of apoptotic neutrophils was assayed in WT, Mc1r2/2, and Mc3r2/2 macrophages (E) and in WT in the presence of the ERK1/2 inhibitor FR180204 at 1 mM (F). Apoptotic neutrophils were added to the macrophages at a ratio of 1:2 on AP1189-pretreated macrophages (as in D), and 1 h later, myeloperoxidase staining was performed to visualize ingested neutrophils. Data are the mean 6 SEM of three independent experiments. *p , 0.05 versus control (WT [A–C] or vehicle [D–F]). FIGURE 4. Effects of MC compounds on melanogenesis. (A) B16-F10 cells were treated with AP1189 or the pan-agonists aMSH and NDP-aMSH for 1 d (monolayer cells are shown; original magnification 340). At day 3, cells were collected by centrifugation and pellets photographed. (B) Melanin production was quantified spectrophotometrically by measuring absorbance at 405 nM, 72 h after drug stimulation. aMSH was used at 10 mM and NDPaMSH at 100 nM. Data are the mean 6 SEM of n = 8, representative of two independent experiments. ***p , 0.01 versus vehicle (Veh). The Journal of Immunology 3385 by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from major obstacle in this study derives from the absence of selective MC3 agonists devoid of off-target effects associated particularly with MC1 and MC4 activation. In this article, we characterize the orally active small molecule AP1189 as a biased agonist with the additional advantage of targeting both tissue-protective MC1 and MC3 receptors. AP1189 activation of MC receptors did not induce cAMP accumulation, the canonical pathway ascribed to MC agonists and used for drug screening programs (e.g., see Pantel et al. [14]). AP1189 interaction with MC1 and MC3 activated a second pathway centered on ERK1/2 phosphorylation, as well as intracellular Ca2+ mobilization. Although somehow neglected, these two signaling pathways have been described by downstream activation of most MC receptors (32). The immediate aspect we addressed was the analysis of the consequences of this unusual bent toward the currently considered noncanonical pathways. Eumelanogenesis is unquestionably dependent on cAMP induction by MC1 (33) whereas ERK1/2 phosphorylation seems to meFIGURE 5. In vivo anti-inflammatory actions of AP1189. Acute peritonitis was induced with one single injection of 1 mg zymosan, and leukocyte infiltration was analyzed by flow cytometry. (A–C) Prophylactic experimental design: AP1189 was administered 30 min before zymosan injection and cells were analyzed at the 4-h time point. (D) Therapeutic design: AP1189 was administered 2 h after zymosan, and cells were analyzed at the 6-h time point. (E) AP1189 promotes resolution. AP1189 (1 mg/kg) or vehicle were injected i.p. at 12 h postzymosan, and cells were analyzed at the 22- or 44-h time point. Neutrophils infiltration and resolution indices are shown: Cmax = maximal neutrophil infiltration; Tmax = time when maximal neutrophil infiltration is achieved; T50 = time when maximal neutrophil infiltration is reduced by 50%; Ri = time from maximal response to T50. (F and G) Total cells numbers and monocytes/macrophages values from the experiment in (E). AP1189 was administered i.p. (A and E–G), i.v. (B and C), or orally [p.o., (D)]. C57BL/6 mice were used in (A) and (E)–(G). BALB/c mice were used in (B)–(D). Data are the mean 6 SEM of n = 5–6 per group. *p , 0.05 versus control (vehicle). FIGURE 6. Antiarthritic actions of AP1189 in the K/BxN serum transfer model. Arthritis was induced on C57BL/6 mice by two i.p. injections of arthritogenic serum on days 0 and 2. From day 2, AP1189 or vehicle (PBS) were administered orally once daily. Clinical score (A), paw swelling (B), disease incidence (C), number of mice with all paws affected (D), and the number of paws per mouse severely affected (E) were recorded over 8 d. (F and G) Paraffin tissue sections were stained with H&E and fast green and safranin O. Sections were graded from 0 (no disease) to 3 (severe) based on the degree of synovitis (purple staining in the H&E sections) and cartilage degradation (loss of red coloration in the safranin O sections, arrows). Representative images are shown (original magnification 34). Data are the mean 6 SEM of n = 6 per group. *p , 0.05 versus control (vehicle). B, bone; S, synovitis. 3386 BIASED AGONISM AT MC1 AND MC3 REDUCES INFLAMMATION by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from diate the anorexigenic effects of MC4 (34); but whether ERK1/2 phosphorylation occurs downstream of cAMP is controversial. Inhibition of NF-kB activation upon MC activation also has been linked to the cAMP pathway (35). However, anti-inflammation via ERK1/2 activation, in conjunction or not with cAMP induction, has also been suggested (36, 37). In this respect, we show that AP1189 reduced cytokines release by zymosan-stimulated macrophages and promoted both phagocytosis and efferocytosis by this cell type. These effects were produced with distinct profiles when using Mc1r2/2 and Mc3r2/2 cells, suggesting nonredundant engagement for both receptors by AP1189. The protective actions of AP1189 were absent in Mc3r2/2 cells for IL-1b, IL-6, and TNF-a release and efferocytosis. With Mc1r2/2 cells, only the effects on TNF-a release and efferocytosis were prevented. In addition, promotion of efferocytosis by AP1189 was entirely dependent on ERK1/2 activation, as observed by using a selective inhibitor. As predicted, the biased signaling dissociated from cAMP also prevented the unwanted pigmentary effects on melanocytes, including eumelanin production and formation of dendrites. These findings, together with the fact that the in vivo experiments were performed in BALB/c mice (Fig. 5B–D), exclude any involvement of melanogenesis and melanin, a molecule with protective antioxidant properties (38), in the actions of AP1189. We have pioneered the concept that therapeutic innovation for inflammatory diseases could derive from appreciation and exploitation of the mechanisms operative during the resolution phase of inflammation (39, 40). Such an approach, we propose, would yield therapeutics that would not be resolution toxic by repressing or delaying tissue repair processes and the ensuing restoration of homeostasis (40). To this end, we took advantage of the resolution indices, introduced by Serhan’s group, to quantify the impact of novel drugs in the resolution phase of inflammation (20). When administered at the peak of neutrophil infiltration, that is, right before the beginning of resolution, AP1189 promoted resolution. Analysis of the resolution indices indicated that in AP1189-treated mice, recovery was achieved three times faster than in vehicletreated animals. Monocyte/macrophage counts were also reduced by AP1189. This effect could be because of reduced infiltration, because we observed that AP1189 at 10 mg/kg afforded a 37% reduction of peritoneal infiltration of this cell type after zymosan (Supplemental Fig. 4A). However, increased efflux of macrophages after increased efferocytosis could also contribute to the lower counts quantified at a given time point (Supplemental Fig. 4B). Exciting results were also obtained with the oral treatment with AP1189 in the serum transfer model of arthritis. This arthritogenic serum contains anti–glucose-6-phosphate-isomerase Abs that, when passively transferred to naive mice, form immunocomplexes on the cartilage and synovial surface, which attract and activate neutrophils and mast cells initiating joint inflammation (41). In line with data produced with classical MC agonists such as AP214 (18) or DTrp8 -gMSH (21, 31), oral administration of AP1189 reduced clinical score, paw swelling, incidence of severe disease incidence, and the overall disease severity. Microscopically, AP1189 significantly decreased leukocyte infiltration and synovitis, although no significant changes were observed on cartilage degradation. These findings, together with the wellknown protective effects of ACTH in human joint diseases, present the MC AP1189 as a promising oral candidate for pathologies of the joint including RA. MC receptors are products of genes that are very polymorphic, thus making it necessary to determine whether gene variants could impact on the pharmacological profile. In this vein, Herraiz et al. (42) found that red hair variants of MC1 presented reduced cAMP response to NDP-aMSH, but normal ERK1/2 response. Moreover, Doyle et al. (37) showed that red hair variants do not impact on the anti-inflammatory properties, measured as inhibition of NF-kB activation, of the MC1 selective small molecule BMS-470539. These studies indicate to us that although MC receptor polymorphism must be considered and tested for each MC candidate agonist, fully active molecules on MC1 variants are plausible, hence the low activity elicited by the endogenous ligand does not necessarily predict low activity for synthetic molecules. Indeed, a similar situation occurs for MC4: Haslach et al. (43) recently identified a number of peptides with full agonistic activity at lossof-function obesity-related MC4 variants. Another matter of interest derived from the work presented in this article will be the identification of molecular or chemical determinants that lead to biased agonism. AP1189 was not able to displace NDP-aMSH from binding to MC3 but was able to induce ERK1/2 phosphorylation and Ca2+ mobilization. The same was essentially true also for MC1, where apparent competition occurred at concentrations .1,000-fold higher than those active for ERK1/2 phosphorylation. Altogether these studies suggest that AP1189 binds to an allosteric site, hence it acts as a biased allosteric agonist, explaining the differential activation profile observed when compared with classical “orthosteric” agonists. Elucidation of these determinants might guide a rational design of a new class of drugs combining allosterism and ligand bias. We note how Ca2+ mobilization, albeit not novel for MC receptor agonists, is yet to be functionally linked to the anti-inflammatory actions of MC-based compounds. Our data on the biased agonist AP1189 also suggest the urgency for a redesign of current MC-based drug discovery approaches (by including noncanonical pathways) and possibly even reassessment of known compounds. In fact, AP1189 could have been mistakenly described as a weak antagonist at MC1, active at micromolar concentrations, if cAMP signaling and binding properties were solely studied. Similar conclusions have been suggested for the endogenous antagonist, or more correctly inverse agonist, agoutirelated protein (AGRP). AGRP can actually initiate Gi/o protein– induced signaling (44), as well as internalization of MC3 and MC4, a receptor turn-off mechanism attributed to agonistic activity (45). More recently, AGRP has been identified as a biased agonist at MC4 causing, indeed, activation of the ERK1/2 pathway (46). In summary, we report in this article a novel strategy to harness the tissue-protective properties of MC receptors in inflammatory settings. Our data indicate that, in addition to a redefinition of efficacy (quality/quantity), drug selectivity needs to be reconsidered in terms of “receptor” or “pathway” selectivity, given the impact this may have on the development and characterization of novel therapeutics and subsequent medicinal chemistry programs. We also show that ERK1/2 phosphorylation downstream MC1 and MC3 activation leads to proresolving and anti-inflammatory actions, despite absence of cAMP signaling. New knowledge from basic research needs to be better translated into industry and incorporated into the drug-discovery process: despite that the existence of ligand bias has been known for .20 y, it is only now that its relevance is being appreciated. We propose that in the MC receptor discovery arena, biased agonism is a novel approach that can lead to improved therapeutics, perhaps like AP1189 acting on two nonredundant anti-inflammatory targets at once, yet likely devoid of side effects associated with cAMP activation. Disclosures T.E.N.J. is shareholder in SynAct Pharma and owns intellectual property on AP1189. The Journal of Immunology 3387 by guest on May 26, 2021 http://www.jimmunol.org/ Downloaded from References 1. Cone, R. D. 2006. Studies on the physiological functions of the melanocortin system. Endocr. Rev. 27: 736–749. 2. Bo¨hm, M., and S. Gra¨ssel. 2012. Role of proopiomelanocortin-derived peptides and their receptors in the osteoarticular system: from basic to translational research. Endocr. Rev. 33: 623–651. 3. Catania, A. 2008. Neuroprotective actions of melanocortins: a therapeutic opportunity. Trends Neurosci. 31: 353–360. 4. Getting, S. J. 2006. Targeting melanocortin receptors as potential novel therapeutics. Pharmacol. Ther. 111: 1–15. 5. Catania, A., V. Gerloni, S. Procaccia, L. Airaghi, M. G. Manfredi, C. Lomater, L. Grossi, and J. M. Lipton. 1994. The anticytokine neuropeptide alphamelanocyte-stimulating hormone in synovial fluid of patients with rheumatic diseases: comparisons with other anticytokine molecules. Neuroimmunomodulation 1: 321–328. 6. Rajora, N., G. Ceriani, A. Catania, R. A. Star, M. T. Murphy, and J. M. Lipton. 1996. alpha-MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line. J. Leukoc. Biol. 59: 248–253. 7. Buckley, C. D., D. W. Gilroy, C. N. Serhan, B. Stockinger, and P. P. Tak. 2013. The resolution of inflammation. Nat. Rev. Immunol. 13: 59–66. 8. Wallace, J. L. 2014. Exploiting endogenous anti-inflammatory pathways as a therapeutic approach to multiorgan inflammatory disease. Am. J. Pathol. 184: 2154–2155. 9. Hench, P. S., E. C. Kendall, C. H. Slocumb, and H. F. Polley. 1950. Effects of cortisone acetate and pituitary ACTH on rheumatoid arthritis, rheumatic fever and certain other conditions. Arch. Intern. Med. (Chic). 85: 545–666. 10. Getting, S. J., H. C. Christian, R. J. Flower, and M. Perretti. 2002. Activation of melanocortin type 3 receptor as a molecular mechanism for adrenocorticotropic hormone efficacy in gouty arthritis. Arthritis Rheum. 46: 2765–2775. 11. Montero-Melendez, T., H. B. Patel, and M. Perretti. 2011. Role of melanocortin receptors in the regulation of gouty inflammation. Curr. Rheumatol. Rep. 13: 138–145. 12. Mapa, J. B., and M. H. Pillinger. 2010. New treatments for gout. Curr. Opin. Investig. Drugs 11: 499–506. 13. Wootten, D., A. Christopoulos, and P. M. Sexton. 2013. Emerging paradigms in GPCR allostery: implications for drug discovery. Nat. Rev. Drug Discov. 12: 630–644. 14. Pantel, J., S. Y. Williams, D. Mi, J. Sebag, J. D. Corbin, C. D. Weaver, and R. D. Cone. 2011. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay. Eur. J. Pharmacol. 660: 139–147. 15. Kenakin, T., and A. Christopoulos. 2013. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat. Rev. Drug Discov. 12: 205– 216. 16. Reiter, E., S. Ahn, A. K. Shukla, and R. J. Lefkowitz. 2012. Molecular mechanism of b-arrestin-biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52: 179–197. 17. Mastrofrancesco, A., A. Kokot, A. Eberle, N. C. Gibbons, K. U. Schallreuter, E. Strozyk, M. Picardo, C. C. Zouboulis, T. A. Luger, and M. Bo¨hm. 2010. KdPT, a tripeptide derivative of alpha-melanocyte-stimulating hormone, suppresses IL-1 beta-mediated cytokine expression and signaling in human sebocytes. J. Immunol. 185: 1903–1911. 18. Montero-Melendez, T., H. B. Patel, M. Seed, S. Nielsen, T. E. Jonassen, and M. Perretti. 2011. The melanocortin agonist AP214 exerts anti-inflammatory and proresolving properties. Am. J. Pathol. 179: 259–269. 19. Ortega-Go´mez, A., M. Perretti, and O. Soehnlein. 2013. Resolution of inflammation: an integrated view. EMBO Mol. Med. 5: 661–674. 20. Bannenberg, G. L., N. Chiang, A. Ariel, M. Arita, E. Tjonahen, K. H. Gotlinger, S. Hong, and C. N. Serhan. 2005. Molecular circuits of resolution: formation and actions of resolvins and protectins. J. Immunol. 174: 4345–4355. 21. Patel, H. B., M. Bombardieri, A. L. Sampaio, F. D’Acquisto, M. Gray, P. Grieco, S. J. Getting, C. Pitzalis, and M. Perretti. 2010. Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis. FASEB J. 24: 4835–4843. 22. Daoussis, D., I. Antonopoulos, and A. P. Andonopoulos. 2014. ACTH as a treatment for acute crystal-induced arthritis: update on clinical evidence and mechanisms of action. Semin. Arthritis Rheum. 43: 648–653. 23. Schlesinger, N. 2008. Overview of the management of acute gout and the role of adrenocorticotropic hormone. Drugs 68: 407–415. 24. Groer, C. E., K. Tidgewell, R. A. Moyer, W. W. Harding, R. B. Rothman, T. E. Prisinzano, and L. M. Bohn. 2007. An opioid agonist that does not induce mu-opioid receptor—arrestin interactions or receptor internalization. Mol. Pharmacol. 71: 549–557. 25. Getting, S. J., C. W. Lam, A. S. Chen, P. Grieco, and M. Perretti. 2006. Melanocortin 3 receptors control crystal-induced inflammation. FASEB J. 20: 2234– 2241. 26. Hench, P. S. 1950. Introduction: cortisone and ACTH in clinical medicine. Proc. Staff Meet. Mayo Clin. 25: 474–476. 27. Gong, R. 2012. The renaissance of corticotropin therapy in proteinuric nephropathies. Nat. Rev. Nephrol. 8: 122–128. 28. Berkovich, R., and M. A. Agius. 2014. Mechanisms of action of ACTH in the management of relapsing forms of multiple sclerosis. Ther. Adv. Neurol. Disord. 7: 83–96. 29. Leoni, G., M. B. Voisin, K. Carlson, S. Getting, S. Nourshargh, and M. Perretti. 2010. The melanocortin MC(1) receptor agonist BMS-470539 inhibits leucocyte trafficking in the inflamed vasculature. Br. J. Pharmacol. 160: 171–180. 30. Kang, L., K. W. McIntyre, K. M. Gillooly, Y. Yang, J. Haycock, S. Roberts, A. Khanna, T. F. Herpin, G. Yu, X. Wu, et al. 2006. A selective small molecule agonist of the melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice. J. Leukoc. Biol. 80: 897– 904. 31. Montero-Melendez, T., M. F. Madeira, L. V. Norling, A. Alsam, M. A. Curtis, T. A. da Silva, and M. Perretti. 2014. Association between periodontal disease and inflammatory arthritis reveals modulatory functions by melanocortin receptor type 3. Am. J. Pathol. 184: 2333–2341. 32. Garcı´a-Borro´n, J. C., B. L. Sa´nchez-Laorden, and C. Jime´nez-Cervantes. 2005. Melanocortin-1 receptor structure and functional regulation. Pigment Cell Res. 18: 393–410. 33. Busca`, R., and R. Ballotti. 2000. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res. 13: 60–69. 34. Sutton, G. M., B. Duos, L. M. Patterson, and H. R. Berthoud. 2005. Melanocortinergic modulation of cholecystokinin-induced suppression of feeding through extracellular signal-regulated kinase signaling in rat solitary nucleus. Endocrinology 146: 3739–3747. 35. Sarkar, A., Y. Sreenivasan, and S. K. Manna. 2003. alpha-Melanocyte-stimulating hormone induces cell death in mast cells: involvement of NF-kappaB. FEBS Lett. 549: 87–93. 36. Ottani, A., M. Galantucci, E. Ardimento, L. Neri, F. Canalini, A. Calevro, D. Zaffe, E. Novellino, P. Grieco, D. Giuliani, and S. Guarini. 2013. Modulation of the JAK/ERK/STAT signaling in melanocortin-induced inhibition of local and systemic responses to myocardial ischemia/reperfusion. Pharmacol. Res. 72: 1–8. 37. Doyle, J. R., J. P. Fortin, M. Beinborn, and A. S. Kopin. 2012. Selected melanocortin 1 receptor single-nucleotide polymorphisms differentially alter multiple signaling pathways. J. Pharmacol. Exp. Ther. 342: 318–326. 38. Denat, L., A. L. Kadekaro, L. Marrot, S. A. Leachman, and Z. A. Abdel-Malek. 2014. Melanocytes as instigators and victims of oxidative stress. J. Invest. Dermatol. 134: 1512–1518. 39. Perretti, M. 1997. Endogenous mediators that inhibit the leukocyte-endothelium interaction. Trends Pharmacol. Sci. 18: 418–425. 40. Serhan, C. N., S. D. Brain, C. D. Buckley, D. W. Gilroy, C. Haslett, L. A. O’Neill, M. Perretti, A. G. Rossi, and J. L. Wallace. 2007. Resolution of inflammation: state of the art, definitions and terms. FASEB J. 21: 325–332. 41. Ditzel, H. J. 2004. The K/BxN mouse: a model of human inflammatory arthritis. Trends Mol. Med. 10: 40–45. 42. Herraiz, C., C. Jime´nez-Cervantes, P. Zanna, and J. C. Garcı´a-Borro´n. 2009. Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways. FEBS Lett. 583: 3269–3274. 43. Haslach, E. M., H. Huang, M. Dirain, G. Debevec, P. Geer, R. G. Santos, M. A. Giulianotti, C. Pinilla, J. R. Appel, S. R. Doering, et al. 2014. Identification of tetrapeptides from a mixture based positional scanning library that can restore nM full agonist function of the L106P, I69T, I102S, A219V, C271Y, and C271R human melanocortin-4 polymorphic receptors (hMC4Rs). J. Med. Chem. 57: 4615–4628. 44. B€uch, T. R., D. Heling, E. Damm, T. Gudermann, and A. Breit. 2009. Pertussis toxin-sensitive signaling of melanocortin-4 receptors in hypothalamic GT1-7 cells defines agouti-related protein as a biased agonist. J. Biol. Chem. 284: 26411–26420. 45. Breit, A., K. Wolff, H. Kalwa, H. Jarry, T. B€uch, and T. Gudermann. 2006. The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors. J. Biol. Chem. 281: 37447–37456. 46. Mo, X. L., and Y. X. Tao. 2013. Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant huma

[macgyver]

Sorry to interject, but I think Mr Svensson was out of line for attacking Uhoh on this forum. There has been enough of that in the past here and it would be disappointing to go back to that. Also pointless trying to demean someone by calling them an "Aspie", why is that a derogatory term? Elon Musk seems to be doing ok...

There isn't any doubt in my mind that eventually we will see an approved melanocortin agonist from a competing company. There are drugs approved across the world with negligible benefits so I can certainly see such an agonist drug approved if safety holds even if it only provides a negligible benefit. The potential for melanocortin therapies is vast, too vast for Clinuvel to secure and I'm not even sure PW wants to do that. Trying to do so is folly and doesn't make good business sense in the long term if you lose focus and spread yourself too thin. I'd have a problem with PW's strategy if they didn't pursue atherosclerosis/CVD at some point in the future because of its obvious financial windfall if proven successful, just look at the statin drug market and how much that is valued at. 

Its pleasing actually to see another pharmaceutical company attempt a foray into this area, I think its pretty clear to everybody how important pursuing CVD is. And I wouldn't like to write SynAct off just yet. If their drug has even a slight rate of efficacy, it would have an easier time of getting approval as CVD is one of the top killers globally. Generally with these types of indications, the need trumps concerns about weak efficacy. But of course it doesn't trump safety so that is the current problem for SynAct and Mitsubishi Tanabe. The company that brings a safe agonist to market (besides Palatin, Clinuvel or Acthar) are going to do very well imo.

To end, it would be extremely disappointing to see this forum shut down. Its too important with its immense repository of related information to dismiss, and the effort put into it by Uhoh needs to be recognised. Strong debate is always healthy, attacking is not.

[Uhohinc]

SynAct Pharma, has ZERO employees....................none. As reported in it's latest annual report.  SynAct has ZERO intention of entering a phase III in anything, and its managers  owners many who came from Action Pharma, state their intent is to sell the rights after a phase II.  I do not know how much money it has in the bank, but in the annual the claim is made enough to make it thru a phase IIa............................

[Uhohinc]

https://news.cision.com/synact/r/synact-carries-out-a-directed-share-issue-and-raises-proceeds-of-sek-80-million---english-version---,c3280090     I think the Claes was trolling to try and pump up Synactin on gg expecting to get more investors............Typical pumper dumper,  "Wait and see" but he could not back up anything he claimed.  He got angry ..............not because of me or my posts or citing or copy and paste...........he got himself angry and emotive, not because I saw incongruities, but because he saw them for himself, and these did not fit in with his construct of getting rich on his Synactin stock with Covid and ARDS..........his dream is an delusion, his emperor has no clothes.......I poked a big hole in his dream, and that he could not deal with..............that made me ignorant (thank you very much can I have some more of that )   which I think is a complement as I am ignorant...............but I have google  which makes me less ignorant then...................If you come on a public website and expose your self and dish it out, be prepared to take it too.  My ego is not hurt by anything he wrote, but his ego and sense of self he walked right into a big mirror and shattered his delusion himself and saw himself .  I would rather be ignorant with access to the web, than a know it all..........

The Aspie comment, I find humorous, did not irk me, it showed me the man can not handle a discourse not favorable to him very long, he lost it, lost his original frame that started with his first post, lost any debate or reference to facts or abstracts, lost his temper, lost his mind , and will lose his money. or not make as much as his cherry picked delusion........better him then me.....

 The insights into the subconscious we all mostly repress unintentionally or do not even recognize or can use the cognitive frontal lobe to not verbalize, come out  when we laugh and release that stress in a joke or comment or observation, but a big truth comes out when one emotionally lets the limbic true thoughts take over and bespoke in anger., .............................This Claus would not identify himself, his interest in melanocortins, or why he carries the water for Synactin..........he had a internet version of road rage,  He may wise up, as his frustration showed when he could not himself explain the incongruities in the patent.......maybe he should invest by reading his horoscope daily. 

As for Covid, everyone has an opinion , but as far as I can see one who thinks Covid is going to come back or can be monetized is delusioned.......Sure, I have posted on ARDS and the advantages of melanocortins appear in the lungs................but having one of your board members start a Covid trial in Brazil is Bizzare.   And Synactin has no employees, not even a lady to open the mail or answer the phone.............the officers and management give out their personal phone and email......The RArthitis trial phase II had 12 patients on the drug................thats all,  that tells me Synactin has no serious thinking of submittal to FDA.....and that is not conjecture, the company states it has no intent of entering a phase III, it is all about selling the dream of big pharma "may" fish up the likes of a Mitsubishi Tanabe, which bought its MT-7117 oral melanocortin....You give me 12 people with RA, and tout your improvement over the placebo by just two or four patients........thats ignorant.........A over the counter NASAID or a dozen drugs already on market do more than that.............and probably what was not excluded criteria....And the so called improvement was not much..........And where is the statisticals. What is that p value Claus...................

Synactin appears to be a trail of owners who came from Action, who acquired from Mallinkrodt something who acquired from Zealand pharma........and its all followed on gg, even when Martin Skhreli attempted to buy the acth drug in compete to Abbie Vie.........I bring this up because Syactin has a trial in kidney, but so was the drug  and Acth abruptly stopped in trial, and it is not known to me why.....

That being said, i have an open mind, and will watch this AP1189 from my researched reality.......

[Vallaurix_CUV]

.

[Claes Svensson]

Wow. You did it again! Alot of words, alot of time wasted.

It is now clear that you have ZERO understanding of what is going on. The ignorant american shows himself to be even more ignorant. The world doesnt revolve around you. In some other parts of the world, things are done differently.

The plan has ALWAYS, since day 1, been to take this candidate through phase2-studys in different inflammatory diseases, and then sell/license it. Nothing new, and nothing weird. The team co-owns a candidate with Abbvie... But naah.. Its all probably fake.. "Just trying to sell the dream"...

" RArthitis trial phase II had 12 patients on the drug................thats all"... You told me you could read?

So the RA-study only has 12 patients? Only a retard would think that after looking into the facts. The study is to screen up to 135 patients, estimated inclusion between 90-115 patients.

Phase1 - drug tested on healthy volunteers. Phase 2a, still a safety study since they now try it on sick patients. After the first 12 patients had been recruited and dosed, they saw that the 50mg dose of AP1189 was safe and well tolerated

and they could then start dosing with 100mg-dose.

Aspirin? Against severe rheumatoid arthritis? Come on, theres gotta be some limit of your stupidity. Severe RA is treated with MTX/DMARDS, biologics, JAK-inhibitors etc... Aspirin? Wow...
"which may be attributed to AP1189" - for obvious reasons dumbass, its a double blind study. But some patients have reached 80% decrease in CDAI-score. Cant give credit to either MTX or placebo on that one buddy.

No employes, all consultants with high expertise and a track record not very often seen. Slim organization.
The management and board owns +20% of the shares, in lockup since IPO in 2016, and the lockup-period goes all through 2021 and over the 3 study results.

Get your story straight. Questcor bought Acthar Gel. Questcor bought TXPs candidate. Mallinkrodt then bought Questcor and halted all other studys and programs and only focused on Acthar Gel.

Everyone here can fact check your statements, and see that you are full of shit. 12 patients.... Wow.. Dont know if stupid, or just trollin'...

Good luck beeing taken serious in the future. Now go work on thoose google skills, looks like you need it!

[Uhohinc]

Words......... later in this response I am going to give you some word that you are to stupid to want to read and understand.........Fuck you very much would you like some more please............thats the second time you come back to read my words, then state I write a lot of words.......Thats why you came here, for more of my words. You wanted me and came to gg out of your getting close to desperation, a  need me for affirmation. To do your homework, Words to assist you in the thinking beyond you can not do. You came to gg to get the opinion you hoped for.....but if you are already in the hope frame of mind more so than confident states of mind and positive emotions.........You came to me and your my troll bitch. 

You are so full of anxiety about your little not to good drug, not too good stock investment. You already know in your head your wrong..................but you are full of hope...And you hope it will pay off, and you hoped I would give you something to glum onto with some more of that empty region in your head that after all other brain cognitive and emotions goes to one of the last mental states of hope.........You came here on gg because you thought some Asperger smart guy would throw you a alternative to the useless emotion hope..........And you hoped I would do the research, read the words, and confirm what your heart wants but your head knows just a little voice in your head is a looooooooooong shot.....And I shot your logic, your research, your dream and your hope down with a big reality.....Your free ride response is over.........And I purposely let you wait for this reply to let your anticipation, your dopamine motivation, your cortisol, your stress, and others I be ignorant for you........As for the only 12 patients on the Ap1189, these were interim release results, of what was reported to be a double blind trial where no one knows the results, but now go figure how they publiced them and then they changed the trial because it was not showing the result they, and you have delusioned away that is not in an orphan disease and has fewer patients than EPP... And they know it will not ever get FDA or EMA approval for a trial, they tried, so they are pretending they have no intention of running a legitimate trial under the strict standards, and no intention of ever selling the drug........they are going to raise money and sell a story with a once upon a time story that you already bought  by naming the company after the drug of another company and throwing words next to it like you use Big Pharma, Pfizer, ...and SynAct needs more suckers like you to buy into their Forest Gump shrimp AP1189.........

You are not just an idiot, your a big fucking idiot........do you know what a 2 is and a 2a is .........dumbshit........in case that does not translate to Swiss.....that is dumbshit....Do you see where the result you quote from my quote above which I got as a quote from SynAct blind trial not completed interim resultssss........it  has the fucking word "may"...................well that is one word that says the drug did not work, and says the drug did not fail, but we do not want to loose are stupid people like Claes because he needs the words to keep is dream in his head and his money folowing his dream............................I can not ague over a p value...................there is not one.....you phucking idiot.

You show me a drug company with a  plan to not go commercial, not do a phase III trial, and I will show you a company with no phase III and nothing commercial.......

Are you the only fucking idiot in the world that thinks Synact is going to do a trial in Brazil conducted by a paid whore of Synact under zero regulatory collaborations and approvals before being put in a human in USA or Europe.............and if Santa Clause and God and a pink Unicorn that shits gold nuggets  can get hired as the first employees of SynActin can get it done in record time ....................If you are still here, and still a phucking idiot, reading my words, then Synactin will corner the market on Covid ARDS in 2029 just before such patent runs out........

Read what you wrote here and see where your head is..................................

"The plan has ALWAYS, since day 1, been to take this candidate through phase2-studys in different inflammatory diseases, and then sell/license it. Nothing new, and nothing weird. The team co-owns a candidate with Abbvi"

The drug is a ripoff of Mitsubishi Tanabi  trying get credability and legitimacy by association of only words with the ACTH drug but it is really like you said.........it is shiney and sparkly to get you interested but your future buyer does not exist now, and I do not think any one that can get to top of pharma for your plan to Phfailase II will be as stupid as ..........................Claess and if you had showed some class Claes and not responded in this discourse from your fucked up emotions and financial desperate hopes...........you got my free opinion.........Ignorant American good buy........

[Uhohinc]

by the way I do not have Aspieburger nor a Swiss Army knife....

[macgyver]

No indeed, I'm the only one here with a Swiss Army knife......and a roll of duct tape:)

[Claes Svensson]

Hahahaahaha roflmao! Wow you're stupid, big time. Once again, you come here as a retard with false and made up information. Don't know if you are trying to fool yourself or anyone else...
And once again I will have to set the facts straight and show the world what a lying little cunt you are :)
You really think that I'm here for your opinion? HAHAHA! You? I know more than you ever will on this subject :)

I came here because I saw this "AP1189 a Erk1/2 pathway without Mcr1 or Mcr3 activation" - This headline is so dumb and retarded. And then you cite the preclinical article that clearly states that "AP1189 is a biased agonist that activates MC1R and MC3R". Later the article tells that activation of Mc1r is through the ERK1/2 pathway instead of the usual cAMP thus not inducing melanogenesis. But still you thought your headline was correct? You dont see the faults? Smart... That's why I asked if you could READ. Then you went full retard and showed more of your smartness - Aspirin against severe RA. Nice thinking, wonder why rheumatologist's havent thought of that. Your logic is fucked dude, just kill yourself.

Uhohinc said -"MC1R activation doesnt induce melanogenesis". -- https://pubmed.ncbi.nlm.nih.gov/8612494/ You got owned once again, bitch. You are my bitch now, you understand that?

Uhohinc said -"The RArthitis trial phase II had 12 patients on the drug".  --  The right answer is "The study is to screen up to 135 patients, estimated inclusion between 90-115 patients"
https://synactpharma.com/en/synact-pharma-expands-ap1189-begin-study-in-rheumatoid-arthritis-uk-version/ Data's so strong that expanding from 90 to ~105 patients will show STATISTICAL SIGNIFICANCE, and not just only Proof of Concept.

This already in a Ph2a! But I don't expect you to understand the difference Mr Uhohincompletobraino. Ph2a = Safety in unhealthy patients + efficacy that might give Proof of Concept. Ph2b = statistical significance on efficacy.

SynAct expects to deliver statistical significance already in Ph2a, on only ~105 patients.

Then Uhohinc like a lying little whore changed his statement. Not admitting he was wrong or lying and said -"As for the only 12 patients on the Ap1189, these were interim release results". -- The right answer is "The interim was on 26 patients".
The independent DATA SAFETY MONITORING BOARD said "OK GO" for 100mg after first 12pat was dosed on 50mg - and the interim included 26 patients and both dosages.
https://synactpharma.com/en/synact-pharma-announces-positive-interim-phase-2-data-of-ap1189-in-rheumatoid-arthritis/

Once again, OWNED. 100mg Ap1189 shows 70% better effect than MTX alone. 44%x1,7=75%.

But Uhohinc coninues to be a retard -"it has the fucking word "may"- Yeah DUMBASS. WHEN THEY COMMENTED ON THE THE BLIND DATA before interim they said MAY. Anything else would have been unproffesional dumbass.

2xBlind data + interim data + historical data on MTX and Placebo tells the tale. https://www.bildtagg.se/bild/buqzk6io0w7x65r3jk7unij = you get owned once again bitch.

You said Peretti was a stellar guy?  https://www.researchgate.net/publication/283014434_Resolution_Pharmacology_Opportunities_for_Therapeutic_Innovation_in_Inflammation#pf8

Mimetics of Effectors of Resolution

ACTH has long been known to be effective in controlling clinical signs of arthritides, including RA

and gout. This work has been validated in controlled clinical studies including hospitalised

patients suffering from severe gout [21,35]. ACTH is being trialled for efficacy in multiple sclerosis

and systemic lupus erythematosus (Table 1). Modifications of /MSH, a 13-amino acid peptide

derived from ACTH, have inspired a series of peptides trialled in distinct settings. Recently, a

synthetic analogue of /MSH (afamelanotide or Scenesse®) has been approved by the European

Medicine Agency (EMA) for erythropoietic protoporphyria. This represents a success for mel-

anocortin biology [36]. The skin darkening associated with melanocortin peptides is often

perceived as an unwanted side effect by these peptides, but it is clearly beneficial in a condition

characterised by light intolerance. More examples of drug development approaches based on

melanocortin peptides are reported in Table 1.

Small molecule agonists at melanocortin receptors have also been developed. Receptor

subtypes MC1, MC3, and MC5 present particular interest as anti-inflammatory targets.

AP1189 is a small molecule that acts as a biased agonist, because it does

activate ERK1/2 and Ca2+pathways but not the canonical cAMP. The relevance of this unusual

activity is that the side effects associated with skin darkening (MC

1–cAMP-dependent) are avoided [56.]

You must suffer from some kind of "I aM an AmUuriCaan I KnoW EveRYthINg Derp DerP"-syndrome.

As you know and pointed out, SynAct Pharma is a small company. How the fuck do you think they will be able to do a PhaseIII-study on their own? That aint the business model and has never been.
Uhohinc replies -"BUt tHIS Is NOt tHe AMeriCaN WaY, ThaT cAnt Be GoOd - I KnoW EveRYtHIng"

Thomas Jonassen, Thomas Boesen, Mauro Peretti, Mauro Martins Teixeira (Teixeira in the Brazilian FDA) Trinidad Melendez - all top scientists on melanocortins and on inflammation are behind SynAct Pharma. Peretti is on the Scientific Board of Synact. But naah, Uhohincompletobraino knows best. The one who appears to be a complete illiterate and cant even understand what he's reading. 12 patients interim hahaha! Get your facts straight before coming here like a complete retard.

"August 28, 2020

SynAct Pharma AB (“SynAct Pharma”) hereby announces that the company together with Prof Mauro Teixeira, MD, PhD, Universidade Federal de Minas, Belo Horizonte, Brazil and Prof Mauro Perretti, PhD William Heavy Research Institute, Barts and the London School of Medicine, Queen Mary University, London, UK has established a scientific and clinical collaboration, RESOVIR (resolution in viral inflammation), aimed to investigate pharmacological promotion of inflammatory resolution as a novel approach to control viral infections. The first step of the collaboration will be to run an exploratory clinical study in Covid-19 infected patients aimed to investigate repeated dosing of SynAct Pharma´s clinical stage compound AP1189. Later the collaboration will investigate the potential of promoting inflammatory resolution in other viral diseases such as Dengue and Influenza virus."

SynAct is only providing AP1189 for this study hahaha, cost's are almost none! Paid whore? Hahaha, come on, you can do better than that? You big InTeLlEcTuaL AMerICanO!

You clearly have no idea how patents work
SynAct will have the rights for the combination treatment in RA with Ap1189+MTX till 2041.
Patent for treating ARDS with AP1189 was applied on April 3, 2020. So patent is on the way. Takes ~18 months before application is published and searchable in databases.

Patent for treatment against Nephrotic Syndrome got "intention to grant" a few weeks ago.

If anyone else wants to make money on AP1189 after the original patent expires in 2034 (US), they will then have to do their own studies Ph1 Ph2 Ph3 in that specific disease. And that also adds some extra years to it.

A competitor can't manufacture AP1189 in 2034 and sell it as a RA-monotherapy without first doing monotherapy studies. Do you understand? Probably not...

Kempen - One of the highest regarded biotech analyst firms said this about AP1189

”AP1189 is developed as an add-on to MTX to reduce inflammation and support resolution and reduce the need for 2L treatment and/or reduce MTX dose. AP1189 COULD BE POSITIONED AS THE BEST ORAL THERAPY FOR RA IN TERMS OF EFFICACY/SAFETY PROFILE (SAFER THAN JAK’s). --> https://www.bildtagg.se/bild/l9xz3l17mfq3tulmd7u0f8gd

https://www.kempen.com/en/securities/life-sciences-and-healthcare

But Uhohimretardo knows best I guess! Not Peretti and the other top scientist's involved.... Not James Knight that sold Acthar Gel for 5.6billion$ that recently joined SynAct...

My guess is fuckoh is disappointed since he can't buy shares in SynAct, and he knows that "Clinuvel Afamelanotide SCENESSE" is an inferior substance that soon will be put on the shelf.

All that you have brought to this discussion has been bullshit and lies. Deliberate misunderstandings presented as facts and so on.
Looks like someone (you) is desperately is looking for confirmation bias. Twisting the facts to match your opinion etc...

And now everyone got to see how stupid you are :) Good job :)

[Uhohinc]

Words.....look who gots the words now  My Swisshole bitch is back for more   (I assume Swiss ? since you will not identify why or what you want on a stock website for Clinuvel info}.............I would feel sorry for your dog if you had one........ with you its real anger and me I get to bully the bully............You are engulfed and full of social anger and if you can not engage on the internet in a normal civil discourse then you probably can not have a real in person social discourse with a live man or female without that aggression response that you have and cause interactively, you have no respect for anyone, you make fun of Asperger's, you call others retards,you have emotional negative to Americans, you have a childish defense aggression to anyone who does not see things your way,  the comment that I am a lying cunt is indicative of YOUR anger issue at females and probably a not so good relation with your own mother..Being you have the entire repoirtoire of all the words for a verbal conflict which strongly leads to my opinion you are not just socially maladjusted to normal human interactions, but you have a problem not only formulating normal interpersonal relations with heteros on the internet but in real life you can not formulate any kind of one on one relationship with a female.   Since no female will fuck you unless transactionally.......I  enjoy fucking with you.....

but attempting to argue or debate the facts, the drug, the trials, is just useless with you.........you will only use the facts, see only what you want to see, read into the words and concepts that you only want to see that fit this construct in your mind  (dream) that is based on your center emotion over others.......anger.....You appear to not graduate rheostaticly respond like normal social humans..hru the normal mental states like a normal person...............your brain goes directly to the fight or flight and now for the real bad news....I could counter your statements with more facts, but you will use YOUR logic that omits, amplifys, trivializes or out right makes up anything or nothing to suit your delusion. This delusion is YOUR defense mechanism because you can not deal with realities that others see and now me when I point out how your thought processing refuses to deal with incongruities not just in the patent, but your everyday life counter to your disassociations. 

Lets just pretend you are right,,,,you should be happy, everything in your head........yea its going to come true.....money just makes one more of what one already is.........generally a contented happy most of the time person is more so with more money.........and a miserable, a maladjusted childhood, a person going from one social or sexual unhealthy state like yourself becomes more of a centering on anger and more on the social outskirts..........................you lose either way  as you are and will be a miserable angry unhappy cahoot.........even if someone or company or idiot like you with access to $5billion  (not many like you with money) as you say comes along and buys a phase II worthless very very very small statistically useless trial that is not double blind when the management can release any kind of result on the 12 patients on the drug before it is done, mix it up with other drugs for RA and put out a press release stating these results "may" be that the drug shows efficacy.......(only a gullible dummy shithead for brains like Claes does not understand the reason for the trial in the first place is because a drug may show efficacy. So it is not too good a result, interim, when after half way thru the trial it still only "may" show effficacy as opposed to SHOWING efficacy.......you "may" think other wise but he carefully prefaced the trial interim with that word......"may"......

Here is some more info that is going to not just fuck up your day, but your dream..........the chemical structure  Ap1189 of SynAct is no secret, its a quick throw together and half a dozen other pharma have it on their back of lab and some have ip rights mostly expired........I hope your not just an angry man, but a young angry man, as no pharma is going to buy these not even credible in size or protocol trials, and then they are probably not going to buy the drug when they can pick it off the shelf and make themselves, or not pay much, and then they are going to have to do some real clinical trials............you pick 5 or 10 years.............til then this is your dream you tried to get me to buy into........

And I have a very very important resource of info on AP1189 of information, that I would normally have posted. and it is in my opinion which you come here to gg because you value my opinion, is that this info is so detrimental to the future and maybe present too, of Ap1189 that no investor with any common sense would read and buy into the Ap1189 drug or stock of SynActin....................it is a huge RED FLAG...........but since your an asshole, and my  bitch,,,,,,,go find it yourself.........

[Uhohinc]

More information on the fraud of Claes Svensson, this is a fake name. The  name is   Thomas Jonassen      CSO, SynAct Pharma    t...@synactpharma.com  

He is on the board of SynAct and holds the stock just issued, prior to coming on here trying to interest investors who know a little about melanocortins into pumping up his stock ......its a dump and pump......No doubt he will probably be on share tease, as he is trying to get a social stock board about SynAct to proliferate, some of his posts are just him and his only posts. He is fishing for more suckers to think they are going to sell for Billions.........................it may have backfired for him with Clinuvel investors now.........interesting how on May 21, 2021 SynAct had to make some legal filings and changes to absolve Thomas Jonassen of prior activities not looking to legal.   Are you still coming back Thomas ?

[Claes Svensson]

Wow, your retardism has no boundaries. I am not Thomas J dumbass. I am a Swedish shareholder. Thomas J has better things to do than to discuss shit like this with a retardo americano. He's probably on the phone with the "stellar" guy Peretti :)
You americans can't even invest in SynAct Pharma haha, so that would be alot of time wasted. I came here because you had no idea what you were talking about. You described AP1189 totally wrong, thats all you stupid fuck.
Haha, the neverending story of lies, so now the annual meeting with standard protocols in accordance with swedish laws are fishy?

Just shut the fuck up with your bullshit. No one is interested in Clinuvel. Not anyone who's smart anyways since a-MSH will never be a thing due to the side effects.

I googled your nickname btw - GET A LIFE.

[Uhohinc]

Your not a Swede, I have known many, and they are not raised to be like you, unless maybe you had a head injury, which is possible ?           "But anyways"..........I almost started to pity you, when my mother said I used to be pissy namecalling argumentative puckered asshole like you too. But I said mommy, how come I do not recall being like Thomas Jon(ass)en of SynAct Pharma......................and my mommy said I grew out of it by age 4. She said I specially got onry when I sat on some Ikea furniture that false apart when you take it out of the box. I can see you even have ass in your name almost twice. Maybe your ancestry was trying to tell you something because you do live up to your name here as far as I can see. You clearly are a USDA prime ASS Thomass JonASSen.

Thom(ass) is this your normal personality or do you drink all the time. ?

Thomass Jonassen, coming on these websites attempting to stir up interest on your dead SynActin websites with your multiple fake names fake writing responses is not going to work here. And I will be over there. Thomass Jonassen with your personality, you would be making more money working at the Ikea customer service telephone responding to disgruntled Ikea  customers who can not follow the instructions and are missing a screw.  That or a ABBA tribute band to an ABBA tribute band.

Since Thomas Jon(ass)en you are so good at evaluating msh, tell me what kind of revenues do you expect for SynActin when it comes to market with the trial it started this March in Covid 19 for patients with respiratory needs............yesssereeee SynActin is going to have a monopoly and corner every Covid 19 patient on a ventilator in the year..........maybe 2025.........SynActin could even sell their Ap1189 to the patients with the 1918 influenza epidemic, and maybe the 1968 Hong Kong Flue, and the 1976 Swine flue, and the 2015 Eboli outbreak, and Zika virus or 2004 Sars virus......

.I will meet you when the Titanic finally gets to port in New York, its a little late, and drinks are on me at Sardi's with all the Unicorn urine you can swallow with no ice.  Maybe you can show me all the money you thought youe were going to make in  SynActin sales made with yesterdays pandemic.........I will pay, as I already know you will not make or have any money left from your investing in SynAct and all those patients with Covid that will not exist, that did not take Ap1189 that was not approved, that SynAct was not bought out..................Maybe SynActin can change Ap1189 to erectile dysfunction or female libido....Being tha Ap1189 is a pill, I sure hope you and your partner take it with alot of water so you do not get it stuck in your throat and get a stiff neck.  Wait, can you read that patent for Ap1189 again what does the drug do, and not do, at the same time....."But Any ways".....................

SynActin stock is going to sink for the same reason the Swedish Kings ship sank the moment it was put in water......................know it all holes who are at the top with too big of a head and think they have it all figured out, and do not heed the ignorant voices whispering in their ear (or on google groups) and already in their repressed head,,,,,,,,,,,you missed something Thom(ass) Jon(ass)en you overlooked something,,,,,,,

By the way, I do not think you found the Big RED FLAG on Ap1189 and a smaller red flag not as much imperitives.......

I guess as you say, you really have nothing better to do ....." Thomas J has better things to do than to discuss shit like this with a retardo americano. "    

You set yourself up for that one,,,,,I guess it is all that social intelligence you have conflicting with all that emotional intelligence control you do not have.

Perreti is legitimate, as is his research and his name, but merely associating yourself and buying his name to put on the board only goes so far, it does not legitimatize the worthless trials, the imitation drug, and the worthless stock investment in SynActin.  In fact it shows desperation which adds to the gibber patent, the legal problems filing last week to attempt to absolve yourself and others on the SynAct board, and the absolutely ridiculous trials and protocolls. Particularly the next leg of the trial is going to subscribe patients who will later not be included in the trial results, to create a useless result.......

"But Anyways",  for those reading here which is just another deflection by Thomass JonASSen is that double blind means no one knows the results or who is on the drug or the placebo, not even the person administering the drug to the patient, nor do they know who got the placebo, so if the ongoing trial is really blind,  the data and info can not be ascribed to the control or any patient, so how can an official press release of so far 12 patients on the Ap1189 be divulging a result as positive, and the ceo stating "the drug may be helping in RA" 

"But anyways" the more I look into SynActin, the more red flags.................and it shows desperation for a SynActin board member to come on to investment boards in attempting to manipulate retail stock investors..........its not that Thomass Jonassen has "nothing better to do", he is desperate to do it.....

Anytime you want to come back here and be my internet bitch and have this American ignorant retard intercourse with you ......about your being fucked in your invest in :SynActin,,, I am here...

By the way, do you have that Swedish Phish gummy candy in the same Kool Aid flavor those followers of that cult drank ????

[Uhohinc]

  And so you can not delete, I copied your reply.....Well for everyone else that follows Clinuvel, this was an easy bait  mostly because Thomas Jonassen has a BIG EGO and he basicly trapped himself and all I did was walk him thru, and he though use of a fake name Claes Svensson whom is really a director and priciple in management  of SynActin who is the real name of Thomas Jonassen.  

I have highlighted in yellow, but if you read all he wrote, and below I put his very first post next here, the rest may not be chrono.................But he being the actual liar, he could not keep the lies and story going though he being the tree in the forest that could not maintain the false frame the more he divulged.....My first clue he is Thomas J is because he intentionally tried to throw us all off by not including Thomas J with the full name, and I think for most anyone reading here if you read in context, what he states, what he does not state, what he does not answer or does answer (I am not Thomas Dumbass, I am a Swede) how he deflects, attempts to redirect, and puts on a act of being an outrageous internet investor troll, but look at the context of when he does let his not so careful act stay in the fake frame as Claes who is really Thomas Jonassen. Because he also knows he has crossed a legal line in many respects. Also he makes statements that only an insider very close of personal knowledge and not public knowledge would or could know.....To be fair in understanding the entire context, one should follow Thomas J and my own posts to see how he first starts out so happy to share with us here on gg, but his tone changes completely when I start pointing out things that in the press release and the patent do not make sense. There is a line, and it is not fine, when an enthusiastic internet stock board person responds and when a manipulatior from inside the company that is just going public, just raising money, just moving into bigger exchanges, and is pumping up a dump.....Thomas Jonassen working on SynAct board  put out that press release and was so anxious to see what result or where it would go, which I found by googl,ing and Thomas Jonassen wanted to see who out there picked up,,,, and he will be putting more out there. Not the first time, and will not be the last time that manipulation and use or abuse of info can be used......we even had the real Florian Homm post on GG, and I am certain shorters are all over these boards. You can be sure, that if Clinuvel has such large amount of numerical short sale contracts, it has extremely clever and not obvious short sale manipulators.

Below is fake Claes but really Thomas Jonassen first post..........and then others.

Wait and see! Melanocortin receptors 1 and 3 are the most important when regulating the inflammatory response. The key is to switch macrophages into M2-phenotype, Mc3r helps alot!

SynAct has good preclinical data in ARDS already, dating 10 years back. The covid19-situation gave a great opportunity to do human trials - SynAct are front runners in inflammation resolution.

Lots of data supporting melanocortins in the treatment of ARDS - central downregulation of the inflammatory response includes the lungs :)

One would think that achieving activation of Mc1R without cAMP and side effects, was a matter of company secrets. They are the only ones that know how. Thomas J is in the top5 on theese receptors!Claes Svensson<classesve...@gmail.com>

May 29, 2021, 1:42:05 PM (yesterday) 

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to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9

Wow, your retardism has no boundaries. I am not Thomas J dumbass. I am a Swedish shareholder. Thomas J has better things to do than to discuss shit like this with a retardo americano. He's probably on the phone with the "stellar" guy Peretti :)
You americans can't even invest in SynAct Pharma haha, so that would be alot of time wasted. I came here because you had no idea what you were talking about. You described AP1189 totally wrong, thats all you stupid fuck.
Haha, the neverending story of lies, so now the annual meeting with standard protocols in accordance with swedish laws are fishy?

Just shut the fuck up with your bullshit. No one is interested in Clinuvel. Not anyone who's smart anyways since a-MSH will never be a thing due to the side effects.

I googled your nickname btw - GET A LIFE.

Claes Svensson<classesve...@gmail.com>

May 29, 2021, 4:46:35 AM (2 days ago) 

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to Clinuvel Afamelanotide SCENESSE senescence CUV ASX.CUV CLVLY ur9

Hahahaahaha roflmao! Wow you're stupid, big time. Once again, you come here as a retard with false and made up information. Don't know if you are trying to fool yourself or anyone else...
And once again I will have to set the facts straight and show the world what a lying little cunt you are :)
You really think that I'm here for your opinion? HAHAHA! You? I know more than you ever will on this subject :)

I came here because I saw this "AP1189 a Erk1/2 pathway without Mcr1 or Mcr3 activation" - This headline is so dumb and retarded. And then you cite the preclinical article that clearly states that "AP1189 is a biased agonist that activates MC1R and MC3R". Later the article tells that activation of Mc1r is through the ERK1/2 pathway instead of the usual cAMP thus not inducing melanogenesis. But still you thought your headline was correct? You dont see the faults? Smart... That's why I asked if you could READ. Then you went full retard and showed more of your smartness - Aspirin against severe RA. Nice thinking, wonder why rheumatologist's havent thought of that. Your logic is fucked dude, just kill yourself.

Uhohinc said -"MC1R activation doesnt induce melanogenesis". -- https://pubmed.ncbi.nlm.nih.gov/8612494/ You got owned once again, bitch. You are my bitch now, you understand that?

Peretti and Thomas J are friends and colleagues since many years. They did the preclinical trials together (the one you cited recently) on AP1189. Mauro Martins Teixeira, NO1 on inflammation in South America is the one doing the ARDS/covid19-study in Brazil at the moment. But naah.. They probably know less than you hahaha!

Just recently this was announced:

“The project is a natural next step in our long-standing collaboration with Dr Trini-Montero Melendez and Professor Mauro Perretti. Our investment of ~£400,000 is to enable an important task of the project, i.e. to link clinical outcome of treatment with our AP1189 compound to MCR1 polymorphism as it would give the possibility to apply a rational approach for patient selection for future clinical studies. In addition, new insight into pharmacology of specific MCR1 variants can be used for identification of next generation compounds”, says Thomas Jonassen, CSO SynAct Pharma."

Not matching up? Your brain isnt matching up. Get a clue. The patent cover all kind of formulations, one of them making up AP1189, another one AP1030 towards diabetes etc. They have peptides, small molecules in their pipeline.

Peretti is on the scientific board of SynAct. They have Thomas Boesen on board. The team behind SynAct is top notch. James Knight who sold Acthar Gel to Questcor is on board as portfolio manager etc.

But dont worry, they aint after to steal the market from your Scenesse. They are focused on RA, PSA, IBD and now also Covid19 induced ARDS.

Wait and see! Melanocortin receptors 1 and 3 are the most important when regulating the inflammatory response. The key is to switch macrophages into M2-phenotype, Mc3r helps alot!

SynAct has good preclinical data in ARDS already, dating 10 years back. The covid19-situation gave a great opportunity to do human trials - SynAct are front runners in inflammation resolution.

Lots of data supporting melanocortins in the treatment of ARDS - central downregulation of the inflammatory response includes the lungs :)

One would think that achieving activation of Mc1R without cAMP and side effects, was a matter of company secrets. They are the only ones that know how. Thomas J is in the top5 on theese receptors!

[Uhohinc]

from the original article that I posted that was submitted by Thomas E. N. Jonassen aka T.E.N.J. aka Claes Svensson the outrageous behavior and childish use of cunt, etc...was part of the act........who would expect the CSO I assume Chief Scientific Officer, to behave this way.......thats why he did it ..................."I'am just a Swedish investor"  his entire response was so awkward to all but him...... to try and pretense he is not on the investor stock boards........What is the first best way to get real retail investors to participate in a stock forum........drama.

Disclosures

T.E.N.J. is shareholder in SynAct Pharma and owns intellectual property on AP1189.

[Uhohinc]

https://clinicaltrials.gov/ct2/history/NCT04004429?A=1&B=5&C=merged#StudyPageTop...........2 years into trial, massive changes..............if you have RA, take a NAISD that was excluded.

[Uhohinc]

SynAct Pharma doses first subject in study with new AP1189 tabletsFRI, OCT 15, 2021 15:34 CET

SynAct Pharma AB (“SynAct”) today announced that the first subject has been dosed in a clinical study in healthy volunteers to describe the pharmacokinetics of AP1189 following administration of a new tablet formulation aimed for once daily dosing in further clinical development of the compound.

The study is a single center, open label, 3-part pharmacokinetic study, with 12 healthy subjects in each part. The primary objective of the first part is to determine the relative bioavailability of AP1189 after dosing with the newly developed tablets versus the oral suspension used so far in clinical trials.

In the second and third part of the study, the food effect on the pharmacokinetics, which is a regulatory requirement, and proportionality following dosing at different dose levels will be evaluated.

“This is a crucial step in the development of AP1189. We are eager to see the results of dosing AP1189 as tablets to humans. We have succeeded in making AP1189 tablets that meet our expectations. Importantly, the manufacturing process allow for further development, including scaling as will be required for clinical development in later phases.”, said Thomas Boesen, COO, SynAct Pharma.

Preclinical testing of the tablets supports that the pharmacokinetic profile of AP1189 in plasma is comparable or better after dosing of tablets than after dosing of the suspension currently used in the phase 2a clinical development, and when confirmed in humans, the tablets will be used instead of the suspension in future clinical trials.

“The new tablets will enable supply of AP1189 to a wider population of patients, with the aim to enhance safety and compliance. The new formulation shows unique characteristics with regard to compound release that has been covered in the IP application we filed in June and will give an additional level of protection on the AP1189 product once granted.”, said Thomas Jonassen, CSO.  

 

The information was submitted, through the agency of the contact person below, for publication on October 15, 2021 

For further information aboutSynActPharma AB, please contact:  

Jeppe Øvlesen                                     Thomas Jonassen 
CEO, SynAct Pharma AB                 CSO, SynAct Pharma AB 
Phone: +45 28 44 75 67                   Phone: +45 40 15 66 69  
Mail: j...@synactpharma.com           Mail: t...@synactpharma.com 

AboutSynActPharma AB  

SynAct Pharma AB conducts research and development in inflammatory diseases. The company has a platform technology based on a new class of drug candidates aimed at acute deterioration in chronic inflammatory diseases with the primary purpose of stimulating natural healing mechanisms. For more information: www.synactpharma.com. 

About AP1189

The mechanism of action of SynAct Pharma ́s lead compound AP1189 is to promote resolution of inflammation through melanocortin receptor activation directly on macrophages, thereby reducing the pro-inflammatory activity of macrophages and by stimulating macrophage efferocytosis, a specific ability to clear inflammatory cells (J Immun 2015, 194:3381-3388). This effect has shown to be effective in disease models of inflammatory and autoimmune diseases and the clinical potential of the approach is currently tested in two clinical phase 2a studies in patients with active rheumatoid arthritis and in nephrotic syndrome.

https://clinicaltrials.gov/ct2/show/NCT04004429?term=AP1189&draw=2&rank=1)

https://clinicaltrials.gov/ct2/show/NCT04456816?term=AP1189&draw=2&rank=2

Jeppe Øvlesen                                     Thomas Jonassen 
CEO, SynAct Pharma AB                 CSO, SynAct Pharma AB 
Phone: +45 28 44 75 67                   Phone: +45 40 15 66 69  
Mail: j...@synactpharma.com           Mail: t...@synactpharma.com

SynAct Pharma AB conducts research and development in inflammatory diseases. The company has a platform technology based on a new class of drug candidates aimed at acute deterioration in chronic inflammatory diseases with the primary purpose of stimulating natural healing mechanisms. For more information: www.synactpharma.com. 

Tags:

Synact Pharma AP1189

[Uhohinc]

The resomelagon (AP1189) Development Program

Our Lead Program: Resomelagon (AP1189)

SynAct’s drug candidate, resomelagon (AP1189), is a once-daily oral selective melanocortin agonist. resomelagon (AP1189) selectively stimulates the melanocortin receptors that are directly involved in inflammation and its resolution without stimulating the adrenal glands to release cortisol. This selectivity enables resomelagon (AP1189) to exert its anti-inflammatory and immune resolution effects in a steroid-free manner without the significant safety, tolerability, and side effect issues associated with adrenocorticotropic hormone (ACTH) based therapies. Resomelagon (AP1189) is also a biased agonist that does not stimulate melanocortin pathways that are responsible for off-target activity like skin hyperpigmentation.

The company is evaluating resomelagon (AP1189) in three Phase 2 clinical programs: rheumatoid arthritis (RA), idiopathic membranous nephropathy (iMN), a form of nephrotic syndrome, and virus-induced respiratory insufficiency (VIRI) like that seen in COVID-19. In 2021, SynAct successfully completed Phase 2a trials in early severe RA and in hospitalized patients with COVID-19-induced respiratory insufficiency. Also in 2021, SynAct successfully tested a new oral solid tablet formulation of resomelagon (AP1189) in healthy volunteers and filed additional composition patents that should provide molecule exclusivity past 2040.

In 2022, the Company initiated two new Phase 2 clinical trials in RA: EXPAND a Phase 2b trial in newly diagnosed RA patients experiencing severe disease activity and RESOLVE a Phase 2a/b trial in RA patients experiencing an incomplete or loss of response to methotrexate. In addition, the ongoing Phase 2a iMN trial was amended in 2022 to introduce the new oral tablet dosage form and to increase the treatment period to 3 months. Topline results from all three studies are expected in the second half of 2023.

Phase 1 Development

In the Phase 1 clinical assessment, 2 weeks of once-daily dosing of resomelagon (AP1189) supported continued development as a once-daily orally dosed medication. The plasma concentration needed induce pharmacological efficacy was reached within 1.5 hours of dosing and daily exposure is increased until steady state has been reached following 5-6 days of treatment after which no further drug accumulation was observed.

Importantly, no treatment limiting adverse events were identified following multiple dosing and doses used in the ongoing Phase 2a studies (50 and 100 mg) were found to be safe and well tolerated.

• Pipeline
  • Pipeline Overview
  • The resomelagon (AP1189) Development Program
    • Rheumatoid Arthritis
    • Membranous Nephropathy
    • Virus Induced Respiratory Insufficiency
  • Peptide Agonists
  • Ongoing clinical studies

About Synact Pharma

SynAct Pharma is a clinical stage biotechnology company focused on resolving inflammation with melanocortin biology. Selective activation of the melanocortin system can help the immune system resolve excessive or chronic inflammation.

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