Pharmacokinetics And Biodistribution

[Serafin Sonnier]

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Pharmacokinetic properties of oligonucleotides are largely driven by chemistry of the backbone and thus are sequence independent within a chemical class. Tissue bioavailability (% of administered dose) is assisted by plasma protein binding that limits glomerular filtration and ultimate urinary excretion of oligonucleotides. The substitution of one non-bridging oxygen with the more hydrophobic sulfur atom (phosphorothioate) increases both plasma stability and plasma protein binding and thus, ultimately, tissue bioavailability. Additional modifications of the sugar at the 2' position, increase RNA binding affinity and significantly increase potency, tissue half-life and prolong RNA inhibitory activity. Oligonucleotides modified in this manner consistently exhibit the highest tissue bioavailability (>90%). Systemic biodistribution is broad, and organs typically with highest concentrations are liver and kidney followed by bone marrow, adipocytes, and lymph nodes. Cell uptake is predominantly mediated by endocytosis. Both size and charge for most oligonucleotides prevents distribution across the blood brain barrier. However, modified single-strand oligonucleotides administered by intrathecal injection into the CSF distribute broadly in the CNS. The majority of intracellular oligonucleotide distribution following systemic or local administration occurs rapidly in just a few hours following administration and is facilitated by rapid endocytotic uptake mechanisms. Further understanding of the intracellular trafficking of oligonucleotides may provide further enhancements in design and ultimate potency of antisense oligonucleotides in the future.

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Cell-penetrating peptides (CPPs) are small peptides (shorter than 30 amino acids), often cationic, with a capacity to penetrate the cell membrane1. The very first report of CPPs arose with the observation that the HIV-trans-activator of transcription (TAT) protein could reach the nucleus of cultured cells, resulting in target gene expression2,3. Subsequent studies showed that CPPs enhance cellular concentrations of cargos such as peptides, proteins, and DNA- or RNA-based medicines4,5,6,7,8. However, most studies with CPPs have been caried out in vitro. As with any drug, CPPs must also display acceptable pharmacokinetic (PK) and toxicity profiles to be translated toward in vivo and clinical uses.

Over 25 clinical trials involving CPPs are in progress including a few in Phase III9, the vast majority of them intended for application in oncology10. Yet, no CPP-based drugs have been approved by the US Food and Drug Administration (FDA). Although the efficacy of CPPs in bioassays have been the focus of many studies11,12,13,14, the knowledge on their PK properties and biodistribution in vivo is limited. The bulk of the work on CPPs has focused on a small number of entities, with TAT and penetratin representing 44% and 23% of the studies, respectively15,16,17,18,19. Biodistribution studies of different CPPs have shown preferential accumulation in highly perfused capillary-rich organs, such as the lungs, spleen, kidneys and liver19. Available PK studies suggest that CPPs undergo rapid liver and renal clearance, thereby reducing their plasma area under the curve and limiting subsequent target engagement.19,20,21,22,23. Regarding the safety profile of CPPs, most studies report that toxicity remains relatively low but may vary according to the types of CPP and cargos24,25,26,27.

Here, we have studied a novel CPP comprising both a protein transduction domain (PTD) and an endosomal escape domain (EED) named Shuttle cell-penetrating peptide (S-CPP). This S-CPP has already been shown to undergo rapid, safe, and efficient cytosolic delivery of functional proteins into 20 mammalian cell types in vitro32,33. One of the major limitations in the development of CPPs is their entrapment with their cargo inside endosomes during intracellular trafficking, leading to lysosomal degradation34. EEDs can interact with the endosomal membrane to cause its degradation, its destabilization or pore formation, allowing leakage of endosomal content into the cytoplasm35,36,37. In addition, we have investigated the effect of the conformation of S-CPP by comparing its L and D enantiomers. D-amino acids are generally expected to protect from enzymatic activity and are thus more likely to increase peptide stability in vivo38,39. However, the CPPsite 2.0 database ( ) shows that, out of 1850 sequences of referenced CPPs, less than 350 relate to D forms, suggesting that PK features of D-CPPs may be understudied. In summary, we have carried out PK and biodistribution studies of a S-CPP, alone or non-covalently combined to an immunoglobulin G (IgG) cargo, in its two enantiomeric forms, after systemic injection in animals. We have also evaluated toxicity from hematological, biochemical and inflammatory standpoints after repeated high-dose systemic injections in rodents. Finally, brain uptake of S-CPPs across the BBB was evaluated using in situ cerebral perfusion.

In order to elucidate whether combination with S-CPP alters the biodistribution of a large cargo, we compared the VD in organs 1 h after systemic administration of 3H-IgG, combined or not to unlabeled L-S-CPP, D-S-CPP, or a scrambled peptide (Fig. 4). Combining the 3H-IgG with D-S-CPP led to significantly higher plasma concentrations at 1 h post injection, when compared to 3H-IgG combined with the scrambled control peptide. This effect was not significant for L-S-CPP. The combination of 3H-IgGantiNUP to S-CPPs increased its VD in the liver but decreased its VD in the muscle (Fig. 4). A higher relative distribution was also seen in the spleen but only for D-S-CPP (Fig. 4). No other significant difference was measured in other organs (Fig. 4). These data suggest that S-CPPs induced a preferential distribution of IgG in the liver and possibly spleen, but had the opposite effect in the muscle.

Serum cytokine quantification using multiplex ELISA was also evaluated. A large panel of cytokines were measured in triplicates in plasma samples: TNF-α, IFN-ɣ, GM-CSF, IL-1, IL-2, IL-5, IL-4 and IL-10. Most pro-inflammatory cytokines or anti-inflammatory cytokines were below detection threshold. Therefore, despite the high doses used (in the mg/kg range), S-CPPs did not induce systemic inflammation or immunogenicity (Table 3).

After intravenous administration, we observed that both S-CPPs displayed a fast distribution phase of less than 3 min. This is consistent with a quick distribution in organs due to the small size and relative lipophilicity of the CPPs19. Although a significant portion was rapidly removed from the blood, S-CPPs then displayed a longer and slower elimination phase (T1/2 over 450 min), whichwas uncovered because of the use of a two-compartment PK model. True in vivo PK studies of CPPs in the literature are scarce. For example, one study using a panel of ten CPPs report half-lives ranging from 72 (penetratin) to 528 (TAT) min to over 72 h by measuring their stability in vitro at 37 C in human serum19, which does not take into account tissue distribution and metabolism, as well as excretion. On the other hand, Lee et al. reported T1/2 values of 0.87 and 107 min for the distribution and elimination phases, respectively, after i.v. injection of TAT-biotin in rats19,40,41. The present results are in line with these studies and suggest that S-CPPs alone may display a sufficient residence time to exert a pharmacological effect in target tissue.

Very few studies have compared CPP enantiomers in vivo19,22,42,43. The rationale for their comparison here was that stereoselective interactions with liver enzymes or the cell membrane might differ between L-S-CPP and D-S-CPP44,45. For example, it has been postulated that D-form CPPs may be more resistant to degradation by enzymes compared to L-forms, thereby increasing their stability in vivo38,39. Here, the D-form exhibited slightly lower T1/2 and AUC, as well as higher clearance and Vc, compared to the L-form, although these differences did not reach statistical significance. Thus, our results do not suggest obvious advantages of using either enantiomer to improve PK parameters, although biodistribution was affected (see below).

As S-CPPs are designed to deliver protein-based cargo, PK parameters of intravenously injected immunoglobulin G (IgG), in the presence or not of S-CPPs, were investigated. As expected, the calculation of PK parameters of IgG required a single-compartment model because of the slow distribution and stability of IgG in the blood in vivo. Comparison of IgG alone or combined to L-S-CPP did not reveal significant differences in PK parameters such as AUC, clearance or VD. This suggests that the combination to CPPs did not modify cargo functionality. Thus, the PK data obtained in this study is consistent with what is expected for IgG, with a half-life of days to weeks in the mouse46,47. Interestingly, with regards to biodistribution, D-S-CPP increased the plasma concentration of IgG 1 h post-injection in mouse when compared to the scramble peptide. This indicates that IgG/S-CPP formulations may present an advantageous circulating time, increasing the propensity to reach their intracellular target after systemic administration.

In future clinical applications, S-CPPs are unlikely to be utilized alone and their efficacy will ultimately be determined by the ability of their cargo to reach target organs22,32. The biodistribution of IgGantiNUP was investigated with or without combined S-CPPs or a scrambled peptide. The combination with D-S-CPP increased plasma concentrations of the IgG at 1 h post-injection, suggesting a slower elimination not apparent in the previous PK experiment. The most striking observation was that both S-CPPs induced a preferential distribution of IgG in the liver but had the opposite effect in the muscle. A higher accumulation of IgG combined with D-S-CPP was also observed in the spleen compared to the scramble peptide. There is published evidence that D-penetratin improved nasal absorption of interferon beta (IFN-β) better than the L form after intranasal administration48. Here, concentrations of cargo IgG were estimated to be between 0.3 and 0.5 nM in the liver and spleen 1 h after administration. It should be reminded, however, that combining a CPP to a cargo smaller than an IgG may have had more impact on its distribution. Nevertheless, such an increase in accumulation of S-CPP/IgG complexes in the liver and spleen, while avoiding the muscle, may be therapeutically valuable for certain indications.

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