Abstract INTRODUCTION The melanocortin system plays an important role in resolving inflammatory processes. The melanocortin 1 receptor (MC1R)–specific agonist PL8177 has demonstrated MC1R binding affinity and functional activity that mirrors that of α-melanocyte–stimulating hormone. The effect of PL8177 on inflammation, cell population composition, and gene and protein expression was investigated in colons from a dextran sulfate sodium (DSS)–induced rat model of colitis. METHODS Colitis was induced by 5% DSS in drinking water. Rats received vehicle control (placebo) capsules, oral PL8177 capsules (20, 50, and 100 μg per animal), or mesalazine (positive control). Colons were harvested on day 8. Samples were analyzed for cytokines, and by single nuclei RNA-seq and data-independent acquisition tandem mass spectrometry phosphoproteome profiling. Colitis was assessed by diarrhea and rectal bleeding, colon length shortening, colon weight gain, and histopathology. Total colitis index assessed inflammatory damage. RESULTS PL8177 50 μg showed a significant (P<0.05) improvement in colon weight (53% reduction), stool consistency, and fecal occult blood score vs vehicle, and there was significant improvement in the total colitis index for the PL8177 100-μg group vs vehicle. All PL8177 cohorts showed greater improvement in the total colitis index than mesalazine. Mesalazine produced a marked reduction in colon length, but only moderate improvement (35%) in colon weight. Single nuclei RNA-seq showed that in the PL8177 100-μg group, relative cell populations and key gene expression levels were closer to those of healthy controls PL8177 100-μg treatment caused a relative decrease in immune cells and an increase in enterocytes and enteric glial cells compared to vehicle control. Subclustering analysis revealed significant differences between PL8177 100-μg and vehicle populations. Although both showed the presence of macrophages, vehicle-treated colons were primarily M1 macrophages involved in inflammation. In PL8177 100 μg–treated colons, macrophages were primarily pro-resolution M2. Proteomic analysis showed that PL8177-treated colons are more similar to sham colons than to vehicle colons. Also after treatment with PL8177, Smad 2 and 3, proteins known to promote intestinal homeostasis showed expression similar to that seen in the sham group, a result not seen in vehicle-treated colons. DISCUSSION Oral PL8177 treatment showed significant improvement in anatomical markers of colitis vs the vehicle and mesalazine control groups, supporting the aim of treating inflammatory bowel disease (IBD). Transcriptomics and proteomics data show that oral PL8177 treatment causes diseased colons to move toward the healthy state and to resolve inflammation. Resolving inflammation—rather than blocking it—provides the possibility of efficacy coupled with safety in treating IBD.

Introduction: Melanocortin-1 receptors (MC1Rs), found in many tissues including the colon, promote the resolution of inflammation. High potency and no detected systemic absorption make the MC1R agonist PL8177 a promising candidate for oral gut restricted ulcerative colitis (UC) treatment. Methods: Oral PL8177 capsules were tested in 2 rat UC models and evaluated for distribution, in vivo, in rats, dogs, and humans. UC was induced in rats by dinitrobenzene sulfonic acid (DNBS) or dextran sulfate sodium (DSS). BID doses of 10, 20, 50, 100 or 200 µg PL8177 were compared with vehicle control (placebo) in DNBS and DSS rats. Colitis was assessed by diarrhea and rectal bleeding, and by changes in colon length and weight, and histopathological assessment on termination. Distribution of PL8177 within the GI tract after a single PL8177 dose was investigated in rats and dogs. A phase 0 study using a single oral microdose (70 µg) of [14C]-labeled PL8177 investigated the release of PL8177 in the colon of healthy men. A phase 2 double-blind, placebo-controlled, study will evaluate the safety, tolerability and efficacy of oral PL8177 in adults with UC. Results: Rats treated with 10-200 µg oral PL8177 demonstrated lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle. Histopathology analysis showed PL8177 treatment resulted in the maintenance of intact colon structure and barrier, and reduced immune cell infiltration. In rats and dogs, PL8177 was detected at higher amounts in the colon vs upper GI tract. [14C]-PL8177 and the main metabolite were not detected in human plasma and urine, suggesting that the parent drug [14C]PL8177 was released from the polymer formulation and metabolized within the GI tract. The most efficacious doses (P< 0.05 vs vehicle) in the rat studies were 20 and 50 µg, which provide the basis for estimating suitable doses for the phase 2 trial. Colonic surface area for humans is ∼3,000 greater than rat. Considering species-specific MC1R receptor sensitivity differences and other variables, such as GI transit time, pH, and proteolytic enzyme activity, a dose of 20 mg/day was estimated to be the best choice for observation of efficacy in the phase 2 trial. Conclusion: Collectively, these findings support further development of PL8177 as a treatment option for GI inflammatory diseases in humans. This will be further examined in the phase 2 trial in humans with UC.

Introduction: The effect of PL8177, a melanocortin 1 receptor-specific agonist, on inflammation, cell population composition, and gene and protein expression was investigated in colons from a dextran sulfate sodium (DSS)–induced rat model of colitis. Methods: Colitis was induced by 5% DSS in drinking water. Rats received vehicle control (placebo) capsules, oral PL8177 capsules (20, 50, and 100 μg per animal), or mesalazine (positive control). Colons were harvested on day 8. Samples were analyzed for cytokines, and by single nuclei RNA-seq and data-independent acquisition tandem mass spectrometry. Colitis was assessed by diarrhea and rectal bleeding, colon length shortening, colon weight gain, and histopathology. Total colitis index assessed inflammatory damage. Results: PL8177 50 μg showed a significant (P< 0.05) improvement in colon weight (53% reduction), stool consistency, and fecal occult blood score vs vehicle, and there was significant improvement in the total colitis index for the PL8177 100-μg group vs vehicle. All PL8177 cohorts showed greater improvement in the total colitis index than mesalazine. Mesalazine produced a marked reduction in colon length, but only moderate improvement (35%) in colon weight. Single nuclei RNA-seq showed that in the PL8177 100-µg group, relative cell populations and key gene expression levels were closer to those of healthy controls. Subclustering analysis revealed significant differences between PL8177 100-µg and vehicle populations. Although both showed the presence of macrophages, vehicle-treated colons were primarily M1 macrophages involved in inflammation. In PL8177 100 µg–treated colons, macrophages were primarily pro-resolution M2. Proteomic analysis showed that PL8177-treated colons are more similar to sham colons than to vehicle colons. Also after treatment with PL8177, a protein known to promote intestinal homeostasis and a blood-based biomarker protein showed expression similar to that seen in the sham group, a result not seen in vehicle-treated colons. Conclusion: Oral PL8177 treatment showed significant improvement in anatomical markers of colitis vs the vehicle and mesalazine control groups, supporting the aim of treating inflammatory bowel disease (IBD). Transcriptomics and proteomics data show that oral PL8177 treatment causes diseased colons to move toward the healthy state and to resolve inflammation. Resolving inflammation—rather than blocking it—provides the possibility of efficacy coupled with safety in treating IBD.

Purpose: The melanocortin receptor pan-agonist PL9643, a potential therapy for ocular diseases, was investigated in a phase 2, 12-week study in patients with dry eye disease (DED). Methods: This was a placebo-controlled study evaluating efficacy and safety of thrice-daily PL9643. Placebo (vehicle) was similar to tears. Primary endpoints were intra-patient changes in inferior corneal fluorescein staining and ocular discomfort after 12 weeks. Secondary endpoints were changes in additional DED signs or symptoms. Multiple secondary endpoints were not adjusted for multiplicity. Patients with moderate or severe DED were analyzed in addition to the overall intent-to-treat (ITT) population. Results: In the ITT population (n = 160) the PL9643 group did not demonstrate significant treatment difference versus placebo at week 12/day 85 for the primary endpoints (P > 0.05). In patients with moderate or severe DED (n = 53), PL9643 treatment demonstrated either nominally significant (P < 0.05) or trending (P < 0.1) improvement over placebo in mean change from baseline at week 12/day 85 in several sign endpoints, including fluorescein staining in inferior, superior, corneal sum, and total sum regions; Lissamine Green staining in temporal, nasal, conjunctival sum, and total sum regions; and tear film breakup time. Conjunctival redness also showed (nonsignificant) improvement at week 12/day 85. There were no drug-related adverse events (AEs) and no drug-related discontinuations. Conclusions: PL9643 showed no significant efficacy for the ITT population; however, efficacy results across several signs and symptoms in the subpopulation of moderate to severe DED patients, the low number of ocular AEs, and no tolerability issues suggest that PL9643 shows promise as a therapeutic for DED. Clinical Trial Registration number: NCT04268069.

IntroductionPL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed. This formulation was tested in 2 rat ulcerative colitis models and evaluated for distribution, in vivo, in rats, dogs, and humans. MethodsThe rat models of colitis were induced by treatment with 2,4-dinitrobenzenesulfonic acid or dextran sulfate sodium. Single nuclei RNA sequencing of colon tissues was performed to characterize the mechanism of action. The distribution and concentration of PL8177 and the main metabolite within the GI tract after a single oral dose of PL8177 was investigated in rats and dogs. A phase 0 clinical study using a single microdose (70 µg) of [14C]-labeled PL8177 investigated the release of PL8177 in the colon of healthy men after oral administration.ResultsRats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. Histopathology analysis resulted in the maintenance of intact colon structure and barrier, reduced immune cell infiltration, and increased enterocytes with PL8177 treatment. Transcriptome data show that oral PL8177 50 µg treatment causes relative cell populations and key gene expressions levels to move closer to healthy controls. Compared with vehicle, treated colon samples show negative enrichment of immune marker genes and diverse immune-related pathways. In rats and dogs, orally administered PL8177 was detected at higher amounts in the colon vs upper GI tract. [14C]-PL8177 and the main metabolite were detected in the feces but not in the plasma and urine in humans. This suggests that the parent drug [14C]-PL8177 was released from the polymer formulation and metabolized within the GI tract, where it would be expected to exert its effect. ConclusionCollectively, these findings support further research into the oral formulation of PL8177 as a possible therapeutic for GI inflammatory diseases in humans.

The melanocortin 1 receptor (MC1r)–specific agonist PL8177 and its main metabolite PL8435 have demonstrated MC1r binding affinity and functional activity that mirrors that of α-melanocyte stimulating hormone (a-MSH). a-MSH has been demonstrated to be effective in reducing inflammation in numerous experimental models. This study investigates the effects of PL8177 on inflammation, cell population composition, and gene and protein expression in colons from a DSS–induced rat model of colitis. The objective is to determine the effectiveness of PL8177 in this model and to characterize its underlying mechanism of action. Male Wistar rats received 5% DSS in drinking water for 3 days to induce colitis. Rats in the sham group received drinking water only (each group n=6). The other groups received vehicle control (placebo)–filled capsules; PL8177-filled capsules at 20, 50, and 100 μg/animal (by oral gavage); or oral mesalazine (positive control). At termination on day 8, 24 hours after the last dosing, colon tissues were harvested, dissected and snap frozen with liquid nitrogen. Colon samples were analyzed for cytokine levels, single nuclei RNA-seq, and data-independent acquisition tandem mass spectrometry (LC/MS DIA). Colitis was assessed by a disease activity index (diarrhea and rectal bleeding), colon length shortening, colon weight gain, and histopathological assessment. The total colitis index, was used to assess inflammatory damage. Colitis was induced in rats treated with DSS. Oral PL8177 50 μg/animal treatment showed a significant improvement in colon weight (53% reduction), stool consistency, and fecal occult blood score compared to vehicle. There was a significant (P<0.05) improvement in the total colitis index for the PL8177 100-μg group vs vehicle control group. All PL8177-cohorts showed greater improvement in the total colitis index compared to the mesalazine-treated cohort. PL8177 50 μg/animal showed a significant improvement in colon weight (53% reduction), while mesalazine treatment was associated with significant reduction in colon length, but only moderate improvement (35%) in colon weight. Changes in the cellular population compositions, gene expression changes and protein level data will be presented and discussed at the conference. Oral PL8177 treatment of inflamed colon showed significant improvement in markers of colitis in the rat model compared to the placebo and mesalazine control groups supporting the aim of treating inflammatory bowel disease in humans. Genomic and proteomic data will be interpreted in the context of disease change to identify alterations in immune and cellular states of the experimental colons.

Background Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC1) as reported in several experimental settings. As such, MC1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis. Methods PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177. Results PL8177 activates mouse and human MC1 with apparent EC50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint. Conclusion These results demonstrate that the MC1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis. Significance Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC1 receptor, as a promising strategy to manage chronic inflammatory diseases.

PL8177 is a selective melanocortin 1 receptor agonist in development for the treatment of various immunologic and inflammatory conditions. Here we describe the pharmacokinetics of PL8177 after subcutaneous (sc) delivery in animals and humans. Mice, rats, and dogs were administered sc PL8177 at single doses of 1.0 and 3.0 mg/kg (mice); 1.0, 5.0, and 25.0 mg/kg/day (rats); or 1.5, 8.0, and 40.0 mg/day (dogs). Blood was collected over 24 h (mice) or 28 days (rats and dogs). Safety and pharmacokinetics of single and multiple sc doses were also examined in human volunteers. Two dose levels were tested in two dosing cohorts of 1.0 and 3.0 mg/day for 7 days. Blood samples were collected through Day 1 and on Days 2 to 6 at peak and trough times based on analysis of the first two single-dose cohorts. In mice, 3 mg/kg PL8177 resulted in an area under the plasma concentration–time curve from 0 to infinity (AUC∞) of 1727 ng·h/mL, a maximum plasma concentration (Cmax) of 2440 ng/mL, an elimination half-life (t½) of 0.5 h, and a time to maximum concentration (tmax) of 0.25 h. Results for the 1-mg/kg dose were generally proportional. In rats, mean tmax values were independent of dose and ranged from 0.25 to 1.0 h for single and multiple dosing. Cmax values ranged from 516 to 695 ng/mL (1-mg/kg dose) and from 666 to 1180 ng/mL (25-mg/kg dose). In dogs, mean tmax values ranged from 0.4 to 1.3 h for single and multiple dosing. Values for tmax decreased with increasing dose and mean plasma Cmax increased less than dose proportionally (96–129 ng·h/mL [1.5 mg], 275–615 ng·h/mL [8.0 mg], and 633–1280 ng·h/mL [40.0 mg]). In humans, PL8177 was observed in the plasma within 15 min after a single dose and persisted for up to 48 h at higher doses. The tmax was 30–45 min (single dose) and 15–45 min (multiple doses). In multiple-dose studies, maximum steady-state plasma concentration (Cmax,ss) and AUC∞ increased with dose. Geometric mean Cmax,ss values were 20.1 ng/mL (1.0 mg) and 57.2 ng/mL (3.0 mg). AUC∞ values were 54.3 ng·h/mL (1.0 mg) and 199 ng·h/mL (3.0 mg). Unchanged PL8177 excreted in the urine was ≤ 1%, and accumulation was minimal. PL8177 administration resulted in a consistent pharmacokinetic profile. The measured exposure levels resulted in pharmacologically active PL8177 concentrations at the targeted MC1R. Rapid absorption was seen in healthy volunteers, and multiple-dose administration over 7 days resulted in pharmacokinetic characteristics similar to those observed after single-dose administration. Results support the continued development of PL8177 to treat immunologic and inflammatory conditions.

Background: The melanocortin α-melanocyte stimulating hormone (α-MSH), an endogenous peptide with high affinity for the melanocortin 1 receptor (MC1r), has demonstrated prevention and reversal of intestinal and ocular inflammation in animal models. Preclinical studies were performed to determine whether two MC1r receptor agonists, PL-8177 and PL-8331, exhibit actions and efficacy similar to α-MSH in preventing and reversing intestinal and ocular inflammation. Methods: Both PL-8177 and PL-8331 were assessed in a Eurofins LeadProfilingScreen selectivity panel including 72 in vitro assays. PL-8177 and PL-8331 were evaluated in an in vitro assay using human whole blood stimulated by lipopolysaccharide to determine inhibition of tumor necrosis factor alpha (TNF-α); for comparison, adrenocorticotropic hormone (ACTH) and α-MSH were used as positive controls. PL-8177, dosed at 0.5, 1.5, and 5.0 μg, was assessed in a cannulated rat model of dinitrobenzene sulfonic acid (DNBS)-induced bowel inflammation versus vehicle and oral sulfasalazine. PL-8177 was also dosed at 0.3 mg/kg/mouse injected intraperitoneally versus untreated controls and α-MSH treatment in mice with experimental autoimmune uveitis (EAU). PL-8331 at 3 doses, 3 times daily, was evaluated in a murine model of scopolamine-induced dry eye disease (SiccaSystemTM model), versus twice-daily Restasis® and Xiidra®. Results: Both PL-8177 and PL-8331 demonstrated no significant activity at the 1 μm concentration in any of the 72 in vitro assays. PL-8177 and PL-8331 inhibited lipopolysaccharide-induced TNF-α to a similar degree as ACTH and α-MSH. In the DNBS rat model of bowel inflammation, PL-8177 was significantly superior to untreated controls at all 3 doses (P < 0.05) in reducing bowel inflammation parameters, with effects similar to sulfasalazine. In the murine EAU model, PL-8177 significantly reduced retinal inflammation scores versus untreated controls (P = 0.0001) over 3–5 weeks, and to a similar degree as α-MSH. In the murine scopolamine-induced model of dry eye disease, PL-8331 reduced corneal fluorescein staining scores at all doses, significantly (P = 0.02) for the highest dose (1 × 10-5 mg⋅mL-1), and similarly to Restasis®; Xiidra® demonstrated no effect. Conclusion: The MC1r receptor agonists PL-8177 and PL-8331 exhibited actions similar to those of α-MSH in preventing and reversing intestinal and ocular inflammation in preclinical disease models.