Search

The melanocortin 4 receptor (MC4R) plays a central role in appetite regulation, and agonistic activity at this receptor promotes satiety. Results from two randomized controlled clinical trials examine the effects of bremelanotide's agonism at MC4R on caloric intake and body weight.Premenopausal women with a body mass index30 kg/mIn Study A, 27 of 30 bremelanotide subjects (90.0%) completed the trial and exhibited a significantly greater reduction in body weight after 16 days versus placebo [least squares mean difference (95% CI), -1.3 (-1.9 to -0.8) kg; p .0001]. Mean caloric intake in bremelanotide subjects was decreased versus placebo, with a magnitude of reduction of approximately 400 kcal/day throughout Study A (p .01). In Study B, 15 of 27 subjects (55.6%) completed all three phases. Significantly greater reduction of mean body weight occurred in twice-daily bremelanotide subjects versus placebo (1.7 vs. 0.9 kg, respectively, p .001). Total caloric intake reduction was significantly greater in the bremelanotide groups versus placebo (mean difference range: 398-469 kcal; p .0001).Agonist activity at the MC4R may aid in reducing caloric intake and weight loss in obese women.

Background and Objective 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. Methods 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. Results 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. Conclusion 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 For the treatment of female sexual dysfunction, the most relevant outcome measures are patient-reported treatment effects and changes in symptoms, underscoring the need for reliable, validated patient-reported outcome (PRO) instruments. The aim of this study was to evaluate the psychometric characteristics (validity and reliability) of the Female Sexual Distress Scale-Desire/Arousal/Orgasm (FSDS-DAO) PRO measure, which was adapted from the validated FSDS-Revised (FSDS-R) questionnaire and added 2 questions involving arousal and orgasm. Methods Psychometric analyses were based on the data from a multicenter phase 2b dose-finding study that compared the safety and efficacy of bremelanotide versus placebo and were conducted in the evaluable modified intent-to-treat population (N = 325) from that study. Psychometric evaluation of the new items in the FSDS-DAO included confirmatory factor analyses, tests of internal consistency and test–retest reliability, examinations of convergent and discriminant validity, and determination of responsiveness. The validity of the FSDS-DAO was evaluated based on previously developed instruments, including the Female Sexual Function Index (FSFI), General Assessment Questionnaire (GAQ), Women’s Inventory of Treatment Satisfaction (WITS-9), and Female Sexual Encounter Profile-Revised (FSEP-R). Results Confirmatory factor analyses demonstrated that the FSDS-DAO items fit very well (Bentler’s comparative fit index of 0.929). Cronbach’s α for the FSDS-DAO total score was ≥ 0.91 at Visits 1, 2, 5, and 12, demonstrating adequate internal consistency reliability. Test–retest reliability was acceptable with an intra-class coefficient of 0.61 and a Spearman’s correlation coefficient score of 0.62 between Visits 1 and 2 (4 weeks). Acceptable construct validity was demonstrated by significant correlations with related PRO scales in the expected directions and magnitude. For example, participants reporting the worst levels of sexual function on the FSFI also showed the worst FSDS-DAO scores at Visits 5 and 12. The FSDS-DAO total score was responsive to change. Conclusions Evidence supports the validity and reliability of the FSDS-DAO for assessing sexually related distress in women with female sexual arousal disorder and/or hypoactive sexual desire disorder; the addition of the arousal and orgasm items did not impact the validity and reliability of the measure. Clinical Trial Registration ClinicalTrials.gov NCT01382719.

Hypoactive sexual desire disorder (HSDD) is a common female sexual dysfunction and is estimated to affect approximately 10% of women in the United States. It has been suggested that HSDD is associated with an imbalance of hormone and neurotransmitter levels in the brain, resulting in decreased excitation, increased inhibition, or a combination of both. Evidence suggests neurotransmitters, including dopamine (DA), norepinephrine, and serotonin, as well as hormones such as estradiol and testosterone, contribute to female sexual desire and response. Current treatments for HSDD include psychotherapy, and two US Food and Drug Administration-approved medications for premenopausal women: flibanserin, a serotonin mixed agonist and antagonist, and bremelanotide, a melanocortin receptor (MCR) agonist. Melanocortins are endogenous neuropeptides associated with the excitatory pathway of the female sexual response system. MCRs are found throughout the body, including the brain. Bremelanotide is an MCR agonist that nonselectively activates several of the receptor subtypes, of which subtype 4 (MC4R) is the most relevant at therapeutic doses. MC4R is predominantly expressed in the medial preoptic area (mPOA) of the hypothalamus in the brain, and is important for female sexual function. Animal studies suggest that bremelanotide may affect female sexual desire by activating presynaptic MC4Rs on neurons in the mPOA of the hypothalamus, leading to increased release of DA, an excitatory neurotransmitter that increases sexual desire. This review presents what is known about the mechanism of action of bremelanotide in the context of treating HSDD.

Background Responder analyses are used to determine whether changes that occur during a clinical trial are clinically meaningful; for subjective endpoints such as those based on patient-reported outcomes (PROs), responder analyses are particularly useful. Aim To identify the minimal clinically important difference (MCID) for selected scores on questionnaires assessing female sexual functioning and to use these differences to analyze the response in a large, controlled, phase 2b, dose-finding study of bremelanotide in premenopausal women with hypoactive sexual desire disorder (HSDD) and mixed HSDD/female sexual arousal disorder (FSAD). Methods The responder analyses were performed for the change from baseline to end of study for a total of 7 endpoints. Each PRO endpoint was assessed using at least 1 of 4 types of responder analyses: a planned analysis anchored to MCIDs based on expert estimates (historical anchors); post hoc analyses based on self-reported global benefit; receiver operating characteristic (ROC) curves; and cumulative distribution function. The prespecified analysis groups were all female sexual dysfunction (FSD)-based diagnoses (all study participants), those with HSDD alone, and a combined group of those with FSAD alone plus those with mixed HSDD/FSAD. Post hoc analyses were also performed for subjects with mixed HSDD/FSAD with a primary diagnosis of HSDD. Outcomes MCIDs based on the ROC curves for changes in Female Sexual Function Index–desire domain, Female Sexual Distress Scale–Desire/Arousal/Orgasm (FSDS-DAO) total score, FSDS-DAO item 13 and 14 scores, and number of satisfying sexual events. Results Outcomes matched those based on input from clinical experts. For all 7 endpoints, responder rates at the 1.75 mg dose in the overall modified intention-to-treat population achieved statistical significance compared with placebo (P ≤ .03). Clinical Implications These responder definitions were subsequently used in the bremelanotide phase 3 registration studies (RECONNECT) that evaluated the safety and efficacy of the bremelanotide 1.75 mg subcutaneous dose in premenopausal women with HSDD. Strengths & Limitations MCIDs for this study were based on changes from a single-blind phase to account for changes due to the placebo effect. These analyses were restricted to a study population composed only of premenopausal women with a clinical diagnosis of HSDD and/or FSAD and were based on data from the same clinical trial. Conclusion Bremelanotide was safe and well tolerated and demonstrated significant improvement in efficacy vs placebo in the phase 2b trial. The multiple responder analyses offer a valuable approach for determining clinically important effects of bremelanotide for HSDD and FSAD.

INTRODUCTION 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. METHODS 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. RESULTS 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 CONCLUSION 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.

Aim: Evaluate efficacy/safety of bremelanotide (BMT), a melanocortin-receptor-4 agonist, to treat female sexual dysfunctions in premenopausal women. Methods: Patients randomized to receive placebo or BMT 0.75, 1.25 or 1.75 mg self-administered subcutaneously, as desired, over 12 weeks. Primary end point was change in satisfying sexual events/month. Secondary end points included total score changes on female sexual function index and female sexual distress scale-desire/arousal/orgasm. Results: Efficacy data, n = 327. For 1.25/1.75-mg pooled versus placebo, mean changes from baseline to study end were +0.7 versus +0.2 satisfying sexual events/month (p = 0.0180), +3.6 versus +1.9 female sexual function index total score (p = 0.0017), −11.1 versus −6.8 female sexual distress scale-desire/arousal/orgasm total score (p = 0.0014). Adverse events: nausea, flushing, headache. Conclusion: In premenopausal women with female sexual dysfunctions, self-administered, as desired, subcutaneous BMT was safe, effective, and well tolerated (NCT01382719).

Melanocortinergic agents are currently being investigated for a possible therapeutic role in male and female sexual dysfunction. These investigations were sparked by findings that systemic administration of a synthetic analog of alpha-MSH, MT-II, causes penile erections in a variety of species, including humans. Several other melanocortinergic agents including HP-228, THIQ, and bremelanotide (PT-141) have since been shown to have erectogenic properties thought to be due to binding to melanocortin receptors in the central nervous system, particularly the hypothalamus. Bremelanotide, a nasally administered synthetic peptide, is the only melanocortinergic agent that has been clinically studied in both males and females. Data from Phase II clinical trials of bremelanotide support the use of melanocortin-based therapy for erectile dysfunction. Studies using animal models have demonstrated that pre-copulatory behaviors in female rats analogous to sexual arousal are evoked, and preliminary clinical data also suggest a role in promoting sexual desire and arousal in women. Based on bremelanotide clinical experience, administration of a melanocortin agonist is well tolerated and not associated the hypotension observed with phosphodiesterase-5 inhibitors currently used to treat erectile dysfunction. This review discusses investigations of melanocortin agonists for the treatment of sexual dysfunction with emphasis on proposed sites and mechanisms of action in the central nervous system that appear to be involved in melanocortinergic modulation of sexual function. Current research validates use of melanocortinergic agents for the treatment of both male and female sexual dysfunction.

Sexual dysfunction affects millions of men and women worldwide. Preclinical and clinical studies demonstrate and confirm that melanocortin receptor agonists such as bremelanotide (PT141, a synthetic alpha-melanocyte stimulating hormone analog) improve male and female sexual function presumptively via action on receptors in the central nervous system, particularly within the hypothalamus. Bremelanotide initiates penile erections in males. A recent pilot study demonstrates that bremelanotide also increases sexual arousal and desire in females. Intranasally administered bremelanotide is well tolerated.

While previous studies have implicated the tyrosine protein kinase p60fyn in antigenic activation of T lymphocytes, it is clear that signal transduction initiated through the antigen receptor requires the concerted actions of several proteins. Here, we report our finding that the activation of p60fyn following TcR cross-linking results in the tyrosine phosphorylation of two Fyn-associated proteins of 82 and 116 kDa. In the cells analyzed, p116 appears to represent one of the major substrates of T-cell antigen receptor-mediated tyrosine phosphorylation. In activated T-cells, the interaction of these proteins is specific for p60fyn since neither p56lck nor p62c-yes were found to detectably associate with p82 or p116. Furthermore, the p60fyn-p82/p116 complex could be dissociated and then reconstituted in vitro using purified recombinant Fyn. Using this technique we demonstrated that both p82 and p116 were capable of binding to the Fyn SH2 domain while p82 was to some extent capable of independent binding to the Fyn SH3 domain. An association between p60fyn and phosphoproteins possibly related to the T-cell p82 and p116 was also observed in other hematopoietic cells. Thus, the activation-induced phosphorylation of the p60fyn-associated proteins p116 and p82 and the wide distribution of potentially similar p60fyn-associated proteins in hematopoietic cells suggest that p116 and p82 may play a role as physiological substrates and/or regulators of p60fyn.

Tyrosine phosphorylation is thought to be one of the earliest steps in antigenic activation of T cells. Three nonreceptor tyrosine kinases, p56lck, p60fyn, and ZAP-70, are known to be involved in T cell receptor (TCR) signaling, albeit their functional roles appear to be different. Whereas p60fyn and ZAP-70 are functionally associated with the T cell antigen receptor, p56lck is essential for TCR signaling without being directly coupled to the TCR. We have studied a mutant variant of the Jurkat T cell line (J32–3.2), in which basal activities of p56lck and p60fyn are 2- to 2.5-fold reduced relative to those in its parental line (J32) while basal activity of ZAP-70 remains unchanged, and compared responses of J32–3.2 and J32 to TCR stimulation. We have demonstrated that tyrosine phosphorylation following CD3 cross-linking in J32–3.2 cells was extremely short-lived and thus insufficient for the induction of subsequent physiological responses. This was at least partially due to the diminished tyrosine kinase activity in these cells. A decrease in the activity of src-related kinases was caused primarily by their lower expression, whereas expression of ZAP-70 was unchanged but its response to CD3 crosslinking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 -chain, recently observed in J32–3.2. These data are consistent with the idea that sir-related kinases phosphorylate the -chain, which in turn recruits ZAP-70 required to sustain the signal. J. Leukoc. Biol. 55: 289–298; 1994.

A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. By SDS gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 Da. p56lck purified in this manner retained a high level of activity, phosphorylated an exogenous substrate on tyrosine residues, and underwent autophosphorylation on tyrosine residues. The Km (approximately 0.33 mmol) and Vmax (approximately 83 pmol min-1 mg-1) values were also determined by using enolase as a substrate.

The Src family of tyrosine protein kinases represent an expanding class of closely related intracellular enzymes that participate in the signal transduction pathways of a variety of surface receptors. One of the more surprising aspects of the information relating Src protein kinases to receptor signaling is the apparent diversity of receptor types with which the Src-related enzymes are reported to interact physically or functionally. Traditional biochemical and genetic approaches have yielded much information regarding the interactions between the Src tyrosine protein kinases and other cellular proteins in defined cell types, and emerging technologies, most notably homologous recombination in embryonal stem cells to achieve gene "knockouts," are providing new insights into the participation of the Src-related gene products in signal transduction and development.