MC4R ( EC50 = 0.27 nM ); MC4R ( Ki = 2.1 nM )

Setmelanotide/BIM-22493 and BIM-22511 exhibit agonist activity at the MC1R, MC3R and MC4R (Table 1). Receptor affinity (Ki) and activity (EC50) data of both ligands at human MC1R, MC3R, MC4R and MC5R are shown in Table 1. Both compounds exhibit activity at the MC1R-MC4R, but exhibit weak activity at MC5R (EC50>1 μM) and are inactive at the mouse MC2R (EC50>10 μM). To determine whether agonist activity is also observed in the rodent, researchers assessed Ki and EC50 in rat MC4R Ki and EC50 data for BIM-22493 and BIM-22511 were comparable in rat and human MC4R (Table 1).

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (Setmelanotide/BIM-22493, BIM-22511) that cross the blood-brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (approximately 50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (approximately 50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.[1]

---

Treatment with a highly-selective novel MC4R agonist (Setmelanotide/BIM-22493 or RM-493) resulted in transient decreases in food intake (35%), with persistent weight loss over 8 weeks of treatment (13.5%) in a diet-induced obese nonhuman primate model. Consistent with weight loss, these animals significantly decreased adiposity and improved glucose tolerance. Importantly, we observed no increases in blood pressure or heart rate with BIM-22493 treatment. In contrast, treatment with LY2112688, an MC4R agonist previously shown to increase blood pressure and heart rate in humans, caused increases in blood pressure and heart rate, while modestly decreasing food intake. These studies demonstrate that distinct melanocortin peptide drugs can have widely different efficacies and side effects[2].

Radioligand Binding Assay to human MC1R, MC3R, MC4R and MC5R [1]
Cell membranes were prepared from CHO-K1 cells stably expressing the human melanocortin receptor subtypes (MC1R, MC3R, MC4R and MC5R). They were incubated at 1-10 μg protein/well in 50 mM Tris-HCl, pH 7.4, containing 0.2% BSA, 5 mM MgCl2, 1 mM CaCl2 and 0.1 mg/mL bacitracin, with increasing concentrations of compound to be tested and 0.1-0.3 nM [125I]-NDP-α-MSH for 90-120 min at 37°C, depending on the receptor subtype. Bound from free [125I]-NDP-α-MSH was separated by filtration through GF/C glass fiber filters presoaked with 0.1 % (w/v) PEI. Filters were washed three times with 50 mM Tris-HCl, pH 7.4, at 0-4°C and assayed for radioactivity using Perkin Elmer Topcount scintillation counter.

Intracellular cAMP Assay [2]
Intracellular cAMP levels were determined by an electrochemiluminescence (ECL) assay. CHO-K1 cells stably expressing the human MC1R, MC3R, MC4R and MC5R were suspended in RMPI 1640 containing 0.5 mM IBMX, and 0.2% BSA. They were dispensed (7,000 cells per well) in Multi-Array plates (MSD) containing integrated carbon electrodes and coated with anti-cAMP antibody. Increasing concentrations of compound were added for 40 min incubation at 37°C. Then, the cells were lysed and 2.5 nM TAG ruthenium-labeled cyclic AMP (MSD) was added. After 90 minutes, cyclic AMP levels were determined by ECL detection using Sector Imager 6000 reader (MSD). NDP-MSH was used as the reference compound. Maximal cAMP stimulation level was 12 to 20 folds higher than basal level, depending on receptor subtype.

Osmotic Mini-pump Implantation [1]
Mice were surgically implanted with 14d osmotic mini pumps (Alzet). Pumps contained either 0.9% saline, or agonist dissolved in 0.9% saline plus 0.1% BSA. The α-MSH analogs (Setmelanotide/BIM-22493, BIM-22511) were synthesized by Biomeasure Inc., IPSEN (Milford, MA). Mice were anesthetized with isoflurane gas, an area on the dorsal surface in the interscapular region shaved and sterilized for surgery. A small incision was then made above the scapula and blunt forceps used to make a small space in the interscapular region. After insertion of the pumps, the incision was closed using a metal clip.

---

Glucose Tolerance Test [1]
After fasting overnight, mice were weighed and pre-injected with the melanocortin agonist SetmelanotideBIM-22493. Baseline blood glucose was measured preinjection using a small sample of blood taken from a tail nick using a OneTouch Glucometer, and 2 g/kg body weight of D-glucose injected by i.p. Blood glucose was measured at 15, 30, 60, and 120 minutes post injection. Mice were not restrained during the test.

---

Experiment 1: Chronic treatment in DIO animals. [1]
The study used 12 mature adult (age 9–11 years) male rhesus macaques, with body weights ranging from 9 to 19 kg. Monkeys were maintained in single-housing cages and fed a high-fat diet (HFD: 32% calories from fat; Custom Diet 5A1F; Test Diet, Richmond, IN) daily plus calorically dense enrichment. These animals had been maintained on the HFD for approximately 1.5 years before these studies. Nine animals were obese, insulin-resistant, and hypertensive, classified as diet-sensitive (Table 1). Three animals maintained normal body weight, adiposity, and blood pressure, and were classified as diet-resistant. Two-week minipumps (model 2ML2; Alzet, Cupertino, CA) were implanted subcutaneously in the scapular region under ketamine sedation (5 mg/kg). The animal received minipumps containing vehicle (0.9% saline, 2% heat-inactivated nonhuman primate [NHP] serum and 5% N,N-dimethylacetamide) for 4 weeks (pumps were exchanged after 2 weeks) to obtain baseline values. On study day 0, a 2-week minipump was implanted in all animals containing 0.5 mg/kg/day of Setmelanotide/BIM-22493 (dissolved in 0.9% saline, 2% heat-inactivated NHP serum, and 5% N,N-dimethylacetamide). For an overview of the study design, see Supplementary Fig. 1.

---

Experiment 2: Effect of melanocortin agonist LY2112688 on food intake and heart rate. [1]
After full recovery from the Setmelanotide/BIM-22493 treatment (more than 8 weeks after completion of 0.17 mg/kg/day treatment), 8 diet-sensitive animals with working telemetry devices were selected from the 12 animals described in experiment 1. These animals received three consecutive weekly therapies, each separated by 1 week of vehicle treatment, in the following order: LY2112688 (0.17 and 0.5 mg/kg/day), followed by Setmelanotide/BIM-22493 (0.5 mg/kg/day) for comparison. Food intake over the week of active treatment and cardiovascular measurements were recorded

Absorption, Distribution and Excretion
The time to peak concentration (Tmax) of semeralanolide is 8 hours. Following subcutaneous injection of 3 mg semeralanolide, 39% is excreted unchanged in the urine. The apparent volume of distribution of semeralanolide is 48.7 L. The estimated clearance of 3 mg semeralanolide from subcutaneous injection is 4.86 L/h. Metabolisms/Metabolites Semeralanolide is expected to be metabolized into small peptides and amino acids. Biological Half-Life The elimination half-life of semeralanolide is approximately 11 hours.

Hepatotoxicity
In a small, open-label clinical trial of sermelanotide for hereditary obesity, no abnormalities in serum transaminase or bilirubin levels were reported. One patient developed cholecystitis, but this was considered unrelated to the treatment. Since sermelanotide was approved and introduced into clinical use, no clinically significant liver injury has been published attributable to sermelanotide treatment, although elevated serum transaminase levels have been reported during treatment. Therefore, sermelanotide treatment has not been associated with clinically significant liver injury, but its overall clinical experience is limited. Probability Score: E (Unlikely to be a cause of clinically significant liver injury). Protein Binding The protein binding rate of sermelanotide is 79.1%.

[1]. Analysis of the therapeutic functions of\nnovel melanocortin receptor agonists in MC3R- and MC4R-deficient\nC57BL/6J mice. Peptides. 2009 Oct;30(10):1892-900

[2]. Chronic treatment with a melanocortin-4\nreceptor agonist causes weight loss, reduces insulin resistance, and\nimproves cardiovascular function in diet-induced obese rhesus macaques.\nDiabetes. 2013 Feb;62(2):490-7.

[3]. Melanocortin-4 receptor modulators for the treatment of obesity: a\npatent analysis (2008-2014). Pharm Pat Anal. 2015;4(2):95-107.

Setmelanotide is the first drug approved for the treatment of patients with proopiotropic melanocortinogen, proprotein subunit protease/kexin type 1, or leptin deficiency. It is a melanocortin 4 receptor agonist. Early attempts to use drugs that stimulate the MC4R (such as LY2112688) were effective in reducing weight, but also caused increased blood pressure and heart rate. Other early treatments included gastric bypass surgery. Patients taking setmelanotide lost an average of 0.6 kg per week. Imcivree received Orphan Drug Designation from the EMA on November 19, 2018, and FDA approval on November 25, 2020. It was approved by Health Canada on May 4, 2023. Setmelanotide is a melanocortin 4 receptor agonist. Semeranotide's mechanism of action is as a melanocortin 4 receptor agonist. Semeranotide is a melanocortin 4 receptor agonist used for the treatment of chronic weight management in adults and children with a rare genetic obesity caused by a defect in the melanocortin pathway gene. Semeranotide treatment is not associated with elevated serum transaminases or bilirubin, or clinically significant liver injury. See also: Semeranotide acetate (its active ingredient). Semeranotide hydrochloride (active ingredient).

---

Indications
Semeranotide is indicated for chronic weight management in patients aged 6 years and older with obesity due to deficiency of proopiotropic melanocytocorticosteroid (POMC), proteolytic enzyme/kexin type 1 (PCSK1) deficiency, or leptin receptor (LEPR) deficiency. This obesity requires a POMC, PCSK1, or LEPR gene mutation confirmed by an approved test, and these mutations must be interpreted as pathogenic, possibly pathogenic, or of unknown significance. These conditions affect the MC4R signaling pathway. Semeranotide is also indicated for chronic weight management in patients aged 6 years and older with obesity due to Budd-Bead syndrome. The drug is reportedly ineffective in patients with benign or possibly benign POMC, PCSK1, or LEPR variants, as well as other types of obesity not listed above. Imcivree® is indicated for the treatment of obesity and hunger control associated with genetically confirmed Bardet-Biedl syndrome (BBS), loss of function of biallelic proopiotropic melanocorticoid hormone (POMC) (including PCSK1), or loss of function of biallelic leptin receptor (LEPR), in adults 6 years of age and older and children. Treatment of appetite and general nutritional disorders. Mechanism of Action: Ghrelin and other hunger signals in the gastrointestinal tract stimulate appetite-stimulating neurons, thereby stimulating the release of spiky-associated proteins. Spiky-associated proteins inhibit the activation of melanocortin 4 receptor (MC4R) until satiety signals such as insulin or leptin stimulate anorexia neurons. Insulin and leptin stimulate the production of POMC-derived melanocortin peptide α-melanocyte-stimulating hormone, a ligand of MC4R. Both orexin neurons contain prohormone-converting enzyme 1/3 (PC1/3), an enzyme encoded by the proprotein subtilisin/kexin type 1 gene. PC1/3 can activate various peptide hormone precursors, including alpha-melanocyte-stimulating hormone. Semelanotide, a peptide derived from opioid melanocytocorticotropic hormone, is an agonist of MC4R. It is an MC4R agonist approximately 20 times more potent than endogenous alpha-melanocyte-stimulating hormone, with an EC50 of 0.27 nM. By directly activating MC4R, defects in upstream genes of the MC4R signaling pathway cannot suppress satiety, thereby reducing food intake and achieving weight loss. MC4R is a G protein-coupled receptor (G-PCR) composed of 332 amino acids. Although the mechanism by which setmelanotide treatment lacks cardiovascular adverse effects is not fully understood, it is believed that early MC4R antagonists activated multiple G protein signaling pathways. Early drugs targeting G-PCR either bind to highly conserved ortho-binding sites with high affinity or bind to less conserved allosteric sites with high specificity. Setmelanotide is an atypical dual-site ligand that can interact with both ortho-binding sites and putative allosteric binding sites, thus possessing both high affinity and specificity. Setmelanotide is a protein drug that was first approved in 2020 for the treatment of obesity, with the highest clinical trial stage being Phase IV (covering all indications), and has 4 investigational indications. In summary, these experiments demonstrate the application of two novel melanocortin receptor agonists. Our data suggest that MC4R is essential for weight loss using these compounds. However, the activity of other melanocortin receptors may be independent of MC4R and weight loss, significantly improving insulin sensitivity. [1] In summary, we describe an MC4R agonist, BIM-22493 (also known as RM-493), which, after long-term subcutaneous infusion, reduced food intake and fat accumulation and resulted in a significant 13.5% reduction in total body weight. Studies have shown improvements in glucose homeostasis, insulin sensitivity, and leptin levels. In addition, we found that blood pressure or heart rate did not change immediately after administration via a micropump following activation of the melanocortin system with BIM-22493; in fact, cardiovascular function was generally improved in the long term after weight loss. Further human studies are needed to determine the effectiveness of BIM-22493 as an anti-obesity therapy. [2]

C₄₉H₆₈N₁₈O₉S₂

1117.31

1116.485

920014-72-8

Setmelanotide TFA;1504602-49-6; 1301120-74-0 (pamoate); Setmelanotide acetate;2759937-80-7

11993702

Ac-Arg-Cys(1)-D-Ala-His-D-Phe-Arg-Trp-Cys(1)-NH2; N-acetyl-L-arginyl-L-cysteinyl-D-alanyl-L-histidyl-D-phenylalanyl-L-arginyl-L-tryptophyl-L-cysteinamide (2->8)-disulfide

RCAHFRWC

Typically exists as White to off-white solid at room temperature

1.5±0.1 g/cm3

1.728

-2.36

15

14

18

78

2130

8

C([C@H]1C(=O)N[C@H](C(=O)N)CSSC[C@H](NC(=O)[C@@H](NC(=O)C)CCCNC(N)=N)C(=O)N[C@H](C)C(=O)N[C@@H](CC2NC=NC=2)C(=O)N[C@H](CC2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C1=CNC2C=CC=CC1=2

HDHDTKMUACZDAA-PHNIDTBTSA-N

InChI=1S/C49H68N18O9S2/c1-26-41(70)63-37(20-30-22-55-25-59-30)46(75)64-35(18-28-10-4-3-5-11-28)44(73)62-34(15-9-17-57-49(53)54)43(72)65-36(19-29-21-58-32-13-7-6-12-31(29)32)45(74)66-38(40(50)69)23-77-78-24-39(47(76)60-26)67-42(71)33(61-27(2)68)14-8-16-56-48(51)52/h3-7,10-13,21-22,25-26,33-39,58H,8-9,14-20,23-24H2,1-2H3,(H2,50,69)(H,55,59)(H,60,76)(H,61,68)(H,62,73)(H,63,70)(H,64,75)(H,65,72)(H,66,74)(H,67,71)(H4,51,52,56)(H4,53,54,57)/t26-,33+,34+,35-,36+,37+,38+,39+/m1/s1

(4R,7S,10S,13R,16S,19R,22R)-22-[[(2S)-2-acetamido-5-(diaminomethylideneamino)pentanoyl]amino]-13-benzyl-10-[3-(diaminomethylideneamino)propyl]-16-(1H-imidazol-5-ylmethyl)-7-(1H-indol-3-ylmethyl)-19-methyl-6,9,12,15,18,21-hexaoxo-1,2-dithia-5,8,11,14,17,20-hexazacyclotricosane-4-carboxamide

RM-493 ;Imcivree; RM493; Setmelanotide; 920014-72-8; BIM-224,93; Setmelanotida; Setmelanotide acetate; UNII-N7T15V1FUY; N7T15V1FUY; BIM 22493; IRC-022493; RM493; BIM22493; IRC 022493; RM 493; BIM-22493; IRC022493

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~89.5 mM)
Water: ~100 mg/mL
Ethanol: ~100 mg/mL

5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 5.0mg/ml (4.48mM) (Please use freshly prepared in vivo formulations for optimal results.)