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

Setmelanotide/BIM-2493和BIM-2511在MC1R、MC3R和MC4R上表现出激动剂活性（表1）。表1显示了两种配体在人MC1R、MC3R、MC4R和MC5R上的受体亲和力（Ki）和活性（EC50）数据。这两种化合物在MC1R-MC4R上都表现出活性，但在MC5R上表现出较弱的活性（EC50>1μM），在小鼠MC2R上没有活性（EC50>10μM）。为了确定在啮齿动物中是否也观察到激动剂活性，研究人员评估了大鼠MC4R中的Ki和EC50。BIM-2493和BIM-2511的Ki数据和EC50数据在大鼠和人类MC4R中具有可比性（表1）。

黑素皮质激素受体激动剂在大脑中起作用，调节食物摄入和体重，并且独立于这些作用，影响胰岛素敏感性。这些实验研究了在外周给药时穿过血脑屏障的新型非选择性黑皮质素受体激动剂（Setmelanotide/BIM-2493、BIM-2511）的功能。用外周注射的黑皮质素激动剂治疗饮食诱导的肥胖C57BL/6J（B6）小鼠，可改善肥胖、高胰岛素血症（约50%）和脂肪肝疾病。使用B6黑皮质素-3和黑皮质素-4受体敲除小鼠（MC3RKO、MC4RKO）确定功能的特异性。用于这些测试的Chow喂食MC4RKO，而不是MC3RKO，表现出肥胖、高胰岛素血症和严重的肝脂肪变性，与参与脂肪生成的胰岛素刺激基因表达增加有关。与急性BIM-22493治疗相关的食物摄入量减少，以及与BIM-22511治疗14天相关的体重减轻，需要功能性MC4R，但不需要MC3R。然而，尽管BIM-22511治疗14天对MC4RKO的体重没有影响，甚至增加了累积食物摄入量，但仍然观察到空腹胰岛素显著降低（约50%）。尽管降低了胰岛素水平，但BIM-22511的慢性治疗并没有改善MC4RKO的肝脂肪变性，也没有影响肝脂肪生成基因的表达。总之，这些结果表明，外周给药的黑皮质素受体激动剂通过独立的途径调节体重、肝脏代谢和葡萄糖稳态。MC4R是黑皮质素激动剂诱导的体重减轻和肝脏代谢改善所必需的，但不是改善高胰岛素血症所必需的。具有MC4R活性的激动剂至少部分通过导致体重减轻来改善葡萄糖稳态，但其他黑皮质素受体可能具有治疗与肥胖相关的葡萄糖稳态异常的潜力。[1]

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在饮食诱导的肥胖非人类灵长类动物模型中，使用高选择性新型MC4R激动剂（Setmelanotide/BIM-2493或RM-493）治疗导致食物摄入量短暂减少（35%），治疗8周后体重持续减轻（13.5%）。与体重减轻一致，这些动物显著降低了肥胖，改善了葡萄糖耐量。重要的是，我们没有观察到BIM-22493治疗的血压或心率增加。相比之下，用LY2112688治疗，一种MC4R激动剂，以前被证明可以增加人类的血压和心率，导致血压和心率升高，同时适度减少食物摄入量。这些研究表明，不同的黑皮质素肽药物可能具有截然不同的疗效和副作用[2]。

人MC1R、MC3R、MC4R和MC5R的放射配体结合分析[1]
细胞膜由稳定表达人黑皮质素受体亚型（MC1R、MC3R、MC4R和MC5R）的CHO-K1细胞制备。它们在pH 7.4的50 mM Tris-HCl中以1-10μg蛋白质/孔的浓度孵育，含有0.2%BSA、5 mM MgCl2、1 mM CaCl2和0.1 mg/mL杆菌肽，待测化合物浓度增加，0.1-0.3 nM[125I]-NDP-α-MSH在37°C下孵育90-120分钟，具体取决于受体亚型。通过用0.1%（w/v）PEI预浸的GF/C玻璃纤维过滤器过滤，分离游离[125I]-NDP-α-MSH。在0-4°C下用50 mM Tris-HCl（pH 7.4）洗涤过滤器三次，并使用Perkin-Elmer Topcount闪烁计数器测定放射性。

细胞内cAMP测定[2]
通过电化学发光（ECL）测定细胞内cAMP水平。将稳定表达人MC1R、MC3R、MC4R和MC5R的CHO-K1细胞悬浮在含有0.5 mM IBMX和0.2%BSA的RMPI 1640中。将它们分配（每孔7000个细胞）在含有集成碳电极并涂有抗cAMP抗体的多阵列板（MSD）中。在37°C下孵育40分钟，加入越来越高浓度的化合物。然后，裂解细胞并加入2.5nM TAG钌标记的环AMP（MSD）。90分钟后，使用Sector Imager 6000阅读器（MSD）通过ECL检测测定环AMP水平。NDP-MSH用作参考化合物。最大cAMP刺激水平比基础水平高12至20倍，具体取决于受体亚型。

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.

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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.

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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.

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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
Setmelanotide has a Tmax of 8 hours.
A 3mg subcutaneous dose of setmelanotide is 39% eliminated in the urine as the unchanged parent compound.
The apparent volume of distribution of setmelanotide is 48.7 L.
A 3mg subcutaneous dose of setmelanotide has an estimated clearance of 4.86 L/h.

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Metabolism / Metabolites
Setmelanotide is expected to be metabolized to small peptides and amino acids.

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Biological Half-Life
The elimination had life of setmelanotide is approximately 11 hours.

Hepatotoxicity
In the small open label clinical trials of setmelanotide for genetic forms of obesity, there were no reports of abnormalities of serum aminotransferase or bilirubin levels. One patient developed cholecystitis, but it was considered unrelated to therapy. Since its approval and clinical use, there have been no published cases of clinically apparent liver injury attributed to setmelanotide therapy, but elevations in serum aminotransferase levels during therapy have been described. Thus, setmelanotide therapy has not been linked to clinically apparent liver injury, but the total clinical experience with its use is limited.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Setmelanotide is 79.1% protein bound.

[1]. Analysis of the therapeutic functions of novel melanocortin receptor agonists in MC3R- and MC4R-deficient C57BL/6J mice. Peptides. 2009 Oct;30(10):1892-900.

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

Setmelanotide is the first available treatment for patients with pro-opiomelanocortin, proprotein subilisin/kexin type 1, or leptin deficiencies. It is an agonist of the melanocortin 4 receptor. Earlier attempts at agonizing MC4R (such as LY2112688) lead to successful weight loss, but also an increase in blood pressure and heart rate. Other earlier treatments for these patients included gastric bypass surgery. Patients taking setmelanotide experienced an average weight loss of 0.6 kg/week. Imcivree was granted EMA orphan designation on November 19, 2018 and FDA approval on November 25, 2020. On May 4, 2023, it was approved by Health Canada.
Setmelanotide is a Melanocortin 4 Receptor Agonist. The mechanism of action of setmelanotide is as a Melanocortin 4 Receptor Agonist.
Setmelanotide is a melanocortin 4 receptor agonist that is used for chronic weight management for adults and children with rare genetic forms obesity due to gene defects in the melanocortin pathway. Setmelanotide therapy has not been associated serum aminotransferase or bilirubin elevations or to instances of clinically apparent liver injury.
See also: Setmelanotide acetate (is active moiety of); Setmelanotide Hydrochloride (is active moiety of).

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Drug Indication
Setmelanotide is indicated for chronic weight management in patients 6 years and older with obesity due to pro-opiomelanocortin (POMC) deficiency, proprotein subtilisin/kexin type 1 (PCSK1) deficiency, or leptin receptor (LEPR) deficiency as determined by an approved test demonstrating variants in POMC, PCSK1, or LEPR genes that are interpreted as pathogenic, likely pathogenic, or of uncertain significance. These conditions affect the MC4R signalling pathway. Setmelanotide is also indicated for chronic weight management in patients 6 years and older with obesity due to Bardet-Biedl syndrome. The drug is not reported to be effective in patients with POMC, PCSK1, or LEPR variants classified as benign or likely benign, as well as other types of obesity not listed above.
Imcivree is indicated for the treatment of obesity and the control of hunger associated with genetically confirmed Bardet Biedl syndrome (BBS), loss-of-function biallelic pro-opiomelanocortin (POMC), including PCSK1, deficiency or biallelic leptin receptor (LEPR) deficiency in adults and children 6 years of age and above.
Treatment of appetite and general nutrition disorders

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Mechanism of Action
Grehlin and other hunger signals from the gastrointestinal tract stimulate orexigenic neurons, stimulating the release of agouti-related protein. Agouti-related protein inhibits melanocortin 4 receptor (MC4R) activation until satiety signals such as insulin or leptin stimulate anorexigenic neurons. Insulin and leptin stimulate production of the POMC-derived melanocortin peptide α-melanocyte simulating hormone, which is a ligand of MC4R. Orexigenic and anorexigenic neurons contain prohormone convertase 1/3 (PC1/3), which is encoded by the gene proprotein subtilisin/kexin type 1. PC1/3 preforms activation cleavage of a number of peptide hormone precursors, including α-melanocyte simulating hormone. Setmelanotide is a pro-opiomelanocortin derived peptide that is an agonist of MC4R. It is an approximately 20-fold more potent agonist of MC4R than endogenous α-melanocyte stimulating hormone, with an EC₅₀ of 0.27 nM. By directly agonizing MC4R, upstream genetic deficiencies in the MC4R signalling pathway cannot inhibit satiety, food intake is decreased, and weight loss is achieved. MC4R is a 332 amino acid G-protein coupled receptor (G-PCR). Although the lack of cardiovascular adverse effects with setmelanotide treatment are not well understood, it is believed that earlier MC4R antagonists activated multiple G-protein signalling pathways. Earlier drugs that targeted G-PCRs either bound with high affinity to the highly conserved orthosteric binding site, or with high specificity to less conserved allosteric sites. Setmelanotide is an atypical bitopic ligand that interacts with both the orthosteric and putative allosteric binding site, allowing for both high affinity and specificity.
SETMELANOTIDE is a Protein drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2020 and is indicated for obesity and has 4 investigational indications.

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In summary, these experiments have demonstrated the use of two novel melanocortin receptor agonists. Our data suggest that MC4Rare necessary for weight loss associated with the use of these compounds. However, activity at other melanocortin receptors may be responsible for significant improvements insulin sensitivity independently of the MC4R and of weight loss.[1] In conclusion, we have described a MC4R agonist, BIM-22493 (also known as RM-493), which, after chronic subcutaneous infusion, reduced food intake and adiposity and resulted in an impressive 13.5% decrease in overall body weight. An associated improvement was noted in glucose homeostasis, insulin sensitivity, and leptin level results. Further, we report that activation of the melanocortin system using BIM-22493 does not result in immediate changes to the blood pressure or heart rate when the agonist is administered via a minipump, and in fact, that there is an overall chronic improvement of cardiovascular function secondary to weight loss. Future studies in human subjects will be needed to define the effectiveness of BIM-22493 as an antiobesity therapeutic.[2]

C51H72N18O11S2

1177.36

1176.50693

C, 51.44; H, 6.19; N, 20.38; O, 16.81; S, 5.18

2759937-80-7

Setmelanotide;920014-72-8;Setmelanotide TFA; 1301120-74-0 (pamoate); 1504602-49-6

162341716

Ac-Arg-Cys(1)-D-Ala-His-D-Phe-Arg-Trp-Cys(1)-NH2.CH3CO2H; N-acetyl-L-arginyl-L-cysteinyl-D-alanyl-L-histidyl-D-phenylalanyl-L-arginyl-L-tryptophyl-L-cysteinamide (2->8)-disulfide acetic acid

RCAHFRWC

Typically exists as solids at room temperature

537 Ų

16

16

18

82

2160

8

S1CC(C(N)=O)NC([C@H](CC2=CNC3C=CC=CC2=3)NC([C@H](CCC/N=C(\N)/N)NC([C@@H](CC2C=CC=CC=2)NC(C(CC2=CN=CN2)NC([C@@H](C)NC(C(CS1)NC([C@H](CCC/N=C(\N)/N)NC(C)=O)=O)=O)=O)=O)=O)=O)=O.OC(C)=O

BAJXLDXHOOSXOU-GZRAWZNHSA-N

InChI=1S/C49H68N18O9S2.C2H4O2/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;1-2(3)4/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);1H3,(H,3,4)/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;acetic acid

Setmelanotide acetate; Setmelanotide (acetate); 1504602-49-6; 2759937-80-7;

2934.99.9001

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。