MC1R/melanocortin receptor

The overall objective of these studies was to develop a controlled-release formulation of Afamelanotide/Melanotan-I (MT-I) containing poloxamer 407 (P407). Various aqueous formulations were evaluated containing MT-I and 25% w/v P407 alone, or with one of the following additives present, i.e., poly(vinylpyrrolidone) 10 000 (PVP), methylcellulose (MC), or hydroxypropyl methylcellulose (HPMC). The in-vitro release profiles of MT-I from the P407 formulations and the dissolution of the gel were obtained simultaneously using a membraneless in-vitro model. These data were obtained at 37 °C and room temperature (24 °C). It was observed that the PVP-containing P407 formulations of MT-I accelerated the dissolution of gel and the release of the peptide compared to the control formulation. The formulations containing MC or HPMC exhibited the slowest dissolution rates and release of MT-I. The same rank order was observed for the dissolution and release profiles of MT-I from the various formulations at both temperatures [4].

Melanotropic peptides exert photoprotective effects in humans in vivo, and ongoing clinical trials are exploring whether this may translate into tangible clinical benefits in the photoprotection of photosensitivity disorders and nonmelanoma skin cancer. There are safety concerns regarding the unregulated use of chemicals labelled as melanotan I/Afamelanotide and II, including risks of virus transmission through needle sharing and of diagnostic confusion through distortion of the appearance of pigmented skin lesions, and advice should be given against this recreational use. Future research will clarify the scope of characterized α‐MSH analogues as a therapeutic tool, when applied in controlled dosage and defined conditions.[2]

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In an open‐label study of five subjects with EPP over 4 months, patients received a subcutaneous implant of Afamelanotide at day 7 and at week 8. Skin melanin density increased when compared with baseline, accompanied by an increase in tolerance to photoprovocation, assessed by time taken to reach intolerable pain on light exposure. This is a subjective end‐point, and randomized controlled studies are ongoing, further to explore the potential for treatment of EPP with Afamelanotide. The mechanism of action of afamelanotide in EPP could potentially involve more than melanization alone, and thus the measure of protection could vary depending on photosensitivity disorder. While increased melanization will reduce penetration of optical radiation and may thus increase symptom threshold, the parent α‐MSH molecule is reported experimentally to possess antioxidant, anti‐inflammatory and immunomodulatory properties, as discussed earlier. [2]

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Ongoing trials are evaluating Afamelanotide in hospital‐based patients with SU and PLE. SU is a rare immune‐mediated photosensitivity disorder triggered by UVR and/or visible light, with a large impact on quality of life. First‐line treatment is antihistamines, avoidance of exposure and photoprotection. Other treatment strategies are ‘skin hardening’ through repeated exposure to UVR, beneficial in some, and plasmapheresis, effective in some patients but often a difficult modality to procure. PLE, on the other hand, is a common inflammatory photosensitivity disorder, with evidence of underlying immune mediation, the moderate to severe forms of which are associated with significant morbidity. Exposed sites are less sensitive to rash provocation; this may be due to skin ‘hardening’, whereby these areas of skin are more pigmented and thickened and hence more tolerant of UVR. The pigmentation may be replicated, without the UVR exposure that triggers the condition, using [Nle4‐D‐Phe7]‐α‐MSH [2].

Absorption
Afameranotide is administered as a subcutaneous implant, allowing for a slow release of the active drug. Most of the dose is released within the first 48 hours, with over 90% released by day 5. Plasma concentrations of afameranotide decrease slowly over several days after administration—by day 10, plasma concentrations are undetectable in most clinical trial participants. Following a single subcutaneous implantation, the median time to peak concentration (Tmax) was 36 hours, the mean peak concentration (Cmax) was 3.7 ± 1.3 ng/mL, and the mean area under the curve (AUC0-∞) was 138.9 ± 42.6 hr·ng/mL.
Elimination Route
Only trace amounts of unmetabolized afameranotide are recovered in the urine after administration, indicating extensive metabolism and likely elimination primarily via feces or bile.
Volume of Distribution
The apparent volume of distribution after intravenous administration of afameranotide is approximately 0.54 L/kg.
Clearance
Limited data are available regarding the plasma clearance of afameranotide. Plasma drug concentrations are typically undetectable by day 10 after subcutaneous implantation of afameranotide.

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Metabolism/Metabolites
Detailed information on the metabolism and metabolites of afameranotide is scarce. This drug is more resistant to degradation by serum and proteolytic enzymes compared to its endogenous counterpart, α-MSH, but given its short half-life, its hydrolysis is presumed to be relatively rapid. Studies suggest that afameranotide may degrade in the same way as α-MSH, but at a much slower rate, or possibly via endocytosis or intracellular degradation by nonspecific proteases.

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Biological Half-Life
The half-life of afameranotide is approximately 30 minutes. The apparent half-life after administration of the subcutaneous sustained-release implant is 15 hours.

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Mechanism of Action
Erythropoietic porphyria (EPP) patients lack ferrous chelate synthase (FECH), an enzyme involved in the final step of heme biosynthesis. FECH is essential for the insertion of iron into protoporphyrin IX (PPIX) to generate heme; FECH deficiency leads to PPIX accumulation (especially in the liver and superficial skin blood vessels). PPIX molecules are photodynamic—exposure to ultraviolet radiation causes these molecules to form reactive oxygen species, leading to tissue damage. Alfameranotide mimics endogenous alpha-melanocyte-stimulating hormone (α-MSH), which is typically released after UV-induced skin damage. Both alfameranotide and α-MSH bind to the melanocortin-1 receptor (MC1R) on melanocytes, thereby stimulating the synthesis of the photoprotective compound eumelanin. Eumelanin is encapsulated in small vesicles called melanosomes, which are then distributed to surrounding keratinocytes. Melanosomes aggregate above the nuclei of these keratinocytes, thus protecting them from UV damage. The production of endogenous α-MSH requires skin damage caused by ultraviolet radiation, while afameranolide can increase the biosynthesis of eumelanin without ultraviolet radiation. Afameranolide can also activate the MC1R signaling pathway and initiate other protective processes, including enhancing antioxidant activity, promoting DNA repair and secreting immunomodulatory proteins such as interleukin-10.

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The in vivo release kinetics of the selected formulation were analyzed after intraperitoneal administration in guinea pigs. Plasma concentration-time curves showed that the peptide release time in the gel formulation was longer than that in the intraperitoneal solution form of afameranolide/MT-I. Based on in vitro and in vivo experimental results, the MT-I P407 formulation with MC or HPMC as additives showed potential as a controlled-release delivery system for MT-I[4].

Hepatotoxicity
Erythropoietic protoporphyria and X-linked protoporphyria are rare genetic disorders. The pivotal trials of afamelanotide enrolled a limited number of patients, therefore the full extent of its hepatotoxicity may not be fully understood. Furthermore, some untreated erythropoietic/X-linked protoporphyria patients have liver disease, characterized by mildly fluctuating elevations in serum transaminase levels; in fact, 2% to 5% of patients develop severe liver disease with cholestasis and cirrhosis, some of which may be due to gallstones, a common symptom in erythropoietic/X-linked protoporphyria patients, while others may be due to alcohol or other underlying liver conditions. Against this backdrop, while mild elevations in serum enzymes were observed in clinical studies of afamelanotide, their incidence was similar to that in the placebo group, and none reached the levels indicative of drug-induced liver injury. In three prospective placebo-controlled trials of afameranotide for the treatment of erythropoietic protoporphyria/X-linked protoporphyria, no clinically significant liver injury events occurred during treatment, but a small number of patients showed evidence of complications such as chronic liver disease, cirrhosis, or gallstones during follow-up. These events occurred long after afameranotide discontinuation and were thought to be caused by primary liver disease or concurrent excessive alcohol consumption. In fact, afameranotide treatment can slightly decrease protoporphyrin levels and may improve the course of chronic liver injury associated with erythropoietic protoporphyria/X-linked protoporphyria. Since its approval and widespread use, there have been no reports of clinically significant liver injury caused by afameranotide. Afameranotide is a melanocortin-1 receptor agonist that stimulates melanin production in the skin and is used to relieve photosensitive pain and itching in patients with erythropoietic protoporphyria and X-linked protoporphyria. Alfamelanotide was not associated with elevated serum transaminase levels during treatment, nor with specific cases of acute liver injury accompanied by symptoms and jaundice.

[1]. Discovery and development of novel melanogenic drugs. Melanotan-I and -II. Pharm Biotechnol. 1998:11:575-95.

[2]. Melanotropic peptides: more than just 'Barbie drugs' and 'sun-tan jabs'? Br J Dermatol. 2010 Sep;163(3):451-5.

[3]. Atypical melanocytic naevi following melanotan injection. Ir Med J. 2013 May;106(5):148-9.

[4]. Controlled-release delivery system for the alpha-MSH analog melanotan-I using poloxamer 407. J Pharm Sci. 1996 Sep;85(9):915-9.

Drug Indication
Prophylaxis of phototoxicity in adults with erythropoietic protoporphyria (EPP). Alfameranotide is a 13-amino acid peptide and an analog of alpha-melanocyte-stimulating hormone (α-MSH). It is approved as a dermatological drug for the prevention of phototoxicity in adults with erythropoietic protoporphyria. It has dermatological drug use. See also: Alfameranotide (preferred). Alfameranotide is a first-in-class synthetic 13-amino acid peptide and an analog of endogenous alpha-melanocyte-stimulating hormone (α-MSH). It differs from endogenous α-MSH by only two amino acids—these structural differences enhance its biological activity by conferring higher target affinity and a longer biological half-life. Alfamelanotide is currently the only approved drug for the treatment of erythropoietic protoporphyria. It was approved in the European Union in December 2014 and by the US FDA in October 2019. Despite its relatively short approval time, afamelanotide has been marketed as an orphan drug in the US and EU since 2008. Alfamelanotide is a melanocortin-1 receptor agonist that stimulates melanin production in the skin, used to relieve pain and itching caused by light exposure in patients with erythropoietic protoporphyria and X-linked protoporphyria. No elevation of serum transaminases or specific acute liver injury with symptomatic jaundice was observed during afamelanotide treatment. Alfamelanotide is a synthetic peptide and an analog of naturally occurring α-melanocyte-stimulating hormone (α-MSH), possessing potential photoprotective activity. Alfamelanotide mimics the action of α-MSH, stimulating melanocytes to increase melanin production and release. Increased melanin production in melanocytes may help combat UVR-induced cellular DNA damage, membrane protein oxidation, and alterations in intracellular signaling processes. Endogenous α-MSH is released by skin cells under UVR irradiation, stimulating melanocytes to produce and release melanin. While UVR is a major cause of skin aging and cancer, there is increasing media and scientific journal coverage of the public seeking novel tanning strategies that do not require UVR exposure. This involves subcutaneous injections of unregulated products labeled “Melanotan I” and/or “Melanotan II,” synthetic analogs of α-melanocyte-stimulating hormone (α-MSH), available online, in tanning salons, and at gyms. The Medicines and Healthcare products Regulatory Agency (MHRA) has recently raised public awareness of the potential impurities in these products and the public health risks of bloodborne viral transmission from shared needles. Dermatologists should also be aware that these medications may complicate the clinical presentation of patients with pigmented lesions; individuals experiencing a sudden darkening of skin and a rapid increase in mole pigmentation should be suspected of using these medications. Meanwhile, the regulated α-MSH analog alfameranotide (Clinuvel Pharmaceuticals Ltd, Melbourne, Australia) has shown promising potential for photoprotection and is currently undergoing phase II and III clinical trials in patients with photosensitive diseases and non-melanoma-prone skin cancer populations. The photoprotective effects and other biological effects of α-MSH analogs remain to be fully determined. [2]

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Meranotane is a synthetic analog of α-melanocyte-stimulating hormone (α-MSH) that stimulates melanin production. It is sold online and in tanning salons as a quick “tanning injection.” We report a case of a patient who developed multiple new atypical nevi within a week of receiving two melananotane injections. This case highlights the potential risk of melananotane stimulating dysplastic nevi or potentially inducing malignant melanoma. [3]

C80H115N21O21

827.03

1705.857

C, 55.22; H, 6.79; N, 16.10; O, 21.89

1566590-77-9

Melanotan I;75921-69-6

131839615

Typically exists as solid at room temperature

24

24

51

122

3390

12

O=C([C@H](CCCCN)NC(CNC([C@H](CC1=CNC2C=CC=CC1=2)NC([C@H](CCCNC(=N)N)NC([C@@H](CC1C=CC=CC=1)NC([C@H](CC1=CNC=N1)NC([C@H](CCC(=O)O)NC([C@H](CCCC)NC([C@H](CO)NC([C@H](CC1C=CC(=CC=1)O)NC([C@H](CO)NC(C)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)N1CCC[C@H]1C(N[C@@H](C(N)=O)C(C)C)=O.OC(C)=O

LBIUKNXYUXOWFF-CRYSLBJVSA-N

InChI=1S/C78H111N21O19.C2H4O2/c1-5-6-19-52(91-75(116)61(41-101)97-72(113)57(34-46-24-26-49(103)27-25-46)94-74(115)60(40-100)88-44(4)102)68(109)92-54(28-29-64(105)106)70(111)96-59(36-48-38-83-42-87-48)73(114)93-56(33-45-16-8-7-9-17-45)71(112)90-53(22-14-31-84-78(81)82)69(110)95-58(35-47-37-85-51-20-11-10-18-50(47)51)67(108)86-39-63(104)89-55(21-12-13-30-79)77(118)99-32-15-23-62(99)76(117)98-65(43(2)3)66(80)107;1-2(3)4/h7-11,16-18,20,24-27,37-38,42-43,52-62,65,85,100-101,103H,5-6,12-15,19,21-23,28-36,39-41,79H2,1-4H3,(H2,80,107)(H,83,87)(H,86,108)(H,88,102)(H,89,104)(H,90,112)(H,91,116)(H,92,109)(H,93,114)(H,94,115)(H,95,110)(H,96,111)(H,97,113)(H,98,117)(H,105,106)(H4,81,82,84);1H3,(H,3,4)/t52-,53-,54-,55-,56+,57-,58-,59-,60-,61-,62-,65-;/m0./s1

(4S)-4-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-hydroxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-5-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-6-amino-1-[(2S)-2-[[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxohexan-2-yl]amino]-2-oxoethyl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid;acetic acid

Afamelanotide acetate; 1XCC161YKC; Afamelanotide acetate [USAN]; scenesse; Afamelanotide triacetate; CUV1647 ACETATE; CUV-1647 ACETATE; ...; 1566590-77-9;

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)

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)