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
Afamelanotide is administered as a subcutaneous implant that slowly elutes active drug. Most of the dose is released within the first 48 hours, with >90% released by day 5. Plasma levels of afamelanotide decrease slowly over the course of several days following administration - by day 10, plasma levels were undetectable in most clinical trial subjects. Following administration of a single subcutaneous implant, the median Tmax was 36 hours, the mean Cmax was 3.7 ± 1.3 ng/mL, and the mean AUC0-∞ was 138.9 ± 42.6 hr.ng/mL.

Route of Elimination
Minimal amounts of unchanged afamelanotide are recovered in the urine following administration, suggesting the drug is extensively metabolized and most likely eliminated primarily via fecal or biliary route.

Volume of Distribution
The apparent volume of distribution of afamelanotide following intravenous administration is approximately 0.54 L/kg.

Clearance
Data regarding plasma clearance of afamelanotide are limited. Plasma drug levels are typically undetectable at day 10 following subcutaneous administration of the afamelanotide implant.

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Metabolism / Metabolites
Details regarding the metabolism and metabolites of afamelanotide are sparse. The drug is more resistant to degradation by serum and proteolytic enzymes than its endogenous counterpart, α-MSH, but presumably undergoes a relatively rapid hydrolysis given its short half-life. It has been suggested that afamelanotide may be degraded in the same manner as α-MSH but at a much slower rate, or may instead be degraded intracellularly via endocytosis or non-specific proteases.

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Biological Half-Life
The half-life of afamelanotide is approximately 30 minutes. The apparent half-life following administration of a slow-release subcutaneous implant is 15 hours.

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Mechanism of Action
Patients with erythropoietic porphyria (EPP) have a deficiency of ferrochelatase (FECH), an enzyme involved in the final step of heme biosynthesis. FECH is required to insert iron into protoporphyrin IX (PPIX) to generate heme, and a deficiency in FECH results in accumulation of PPIX (particularly in the liver and superficial skin vasculature). PPIX molecules are photodynamic - exposure to UV radiation causes these molecules to form reactive oxygen species that lead to subsequent tissue damage. Afamelanotide mimics endogenous alpha melanocyte-stimulating hormone (α-MSH), a hormone typically released in response to UV-induced skin damage. Both afamelanotide and α-MSH bind to the melanocortin-1 receptor (MC1R) on melanocytes which stimulates the synthesis of eumelanin, a photoprotective compound. Eumelanin is incorporated into small vesicles called melanosomes which are then distributed to surrounding keratinocytes. Melanosomes are concentrated above the nucleus of these keratinocytes, thus protecting them from UV-induced damage. While endogenous α-MSH requires UV-induced skin damage in order to be produced, afamelanotide increases eumelanin biosynthesis independent of UV exposure. Activation of MC1R signalling by afamelanotide also instigates other protective processes, including an increase in antioxidant activity, DNA repair, and secretion of immunomodulatory proteins such as interleukin-10.

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The in-vivo release kinetics of selected formulations were analyzed in guinea pigs following intrap-eritoneal administration. The plasma concentration–time profiles showed an extended release of the peptide formulated with gel compared to the intraperitoneal administration of Afamelanotide/MT-I in solution. On the basis of the in-vitro and in-vivo results, the P407 formulations of MT-I with MC or HPMC as an additive showed potential for use as a controlled-release delivery system for MT-I [4].

Hepatotoxicity
Erythropoietic protoporphyria and X-linked protoporphyria are rare genetic diseases, and the pivotal trials of afamelanotide were conducted in a limited number of patients, so the full spectrum of hepatotoxicity may not be fully known. Furthermore, a proportion of untreated persons with EPP/XLPP have liver disease with mild, fluctuating elevations of serum aminotransferase levels; indeed, 2% to 5% develop significant liver disease with cholestatic features and cirrhosis, some of which may be due to gallstones which are common in patients with EPP/XLPP and others due to alcohol or other underlying liver diseases. Against this background, the clinical studies of afamelanotide were accompanied by minor serum enzyme elevations, but in rates that were similar to those receiving placebo and none reaching levels considered indicative of drug induced liver injury. In the three prospective placebo-controlled trials of afamelanotide in EPP/XLPP, there were no episodes of clinically apparent liver injury that arose during therapy, but a few patients developed evidence of chronic liver disease, cirrhosis, or complications of gallstones during follow up. These episodes arose long after afamelanotide was stopped and were considered due to the underlying liver disease or concurrent excessive alcohol use. Indeed, afamelanotide treatment is associated with slight decreases in protoporphyrin levels and may ameliorate the course of the EPP/XLPP-associated chronic liver injury. Since, its approval and more widescale use, there have been no reports of clinically apparent liver injury attributed to afamelanotide.

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Afamelanotide is a melanocortin-1 receptor agonist that stimulates melanin production in the skin and is used to decrease pain and itching from light exposure in patients with erythropoietic protoporphyria and X-linked protoporphyria. Afamelanotide has not been linked to serum aminotransferase elevations during therapy nor to instances of idiosyncratic acute liver injury with 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
Prevention of phototoxicity in adult patients with erythropoietic protoporphyria (EPP).
Afamelanotide is a polypeptide comprising of 13 amino acids which is an analogue of alpha-melanocyte stimulating hormone. It is approved as a dermatologic drug for the prevention of phototoxicity in adult patients with erythropoietic protoporphyria. It has a role as a dermatologic drug.
See also: Afamelanotide (preferred).

Afamelanotide is a first-in-class, synthetic, 13-amino acid peptide analogue of the endogenous alpha melanocyte-stimulating hormone (α-MSH). It differs structurally from its endogenous counterpart by only two amino acids - these structural differences improve biological efficacy by imparting a greater affinity for its target and a longer biological half-life. Afamelanotide is currently the only approved drug therapy used in the management of erythropoietic protoporphyria, having received approval in the EU in December 2014 and subsequent FDA approval in October 2019. Despite its relatively recent approval, afamelanotide has been available for use as an orphan drug in both the US and EU since 2008.

Afamelanotide is a melanocortin-1 receptor agonist that stimulates melanin production in the skin and is used to decrease pain and itching from light exposure in patients with erythropoietic protoporphyria and X-linked protoporphyria. Afamelanotide has not been linked to serum aminotransferase elevations during therapy nor to instances of idiosyncratic acute liver injury with symptoms and jaundice.

Afamelanotide is a synthetic peptide analogue of the naturally occurring alpha-melanocyte stimulating hormone (a-MSH) with potential photoprotective activity. Mimicking the action of a-MSH, afamelanotide stimulates melanocytes to increase the production and release of melanin. Increased melanocyte melanin may protect against ultraviolet radiation (UVR)-initiated cellular DNA damage, oxidation of membrane proteins, and alterations in intracellular signaling processes in epidermal cells. Endogenously, a-MSH is released by skin cells in response to UVR exposure, stimulating melanocytes to produce and release melanin.

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While ultraviolet radiation (UVR) is a major cause of skin ageing and carcinogenesis, public pursuit of a novel tanning strategy circumventing the need for UVR is increasingly reported in the media and scientific press. This involves the subcutaneous self-administration of unregulated products labelled as melanotan I and/or II, synthetic analogues of α-melanocyte stimulating hormone (α-MSH), as obtained via the internet, tanning salons and gyms. The Medicines and Healthcare products Regulatory Authority has recently raised awareness of the public health risk of transmission of blood-borne viruses from the needle sharing that may occur, and of the potential impurity of these products. Dermatologists should also be aware that these agents can complicate the clinical presentation of patients with pigmented lesions; their use may be suspected in unexpectedly tanned individuals with rapidly pigmenting naevi. Meanwhile, the regulated α-MSH analogue afamelanotide (Clinuvel Pharmaceuticals Ltd, Melbourne, Australia) is showing promise for its photoprotective potential, and is undergoing phase II and III clinical trials in people with photosensitivity disorders and those prone to nonmelanoma skin cancer. The photoprotective and other biological effects of α-MSH analogues await full determination.[2]

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Melanotan is a synthetic analogue of alpha melanocyte stimulating hormone (a-MSH) that stimulates melanogenesis. It is sold on the internet and tanning salons as a quick 'tanning jab'. We report a patient who developed multiple new onset atypical naevi within one week of receiving two Melanotan injections. This case highlights the potential risk of Melanotan in stimulating dysplastic naevi or possibly 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.)