RESEARCH USE ONLY — QUALIFIED LABORATORY PERSONNEL ONLY: This article is for controlled laboratory research only. Personal or home use is prohibited.
The material described is not for human use or veterinary administration. Any mention of reconstitution, dilution, sample transfer, or experimental exposure refers only to in vitro work or laboratory animal studies conducted under institutionally approved protocols.
It must not be used as a dosing or administration guide.
Melanotan I, also known as NDP-α-MSH or afamelanotide, is a synthetic 13-residue analogue of α-MSH. Research-grade Melanotan I is used in receptor, signaling, and melanocyte studies.
Separately, afamelanotide is the active ingredient in SCENESSE, an FDA-approved prescription implant for erythropoietic protoporphyria (EPO). The research material discussed on this page is not that approved drug product and is not intended for human or veterinary use.[1][2]
The anhydrous free peptide has the formula C78H111N21O19, an average molecular mass of approximately 1,646.85 Da, and a neutral monoisotopic mass of approximately 1,645.8365 Da. The observed LC-MS m/z will depend on the charge state, adducts, and data-processing conventions; the average molecular mass should not be used as a direct HRMS target.[1]
Basic Information
| Item | Information |
|---|---|
| Other names | Afamelanotide; NDP-α-MSH; [Nle4, D-Phe7]-α-MSH |
| Sequence | Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 |
| Length | 13 amino-acid residues |
| CAS number | 75921-69-6 |
| Molecular formula (anhydrous free peptide) | C78H111N21O19 |
| Average molecular mass (anhydrous free peptide) | Approximately 1,646.85 Da |
| Monoisotopic mass (anhydrous free peptide) | 1,645.8365 Da[1] |
| Structure | Linear peptide with an acetylated N-terminus and an amidated C-terminus |
Melanotan I differs from natural α-MSH at two positions. Norleucine replaces methionine at position 4, which removes the sulfur-containing methionine side chain and lowers the risk of that specific oxidation pathway.
D-phenylalanine replaces L-phenylalanine at position 7. In the original laboratory report, these changes increased resistance to serum-enzyme breakdown and gave longer-lasting activity in the assays and animal models used in that study.[3]
The formula and molecular masses above apply to the anhydrous free peptide. A supplied batch may be an acetate form; the approved SCENESSE label describes afamelanotide acetate as C78H111N21O19 · xC2H4O2 with 3 ≤ x ≤ 4, meaning the acetate content is not a fixed 1:1 stoichiometry.
Merely stating “acetate salt” or “TFA salt” without quantified counterion content may be insufficient for accurate gravimetric molarity calculations. Use the batch-specific peptide assay/content value together with the quantified counterion and water content from the COA, rather than treating the total powder weight as pure free peptide.[12]
How Melanotan I Is Studied
Melanotan I is active at MC1R, MC3R, MC4R, and MC5R in receptor-assay systems, while MC2R is pharmacologically distinct and is primarily activated by ACTH.
It should not be called a selective MC1R-only ligand.
The measured potency changes with the receptor type, species, cell system, test method, and readout, so a single fold-potency number should not be used as a universal value.[4]
Human MC1R is a 317-amino-acid G-protein-coupled receptor.[5] A simplified canonical signaling pathway linked to this receptor is:[6]
MC1R/Gs activation → adenylyl cyclase → increased cAMP → PKA/CREB signaling → altered MITF expression or activity → changes in melanogenic genes.
This is not the only signaling route associated with MC1R; the response can also involve ERK/MAPK and other pathways depending on the cell background, receptor variant, ligand concentration, and exposure time.
MITF helps control genes such as tyrosinase, DCT, and TYRP1, but the size and timing of the response can change between cell lines and test conditions.[7] DCT converts dopachrome to DHICA during the eumelanin pathway.[8]
TYRP1 also takes part in pigment biology, but its exact enzyme activity is not the same in every species—human TYRP1 does not exhibit the same DHICA oxidase activity as mouse Tyrp1—so results from mouse and human systems should not be treated as identical.[9]
Common Research Uses
- Receptor assays: measuring receptor binding, cAMP production, or reporter activity in cells that express a stated melanocortin receptor.
- Melanocyte studies: measuring tyrosinase activity, pigment-related proteins, total pigment, or other defined cell endpoints. Published cultured-human-melanocyte work has linked melanocortin receptor binding with cAMP, tyrosinase activity, and pigment-related responses under specific in vitro conditions.[10]
- Animal studies: studying receptor or pigment biology under an institutionally approved animal-use protocol. This article does not provide animal-treatment, veterinary, route, or dose instructions.
Each study should clearly report the receptor or model used, the cell line or species, peptide concentration, exposure time, controls, test method, and measured endpoint.
Results from one setup should not be presented as proof of what will happen in another setup.
How to Read the COA
A COA describes the quality results for one batch. It does not prove that the material is safe or effective for human, cosmetic, clinical, or veterinary use.
The test method must be suitable for what it is being used to measure. ICH Q2(R2) explains that identity, purity, impurities, assay, accuracy, precision, and range are separate analytical questions and may require different checks; these principles are cited as analytical-quality references and do not mean that a research-use-only material has been manufactured or released under a pharmaceutical GMP specification.[11]
| COA item | What it means |
|---|---|
| Identity | The sequence and terminal changes should match the intended peptide. At least two orthogonal methods are recommended (e.g., LC-MS intact mass plus LC-MS/MS sequencing or amino-acid analysis). Retention time alone is not enough to prove full identity. |
| HPLC purity | The HPLC area percentage shows the relative detector response of peaks under that HPLC method. Unless relative response factors have been established for each impurity, area normalization is not necessarily equivalent to molar or mass percentage. It is not the same as peptide content or the percentage of peptide in the total powder weight. |
| Mass spectrometry | The measured mass should match the expected mass within the method limit. The expected result depends on the charge state, adducts, and mass convention (average or monoisotopic). The average molecular mass of 1,646.85 Da should not be used as a direct high-resolution MS target. |
| D-Phe confirmation | D-Phe and L-Phe are isobaric, so intact-mass MS alone cannot confirm stereochemistry. Confirmation requires a validated stereoselective method, such as chiral LC or GC, suitable derivatization with amino-acid analysis, or another qualified stereochemical method, generally used with an appropriately characterized reference standard. |
| Peptide content / assay | This is separate from HPLC purity. Water, counterions, residual solvents, and other non-peptide material can change how much peptide is present in a weighed sample. The batch-specific peptide content should be reported with the calculation basis (e.g., as-is, anhydrous, or free-peptide-equivalent) and method. |
| Counterion content | Acetate, TFA, or other counterions should be reported with identity and quantitative content. Afamelanotide acetate is not a fixed 1:1 stoichiometric salt; the approved drug label specifies 3 ≤ x ≤ 4 acetate equivalents. Counterion content directly affects the mass basis for molar concentration calculations. |
| Water and residual solvents | Water (e.g., by Karl Fischer) and residual solvents require separate validated methods and acceptance limits. These affect the weighed mass and should not be inferred from HPLC. |
| Related impurities | Possible impurities can include shortened sequences, missing residues, incomplete deprotection, epimers, oxidation products, aggregates or oligomers, and other process or storage by-products. |
| Endotoxin and bioburden | For cell-based assays or animal studies, endotoxin testing (LAL, rFC, or another validated method with stated units and spike-recovery data) and, where relevant, bioburden or sterility testing should be considered according to the experimental application. |
The EMA guideline for synthetic peptides (EMA/CHMP/CVMP/QWP/367182/2025, effective 1 June 2026) addresses manufacturing, characterization, specifications, and analytical control for synthetic peptides used in medicinal products. Its principles may be used as quality-reference benchmarks for research peptides, but this guideline is written for drug development and does not by itself establish mandatory specifications for research-use-only materials.[12]
There is no single HPLC purity number that makes a batch suitable for every experiment.
The required level depends on the test, the sensitivity of the readout, and which impurities are present.
Storage and Reconstitution
- Lyophilized powder: Unless batch-specific stability data say otherwise, store at −20°C in a tightly closed container, protected from light and moisture. The EMA guideline notes that peptides are commonly stored refrigerated (5 ± 3°C) or frozen (−20 ± 5°C), but specific conditions and retest periods must be supported by stability data.
- Before opening: Let the sealed container warm toward room temperature if condensation may form.
- Reconstitution: Choose the solvent appropriate to the experimental use: HPLC-grade or equivalent purified water for analytical work, sterile low-endotoxin or cell-culture-grade solvent for cell-based assays, and a validated buffer system for biological activity experiments. This is laboratory sample preparation only.
- Prepared solutions: Do not assume one storage time works for every solution. Stability can change with pH, concentration, buffer, temperature, light, oxygen, container material, and freeze-thaw cycles. Low-concentration peptide solutions may also experience adsorptive loss to container surfaces.
- Aliquots: When suitable for the assay, small laboratory aliquots can reduce repeated opening and freeze-thaw exposure.
Limits of the Data
Binding strength, cell activity, breakdown rate, and the length of a downstream response are different measurements. Terms such as “highly selective,” “1,000-fold more potent,” “ultralong,” or “stable for days” should only be used when the exact test system and original source are given.
All conclusions in this article are limited to the stated in vitro system or laboratory animal model and must not be turned into human, medical, cosmetic, personal-use, or veterinary claims.
Research Use Only Disclaimer
Research Use Only: The Melanotan I / afamelanotide research material supplied by NEXALUME LABS is intended and labeled solely for laboratory research use. It is not the approved SCENESSE (afamelanotide) implant drug product and is not intended, manufactured, tested, or released for human or veterinary administration.
This article provides general scientific information regarding standard laboratory in vitro and in vivo research contexts only, and does not constitute a validated experimental protocol, medical guidance, clinical guidance, veterinary guidance, or treatment advice.
