PT-141 (Bremelanotide): Research Applications and Quality Assessment

PT-141 (Bremelanotide): Research Applications and Quality Assessment

PT-141, also known as Bremelanotide, is a synthetic melanocortin peptide analog developed from Melanotan II. Unlike Melanotan II, which primarily targets skin pigmentation, PT-141 exhibits a more pronounced effect on sexual arousal. This article provides a comprehensive overview of PT-141, focusing on its molecular structure, mechanism of action, research applications, crucial quality markers, common impurities, and appropriate storage conditions. This information is vital for researchers seeking to utilize PT-141 in their studies and ensuring the reliability and reproducibility of their results.

Molecular Structure and Properties

Bremelanotide is a cyclic heptapeptide with the amino acid sequence Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Gly]-NH2. Its molecular formula is C₅₀H₆₈N₁₄O₁₀, and its molecular weight is approximately 1025.2 Da. The cyclic structure is formed by a lactam bridge between the Asp and Gly residues. The presence of non-natural amino acids like Nle (norleucine) and D-Phe (D-phenylalanine) contributes to its increased resistance to enzymatic degradation compared to native melanocortin peptides.

Mechanism of Action

PT-141 acts as a non-selective agonist of melanocortin receptors (MC1R, MC3R, MC4R, and MC5R), though its primary effects are attributed to its interaction with MC3R and MC4R in the central nervous system. These receptors are involved in various physiological processes, including sexual function, inflammation, and energy homeostasis. Specifically, the activation of MC4R in the hypothalamus is believed to be the key mediator of PT-141's pro-sexual effects. It bypasses the vascular mechanisms associated with PDE5 inhibitors (like sildenafil), making it a potential research avenue for addressing sexual dysfunction independent of blood flow issues.

Research Applications

PT-141 has been investigated in preclinical and clinical research for various potential applications, primarily focusing on sexual dysfunction:

• Female Sexual Dysfunction (FSD): PT-141 was approved as a treatment for hypoactive sexual desire disorder (HSDD) in premenopausal women under the brand name Vyleesi. Research continues to explore its efficacy and safety in different patient populations and formulations.
• Male Erectile Dysfunction (ED): While not as widely studied as for FSD, research has explored PT-141's potential in treating ED, particularly in cases where PDE5 inhibitors are ineffective or contraindicated. Studies have explored its efficacy in men with diabetes and post-prostatectomy ED.
• Melanocortin Receptor Research: PT-141 serves as a valuable tool for researchers studying the melanocortin receptor system. Its relatively high affinity for MC3R and MC4R allows for investigation of these receptors' roles in various physiological processes beyond sexual function.
• Inflammation: Some studies suggest that melanocortin receptor activation may have anti-inflammatory effects. PT-141, as a melanocortin receptor agonist, could be used in research exploring this potential application.
• Obesity and Metabolic Disorders: The melanocortin system plays a role in energy homeostasis. Research is investigating the potential of melanocortin agonists, including PT-141, in the treatment of obesity and related metabolic disorders.

Quality Markers to Look For

Ensuring the quality of PT-141 is paramount for reliable and reproducible research. Several key quality markers should be assessed:

• Peptide Purity: Purity refers to the percentage of the desired peptide in the sample. A minimum purity of 98% is generally recommended for research purposes. High-performance liquid chromatography (HPLC) is the standard method for determining peptide purity. The HPLC chromatogram should show a single major peak corresponding to PT-141, with minimal presence of other peaks representing impurities.
• Amino Acid Analysis (AAA): AAA confirms the correct amino acid composition of the peptide. This analysis involves hydrolyzing the peptide into its constituent amino acids and then quantifying each amino acid. The molar ratios of the amino acids should match the expected ratios based on the peptide sequence. Deviations from expected ratios can indicate errors in peptide synthesis or degradation.
• Mass Spectrometry (MS): MS is used to confirm the molecular weight of the peptide. The observed molecular weight should match the theoretical molecular weight of PT-141 (1025.2 Da) within a narrow tolerance (typically +/- 1 Da). MS can also detect the presence of post-translational modifications or other unexpected modifications. Techniques like MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) and ESI-MS (Electrospray Ionization Mass Spectrometry) are commonly used.
• Peptide Content: Peptide content refers to the actual amount of peptide present in the sample, taking into account factors like water content and counter-ion content. This is typically expressed as a percentage. Peptide content is determined by quantitative amino acid analysis or by UV spectrophotometry using a predetermined extinction coefficient. Knowing the peptide content is crucial for accurate dosing.
• Water Content: Peptides are hygroscopic and can absorb water from the atmosphere. High water content can affect the accuracy of dosing and the stability of the peptide. Water content is typically determined by Karl Fischer titration. A water content of less than 5% is generally desirable.
• Counter-ion Content: Peptides are often synthesized as salts (e.g., acetate salt) to improve their solubility and stability. The counter-ion content should be specified and quantified. This information is necessary for accurate dosing calculations. Ion chromatography (IC) is commonly used to determine counter-ion content.
• Endotoxin Levels: Endotoxins are bacterial lipopolysaccharides (LPS) that can cause pyrogenic reactions. Endotoxin levels should be minimized, especially if the peptide is intended for in vivo studies. The Limulus Amebocyte Lysate (LAL) assay is used to detect and quantify endotoxins. Endotoxin levels should be below 10 EU/mg (Endotoxin Units per milligram) for research purposes, and even lower for in vivo applications.
• Solubility: The peptide should be readily soluble in appropriate solvents, such as sterile water or phosphate-buffered saline (PBS), at the desired concentration. Insoluble peptides can lead to inaccurate dosing and inconsistent results. Solubility testing should be performed to ensure that the peptide dissolves completely and remains in solution.

Common Impurities

Peptide synthesis is not a perfect process, and several impurities can be present in the final product. Identifying and minimizing these impurities is essential for obtaining reliable research results. Common impurities include:

• Truncated Sequences: These are peptides that are missing one or more amino acids from the desired sequence. They arise from incomplete coupling during peptide synthesis.
• Deletion Sequences: These peptides are missing one or more amino acids from within the sequence. They can occur due to incomplete deprotection or side reactions during synthesis.
• Modified Sequences: These are peptides that contain modified amino acids, such as oxidized methionine or D-amino acids at incorrect positions.
• Protecting Group Derivatives: Incompletely removed protecting groups from amino acid side chains can remain attached to the peptide.
• Diketopiperazine (DKP) Formation: This is a cyclization reaction that can occur at the N-terminus of dipeptides, leading to a cyclic dipeptide impurity.
• Aggregated Peptides: Peptides can aggregate in solution, especially at high concentrations. Aggregation can reduce the bioavailability of the peptide and affect its activity.
• Solvents and Reagents: Residual solvents and reagents used during peptide synthesis and purification can be present in the final product.

Storage Requirements

Proper storage is crucial for maintaining the stability and integrity of PT-141. The following storage conditions are recommended:

• Lyophilized Form: Store lyophilized PT-141 at -20°C or -80°C in a tightly sealed container. Protect from moisture and light. Under these conditions, the peptide can be stable for several years.
• Solution Form: Solutions of PT-141 are less stable than the lyophilized form. Store solutions at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles. The stability of the solution will depend on the solvent and concentration, but it is generally recommended to use solutions within a few weeks. Adding a small amount of a cryoprotectant, such as glycerol (5-10%), can improve the stability of frozen solutions.
• Avoid Repeated Freeze-Thaw Cycles: Repeated freeze-thaw cycles can degrade the peptide. Aliquot the peptide into single-use vials to avoid this.
• Desiccants: Store the peptide with a desiccant to minimize moisture absorption.
• Inert Atmosphere: Storing the peptide under an inert atmosphere (e.g., argon or nitrogen) can help prevent oxidation.

Sourcing Considerations

Selecting a reputable supplier is critical for obtaining high-quality PT-141. Consider the following factors when sourcing PT-141:

• Supplier Reputation: Choose a supplier with a proven track record of providing high-quality peptides. Look for suppliers with ISO 9001 certification or other quality management systems.
• Certificate of Analysis (CoA): A CoA should be provided for each batch of PT-141. The CoA should include detailed information on the purity, amino acid analysis, mass spectrometry, water content, counter-ion content, and endotoxin levels. Carefully review the CoA to ensure that the peptide meets your quality requirements.
• Manufacturing Process: Inquire about the supplier's manufacturing process, including the synthesis method, purification techniques, and quality control procedures.
• Customer Support: Choose a supplier that provides excellent customer support and is responsive to your questions and concerns.
• Price: While price is a factor, it should not be the sole determinant. Prioritize quality over price to ensure the reliability of your research.

Comparing Quality Metrics from Different Suppliers (Example)

Note: TFA (trifluoroacetic acid) as a counter-ion can sometimes interfere with certain biological assays. Acetate is generally preferred.

Practical Tips for Researchers

• Always verify the CoA: Do not rely solely on the supplier's claims. Carefully review the CoA and compare the reported values to your own quality requirements.
• Perform independent testing: If possible, consider performing independent testing of the peptide to confirm its quality. This is especially important for critical experiments.
• Use appropriate solvents: Use high-quality solvents that are compatible with the peptide and the intended application.
• Filter sterilize solutions: Filter sterilize peptide solutions using a 0.22 ?m filter to remove any bacteria or particulate matter.
• Handle with care: Handle the peptide with care to avoid contamination or degradation. Use sterile techniques and wear gloves.
• Monitor stability: Monitor the stability of the peptide solution over time. If you observe any signs of degradation, such as discoloration or precipitation, discard the solution.

Key Takeaways

• PT-141 (Bremelanotide) is a melanocortin receptor agonist with potential research applications in sexual dysfunction, inflammation, and metabolic disorders.
• Key quality markers to assess include peptide purity (HPLC), amino acid analysis (AAA), mass spectrometry (MS), peptide content, water content, counter-ion content, and endotoxin levels.
• Common impurities include truncated sequences, deletion sequences, modified sequences, and residual solvents.
• Store lyophilized PT-141 at -20°C or -80°C, protect from moisture and light, and avoid repeated freeze-thaw cycles of solutions.
• Choose a reputable supplier that provides a detailed Certificate of Analysis and has a proven track record of providing high-quality peptides.
• Always verify the CoA and consider performing independent testing to confirm the quality of the peptide.