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 receptor agonist with a growing body of research exploring its potential applications. This article provides a comprehensive overview of PT-141, focusing on its structure, mechanism of action, research applications, critical quality assessment parameters, common impurities, and storage recommendations. This information is intended to assist researchers in making informed decisions regarding sourcing and utilizing PT-141 in their studies.

Molecular Structure and Properties

Bremelanotide is a synthetic cyclic heptapeptide analogue of alpha-melanocyte-stimulating hormone (?-MSH). Its chemical formula is C₅₀H₆₈N₁₄O₁₀, and its molecular weight is approximately 1025.2 Da. The cyclic structure, formed by a lactam bridge, confers enhanced receptor selectivity and stability compared to linear ?-MSH analogues.

The sequence is Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂. Key features include:

• Nle (Norleucine): A non-natural amino acid analogue of methionine, often used to improve metabolic stability.
• Cyclization: The Asp-Lys lactam bridge creates a constrained conformation, optimizing receptor binding.
• D-Phe (D-Phenylalanine): The D-amino acid substitution contributes to resistance against enzymatic degradation.

Mechanism of Action

PT-141 exerts its effects primarily by activating melanocortin receptors (MCRs), specifically MC1R and MC4R. While MC1R is predominantly involved in melanogenesis and pigmentation, MC4R plays a crucial role in sexual function, appetite, and energy homeostasis. Bremelanotide's affinity for MC4R is believed to be the primary driver of its pro-sexual effects.

Upon binding to MC4R, PT-141 stimulates intracellular signaling cascades, including the activation of adenylyl cyclase and the subsequent increase in cyclic AMP (cAMP) levels. This cascade ultimately leads to increased neuronal activity in brain regions associated with sexual arousal and desire. Importantly, unlike PDE5 inhibitors (e.g., Sildenafil), PT-141 acts directly on the central nervous system, bypassing the need for direct physical stimulation.

Research Applications

PT-141 has been investigated in various research settings, primarily focusing on sexual dysfunction. Some key areas of research include:

• Female Sexual Dysfunction (FSD): Clinical trials have demonstrated PT-141's efficacy in treating hypoactive sexual desire disorder (HSDD) in premenopausal women. Studies have focused on understanding the dose-response relationship, long-term safety, and potential benefits in different subgroups of women with FSD.
• Erectile Dysfunction (ED): While PT-141's primary clinical application is for FSD, research has explored its potential benefits in treating ED, particularly in cases where PDE5 inhibitors are ineffective or contraindicated. Studies suggest that PT-141 may offer an alternative mechanism of action for improving erectile function.
• Melanocortin Receptor Signaling: PT-141 serves as a valuable tool for researchers studying the role of melanocortin receptors in various physiological processes, including sexual function, energy balance, and inflammation. In vitro and in vivo studies using PT-141 can help elucidate the specific signaling pathways and downstream effects mediated by MC1R and MC4R activation.
• Obesity Research: Given MC4R's role in appetite regulation, some research has explored the potential of PT-141 or related melanocortin agonists as therapeutic agents for obesity. However, further investigation is needed to determine the long-term efficacy and safety of this approach.

Quality Assessment Markers

Ensuring the quality of PT-141 is paramount for reliable and reproducible research results. Several key quality markers should be carefully evaluated when sourcing and utilizing this peptide.

1. Peptide Purity

Purity refers to the percentage of the desired peptide sequence in the final product, relative to other peptide-related impurities. High purity is crucial to minimize off-target effects and ensure accurate interpretation of research findings. Purity is typically determined by analytical High-Performance Liquid Chromatography (HPLC).

• HPLC Analysis: Reverse-phase HPLC (RP-HPLC) is the standard method for assessing peptide purity. A C18 column is commonly used, with a gradient of acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA).
• Acceptable Purity Level: For research purposes, a purity level of ? 98% is generally recommended. Lower purity levels may introduce confounding factors and compromise the validity of the results.
• Certificate of Analysis (CoA): Always request a CoA from the supplier, detailing the HPLC analysis and purity determination. Examine the chromatogram to ensure that the peak corresponding to PT-141 is well-defined and free from significant contaminating peaks.

2. Peptide Identity

Verifying the identity of the peptide ensures that the product actually contains the correct amino acid sequence. Mass spectrometry (MS) is the gold standard for confirming peptide identity.

• Mass Spectrometry (MS): MS analysis, such as MALDI-TOF or ESI-MS, provides a precise measurement of the peptide's molecular weight. The measured mass should match the theoretical mass of PT-141 (1025.2 Da) within a tolerance of ± 1 Da.
• MS/MS Sequencing: For more definitive confirmation, tandem mass spectrometry (MS/MS) can be used to fragment the peptide and analyze the resulting fragment ions. This provides sequence-specific information, confirming the correct amino acid sequence.
• CoA Verification: The CoA should include MS data confirming the peptide's identity. Carefully review the MS spectrum to ensure that the major peak corresponds to the expected molecular weight.

3. Peptide Content

Peptide content refers to the actual amount of peptide present in the product, taking into account factors such as residual water and counterions (e.g., acetate). Content is typically expressed as a percentage and is determined by quantitative amino acid analysis (AAA) or elemental analysis.

• Amino Acid Analysis (AAA): AAA involves hydrolyzing the peptide into its constituent amino acids and quantifying each amino acid using HPLC. This provides an accurate measure of the peptide content and can also detect the presence of any incorrect amino acids.
• Elemental Analysis: Elemental analysis measures the percentage of carbon, hydrogen, and nitrogen in the sample. This data can be used to estimate the peptide content, although it is less specific than AAA.
• Acceptable Content Level: The peptide content should be clearly stated on the CoA. A content level of 80-95% is generally considered acceptable, depending on the specific manufacturing process and counterion composition.

4. Water Content

Water content (also known as moisture content) refers to the amount of water present in the peptide sample. Excessive water content can lead to peptide degradation and inaccurate concentration measurements.

• Karl Fischer Titration: Karl Fischer titration is the standard method for determining water content. This technique involves a chemical reaction that selectively reacts with water, allowing for its quantification.
• Acceptable Water Content: The water content should be kept as low as possible, ideally below 5%. Higher water content may indicate improper storage or handling.
• CoA Information: The CoA should include the water content as determined by Karl Fischer titration.

5. Counterion Content

Peptides are often synthesized with counterions (e.g., acetate, TFA) to improve their solubility and stability. However, excessive counterion content can affect the accuracy of concentration measurements and may potentially interfere with biological assays.

• Ion Chromatography: Ion chromatography is used to quantify the amount of counterions present in the peptide sample.
• Acceptable Counterion Content: The counterion content should be specified on the CoA. While there is no single "acceptable" level, it is important to be aware of the counterion and its potential effects on your experiments.

6. Endotoxin Levels

Endotoxins, also known as lipopolysaccharides (LPS), are components of the outer membrane of Gram-negative bacteria. Even trace amounts of endotoxins can elicit strong inflammatory responses and interfere with cell-based assays. For studies involving cell culture or in vivo administration, it is crucial to ensure that the peptide is endotoxin-free.

• Limulus Amebocyte Lysate (LAL) Assay: The LAL assay is the standard method for detecting and quantifying endotoxins. This assay utilizes a lysate from horseshoe crab blood, which clots in the presence of endotoxins.
• Acceptable Endotoxin Level: For cell culture applications, the endotoxin level should be below 10 EU/mg (Endotoxin Units per milligram). For in vivo studies, even lower levels (e.g., < 1 EU/mg) may be required, depending on the route of administration and the sensitivity of the animal model.
• CoA Requirement: The CoA should include the endotoxin level as determined by the LAL assay.

Common Impurities

Peptide synthesis is not a perfect process, and several impurities can arise during manufacturing. Understanding these potential impurities is crucial for interpreting analytical data and troubleshooting unexpected results.

• Deletion Sequences: These are peptide sequences missing one or more amino acids due to incomplete coupling during synthesis.
• Truncated Sequences: These are peptide sequences that are prematurely terminated during synthesis.
• Modified Amino Acids: These are amino acids that have been unintentionally modified during synthesis or purification (e.g., oxidation of methionine).
• Diastereomers: These are isomers that differ in the configuration of one or more chiral centers.
• Protecting Group Adducts: These are protecting groups that have not been completely removed during deprotection steps.
• Solvents and Reagents: Residual solvents and reagents used during synthesis and purification can also be present as impurities.

Storage Recommendations

Proper storage is essential to maintain the integrity and stability of PT-141. The following recommendations should be followed:

• Storage Temperature: Store PT-141 at -20°C or -80°C in a tightly sealed container.
• Desiccation: Store the peptide under anhydrous conditions using a desiccant.
• Light Protection: Protect the peptide from light exposure.
• Solubilization: When reconstituting the peptide, use sterile, endotoxin-free water or buffer. Avoid using buffers containing reducing agents, as these may degrade the peptide. Aliquot the reconstituted peptide into single-use vials to avoid repeated freeze-thaw cycles.
• Stability in Solution: The stability of PT-141 in solution depends on the buffer, pH, and concentration. Generally, it is best to use freshly prepared solutions whenever possible. If storage of solutions is necessary, store them at -20°C or -80°C.

Practical Tips for Researchers

• Source from Reputable Suppliers: Choose suppliers with a proven track record of producing high-quality peptides. Look for suppliers that provide comprehensive CoAs and are willing to answer technical questions.
• Request a Sample for Testing: Before purchasing a large quantity of PT-141, request a small sample for testing. Perform your own HPLC analysis and mass spectrometry to verify the purity and identity of the peptide.
• Optimize Storage Conditions: Carefully follow the recommended storage conditions to minimize peptide degradation.
• Monitor Peptide Stability: Periodically monitor the purity and stability of your PT-141 stock solutions using HPLC.
• Consider Peptide Modifications: If your research requires enhanced stability or specific targeting, consider using modified versions of PT-141, such as PEGylated peptides or peptides conjugated to targeting ligands.

Comparison of Quality Markers

Key Takeaways

• PT-141 (Bremelanotide) is a synthetic melanocortin receptor agonist with research applications in sexual dysfunction, obesity, and melanocortin receptor signaling.
• High purity (? 98%) and confirmed identity by mass spectrometry are crucial quality markers for reliable research results.
• Pay close attention to peptide content, water content, endotoxin levels, and counterion composition, as these factors can affect peptide stability and experimental outcomes.
• Store PT-141 at -20°C or -80°C under anhydrous conditions, protected from light.
• Source PT-141 from reputable suppliers and request a Certificate of Analysis (CoA) detailing all relevant quality parameters.