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 diverse range of research applications, primarily focusing on sexual dysfunction. Unlike traditional treatments like sildenafil that target vascular mechanisms, PT-141 acts directly on the central nervous system, influencing sexual desire and arousal. This mechanism makes it a valuable tool for investigating the neurobiological aspects of sexual behavior in both males and females. This article will delve into the molecular characteristics of PT-141, its mechanism of action, its applications in research, critical quality markers to assess, common impurities to be aware of, and optimal storage conditions. We aim to provide researchers with the necessary information to critically evaluate the quality of PT-141 and ensure reliable research outcomes.

Molecular Structure and Properties

Bremelanotide is a cyclic heptapeptide analog of ?-Melanocyte-Stimulating Hormone (?-MSH). Its amino acid sequence is Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2. The cyclic structure is formed by a lactam bridge between the Asp and Lys residues. The molecular weight of PT-141 is approximately 1025.2 Da. The presence of non-natural amino acids, such as norleucine (Nle) and D-phenylalanine (D-Phe), enhances its metabolic stability and receptor binding affinity. The acetylation (Ac-) at the N-terminus further protects the peptide from enzymatic degradation. Its structural formula is:

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Key Structural Features:

• Cyclic Structure: Enhances binding affinity and metabolic stability.
• Non-Natural Amino Acids: Improve resistance to enzymatic degradation.
• Acetylation: Protects the N-terminus from degradation.

Mechanism of Action

PT-141 exerts its effects primarily through activation of the melanocortin receptors (MC1R, MC3R, MC4R, and MC5R). While it exhibits affinity for all four receptors, its primary target for sexual function is believed to be the MC4R in the central nervous system. Activation of MC4R triggers a cascade of intracellular signaling events, leading to increased dopamine release and modulation of neuronal activity in brain regions associated with sexual desire and arousal. Unlike PDE5 inhibitors, PT-141 does not directly affect vasodilation. Instead, it modulates central nervous system pathways involved in sexual motivation and behavior.

Receptor Binding Affinities (Approximate Ki Values):

Receptor	Ki (nM)
MC1R	~10
MC3R	~2
MC4R	~1
MC5R	~20

Practical Tip: When designing experiments, consider the potential for off-target effects due to PT-141's activity at other melanocortin receptors. Dose-response studies are crucial to determine the optimal concentration for targeting MC4R while minimizing potential confounding effects from other receptors.

Research Applications

PT-141 has been extensively studied for its potential in treating sexual dysfunction, particularly hypoactive sexual desire disorder (HSDD) in women and erectile dysfunction (ED) in men. Its mechanism of action, targeting the central nervous system rather than peripheral vasodilation, offers a unique approach to addressing these conditions. Beyond sexual dysfunction, research is exploring the potential of PT-141 in other areas, including:

• Treatment of Melanoma: MC1R is expressed in melanoma cells, and PT-141 may have potential as a targeted therapy.
• Management of Inflammatory Conditions: Melanocortin receptors are involved in regulating immune responses, and PT-141 may have anti-inflammatory effects.
• Modulation of Appetite and Metabolism: MC4R plays a role in energy homeostasis, and PT-141 may influence appetite and metabolism.

Specific Research Areas:

• Neurobiological Mechanisms of Sexual Desire: PT-141 is used to investigate the neural circuits and neurotransmitters involved in sexual motivation and arousal.
• Pharmacological Modulation of Melanocortin Receptors: PT-141 serves as a tool to study the function and regulation of melanocortin receptors in various tissues and organ systems.
• Development of Novel Therapeutics for Sexual Dysfunction: PT-141 has paved the way for the development of other melanocortin receptor agonists for treating sexual disorders.

Quality Markers to Look For

Ensuring the quality of PT-141 is paramount for obtaining reliable and reproducible research results. Several key quality markers should be assessed when evaluating PT-141 from different suppliers:

1. Peptide Purity

Purity refers to the percentage of the desired peptide sequence in the sample. High purity is crucial to minimize the impact of impurities on experimental outcomes. Purity is typically determined by reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at 214 nm (peptide bond absorbance) and 280 nm (aromatic amino acid absorbance). A purity level of ? 98% is generally considered acceptable for research purposes. However, for highly sensitive experiments, higher purity (? 99%) may be required.

HPLC Method Considerations:

• Column: C18 column (e.g., 4.6 x 250 mm, 5 ?m particle size)
• Mobile Phase: Gradient of acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA)
• Flow Rate: 1.0 mL/min
• Detection: UV at 214 nm and 280 nm

Practical Tip: Always request a Certificate of Analysis (CoA) from the supplier, which should include the RP-HPLC chromatogram and the reported purity value. Carefully examine the chromatogram for any significant impurity peaks.

2. Peptide Identity

Identity confirmation ensures that the peptide sequence is correct. Mass spectrometry (MS) is the gold standard for confirming peptide identity. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS or electrospray ionization (ESI) MS are commonly used techniques. The measured molecular weight should match the theoretical molecular weight of PT-141 (1025.2 Da) within a reasonable tolerance (e.g., ± 1 Da).

MS Method Considerations:

• Ionization Mode: Positive mode
• Mass Range: Appropriate for the peptide's molecular weight
• Calibration: Use appropriate mass standards for accurate mass measurement

Practical Tip: The CoA should include the MS spectrum and the reported molecular weight. Verify that the measured molecular weight matches the theoretical value and that the isotopic distribution pattern is consistent with the expected peptide sequence.

3. Peptide Content

Peptide content refers to the actual amount of peptide in the sample, taking into account factors such as residual water and counterions (e.g., TFA). Peptide content is typically determined by amino acid analysis (AAA) or quantitative UV spectrophotometry. AAA involves hydrolyzing the peptide into its constituent amino acids and quantifying each amino acid using HPLC. UV spectrophotometry relies on the absorbance of aromatic amino acids at 280 nm. A peptide content of ? 80% is generally considered acceptable.

AAA Considerations:

• Hydrolysis: Acid hydrolysis (e.g., 6N HCl at 110°C for 24 hours)
• Derivatization: Pre- or post-column derivatization with reagents like o-phthalaldehyde (OPA) or phenylisothiocyanate (PITC)
• Quantification: HPLC with UV or fluorescence detection

UV Spectrophotometry Considerations:

• Extinction Coefficient: Calculate the theoretical extinction coefficient based on the amino acid sequence (e.g., using ExPASy ProtParam tool).
• Wavelength: Measure absorbance at 280 nm.
• Purity Correction: Account for the purity of the peptide as determined by HPLC.

Practical Tip: Peptide content is often overlooked but is crucial for accurate dosing. A peptide with 98% purity but only 70% peptide content will require a significantly higher mass to achieve the desired concentration.

4. Water Content

Water content, also known as moisture content, can affect the stability and weight of the peptide. It is typically determined by Karl Fischer titration. A water content of ? 10% is generally acceptable.

Karl Fischer Titration Considerations:

• Reagent: Use a suitable Karl Fischer reagent (e.g., pyridine-free reagent).
• Solvent: Dissolve the peptide in a suitable solvent (e.g., methanol or dimethylformamide).
• Titration: Perform the titration according to the manufacturer's instructions.

Practical Tip: High water content can lead to peptide degradation over time. Store peptides under desiccated conditions to minimize water absorption.

5. Counterion Content

Peptides are often synthesized with counterions (e.g., TFA, acetate) to improve solubility and stability. The counterion content should be reported on the CoA. TFA is the most common counterion, and its presence can affect the peptide's biological activity. Ideally, the TFA content should be minimized or replaced with a more biocompatible counterion (e.g., acetate).

Counterion Determination:

• Ion Chromatography (IC): For quantifying TFA or acetate.
• NMR Spectroscopy: Can provide information about the presence and quantity of counterions.

Practical Tip: If TFA is a concern, consider purchasing peptides with acetate or another biocompatible counterion. Alternatively, TFA can be removed by reverse-phase HPLC using volatile buffers (e.g., ammonium acetate or ammonium bicarbonate).

6. Endotoxin Levels

Endotoxins, also known as lipopolysaccharides (LPS), are components of the cell walls of Gram-negative bacteria. They can cause potent inflammatory responses and interfere with cell-based assays. Endotoxin levels should be minimized, especially for in vitro and in vivo studies. Endotoxin levels are typically measured using the Limulus Amebocyte Lysate (LAL) assay. An endotoxin level of ? 10 EU/mg (Endotoxin Units per milligram) is generally considered acceptable for research purposes.

LAL Assay Considerations:

• Method: Use a commercially available LAL assay kit.
• Sample Preparation: Follow the manufacturer's instructions for sample preparation.
• Controls: Include appropriate positive and negative controls.

Practical Tip: Request endotoxin testing from the supplier, especially if the peptide is intended for cell culture or animal studies. Use endotoxin-free water and reagents when handling peptides.

Common Impurities

Peptide synthesis is not a perfect process, and several impurities can be present in the final product. Common impurities include:

• Truncated Sequences: Peptides with missing amino acids due to incomplete coupling reactions.
• Deletion Sequences: Peptides with one or more amino acids deleted during synthesis.
• Modified Sequences: Peptides with amino acid modifications (e.g., oxidation, deamidation).
• Diastereomers: Peptides with incorrect stereochemistry at one or more chiral centers.
• Protecting Group Derivatives: Peptides with residual protecting groups from the synthesis.
• Solvents and Reagents: Residual solvents and reagents used during synthesis and purification.

Impact of Impurities:

• Reduced Potency: Impurities can reduce the overall potency of the peptide.
• Off-Target Effects: Impurities can interact with unintended targets and cause off-target effects.
• Toxicity: Some impurities can be toxic to cells or animals.
• Assay Interference: Impurities can interfere with analytical assays.

Storage Requirements

Proper storage is essential to maintain the stability and integrity of PT-141. The following storage conditions are recommended:

• Temperature: Store at -20°C or -80°C in a freezer.
• Desiccation: Store under desiccated conditions to minimize water absorption.
• Light Protection: Protect from light by storing in a dark container or wrapping with foil.
• Solvent: If reconstituting the peptide, use a sterile, endotoxin-free solvent (e.g., sterile water or PBS). Avoid repeated freeze-thaw cycles. Aliquot the reconstituted peptide into smaller volumes to minimize degradation.
• Storage Duration: Lyophilized peptides can be stored for several years at -20°C or -80°C. Reconstituted peptides should be used within a few weeks or months, depending on the storage conditions and the peptide's stability.

Practical Tip: Date and label all peptide vials with the date of receipt, reconstitution, and any other relevant information. Keep a detailed record of storage conditions and handling procedures.

Key Takeaways

• PT-141 (Bremelanotide) is a melanocortin receptor agonist used in research for sexual dysfunction and other applications.
• Assess peptide purity (? 98% by RP-HPLC), identity (by MS), and content (? 80% by AAA or UV spectrophotometry).
• Monitor water content (? 10% by Karl Fischer titration) and endotoxin levels (? 10 EU/mg by LAL assay).
• Be aware of common impurities such as truncated sequences, modified sequences, and residual solvents.
• Store lyophilized peptides at -20°C or -80°C under desiccated conditions and protect from light.
• Reconstituted peptides should be used promptly and avoid repeated freeze-thaw cycles.