Peptide drugs are compounds composed of 10 to 50 amino acids, with molecular weights typically ranging between small molecule drugs (less than 500 Da) and protein drugs (greater than 5000 Da), thus combining the advantages of both drug categories. These drugs possess significant physiological activity and have been widely applied in the medical field. Since 1922, when insulin extracted from bovine and porcine pancreases was first used for therapeutic purposes, significant advancements have been made in the development of peptide drugs. In 1954, scientists successfully achieved the chemical synthesis of peptide drugs, and in 1963, the solid-phase peptide synthesis method was invented. These breakthroughs paved the way for the production and research of peptide medications. Currently, more than 80 peptide drugs have been approved for market use, with 85% of the market share concentrated in the treatment areas of chronic diseases such as cancer and diabetes.
Peptide drugs offer numerous advantages, including high target affinity, low off-target probability, easy degradation (high plasma clearance rate), hydrolysis products being amino acids (with toxicity of metabolic products typically not a concern), high approval rates, and shorter development cycles. However, despite these advantages, peptide drugs also have certain limitations, such as poor stability and restricted administration routes (mainly via injection). These challenges remain key issues to overcome in current research and development.
The structural diversity and complexity of peptide drugs are determined by their amino acid sequences, composition, length, and the three-dimensional conformation of the peptide chain. Their activity and stability can be influenced by the chemical properties of the amino acid side chains, such as hydrophobicity, charge, and polarity. These properties determine the peptide's solubility, interaction with biological targets, and pharmacokinetic characteristics in the body. These structural variations enable peptide drugs to have broad application potential in treating various diseases. Popular targets are concentrated on molecules associated with major diseases, covering areas such as cancer, inflammation, metabolism, diabetes, and rare diseases.
Table 1: Summary of Representative FDA-approved Peptide Drugs Since 2019
| Drug | Year | Manufacturer | Target | Indication |
|---|---|---|---|---|
| Trofinetide | 2023 | Acadia | IGF-1 class mimic | Rett syndrome |
| Rezafungin | 2023 | Cidara | Fungicide class antifungal | Invasive fungal infections |
| Posluma | 2023 | Blue Earth Diagnostics | PSMA-targeted imaging | Prostate cancer diagnosis |
| Aphexda | 2023 | BioLineRx | CXCR4 inhibitors | Multisystem inflammatory disease |
| Zilbrysq | 2023 | UCB | Complement C5 inhibitor | Full-body skeletal muscle atrophy |
| Paxlovid | 2023 | Pfizer | 3C-like proteinase inhibitor | Moderate to severe COVID-19 in children |
| Tirzepatide | 2022 | Lilly | GIP and GLP-1 receptor activator | Type 2 diabetes |
| Pluvicto | 2022 | Novartis | PSMA-targeted prostate cancer | Metastatic castration-resistant prostate cancer (mCRPC) |
| Terlipressin | 2022 | Mallinckrodt | Vasopressin analog | Type 1 hepatorenal syndrome |
| Dasiglucagon | 2021 | Zealand | Glucagon receptor (GCGR) activator | Severe hypoglycemia in children over 6 years |
| Setmelanotide | 2020 | Rhythm | MC4R receptor agonist | Rare genetic obesity disorders |
| Afamelanotide | 2021 | CLNUVEL | MC1R receptor activator | Erythropoietic protoporphyria |
| Bremelanotide | 2019 | Paladin | MC4R receptor activator | Hypoactive sexual desire disorder (HSDD) |
Peptide drugs exhibit relatively poor in vivo stability and face challenges in crossing physiological barriers. Therefore, non-gastrointestinal administration routes, such as injections, nasal, and pulmonary administration, are commonly used. The distribution of these drugs occurs slowly, primarily through blood-tissue fluid convection in the paracellular route and cellular endocytosis to peripheral tissues. Peptide drugs generally have low transmembrane permeability and are largely confined to plasma and interstitial spaces.
Peptide drugs are eliminated through intracellular protease degradation and endocytosis, with the majority being excreted by the kidneys. However, in most cases, due to their larger molecular size, rapid elimination of these drugs primarily occurs via intracellular enzymatic processes. When designing pharmacokinetic studies for peptide drugs, it is important to consider the peptide's stability, molecular weight, administration route, and in vivo distribution. Based on the pharmacokinetic characteristics of peptides, the challenges in bioanalysis mainly include issues with peptide stability, limitations of administration routes, complex dose design, and the high plasma protein binding rate of peptides, which can affect their distribution and clearance. Furthermore, bioanalysis must address the complexity of sample preparation to ensure accurate measurement of peptide concentrations and develop highly sensitive and specific detection methods. These challenges require researchers to carry out meticulous experimental design and optimization to ensure reliable and accurate pharmacokinetic data.
Due to the numerous challenges associated with in vivo detection of peptide drugs, the analytical methods for these drugs must have high specificity, extreme sensitivity, and the ability to distinguish between target peptide molecules and interfering substances.
Peptide drugs have multiple sample preparation methods, including protein precipitation, liquid-liquid extraction, solid-phase extraction, and enzymatic methods.
Table 2: Comparison of Different Sample Preparation Methods for Peptide Drugs
| Sample Preprocessing Method | Advantages | Disadvantages |
|---|---|---|
| Protein Precipitation | High recovery rate, easy operation | Low recovery rate, difficult to meet fixed volume requirements |
| Liquid-Liquid Extraction | High recovery rate | Complex operation, requires selecting appropriate extraction solvents |
| Solid-Phase Extraction | High recovery rate, cleaner samples | More complex operation, requires selecting the correct column, and washing |
Common Issues in Sample Preparation
Protein precipitation is a commonly used method; however, due to the nature of peptide drugs, phenomena such as adsorption and plasma protein binding can affect recovery rates. In sample preparation, various approaches can be employed to optimize recovery and purity based on the unique properties of peptide drugs. For mildly basic peptides, a precipitating agent can be directly added to facilitate protein precipitation, effectively removing proteins and purifying the peptide. On the other hand, mildly acidic peptides tend to experience adsorption and plasma protein binding.
To address the adsorption phenomenon, the following methods can be applied:
To tackle plasma protein binding, several strategies can be utilized:
In summary, different sample preparation methods are suitable for peptides with different characteristics. By selecting the appropriate method, recovery rates and purity can be significantly enhanced, laying a solid foundation for subsequent analysis and research.
Common Issues in Sample Detection
When using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect peptide drugs, common issues such as excessive residue, poor method specificity, or high baseline noise may arise. In such cases, adjustments to sample preparation methods and detection techniques are necessary.
Excessive residue can compromise the accuracy and reproducibility of the analysis. Here are strategies to address this issue:
Improving the specificity of peptide drug detection is key to ensuring accurate analysis. Strategies to improve specificity include:
High baseline noise is typically caused by sample complexity, system contamination, or improper operational conditions. To address this, consider the following methods:
Additionally, in LC-MS/MS analysis, replacing the acid in the mobile phase can enhance response. In summary, based on the distinct properties of peptide drugs, choosing appropriate sample preparation methods and mobile phase compositions can optimize bioanalytical results. These strategies help refine LC-MS/MS analysis, improving the accuracy and reliability of detection.
The immunogenicity of peptide drugs refers to their ability, or the ability of their metabolites, to induce immune responses or immune-related events against self or related proteins. Compared to large molecules, peptide drugs typically have lower immunogenicity. The risk of immunogenicity can be assessed based on the dosage, frequency, and duration of administration. For common small peptides, they generally fall under the low-risk category, and immunogenicity studies are often not required in the preclinical phase. This represents one of the advantages of peptide drugs.
Although antibody detection methods are well-established, there are still several challenges during the analysis process. For instance, small-molecule drugs may not be able to directly coat plates. This issue can be resolved by conjugating the peptide to a larger molecular carrier. Common carriers include proteins, peptide polymers, macromolecular polymers, and certain particles, such as bovine serum albumin (BSA), human serum albumin (HSA), rabbit serum albumin, ovalbumin (OVA), and keyhole limpet hemocyanin (KLH). Additionally, the complex composition of matrices like serum can affect the capture of target antibodies, as multiple immunoglobulins in the matrix may interfere with the process. This can be addressed by adjusting the minimum required dilution (MRD), optimizing testing methods, trying various detection systems and methods, such as the ACE method.
In direct or indirect immunoassays, the labeled secondary antibodies may face challenges in labeling or exhibit low labeling efficiency. To enhance labeling efficiency, approaches like chemically synthesizing labeled secondary antibodies, using universal antibodies, switching to more sensitive ECL platforms, or employing higher labeling success rate reagents like Ru can be applied. These solutions contribute to improving the accuracy and reliability of immunoassays.
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