Peptide drugs have gained considerable interest due to their potential across a spectrum of diseases, including metabolic disorders, cardiovascular issues, cancer, and infectious diseases. The recent blockbuster drug glucagon-like peptide-1 (GLP-1) hormone stands as a great example of the efficacy and promise offered by peptide therapeutics in clinical practice.

Preclinical testing is crucial for peptides to evaluate their safety, efficacy, and pharmacokinetics before they enter clinical trials. It helps identify any potential toxic effects, optimal dosing regimens, and therapeutic potential, ensuring that only promising candidates proceed to human trials.

Preclinical DMPK research on peptides mainly focuses on stability, permeation, delivery carrier, and tissue distribution studies. Bioanalytical method development focuses on detecting and quantifying the peptide in biological samples with high specificity and sensitivity.

Peptide development and testing face numerous challenges, including the need for highly sensitive and specific analytical methods to detect and quantify these larger, structurally complex molecules in biological matrices. Peptides are physiologically active, requiring small dosages and resulting in low in vivo drug concentrations amid many endogenous interfering substances. Mass spectrometry is complicated by the generation of multiple charged ions and dispersed ions of different valence states. Additionally, peptides exhibit issues such as non-specific adsorption, low stability, and high protein binding ratios, which complicate sample processing and detection. Their high polarity and poor permeability hinder their ability to cross bio-membrane barriers, reducing oral bioavailability. Peptides also suffer from poor metabolic stability, multiple metabolic pathways, and short half-lives.

Preclinical studies are crucial for evaluating the properties and behavior of peptides to ensure their safety and efficacy before clinical trials. These studies encompass several stages, each focusing on different aspects: 

• Early screening stage: Mainly investigating the in vitro properties of peptide molecules (including plasma stability, metabolic stability, permeation, and protein binding) and guiding molecular structure optimization. The interactions and enzyme phenotyping of peptides with low molecular weight are evaluated as well.
• PCC stage: This stage focuses on the metabolic differences of species and the in vivo PK study of peptides. It aims to select the appropriate species for relevant research by considering both pharmacodynamics and toxicology. The study of various delivery carriers makes it possible to achieve the desired in vivo exposure characteristics.
• IND stage: In this stage, single dose escalation and repeated administration PK in animals will be studied. The excretion pathway (mass balance), tissue distribution, and in vivo metabolites identification also need be evaluated.

Immunogenicity tests are particularly important for peptides due to the potential for peptides to elicit immune responses. Acute and chronic toxicity studies are conducted to determine the toxic effects over different exposure periods. Genotoxicity studies evaluate any risk of genetic damage, and safety pharmacology studies monitor vital functions such as cardiovascular, respiratory, and central nervous systems.

For Drug Metabolism and Pharmacokinetics (DMPK) studies on peptides, it is important to consider the peptide’s stability, bioavailability, and routes of administration. The potential for enzymatic degradation, the impact of formulation on absorption, and the peptide’s distribution and elimination pathways are also critical factors.

Preclinical testing of peptides must adhere to regulatory guidelines such as those from the FDA, EMA, and ICH, which include Good Laboratory Practice (GLP) standards. Specific guidelines for biologics, including peptides, cover aspects such as immunogenicity (ICH S6(R1)), toxicology studies (ICH M3(R2)), and the evaluation of safety pharmacology (ICH S7A).

Pharmacological concerns specific to peptides include their rapid degradation by proteases in the body, leading to poor bioavailability and short half-lives. Peptides can also be immunogenic, potentially triggering unwanted immune responses. Their specificity to target receptors may cause off-target effects, and they often require advanced delivery systems to achieve therapeutic levels.

Efficacy tests for peptides involve in vitro assays to assess their interaction with target receptors or enzymes and to measure biological activity. In vivo studies use animal models of the target disease to evaluate therapeutic outcomes, such as reduction in disease biomarkers, improvement in clinical symptoms, and overall survival rates, providing a comprehensive assessment of the peptide’s therapeutic potential.

Peptides require the development and validation of robust analytical methods tailored to their unique characteristics. This may involve optimizing sample preparation techniques, selecting appropriate analytical platforms such as liquid chromatography-mass spectrometry (LC-MS) or enzyme-linked immunosorbent assay (ELISA), and implementing stringent quality control measures to ensure the accuracy and reliability of peptide quantification in preclinical studies.