Peptide-Drug Conjugates (PDCs) are composed of a cytotoxic payload, a homing peptide, and a linker between the peptide and the cytotoxic payload. After antibody-drug conjugates, they are the next generation of targeted therapeutics drug with enhanced cellular permeability and improved drug selectivity. They pose a great opportunity to target cancer, metabolic diseases, and more. While the potential is significant, development challenges – like poor stability, inferior targeting and potential payload toxicity – can slow the preclinical and clinical development process. WuXi AppTec Drug Metabolism and Pharmacokinetics (DMPK) Service Department has established a robust DMPK research program to develop PDCs. This program has been built with our collective expertise of in vitro ADME, in vivo pharmacokinetics, bioanalysis and metabolite identification coupled with extensive research strategy, data interpretation and cross department cooperation to significantly reduce time and costs.
Solubility test in different excipients
In vivo absorption study through PK
Protein binding in plasma and tissues
In vitro partition between whole blood and plasma
Tissue distribution with cold/hot compounds or through QWBA approach
In vitro metabolic stability
Bio-transformation through in vitro and in vivo metabolite identification
Payload release study with in vitro models
In vivo excretion study to explore excretion pathway
In vitro CYP450 DDI evaluation
In vitro transporter DDI evaluation
A PDC is a conjugate of a peptide and payload. It is necessary to conduct DMPK research for both the PDC and payload.
Neither the FDA nor the ICH has specific guidelines for DMPK research on PDCs.
If the payload is highly toxic, it is impossible for the PDC to perform absorption, metabolism and excretion (AME) studies with a radiolabeled PDC in human.
The efficacy of a PDC is closely related to the release and concentration of the payload in the target tissue.
The toxicity of a PDC is directly related to the release and concentration of the payload in the non-target tissues.
Toxicological species of a PDC is related to the metabolism similarity of a PDC in humans and other species.
Drug-drug interactions of a PDC are closely associated with the exposure, metabolism, and elimination of a PDC and payload in the tissues.
Various in vitro payload release models.
Metabolite identification of a PDC and payload in different matrices using high-resolution mass spectrometry.
In vivo PK study on a PDC utilizing LC/MS and MSD platforms.
Tissue distribution and excretion studies with a radiolabeled PDC.
Tissue distribution of a PDC in healthy or tumor-bearing rodent species using QWBA method.
The screening stage mainly focuses on the in vitro and in vivo stability optimization of homing peptides and PDC, including the stability in whole blood (or plasma), liver S9 and kidney homogenate. In addition, in vitro and in vivo metabolite identification is often conducted to further optimize PDC structures, thus improving the stability and prolonging the in vivo half-life. Tissue distribution studies in rodent species are often recommended in early stages to assess the potential toxicity of payloads.
Depending on whether and how the linker is cleaved, payload release is suggested to perform in vitro in different matrices to mimic in vivo release. When conducting in vivo experiments, the concentrations of both PDC and payload in the samples will be detected. Detecting ADA levels in the repeated-dose experiments is recommended as well as tissue distribution in the PD species to evaluate the targeting capabilities of the PDC.
It is necessary to conduct a comprehensive in vitro ADME assays and PK study in at least one species for payload if the DMPK properties are unknown. In addition, sponsors often find it helpful to evaluate the tissue distribution and mass balance in rodents with a radiolabeled PDC or using a QWBA approach. We also recommend drug-drug interaction and metabolism studies in other matrices of PDC.
Customer First and Customer CentricWe have a specialized and dedicated service model. Each client will be connected to a dedicated study director who will provide comprehensive management services for the pharmacokinetic project from drug discovery to the clinical phase.
Extensive Experience in Customizing Study DesignsWe have extensive experience in completing many successful PDC projects – allowing us to offer customized study design for clients.
Cross-Department Cooperation and High EfficiencyOur DMPK team works closely with chemistry and biology departments internally to facilitate smooth execution of the project and shorten the turnaround time.
Comprehensive Capabilities to Deal with PDC ChallengesWe are able to conduct DMPK studies for the payload and PDC. In addition, we have the comprehensive capacity to carry out bioanalysis, in vitro and in vivo metabolite identification, and radiolabeled ADME experiments of PDC.
After we received the request from the client, we took the initiative to ask the client about the linker type of the PDC. According to our experience, special attention needs to be paid to linker type. For example, attention should be given to the pH value of the mobile phase on the detection instrument in the case of pH-sensitive linker. For this PDC, the homing peptide was linked to p-aminobenzyl carbamate (PABC) and MMAE via Val-Cit dipeptides. To our knowledge, the CES1c enzyme from mouse plasma probably hydrolyzes this linker to release MMAE. If the mouse plasma is directly collected following routine procedure, freeze-thaw stability issue probably arises during bioanalysis, resulting in unreliable concentration of MMAE and the PDC. Due to the release of MMAE hydrolyzed by CES1c enzyme from mouse plasma, the concentration of MMAE will increase while the concentration of PDC will decrease during the time of plasma sample processing. Therefore, we suggested that our client began with method development. Based on method development, direct protein precipitation in the plasma afforded the supernatant immediately after collection, and specific absorption inhibitor Triton X-100 was added to remove non-specific binding.
Reliable analysis results were attained after following our suggestion. During the bioanalysis of the PDC samples, special attention should be given to the issues such as stability and non-specific binding.
 Bethany M. Cooper et al. Peptides as a platform for targeted therapeutics for cancer: peptide–drug conjugates (PDCs). Chemical Society Reviews. 2021, 50, 1480.