The DMPK group was established within the Therapeutic Development Branch in the Division of Preclinical Innovation (DPI) to address issues related to drug absorption, biodistribution and elimination via metabolism or excretion. The DMPK group has supported diverse projects across DPI, contributing significantly to all stages of translational research at NCATS, from early probe development in drug discovery to Phase II clinical trials. Our major capabilities include: 1. In vitro ADME high-throughput screening (Tier I HTS assays) on solubility, permeability and microsomal stability for all small molecule compounds registered at NCATS ( 3000 compounds/year). 2. Customized in vitro ADME assays (Tier II assays) as required by projects specific needs. The common Tier II assays include metabolic stability in different species, metabolite identification (MetID), aldehyde oxidase stability in cytosol fraction, plasma stability for prodrugs and biologics, blood/plasma partition, CYP inhibition, and transporter assessments in Caco-2 and MDKC cells. 3. PK studies in lab animals and bioanalytical measurements of drug concentrations in different biological fluids (e.g., blood, plasma, urine) and tissue extracts. 4. High definition mass spectrometry for quantitation of small molecules and peptides, and structure identification of metabolites. 5. Bioanalytical method development for therapeutic macromolecules, such as recombinant human protein and engineered proteins. 6. Pharmacokinetic parameter calculation and simulation. Examples of the DMPK group contributions to recent projects within the Therapeutic Development Branch include: Proof of Concept (POC) Study for Creatine Transporter Deficiency (CTD): Cyclocreatine (Ccr) has been proposed as a drug candidate for the treatment of CTD. Our in vitro ADME data on membrane permeability did not support the original therapeutic hypothesis of passive diffusion of Ccr into the brain in the absence of the creatine transporter (CT). To address this issue, we designed a series of PK and tissue distribution studies with radiolabeled Ccr in CT KO mice. Our data demonstrated that 14-CCcr enters the brain after oral administration but at a very low level. However, once in the brain tissue, 14-CCcr is eliminated very slowly with a long half-life (t1/2) of 28 days. Thus, after multiple dose treatment, Ccr will accumulate in the brain to yield a concentration high enough to provide the beneficial effects observed in the CT KO mice. This proof of concept study enhanced our understanding of brain uptake for Ccr and led the team to advance this project to clinical trials. Applying Metabolite Identification (MetID) to Guide Structure Optimization: To develop a novel therapy for fibrodysplasia ossificans progressiva (FOP), a lead compound (LDN-189) was proposed as a development candidate by our collaborator. However, based on our past experience with similar chemical structures, we immediately realized that this molecule could have metabolic issues associated with toxicity. To confirm this, we conducted extensive MetID experiments, and several metabolic liable spots (red flags) were identified. The formation of major metabolite, NIH-Q55, was mediated by aldehyde oxidase (AO), an enzyme known to be species-dependent. AO enzyme activity (greatest to least) could be ranked in the order of monkey > human > rodents. Dog, a common non-rodent species used for drug safety evaluation, totally lacks AO activity. The AO-mediated metabolism could also contribute to a large variability in exposures observed among different human subjects. In addition to the AO-mediated metabolism, the piperazinyl moiety was the target of NADPH-dependent metabolism, resulting in reactive iminium intermediates as confirmed through chemical trapping experiments. Two aniline metabolites were also detected, which brought up concerns about drug safety. These findings provided valuable information to the chemists, guiding subsequent structure modifications to generate safer drug candidates. By blocking the AO-mediated metabolic site and changing the piperazinyl moiety, the chemists were able to develop new lead compounds with more metabolic stability and less toxicity, while retaining pharmacological activity. Currently, the lead collaborators on this project are on track to file an Investigational New Drug (IND) application with the Food and Drug Administration (FDA) to begin clinical trials. Optimization of Dosing Regimen for Fungal Infection Treatment: VT-1129 is a potent antifungal agent with a slow absorption phase followed by a slow elimination phase in the PK profile after oral administration. In order to deliver a high initial drug concentration for a quick kill on fungus while avoiding significant drug accumulation in tissues (especially liver) after multiple-dose treatment, we proposed a dosing regimen with a high loading dose, followed by low daily maintenance doses. A series of simulations were conducted with different dosage combinations. The predicted drug concentrations based on simulations were confirmed by in vivo measured concentrations in mice. The optimized dosing regimen was successfully used in subsequent mouse efficacy studies, supporting a successful IND application to the FDA.
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