ADME Services to Enable Informed Decision Making in Drug Discovery & Development

ISSX 2019 Presentation

In this presentation, two case studies will be presented, including metabolite profiling of low-turnover drugs and addressing factors confounding accurate human plasma protein binding data.

Case Study 1: To provide a potential solution for profiling low-turnover drugs, the HuREL® human hepatocyte coculture model was evaluated and results were recently published (Drug Metab Dispos 46(11) 2018). Metabolites of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HuREL® hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in suspended hepatocytes, whereas incubations conducted in the HuREL® coculture model resulted in more robust metabolic turnover, with continuous metabolite production observed for up to 7 days of incubation, covering cytochrome P450, aldehyde oxidase, and UGT-mediated pathways. The predominant metabolites correlated well with the most abundant metabolites reported in human clinical studies.

Case Study 2: Fraction unbound (fu) is a critical drug distribution parameter commonly utilized for modeling efficacious dosage and safety margin predictions. Following identification of a consistent over-estimation of fu for a known alpha-1-acid glycoprotein (AAG) binding drug (verapamil), it was suspected that a contaminant in the sourced plasma may be the culprit (AAPS J 21(1) 2018). Di-(2-ethylhexyl) phthalate (DEHP), a plasticizer known to be used in the manufacture of blood collection bags, was identified in plasma obtained through three common techniques. Blood stored in Terumo bags, blood collected in Terumo bags, but immediately transferred to conical vials, and vacutainer collected blood yielded DEHP concentrations of 300–1000 μM, 1–10 μM, and 0.1–2 μM, respectively. The concentration of DEHP corresponded with the fu of 13 tested drugs in DEHP-spiked plasma increasing between 2- and 5-fold. Additionally, DEHP was discovered to leach from the Terumo bag, with concentrations increasing 10-fold over a 7-day test period. In summary, the presence of DEHP in commercially available blood collection bags confounds in vitro fu estimation for drugs that bind primarily to AAG. It is thus recommended that vacutainer collected human plasma, which contains negligible DEHP, be used for the most accurate estimation of fu in human plasma.