Alfirevic, A. | bioRxiv Science

bioRxiv2023

Role of transporters and enzymes in metabolism and distribution of 4-chlorokynurenine and metabolites

4-chlorokynurenine (4-Cl-KYN) is in clinical development for potential CNS indications. We have sought to further understand the distribution and metabolism of 4-Cl-KYN as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite had the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.

pharmacology and toxicology↗

bioRxiv2022

The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury: a translational in vitro study

BackgroundEvidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here we describe the generation of HepG2-derived transmitochondrial cybrids in order to investigate the impact of mtDNA variation upon mitochondrial (dys)function and susceptibility to iDILI against a constant nuclear background. In this study, cybrids were created to contain mitochondrial genotypes of haplogroup H and haplogroup J for comparison. MethodsBriefly, HepG2 cells were depleted of mtDNA to make rho zero cells before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 distinct transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide and tolcapone, by ATP assays and extracellular flux analysis. FindingsOverall, baseline mitochondrial function was similar between haplogroups H and J. However, haplogroup specific responses to mitotoxic drugs were observed; haplogroup J was more susceptible to the inhibition of respiratory complexes I and II, and also to the effects of tolcapone, an uncoupler of mitochondrial respiration. ConclusionsThis study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest, thus providing a practical and reproducible system to investigate the cellular consequences of variation in mitochondrial genome against a constant nuclear background. Additionally the results support that that inter-individual variation in mitochondrial genotype and haplogroup may be a factor in determining sensitivity to mitochondrial toxicants. FundingThis work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

pharmacology and toxicology↗