Minipig and Human Metabolism of Aldehyde Oxidase Substrates: In Vitro–In Vivo Comparisons

Abstract

The importance of aldehyde oxidase (AOX) is becoming increasingly recognized in the prediction of human pharmacokinetic parameters from animal data. The objectives of these studies were to ascertain whether an in vitro-in vivo correlation existed in the clearance and metabolic pathways of AOX substrates and to establish whether the minipig represented an appropriate non-rodent model for man in the pre-clinical development of drugs metabolized by AOX. Using the AOX substrates carbazeran, 6-deoxypenciclovir and zaleplon, clearance was estimated from in vitro depletion experiments with minipig and human liver cytosol and microsomes and scaled before comparison with data generated in parallel in vivo studies in minipigs. In vitro and in vivo metabolic pathways were characterized by LC-MS/MS. Scaling of in vitro metabolism data to predict in vivo clearance underestimated in vivo values, although the rank order of clearance for the three compounds was preserved. Prediction of human in vivo clearance from scaled minipig in vivo data produced results which correlated well with published clinical values. Overall, this study is the first to compare minipig in vitro metabolism data with in vivo pharmacokinetic data for compounds metabolized by AOX and provides a scientific rationale for the selection of this species as a model for humans in the development of drugs which are substrates of AOX.

Evaluation of Carbazeran 4-Oxidation and O6-Benzylguanine 8-Oxidation as Catalytic Markers of Human Aldehyde Oxidase: Impact of Cytosolic Contamination of Liver Microsomes

“…Other experimental evidence of cytosolic contamination of our microsomal preparations include: 1) the functional activity of AOX-1, as assessed by another AOX-1-catalyzed reaction, O 6 -benzylguanine 8-oxidation, was detected in human liver microsomal incubations without the addition of NADPH, and this occurred in multiple pools of human liver microsomes obtained from multiple commercial suppliers; 2) an AOX-1 inhibitor (hydralazine) almost completely abolished the two AOX-1-mediated reactions (carbazeran 4-oxidation and O 6benzylguanine 8-oxidation) in human liver microsomal incubations; and 3) another well characterized cytosolic enzyme activity (DHEA sulfonation catalyzed by SULT2A1, SULT2B1, and SULT1E1) was also quantified in our pools of human liver microsomes. Previous studies detected AOX-1-catalyzed metabolites from carbazeran (Wilkinson et al, 2017), VU0409106 (Crouch et al, 2016), and SGX523 (Diamond et al, 2010) in human liver microsomes but did not quantify microsomal AOX-1 protein content and postulated the metabolite formation was the result of contamination of microsomes with cytosol (Crouch et al, 2016;Wilkinson et al, 2017). Overall, by using various experimental approaches to address the issue of cytosolic contamination of our panel of human liver microsomes, our results indicate that microsomal cytochrome P450 does not catalyze carbazeran 4-oxidation.…”

“…Carbazeran 4-oxidation is used as a catalytic marker of this cytosolic enzyme (Zientek et al, 2010;Hutzler et al, 2012;Fu et al, 2013). However, it is not certain whether carbazeran 4-oxidation is an enzyme-selective catalytic marker of AOX-1, because of a study reporting that a general inhibitor of cytochrome P450, 1-aminobenzotriazole (1-ABT), decreased the in vitro intrinsic clearance of carbazeran in human liver microsomes, although the extent of the decrease was marginal and occurred within a single pool of human liver microsomes (Wilkinson et al, 2017). O 6 -Benzylguanine 8-oxidation is also used as a catalytic marker of AOX-1 (Zientek et al, 2010;Hutzler et al, 2012;Barr et al, 2015).…”

Successful and Unsuccessful Prediction of Human Hepatic Clearance for Lead Optimization

“…The reaction requires addition of the cofactor uridine diphosphate glucuronic acid (UDPGA) as well as a pore-forming agent such as alamethicin, as the catalytic active site of UGTs is located within the lumen of the endoplasmic reticulum . It has also been reported by a number of investigators that cytosolic contamination of microsomal fractions can occur as a result of the preparation process, resulting in appreciable nonmicrosomal metabolism by enzymes such as aldehyde oxidase. − In fact, many investigators choose to perform tier 1 stability assays using the S9 fraction (supplemented with NADPH) to capture both the microsomal Phase I CYP-mediated metabolism as well as contributions of Phase I and II cytosolic enzymes such as esterases, aldehyde oxidase, xanthine oxidase, glutathione S-transferase, and sulfotransferase …”

Porcine Prediction of Pharmacokinetic Parameters in People: A Pig in a Poke?

“…The results showed that in vivo CL was underpredicted by IVIVE, generally similar to that observed for compounds with elimination mechanisms via other metabolic pathways. In vivo-based allometry, with an exponent fixed at 0.75 (between minipigs and humans), seemed to predict well the human in vivo CL, supporting the use of minipigs for predicting human PK for AOX substrates (Wilkinson et al, 2017). The in vitro intrinsic CL (CL int ) of five AOX substrates in multiple species, including humans and minipigs, were evaluated.…”