Development of a New Practical Approach to Perform Single‐Mouse Pharmacokinetic Study

Abstract

Single‐animal pharmacokinetic (PK) study has largely been limited to rats and larger animals. However, given the increasingly large body of research using transgenic and immunocompromised mouse models, and an economical advantage, a single‐mouse PK model is highly warranted. Given a high sensitivity of LC‐MS/MS in quantification of drugs and metabolites, we have established a practical and standard protocol for series blood samplings from single mouse via tail vein at multiple time points towards a complete PK profile. CD‐1 Mice were given a cocktail of cytochrome P450 (CYP) isoform‐specific probe substrates per os, consisting of midazolam (MDZ), dextromethorphan (DXM), phenacetin (PHE), diclofenac (DIC), and chlorzoxazone (CHL). CYP inhibitors, ketoconazole (KTZ) and α‐napthoflavone (α‐NF), were administered by intraperitoneal injection 1 hour prior to dosing; CYP inducers, pregnenolone 16α‐carbonitrile (PCN) and 3‐methyl cholanthrene (3‐Me), were administered 3 days prior to dosing. Blood (10–20 μl per time point) was collected by tail vein puncture at 7–10 different time points over 3 hours and plasma was prepared for LC‐MS/MS quantification of MDZ, DXM, PHE, DIC, and CHL, and CYP isoform‐specific metabolites 1′‐OH‐MDZ, DXO, ACE, 4′‐OH‐DIC, and 6′‐OH‐CHL, respectively.A small volume (3 μl) of plasma at each time point was found to be sufficient to produce a complete PK profile in a single mouse. Visual inspection and non‐compartmental PK modeling showed that formation of 1′‐OH‐MDZ and ACE was sharply and significantly reduced in mice pre‐treated with KTZ and α‐NF, respectively, suggesting that these compounds preferentially inhibit MDZ 1′‐hydroxylase and PHE O‐deethylase, respectively, in mice in vivo. Additionally, plasma MDZ concentrations and all PK parameters (Cmax, AUC, t1/2) were significantly altered in mice administered PCN. Interestingly, this was not reflected by an increase of 1′‐OH‐MDZ levels, yet by a decrease in plasma 1′‐OH‐MDZ and Cmax and AUC values, suggesting that formation of other metabolites may be induced. Furthermore, plasma PHE and all PK parameters were significantly reduced to near negligible concentrations in mice administered 3‐Me, but plasma ACE, Cmax, and AUC were also slightly attenuated relative to control, suggesting that other metabolic pathways may be induced. Lastly and surprisingly, plasma MDZ concentrations were also reduced in mice administered 3‐Me, but to a much lesser extent, suggesting that 3‐Me is a promiscuous inhibitor to MDZ 1′‐hydroxylation in mice.This approach provides a useful and practical means to conduct single mouse PK research and may prove useful for studying drug metabolism in widely‐used laboratorial mouse models. Additionally, our data suggest alternative roles for classical human CYP inhibitors and inducers in a mouse model.Support or Funding InformationJ.L.J., Y.T., and A.‐M. Y. were supported by a National Institutes of Health National Cancer Institute grant [U01CA175315].