ABSTRACT:To understand the rate-limiting process of oral drug absorption, not only total bioavailability (F) but also intestinal (F a ⅐ F g ) and hepatic (F h ) availability after oral administration should be evaluated. Usually, F a ⅐ F g of drug is calculated from pharmacokinetic parameters after intravenous and oral administration. This approach is influenced markedly by the estimated value of F h , which varies with the hepatic blood flow used in the calculations. In this study, portal vein-cannulated rats were used to calculate the F a ⅐ F g of drugs from a single oral dosing experiment without data from intravenous injection. Portal vein-cannulated rats were prepared by a new operative method that enables stable portal vein blood flow. This surgery had no effects on hepatic blood flow and metabolic activity. Our method for calculating F a ⅐ F g was validated by determining both portal and systemic plasma concentration profiles of various drugs possessing different pharmacokinetic properties after oral administration to the portal vein-cannulated rats. Simulation of portal and systemic plasma concentrations by physiologically based pharmacokinetic modeling indicated that the balance of the absorption rate constant (k a ) and elimination rate constant (k e ) resulted in different patterns in portal and systemic plasma concentration-time profiles. This study is expected to provide a new experimental animal model that enables identification of the factors that limit oral bioavailability and to provide pharmacokinetic information on the oral absorption process of drugs during drug discovery.

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Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes.

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Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.

The purpose of this study was to evaluate the impact of intestinal efflux transporters on the in vivo oral absorption process. Three model drugs-fexofenadine (FEX), sulfasalazine (SASP), and topotecan (TPT)-were selected as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and P-gp and BCRP substrates, respectively. The drugs were orally administered to portal veincannulated rats after pretreatment with zosuquidar (ZSQ), P-gp inhibitor, and/or Ko143, BCRP inhibitor. Intestinal availability (Fa·Fg) of the drugs was calculated from the difference between portal and systemic plasma concentrations. When rats were orally pretreated with ZSQ, Fa·Fg of FEX increased 4-fold and systemic clearance decreased to 75% of the control. In contrast, intravenous pretreatment with ZSQ did not affect Fa·Fg of FEX, although systemic clearance decreased significantly. These data clearly show that the method presented herein using portal vein-cannulated rats can evaluate the effects of intestinal transporters on Fa·Fg of drugs independently of variable systemic clearance. In addition, it was revealed that 71% of FEX taken up into enterocytes underwent selective efflux via P-gp to the apical surface, while 79% of SASP was effluxed by Bcrp. In the case of TPT, both transporters were involved in its oral absorption. Quantitative analysis indicated a 3.5-fold higher contribution from Bcrp than P-gp. In conclusion, the use of portal vein-cannulated rats enabled the assessment of the impact of efflux transporters on intestinal absorption of model drugs. This experimental system is useful for clarifying the cause of low bioavailability of various drugs.

PV rats enabled in vivo quantitative assessment of intestinal first-pass metabolism by CYP3A and UGT. This method is useful for clarifying the cause of low bioavailability.