•Drug-induced liver injury (DILI) is a major safety concern in drug development.•The utility of HepaRG cells as a model for DILI risk assessment was investigated.•HepaRG cells exhibited a 67% ...sensitivity and 73% specificity at 100-fold Cmax.•HepaRG cells distinguished relatively safe drugs from their high-risk analogs.•HepaRG cells may be of use for DILI risk assessment.
The utility of HepaRG cells as an in vitro cell-based assay system for assessing drug-induced liver injury (DILI) risk was investigated. Seventeen DILI-positive and 15 DILI-negative drugs were selected for the assay. HepaRG cells were treated with each drug for 24h at concentrations that were 1.6-, 6.3-, 25-, and 100-fold the therapeutic maximum plasma concentration (Cmax). After treatment, the cell viability, glutathione content, caspase 3/7 activity, lipid accumulation, leakage of lactate dehydrogenase, and albumin secretion were measured. The sensitivity and specificity were calculated to assess the ability of the assay to predict DILI. Our multiparametric assay using HepaRG cells exhibited a 67% sensitivity and 73% specificity at a 100-fold concentration of Cmax and a 41% sensitivity and 87% specificity at a 25-fold concentration of Cmax. When a 25-fold Cmax cut-off was applied, approximately 70% of drugs exhibiting positive responses were classified into the high DILI risk category. HepaRG cells distinguished relatively safe drugs from their high-risk analogs. Our study indicates that HepaRG cells may be of use to (1) prioritize drug analogs, (2) analyze the mechanism of DILI, and (3) assess the risk for DILI in the early drug discovery stage.
In this study, advantages of minipigs to use in preclinical study for new drug development were evaluated in terms of prediction of human pharmacokinetic (PK) parameters of various drugs. Fourteen ...model drugs having diverse physicochemical properties were selected and intravenously administered to mice, rats and minipigs to obtain their PK parameters. The human volume of distribution (Vd) and clearance (CL) of model drugs were predicted from PK parameters in each animal species.
When Vd of 14 drugs in each species were directly compared with those in humans, minipigs showed the highest correlation. Correction of Vd with an unbound fraction of drugs in tissues further improved the correlation. Allometric scaling that included minipig data resulted in high accuracy in the prediction of human Vd, clearly indicating an importance of minipig data. Minipigs also showed the high predictability of human CL. The prediction of human CL by allometric scaling showed a high accuracy when the data of minipigs were included.
In conclusion, potential advantages of minipigs for predicting human Vd and CL were clearly demonstrated. Reliable prediction of human PK from data of minipigs appears to be possible in preclinical PK study, without relying on PK analysis in other species.
Purpose
To evaluate the impact of intestinal first-pass metabolism (Fg) by cytochrome P4503A (CYP3A) and uridine 5’-diphosphate-glucuronosyltransferases (UGT) on
in vivo
oral absorption of their ...substrate drugs.
Methods
CYP3A and UGT substrates were orally administered to portal-vein cannulated (PV) rats to evaluate their intestinal availability (Fa · Fg). In the case of CYP3A substrates, vehicle or 1-aminobenzotriazole (ABT), a potent inhibitor of CYP enzymes, was pretreated to assess Fg separately from Fa (Enzyme-inhibition method). On the other hand, since potent inhibitors of UGT have not been identified, Fg of UGT substrate was calculated from total amount of metabolites generated in enterocytes (Metabolite-distribution method).
Results
After oral administration of CYP3A substrates in ABT-pretreated rats, the portal and systemic plasma concentrations of the metabolite were nearly the same, indicating almost complete inhibition of intestinal CYP3A-mediated metabolism. Using Enzyme-inhibition method, Fg of midazolam (1 mg/kg) was calculated as 0.71. Additionally, total amount of raloxifene-6-glucuronide generated in enterocytes after oral administration of raloxifene was estimated using Metabolite-distribution method and Fg of raloxifene (0.98 μmol/kg) was calculated as 0.21.
Conclusions
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.
To understand the rate-limiting process of oral drug absorption, not only total bioavailability (F) but also intestinal (F(a) · F(g)) and hepatic (F(h)) availability after oral administration should ...be evaluated. Usually, F(a) · 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) · 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) · 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.
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 vein-cannulated 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.
Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey ...P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.
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.
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Although anatomical and structural similarities between the skin of minipigs and humans are often reported, few percutaneous pharmacokinetic studies have been conducted in minipigs. ...The objective of this study was to clarify the usefulness of minipigs for estimating the percutaneous absorption of various drugs in humans. The absorption of several marketed drugs was observed in mice, rats and minipigs both in vivo and in vitro, and results were compared with those in humans. For all six model drugs, after percutaneous administration in vivo, fraction of dose absorbed (F) from the skin was the lowest in minipigs among the four species studied, including humans. In vitro drug permeation results were similar, with the lowest permeability observed in minipigs. However, combined use of both in vitro permeation and in vivo absorption data from minipigs using triple pack approach resulted in better prediction of human F values than data obtained from mice. These results suggest some qualitative, but not quantitative, similarities between the drug absorption process across the skin of minipigs and humans. In conclusion, minipigs appear to be a promising model animal for predicting percutaneous drug absorption in humans, however, more in vivo and in vitro studies are needed to improve predictability.
