Masashi Yabuki

Evaluation of the potential for drug-induced liver injury based on in vitro covalent binding to human liver proteins

Prediction of idiosyncratic drug-induced liver injury (DILI) is difficult, and the underlying mechanisms are not fully understood. However, many drugs causing DILI are considered to form reactive metabolites and covalently bind to cellular macromolecules in the liver. The objective of this study was to clarify whether the risk of idiosyncratic DILI can be estimated by comparing in vitro covalent binding (CB) levels among 12 positive compounds (acetaminophen, alpidem, bromfenac, carbamazepine, diclofenac, flutamide, imipramine, nefazodone, tacrine, ticlopidine, tienilic acid, and troglitazone) for DILI and 12 negative compounds (acetylsalicylic acid, caffeine, dexamethasone, losartan, ibuprofen, paroxetine, pioglitazone, rosiglitazone, sertraline, theophylline, venlafaxine, and zolpidem). After incubation with human liver microsomes in the presence of NADPH, there was a large overlap in the distribution of CB amounts between the positive and negative groups. On addition of UDP-glucuronic acid (UDPGA) as a cofactor for glucuronidation, the CB levels of bromfenac and diclofenac were increased. With addition of nucleophilic glutathione (GSH), values for most compounds were decreased. However, separation of the two groups on the basis of CB could not be improved by UDPGA or GSH. Furthermore, CB with human hepatocytes also failed to discriminate positive from negative compounds. Therefore, the CB amount alone is not sufficient for risk assessment of DILI. In contrast, when the CB amount was multiplied by the maximum daily dose, which may reflect maximum hepatic exposure, the two groups did become discriminated. Taken together, our findings suggest that the combination of CB amount and daily dose can estimate the risk of idiosyncratic DILI.

A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes

Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations. Copyright

Prediction of the intestinal first-pass metabolism of CYP3A and UGT substrates in humans from in vitro data.

This study aimed to establish a practical and simplified method of predicting intestinal availability in humans (F(g,human)) at the drug discovery stage using in vitro metabolic clearance values and permeability clearance values. A prediction model for F(g,human) of 19 CYP3A substrates and 5 UGT substrates was constructed based on the concept that the permeability clearance values mean the permeability across the basal membrane with a pH of 7.4 on both sides. Permeability clearance values were obtained by parallel artificial membrane permeability assay (PAMPA) at pH 7.4. PAMPA is widely used in the pharmaceutical industry as the earliest primary screening stage and enables estimation of the kinetics of transport by passive diffusion. For CYP3A substrates, the metabolic clearance was obtained from in vitro intrinsic clearance values in human intestinal or hepatic microsomes (CL(int,HIM) or CL(int,HLM), respectively). Using metabolic clearances corrected by the ratio of CL(int,HIM) to CL(int,HLM), HLM showed equivalent predictability to that of HIM for CYP3A substrates. For UGT substrates, the clearance was obtained from alamethicin-activated HIM using one incubation with both NADPH and UDPGA cofactors. The method proposed in this study could predict F(g,human) for the compounds investigated and represents a simplified method based on a new concept applicable to lower permeability compounds.

Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs

Abstract 1.   Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds. 2.   Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A. 3.   Hepatic intrinsic clearance (CLint) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CLint values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CLint values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans. 4.   In conclusion, dogs showed the most similar metabolic activity to humans.

Prediction of the Intestinal First-Pass Metabolism of CYP3A Substrates in Humans Using Cynomolgus Monkeys

To select high bioavailability compounds, it is necessary to predict the first-pass metabolism in the intestine. However, in vitro-in vivo predictions of the intestinal metabolism have proven both challenging and less definitive. The purpose of this study was to investigate prediction of intestinal first-pass metabolism in humans using cynomolgus monkeys. First, we investigated intrinsic metabolic activities in intestinal microsomes of monkeys (MIM) and humans (HIM) (CLint, MIM and CLint, HIM, respectively) of 18 CYP3A substrates. The CLint, MIM values were found to be relatively high and showed excellent correlation with the CLint, HIM values. Subsequently, we determined the plasma concentrations of 9 CYP3A substrates (buspirone, carbamazepine, diazepam, felodipine, midazolam, nicardipine, nifedipine, saquinavir, and verapamil) in monkeys after an oral dose of 2 mg/kg with or without an oral dose of 5 mg/kg ketoconazole and calculated AUC(+vehicle)/AUC(+ketoconazole), defined as Fg, monkey(observed); we confirmed that the dose of ketoconazole inhibited only intestinal CYP3A metabolism by preliminary in vitro and in vivo experiments using ketoconazole. The Fg, monkey(observed) was lower than the Fg, human(observed) for most compounds, but moderate correlation was observed. Furthermore, using these data, we established a new methodology to estimate Fg, human(predicted) more precisely on the basis of the assumption that intestinal physiological conditions other than intrinsic metabolic activity would be the same between monkeys and humans. In conclusion, the in vivo model using cynomolgus monkeys in this study is useful for prediction of intestinal first-pass metabolism by CYP3A in humans because it was able to predict Fg, human of all nine compounds investigated.

Species Differences in Intestinal Metabolic Activities of Cytochrome P450 isoforms between Cynomolgus Monkeys and Humans

The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans.

Dehydroepiandrosterone sulfate, a useful endogenous probe for evaluation of drug–drug interaction on hepatic organic anion transporting polypeptide (OATP) in cynomolgus monkeys

Since drug-drug interaction (DDI) can affect organic anion-transporting polypeptide (OATP) and cause clinical events, prediction of such DDI is important in early clinical development. Although statins are useful probes for OATP-mediated DDI, endogenous probes would be more practical for predicting such DDI. In this study, we investigate the possible use of dehydroepiandrosterone sulfate (DHEAS), an endogenous OATP substrate, in predicting OATP-mediated DDI in cynomolgus monkeys as a first step toward in human assessment. In in vitro experiments, both human and cynomolgus monkey hepatocytes showed a time- and temperature-dependent DHEAS uptake. Rifampicin (RIF), a typical OATP inhibitor, inhibited this uptake, indicating the involvement of OATP in DHEAS uptake. In in vivo experiments, the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of DHEAS were significantly increased following administration of RIF 10 mg/kg, although the extent of this increase was lower than that observed with the test-statins used in this study. However, based on the results of in vitro hepatic DHEAS uptake, changes in DHEAS concentration are expected to be more prominent in human than in monkey. This shows for the first time that DHEAS may be used as endogenous probe for predicting OATP-mediated DDI.

Transport of biguanides by human organic cation transporter OCT2.

Biguanides have the severe side effect of lactic acidosis. Although both metformin and phenformin are biguanide derivatives, there is a difference in the frequency at which they induce lactic acidosis. However, the reasons for the difference are not clear. Metformin has been reported to be mainly excreted into urine by human organic cation transporter 2 (hOCT2). The present study was designed to investigate the renal transport of metformin and phenformin, focusing on hOCT2, using hOCT2-expressing oocytes. Both biguanides were found to be good substrates for hOCT2. However, phenformin exhibited a higher affinity and transport activity than metformin. The Km values for metformin and phenformin were 235 and 37.4 μM, with CL(int) (V(max)/K(m)) values of 71.9×10⁻³ μL/min per oocyte and 209×10⁻³ μL/min per oocyte, respectively. This is the first report that has compared the transport profiles of these biguanides in hOCT2-expressing oocytes. The results suggest that plasma concentration of phenformin in subjects carrying hOCT2 variant may be higher compared to reference subjects, as reported in metformin. In addition, the relationship between plasma concentration of these biguanides and blood lactate level as well as the possible reasons for the difference in the associated frequency of occurrence of lactic acidosis are discussed.

Significance of Reductive Metabolism in Human Intestine and Quantitative Prediction of Intestinal First-Pass Metabolism by Cytosolic Reductive Enzymes

The number of new drug candidates that are cleared via non-cytochrome P450 (P450) enzymes has increased. However, unlike oxidation by P450, the roles of reductive enzymes are less understood. The metabolism in intestine is especially not well known. The purposes of this study were to investigate the significance of reductive metabolism in human intestine, and to establish a quantitative prediction method of intestinal first-pass metabolism by cytosolic reductive enzymes, using haloperidol, mebendazole, and ziprasidone. First, we estimated the metabolic activities for these compounds in intestine and liver using subcellular fractions. Metabolic activities were detected in human intestinal cytosol (HIC) for all three compounds, and the intrinsic clearance values were higher than those in human liver cytosol for haloperidol and mebendazole. These metabolic activities in HIC were NADPH- and/or NADH-dependent. Furthermore, the metabolic activities for all three compounds in HIC were largely inhibited by menadione, which has been used as a carbonyl reductase (CBR)–selective chemical inhibitor. Therefore, considering subcellular location, cofactor requirement, and chemical inhibition, these compounds might be metabolized by CBRs in human intestine. Subsequently, we tried to quantitatively predict intestinal availability (Fg) for these compounds using human intestinal S9 (HIS9). Our prediction model using apparent permeability of parallel artificial membrane permeability assay and metabolic activities in HIS9 could predict Fg in humans for the three compounds well. In conclusion, CBRs might have higher metabolic activities in human intestine than in human liver. Furthermore, our prediction method of human Fg using HIS9 is applicable to substrates of cytosolic reductive enzymes.

Blonanserin, a novel atypical antipsychotic agent not actively transported as substrate by P-glycoprotein.

Although blonanserin, a novel atypical antipsychotic agent with dopamine D(2)/serotonin 5-HT(2A) antagonistic properties, displays good brain distribution, the mechanism of this distribution has not been clarified. P-glycoprotein [(P-gp) or multidrug resistance protein 1 (MDR1)] is an efflux transporter expressed in the brain and plays an important role in limiting drug entry into the central nervous system (CNS). In particular, P-gp can affect the pharmacokinetics and efficacy of antipsychotics, and exacerbate or soothe their adverse effects. In this study, we conducted in vitro and in vivo experiments to determine whether blonanserin is a P-gp substrate. Risperidone and its active metabolite 9-hydroxyrisperidone, both of which are P-gp substrates, were used as reference drugs. Affinity of blonanserin, risperidone, and 9-hydroxyrisperidone for P-gp was evaluated by in vitro transcellular transport across LLC-PK1, human MDR1 cDNA-transfected LLC-PK1 (LLC-MDR1), and mouse Mdr1a cDNA-transfected LLC-PK1 (LLC-Mdr1a). In addition, pharmacokinetic parameters in the brain and plasma (B/P ratio) of test compounds were measured in mdr1a/1b knockout (KO) and wild-type (WT) mice. The results of in vitro experiments revealed that P-gp does not actively transport blonanserin as a substrate in humans or mice. In addition, blonanserin displayed comparable B/P ratios in KO and WT mice, whereas B/P ratios of risperidone and 9-hydroxyrisperidone differed markedly in these animals. Our results indicate that blonanserin is not a P-gp substrate and therefore its brain distribution is unlikely to be affected by this transporter.