Regular Article the Quantitative Prediction of in Vivo Enzyme-Induction Caused by Drug Exposure from in Vitro Information on Human Hepatocytes

Drug Metab. Pharmacokinet. 20 (4): 236–243 (2005).

Regular Article The Quantitative Prediction of In Vivo Enzyme-Induction Caused by Drug Exposure from In Vitro Information on Human Hepatocytes

Motohiro KATO1,KojiCHIBA2,MasatoHORIKAWA3 and Yuichi SUGIYAMA4 1Chugai Pharmaceutical Co. Ltd., Gotemba, Japan 2Pˆzer Japan Inc., Tokyo, Japan 3Nissan Chemical Industries Ltd., Saitama, Japan 4Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: There have been no reports of the quantitative prediction of induction for drug-metaboliz- ing enzymes in humans. We have tried to predict such enzyme induction in humans from in vitro data obtained using human hepatocytes. The in vitro and in vivo data on enzyme induction by inducers, such as rifampicin, phenobarbital and omeprazole, were collected from the published literature. The degree of enzyme induction in humans was compared with that predicted from in vitro data on human hepatocytes. Using the in vivo data, we calculated the hepatic intrinsic clearance of typical CYP substrates, such as midazolam and caŠeine, before and after inducer treatment and estimated the induction ratios of hepatic intrinsic clearance following treatment. In the in vitro studies, the amount of mRNA or enzyme and enzyme activity in human hepatocytes, with or without an inducer, were compared and the induction ratios were estimated. The unbound mean concentration was taken as an index of drug exposure and the induction ratios in the in vivo and in vitro studies were compared. The unbound mean concentrations of inducers used in the in vitro studies were higher than those in the in vivo studies. The maximum induction ratios by inducers in the in vitro studies were higher than those in the in vivo studies. The induction ratio for rifampicin, omeprazole, troglitazone, dexamethasone and phenobarbital increased as the unbound mean concentration increased to reach a constant value. The induction of CYP3A and 1A was analyzed by the Emax model. The maximum induction ratio (Emax) and the concentration at half maximum induction (EC50) for rifampicin, omeprazole, troglitazone, dexamethasone and phenobarbital were 12.3, 0.847 mmolWL, 2.36, 0.225 mmolWL, 6.86, 0.002 mmolWL, 8.30, 9.32 mmolWL, and 7.62, 58.4 mmolWL, respectively. The Emax and EC50 of omeprazole for CYP1A were 12.02 and 0.075 mmolWL, respectively. The predicted induction ratio of all those inducers, except for omeprazole, based on the Emax and EC50 values obtained from the in vitro data were similar to the observed values. On the whole, a good correlation between the observed and predicted induction ratio of omeprazole was observed (r＝0.768, pº0.05), although the predicted induction ratio was higher than the observed value. In conclu- sion, the present study suggests that it is possible to predict quantitatively the CYP3A enzyme induction from hepatocyte data.

Key words: enzyme induction; hepatocyte; CYP3A; CYP1A

the drug concentration. The prediction of drug-drug Introduction interactions during the drug discovery stage would help Many drug-drug interactions caused by co-adminis- avoid such interactions. Although the use of human tration of drugs in clinical situations have been report- microsomes and recombinant CYP enables the predic- ed. These involve inhibition and induction of drug tion of enzyme inhibition,1,2) no prediction method for metabolizing enzymes. Enzyme inhibition causes ad- enzyme induction has been established yet. Enzyme verse eŠects increasing the drug concentration while induction has been estimated using animals treated with enzyme induction causes reduced e‹cacy by lowering the drug candidates and animal studies need relatively

Received; February 4, 2005, Accepted; June 4, 2005 To whom corrspondence should be addressed: Motohiro KATO,Ph.D.,Pre-clinical Research Dept. Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. Tel. ＋81-0550-87-6708, Fax. ＋81-0550-87-5397, E-mail: katomth＠chugai-pharm.co.jp

236 The Prediction of In Vivo Enzyme-Induction 237 large amounts of candidates and, hence, such studies Clint was calculated using the following equation are not suitable for screening. In addition, it has been based on the well-stirred model. reported that there are species diŠerences in enzyme CLint＝CLhQhRbWsfp (Qh-CLh)t (4) induction.3) Therefore, enzyme induction in humans has been estimated using primary cultured human hepato- where Qh, Rb and fb are the blood ‰ow rate, blood to cytes.4–7) Recently, it has become clear that the induction plasma concentration ratio and blood unbound frac- of CYPs contributes to a variety of nuclear receptors tion,respectively.RbandQhwereassumedtobe1and and a reporter gene assay of nuclear receptors has been 1610 mLWmin, respectively. developed for enzyme induction.8–12) Although it is The pharmacokinetic parameters of rifampicin, possible to estimate the enzyme induction potency of a omeprazole, lansoprazole, carbamazepine, dexametha- candidate drug using human hepatocytes, quantitative sone, phenobarbital and sulˆnpyrazone in humans were prediction of enzyme induction in clinical situations has also obtained from Goodman & Gilman's Textbook.46) not been possible. Omeprazole and lansoprazole induce The parameters of troglitazone were obtained from the CYP1A and 3A in hepatocytes13) and omeprazole is report by Izumi et al..47) metabolized by CYP2C19 and CYP3A. The AUC of The mean unbound plasma concentrations (Css,u) omeprazole in poor metabolizers of CYP2C19 is 4-fold were calculated from equation 5. greater than that in extensive metabolizers. Omeprazole Css,u＝fp doseWCLWT＝fp AUCWT(5) induces CYP1A in poor metabolizers but not in extensive metabolizers at clinical dosages.14,15) If a drug where fp and T are the plasma unbound fraction and has the ability to induce an enzyme and there is low dosage interval. exposure to the drug in clinical situations, treatment Calculation of in vitro data: The induction ratios of with the drug will not induce the enzyme. The quantita- mRNA (RT-PCR), enzyme activity (testosterone 6-beta tive prediction of enzyme induction is important for hydroxylation activity etc.) and enzyme amount successful drug development and the present study was (Western blot) for CYP without an inducer to the values carried out to investigate whether enzyme induction can with an inducer were calculated. The mean unbound be quantitatively predicted using human hepatocytes. plasma concentrations (Css,u) in medium were calculat- ed to compare the induction ratios at the same concen- Methods tration under in vivo and in vitro conditions. The Css of Data collection: Data on the human pharmacoki- an inducer was calculated by dividing the AUC by the netics of a number of drugs metabolized by CYPs after medium change interval. The hepatic intrinsic clearance treatment with inducers, rifampicin, omeprazole, lan- was calculated from equations 3 and 4. Body weight, soprazole, carbamazepine, dexamethasone, phenobar- liver weight, hepatic blood ‰ow rate, cell numberWg liver bital, troglitazone, phenytoin, and sulˆnpyrazone, were and Rb were assumed to be 70 kg, 1695 g, 0.95 mLW collected16–37) along with induction data of those drugs in minWg, 120×106 cellsWg liver and 1, respectively. These human hepatocytes.4–7, 38–45) values were used and the intrinsic clearance per cell Calculation of in vivo data: Theincreaseinthe (CLint, cell) was obtained. ratios of the hepatic intrinsic clearances of typical CYP The intrinsic clearance per well (CLint,well) was substrates for CYP1A and 3A were calculated. In the calculated by multiplying CLint,cell by the cell number case of intravenous data, the total body clearance of per well. The elimination rate constant (k) of an inducer substrate was calculated from the AUC using equation from medium was calculated from equation 6. 1. For oral administration, CL was calculated from the k＝fm CLint,wellWV(6) elimination half-life. The pharmacokinetic model for speciˆc substrates was assumed to be the one compart- where fm and V are the unbound fraction in the medium ment model and the distribution volume and renal and medium volume. clearance were assumed not to be aŠected by treatment It was assumed that there was no diŠerence between with inducers. These drug parameters were obtained the binding of an inducer to human albumin and bovine from Goodman & Gilman's textbook, The Pharmaco- albumin. The nPtWKd was calculated using equation 7 logical Basis of Therapeutics, 9th ed..46) from the human plasma unbound fraction. Pt, n and Kd are the protein concentration, number of binding sites CL＝DoseWAUC (1) and dissociation constant, respectively. The human CL＝0.693 VdWT1W2 (2) albumin concentration (M.W.:67,000) was assumed to The hepatic clearance (CLh) was estimated from the be 500 mmolWL. The unbound fraction in medium was CL and renal clearance (CLr) using equation 3. calculated by correcting human nPtWKd by the ratio of the bovine albumin concentration in medium to the CLh＝CL-CLr (3) human albumin concentration in plasma (a) using 238 Motohiro KATO et al.

Fig. 1. The method for predicting in vivo induction from in vitro data.

equation 8. program MULTI48) in which each data point was not weighted. Bartlett analysis showed that there was no nPtWKd＝(1-fp)Wfp (7) signiˆcant diŠerence among the variance of the induc- fm＝1W(1＋anPtWKd) (8) tion ratios at each concentration. The unbound mean concentration in medium was R＝1＋Emax Css,uW(EC50＋Css,u) (13) calculated using equation 9. The induction ratios in humans after inducer treatment Css,u＝fm AUC T＝fm C (1-exp(-k T)) k T(9) W 0 WW were predicted using the EC50, Emax, and in vivo Css,u

C0 and T are the initial concentration and the medium values obtained by equation 13. change time, respectively. The method for predicting in vivo induction from Analysis of enzyme induction: The Emax model in vitro data is shown in Fig. 1. was used in which an inducer stimulates the production Results of mRNA. The following equations express the mass balance Table 1 shows the induction ratio of CLint for equations of mRNA and enzyme. speciˆc CYP substrates after treatment with inducers. Treatment with rifampicin, 600 mgWday, caused a 1.47- dmRWdt＝vsyn＋vsyn Emax CW(EC50＋C)- kdeg1 mR (10) to 5.35-fold induction of CYP3A. The induction ratio dEWdt＝ksyn mR-kdeg2 E(11)of CYP3A for treatment with phenobarbital, 100 mgW where vsys, Emax, EC50, kdeg1,kdeg2 and ksyn are the kg, was 3.08. The induction ratios for the other inducers synthesis rate of mRNA, maximum induction ratio, were less than this value. Treatment with omeprazole, concentration at the half maximum induction, degrada- 40 mgWday, in CYP2C19 poor metabolizers caused a tion rate constant of mRNA, the degradation rate 1.62-fold induction of CYP1A. The unbound mean constant of enzyme and translation rate constant of concentrations of 8 inducers in the in vitro studies were mRNA to enzyme, respectively. higher than those in the in vivo studies. The induction Equations 10 and 11 were solved under steady-state ratios for inducers at the highest concentration in the conditions. Equation 12 was obtained. in vitro studies were also higher than those in the in vivo studies (Fig. 2). The induction ratio of rifampicin, Rss R ＝Ess E ＝1＋Emax Css (EC50＋Css) (12) W 0 W0 W omeprazole, troglitazone, dexamethasone and pheno- where R0 and E0 are the initial amounts of mRNA and barbital increased as the unbound mean concentration enzyme, respectively. increased, reaching a constant value at high concentra- To estimate the EC50 and Emax values of inducers, tions (Fig. 2). The induction of CYP3A and 1A was the induction ratios(R) obtained from in vitro studies analyzed by the Emax model. Table 2 shows the were ˆtted to equation 13 using the nonlinear regression maximum induction ratio (Emax) and the concentration The Prediction of In Vivo Enzyme-Induction 239

Table 1. Enzyme induction by various inducers in humans

inducer dose priod substrate CYP induction ratio Ref. rifampicin 600 mgWday 5 days midazolam 3A 5.35 16 600 mgWday 7 days quinidine 3A 4.84 17 600 mgWday cyclosporine 3A 1.54 18 600 mgWday 18 days tacrolimus 3A 1.47 19 600 mgWday 7 days nifedipine 3A 4.86 20 600 mgWday (PM$) 9 days propafenone 2D6,3A 3.1 21 600 mgWday 5 days zolpidem 3A 1.75 22 600 mgWday 5 days triazolam 3A 3.04 23 600 mgWday 14 days 6-OHC*Wcortisol 3A 3.20 24 600 mgWday 14 days 6-OHC*Wcortisol 3A 3.71 25 1200 mgWday 6-OHC*Wcortisol 3A 5.90 carbamazepine 400–600 mgWday 3 weeks omeprazole 3A4 2.44 26 2C19 0.88 sulˆnpyrazone 800 mgWday 7 days verapamil 3A 1.3 27 phenytoin 300–400 mgWday 10 days cyclosporine 3A 2.79 28 300–400 mgWday 9 days cyclosporine 3A 1.81 29 dexamethasone 1.5 mgWday 4 days triazolam 3A 1.24 30 troglitazone 400 mgWday 6-OHC*Wcortisol 3A 2.2 31 phenobarbital 15 mgWday 26 days 6-OHC*W17-OHCS** 3A 1.09 32 30 mgWday 6-OHCW17-OHCS 3A 1.59 100 mgWday 14 days antipyrine 3A, 1A2, 2B6, 2C9 1.87 33 100 mgWday 6-OHC*W17-OHCS** 3A 3.08 omeprazol 40 mgWday PM# 7 days caŠeine 1A 1.62 34 40 mgWday EM## 7 days caŠeine 1A 1.06 120 mgWday EM## 7 days caŠeine 1A 1.26 35 120 mgWday EM## 7 days 6-OHC*Wcortisol 3A 1.14 40 mgWday EM## 7 days 6-OHC*Wcortisol 3A 1.03 40 mgWday EM## 7 days caŠeine 1A 1.17 36 40 mgWday PM# caŠeine 1A 1.32 120 mgWday EM## caŠeine 1A 1.41 40 mgWday PM# 7 days caŠeine 1A 1.39 37 40 mgWday EM## caŠeine 1A 1.12 20 mgWday EM## 7 days caŠeine 1A 0.98 15

*:6b hydroxy cortisol **: 17-hydroxy corticosteroids $: 2D6 poor metabolizer #: 2C19 poor metabolizer ##: 2C19 extensive metabolizer

at half-maximum induction (EC50) for rifampicin, treatment reached a steady-state after 5 days.24) The omeprazole, troglitazone, dexamethasone and pheno- inducers were administered for more than 5 days. The barbital. The Emax and EC50 of omeprazole for in vivo data appear to suggest that induction had CYP1A were 12.02 and 0.075 mmolWL, respectively reached a steady-state in every case. Enzyme induction (Fig. 2, Table 2). The predicted induction ratio of is commonly estimated from this reduction in AUC. CYP3A for all the inducers, except for omeprazole, The AUC of midazolam and triazolam after multiple basedontheEmaxandEC50obtainedfromin vitro dosing of rifampicin fell to about 1W20 that before data were similar to the observed in vivo values treatment.16,23) However, this phenomenon cannot be (Fig. 3A). The predicted induction ratio of omeprazole explained only by induction of hepatic CYP3A. It has was higher than the observed value. However, a good been reported that rifampicin induces not only hepatic correlation was found between the observed and CYP3A but also intestinal CYP3A.49) Our previous predicted induction ratios of omeprazole (r＝0.768, report indicated that multiple administration of rifam- pº0.05)(Fig. 3B). picincausedamarkedreductionintheintestinal availability of CYP3A substrates metabolized by intesti- Discussion nal ˆrst-pass metabolism, such as triazolam and Multiple administrations of inducers cause a reduc- midazolam.50) Therefore, in the present study, the tion in the AUC of a drug. The induction by rifampicin change in hepatic CLint was estimated from the half-life 240 Motohiro KATO et al.

Fig. 2. Relationship between the unbound steady-state drug concentration and induction ratio of CYPs under in vivo and in vitro conditions. Open squares and closed circles represent the induction ratio in vivo and in vitro, respectively. The solid line represents the ˆtted line. The inducers of CYP3A are rifampicin (A), omeprazole (B), troglitazone (D), dexamethazone (E), phenobarbital (F) carbamazepine (G) and sulˆnpyrazone (H). The inducer of CYP1A is omeprazole (C).

Table 2. The parameters for enzyme induction using human hepatocytes

inducer CYP EC50 (mmolWL) Emax

rifampicin 3A 0.847±0.749 12.3±3.4 omeprazole 3A 0.225±0.15 2.36±0.92 1A 0.056±0.074 13.6±5.9 troglitazone 3A 0.002±0.0005 6.86±0.46 dexamethasone 3A 9.32±2.29 8.30±0.65 phenobarbital 3A 58.4±95.8 7.62±1.82

Fig. 3. Relationship between the predicted induction ratio in hu- mans from in vitro data and the observed induction ratio of CYP3A and not the AUC. The induction ratio of CYP3A (A) and CYP1A (B) in humans after inducer treatments. estimated from the change in CLint for midazolam and Open circles and vertical bars represent the mean and SD of induction triazolam after treatment with rifampicin, 600 mgWday, ratios for rifampicin in Table 1.Closedcircles,opensquares,closed was 5.35 and 3.04, respectively. These values were squares and closed triangles represent the induction ratio for comparable with the induction ratio estimated from the dexamethasone, omeprazole, phenobarbital and troglitazone. The solid line indicates a 1:1 correspondence. urinary 6-beta-hydroxy cortisolWcortisol ratio and other substrates, such as quinidine and nifedipine (Table 1). These results suggest that the contribution of intestinal The Prediction of In Vivo Enzyme-Induction 241 metabolism to the total body clearance might be minor and in vivo is concerned. If a metabolite is a potential although metabolism in the small intestine contributes inducer, there may be a diŠerence in the exposure of markedly to the ˆrst-pass metabolism. The induction of metabolite in vitro and in vivo. A correlation between hepatic CYP3A by treatment with rifampicin, 600 mgW the observed and predicted induction ratio for CYP1A day, was 2- to 5-fold. The marked reduction in the AUC by omerazole was observed which suggested that 1A of some substrates, such as midazolam and triazolam, induction might be predictable following some correc- could be due to the induction of intestinal metabolism. tions. In the present study, the induction ratio of mRNA, Marked individual and experimental diŠerences in the enzyme amount and enzyme activity were collected in vitro studies were observed (Fig. 2). The prediction of because good correlations were observed among these enzyme induction from in vitro data depends on the parameters.38,43) These correlations suggest that the Emax value. The Emax values of rifampicin, troglita- induction of mRNA and enzyme reach a steady-state. zone, dexamethasone and phenobarbital were 6.89–12.3 The maximum induction ratio of CYP3A by rifampicin and, overall, similar. The coincidences between the in in vitro studies was 32-fold. This was higher than that observed and predicted induction ratio might be due to in in vivo studies (Fig. 2). To clarify whether the similar Emax values. Although some individual diŠer- diŠerence in enzyme induction under in vivo and in vitro ences in the induction ratio for taxol and rifampicin conditions is due to exposure to the inducer, the degree were observed, there was a good correlation between the of enzyme induction at the same unbound concentration induction ratio for taxol and rifampicin. This result was compared. In some in vitro studies, the medium suggests the possibility of quantitative prediction fol- contained bovine albumin. It was assumed that there lowing a correction using the Emax of a stable metabol- was no species diŠerence in bovine and human protein ic inducer, such as rifampicin or phenobarbital. The binding. The unbound fraction of all the inducers in the coincidences between the observed and predicted induc- medium was more than 0.6 except for omeprazole. The tion ratios in the present study suggest that the suitable eŠect of this assumption may be minor. The concentra- Emax for CYP3A4 in human hepatocyte inducers might tionsofinducersinallthein vitro studies were higher be approximately 10 to correct for the diŠerence than those in the in vivo studies. Induction studies under between in vivo and in vitro conditions. conditions mimicking those in vivo might be needed to Recently, the reporter gene assay has been used to predict enzyme induction quantitatively. determine enzyme induction instead of hepatocytes.11,12) The induction ratio for all inducers increased as the The EC50 of rifampicin from human hepatocytes, unbound mean concentration increased, reaching a which was 0.847 mmolWL, was comparable with the constant value at high concentrations in the in vitro EC50 obtained from the reporter gene assay, which was studies (Fig. 2). The induction of CYP3A and 1A was 0.71 mmolWL.11) There was a good correlation between analyzed by the Emax model. The predicted induction the induction ratios using human hepatocytes and the ratio of those inducers of CYP 3A, based on parameters induction ratios obtained from reporter gene assay of obtained from in vitro data, were similar to the fourteen inducers, except ritonavir and troleandmycin observed values (Fig. 3A). This study suggests that (r＝0.864).12) Thereportergeneassaymightbeuseful enzyme induction in vivo may be predictable. Further for the future quantitative prediction of enzyme studies are needed because this analysis involved a induction. number of assumptions and there was considerable In the present study, many reports of enzyme induc- variation in the data used. The prediction of enzyme tion in humans were subjected to detailed examination induction of CYP1A by omeprazole from human and most concerned rifampicin. The treatments with hepatocytes was overestimated (Fig. 3B). This overesti- rifampicin, phenobarbital and carmabazepine markedly mation might be due to the assumption that an inducer reduced the AUC of the co-administered drug. The in the medium was reduced by metabolism. The CLint induction eŠect of other inducers was weaker than that of omeprazole was the highest of all the inducers. of rifampicin. Comparison with the induction eŠect of Although the enzyme activity of hepatocyes in the rifampicin might be important. A compound that has a absence of an inducer falls with time, the enzyme stronger induction eŠect than rifampicin may not be activity was assumed to be constant. In the case of suitable for use as a drug. rifampicin, the eŠect of this assumption may be minor. In conclusion, the present study suggests that it is On the other hand, for omeprazole, this assumption possible to predict quantitatively CYP3A enzyme may in‰uence the exposure. 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