In vitro metabolism of alectinib, a novel potent ALK inhibitor, in human: contribution of CYP3A enzymes.

1. The in vitro metabolism of alectinib, a potent and highly selective oral anaplastic lymphoma kinase inhibitor, was investigated. 2. The main metabolite (M4) in primary human hepatocytes was identified, which is produced by deethylation at the morpholine ring. Three minor metabolites (M6, M1a, and M1b) were also identified, and a minor peak of hydroxylated alectinib (M5) was detected as a possible precursor of M4, M1a, and M1b. 3. M4, an important active major metabolite, was produced and further metabolized to M6 by CYP3A, indicating that CYP3A enzymes were the principal contributors to this route. M5 is possibly produced by CYP3A and other isoforms as the primary step in metabolism, followed by oxidation to M4 mainly by CYP3A. Alternatively, M5 could be oxidized to M1a and M1b via an NAD-dependent process. None of the non-CYP3A-mediated metabolism appeared to be major. 4. In conclusion, this study suggests that involvement of multiple enzymes in the metabolism of alectinib reduces its potential for drug-drug interactions.

A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.

Tofogliflozin Salt Cocrystals with Sodium Acetate and Potassium Acetate.

We investigated the salt cocrystals formed by tofogliflozin with sodium acetate and potassium acetate by determining the crystal structures of the salt cocrystals and characterizing the solid states. The salt cocrystal screening using the slurry method and the liquid-assisted grinding method resulted in the formation of tofogliflozin-sodium acetate 1 : 1 and tofogliflozin-potassium acetate 1 : 1 salt cocrystals. Single-crystal X-ray diffraction revealed that, although each salt cocrystal belongs to a different space group, both of the salt cocrystals have almost similar structural features, including the conformation of tofogliflozin molecules, the coordination to Na+/K+ ions, and hydrogen bonds. The salt cocrystals exhibited extreme hygroscopicity with deliquescence, which is also a property of sodium acetate and potassium acetate. In addition, tofogliflozin-potassium acetate salt cocrystal had two polymorphs, which were enantiotropically related.

Simple Evaluation Method for CYP3A4 Induction from Human Hepatocytes: The Relative Factor Approach with an Induction Detection Limit Concentration Based on the Emax Model.

We investigated the robustness and utility of the relative factor (RF) approach based on the maximum induction effect (Emax) model, using the mRNA induction data of 10 typical CYP3A4 inducers in cryopreserved human hepatocytes. The RF value is designated as the ratio of the induction detection limit concentration (IDLC) for a standard inducer, such as rifampicin (RIF) or phenobarbital (PB), to that for the compound (e.g., RFRIF is IDLCRIF/IDLCcpd; RFPB is IDLCPB/IDLCcpd). An important feature of the RF approach is that the profiles of the induction response curves on the logarithmic scale remain unchanged irrespective of inducers but are shifted parallel depending on the EC50 values. A key step in the RF approach is to convert the induction response curve by finding the IDLC of a standard inducer. The relative induction score was estimated not only from Emax and EC50 values but also from those calculated by the RF approach. These values showed good correlation, with a correlation coefficient of more than 0.974, which revealed the RF approach to be a robust analysis irrespective of its simplicity. Furthermore, the relationship between RFRIF or RFPB multiplied by the steady-state unbound plasma concentration and the in vivo induction ratio plotted using 10 typical inducers gives adequate thresholds for CYP3A4 drug-drug interaction risk assessment. In light of these findings, the simple RF approach using the IDLC value could be a useful method to adequately assess the risk of CYP3A4 induction in humans during drug discovery and development without evaluation of Emax and EC50.

Preclinical evaluation of the potential for cytochrome P450 inhibition and induction of the selective ALK inhibitor, alectinib.

1. A novel selective anaplastic lymphoma kinase (ALK) inhibitor, alectinib, has shown remarkable efficacy and safety in patients with ALK-positive non-small-cell lung cancer (NSCLC). The purpose of this study was to evaluate in vitro the potential to inhibit and induce cytochrome P450 (CYP) isoforms for alectinib and its major metabolite M4. 2. Alectinib and M4 did not show the meaningful direct inhibition of six major CYP isoforms (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) in human liver microsomes (HLM). Alectinib, but not M4, competitively inhibited CYP2C8, by which few marketed drugs are exclusively metabolized, with an inhibition constant of 1.98 µM. 3. Out of the seven CYP isoforms in HLM, alectinib and M4 showed time-dependent inhibition (TDI) of only CYP3A4, which suggests low TDI potential due to low inactivation efficiency. 4. Alectinib exhibited quite smaller induction of mRNA expression of CYP1A2, 2B6 and 3A4 genes in human hepatocytes compared to the respective positive controls, suggesting a low potential of enzyme induction. 5. In summary, the risk of alectinib causing drug-drug interactions with coadministered drugs is expected to be low due to the weak potential of CYP inhibition and induction estimated in the preclinical studies.

Intracellular Dynamics and Fate of a Humanized Anti-Interleukin-6 Receptor Monoclonal Antibody, Tocilizumab.

Tocilizumab (TCZ), a humanized anti-interleukin-6 (IL-6) receptor (IL-6R) monoclonal antibody, abrogates signal transducer protein gp130-mediated IL-6 signaling by competitively inhibiting the binding of IL-6 to the receptor, and shows clinical efficacy in autoimmune and inflammatory diseases. Despite accumulating evidence for therapeutic efficacy, the behavior and fate of TCZ at the cellular level remain largely unknown. To address this, we evaluated the endocytosis and intracellular trafficking of IL-6R in HeLa cells. The results of our study provide evidence that IL-6R is constitutively internalized from the cell surface by ligand or TCZ binding and the expression of gp130 in an independent manner and is targeted via endosomes without being significantly directed to the recycling pathway to, and degraded in, lysosomes. Furthermore, the cytoplasmic tail of IL-6R is required for constitutive endocytosis of the receptor, which is mediated by the clathrin and AP-2 complex. We further demonstrate that FcRn, whose function is to regulate the serum persistence of IgG, is confined primarily to early/recycling endosomes and rapidly transits between these compartments and late endosomes/lysosomes without being degraded. Importantly, the expression of FcRn induces the segregation of TCZ from IL-6R, resulting in extensive colocalization of TCZ and FcRn in IL-6R-depleted endosomal compartments. Collectively, our results suggest that FcRn can accelerate the retrieval of the internalized TCZ, not only from endosomes but also from lysosomes. Our findings provide new insight into the mechanism by which the antibody internalized into cells is rescued from lysosomal degradation and into how its serum levels are maintained.

A trial proteomics fingerprint analysis of HepaRG cells by FD-LC-MS/MS.

A proteomics profile analysis was performed on a human hepatocyte carcinoma cell line (HepaRG) by using the FD-LC-MS/MS method. One hundred and fifty-eight proteins were newly identified for the first time of which 10 were found to be specific to human hepatocytes. These proteins are a "proteomics fingerprint" that can be used to characterize HepaRG cells.

In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP.

Abstract 1. The metabolism and drug-drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

A useful model capable of predicting the clearance of cytochrome 3A4 (CYP3A4) substrates in humans: validity of CYP3A4 transgenic mice lacking their own Cyp3a enzymes.

The accurate prediction for the body clearance of a novel drug candidate by humans during the preclinical stage contributes to its successful development. To improve the predictability of human hepatic clearance, we focused on CYP3A4, which is involved in the metabolism of more than 50% of all currently marketed drugs. In this study, we investigated the validity of the in vivo model using transgenic mice carrying the human CYP3A4 gene and lacking their own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward its substrates in liver microsomes was similar in CYP3A4-Tg mice and humans. As for the clearance, six CYP3A4 substrates (alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and quinidine) were given intravenously to CYP3A4-Tg mice, and their hepatic intrinsic clearance (CLint,h) was evaluated. A regression analysis of the data obtained indicated that the CLint,h values of six substrates in CYP3A4-Tg mice were highly correlated with those in humans (R(2) = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially expressed CYP3A4 to the overall metabolism in the mice. From these findings, it is reasonable to expect that the CLint,h of a particular drug in humans is predictable by applying the CLint,h obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was evaluated and found to be within a range of 2-fold of the regression value. These results suggest that the CYP3A4-Tg mouse model has the potential to accurately predict the human hepatic clearance of CYP3A4 substrates.

Application of human FcRn transgenic mice as a pharmacokinetic screening tool of monoclonal antibody.

1. For drug discovery, useful screening tools are essential to select superior candidates. Here, we evaluated the applicability of transgenic mice expressing human neonatal Fc receptor (FcRn) (hFcRn Tgm) as a pharmacokinetic screening tool of therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins that overcomes the species difference in FcRn binding. 2. Marketed 11 mAbs and 2 Fc-fusion proteins were intravenously administered to hFcRn Tgm and WT mice. The half-lives in hFcRn Tgm and WT mice were compared with those in human obtained from literature. The linear half-lives in human and monkey were also calculated by nonlinear pharmacokinetic analysis. For comparison, correlations of half-lives between monkey and human were also evaluated. 3. The half-lives of mAbs and Fc-fusion proteins after intravenous administration ranged from 1.1 to 13.2 days in hFcRn Tgm and from 1.2 to 30.3 days in WT mice. The half-lives in human correlated more closely with those in hFcRn Tgm than in WT mice and monkey. 4. Our results suggest that hFcRn Tgm are a valuable and useful tool for pharmacokinetic screening of mAbs and Fc-fusion proteins in the preclinical stage. Furthermore, we believe that hFcRn Tgm are broadly applicable to preclinical pharmacokinetic screening of mAbs-based therapeutics.

Comprehensive and temporal analysis of secreted proteins in the medium from IL-6 exposed human hepatocyte.

We have previously identified intracellular secretory acute phase response (sAPR) proteins in human hepatocytes following interleukin-6 (IL-6) induction by fluorogenic derivatization (FD)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS). In this report, we utilized this method, which uses 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as the FD reagent, to comprehensively and time-dependently analyze secreted proteins in the medium, including sAPR proteins. Since DAABD-Cl selectively reacts with thiol moieties of cysteinyl residues, direct derivatization, high-resolution LC separation and identification of the secreted proteins in the culture medium were successfully achieved without a pretreatment step. As a result, 14 sAPR proteins were identified simultaneously during a 72 h induction by IL-6. The secretion levels of 11 proteins increased, whereas the secretion levels of three important transport proteins decreased (albumin, retinol-binding protein 4 and transthyretin). In addition, the secretion level of a haptoglobin was found to increase significantly between 0 and 6 h by 1.88-fold compared with the control sample. The secretion levels of four cytoplasmic proteins increased: LDH, a known marker for cell damage, and GSTA1, FABP1 and ADH1B, which are marker proteins for hepatocellular damage. The secretion levels of the other two newly identified cytoplasmic proteins, profilin-1 and SOD2, were also found to increase, suggesting that these two proteins represent novel markers for cell damage. These results suggest that the FD-LC-MS/MS proteomics method can be used to analyze comprehensively and time-dependently the secreted proteins and thereby can offer information that aids our understanding of the dynamics of protein secretion affected by the exposure of cytokines such as IL-6.

In vitro-in vivo correlation of the inhibition potency of sodium-glucose cotransporter inhibitors in rat: a pharmacokinetic and pharmacodynamic modeling approach.

To evaluate the relationship between the in vitro and in vivo potency of sodium-glucose cotransporter (SGLT) inhibitors, a pharmacokinetic and pharmacodynamic (PK-PD) study was performed using normal rats. A highly selective SGLT2 inhibitor, tofogliflozin, and four other inhibitors with different in vitro inhibition potency to SGLT2 and selectivity toward SGLT2, versus SGLT1 were used as test compounds, and the time courses for urinary glucose excretion (UGE) and the plasma glucose and compound concentrations were monitored after administration of the compounds. A PK-PD analysis of the UGE caused by SGLT inhibition was performed on the basis of a nonlinear parallel tube model that took into consideration the consecutive reabsorption by different glucose transport properties of SGLT2 and SGLT1. The model adequately captured the time course of cumulative UGE caused by SGLT inhibition; then, the in vivo inhibition constants (Ki) of inhibitors for both SGLT1 and SGLT2 were estimated. The in vivo selectivity toward SGLT2 showed a good correlation with the in vitro data (r = 0.985; P < 0.05), with in vivo Ki values for SGLT2 in the range of 0.3-3.4-fold the in vitro data. This suggests that in vitro inhibition potency to both SGLT2 and SGLT1 is reflected in vivo. Furthermore, the complementary role of SGLT1 to SGLT2 and how selectivity toward SGLT2 affects the inhibitory potency for renal glucose reabsorption were discussed using the PK-PD model.

A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy.

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)2D3 had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)2D3. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D3 analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.

A new approach to predicting human hepatic clearance of CYP3A4 substrates using monkey pharmacokinetic data.

Focusing on the genetic similarity of CYP3A subfamily enzymes (CYP3A4 and CYP3A5) between monkeys and humans, we have attempted to provide a single-species approach to predicting human hepatic clearance (CLh) of CYP3A4 substrates using pharmacokinetic parameters in cynomolgus monkeys following intravenous administrations. 2. Hepatic intrinsic clearance (CLint,h) of six CYP3A4 substrates (alprazolam, clonazepam, diltiazem, midazolam, nifedipine, and quinidine), covering a wide range of clearance, in monkeys correlated well with that cited in literature for humans (R = 0.90) with a simple equation of Y = 0.165X (Y: human CLint,h, X: monkey CLint,h, represented in mL/min/kg). 3. To verify the predictability of human CLint,h, monkey CLint,h of a test set of CYP3A4 substrates cited in literature (dexamethasone, nifedipine, midazolam, quinidine, tacrolimus, and verapamil) was applied to the equation and human CLint,h was calculated. The human CLint,h of all the substrates was predicted within 3-fold error (fold error: 0.35-2.77). 4. The predictability of human CLh by our method was superior to common in vivo prediction methods (allometry and liver blood flow method). These results suggest that human hepatic clearance of CYP3A4 substrates can be predicted by applying cynomolgus monkey CLint,h obtained following intravenous administrations in each laboratory to the simple equation.

Alteration of intracellular secretory acute phase response proteins expressed in human hepatocyte induced by exposure with interleukin-6.

Interleukin-6 (IL-6) is a principal proinflammatory cytokine inducing the acute phase response in various tissues, including liver. Here, we adopt the FD-LC-MS/MS method, consisting of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification of proteins by LC-tandem mass spectrometry (MS/MS), to reveal how exposure to IL-6 alters temporally the intracellular secretory acute phase response (sAPR) proteins expressed in human hepatocytes as compared to non-exposure. Nine altered sAPR proteins were identified in cultures in response to IL-6. Seven of them (serum amyloid A protein, haptoglobin, fibrinogen α chain, fibrinogen ß chain, fibrinogen γ chain, α(1)-acid glycoprotein and α(1)-antitrypsin) were significantly increased and two (ß(2)-glycoprotein 1 and transferrin) were significantly decreased in response to IL-6. In addition, the transmission speed of transferrin might be much faster than the other sAPR proteins. These results suggest a different molecular mechanism for protein synthesis and the secretory pathway among the sAPR proteins. In this study, we observed the simultaneously and temporally altered expression of sAPR proteins which had been induced by exposure to IL-6 in human hepatocytes, in contrast to previous reports, in all of which the proteins were tested from the time they were secreted into the medium from the cells.

Pharmacokinetic and pharmacodynamic modeling of the effect of an sodium-glucose cotransporter inhibitor, phlorizin, on renal glucose transport in rats.

A pharmacokinetic and pharmacodynamic (PK-PD) model for the inhibitory effect of sodium-glucose cotransporter (SGLT) inhibitors on renal glucose reabsorption was developed to predict in vivo efficacy. First, using the relationship between renal glucose clearance and plasma glucose level in rats and both the glucose affinity and transport capacity obtained from in vitro vesicle experiments, a pharmacodynamic model analysis was performed based on a nonlinear parallel tube model to express the renal glucose transport mediated by SGLT1 and SGLT2. This model suitably expressed the relationship between plasma glucose level and renal glucose excretion. A PK-PD model was developed next to analyze the inhibitory effect of phlorizin on renal glucose reabsorption. The PK-PD model analysis was performed using averaged concentrations of both the drug and glucose in plasma and the corresponding renal glucose clearance. The model suitably expressed the concentration-dependent inhibitory effect of phlorizin on renal glucose reabsorption. The in vivo inhibition constants of phlorizin for SGLT in rats were estimated to be 67 nM for SGLT1 and 252 nM for SGLT2, which are similar to the in vitro data reported previously. This suggests that the in vivo efficacy of SGLT inhibitors could be predicted from an in vitro study based on the present PK-PD model. The present model is based on physiological and biochemical parameters and, therefore, would be helpful in understanding individual differences in the efficacy of an SGLT inhibitor.

Quantitative prediction of mechanism-based inhibition caused by mibefradil in rats.

It was previously demonstrated that mibefradil, which shows mechanism-based inhibition in humans, also caused drug-drug interactions (DDIs) with midazolam (MDZ) in rats. In this study, we aimed to quantitatively predict the DDIs observed in rats using a physiologically based pharmacokinetic (PBPK) model from in vitro inactivation parameters. For more precise predictions, contribution ratios of cytochrome P450 (P450) isozymes involved in MDZ metabolism and inactivation parameters of mibefradil against each isozyme were incorporated in the predictive model. The evaluation of metabolic rate using recombinant P450s suggested that CYP3A2 and CYP2C11 contributed to 89 and 11% of MDZ metabolism, respectively. Inactivation studies of mibefradil against the two isozymes showed that the maximal inactivation rate constants (k(inact)) were considerable in both isozymes (0.231-0.565 min(-1)), whereas the inhibitor concentration producing half the k(inact) (K(I, app)) of CYP3A2 (0.263-0.410 µM) was a good deal lower than that for CYP2C11 (6.82-11.4 µM). As a result of predicting the DDIs using the PBPK model, predicted increases in areas under the concentration-time curve of MDZ with coadministration of mibefradil (284 and 510% at 6 and 12 mg/kg mibefradil, respectively) closely corresponded to the observed values (226 and 545%, respectively). From those results, it was thought that the construction of a predictive model for DDIs using the PBPK model in detail would enable us to quantitatively predict in vivo DDIs from in vitro data. This approach to predict DDIs on the basis of the contributing isozymes would be important for predicting clinical DDIs of drugs metabolized by multiple enzymes.

Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis.

Novel vitamin D(3) analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked 1α-hydroxy group and/or 3ß-hydroxy group of 1α,25-dihydroxyvitamin D(3). A highly potent analog, 6a, with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound 6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.

Antigen-dependent internalization is related to rapid elimination from plasma of humanized anti-HM1.24 monoclonal antibody.

Anti-HM1.24 monoclonal antibody (AHM) is a humanized anti-HM1.24 monoclonal antibody that binds to the HM1.24 antigen, a protein that is highly expressed in multiple myeloma cells. The pharmacokinetics of AHM was determined in experiments in which AHM was administered intravenously to cynomolgus monkeys. The area under the plasma concentration-time curve increased by more than the dose ratio between 2 and 20 mg/kg, and nonlinear pharmacokinetics was observed. The elimination half-life of AHM from the plasma was 7.56 h at 2 mg/kg and 28.6 h at 20 mg/kg, which was shorter than that observed for other therapeutic humanized monoclonal antibodies, such as trastuzumab and bevacizumab. Although antibodies to AHM were detected in all monkeys on or after 10 days of administration, there was a temporal disassociation between the rapid elimination of AHM and the appearance of anti-AHM antibodies. HM1.24 antigen-dependent internalization and intracellular metabolism of AHM were investigated in peripheral blood mononuclear, KPMM2, and U937 cells. In all cases, AHM was rapidly internalized from the cell surface; this internalization was significantly prevented by phenylarsine oxide in KPMM2 cells, an inhibitor of receptor-mediated endocytosis, and the internalized AHM was subsequently degraded within the cells. Furthermore, immunofluorescence microscopy revealed that the internalized AHM is delivered to and degraded in late endosomes/lysosomes. Taken together, our results suggest that the rapid elimination of AHM from plasma in monkey is due to HM1.24 antigen-dependent internalization followed by delivery to the lysosomes.

Risk of CYP2C9 induction analyzed by a relative factor approach with human hepatocytes.

By using the Relative Factor (RF) method-a method that can simply assess cytochrome P450 (CYP) induction risk based on a maximum induction effect model-we evaluated the risk of CYP2C9 induction and examined its relationship with risk of CYP3A4 induction. In cryopreserved human hepatocytes, the magnitude of CYP2C9 induction by eight drugs known to induce CYP3A4 was lower than the magnitude of CYP3A4 induction, but the magnitudes of induction of both were correlated. The RF values determined for CYP2C9 had a one-to-one linear relationship with values determined for CYP3A4, supporting reports that the induction mechanism of both enzymes is the same. Furthermore, clinical CYP2C9 induction data of compounds reported to induce CYP2C9 clinically were shown to be lower than those of CYP3A4. The thresholds for CYP2C9 induction risk assessment by the RF approach were determined to be at higher steady-state plasma concentrations than those for CYP3A4. Based on these results, induction of CYP2C9 was correlated with that of CYP3A4, and induction risk could be evaluated by the RF method using hepatocytes. The CYP2C9 induction risk of a compound was confirmed to be lower than its CYP3A4 induction risk.