Enzyme-mediated drug-drug interactions: a review of in vivo and in vitro methodologies, regulatory guidance, and translation to the clinic.

Enzyme-mediated pharmacokinetic drug-drug interactions can be caused by altered activity of drug metabolizing enzymes in the presence of a perpetrator drug, mostly via inhibition or induction. We identified a gap in the literature for a state-of-the art detailed overview assessing this type of DDI risk in the context of drug development. This manuscript discusses in vitro and in vivo methodologies employed during the drug discovery and development process to predict clinical enzyme-mediated DDIs, including the determination of clearance pathways, metabolic enzyme contribution, and the mechanisms and kinetics of enzyme inhibition and induction. We discuss regulatory guidance and highlight the utility of in silico physiologically-based pharmacokinetic modeling, an approach that continues to gain application and traction in support of regulatory filings. Looking to the future, we consider DDI risk assessment for targeted protein degraders, an emerging small molecule modality, which does not have recommended guidelines for DDI evaluation. Our goal in writing this report was to provide early-career researchers with a comprehensive view of the enzyme-mediated pharmacokinetic DDI landscape to aid their drug development efforts.

Effect of lemborexant on pharmacokinetics of clozapine: A potential drug-drug interaction mediated by time-dependent inhibition of CYP3A4.

Clozapine (CLZ) is extensively used for treatment-resistant schizophrenia (TRS) with caution to avoid serious adverse events such as agranulocytosis and drug-drug interactions (DDIs). In the current report, we present a case of a 35-year-old male non-smoking TRS patient whose steady-state plasma trough concentrations (Ctrough ) of CLZ and its active metabolite, N-desmethylclozapine (NDMC), were significantly increased after initiating oral administration of lemborexant (LEM), a dual orexin receptor antagonist, for the treatment of insomnia. The patient experienced oversedation with sleepiness and fatigue while maintaining high levels of Ctrough of CLZ. The increased concentrations of CLZ returned to normal ranges after the discontinuation of LEM dosing, implying a pharmacokinetic DDI between CLZ and LEM. To gain insight into possible mechanisms, we performed in vitro assays of CYP1A2- and CYP3A4-mediated CLZ metabolism by measuring the formations of NDMC and clozapine N-oxide (CNO). In accordance with previous studies, the incubation of CLZ with each enzyme resulted in the production of both metabolites. LEM had only a weak inhibitory effect on CYP1A2- and CYP3A4-mediated CLZ metabolism. However, the preincubation of LEM with CYP3A4 in the presence of NADPH showed a significant enhancement of inhibitory effects on CLZ metabolism with IC50 values for the formations of CNO and NDMC of 2.8 µM and 4.1 µM, respectively, suggesting that LEM exerts as a potent time-dependent inhibitor for CYP3A4. Taken together, the results of the current study indicate that co-medication of CLZ with LEM may lead to increase in exposure to CLZ and risks of CLZ-related adverse events.

Preparation of antibacterial Zn and Ni substituted cobalt ferrite nanoparticles for efficient biofilm eradication.

Zinc (Zn) and, alternatively, nickel (Ni) substituted cobalt ferrite (CF) nanoparticles (NPs) were prepared by sol-gel method. X-ray diffraction analysis revealed the formation of cubic structure of cobalt ferrite. FTIR analysis confirmed the vibrational band located at 550-580 cm-1 that belongs to the M - O bond (M = Ni, and Zn). The alteration of the surface morphology of CF after the addition of Zn and Ni ions was observed from scanning electron microscopic images. The additional peaks in the energy dispersive X-ray diffraction (EDX) analysis spectra were found to correspond to Zn and Ni. The presence of Zn and, alternatively, Ni ions enhanced the biocidal properties of CF NPs against gram negative organisms, in a concentration and time-dependent manner. Furthermore, exposure to CF, CF-Zn and CF-Ni NPs decreased metabolic activity due to the damage of extra polymorphic substances, live/dead cell variation, architecture and surface integrity of the cells. Altogether, the present investigation provides the basis of metal ion substituted metal oxide NPs as anti-biofilm agents against gram-positive and gram-negative bacteria.

Kinetic Modeling of Time-Dependent Enzyme Inhibition by Pre-Steady-State Analysis of Progress Curves: The Case Study of the Anti-Alzheimer's Drug Galantamine.

The Michaelis-Menten model of enzyme kinetic assumes the free ligand approximation, the steady-state approximation and the rapid equilibrium approximation. Analytical methods to model slow-binding inhibitors by the analysis of initial velocities have been developed but, due to their inherent complexity, they are seldom employed. In order to circumvent the complications that arise from the violation of the rapid equilibrium assumption, inhibition is commonly evaluated by pre-incubating the enzyme and the inhibitors so that, even for slow inhibitors, the binding equilibrium is established before the reaction is started. Here, we show that for long drug-target residence time inhibitors, the conventional analysis of initial velocities by the linear regression of double-reciprocal plots fails to provide a correct description of the inhibition mechanism. As a case study, the inhibition of acetylcholinesterase by galantamine, a drug approved for the symptomatic treatment of Alzheimer's disease, is reported. For over 50 years, analysis based on the conventional steady-state model has overlooked the time-dependent nature of galantamine inhibition, leading to an erroneous assessment of the drug potency and, hence, to discrepancies between biochemical data and the pharmacological evidence. Re-examination of acetylcholinesterase inhibition by pre-steady state analysis of the reaction progress curves showed that the potency of galantamine has indeed been underestimated by a factor of ~100.

4-O-galloylalbiflorin inhibits the activity of CYP3A, 2C9, and 2D in human liver microsomes.

The effect of 4-O-galloylalbiflorin on the activity of cytochrome P450 enzymes (CYP450s) is an important factor that may induce drug-drug interaction.The effect of 4-O-galloylalbiflorin on the activity of CYP450s was evaluated in the presence of 0, 2.5, 5, 10, 25, 50, and 100 µM 4-O-galloylalbiflorin in pooled human liver microsomes. The inhibition model and corresponding parameters were assessed b fitting with Lineweaver-Burk plots. The time-dependent study was performed with the incubation time of 0, 5, 10, 15, and 30 min.4-O-galloylalbiflorin significantly inhibited the activity of CYP3A, 2C9, and 2 D in a concentration-dependent manner with the IC50 values of 8.2, 13, and 11 µM, respectively. The inhibition of CYP3A was found to be non-competitive and time-dependent with the Ki value of 4.0 µM and the KI/Kinact value of 2.2/0.030 (µM·min). The inhibition of CYP2C9 and 2 D was not affected by the incubation time but was found to be competitive with the Ki values of 6.7 and 6.6 µM, respectively.The inhibitory effect of 4-O-galloylalbiflorin on the activity of CYP3A, 2C9, and 2 D implying the potential drug-drug interaction between 4-O-galloylalbiflorin and the drugs metabolized by these CYP450s.

Inhibition, crystal structures, and in-solution oligomeric structure of aldehyde dehydrogenase 9A1.

Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD+-dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition. Two crystal structures of ALDH9A1 are reported, including the first of the enzyme complexed with NAD+. One of the structures reveals the active conformation of the enzyme, in which the Rossmann dinucleotide-binding domain is fully ordered and the inter-domain linker adopts the canonical ß-hairpin observed in other ALDH structures. The oligomeric structure of ALDH9A1 was investigated using analytical ultracentrifugation, small-angle X-ray scattering, and negative stain electron microscopy. These data show that ALDH9A1 forms the classic ALDH superfamily dimer-of-dimers tetramer in solution. Our results suggest that the presence of an aldehyde substrate and NAD+ promotes isomerization of the enzyme into the active conformation.

Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting.

Time-dependent inhibition (TDI) may confound drug interaction predictions. Recently, models were generated for an array of TDI kinetic schemes using numerical analysis of microsomal assays. Additionally, a distinct terminal inactivation step was identified for certain mechanism based inhibitors (MBI) following reversible metabolite intermediate complex (MIC) formation. Longer hepatocyte incubations potentially allow analysis of slow TDI and terminal inactivation. In the experiments presented here, we compared the quality of TDI parameterization by numerical analysis between hepatocyte and microsomal data. Rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and sandwich-cultured rat hepatocytes (SCRH) were incubated with the prototypical CYP3A MBI troleandomycin and the substrate midazolam. Data from RLM provided a better model fit as compared to SRH. Increased CYP3A expression after dexamethasone (DEX) induction improved the fit for RLM and SRH. A novel sequential kinetic scheme, defining inhibitor metabolite production prior to MIC formation, improved the fit compared to direct MIC formation. Furthermore, terminal inactivation rate constants were parameterized for RLM and SRH samples with DEX-induced CYP3A. The low expression of CYP3A and experimental error in SCRH resulted in poor data for model fitting. Overall, RLM generated data better suited for elucidation of TDI mechanisms by numerical analysis.

Cytochrome P450-Mediated Metabolic Characterization of a Mono-Carbonyl Curcumin Analog WZ35.

WZ35 is a monocarbonyl analog of curcumin, which had been proved advantage over curcumin in chemical stability and antitumor activity. However, its pharmacokinetic profile has not been determined. In the present study, an ultraperformance liquid chromatography-tandem mass spectrometry assay was developed to detect concentration of WZ35 in rat plasma. Subsequently, pharmacokinetic study showed that the oral bioavailability of WZ35 is 10.56%. Cytochrome P450 (CYP450) plays a major role in metabolizing exogenous substance. The concentration of WZ35 was sharply decreased while incubating with microsome. It's indicated that WZ35 is a substrate of CYP450s. Molecular docking assay showed that WZ35 can combine with CYP2B6 and CYP2C9 to form much more stable complex. The lowest docking energy was generated in complex with CYP2E1. The inhibition of CYP450s by WZ35 was also evaluated. Pan inhibitions of WZ35 on rat CYP3A2, CYP2B1, CYP2C11, CYP2D1, and -CYP2E1 were observed by detecting probe substrates (midazolam, bupropion, tolbutamide, dextromethorphan, chlorzoxazone) and metabolites accordingly. On an average, 80% activities of enzymes were blocked. Mechanistically, the inhibitions of WZ35 on CYP3A2, CYP2B1, CYP2E1 were in a time-dependent manner according to the results of IC50 shift assay. The collective data demonstrated that the oral bioavailability of monocarbonyl analog of curcumin has significantly improved compared to curcumin. It's both the substrate and inhibitor of CYP450s through in a time-dependent mechanism.

In vitro and in vivo methods to assess pharmacokinetic drug- drug interactions in drug discovery and development.

Drug-drug interactions (DDIs) caused by the co-administration of multiple drugs are major safety concerns in the clinic. Several drugs have been withdrawn from the market due to perpetrator or victim DDIs. Strategies have been developed to assess DDI risks early in drug discovery to reduce DDI liabilities. High-to-medium throughput assays are available to identify undesirable scaffolds and to guide structural modifications to minimize DDIs. Definitive methods are used at later stages of drug discovery and development to provide a more accurate measurement of DDI parameters and to enable clinical translations. Physiologically based pharmacokinetic modeling and simulations are powerful tools to accurately predict DDIs and to assess risks in the clinic. Although significant advances have been made over the years, many challenges remain for clinical DDI translations. This includes DDIs involving non-cytochrome P450 enzymes, transporters, enzyme-transporter interplay, indirect effects from biologics, and pharmacodynamic based DDI. This review focuses on methods that are used to assess hepatic DDIs caused by enzyme inhibition and induction.

Staphylococcus aureus evasion proteins EapH1 and EapH2: Residue-level investigation of an alternative binding motif for human neutrophil elastase.

The Staphylococcus aureusExtracellular Adherence Protein (Eap) and its homologs, EapH1 and EapH2, are a family of secreted proteins that potently inhibit the neutrophil serine proteases Neutrophil Elastase (hNE), Cathepsin G, and Proteinase 3. Similarly to EapH1, inhibition of hNE by EapH2 is characterized by a rapid association rate (2.9 × 105 M-1s-1) coupled with a very slow dissociation rate (5.9 × 10-4 s-1), yielding an apparent inhibition constant of 2.11 nM. As with EapH1, inhibition of hNE by EapH2 is also time-dependent in character. A phenylalanine in EapH2 replaces the leucine in EapH1 that sits over the hNE catalytic serine and creates a potential steric clash. Indeed, the EapH1 L59F mutant is severely decreased in its ability to inhibit hNE (~9500-fold). When compared to the EapH1:hNE co-crystal structure, a model of the EapH2:hNE complex predicts an alternative binding motif comprised of EapH2 residues 120-127. These putative interfacing residues were individually mutated and kinetically interrogated. The EapH2 N127A mutant resulted in the largest decrease in hNE inhibition (~200-fold) and loss of the time-dependent characteristic. Surprisingly, the time-dependent characteristic was still abolished in the EapH2 T125A mutant, even though it was less perturbed in hNE inhibition (~25-fold). T125 forms an intra-molecular hydrogen bond to the carbonyl oxygen of N127 in the EapH2 crystal structure. Given these observations, we conclude (i) that EapH2 has an altogether distinct hNE binding motif than EapH1, (ii) that N127 is the main functional determinant in EapH2, and (iii) that T125 serves an ancillary role aiding in the optimal orientation of N127.

Synthesis and structure-activity relationship of 4-alkoxy-thieno[2,3-b]pyridine derivatives as potent alkaline phosphatase enhancers for osteoporosis treatment.

The synthesis and structure-activity relationships of a novel series of 3-aminothieno[2,3-b]pyridine-2-carboxamides were explored. Our efforts were focused on modifying the C-4 substituent of the thienopyridine ring to develop orally available bone anabolic agents. 4-Alkoxy derivatives were found to be novel ALPase enhancers without inhibitory effect on P450 activity. Among these derivatives, compound 6k was orally administered to ovariectomized rats, and it was found to significantly improve areal bone mineral density at a dose of 30 mg/kg/day.

Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions.

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. PKCθ inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34 exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant model.

Time-dependent inhibition (TDI) of CYP1A2 by a CYP3A4-mediated reactive metabolite: proposal for a novel mechanism of irreversible TDI by a non-suicide substrate.

1. The purpose of this study was to clarify the mechanism of DSP-1053 time-dependent inhibition (TDI) for CYP1A2. 2. DSP-1053 inhibited time- and concentration-dependently CYP1A2 activity in human liver microsomes even in a dilution assay. However, DSP-1053 was not metabolized by recombinant human CYP1A2. These findings indicate that the inhibitory effect of DSP-1053 on CYP1A2 does not follow a general mechanism-based inhibition (MBI) because it did not seem to be a suicide substrate. 3. In fact, CYP1A2 was not inhibited with DSP-1053 pre-incubation in recombinant human CYP1A2. On the other hand, CYP1A2 was potently inhibited after pre-incubation with DSP-1053 in a mixture of human recombinant CYP1A2 and CYP3A4. In addition, DSP-1053 TDI of CYP1A2 in human liver microsomes was drastically reduced not only by addition of a CYP3A4 inhibitor, but also by addition of potassium cyanide (KCN), which is a trapping agent for iminium ions. We also confirmed in this study that CYP1A2 suicide inhibition by DSP-1053 metabolites generated by CYP3A4 had only minimal role in DSP-1053 TDI of CYP1A2. 4. In conclusion, a possible mechanism for DSP-1053 TDI of CYP1A2 is that DSP-1053 iminium ion, which is generated by CYP3A4, departs from CYP3A4 without inhibiting it and covalently binds to CYP1A2.

Methoxsalen as an in vitro phenotyping tool in comparison with 1-aminobenzotriazole.

The objective is to evaluate methoxsalen as an in vitro phenotyping tool in comparison to ABT as a nonspecific inactivator of P450 mediated metabolism. The reversible inhibition of methoxsalen and ABT against the P450, FMO, AO, MAO-A and -B, enzymes were evaluated using standard marker probe reactions. The time-dependent inhibition of P450 enzymes was evaluated in human liver microsomes. CES1 activities were determined by monitoring the depletion of known substrate, the clopidogrel. The metabolism of P450 substrates in the presence and absence of methoxsalen or ABT was evaluated in human liver microsomes. Methoxsalen is a direct inhibitor and inhibited the activities (>90%) of all enzymes at a concentration of 300 µM except for CYP2C9. Methoxsalen is also a potent time-dependent inhibitor of all P450 enzymes except for CYP2C19 (moderate) at a concentration of 300 µM. Methoxsalen inhibited the metabolism of P450 substrates in the pre-incubation mode. ABT is a potent TDI of several P450 except for CYP2C19 (47%) and CYP2C9 (27%). The results indicate that methoxsalen is a potent pan P450 inhibitor than ABT and can be a better tool in distinguishing P450 mediated metabolism form non-P450 metabolism in human liver microsomes.

Coumarins and P450s, Studies Reported to-Date.

Cytochrome P450 enzymes (CYPs) are important phase I enzymes involved in the metabolism of endogenous and xenobiotic compounds mainly through mono-oxygenation reactions into more polar and easier to excrete species. In addition to their role in detoxification, they play important roles in the biosynthesis of endogenous compounds and the bioactivation of xenobiotics. Coumarins, phytochemicals abundant in food and commonly used in fragrances and cosmetics, have been shown to interact with P450 enzymes as substrates and/or inhibitors. In this review, these interactions and their significance in pharmacology and toxicology are discussed in detail.

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone.

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M-1 s-1. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.

Improved Predictions of Drug-Drug Interactions Mediated by Time-Dependent Inhibition of CYP3A.

Time-dependent inactivation (TDI) of cytochrome P450s (CYPs) is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to overpredict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human-liver microsomes. The inhibitors evaluated included troleandomycin (TAO), erythromycin (ERY), verapamil (VER), and diltiazem (DTZ) along with the primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (NDD). The complexities incorporated into the models included multiple-binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The resulting inactivation parameters were incorporated into static in vitro-in vivo correlation (IVIVC) models to predict clinical DDIs. For 77 clinically observed DDIs, with a hepatic-CYP3A-synthesis-rate constant of 0.000 146 min-1, the average fold difference between the observed and predicted DDIs was 3.17 for the standard replot method and 1.45 for the numerical method. Similar results were obtained using a synthesis-rate constant of 0.000 32 min-1. These results suggest that numerical methods can successfully model complex in vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach.

A strategy for early-risk predictions of clinical drug-drug interactions involving the GastroPlusTM DDI module for time-dependent CYP inhibitors.

1. A set of reference compounds for time-dependent inhibition (TDI) of cytochrome P450 with available literature data for kinact and KI was used to predict clinical implications using the GastroPlusTM software. Comparisons were made to in vivo literature interaction data. 2. The predicted AUC ratios (AUC+inhibitor/AUCcontrol) could be compared with the observed ratios from literature for all compounds with detailed information about in vivo administration, pharmacokinetics and in vivo interactions (N = 21). For this dataset, the difference between predicted and observed AUC ratios for interactions with midazolam was within twofold for all compounds except one (telaprevir, for which non-CYP-mediated metabolism likely plays a role after multiple dosing). 3. The sensitivity, specificity and accuracy of the GastroPlusTM predictions using a binary classification as no-to-weak interaction versus moderate-to-strong interaction for all compounds with available in vivo interaction data, were 80%, 82% and 81%, respectively (N = 31). 4. As a result of our evaluations of the DDI module in GastroPlusTM, we have implemented an early TDI risk assessment decision tree for our drug discovery projects involving in vitro screening and early GastroPlusTM predictions. Shifted IC50 values are determined and kinact/KI estimated (by using a regression line established with in house-shifted IC50 values and literature kinact/KI ratios), followed by GastroPlusTM predictions.

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.

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human.

1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug-drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment. 2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC50 values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH. 3. In the absence of preincubation, the IC50 values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC50 were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem). 4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.