Identification of reversible OATP1B1 and time-dependent CYP3A4 inhibition as the major risk factors for drug-induced cholestasis (DIC).

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

Improving In Vitro-In Vivo Extrapolation of Clearance Using Rat Liver Microsomes for Highly Plasma Protein-Bound Molecules.

It is common practice in drug discovery and development to predict in vivo hepatic clearance from in vitro incubations with liver microsomes or hepatocytes using the well-stirred model (WSM). When applying the WSM to a set of approximately 3000 Novartis research compounds, 73% of neutral and basic compounds (extended clearance classification system [ECCS] class 2) were well-predicted within 3-fold. In contrast, only 44% (ECCS class 1A) or 34% (ECCS class 1B) of acids were predicted within 3-fold. To explore the hypothesis whether the higher degree of plasma protein binding for acids contributes to the in vitro-in vivo correlation (IVIVC) disconnect, 68 proprietary compounds were incubated with rat liver microsomes in the presence and absence of 5% plasma. A minor impact of plasma on clearance IVIVC was found for moderately bound compounds (fraction unbound in plasma [fup] ≥1%). However, addition of plasma significantly improved the IVIVC for highly bound compounds (fup <1%) as indicated by an increase of the average fold error from 0.10 to 0.36. Correlating fup with the scaled unbound intrinsic clearance ratio in the presence or absence of plasma allowed the establishment of an empirical, nonlinear correction equation that depends on fup Taken together, estimation of the metabolic clearance of highly bound compounds was enhanced by the addition of plasma to microsomal incubations. For standard incubations in buffer only, application of an empirical correction provided improved clearance predictions. SIGNIFICANCE STATEMENT: Application of the well-stirred liver model for clearance in vitro-in vivo extrapolation (IVIVE) in rat generally underpredicts the clearance of acids and the strong protein binding of acids is suspected to be one responsible factor. Unbound intrinsic in vitro clearance (CLint,u) determinations using rat liver microsomes supplemented with 5% plasma resulted in an improved IVIVE. An empirical equation was derived that can be applied to correct CLint,u-values in dependance of fraction unbound in plasma (fup) and measured CLint in buffer.

Use of Pharmacokinetic and Pharmacodynamic Data to Develop the CDK4/6 Inhibitor Ribociclib for Patients with Advanced Breast Cancer.

Ribociclib is an orally bioavailable, selective cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor. CDK4/6 inhibition by ribociclib leads to retinoblastoma tumor suppressor protein (Rb) reactivation, thereby restoring Rb-mediated cell cycle arrest. Ribociclib is approved for the treatment of patients with hormone receptor-positive/human epidermal growth factor receptor-2-negative (HR+/HER2-) advanced breast cancer (ABC), at the dose of 600 mg once daily (QD) during cycles of 21 days on/7 days off, with optional dose reduction to 400 mg and 200 mg. Ribociclib is rapidly absorbed with a median time to reach maximum plasma concentration of 2.4 h, mean half-life of 32.0 h and oral bioavailability of 65.8% at 600 mg. It is eliminated mainly by hepatic metabolism (~ 84% of total elimination), mostly by cytochrome P450 (CYP) 3A4. Age, body weight, race, baseline Eastern Cooperative Oncology Group status, food, mild hepatic impairment, mild-to-moderate renal impairment, proton pump inhibitors, and combination partners (non-steroidal aromatase inhibitors or fulvestrant) have no clinically relevant impact on ribociclib exposure. Ribociclib inhibits CYP3A at 600 mg leading to increased exposure of CYP3A substrates. Strong CYP3A inhibitors or inducers increase or decrease, respectively, ribociclib exposure. Exposure-safety and exposure-efficacy analyses support the clinical benefit of the 600 mg QD starting dose, with potential individualized dose reductions to 400 mg and 200 mg for effective management of the adverse events neutropenia and QTcF interval prolongation, while maintaining efficacy, in patients with HR+/HER2- ABC. Overall, these clinical pharmacology data informed ribociclib dose justification and clinical development, as well as its prescribing information for clinical use in advanced breast cancer patients.

Utilization of OATP1B Biomarker Coproporphyrin-I to Guide Drug-Drug Interaction Risk Assessment: Evaluation by the Pharmaceutical Industry.

Drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptides 1B1/1B3 (OATP1B) can be substantial, however, challenges remain for predicting interaction risk. Emerging evidence suggests that endogenous biomarkers, particularly coproporphyrin-I (CP-I), can be used to assess in vivo OATP1B activity. The present work under the International Consortium for Innovation and Quality in Pharmaceutical Development was aimed primarily at assessing CP-I as a biomarker for informing OATP1B DDI risk. Literature and unpublished CP-I data along with pertinent in vitro and clinical DDI information were collected to identify DDIs primarily involving OATP1B inhibition and assess the relationship between OATP1B substrate drug and CP-I exposure changes. Static models to predict changes in exposure of CP-I, as a selective OATP1B substrate, were also evaluated. Significant correlations were observed between CP-I area under the curve ratio (AUCR) or maximum concentration ratio (Cmax R) and AUCR of substrate drugs. In general, the CP-I Cmax R was equal to or greater than the CP-I AUCR. CP-I Cmax R < 1.25 was associated with absence of OATP1B-mediated DDIs (AUCR < 1.25) with no false negative predictions. CP-I Cmax R < 2 was associated with weak OATP1B-mediated DDIs (AUCR < 2). A correlation was identified between CP-I exposure changes and OATP1B1 static DDI predictions. Recommendations for collecting and interpreting CP-I data are discussed, including a decision tree for guiding DDI risk assessment. In conclusion, measurement of CP-I is recommended to inform OATP1B inhibition potential. The current analysis identified changes in CP-I exposure that may be used to prioritize, delay, or replace clinical DDI studies.

Interlaboratory Variability in the Madin-Darby Canine Kidney Cell Proteome.

Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

Novel insights into bile acid detoxification via CYP, UGT and SULT enzymes.

Bile acid (BA) homeostasis is a complex and precisely regulated process to prevent impaired BA flow and the development of cholestasis. Several reactions, namely hydroxylation, glucuronidation and sulfation are involved in BA detoxification. In the present study, we employed a comprehensive approach to identify the key enzymes involved in BA metabolism using human recombinant enzymes, human liver microsomes (HLM) and human liver cytosol (HLC). We showed that CYP3A4 was a crucial step for the metabolism of several BAs and their taurine and glycine conjugated forms and quantitatively described their metabolites. Glucuronidation and sulfation were also identified as important drivers of the BA detoxification process in humans. Moreover, lithocholic acid (LCA), the most hydrophobic BA with the highest toxicity potential, was a substrate for all investigated processes, demonstrating the importance of hepatic metabolism for its clearance. Collectively, this study identified CYP3A4, UGT1A3, UGT2B7 and SULT2A1 as the major contributing (metabolic) processes in the BA detoxification network. Inhibition of these enzymes by drug candidates is therefore considered as a critical mechanism in the manifestation of drug-induced cholestasis in humans and should be addressed during the pre-clinical development.

Time matters - in vitro cellular disposition kinetics help rationalizing cellular potency disconnects.

Loss in potency is commonly observed in early drug discovery when moving from biochemical to more complex cellular systems. Among other factors, low permeability is often considered to cause such potency disconnects.We developed a novel cellular disposition assay in MDCK cells to determine passive uptake clearance (PSinf), cell-to-medium ratios at steady-state (Kp) and the time to reach 90% steady-state (TTSS90) from a single experiment in a high-throughput format.The assay was validated using 40 marketed drugs, showing a wide distribution of PSinf and Kp values. The parameters generally correlated with transcellular permeability and lipophilicity, while PSinf data revealed better resolution in the high and low permeability ranges compared to traditional permeability data. A linear relationship between the Kp/PSinf ratio and TTSS90 was mathematically derived and experimentally validated, demonstrating the dependency of TTSS90 on the rate and extent of cellular accumulation.Cellular disposition parameters could explain potency (IC50) disconnects noted for seven Bruton's tyrosine kinase degrader compounds in a cellular potency assay. In contrast to transcellular permeability, PSinf data enabled identification of the compounds with IC50 disconnects based on their time to reach equilibrium. Overall, the novel assay offers the possibility to address potency disconnects in early drug discovery.

Pharmacokinetics of asciminib in the presence of CYP3A or P-gp inhibitors, CYP3A inducers, and acid-reducing agents.

Asciminib is a first-in-class inhibitor of BCR::ABL1, specifically targeting the ABL myristoyl pocket. Asciminib is a substrate of CYP3A4 and P-glycoprotein (P-gp) and possesses pH-dependent solubility in aqueous solution. This report summarizes the results of two phase I studies in healthy subjects aimed at assessing the impact of CYP3A and P-gp inhibitors, CYP3A inducers and acid-reducing agents (ARAs) on the pharmacokinetics (PK) of asciminib (single dose of 40 mg). Asciminib exposure (area under the curve [AUC]) unexpectedly decreased by ~40% when administered concomitantly with the strong CYP3A inhibitor itraconazole oral solution, whereas maximum plasma concentration (Cmax ) decreased by ~50%. However, asciminib exposure was slightly increased in subjects receiving an itraconazole capsule (~3%) or clarithromycin (~35%), another strong CYP3A inhibitor. Macroflux studies showed that cyclodextrin (present in high quantities as excipient [40-fold excess to itraconazole] in the oral solution formulation of itraconazole) decreased asciminib flux through a lipid membrane by ~80%. The AUC of asciminib was marginally decreased by concomitant administration with the strong CYP3A inducer rifampicin (by ~13-15%) and the strong P-gp inhibitor quinidine (by ~13-16%). Concomitant administration of the ARA rabeprazole had little or no effect on asciminib AUC, with a 9% decrease in Cmax . The treatments were generally well tolerated. Taking into account the large therapeutic window of asciminib, the observed changes in asciminib PK following multiple doses of P-gp, CYP3A inhibitors, CYP3A inducers, or ARAs are not considered to be clinically meaningful. Care should be exercised when administering asciminib concomitantly with cyclodextrin-containing drug formulations.

Novel Bruton's tyrosine kinase inhibitor remibrutinib: Assessment of drug-drug interaction potential as a perpetrator of cytochrome P450 enzymes and drug transporters and the impact of covalent binding on possible drug interactions.

PURPOSE: Pharmacokinetic drug-drug interactions (DDIs) are investigated to ensure safety for patients receiving concomitant medications. Here, we present a strategy to characterise the DDI potential of remibrutinib, as an inhibitor of drug-metabolising enzymes and drug transporters, and as an inducer. Initial in vitro studies were performed, followed by a biomarker-based assessment of induction in a first in human study, concluded by a clinical study to verify initial results. Remibrutinib is a covalent inhibitor of Bruton's Tyrosine kinase (BTKi) carrying a reactive acrylamide moiety (warhead), thus the potential contribution of covalent binding (off-target) to observed interactions was investigated as this could lead to prolonged and more potent drug interactions. METHODS: DDI assessment was focused on the putative inhibition of key metabolic enzymes (Cytochrome P450, CYP), drug transporters and a potential effect on oral contraceptives (OC) by induction of enzymes that are involved in their clearance (CYP3A4). The impact of covalent binding was assessed by synthesising an identical reference molecule but with an inactivated warhead. RESULTS: An interaction potential of limited clinical relevance was revealed for remibrutinib for CYP enzymes and drug transporters. The reactive warhead of remibrutinib had no impact on CYP enzyme and transporter inhibition, including time-dependent inhibition of CYP3A4, but may increase the induction potential of remibrutinib. CONCLUSIONS: Observed inhibition of metabolic enzymes indicated that remibrutinib is a weak inhibitor of CYP3A4 and CYP2C9 and is not a clinically relevant inhibitor of uptake and efflux transporters, except for intestinal P-glycoprotein and breast cancer resistance protein inhibition. OC may be safely administered and are effective when given with pharmacologically relevant doses of remibrutinib.

A kinase inhibitor which specifically targets the ABL myristate pocket (STAMP), but unlike asciminib crosses the blood-brain barrier.

The ubiquitously expressed ABL1 and ABL2 protein kinases play many important roles in cell function. Although they have been implicated in neuron development, maintenance and signaling, there are no good tool compounds to evaluate the effects of ABL kinase inhibition in the brain. Asciminib is a recently approved drug that specifically and potently inhibits the tyrosine kinase activity of ABL1, ABL2 and that of the chimeric BCR-ABL1 oncoprotein which causes chronic myeloid leukemia. Herein we show that asciminib does not penetrate the intact blood-brain barrier (BBB) following administration to rats, which curtails its utility for assessing the in vivo effects of ABL kinase inhibition in the brain. However, we describe another specific ABL kinase inhibitor, possessing physicochemical characteristics suitable for BBB penetration, and which after administration (either i.v., i.p. or p.o.) to mice achieves substantial, pharmacologically relevant brain concentrations. This bipyridine compound (4) therefore has potential for elucidating the role of ABL kinases in the brain in non-clinical studies.

Pharmacokinetics of Asciminib When Taken With Imatinib or With Food.

Asciminib, a first-in-class, Specifically Targeting the Abelson kinase Myristoyl Pocket (STAMP) inhibitor with the potential to overcome resistance to adenosine triphosphate-competitive tyrosine kinase inhibitors, is being investigated in leukemia as monotherapy and in combination with tyrosine kinase inhibitors including imatinib. This phase 1 study in healthy volunteers assessed the pharmacokinetics of asciminib (40 mg single dose) under 2 conditions: when taken with imatinib (steady state; 400 mg once daily) and a low-fat meal (according to imatinib prescription information), or when taken as single-agent under different food conditions. Asciminib plus imatinib with a low-fat meal increased asciminib area under the plasma concentration-time curve from time 0 to infinity and maximum plasma concentration (geometric mean ratios [90% confidence interval], 2.08 [1.93-2.24] and 1.59 [1.45-1.75], respectively) compared with asciminib alone under the same food conditions. Asciminib plus food decreased asciminib area under the plasma concentration-time curve from time 0 to infinity compared with asciminib taken under fasted conditions (geometric mean ratios: low-fat meal, 0.7 [0.631-0.776]; high-fat meal, 0.377 [0.341-0.417]). Asciminib plus imatinib was well tolerated with no new safety signals. Overall, coadministration of asciminib with imatinib and a low-fat meal results in a moderate increase in asciminib exposure compared with asciminib alone under the same food condition. Food itself decreases asciminib exposure, indicating that single-agent asciminib should be administered in the fasted state to prevent potential suboptimal exposures.

Novel Bruton's Tyrosine Kinase inhibitor remibrutinib: Drug-drug interaction potential as a victim of CYP3A4 inhibitors based on clinical data and PBPK modeling.

Remibrutinib, a novel oral Bruton's Tyrosine Kinase inhibitor (BTKi) is highly selective for BTK, potentially mitigating the side effects of other BTKis. Enzyme phenotyping identified CYP3A4 to be the predominant elimination pathway of remibrutinib. The impact of concomitant treatment with CYP3A4 inhibitors, grapefruit juice and ritonavir (RTV), was investigated in this study in combination with an intravenous microtracer approach. Pharmacokinetic (PK) parameters, including the fraction absorbed, the fractions escaping intestinal and hepatic first-pass metabolism, the absolute bioavailability, systemic clearance, volume of distribution at steady-state, and the fraction metabolized via CYP3A4 were evaluated. Oral remibrutinib exposure increased in the presence of RTV 4.27-fold, suggesting that remibrutinib is not a sensitive CYP3A4 substrate. The rich PK dataset supported the building of a robust physiologically-based pharmacokinetic (PBPK) model, which well-described the therapeutic dose range of 25-100 mg. Simulations of untested scenarios revealed an absence of drug-drug interaction (DDI) risk between remibrutinib and the weak CYP3A4 inhibitor fluvoxamine (area under the concentration-time curve ratio [AUCR] <1.25), and a moderate effect with the CYP3A4 inhibitor erythromycin (AUCR: 2.71). Predictions with the moderate and strong CYP3A4 inducers efavirenz and rifampicin, suggested a distinct remibrutinib exposure decrease of 64% and 89%. Oral bioavailability of remibrutinib was 34%. The inclusion of an intravenous microtracer allowed the determination of all relevant remibrutinib PK parameters, which facilitated construction of the PBPK model. This will provide guidance on the selection or restriction of comedications and prediction of DDI risks.

Predicting Oral Absorption for Compounds Outside the Rule of Five Property Space.

The estimation of the extent of absorption of drug candidates intended for oral drug delivery is an important selection criteria in drug discovery. The use of cell-based transwell assays examining flux across cell-monolayers (e.g., Caco-2 or MDCK cells) usually provide satisfactory predictions of the extent of absorption in vivo. These predictions often fall short of expection for molecules outside the traditional low molecular weight property space. In this manuscript the transwell permeability assay was modified to circumvent potential issues that can be encountered when evaluating the aforementioned drug molecules. Particularly, the addition of albumin in the acceptor compartment to reduce potential binding to cells and the acceptor compartment, improved the predictive power of the assay. Cellular binding and lysosomal trapping effects are significantly reduced for larger molecules, particularly lipophilic bases under these more physiological conditions, resulting in higher recovery values and a better prediction power. The data indicate that lysosomal trapping does not impact the rate of absorption of lipophilic bases in general but is rather an exception. Finally, compounds believed to permeate by passive mechanisms were used in a calibration curve for the effective prediction of the fraction absorbed of molecules of interest in current medicinal chemistry efforts.

Ribociclib Drug-Drug Interactions: Clinical Evaluations and Physiologically-Based Pharmacokinetic Modeling to Guide Drug Labeling.

Ribociclib is approved in combination with endocrine therapy as initial endocrine-based therapy for HR-positive and HER2-negative advanced breast cancer. Ribociclib is primarily metabolized by CYP3A4 and, in vitro, is an inhibitor of CYP3A and CYP1A2. Ritonavir (a strong CYP3A inhibitor) increased ribociclib 400 mg single-dose area under the plasma concentration-time curve (AUC) by 3.2-fold, whereas rifampin (a strong CYP3A inducer) decreased ribociclib AUC by 89% in healthy volunteers (HVs). Multiple 400 mg ribociclib doses increased midazolam (CYP3A substrate) AUC by 3.8-fold and caffeine (CYP1A2 substrate) AUC by 1.2-fold vs. each agent alone. A physiologically-based pharmacokinetic (PBPK) model was developed integrating in vitro, preclinical, and clinical data of HVs and patients with cancer. Data predictions indicated that multiple 600 mg ribociclib doses increased midazolam AUC by 5.85-fold and ritonavir increased ribociclib 600 mg multiple dose AUC by 1.31-fold in cancer patients. Based on pharmacokinetics, safety, and efficacy data, and PBPK modeling, dosing modifications for ribociclib recommend avoiding concurrent use of strong CYP3A inhibitors/inducers, and caution regarding using CYP3A substrates with narrow therapeutic indices.

Physiologically-Based Pharmacokinetic Models for Evaluating Membrane Transporter Mediated Drug-Drug Interactions: Current Capabilities, Case Studies, Future Opportunities, and Recommendations.

Physiologically-based pharmacokinetic (PBPK) modeling has been extensively used to quantitatively translate in vitro data and evaluate temporal effects from drug-drug interactions (DDIs), arising due to reversible enzyme and transporter inhibition, irreversible time-dependent inhibition, enzyme induction, and/or suppression. PBPK modeling has now gained reasonable acceptance with the regulatory authorities for the cytochrome-P450-mediated DDIs and is routinely used. However, the application of PBPK for transporter-mediated DDIs (tDDI) in drug development is relatively uncommon. Because the predictive performance of PBPK models for tDDI is not well established, here, we represent and discuss examples of PBPK analyses included in regulatory submission (the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Pharmaceuticals and Medical Devices Agency (PMDA)) across various tDDIs. The goal of this collaborative effort (involving scientists representing 17 pharmaceutical companies in the Consortium and from academia) is to reflect on the use of current databases and models to address tDDIs. This challenges the common perceptions on applications of PBPK for tDDIs and further delves into the requirements to improve such PBPK predictions. This review provides a reflection on the current trends in PBPK modeling for tDDIs and provides a framework to promote continuous use, verification, and improvement in industrialization of the transporter PBPK modeling.

Siponimod pharmacokinetics, safety, and tolerability in combination with the potent CYP3A4 inhibitor itraconazole in healthy subjects with different CYP2C9 genotypes.

PURPOSE: To evaluate the PK and safety of siponimod, a substrate of CYP2C9/3A4, in the presence or absence of a CYP3A4 inhibitor, itraconazole. METHODS: This was an open-label study in healthy subjects (aged 18-50 years; genotype: CYP2C9 *1*2 [cohort 1; n = 17] or *1*3 [cohort 2; n = 13]). Subjects received siponimod 0.25-mg single dose in treatment period 1 (days 1-14), itraconazole 100 mg twice daily in treatment period 2 (days 15-18), and siponimod 0.25-mg single dose (day 19) with itraconazole until day 31 (cohort 1) or day 35 (cohort 2) in treatment period 3. PK of siponimod alone and with itraconazole and safety were assessed. RESULTS: Overall, 29/30 subjects completed the study. In treatment period 1, geometric mean AUCinf, T1/2, and median Tmax were higher while systemic clearance was lower in cohort 2 than cohort 1. In treatment period 3, siponimod AUC decreased by 10% (geo-mean ratio [90% confidence intervals]: 0.90 [0.84; 0.96]) and 24% (0.76 [0.69; 0.82]) in cohorts 1 and 2, respectively. Siponimod Cmax was similar between treatment periods 1 and 3. In both cohorts, the Cmax and AUC of the metabolites (M17, M3, and M5) decreased in the presence of itraconazole. All adverse events were mild. CONCLUSIONS: The minor albeit significant reduction in plasma exposure of siponimod and its metabolites by itraconazole was unexpected. While the reason is unclear, the results suggest that coadministration of the two drugs would not cause a considerable increase of siponimod exposure independent of CYP2C9 genotype.

Multiple administrations of fluconazole increase plasma exposure to ruxolitinib in healthy adult subjects.

PURPOSE: Ruxolitinib is metabolized by cytochrome P450 (CYP)3A4 and CYP2C9. Dual inhibitors of these enzymes (like fluconazole) lead to increased ruxolitinib exposure relative to a single pathway inhibition of CYP3A4 or CYP2C9. The magnitude of this interaction, previously assessed via physiologically based pharmacokinetic (PBPK) models, was confirmed in an open-label, phase 1 study in healthy subjects. METHODS: The effect of multiple doses (200 mg) of fluconazole on single-dose (10 mg) PK of ruxolitinib was investigated including evaluation of the safety and tolerability. The PK parameters of ruxolitinib alone (reference) were compared to those of ruxolitinib combined with fluconazole (test). The point estimate and corresponding two-sided 90% confidence interval for the difference between means of test and reference parameters were determined. RESULTS: All enrolled subjects (N = 15) completed the study. When coadministered with fluconazole, geometric means of ruxolitinib PK parameters Cmax, AUClast, and AUCinf increased by 47%, 234%, and 232%, respectively, vs ruxolitinib alone. The median Tmax decreased slightly, apparent clearance decreased approximately threefold, and elimination half-life increased approximately 2.5-fold, upon ruxolitinib administration with fluconazole vs ruxolitinib alone. These results were consistent with the prospective predictions from a SimCYP PBPK model. Adverse events (AEs) were reported in six subjects (none were suspected to be related to ruxolitinib); no death or on-treatment serious AE was reported. CONCLUSIONS: Coadministration of ruxolitinib with fluconazole significantly increased ruxolitinib systemic exposure; however, no AEs were attributed to ruxolitinib. Concomitant use of ruxolitinib with fluconazole (dose ≤ 200 mg) may require dose reduction/modification of ruxolitinib.

Prediction of the Impact of Cytochrome P450 2C9 Genotypes on the Drug-Drug Interaction Potential of Siponimod With Physiologically-Based Pharmacokinetic Modeling: A Comprehensive Approach for Drug Label Recommendations.

We predicted the drug-drug interaction (DDI) potential of siponimod in presence of cytochrome P450 (CYP)2C9/CYP3A4 inhibitors/inducers in subjects with different CYP2C9 genotypes by physiologically-based pharmacokinetic (PK) modeling. The model was established using in vitro and clinical PK data and verified by adequately predicting siponimod PK when coadministered with rifampin. With strong and moderate CYP3A4 inhibitors, an increased DDI risk for siponimod was predicted for CYP2C9*3/*3 genotype vs. other genotypes area under the curve ratio (AUCR): 3.03-4.20 vs. ≤ 1.49 for strong; 2.42 vs. 1.14-1.30 for moderate. AUCRs increased with moderate (2.13-2.49) and weak (1.12-1.42) CYP3A4/CYP2C9 inhibitors to the same extent for all genotypes. With strong CYP3A4/moderate CYP2C9 inducers and moderate CYP3A4 inducers, predicted AUCRs were 0.21-0.32 and 0.35-0.71, respectively. This complementary analysis to the clinical PK-DDI studies confirmed the relevant influence of CYP2C9 polymorphism on the DDI behavior of siponimod and represented the basis for the DDI labeling recommendations.

A Systematic In Vitro Investigation of the Inhibitor Preincubation Effect on Multiple Classes of Clinically Relevant Transporters.

Preincubation of a drug transporter with its inhibitor in a cell-based assay may result in the apparent enhancement of the inhibitory potency. Currently, limited data are available on potentiation of transporter inhibition by preincubation (PTIP) for clinically relevant solute-carrier transporters other than OATP1B1 and OATP1B3. Therefore, PTIP was examined systematically using OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, and MATE2-K cell lines. IC50 values of 30 inhibitors were determined with or without 3 hours of preincubation, and compounds with a PTIP ≥2.5× were further characterized by assessing the time course of transport inhibition potency and cellular concentration. For each compound, correlations were calculated between highest observed PTIP and physicochemical properties. PTIP was prevalent among organic cation transporters (OCTs) and organic anion-transporting polypeptides (OATPs) but not among organic anion transporters (OATs) or multidrug and toxin extrusion transporters (MATEs), and most instances of PTIP persisted after controlling for toxicity and nonspecific binding. Occasionally, preincubation in excess of 2 hours was required to attain full inhibitory potency. For four drugs examined, preincubation had the potential to change the in vitro drug-drug interaction risk prediction from "no risk" to "risk" on the basis of current regulatory criteria. Molecular weight and LogD7.4, as well as the ratio of passive cellular accumulation and cellular uptake rate correlated with PTIP; thus, low cellular permeation and a slow build-up of unbound intracellular inhibitor concentration may contribute to PTIP. Taken together, our data suggest that PTIP is partly determined by the physicochemical properties of the perpetrator drug, and preincubation may affect the in vitro predicted drug-drug interaction risk for OCTs as well as OATPs. SIGNIFICANCE STATEMENT: During the development of a novel pharmaceutical drug, in vitro studies are conducted to assess the risk of potential adverse interactions between existing medications a patient may already be taking and the novel compound. The exact way these in vitro assays are performed may influence the outcome of risk assessment. Here we suggest that the interaction risk may be underestimated unless specific assay protocols are modified to include an additional incubation step that allows the test drug to accumulate inside the cells, and demonstrate that adding this step is particularly important for large and hydrophobic drug molecules.

Drug-drug interaction (DDI) assessments of ruxolitinib, a dual substrate of CYP3A4 and CYP2C9, using a verified physiologically based pharmacokinetic (PBPK) model to support regulatory submissions.

Ruxolitinib is mainly metabolized by cytochrome P450 (CYP) enzymes CYP3A4 and CYP2C9 followed by minor contributions of other hepatic CYP enzymes in vitro. A physiologically based pharmacokinetic (PBPK) model was established to evaluate the changes in the ruxolitinib systemic exposures with co-administration of CYP3A4 and CYP2C9 perpetrators. The fractions metabolized in the liver via oxidation by CYP enzymes (fm,CYP3A4 = 0.75, fm,CYP2C9 = 0.19, and fm,CYPothers = 0.06) for an initial ruxolitinib model based on in vitro data were optimized (0.43, 0.56, and 0.01, respectively) using the observed exposure changes of ruxolitinib (10 mg) with co-administered ketoconazole (200 mg). The reduced amount of fm,CYP3A4 was distributed to fm,CYP2C9. For the initial ruxolitinib model with co-administration of ketoconazole, the area under the curve (AUC) increase of 2.60-fold was over-estimated compared with the respective observation (1.91-fold). With the optimized fm values, the predicted AUC ratio was 1.82. The estimated AUC ratios of ruxolitinib by co-administration of the moderate CYP3A4 inhibitor erythromycin (500 mg) and the strong CYP3A4 inducer rifampicin (600 mg) were within a 20% error compared with the clinically observed values. The PBPK modeling results may provide information on the labeling, i.e. supporting a dose reduction by half for co-administration of strong CYP3A4 inhibitors. Furthermore, an AUC increase of ruxolitinib in the absence or presence of the dual CYP3A4 and CYP2C9 inhibitor fluconazole (100-400 mg) was prospectively estimated to be 1.94- to 4.31-fold. Fluconazole simulation results were used as a basis for ruxolitinib dose adjustment when co-administering perpetrator drugs. A ruxolitinib PBPK model with optimized fm,CYP3A4 and fm,CYP2C9 was established to evaluate victim DDI risks. The previous minimal PBPK model was supported by the FDA for the dose reduction strategy, halving the dose with the concomitant use of strong CYP3A4 inhibitors and dual inhibitors on CYP2C9 and CYP3A4, such as fluconazole at ≤200 mg. Fluconazole simulation results were used as supportive evidence in discussions with the FDA and EMA about ruxolitinib dose adjustment when co-administering perpetrator drugs. Thus, this study demonstrated that PBPK modeling can support characterizing DDI liabilities to inform the drug label and might help reduce the number of clinical DDI studies by simulations of untested scenarios, when a robust PBPK model is established.