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Nonalcoholic fatty liver disease (NAFLD), representing a clinical spectrum ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), is rapidly evolving into a global pandemic. Patients with NAFLD are burdened with high rates of metabolic syndrome-related comorbidities resulting in polypharmacy. Therefore, it is crucial to gain a better understanding of NAFLD-mediated changes in drug disposition and efficacy/toxicity. Despite extensive clinical pharmacokinetic data in cirrhosis, current knowledge concerning pharmacokinetic alterations in NAFLD, particularly at different stages of disease progression, is relatively limited. In vitro-to-in vivo extrapolation coupled with physiologically based pharmacokinetic and pharmacodynamic (IVIVE-PBPK/PD) modeling offers a promising approach for optimizing pharmacologic predictions while refining and reducing clinical studies in this population. Use of IVIVE-PBPK to predict intra-organ drug concentrations at pharmacologically relevant sites of action is particularly advantageous when it can be linked to pharmacodynamic effects. Quantitative systems pharmacology/toxicology (QSP/QST) modeling can be used to translate pharmacokinetic and pharmacodynamic data from PBPK/PD models into clinically relevant predictions of drug response and toxicity. In this review, a detailed summary of NAFLD-mediated alterations in human physiology relevant to drug absorption, distribution, metabolism, and excretion (ADME) is provided. The application of literature-derived physiologic parameters and ADME-associated protein abundance data to inform virtual NAFLD population development and facilitate PBPK/PD, QSP, and QST predictions is discussed along with current limitations of these methodologies and knowledge gaps. The proposed methodologic framework offers great potential for meaningful prediction of pharmacological outcomes in patients with NAFLD and can inform both drug development and clinical practice for this population.

AIMS Herbal products, spices and/or fruits are perceived as inherently healthy; for instance, St. John's wort (SJW) is marketed as a natural antidepressant and patients often self-administer it concomitantly with oncology medications. However, food constituents/herbs can interfere with drug pharmacokinetics, with risk of altering pharmacodynamics and efficacy. The objective of this work was to develop a strategy to prioritize herb- or food constituent-drug interactions (FC-DIs) to better assess oncology drug clinical risk. METHODS Physiologically based pharmacokinetic (PBPK) models were developed by integrating in vitro parameters with the clinical pharmacokinetics of food constituents in grapefruit juice (bergamottin), turmeric (curcumin) or SJW (hyperforin). Perpetrator files were linked to verified victim PBPK models through appropriate interaction mechanisms (cytochrome P450 3A, breast cancer resistance protein, P-glycoprotein) and applied in prospective PBPK simulations to inform the likelihood and magnitude of changes in exposure to osimertinib, olaparib or acalabrutinib. RESULTS Reported FC-DIs with oncology drugs were well recovered, with absolute average fold error values of 1.10 (bergamottin), 1.05 (curcumin) and 1.01 (hyperforin). Prospective simulations with grapefruit juice and turmeric showed clinically minor to insignificant changes in exposure (

The role of membrane transporters on pharmacokinetics (PKs), drug-drug interactions (DDIs), pharmacodynamics (PDs), and toxicity of drugs has been broadly recognized. However, our knowledge of modulation of transporter expression and/or function in the diseased patient population or specific populations, such as pediatrics or pregnancy, is still emerging. This white paper highlights recent advances in studying the changes in transporter expression and activity in various diseases (i.e., renal and hepatic impairment and cancer) and some specific populations (i.e., pediatrics and pregnancy) with the focus on clinical implications. Proposed alterations in transporter abundance and/or activity in diseased and specific populations are based on (i) quantitative transporter proteomic data and relative abundance in specific populations vs. healthy adults, (ii) clinical PKs, and emerging transporter biomarker and/or pharmacogenomic data, and (iii) physiologically-based pharmacokinetic modeling and simulation. The potential for altered PK, PD, and toxicity in these populations needs to be considered for drugs and their active metabolites in which transporter-mediated uptake/efflux is a major contributor to their absorption, distribution, and elimination pathways and/or associated DDI risk. In addition to best practices, this white paper discusses current challenges and knowledge gaps to study and quantitatively predict the effects of modulation in transporter activity in these populations, together with the perspectives from the International Transporter Consortium (ITC) on future directions.

The role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-drug interactions (DDIs) and limiting drug absorption as well as restricting the brain penetration of drugs with certain physicochemical properties is well known. P-gp/BCRP inhibition by drugs in the gut has been reported to increase the systemic exposure to substrate drugs. A previous International Transporter Consortium (ITC) perspective discussed the feasibility of P-gp/BCRP inhibition at the blood-brain barrier and its implications. This ITC perspective elaborates and discusses specifically the hepatic and renal P-gp/BCRP (referred as systemic) inhibition of drugs and whether there is any consequence for substrate drug disposition. This perspective summarizes the clinical evidence-based recommendations regarding systemic P-gp and BCRP inhibition of drugs with a focus on biliary and active renal excretion pathways. Approaches to assess the clinical relevance of systemic P-gp and BCRP inhibition in the liver and kidneys included (i) curation of DDIs involving intravenously administered substrates or inhibitors; (ii) in vitro-to-in vivo extrapolation of P-gp-mediated DDIs at the systemic level; and (iii) curation of drugs with information available about the contribution of biliary excretion and related DDIs. Based on the totality of evidence reported to date, this perspective supports limited clinical DDI risk upon P-gp or BCRP inhibition in the liver or kidneys.

ABC transporters (ATP-binding cassette transporter) traffic drugs and their metabolites across membranes, making ABC transporter expression levels a key factor regulating local drug concentrations in different tissues and individuals. Yet, quantification of ABC transporters remains challenging because they are large and low-abundance transmembrane proteins. Here, we analysed 200 samples of crude and membrane-enriched fractions from human liver, kidney, intestine, brain microvessels and skin, by label-free quantitative mass spectrometry. We identified 32 (out of 48) ABC transporters: ABCD3 was the most abundant in liver, whereas ABCA8, ABCB2/TAP1 and ABCE1 were detected in all tissues. Interestingly, this atlas unveiled that ABCB2/TAP1 may have TAP2-independent functions in the brain and that biliary atresia (BA) and control livers have quite different ABC transporter profiles. We propose that meaningful biological information can be derived from a direct comparison of these data sets.

There is an increasing interest in transporter induction (i.e., decreased systemic drug exposure due to increased efflux-limited absorption or transporter-mediated clearance) as a mechanism of drug-drug interactions (DDIs), although evidence of clinical relevance is still evolving. Intestinal P-glycoprotein (P-gp) and hepatic organic anion transporting polypeptides 1B (OATP1B) can be important determinants of drug absorption and disposition, as well as targets for DDIs. Current data indicate that intestinal P-gp protein levels can be induced up to threefold to fourfold in humans primarily with pregnane X receptor (PXR) activators, and that this induction can decrease the systemic exposure of drugs with P-gp efflux-limited absorption (e.g., ≤ 67% decrease in the exposure of total dabigatran following rifampin multiple oral dosing). Evaluation of the clinical relevance of P-gp induction as a DDI mechanism must consider the induction potential of the perpetrator drug for P-gp and attenuation of exposure of the victim drug in the context of its therapeutic window. Practical drug development recommendations are provided herein. Reports are contradictory on OATP1B induction by PXR activators in human hepatocytes and liver biopsies. Some clinical investigations demonstrated that rifampin pretreatment decreased exposure of OATP1B substrates, while other studies found no differences, and the potential involvement of other mechanisms in these observed DDIs cannot be definitively ruled out. Thus, further studies are needed to understand hepatic OATP1B induction and potential involvement of other mechanisms contributing to reduced exposure of OATP1B substrates. This review critically summarizes the state-of-the-art on intestinal P-gp and hepatic OATP1B induction, and highlights implications for drug development.

Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, alternative splicing, post-translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation-mediated changes in transporter function on drug disposition and response.

In drug development, estimating fraction absorbed (F a) in man for permeability-limited compounds is important but challenging. To model F a of such compounds from apparent permeabilities (P app) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the P app across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The P app values of acamprosate and of several paracellular marker molecules were measured. These P app values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (P modeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate P app across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate P app, 1.56 ± 0.28 × 10 -7 cm·s -1, is similar to the P app of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10 -7 cm·s -1), lucifer yellow (1.80 ± 0.35 × 10 -7 cm·s -1), and fluorescein (2.10 ± 0.28 × 10 -7 cm·s -1), and lower than that of atenolol (7.32 ± 0.60 × 10 -7 cm·s -1 mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Q monolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the P app of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The P modeled for all the paracellular markers and acamprosate was dominated by the P para component and matched the experimentally obtained P app. Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B 0,+AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the P modeled estimated well P para, and the paracellular route appears to be the predominant mechanism for acamprosate P app across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.

The aim of this study was to derive region-specific transporter expression data suitable for in vitro-to-in vivo extrapolation (IVIVE) within a physiologically based pharmacokinetic (PBPK) modeling framework. A meta-analysis was performed whereby literary sources reporting region-specific transporter expression obtained via absolute and relative quantification approaches were considered in healthy adult Caucasian individuals. Furthermore, intestinal total membrane protein yield was calculated to enable mechanistic IVIVE via absolute transporter abundances. Where required, authors were contacted for additional information. A refined database was constructed where samples were excluded based on quantification in, non-Caucasian subjects, disease tissue, subjects18 years old, duplicated samples, non-total membrane matrix, pooled matrices, or cDNA. Demographic data were collected where available. The weighted and geometric mean, coefficient of variation, and between-study homogeneity was calculated in each of eight gut segments (duodenum, two jejunum, four ileum, and colon) for 16 transporters. Expression data were normalized to that in the proximal jejunum. From a total of 47 articles, the final database consisted of 2238 measurements for 16 transporters. The solute carrier peptide transporter 1 (PepT1) showed the highest jejunal abundance, while multidrug resistance-associated protein (MRP) 2 was the highest abundance ATP-binding cassette transporter. Transporters displaying significant region-specific expression included the ileal bile acid transporter, which showed 18-fold greater terminal ileum expression compared with the proximal jejunum, while MRP3, organic cation transporter type 1 (OCTN1), and OCT1 showed2-fold higher expression in other regions compared with the proximal jejunum. This is the first systematic analysis incorporating absolute quantification methodology to determine region-specific intestinal transporter expression. It is expected to be beneficial for mechanistic transporter IVIVE in healthy adult Caucasians. SIGNIFICANCE STATEMENT: Given the burgeoning reports of absolute transporter abundances in the human intestine, the incorporation of such information into mechanistic IVIVE-PBPK models could offer a distinct advantage in facilitating the robust assessment of the impact of gut transporters on drug disposition. The systematic and formal assessment via a literature meta-analysis described herein, enables assignment of the regional-specific expression, absolute transporter abundances, interindividual variability, and other associated scaling factors to healthy Caucasian populations within PBPK models. The resulting values are available to incorporate into PBPK models, and offer a verifiable account describing intestinal transporter expression within PBPK models for persons wishing to utilize them. Furthermore, these data facilitate the development of appropriate IVIVE scaling strategies using absolute transporter abundances.

The blood-brain barrier (BBB) maintains brain homeostasis by controlling traffic of molecules from the circulation into the brain. This function is predominantly dependent on proteins expressed at the BBB, especially transporters and tight junction proteins. Alterations to the level and function of BBB proteins can impact the susceptibility of the central nervous system to exposure to xenobiotics in the systemic circulation with potential consequent effects on brain function. In this study, expression profiles of drug transporters and solute carriers in the BBB were assessed in tissues from healthy individuals ( n = 12), Alzheimer's patients ( n = 5), and dementia with Lewy bodies patients ( n = 5), using targeted, accurate mass retention time (AMRT) and global proteomic methods. A total of 53 transporters were quantified, 19 for the first time in the BBB. A further 20 novel transporters were identified but not quantified. The global proteomic method identified another 3333 BBB proteins. Transporter abundances, taken together with the scaling factor, microvessel protein content per unit tissue (BMvPGB also measured here), can be used in quantitative systems pharmacology models predicting drug disposition in the brain and permitting dose adjustment (precision dosing) in special populations of patients, such as those with dementia. Even in this small study, we see differences in transporter profile between healthy and diseased brain tissue.

This review is a summary of factors affecting the drug pharmacokinetics (PK) of dogs versus humans. Identifying these interspecies differences can facilitate canine-human PK extrapolations while providing mechanistic insights into species-specific drug in vivo behavior. Such a cross-cutting perspective can be particularly useful when developing therapeutics targeting diseases shared between the two species such as cancer, diabetes, cognitive dysfunction, and inflammatory bowel disease. Furthermore, recognizing these differences also supports a reverse PK extrapolations from humans to dogs. To appreciate the canine-human differences that can affect drug absorption, distribution, metabolism, and elimination, this review provides a comparison of the physiology, drug transporter/enzyme location, abundance, activity, and specificity between dogs and humans. Supplemental material provides an in-depth discussion of certain topics, offering additional critical points to consider. Based upon an assessment of available state-of-the-art information, data gaps were identified. The hope is that this manuscript will encourage the research needed to support an understanding of similarities and differences in human versus canine drug PK.

Translation of information on drug exposure and effect is facilitated by in silico models that enable extrapolation of in vitro measurements to in vivo clinical outcomes. These models integrate drug-specific data with information describing physiological processes and pathological changes, including alterations to proteins involved in drug absorption, distribution and elimination. Over the past 15 years, quantitative proteomics has contributed a wealth of protein expression data, which are currently used for a variety of systems pharmacology applications, as a complement or a surrogate for activity of the corresponding proteins. In this review, we explore current and emerging applications of targeted and global (untargeted) proteomics in translational pharmacology as well as strategies for improved integration into model-based drug development.

Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important determinants of transporter-mediated drug-drug interactions (DDIs). Current studies assessed the OATP1B1 and OATP1B3-mediated DDI potential of vemurafenib, a kinase inhibitor drug with high protein binding and low aqueous solubility, using R-value and physiologically based pharmacokinetic (PBPK) models. The total half-maximal inhibitory concentration (IC(50,total)) values of vemurafenib against OATP1B1 and OATP1B3 were determined in 100% human plasma in transporter-overexpressing human embryonic kidney 293 stable cell lines. The unbound fraction of vemurafenib in human plasma before (f(u,plasma)) and after addition into the uptake assay plate (f(u,plasma,inc)) were determined by rapid equilibrium dialysis. There was no statistically significant difference between f(u,plasma) and f(u,plasma,inc). Vemurafenib IC(50,total) values against OATP1B1 and OATP1B3 are 175 ± 82 and 231 ± 26 μM, respectively. The R-values [R = 1 + f(u,plasma) × I(in,max)/(f(u,plasma,inc) × IC(50,total))] were then simplified as R=1+I(in,max)/IC(50,total), and were 1.76 and 1.57 for OATP1B1 and OATP1B3, respectively. The simulated pravastatin AUC ratio was 1.28 when a single dose of pravastatin (40 mg) was co-administered with vemurafenib (960 mg, twice daily) at steady-state, compared to pravastatin alone. Both R-value and PBPK models predict that vemurafenib has the potential to cause OATP1B1- and OATP1B3-mediated DDIs.

Nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, is increasing in prevalence. NASH-related alterations in hepatic protein expression (e.g., transporters) and in overall physiology may affect drug exposure by altering drug disposition and elimination. The aim of this study was to build a physiologically-based pharmacokinetic (PBPK) model to predict drug exposure in NASH by incorporating NASH-related changes in hepatic transporters. Morphine and morphine-3-glucuronide (M3G) were used as model compounds. A PBPK model of morphine with permeability-limited hepatic disposition was extended to include M3G disposition and enterohepatic recycling (EHR). The model captured the area under the plasma concentration-time curve (AUC) of morphine and M3G after intravenous morphine administration within 0.82- and 1.94-fold of observed values from three independent clinical studies for healthy adult subjects (n= 6, 10, and 14 individuals). When NASH-related changes in multidrug resistance-associated protein 2 (MRP2) and MRP3 were incorporated into the model, the predicted M3G mean AUC in NASH was 1.34-fold higher compared to healthy subjects, which is slightly lower than the observed value (1.63-fold). Exploratory simulations on other physiological changes occurring in NASH (e.g., moderate decreases in glomerular filtration rate and portal vein blood flow) revealed that the effect of transporter changes was most prominent. Additionally, NASH-related transporter changes resulted in decreased morphine EHR, which could be important for drugs with extensive EHR. This study is an important first step to predict drug disposition in complex diseases such as NASH using PBPK modeling.

Aldehyde oxidase (AO) efficiently metabolizes a range of compounds with N-containing heterocyclic aromatic rings and/or aldehydes. The limited knowledge of AO activity and abundance (in vitro and in vivo) has led to poor prediction of in vivo systemic clearance (CL) using in vitro–to–in vivo extrapolation approaches, which for drugs in development can lead to their discontinuation. We aimed to identify appropriate scaling factors to predict AO CL of future new chemical entities (NCEs). The metabolism of six AO substrates was measured in human liver cytosol (HLC) and S9 fractions. Measured blood-to-plasma ratios and free fractions (in the in vitro system and in plasma) were used to develop physiologically based pharmacokinetic models for each compound. The impact of extrahepatic metabolism was explored, and the intrinsic clearance required to recover in vivo profiles was estimated and compared with in vitro measurements. Using HLC data and assuming only hepatic metabolism, a systematic underprediction of clearance was observed (average fold underprediction was 3.8). Adding extrahepatic metabolism improved the accuracy of the results (average fold error of 1.9). A workflow for predicting metabolism of an NCE by AO is proposed, and an empirical (laboratory-specific) scaling factor of three on the predicted intravenous CL allows a reasonable prediction of the available clinical data. Alternatively, considering also extrahepatic metabolism, an scaling factor of 6.5 applied on the intrinsic clearance could be used. Future research should focus on the impact of the in vitro study designs and the contribution of extrahepatic metabolism to AO-mediated clearance to understand the mechanisms behind the systematic underprediction. SIGNIFICANCE STATEMENT This works describes the development of scaling factors to allow in vitro–in vivo extrapolation of the clearance of compounds by aldehyde oxidase metabolism in humans. In addition, physiologically based pharmacokinetic models were developed for each of the aldehyde oxidase substrate compounds investigated.

Acamprosate is an anionic drug substance widely used in treating symptoms of alcohol withdrawal. It was recently shown that oral acamprosate absorption is likely due to paracellular transport. In contrast, little is known about the eliminating mechanism clearing acamprosate from the blood in the kidneys, despite the fact that studies have shown renal secretion of acamprosate. The hypothesis of the present study was therefore that renal organic anion transporters (OATs) facilitate the renal excretion of acamprosate in humans. The aim of the present study was to establish and apply OAT1 (gene product of SLC22A6) and OAT3 (gene product of SLC22A8) expressing cell lines to investigate whether acamprosate is a substrate or inhibitor of OAT1 and/or OAT3. The studies were performed in HEK293-Flp-In cells stably transfected with SLC22A6 or SLC22A8. Protein and functional data showed that the established cell lines are useful for studying OAT1- and OAT3-mediated transport in bi-laboratory studies. Acamprosate inhibited OAT1-mediated p-aminohippuric acid (PAH) uptake but did not inhibit substrate uptake via OAT3 expressing cells, neither when applied concomitantly nor after a 3 h preincubation with acamprosate. The uptake of PAH via OAT1 was inhibited in a competitive manner by acamprosate and cellular uptake studies showed that acamprosate is a substrate for OAT1 with a Km-value of approximately 700 &micro, M. Probenecid inhibited OAT1-mediated acamprosate uptake with a Ki-value of approximately 13 &micro, M, which may translate into an estimated clinically significant DDI index. In conclusion, acamprosate was identified as a substrate of OAT1 but not OAT3.

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

Morphine has large pharmacokinetic variability, which is further complicated by developmental changes in neonates and small infants. The impacts of organic cation transporter 1 (OCT1) genotype and changes in blood‐flow on morphine clearance (CL) were previously demonstrated in children, whereas changes in UDP‐glucuronosyltransferase 2B7 (UGT2B7) activity showed a small effect. This study, targeting neonates and small infants, was designed to assess the influence of developmental changes in OCT1 and UGT2B7 protein expression and modified blood‐flow on morphine CL using physiologically based pharmacokinetic (PBPK) modeling. The implementation of these three age‐dependent factors into the pediatric system platform resulted in reasonable prediction for an age‐dependent increase in morphine CL in these populations. Sensitivity of morphine CL to changes in cardiac output increased with age up to 3 years, whereas sensitivity to changes in UGT2B7 activity decreased. This study suggests that morphine exhibits age‐dependent extraction, likely due to the developmental increase in OCT1 and UGT2B7 protein expression/activity and hepatic blood‐flow.

Present studies determined the effects of pretreatment with rifampicin, an organic anion-transporting polypeptide (OATP) inhibitor, and the tyrosine kinase inhibitor dasatinib on OATP1B1- and OATP1B3-mediated transport, and evaluated the OATP-mediated drug-drug interaction potential of dasatinib using the static R-value and dynamic physiologically based pharmacokinetic models. Rifampicin and dasatinib pretreatment significantly decreased OATP1B1- and OATP1B3-mediated transport. Rifampicin pretreatment also significantly decreased [ 3 H]-pitavastatin and [ 3 H]-CCK-8 accumulation in human sandwich-cultured hepatocytes. Present studies revealed that estrone-3-sulfate is a less-sensitive OATP1B1 substrate than estradiol-17β-glucuronide in assessing rifampicin pretreatment effects. Pretreatment with rifampicin and dasatinib reduced the inhibition constant (K i ) values against OATP1B1 by 3 and 2.1 fold and against OATP1B3 by 2.4 and 2.1 fold, respectively. The in vitro rifampicin K i values after preincubation are comparable to the estimated in vivo K i reported previously. Models predict that dasatinib has a low potential to cause OATP1B1- and OATP1B3-mediated drug-drug interactions. Time-lapse confocal microscopy demonstrated that rifampicin and dasatinib pretreatment did not affect plasma membrane localization of green-fluorescent protein–tagged OATP1B1 (GFP-OATP1B1) and GFP–OATP1B3 in human embryonic kidney 293 stable cell lines. In summary, we report novel findings that pretreatment with rifampicin and dasatinib potentiates the inhibitory effects toward OATP1B1 and OATP1B3 without affecting plasma membrane levels of the transporters.

Following a meal, a transient increase in splanchnic blood flow occurs that can result in increased exposure to orally administered high-extraction drugs. Typically, physiologically based pharmacokinetic (PBPK) models have incorporated this increase in blood flow as a time-invariant fed/fasted ratio, but this approach is unable to explain the extent of increased drug exposure. A model for the time-varying increase in splanchnic blood flow following a moderate- to high-calorie meal (TV-Q Splanch) was developed to describe the observed data for healthy individuals. This was integrated within a PBPK model and used to predict the contribution of increased splanchnic blood flow to the observed food effect for two orally administered high-extraction drugs, propranolol and ibrutinib. The model predicted geometric mean fed/fasted AUC and C max ratios of 1.24 and 1.29 for propranolol, which were within the range of published values (within 1.0-1.8-fold of values from eight clinical studies). For ibrutinib, the predicted geometric mean fed/fasted AUC and C max ratios were 2.0 and 1.84, respectively, which was within 1.1-fold of the reported fed/fasted AUC ratio but underestimated the reported C max ratio by up to 1.9-fold. For both drugs, the interindividual variability in fed/fasted AUC and C max ratios was underpredicted. This suggests that the postprandial change in splanchnic blood flow is a major mechanism of the food effect for propranolol and ibrutinib but is insufficient to fully explain the observations. The proposed model is anticipated to improve the prediction of food effect for high-extraction drugs, but should be considered with other mechanisms.

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.

Plasma concentrations of dabigatran, an active principle of prodrug dabigatran etexilate (DABE), are increased by renal impairment (RI) or coadministration of a P-glycoprotein inhibitor. Because the combined effects of drug–drug interactions and RI have not been evaluated by means of clinical studies, the decision of DABE dosing for RI patients receiving P-glycoprotein inhibitors is empirical at its best. We conducted virtual drug–drug interactions studies between DABE and the P-glycoprotein inhibitor verapamil in RI populations using physiologically based pharmacokinetic modeling. The developed physiologically based pharmacokinetic model for DABE and dabigatran was used to predict trough dabigatran concentrations in the presence and absence of verapamil in virtual RI populations. The population-based physiologically based pharmacokinetic model provided the most appropriate dosing regimen of DABE for likely clinical scenarios, such as drug–drug interactions in this RI population based on available knowledge of the systems changes and in the absence of actual clinical studies.

Introduction. Acamprosate is a BCS class III, non-metabolized, anionic drug substance for which the excretion mechanism is not fully understood. Intravenous administration in rats of acamprosate together with probenecid, a known substrate and inhibitor of the organic anion transporter (OAT) 1 (SLC22A6) (1), decreased the renal clearance (ClR) of acamprosate in a dose-dependent manner (2). The hypothesis of the present study was that renal OATs expressed in the basolateral membrane of the proximal tubule epithelium (3) could be the carrier contributing to renal excretion of acamprosate and the reason for potential acamprosate-drug (e.g. probenecid) interactions.Aim. To investigate if acamprosate interacts with OAT1 by measuring the concentration-dependent effect of acamprosate on the uptake of [14C]-p-aminohippuric acid ([14C]-PAH), a well-established substrate of OAT1 (4).Method. The apical uptake of 0.5 μCi/mL [14C]-PAH was measured for 5 minutes at 37°C and 100 rpm in the presence of 0 – 33 000 μM acamprosate in human embryonic kidney (HEK)293 cells transiently expressing OAT1 (Corning TransportoCells OAT1), or in a HEK293-Flp-in cell line stably expressing OAT1 (developed in-house). The uptake was in both series measured in parallel in mock-transfected HEK293 cells grown under similar cell culture conditions.Results. A significant, time-dependent and saturable increase of [14C]-PAH apical uptake was observed in the OAT1-transfected HEK293 cells compared to mock-transfected cells. The apical uptake of [14C]-PAH in OAT1-transfected cells was decreased in the presence of acamprosate in a concentration-dependent manner, while no effect of acamprosate was observed in the mock-transfected cells. The mean (+/- SD) inhibition constant for [14C]-PAH uptake (IC50) was 1 042 ± 134 and 902 ± 20 μM in the transiently (n=3) and stably transfected (N=3, n=2) HEK293-OAT1 cells, respectively.Conclusion. Acamprosate inhibits OAT1 in vitro at concentrations close to the maximum unbound plasma concentration of acamprosate, cmax = 154-768 μM (5), obtained after intravenous administration [333-2130 mg].References1. Nigam SK, Bush KT, Martovetsky G, Ahn SY, Liu HC, Richard E, et al. The organic anion transporter (OAT) family: a systems biology perspective. Physiol Rev. 2015;95(1):83-123.2. Zornoza T, Guerri C, Polache A, Granero L. Disposition of acamprosate in the rat: influence of probenecid. Biopharm Drug Dispos. 2002;23(7):283-91.3. Wright SH, Dantzler WH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev. 2004;84(3):987-1049.4. FDA. In Vitro Metabolism and Transporter-Mediated Drug-Drug Interaction Studies - Guidance for Industry. In: CDER, editor. Silver Spring, MD: U.S. Department of Health and Human Services; 2017. p. 1-37.5. Approval package for application number 21-431: Campral clinical pharmacology and biopharmaceutics review: Center for drug evaluation and research; 2004 [cited 2018 08 13]. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2004/21-431_Campral_BioPharmr.pdf.

Morphine shows large interindividual variability in its pharmacokinetics; however, the cause of this has not been fully addressed. The variability in morphine disposition is considered to be due to a combination of pharmacogenetic and physiological determinants related to morphine disposition. We previously reported the effect of organic cation transporter (OCT1) genotype on morphine disposition in pediatric patients. To further explore the underlying mechanisms for variability arising from relevant determinants, including OCT1, a physiologically based pharmacokinetic (PBPK) model of morphine was developed. The PBPK model predicted morphine concentration-time profiles well, in both adults and children. Almost all of the observed morphine clearances in pediatric patients fell within a twofold range of median predicted values for each OCT1 genotype in each age group. This PBPK modeling approach quantitatively demonstrates that OCT1 genotype, age-related growth, and changes in blood flow as important contributors to morphine pharmacokinetic (PK) variability.

This study aimed to derive quantitative abundance values for key hepatic transporters suitable for in vitro–in vivo extrapolation within a physiologically based pharmacokinetic modeling framework. A meta-analysis was performed whereby data on abundance measurements, sample preparation methods, and donor demography were collated from the literature. To define values for a healthy Caucasian population, a subdatabase was created whereby exclusion criteria were applied to remove samples from non-Caucasian individuals, those with underlying disease, or those with subcellular fractions other than crude membrane. Where a clinically relevant active genotype was known, only samples from individuals with an extensive transporter phenotype were included. Authors were contacted directly when additional information was required. After removing duplicated samples, the weighted mean, geometric mean, standard deviation, coefficient of variation, and between-study homogeneity of transporter abundances were determined. From the complete database containing 24 transporters, suitable abundance data were available for 11 hepatic transporters from nine studies after exclusion criteria were applied. Organic anion transporting polypeptides OATP1B1 and OATP1B3 showed the highest population abundance in healthy adult Caucasians. For several transporters, the variability in abundance was reduced significantly once the exclusion criteria were applied. The highest variability was observed for OATP1B3 > OATP1B1 > multidrug resistance protein 2 > multidrug resistance gene 1. No relationship was found between transporter expression and donor age. To our knowledge, this study provides the first in-depth analysis of current quantitative abundance data for a wide range of hepatic transporters, with the aim of using these data for in vitro–in vivo extrapolation, and highlights the significance of investigating the background of tissue(s) used in quantitative transporter proteomic studies. Similar studies are now warranted for other ethnicities.

Aims Use of the anti-tumour antibiotic actinomycin D is associated with development of hepatotoxicity, particularly in young children. A paucity of actinomycin D pharmacokinetic data make it challenging to develop a sound rationale for defining dosing regimens in younger patients. The study aim was to develop a physiologically based pharmacokinetic (PBPK) model using a combination of data from the literature and generated from experimental analyses. Methods Assays to determine actinomycin D Log P, blood:plasma partition ratio and ABCB1 kinetics were conducted. These data were combined with physiochemical properties sourced from the literature to generate a compound file for use within the modelling-simulation software Simcyp (version 14 release 1). For simulation, information was taken from two datasets, one from 117 patients under the age of 21 and one from 20 patients aged 16-48. Results The final model incorporated clinical renal and biliary clearance data and an additional systemic clearance value. The mean AUC0-26h of simulated subjects was within 1.25-fold of the observed AUC0-26h (84 ng h ml(-1) simulated vs. 93 ng h ml(-1) observed). For the younger age ranges, AUC predictions were within two-fold of observed values, with simulated data from six of the eight age/dose ranges falling within 15% of observed data. Simulated values for actinomycin D AUC0-26h and clearance in infants aged 0-12 months ranged from 104 to 115 ng h ml(-1) and 3.5-3.8 l h(-1) , respectively. Conclusions The model has potential utility for prediction of actinomycin D exposure in younger patients and may help guide future dosing. However, additional independent data from neonates and infants is needed for further validation. Physiological differences between paediatric cancer patients and healthy children also need to be further characterized and incorporated into PBPK models.

Relative expression factors (REFs) are used to scale in vitro transporter kinetic data via in vitro-in vivo extrapolation linked to physiologically based pharmacokinetic (IVIVE-PBPK) models to clinical observations. Primarily two techniques to quantify transporter protein expression are available, immunoblotting and liquid chromatography-tandem mass spectrometry. Literature-collated REFs ranged from 0.4 to 5.1 and 1.1 to 90 for intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), respectively. The impact of using human jejunum-Caco-2 REFs for P-gp (REFiP-gp) and BCRP (REFiBCRP), generated from the same samples and using different proteomic methodologies from independent laboratories, on PBPK outcomes was assessed. A 5-fold decrease in REFiP-gp for a single oral dose of digoxin resulted in a 1.19- and 1.31-fold higher plasma area under the curve and Cmax, respectively. All generated REFiP-gp values led to simulated digoxin Cmax values within observed ranges; however, combining kinetic data generated from a different laboratory with the 5-fold lower REFiP-gp could not recover a digoxin-rifampicin drug-drug interaction, emphasizing the necessity to obtain transporter-specific kinetic estimates and REFs from the same in vitro system. For a theoretical BCRP compound, with absorption taking place primarily in the jejunum, a decrease in the REFiBCRP from 2.22 (University of Manchester) to 1.11 (Bertin Pharma) promoted proximal intestinal absorption while delaying tmax 1.44-fold. Laboratory-specific differences in REF may lead to different IVIVE-PBPK outcomes. To understand the mechanisms underlying projected pharmacokinetic liabilities, it is important to assess the potential impact of bias on the generation of REFs on an interindividual basis within a target population.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 mediate hepatic uptake of many drugs including lipid-lowering statins. Current studies determined the OATP1B1/1B3-mediated drug-drug interaction (DDI) potential of mammalian target of rapamycin (mTOR) inhibitors, everolimus and sirolimus, using R-value and physiologically based pharmacokinetic models. Preincubation with everolimus and sirolimus significantly decreased OATP1B1/1B3-mediated transport even after washing and decreased inhibition constant values up to 8.3- and 2.9-fold for OATP1B1 and both 2.7-fold for OATP1B3, respectively. R-values of everolimus, but not sirolimus, were greater than the FDA-recommended cutoff value of 1.1. Physiologically based pharmacokinetic models predict that everolimus and sirolimus have low OATP1B1/1B3-mediated DDI potential against pravastatin. OATP1B1/1B3-mediated transport was not affected by preincubation with INK-128 (10 μM, 1 h), which does however abolish mTOR kinase activity. The preincubation effects of everolimus and sirolimus on OATP1B1/1B3-mediated transport were similar in cells before preincubation with vehicle control or INK-128, suggesting that inhibition of mTOR activity is not a prerequisite for the preincubation effects observed for everolimus and sirolimus. Nine potential phosphorylation sites of OATP1B1 were identified by phosphoproteomics; none of these are the predicted mTOR phosphorylation sites. We report the everolimus/sirolimus-preincubation-induced inhibitory effects on OATP1B1/1B3 and relatively low OATP1B1/1B3-mediated DDI potential of everolimus and sirolimus.

Chronic kidney disease (CKD) differentially affects the pharmacokinetics (PK) of nonrenally cleared drugs via certain pathways (e.g., cytochrome P450 (CYP)2D6); however, the effect on CYP2C8‐mediated clearance is not well understood because of overlapping substrate specificity with hepatic organic anion‐transporting polypeptides (OATPs). This study used physiologically based pharmacokinetic (PBPK) modeling to delineate potential changes in CYP2C8 or OATP1B activity in patients with CKD. Drugs analyzed are predominantly substrates of CYP2C8 (rosiglitazone and pioglitazone), OATP1B (pitavastatin), or both (repaglinide). Following initial model verification, pharmacokinetics (PK) of these drugs were simulated in patients with severe CKD considering changes in glomerular filtration rate (GFR), plasma protein binding, and activity of either CYP2C8 and/or OATP1B in a stepwise manner. The PBPK analysis suggests that OATP1B activity could be decreased up to 60% in severe CKD, whereas changes to CYP2C8 are negligible. This improved understanding of CKD effect on clearance pathways could be important to inform the optimal use of nonrenally eliminated drugs in patients with CKD.

Current formulations of combined oral contraceptives (COC) containing ethinylestradiol (EE) have ≤35 μg due to increased risks of cardiovascular diseases (CVD) with higher doses of EE. Low-dose formulations however, have resulted in increased incidences of breakthrough bleeding and contraceptive failure, particularly when coadministered with inducers of cytochrome P450 enzymes (CYP). The developed physiologically based pharmacokinetic model quantitatively predicted the effect of CYP3A4 inhibition and induction on the pharmacokinetics of EE. The predicted Cmax and AUC ratios when coadministered with voriconazole, fluconazole, rifampicin, and carbamazepine were within 1.25 of the observed data. Based on published clinical data, an AUCss value of 1,000 pg/ml.h was selected as the threshold for breakthrough bleeding. Prospective application of the model in simulations of different doses of EE (20 μg, 35 μg, and 50 μg) identified percentages of the population at risk of breakthrough bleeding alone and with varying degrees of CYP modulation.

In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition.

Digoxin is the recommended substrate for assessment of P-glycoprotein (P-gp)-mediated drug-drug interactions (DDIs) in vivo. The overall aim of our study was to investigate the inhibitory potential of both verapamil and norverapamil on the P-gp-mediated efflux of digoxin in both gut and liver. Therefore, a physiologically-based pharmacokinetic (PBPK) model for verapamil and its primary metabolite was developed and validated through the recovery of observed clinical plasma concentration data for both moieties and the reported interaction with midazolam, albeit a cytochrome P450 3A4-mediated DDI. The validated inhibitor model was then used in conjunction with the model developed previously for digoxin. The range of values obtained for the 10 trials indicated that increases in area under the plasma concentration-time curve (AUC) profiles and maximum plasma concentration observed (Cmax ) values of digoxin following administration of verapamil were more comparable with in vivo observations, when P-gp inhibition by the metabolite, norverapamil, was considered as well. The predicted decrease in AUC and Cmax values of digoxin following administration of rifampicin because of P-gp induction was 1.57- (range: 1.42-1.77) and 1.62-fold (range: 1.53-1.70), which were reasonably consistent with observed values of 1.4- and 2.2-fold, respectively. This study demonstrates the application of permeability-limited models of absorption and distribution within a PBPK framework together with relevant in vitro data on transporters to assess the clinical impact of modulated P-gp-mediated efflux by drugs in development.

Background and Objectives The interplay between liver metabolising enzymes and transporters is a complex process involving system-related parameters such as liver blood perfusion as well as drug attributes including protein and lipid binding, ionisation, relative magnitude of passive and active permeation. Metabolism- and/or transporter-mediated drug–drug interactions (mDDIs and tDDIs) add to the complexity of this interplay. Thus, gaining meaningful insight into the impact of each element on the disposition of a drug and accurately predicting drug–drug interactions becomes very challenging. To address this, an in vitro–in vivo extrapolation (IVIVE)-linked mechanistic physiologically based pharmacokinetic (PBPK) framework for modelling liver transporters and their interplay with liver metabolising enzymes has been developed and implemented within the Simcyp Simulator®. Methods In this article an IVIVE technique for liver transporters is described and a full-body PBPK model is developed. Passive and active (saturable) transport at both liver sinusoidal and canalicular membranes are accounted for and the impact of binding and ionisation processes is considered. The model also accommodates tDDIs involving inhibition of multiple transporters. Integrating prior in vitro information on the metabolism and transporter kinetics of rosuvastatin (organic-anion transporting polypeptides OATP1B1, OAT1B3 and OATP2B1, sodium-dependent taurocholate co-transporting polypeptide [NTCP] and breast cancer resistance protein [BCRP]) with one clinical dataset, the PBPK model was used to simulate the drug disposition of rosuvastatin for 11 reported studies that had not been used for development of the rosuvastatin model. Results The simulated area under the plasma concentration–time curve (AUC), maximum concentration (Cmax) and the time to reach Cmax (tmax) values of rosuvastatin over the dose range of 10–80 mg, were within 2-fold of the observed data. Subsequently, the validated model was used to investigate the impact of coadministration of cyclosporine (ciclosporin), an inhibitor of OATPs, BCRP and NTCP, on the exposure of rosuvastatin in healthy volunteers. Conclusion The results show the utility of the model to integrate a wide range of in vitro and in vivo data and simulate the outcome of clinical studies, with implications for their design. Electronic supplementary material The online version of this article (doi:10.1007/s40262-013-0097-y) contains supplementary material, which is available to authorized users.

A P-glycoprotein (P-gp) IC₅₀ working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC₅₀ determinations. Each laboratory followed its in-house protocol to determine in vitro IC₅₀ values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC₅₀ values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC₅₀ values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC₅₀ values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC₅₀ values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC₅₀ values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC₅₀ determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Itopride, a substrate of FMO3, has been used for the symptomatic treatment of various gastrointestinal disorders. Physiologically based pharmacokinetic (PBPK) modeling was applied to evaluate the impact of FMO3 polymorphism on itopride pharmacokinetics (PK). The Asian populations within the Simcyp simulator were updated to incorporate information on the frequency, activity and abundance of FMO3 enzyme with different phenotypes. A meta-analysis of relative enzyme activities suggested that FMO3 activity in subjects with homozygous Glu158Lys and Glu308Gly mutations (Lys158 and Gly308) in both alleles is ~47% lower than those carrying two wild-type FMO3 alleles. Individuals with homozygous Lys158 and Gly308 mutations account for about 5% of the total population in Asian populations. A CLint of 9 μl/min/pmol was optimised for itopride via a retrograde approach as human liver microsomal results would under-predict its clearance by ~7.9-fold. The developed itopride PBPK model was first verified with three additional clinical studies in Korean and Japanese subjects resulting in a predicted clearance of 52 to 69 l/h, which was comparable to those observed (55 to 88 l/h). The model was then applied to predict plasma concentration-time profiles of itopride in Chinese subjects with wild type or homozygous Lys158 and Gly308 FMO3 genotypes. The ratios of predicted to observed AUC of itopride in subjects with each genotype were 1.23 and 0.94, respectively. In addition, the results also suggested that for FMO3 metabolised drugs with a safety margin of 2 or more, proactive genotyping FMO3 to exclude subjects with homozygous Lys158/Gly308 alleles may not be necessary.