Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

Drug transporters in drug disposition - the year 2022 in review.

On behalf of all the authors, I am pleased to share our third annual review on drug transporter science with an emphasis on articles published and deemed influential in signifying drug transporters' role in drug disposition in the year 2022. As the drug transporter field is rapidly evolving several key findings were noted including promising endogenous biomarkers, rhythmic activity, IVIVE approaches in transporter-mediated clearance, new modality interaction, and transporter effect on gut microbiome. As identified previously (Chothe et Cal. 2021, 2022) the goal of this review is to highlight key findings without a comprehensive overview of each article and to this end, each coauthor independently selected 1-3 peer-reviewed articles published or available online in the year 2022 (Table 1). Each article is summarized in synopsis and commentary with unbiased viewpoints by each coauthor. We strongly encourage readers to consult original articles for specifics of the study. Finally, I would like to thank all coauthors for their continued support in writing this annual review on drug transporters and invite anyone interested in contributing to future versions of this review.

Innovations, Opportunities, and Challenges for Predicting Alteration in Drug-Metabolizing Enzyme and Transporter Activity in Specific Populations.

Drug-metabolizing enzymes and transporters (DMETs) are key regulators of the pharmacokinetics, efficacy, and toxicity of therapeutics. Over the past two decades, significant advancements in in vitro methodologies, targeted proteomics, in vitro to in vivo extrapolation methods, and integrated computational approaches such as physiologically based pharmacokinetic modeling have unequivocally contributed to improving our ability to quantitatively predict the role of DMETs in absorption, distribution, metabolism, and excretion and drug-drug interactions. However, the paucity of data regarding alterations in DMET activity in specific populations such as pregnant individuals, lactation, pediatrics, geriatrics, organ impairment, and disease states such as, cancer, kidney, and liver diseases and inflammation has restricted our ability to realize the full potential of these recent advancements. We envision that a series of carefully curated articles in a special supplementary issue of Drug Metabolism and Disposition will summarize the latest progress in in silico, in vitro, and in vivo approaches to characterize alteration in DMET activity and quantitatively predict drug disposition in specific populations. In addition, the supplementary issue will underscore the current scientific knowledge gaps that present formidable barriers to fully understand the clinical implications of altered DMET activity in specific populations and highlight opportunities for multistakeholder collaboration to advance our collective understanding of this rapidly emerging area. SIGNIFICANCE STATEMENT: This commentary highlights current knowledge and identifies gaps and key challenges in understanding the role of drug-metabolizing enzymes and transporters (DMETs) in drug disposition in specific populations. With this commentary for the special issue in Drug Metabolism and Disposition, the authors intend to increase interest and invite potential contributors whose research is focused or has aided in expanding the understanding around the role and impact of DMETs in drug disposition in specific populations.

Novel insights in drug transporter sciences: the year 2021 in review.

On behalf of the team I am pleased to present the second annual 'novel insights into drug transporter sciences review' focused on peer-reviewed articles that were published in the year 2021. In compiling the articles for inclusion, preprints available in 2021 but officially published in 2022 were considered to be in scope. To support this review the contributing authors independently selected one or two articles that were thought to be impactful and of interest to the broader research community. A similar approach as published last year was adopted whereby key observations, methods and analysis of each paper is concisely summarized in the synopsis followed by a commentary highlighting the impact of the paper in understanding drug transporters' role in drug disposition. As the goal of this review is not to provide a comprehensive overview of each paper but rather highlight important findings that are well supported by the data, the reader is encouraged to consult the original articles for additional information. Further, and keeping in line with the goals of this review, it should be noted that all authors actively contributed by writing synopsis and commentary for individual papers and no attempt was made to standardize language or writing styles. In this way, the review article is reflective of not only the diversity of the articles but also that of the contributors. I extend my thanks to the authors for their continued support and also welcome Diane Ramsden and Pallabi Mitra as contributing authors for this issue (Table 1).[Table: see text].

Predictive In Vitro-In Vivo Extrapolation for Time Dependent Inhibition of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 Using Pooled Human Hepatocytes, Human Liver Microsomes, and a Simple Mechanistic Static Model.

Inactivation of Cytochrome P450 (CYP450) enzymes can lead to significant increases in exposure of comedicants. The majority of reported in vitro to in vivo extrapolation (IVIVE) data have historically focused on CYP3A, leaving the assessment of other CYP isoforms insubstantial. To this end, the utility of human hepatocytes (HHEP) and human liver microsomes (HLM) to predict clinically relevant drug-drug interactions was investigated with a focus on CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Evaluation of IVIVE for CYP2B6 was limited to only weak inhibition. A search of the University of Washington Drug-Drug Interaction Database was conducted to identify a clinically relevant weak, moderate, and strong inhibitor for selective substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, resulting in 18 inhibitors for in vitro characterization against 119 clinical interaction studies. Pooled human hepatocytes and HLM were preincubated with increasing concentrations of inhibitors for designated timepoints. Time dependent inhibition was detected in HLM for four moderate/strong inhibitors, suggesting that some optimization of incubation conditions (i.e., lower protein concentrations) is needed to capture weak inhibition. Clinical risk assessment was conducted by incorporating the in vitro derived kinetic parameters maximal rate of enzyme inactivation (min-1) (kinact) and concentration of inhibitor resulting in 50% of the maximum enzyme inactivation (KI) into static equations recommended by regulatory authorities. Significant overprediction was observed when applying the basic models recommended by regulatory agencies. Mechanistic static models, which consider the fraction of metabolism through the impacted enzyme, using the unbound hepatic inlet concentration lead to the best overall prediction accuracy with 92% and 85% of data from HHEPs and HLM, respectively, within twofold of the observed value. SIGNIFICANCE STATEMENT: Coupling time-dependent inactivation parameters derived from pooled human hepatocytes and human liver microsomes (HLM) with a mechanistic static model provides an easy and quantitatively accurate means to determine clinical drug-drug interaction risk from in vitro data. Optimization is needed to evaluate time-dependent inhibition (TDI) for weak and moderate inhibitors using HLM. Recommendations are made with respect to input parameters for in vitro to in vivo extrapolation (IVIVE) of TDI with non-CYP3A enzymes using available data from HLM and human hepatocytes.

The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N-Acetylgalactosamine-Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.

ADME and DMPK considerations for the discovery and development of antibody drug conjugates (ADCs).

 The therapeutic concept of antibody drug conjugates (ADCs) is to selectively target tumour cells with small molecule cytotoxic drugs to maximise cell kill benefit and minimise healthy tissue toxicity.An ADC generally consists of an antibody that targets a protein on the surface of tumour cells chemically linked to a warhead small molecule cytotoxic drug.To deliver the warhead to the tumour cell, the antibody must bind to the target protein and in general be internalised into the cell. Following internalisation, the cytotoxic agent can be released in the endosomal or lysosomal compartment (via different mechanisms). Diffusion or transport out of the endosome or lysosome allows the cytotoxic drug to express its cell-killing pharmacology. Alternatively, some ADCs (e.g. EDB-ADCs) rely on extracellular cleavage releasing membrane permeable warheads.One potentially important aspect of the ADC mechanism is the 'bystander effect' whereby the cytotoxic drug released in the targeted cell can diffuse out of that cell and into other (non-target expressing) tumour cells to exert its cytotoxic effect. This is important as solid tumours tend to be heterogeneous and not all cells in a tumour will express the targeted protein.The combination of large and small molecule aspects in an ADC poses significant challenges to the disposition scientist in describing the ADME properties of the entire molecule.This article will review the ADC landscape and the ADME properties of successful ADCs, with the aim of outlining best practice and providing a perspective of how the field can further facilitate the discovery and development of these important therapeutic modalities.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Guidelines on Model Fitting and Recommendations on Time Course for In Vitro Cytochrome P450 Induction Studies Including Impact on Drug Interaction Risk Assessment.

Translational and ADME Sciences Leadership Group Induction Working Group (IWG) presents an analysis on the time course for cytochrome P450 induction in primary human hepatocytes. Induction of CYP1A2, CYP2B6, and CYP3A4 was evaluated by seven IWG laboratories after incubation with prototypical inducers (omeprazole, phenobarbital, rifampicin, or efavirenz) for 6-72 hours. The effect of incubation duration and model-fitting approaches on induction parameters (Emax and EC50) and drug-drug interaction (DDI) risk assessment was determined. Despite variability in induction response across hepatocyte donors, the following recommendations are proposed: 1) 48 hours should be the primary time point for in vitro assessment of induction based on mRNA level or activity, with no further benefit from 72 hours; 2) when using mRNA, 24-hour incubations provide reliable assessment of induction and DDI risk; 3) if validated using prototypical inducers (>10-fold induction), 12-hour incubations may provide an estimate of induction potential, including characterization as negative if <2-fold induction of mRNA and no concentration dependence; 4) atypical dose-response ("bell-shaped") curves can be addressed by removing points outside an established confidence interval and %CV; 5) when maximum fold induction is well defined, the choice of nonlinear regression model has limited impact on estimated induction parameters; 6) when the maximum fold induction is not well defined, conservative DDI risk assessment can be obtained using sigmoidal three-parameter fit or constraining logistic three- or four-parameter fits to the maximum observed fold induction; 7) preliminary data suggest initial slope of the fold induction curve can be used to estimate Emax/EC50 and for induction risk assessment. SIGNIFICANCE STATEMENT: Regulatory agencies provide inconsistent guidance on the optimum length of time to evaluate cytochrome P450 induction in human hepatocytes, with EMA recommending 72 hours and FDA suggesting 48-72 hours. The Induction Working Group analyzed a large data set generated by seven member companies and determined that induction response and drug-drug risk assessment determined after 48-hour incubations were representative of 72-hour incubations. Additional recommendations are provided on model-fitting techniques for induction parameter estimation and addressing atypical concentration-response curves.

Evaluation of the drug-drug interaction potential for trazpiroben (TAK-906), a D2/D3 receptor antagonist for gastroparesis, towards cytochrome P450s and transporters.

Trazpiroben (TAK-906), a peripherally selective dopamine D2/D3 receptor antagonist, is being developed for the treatment of patients with gastroparesis. The potential of trazpiroben to act as a perpetrator or a victim for cytochrome P450 (CYP)- or transporter- mediated drug-drug interactions (DDIs) was evaluated following the latest regulatory guidelines.In vitro studies revealed that trazpiroben is metabolised mainly through a non-CYP pathway (56.7%) by multiple cytosolic, NADPH-dependent reductase, such as aldo-keto reductase and short-chain dehydrogenase/reductase including carbonyl reductases. Remaining metabolism occurs through CYP3A4 and CYP2C8 (43.3%). Trazpiroben is neither an inhibitor nor an inducer of major CYP enzymes at a clinically relevant dose. It is a substrate of P-glycoprotein (P-gp) and organic anion transporting polypeptide (OATP) 1B1/1B3, but is not an inhibitor of transporters listed in the DDI guidelines at a clinically relevant dose. This is consistent with findings from CYP3A and P-gp-based clinical assessment showing no substantial change (≤2-fold) in trazpiroben exposure when co-administered with itraconazole.Collectively, trazpiroben has low potential of enzyme-mediated DDIs and is unlikely to act as a perpetrator of transporter-mediated DDIs but there may be a potential to act as a victim of OATP1B1/1B3 DDI that will be evaluated clinically.

Perspectives from the Innovation and Quality Consortium Induction Working Group on Factors Impacting Clinical Drug-Drug Interactions Resulting from Induction: Focus on Cytochrome 3A Substrates.

A recent publication from the Innovation and Quality Consortium Induction Working Group collated a large clinical data set with the goal of evaluating the accuracy of drug-drug interaction (DDI) prediction from in vitro data. Somewhat surprisingly, comparison across studies of the mean- or median-reported area under the curve ratio showed appreciable variability in the magnitude of outcome. This commentary explores the possible drivers of this range of outcomes observed in clinical induction studies. While recommendations on clinical study design are not being proposed, some key observations were informative during the aggregate analysis of clinical data. Although DDI data are often presented using median data, individual data would enable evaluation of how differences in study design, baseline expression, and the number of subjects contribute. Since variability in perpetrator pharmacokinetics (PK) could impact the overall DDI interpretation, should this be routinely captured? Maximal induction was typically observed after 5-7 days of dosing. Thus, when the half-life of the inducer is less than 30 hours, are there benefits to a more standardized study design? A large proportion of CYP3A4 inducers were also CYP3A4 inhibitors and/or inactivators based on in vitro data. In these cases, using CYP3A selective substrates has limitations. More intensive monitoring of changes in area under the curve over time is warranted. With selective CYP3A substrates, the net effect was often inhibition, whereas less selective substrates could discern induction through mechanisms not susceptible to inhibition. The latter included oral contraceptives, which raise concerns of reduced efficacy following induction. Alternative approaches for modeling induction, such as applying biomarkers and physiologically based pharmacokinetic modeling (PBPK), are also considered. SIGNIFICANCE STATEMENT: The goal of this commentary is to stimulate discussion on whether there are opportunities to optimize clinical drug-drug interaction study design. The overall aim is to reduce, understand and contextualize the variability observed in the magnitude of induction across reported clinical studies. A large clinical CYP3A induction dataset was collected and further analyzed to identify trends and gaps. Reporting individual victim PK data, characterizing perpetrator PK and including additional PK assessments for mixed-mechanism perpetrators may provide insights into how these factors impact differences observed in clinical outcomes. The potential utility of biomarkers and PBPK modeling are discussed in considering future directions.

In Vitro Drug-Drug Interaction Evaluation of GalNAc Conjugated siRNAs Against CYP450 Enzymes and Transporters.

Small interfering RNAs (siRNAs) represent a new class of medicines that are smaller (∼16,000 Da) than biologic therapeutics (>150,000 Da) but much larger than small molecules (<900 Da). Current regulatory guidance on drug-drug interactions (DDIs) from the European Medicines Agency, Food and Drug Administration, and Pharmaceutical and Medical Devices Agency provides no recommendations for oligonucleotide therapeutics including siRNAs; therefore, small molecule guidance documents have historically been applied. Over ∼10 years, in vitro DDI investigations with siRNAs conjugated to a triantennary N-acetylgalactosamine [(GalNAc)-siRNA] ligand have been conducted during nonclinical drug development to elucidate the potential clinical DDI liability. GalNAc siRNAs were evaluated as substrates, inhibitors, or inducers of major cytochrome P450s (P450s) and as substrates and inhibitors of transporters. Aggregate analysis of these data demonstrates a low potential for DDI against P450s. Zero of five, 10, and seven are inducers, time-dependent inhibitors, or substrates, respectively, and nine of 12 do not inhibit any P450 isoform evaluated. Three GalNAc siRNAs inhibited CYP2C8 at supratherapeutic concentrations, and one mildly inhibited CYP2B6. The lowest K i value of 28 µM is >3000-fold above the therapeutic clinical C max at steady state, and importantly no clinical inhibition was projected. Of four GalNAc siRNAs tested none were substrates for transporters and one caused inhibition of P-glycoprotein, calculated not to be clinically relevant. The pharmacological basis for DDIs, including consideration of the target and/or off-target profiles for GalNAc siRNAs, should be made as part of the overall DDI risk assessment. If modulation of the target protein does not interfere with P450s or transporters, then in vitro or clinical investigations into the DDI potential of the GalNAc siRNAs are not warranted. SIGNIFICANCE STATEMENT: Recommendations for evaluating DDI potential of small molecule drugs are well established; however, guidance for novel modalities, particularly oligonucleotide-based therapeutics are lacking. Given the paucity of published data in this field, in vitro DDI investigations are often conducted. The aggregate analysis of GalNAc-siRNA data reviewed herein demonstrates that, like new biological entities, these oligonucleotide-based therapeutic drugs are unlikely to result in DDIs; therefore, it is recommended that the need for in vitro or clinical investigations similarly be determined on a case-by-case basis. Given the mechanism of siRNA action, special consideration should be made in cases where there may be a pharmacological basis for DDIs.

Identification and Characterization of a Selective Human Carbonyl Reductase 1 Substrate.

During drug discovery efforts targeting inhibition of cytochrome P450 11B2 (CYP11B2)-mediated production of aldosterone as a therapeutic approach for the treatment of chronic kidney disease and hypertension, (S)-6-(5-fluoro-4-(1-hydroxyethyl)pyridin-3-yl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (1) was identified as a potent and selective inhibitor of CYP11B2. Preclinical studies characterized 1 as low clearance in both in vitro test systems and in vivo in preclinical species. Despite low metabolic conversion, an active ketone metabolite (2) was identified from in vitro metabolite-identification studies. Due to the inhibitory activity of 2 against CYP11B2 as well as the potential for it to undergo reductive metabolism back to 1, the formation and elimination of 2 were characterized and are the focus of this manuscript. A series of in vitro investigations determined that 1 was slowly oxidized to 2 by cytochrome P450s 2D6, 3A4, and 3A5, followed by stereoselective reduction back to 1 and not its enantiomer (3). Importantly, reduction of 2 was mediated by an NADPH-dependent cytosolic enzyme. Studies with human cytosolic fractions from multiple tissues, selective inhibitors, and recombinantly expressed enzymes indicated that carbonyl reductase 1 (CBR1) is responsible for this transformation in humans. Carbonyl reduction is emerging as an important pathway for endogenous and xenobiotic metabolism. With a lack of selective substrates and inhibitors to enable characterization of the involvement of CBR1, 2 could be a useful probe to assess CBR1 activity in vitro in both subcellular fractions and in cell-based systems.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA In Vitro Response Thresholds, Variability, and Clinical Relevance.

The Innovation and Quality Induction Working Group presents an assessment of best practice for data interpretation of in vitro induction, specifically, response thresholds, variability, application of controls, and translation to clinical risk assessment with focus on CYP3A4 mRNA. Single concentration control data and Emax/EC50 data for prototypical CYP3A4 inducers were compiled from many human hepatocyte donors in different laboratories. Clinical CYP3A induction and in vitro data were gathered for 51 compounds, 16 of which were proprietary. A large degree of variability was observed in both the clinical and in vitro induction responses; however, analysis confirmed in vitro data are able to predict clinical induction risk. Following extensive examination of this large data set, the following recommendations are proposed. a) Cytochrome P450 induction should continue to be evaluated in three separate human donors in vitro. b) In light of empirically divergent responses in rifampicin control and most test inducers, normalization of data to percent positive control appears to be of limited benefit. c) With concentration dependence, 2-fold induction is an acceptable threshold for positive identification of in vitro CYP3A4 mRNA induction. d) To reduce the risk of false positives, in the absence of a concentration-dependent response, induction ≥ 2-fold should be observed in more than one donor to classify a compound as an in vitro inducer. e) If qualifying a compound as negative for CYP3A4 mRNA induction, the magnitude of maximal rifampicin response in that donor should be ≥ 10-fold. f) Inclusion of a negative control adds no value beyond that of the vehicle control.

Considerations from the IQ Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Focus on Downregulation, CYP2C Induction, and CYP2B6 Positive Control.

The European Medicines Agency (EMA), the Pharmaceutical and Medical Devices Agency (PMDA), and the Food and Drug Administration (FDA) have issued guidelines for the conduct of drug-drug interaction studies. To examine the applicability of these regulatory recommendations specifically for induction, a group of scientists, under the auspices of the Drug Metabolism Leadership Group of the Innovation and Quality (IQ) Consortium, formed the Induction Working Group (IWG). A team of 19 scientists, from 16 of the 39 pharmaceutical companies that are members of the IQ Consortium and two Contract Research Organizations reviewed the recommendations, focusing initially on the current EMA guidelines. Questions were collated from IQ member companies as to which aspects of the guidelines require further evaluation. The EMA was then approached to provide insights into their recommendations on the following: 1) evaluation of downregulation, 2) in vitro assessment of CYP2C induction, 3) the use of CITCO as the positive control for CYP2B6 induction by CAR, 4) data interpretation (a 2-fold increase in mRNA as evidence of induction), and 5) the duration of incubation of hepatocytes with test article. The IWG conducted an anonymous survey among IQ member companies to query current practices, focusing specifically on the aforementioned key points. Responses were received from 19 companies. All data and information were blinded before being shared with the IWG. The results of the survey are presented, together with consensus recommendations on downregulation, CYP2C induction, and CYP2B6 positive control. Results and recommendations related to data interpretation and induction time course will be reported in subsequent articles.

Evaluation of CYP2B6 Induction and Prediction of Clinical Drug-Drug Interactions: Considerations from the IQ Consortium Induction Working Group-An Industry Perspective.

Drug-drug interactions (DDIs) due to CYP2B6 induction have recently gained prominence and clinical induction risk assessment is recommended by regulatory agencies. This work aimed to evaluate the potency of CYP2B6 versus CYP3A4 induction in vitro and from clinical studies and to assess the predictability of efavirenz versus bupropion as clinical probe substrates of CYP2B6 induction. The analysis indicates that the magnitude of CYP3A4 induction was higher than CYP2B6 both in vitro and in vivo. The magnitude of DDIs caused by induction could not be predicted for bupropion with static or dynamic models. On the other hand, the relative induction score, net effect, and physiologically based pharmacokinetics SimCYP models using efavirenz resulted in improved DDI predictions. Although bupropion and efavirenz have been used and are recommended by regulatory agencies as clinical CYP2B6 probe substrates for DDI studies, CYP3A4 contributes to the metabolism of both probes and is induced by all reference CYP2B6 inducers. Therefore, caution must be taken when interpreting clinical induction results because of the lack of selectivity of these probes. Although in vitro-in vivo extrapolation for efavirenz performed better than bupropion, interpretation of the clinical change in exposure is confounded by the coinduction of CYP2B6 and CYP3A4, as well as the increased contribution of CYP3A4 to efavirenz metabolism under induced conditions. Current methods and probe substrates preclude accurate prediction of CYP2B6 induction. Identification of a sensitive and selective clinical substrate for CYP2B6 (fraction metabolized > 0.9) is needed to improve in vitro-in vivo extrapolation for characterizing the potential for CYP2B6-mediated DDIs. Alternative strategies and a framework for evaluating the CYP2B6 induction risk are proposed.

Contribution of Major Metabolites toward Complex Drug-Drug Interactions of Deleobuvir: In Vitro Predictions and In Vivo Outcomes.

The drug-drug interaction (DDI) potential of deleobuvir, an hepatitis C virus (HCV) polymerase inhibitor, and its two major metabolites, CD 6168 (formed via reduction by gut bacteria) and deleobuvir-acyl glucuronide (AG), was assessed in vitro. Area-under-the-curve (AUC) ratios (AUCi/AUC) were predicted using a static model and compared with actual AUC ratios for probe substrates in a P450 cocktail of caffeine (CYP1A2), tolbutamide (CYP2C9), and midazolam (CYP3A4), administered before and after 8 days of deleobuvir administration to HCV-infected patients. In vitro studies assessed inhibition, inactivation and induction of P450s. Induction was assessed in a short-incubation (10 hours) hepatocyte assay, validated using positive controls, to circumvent cytotoxicity seen with deleobuvir and its metabolites. Overall, P450 isoforms were differentially affected by deleobuvir and its two metabolites. Of note was more potent CYP2C8 inactivation by deleobuvir-AG than deleobuvir and P450 induction by CD 6168 but not by deleobuvir. The predicted net AUC ratios for probe substrates were 2.92 (CYP1A2), 0.45 (CYP2C9), and 0.97 (CYP3A4) compared with clinically observed ratios of 1.64 (CYP1A2), 0.86 (CYP2C9), and 1.23 (CYP3A4). Predictions of DDI using deleobuvir alone would have significantly over-predicted the DDI potential for CYP3A4 inhibition (AUC ratio of 6.15). Including metabolite data brought the predicted net effect close to the observed DDI. However, the static model over-predicted the induction of CYP2C9 and inhibition/inactivation of CYP1A2. This multiple-perpetrator DDI scenario highlights the application of the static model for predicting complex DDI for CYP3A4 and exemplifies the importance of including key metabolites in an overall DDI assessment.

Mass balance, metabolite profile, and in vitro-in vivo comparison of clearance pathways of deleobuvir, a hepatitis C virus polymerase inhibitor.

The pharmacokinetics, mass balance, and metabolism of deleobuvir, a hepatitis C virus (HCV) polymerase inhibitor, were assessed in healthy subjects following a single oral dose of 800 mg of [(14)C]deleobuvir (100 µCi). The overall recovery of radioactivity was 95.2%, with 95.1% recovered from feces. Deleobuvir had moderate to high clearance, and the half-life of deleobuvir and radioactivity in plasma were ∼ 3 h, indicating that there were no metabolites with half-lives significantly longer than that of the parent. The most frequently reported adverse events (in 6 of 12 subjects) were gastrointestinal disorders. Two major metabolites of deleobuvir were identified in plasma: an acyl glucuronide and an alkene reduction metabolite formed in the gastrointestinal (GI) tract by gut bacteria (CD 6168), representing ∼ 20% and 15% of the total drug-related material, respectively. Deleobuvir and CD 6168 were the main components in the fecal samples, each representing ∼ 30 to 35% of the dose. The majority of the remaining radioactivity found in the fecal samples (∼ 21% of the dose) was accounted for by three metabolites in which deleobuvir underwent both alkene reduction and monohydroxylation. In fresh human hepatocytes that form biliary canaliculi in sandwich cultures, the biliary excretion for these excretory metabolites was markedly higher than that for deleobuvir and CD 6168, implying that rapid biliary elimination upon hepatic formation may underlie the absence of these metabolites in circulation. The low in vitro clearance was not predictive of the observed in vivo clearance, likely because major deleobuvir biotransformation occurred by non-CYP450-mediated enzymes that are not well represented in hepatocyte-based in vitro models.

Determination of a Degradation Constant for CYP3A4 by Direct Suppression of mRNA in a Novel Human Hepatocyte Model, HepatoPac.

Accurate determination of rates of de novo synthesis and degradation of cytochrome P450s (P450s) has been challenging. There is a high degree of variability in the multiple published values of turnover for specific P450s that is likely exacerbated by differences in methodologies. For CYP3A4, reported half-life values range from 10 to 140 hours. An accurate value for kdeg has been identified as a major limitation for prediction of drug interactions involving mechanism-based inhibition and/or induction. Estimation of P450 half-life from in vitro test systems, such as human hepatocytes, is complicated by differential decreased enzyme function over culture time, attenuation of the impact of enzyme loss through inclusion of glucocorticoids in media, and viability limitations over long-term culture times. HepatoPac overcomes some of these challenges by providing extended stability of enzymes (2.5 weeks in our hands). As such it is a unique tool for studying rates of enzyme degradation achieved through modulation of enzyme levels. CYP3A4 mRNA levels were rapidly depleted by >90% using either small interfering RNA or addition of interleukin-6, which allowed an estimation of the degradation rate constant for CYP3A protein over an incubation time of 96 hours. The degradation rate constant of 0.0240 ± 0.005 hour(-1) was reproducible in hepatocytes from five different human donors. These donors also reflected the overall population with respect to CYP3A5 genotype. This methodology can be applied to additional enzymes and may provide a more accurate in vitro derived kdeg value for predicting clinical drug-drug interaction outcomes.

Altered CYP2C9 activity following modulation of CYP3A4 levels in human hepatocytes: an example of protein-protein interactions.

Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings.

Bridging in vitro and in vivo metabolism and transport of faldaprevir in human using a novel cocultured human hepatocyte system, HepatoPac.

An increased appreciation of the importance of transporter and enzyme interplay in drug clearance and a desire to delineate these mechanisms necessitates the utilization of models that contain a full complement of enzymes and transporters at physiologically relevant activities. Additionally, the development of drugs with longer half-lives requires in vitro systems with extended incubation times that allow characterization of metabolic pathways for low-clearance drugs. A recently developed coculture hepatocyte model, HepatoPac, has been applied to meet these challenges. Faldaprevir is a drug in late-stage development for the treatment of hepatitis C. Faldaprevir is a low-clearance drug with the somewhat unique characteristic of being slowly metabolized, producing two abundant hydroxylated metabolites (M2a and M2b) in feces (∼40% of the dose) without exhibiting significant levels of circulating metabolites in humans. The human HepatoPac model was investigated to characterize the metabolism and transport of faldaprevir. In human HepatoPac cultures, M2a and M2b were the predominant metabolites formed, with extents of formation comparable to in vivo. Direct glucuronidation of faldaprevir was shown to be a minor metabolic pathway. HepatoPac studies also demonstrated that faldaprevir is concentrated in liver with active uptake by multiple transporters (including OATP1B1 and Na(+)-dependent transporters). Overall, human HepatoPac cultures provided valuable insights into the metabolism and disposition of faldaprevir in humans and demonstrated the importance of enzyme and transporter interplay in the clearance of the drug.