Publications by authors named "Diane Ramsden"

17 Publications

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The Nonclinical Disposition and PK/PD Properties of GalNAc-conjugated siRNA Are Highly Predictable and Build Confidence in Translation to Man.

Drug Metab Dispos 2021 Jun 21. Epub 2021 Jun 21.

Alnylam Pharmaceuticals Inc., United States.

Conjugation of oligonucleotide therapeutics, including small interfering ribonucleic acids (siRNAs) or antisense oligonucleotides (ASOs) 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. While much knowledge has been gained over decades of development there is a paucity of published literature on the DMPK properties of GalNAc-siRNA. With this in mind the goals of this mini-review are to provide an aggregate analysis of these nonclinical 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 ASGPR-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 (RISC) in hepatocytes are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the absorption, distribution, metabolism and excretion (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, 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. 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 PK/PD 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.
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http://dx.doi.org/10.1124/dmd.121.000428DOI Listing
June 2021

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

Xenobiotica 2021 Jun 21;51(6):668-679. Epub 2021 Apr 21.

Global Drug Metabolism and Pharmacokinetics, Takeda Pharmaceuticals International Co, Cambridge, MA, USA.

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.
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http://dx.doi.org/10.1080/00498254.2021.1912438DOI Listing
June 2021

Cardiovascular microphysiological systems (CVMPS) for safety studies - a pharma perspective.

Lab Chip 2021 02;21(3):458-472

Integrative Pharmacology, Integrated Science and Technology, AbbVie, 1 Waukegan Rd, N Chicago, IL 60064, USA.

The integrative responses of the cardiovascular (CV) system are essential for maintaining blood flow to provide oxygenation, nutrients, and waste removal for the entire body. Progress has been made in independently developing simple in vitro models of two primary components of the CV system, namely the heart (using induced pluripotent stem-cell derived cardiomyocytes) and the vasculature (using endothelial cells and smooth muscle cells). These two in vitro biomimics are often described as immature and simplistic, and typically lack the structural complexity of native tissues. Despite these limitations, they have proven useful for specific "fit for purpose" applications, including early safety screening. More complex in vitro models offer the tantalizing prospect of greater refinement in risk assessments. To this end, efforts to physically link cardiac and vascular components to mimic a true CV microphysiological system (CVMPS) are ongoing, with the goal of providing a more holistic and integrated CV response model. The challenges of building and implementing CVMPS in future pharmacological safety studies are many, and include a) the need for more complex (and hence mature) cell types and tissues, b) the need for more realistic vasculature (within and across co-modeled tissues), and c) the need to meaningfully couple these two components to allow for integrated CV responses. Initial success will likely come with simple, bioengineered tissue models coupled with fluidics intended to mirror a vascular component. While the development of more complex integrated CVMPS models that are capable of differentiating safe compounds and providing mechanistic evaluations of CV liabilities may be feasible, adoption by pharma will ultimately hinge on model efficiency, experimental reproducibility, and added value above current strategies.
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http://dx.doi.org/10.1039/d0lc01040eDOI Listing
February 2021

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.

Drug Metab Dispos 2021 Jan 2;49(1):94-110. Epub 2020 Nov 2.

Genentech, South San Francisco, California (S.G.W.); Alnylam Pharmaceuticals, Cambridge, Massachusetts (D.R.); Janssen R&D, Spring House, Pennsylvania (S.D.); Vertex Pharmaceuticals, Boston, Massachusetts (C.F., N.H.); Novartis, East Hanover, New Jersey (H.J.E.); GlaxoSmithKline, King of Prussia, Pennsylvania (L.C.); Pfizer Global Research and Development, Groton, Connecticut (T.C.G.); Pfizer Global Research and Development, Cambridge, Massachusetts (P.D.Y.); Eisai, Cambridge, Massachusetts (Y.A.S.); Corning Life Sciences, Woburn, Massachusetts (G.Z.); Merck & Co., Inc., Kenilworth, New Jersey (D.T., J.P.); and AstraZeneca, Cambridge, Cambridgeshire, United Kingdom (B.J.).

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 (E and EC) 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 E/EC 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.
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http://dx.doi.org/10.1124/dmd.120.000055DOI Listing
January 2021

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.

Drug Metab Dispos 2019 10 22;47(10):1206-1221. Epub 2019 Aug 22.

Alnylam Pharmaceuticals, Cambridge, Massachusetts (D.R.); Vertex Pharmaceuticals, Boston, Massachusetts (C.F., N.H., S.R.); Genentech, South San Francisco, California (J.R.K.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Roche Innovation Center, Basel, Switzerland (N.J.P.); and Merck & Co., Inc., Kenilworth, New Jersey (D.T.).

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.
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http://dx.doi.org/10.1124/dmd.119.087270DOI Listing
October 2019

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

Drug Metab Dispos 2019 10 3;47(10):1183-1194. Epub 2019 Jul 3.

Alnylam Pharmaceuticals, Inc., Cambridge, Massachusetts (D.R., J.-T.W., J.J., V.C., K.A., Y.G., S.D., J.K., S.C.); The Medicines Company, Parsippany, New Jersey (B.Z.); and Sanofi, Waltham, Massachusetts (S.I.).

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 -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 value of 28 M is >3000-fold above the therapeutic clinical 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.
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http://dx.doi.org/10.1124/dmd.119.087098DOI Listing
October 2019

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

Drug Metab Dispos 2018 10 1;46(10):1434-1440. Epub 2018 Aug 1.

Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut

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, ()-6-(5-fluoro-4-(1-hydroxyethyl)pyridin-3-yl)-3,4-dihydro-1,8-naphthyridine-1()-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.
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http://dx.doi.org/10.1124/dmd.118.082487DOI Listing
October 2018

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.

Drug Metab Dispos 2018 Sep 29;46(9):1285-1303. Epub 2018 Jun 29.

Genentech, South San Francisco, California (J.R.K.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Sekisui-XenoTech LLC, Kansas City, Kansas (D.B.B.); Janssen R&D, Spring House, Pennsylvania (S.D.); Vertex Pharmaceuticals, Boston, Massachusetts (C.F., N.H.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Novartis, East Hanover, New Jersey (H.J.E.); GlaxoSmithKline, King of Prussia, Pennsylvania (L.C.); Amgen Inc., Cambridge, Massachusetts (J.G.D.); Sanofi, Waltham, Massachusetts (M.F.); Pfizer Global Research and Development, Groton, Connecticut (T.C.G.); Eisai, Andover, Massachusetts (Y.A.S.); EMD Serono R&D Institute, Inc., Billerica, Massachusetts (R.L.W.); Corning Life Sciences, Woburn, Massachusetts (G.Z.); and Merck & Co., Inc., Kenilworth, New Jersey (D.T.).

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/EC 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.
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http://dx.doi.org/10.1124/dmd.118.081927DOI Listing
September 2018

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.

Drug Metab Dispos 2017 10 23;45(10):1049-1059. Epub 2017 Jun 23.

Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.).

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.
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http://dx.doi.org/10.1124/dmd.116.074567DOI Listing
October 2017

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

Drug Metab Dispos 2016 10 15;44(10):1720-30. Epub 2016 Jul 15.

Pfizer Inc., Groton, Connecticut (O.A.F.); AbbVie Inc., North Chicago, Illinois (M.S.); Merck Research Laboratories, Rahway, New Jersey (J.P.); Bristol-Myers Squibb, Wallingford, Connecticut (M.W.S.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Novartis, East Hanover, New Jersey (H.J.E.); GlaxoSmithKline, King of Prussia, Pennsylvania (L.C.); and University of Maryland School of Pharmacy, Baltimore, Maryland (H.W.).

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.
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http://dx.doi.org/10.1124/dmd.116.071076DOI Listing
October 2016

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

Drug Metab Dispos 2016 Mar 18;44(3):466-75. Epub 2015 Dec 18.

Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut (R.S.S, D.R., J.P.S., L.R., N.T., A.W.J, D.J.T) and Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada (C.C.).

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.
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http://dx.doi.org/10.1124/dmd.115.066985DOI Listing
March 2016

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

Drug Metab Dispos 2015 Sep 15;43(9):1307-15. Epub 2015 Jun 15.

Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut.

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.
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http://dx.doi.org/10.1124/dmd.115.065326DOI Listing
September 2015

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

Antimicrob Agents Chemother 2015 Jan 13;59(1):25-37. Epub 2014 Oct 13.

Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut, USA

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.
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http://dx.doi.org/10.1128/AAC.03861-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291358PMC
January 2015

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

Drug Metab Dispos 2014 Nov 25;42(11):1940-6. Epub 2014 Aug 25.

Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut (D.R., D.J.T., T.S.C.); and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky (T.S.T.).

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.
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http://dx.doi.org/10.1124/dmd.114.057901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201135PMC
November 2014

Generating an in vitro-in vivo correlation for metabolism and liver enrichment of a hepatitis C virus drug, faldaprevir, using a rat hepatocyte model (HepatoPac).

Drug Metab Dispos 2014 Mar 23;42(3):407-14. Epub 2013 Dec 23.

Drug Metabolism & Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut.

Hepatocytes provide an integrated model to study drug metabolism and disposition. As a result of a loss of polarity or a significant decrease in the expression of enzymes and transporters, suspended and sandwich-cultured hepatocytes have limitations in determining hepatocellular drug concentrations. Underprediction of the extent of glucuronidation is also a concern for these hepatocyte models. Faldaprevir is a hepatitis C virus protease inhibitor in late-stage development that has demonstrated significant liver enrichment in in vivo rat models based on quantitative whole-body autoradiography (QWBA) and liver-to-plasma area under-the-curve ratio. In bile duct cannulated rats, the primary biliary metabolite was a glucuronide. Owing to ethical concerns, it is difficult to assess liver enrichment in humans, and a lack of in vitro and in vivo correlation of glucuronidation has been reported. The current study was conducted to verify whether a hepatocyte model, rat HepatoPac, could overcome some of these limitations and provide validity for follow-up studies with human HepatoPac. With rat HepatoPac, liver enrichment values averaged 34-fold and were consistent with rat QWBA (26.8-fold) and in vivo data (42-fold). In contrast, liver enrichment in suspended hepatocytes was only 2.8-fold. Furthermore, the extent of faldaprevir glucuronidation in HepatoPac studies was in agreement with in vivo results, with glucuronidation as the major pathway (96%). Suspended rat hepatocytes did not generate the glucuronide or two key hydroxylated metabolites that were observed in vivo. Overall, our studies suggest that HepatoPac is a promising in vitro model to predict in vivo liver enrichment and metabolism, especially for glucuronidation, and has demonstrated superiority over suspended hepatocytes.
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http://dx.doi.org/10.1124/dmd.113.055947DOI Listing
March 2014

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

Drug Metab Dispos 2014 Mar 23;42(3):394-406. Epub 2013 Dec 23.

Drug Metabolism & Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut.

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.
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http://dx.doi.org/10.1124/dmd.113.055897DOI Listing
March 2014

Enzyme-transporter interplay in the formation and clearance of abundant metabolites of faldaprevir found in excreta but not in circulation.

Drug Metab Dispos 2014 Mar 17;42(3):384-93. Epub 2013 Dec 17.

Drug Metabolism & Pharmacokinetics (Y.L., J.Z., D.R., M.E.T., D.O., J.X., D.J.T.), Chemical Development (C.A.B.), and Analytical Development (N.G.), Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut.

Faldaprevir is a hepatitis C virus protease inhibitor that effectively reduces viral load in patients. Since faldaprevir exhibits slow metabolism in vitro and low clearance in vivo, metabolism was expected to be a minor clearance pathway. The human [(14)C] absorption, distribution, metabolism, and excretion study revealed that two monohydroxylated metabolites (M2a and M2b) were the most abundant excretory metabolites in feces, constituting 41% of the total administered dose. To deconvolute the formation and disposition of M2a and M2b in humans and determine why the minor change in structure [the addition of 16 atomic mass units (amu)] produced chemical entities that were excreted and were not present in the circulation, multiple in vitro test systems were used. The results from these in vitro studies clarified the formation and clearance of M2a and M2b. Faldaprevir is metabolized primarily in the liver by CYP3A4/5 to form M2a and M2b, which are also substrates of efflux transporters (P-glycoprotein and breast cancer resistance protein). The role of transporters is considered important for M2a and M2b as they demonstrate low permeability. It is proposed that both metabolites are efficiently excreted via bile into feces and do not enter the systemic circulation to an appreciable extent. If these metabolites permeate to blood, they can be readily taken up into hepatocytes from the circulation by uptake transporters (likely organic anion transporting polypeptides). These results highlight the critical role of drug-metabolizing enzymes and multiple transporters in the process of the formation and clearance of faldaprevir metabolites. Faldaprevir metabolism also provides an interesting case study for metabolites that are exclusively excreted in feces but are of clinical relevance.
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http://dx.doi.org/10.1124/dmd.113.055863DOI Listing
March 2014
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