Publications by authors named "Niresh Hariparsad"

21 Publications

  • Page 1 of 1

Function and Expression of Bile Salt Export Pump in Suspension Human Hepatocytes.

Drug Metab Dispos 2021 Apr 20;49(4):314-321. Epub 2021 Jan 20.

Drug Metabolism and Pharmacokinetics, Vertex Pharmaceuticals Incorporated, Boston, Massachusetts.

The mechanistic understanding of bile salt disposition is not well established in suspension human hepatocytes (SHH) because of the limited information on the expression and function of bile salt export protein (BSEP) in this system. We investigated the transport function of BSEP in SHH using a method involving in situ biosynthesis of bile salts from their precursor bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). Our data indicated that glycine- and taurine-conjugated CA and CDCA were generated efficiently and transported out of hepatocytes in a concentration- and time-dependent manner. We also observed that the membrane protein abundance of BSEP was similar between SHH and sandwich-cultured human hepatocytes. Furthermore, known cholestatic agents significantly inhibited G-CA and G-CDCA efflux in SHH. Interestingly, cyclosporine A, troglitazone, itraconazole, loratadine, and lovastatin inhibited G-CA efflux more potently than G-CDCA efflux (3- to 5-fold). Because of these significant differential effects on G-CA and G-CDCA efflux inhibition, we determined the IC values of troglitazone for G-CA (9.9 µM) and for G-CDCA (43.1 µM) efflux. The observed discrepancy in the IC was attributed to the fact that troglitazone also inhibits organic anion transporting polypeptides and Na/taurocholate cotransporting polypeptide in addition to BSEP. The hepatocyte uptake study suggested that both active uptake and passive diffusion contribute to the liver uptake of CA, whereas CDCA primarily undergoes passive diffusion into the liver. In summary, these data demonstrated the expression and function of BSEP and its major role in transport of bile salts in cryopreserved SHH. SIGNIFICANCE STATEMENT: BSEP transport function and protein abundance was evident in SHH in the present study. The membrane abundance of BSEP protein was similar between SHH and sandwich-cultured human hepatocytes. The study also illustrated the major role of BSEP relative to basolateral MRP3 and MRP4 in transport of bile salts in SHH. Understanding of BSEP function in SHH may bolster the utility of this platform in mechanistic understanding of bile salt disposition and potentially in the assessment of drugs for BSEP inhibition.
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http://dx.doi.org/10.1124/dmd.120.000057DOI Listing
April 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

Smallest Maximum Intramolecular Distance: A Novel Method to Mitigate Pregnane Xenobiotic Receptor Activation.

J Chem Inf Model 2020 04 16;60(4):2091-2099. Epub 2020 Mar 16.

Vertex Pharmaceuticals, 50 Northern Avenue, Boston, Massachusetts 02210, United States.

Induction of cytochrome P450 isoform 3A4 via activation of the pregnane xenobiotic receptor (PXR) is a concern for pharmaceutical discovery and development, as it can lead to drug-drug interactions. We present a novel molecular descriptor, the smallest maximum intramolecular distance (SMID), which is correlated with PXR activation, and a method for using the SMID descriptor to guide discovery chemists in modifying lead compounds to decrease PXR activation.
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http://dx.doi.org/10.1021/acs.jcim.9b00692DOI Listing
April 2020

A pharmaceutical industry perspective on microphysiological kidney systems for evaluation of safety for new therapies.

Lab Chip 2020 02 28;20(3):468-476. Epub 2020 Jan 28.

Preclinical Safety, AbbVie, 1 Waukegan Rd, N Chicago, IL 60064, USA.

The human kidney contains approximately one million nephrons. As the functional unit of the kidney, the nephron affords an opportunity to approximate the kidney at a microphysiological scale. Recent emergence of physiologically accurate human tissue models has radically advanced the possibilities of mimicking organ biology and multi-organ combinations in vitro. Anatomically, the nephron is one of the most complex, sequentially integrated microfluidic units in the body making the miniaturized microfluidic systems excellent candidates for capturing the kidney biology in vitro. While these models are promising, there are a number of considerations for practical implementation into a drug development paradigm. Opportunities for pharmaceutical industry applications of new MPS models often start with drug safety testing. As such, the intent of this article is to focus on safety and ADME applications. This article reviews biological functions of the kidney and options for characterizing known roles in nephrotoxicity. The concept of "context-of-use" is introduced as a framework for describing and verifying the specific features of an MPS platform for use in drug development. Overall, we present a perspective on key attributes of microphysiological kidney models, which the pharmaceutical industry could leverage to improve confident safety and ADME evaluations of experimental therapies.
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http://dx.doi.org/10.1039/c9lc00925fDOI Listing
February 2020

Microphysiological systems for ADME-related applications: current status and recommendations for system development and characterization.

Lab Chip 2020 02 14;20(3):446-467. Epub 2020 Jan 14.

Pharmaceutical Candidate Optimization, Bristol-Myers Squibb R&D, PO Box 4000, Princeton, NJ 08543-4000, USA.

Over the last decade, progress has been made on the development of microphysiological systems (MPS) for absorption, distribution, metabolism, and excretion (ADME) applications. Central to this progress has been proof of concept data generated by academic and industrial institutions followed by broader characterization studies, which provide evidence for scalability and applicability to drug discovery and development. In this review, we describe some of the advances made for specific tissue MPS and outline the desired functionality for such systems, which are likely to make them applicable for practical use in the pharmaceutical industry. Single organ MPS platforms will be valuable for modelling tissue-specific functions. However, dynamic organ crosstalk, especially in the context of disease or toxicity, can only be obtained with the use of inter-linked MPS models which will enable scientists to address questions at the intersection of pharmacokinetics (PK) and efficacy, or PK and toxicity. In the future, successful application of MPS platforms that closely mimic human physiology may ultimately reduce the need for animal models to predict ADME outcomes and decrease the overall risk and cost associated with drug development.
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http://dx.doi.org/10.1039/c9lc00857hDOI Listing
February 2020

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

Assessment of Transporter-Mediated and Passive Hepatic Uptake Clearance Using Rifamycin-SV as a Pan-Inhibitor of Active Uptake.

Mol Pharm 2018 10 27;15(10):4677-4688. Epub 2018 Aug 27.

Drug Metabolism and Pharmacokinetics , Vertex Pharmaceuticals Incorporated , Boston , Massachusetts 02210 , United States.

The use of in vitro data for the quantitative prediction of transporter-mediated clearance is critical. Central to this evaluation is the use of hepatocytes, since they contain the full complement of transporters and metabolic enzymes. In general, uptake clearance (CL) is evaluated by measuring the appearance of compound in the cell. Passive clearance (CL) is often determined by conducting parallel studies at 4 °C or by attempting to saturate uptake pathways. Both approaches have their limitations. Recent studies have proposed the use of Rifamycin-SV (RFV) as a pan-inhibitor of hepatic uptake pathways. In our studies, we confirm that transport activity of all major hepatic uptake transporters is inhibited significantly by RFV at 1 mM (OATP1B1, 1B3, and 2B1 = NTCP (80%), OCT1 (65%), OAT2 (60%)). Under these incubation conditions, we found that the free intracellular concentration of RFV is ∼175 μM and that several major CYPs and UGTs can be reversibly inhibited. Using this approach, we also determined CL and CL of nine known OATP substrates across three different lots of human hepatocytes. The scaling factors generated for these compounds at 37 °C with RFV and 4 °C were found to be similar. The CL of passively permeable compounds like metoprolol and semagacestat were found to be higher at 37 °C compared to 4 °C, indicating a temperature effect on these compounds. In addition, our data also suggests that incorporation of medium concentrations into CL and CL calculations may be critical for highly protein bound and highly lipophilic drugs. Overall, our data indicate that RFV, instead of 4 °C, can be reliably used to measure CL and CL of drugs.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00654DOI 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

Quantitative Prediction of CYP3A4 Induction: Impact of Measured, Free, and Intracellular Perpetrator Concentrations from Human Hepatocyte Induction Studies on Drug-Drug Interaction Predictions.

Drug Metab Dispos 2017 06 23;45(6):692-705. Epub 2017 Mar 23.

Drug Metabolism and Pharmacokinetics, Vertex Pharmaceuticals Inc., Boston, Massachusetts

Typically, concentration-response curves are based upon nominal inducer concentrations for in-vitro-to-in-vivo extrapolation of CYP3A4 induction. The limitation of this practice is that it assumes the hepatocyte culture model is a static system. We assessed whether correcting for: 1) changes in perpetrator concentration in the induction medium during the incubation period, 2) perpetrator binding to proteins in the induction medium, and 3) nonspecific binding of perpetrator can improve the accuracy of CYP3A4 induction predictions. Of the seven compounds used in this evaluation, significant parent loss and nonspecific binding were observed for rifampicin (29.3-38.3%), pioglitazone (64.3-78.6%), and rosiglitazone (57.1-75.5%). As a result, the free measured EC values (EC) of pioglitazone, rosiglitazone, and rifampicin were significantly lower than the nominal EC values. In general, the accuracy of the induction predictions, using multiple static models, improved when corrections were made for measured medium concentrations, medium protein binding, and nonspecific binding of the perpetrator, as evidenced by 18-29% reductions in the root mean square error. The relative induction score model performed better than the basic static and mechanistic static models, resulting in lower prediction error and no false-positive or false-negative predictions. However, even when the EC value was used, the induction prediction for bosentan, which is a substrate of organic anion transporter proteins, was overpredicted by approximately 2-fold. Accounting for the ratio of unbound intracellular concentrations to unbound medium concentrations (K) (0.5-7.5) and the predicted multiple-dose K (0.6) for bosentan resulted in induction predictions within 35% of the observed interaction.
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http://dx.doi.org/10.1124/dmd.117.075481DOI Listing
June 2017

Evaluation of the Interplay between Uptake Transport and CYP3A4 Induction in Micropatterned Cocultured Hepatocytes.

Drug Metab Dispos 2016 12 21;44(12):1910-1919. Epub 2016 Sep 21.

Drug Metabolism and Pharmacokinetics, Vertex Pharmaceuticals Incorporated, Boston, Massachusetts

Previously we assessed the inductive response of prototypical inducers in hepatocyte monocultures and the long-term coculture model HepatoPac using cryopreserved hepatocytes from the same donors. We noted that the rifampicin EC generated using the HepatoPac model corresponded better to the EC based on clinical data compared with data generated in the monoculture system. We postulated that there may be differences in the functioning of uptake transporters between the two systems that may have led to the EC difference. In this study, we characterized the functional activity of multiple uptake transporters in the two systems using cryopreserved hepatocytes from the same donors. Our data suggest that uptake transporter activity is higher in HepatoPac compared with the monoculture system. As a follow up to this study, we measured the intracellular concentrations of rifampicin and bosentan, which are known substrates of uptake transporters; we observed significantly higher intracellular concentrations of both compounds in HepatoPac relative to the monoculture system. This finding equated to lower cytochrome P450 isoform 3A4 (CYP3A4) EC values in the HepatoPac system compared with the monoculture system for both mRNA and activity. In parallel, no significant EC shift was observed for carbamazepine and phenytoin, which are not known to be substrates of uptake transporters. Our data suggest that next generation liver models such as HepatoPac may be a useful in vitro tool to quantitatively predict drug-drug interactions when it is known that the perpetrator is also a substrate of drug transporters.
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http://dx.doi.org/10.1124/dmd.116.072660DOI Listing
December 2016

VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

Drug Metab Dispos 2016 08 13;44(8):1286-95. Epub 2016 Jun 13.

Drug Metabolism and Pharmacokinetics (C.Z., L.L., S.L., H.T., A.C., N.H.) and Department of Chemistry (F.M.), Vertex Pharmaceuticals Inc., Boston, Massachusetts

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident.
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http://dx.doi.org/10.1124/dmd.116.071100DOI Listing
August 2016

Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes.

Drug Metab Dispos 2016 Feb 9;44(2):250-61. Epub 2015 Dec 9.

Drug Metabolism and Pharmacokinetics, Vertex Pharmaceuticals Incorporated, Boston, Massachusetts.

Long-term coculture models of hepatocytes are promising tools to study drug transport, clearance, and hepatoxicity. In this report we compare the basal expression of drug disposition genes and the inductive response of prototypical inducers (rifampin, phenobarbital, phenytoin) in hepatocyte two-dimensional monocultures and the long-term coculture model (HepatoPac). All the inducers used in the study increased the expression and activity of CYP3A4, CYP2B6 and CYP2C enzymes in the HepatoPac cultures. The coculture model showed a consistent and higher induction of CYP2C enzymes compared with the monocultures. The EC50 of rifampin for CYP3A4 and CYP2C9 was up to 10-fold lower in HepatoPac than the monocultures. The EC50 of rifampin calculated from the clinical drug interaction studies correlated well with the EC50 observed in the HepatoPac cultures. Owing to the long-term stability of the HepatoPac cultures, we were able to directly measure a half-life (t1/2) for both CYP3A4 and CYP2B6 using the depletion kinetics of mRNA and functional activity. The t1/2 for CYP3A4 mRNA was 26 hours and that for the functional protein was 49 hours. The t1/2 of CYP2B6 was 38 hours (mRNA) and 68 hours (activity), which is longer than CYP3A4 and shows the differential turnover of these two proteins. This is the first study to our knowledge to report the turnover rate of CYP2B6 in human hepatocytes. The data presented here demonstrate that the HepatoPac cultures have the potential to be used in long-term culture to mimic complex clinical scenarios.
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http://dx.doi.org/10.1124/dmd.115.067173DOI Listing
February 2016

Lack of a clinically meaningful pharmacokinetic effect of rifabutin on raltegravir: in vitro/in vivo correlation.

J Clin Pharmacol 2011 Jun 17;51(6):943-50. Epub 2010 Sep 17.

Merck & Co, Inc, 126 E. Lincoln Ave, P.O. Box 2000, Rahway, NJ 07065, USA.

Raltegravir is an HIV-1 integrase strand transfer inhibitor with potent activity against HIV-1. A prior investigation of raltegravir coadministered with rifampin demonstrated a decrease in plasma concentrations of raltegravir likely secondary to induction of UGT1A1, the enzyme primarily responsible for the metabolism of raltegravir. Little is known regarding the induction of UGT1A1 by rifabutin, an alternate rifamycin. In vitro characterization of the induction potency of rifampin and rifabutin on UGT1A1 was performed. In vitro studies indicate that rifabutin is a less potent inducer of UGT1A1 messenger RNA expression than is rifampin. A fixed-sequence, 2-period, clinical crossover study was conducted to assess the effect of rifabutin on plasma levels of raltegravir: period 1, 400 mg of raltegravir every 12 hours for 4 days; period 2, 400 mg of raltegravir every 12 hours and 300 mg of rifabutin once daily for 14 days. Geometric mean ratio (GMR) (coadministration of rifabutin and raltegravir vs raltegravir alone) of raltegravir area under the concentration-time curve from 0 to 12 hours post dose (AUC(0-12h)) and the 90% confidence interval (CI) was 1.19 (0.86-1.63); GMR of concentration at 12 hours (C(12h)) and 90% CI was 0.80 (0.68-0.94); and GMR of time to maximal concentration (C(max)) and 90% CI was 1.39 (0.87-2.21). Overall, coadministration of rifabutin did not alter raltegravir pharmacokinetics to a clinically meaningful degree.
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http://dx.doi.org/10.1177/0091270010375959DOI Listing
June 2011

Impact of ritonavir, atazanavir and their combination on the CYP3A4 induction potential of efavirenz in primary human hepatocytes.

Drug Metab Lett 2010 Jan;4(1):45-50

Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0004, USA.

Currently used combinations of anti-HIV drugs, known as Highly Active Antiretroviral Therapy (HAART), have considerably reduced the mortality in patients with AIDS. However, HAART medications such as efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) often cause adverse drug-drug interactions (DDIs) that result from changes in the expression and activity of drug metabolizing enzymes. Since EFV is most commonly used with ATV and RTV, the known CYP inhibitors, we evaluated the effects of combinations of these agents on the CYP3A4 induction by EFV. We determined the induction of CYP3A4 by EFV, RTV, ATV, EFV+RTV, EFV+ATV, EFV+RTV+ATV and rifampicin (RIF) employing primary human hepatocytes from 3 donors. Also, concentration dependent activation of human Pregnane X-receptor (hPXR) which is key transcriptional regulator of CYP3A4 by EFV, RIF and RTV was estimated in transiently transfected LS180 cells. CYP3A4 activity (testosterone-6beta-hydroxylation) was induced by EFV (3 fold) and RIF (4 fold), but was significantly suppressed in the presence of RTV and ATV. All treatments significantly induced the CYP3A4 transcripts (3-25 fold) as quantitated by RT-PCR. hPXR activation data in LS180 cells were consistent with the induction of transcripts and the estimated EC(50) values were 0.87 microM, 0.44 microM and 3.7 microM for RIF, RTV and EFV, respectively. However, in primary hepatocytes the net effect was suppression of EFV mediated CYP3A4 induction by RTV and ATV. This observation corresponds to the clinical observations of attenuated CYP3A4 induction by EFV induction in the presence of RTV and other protease inhibitors (PIs). Our results underscore the limitation of transcriptional activation assays in predicting the net outcome for compounds that exhibit complex interactions resulting from induction and inhibition of CYP enzymes.
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http://dx.doi.org/10.2174/187231210790980453DOI Listing
January 2010

Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes.

Nucleic Acids Res 2009 Mar 7;37(4):1160-73. Epub 2009 Jan 7.

Department of Drug Metabolism and Pharmacokinetics, Merck & Co, Rahway, NJ 07065, USA.

Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.
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http://dx.doi.org/10.1093/nar/gkn1047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651806PMC
March 2009

Comparison of immortalized Fa2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction.

Drug Metab Dispos 2008 Jun 10;36(6):1046-55. Epub 2008 Mar 10.

Department of Drug Metabolism and Pharmacokinetics, Merck & Co., West Point, Pennsylvania 19486, USA.

Fa2N-4 cells have been proposed as a tool to identify CYP3A4 inducers. To evaluate whether Fa2N-4 cells are a reliable surrogate for cryopreserved human hepatocytes, we assessed the basal mRNA expression of 64 drug disposition genes in Fa2N-4 cells. Significant differences were found in the expression of major drug-metabolizing enzymes, nuclear receptors, and transporters between both cell types. Importantly, the expression of constitutive androstane receptor (CAR) and several hepatic uptake transporters was significantly lower (>50-fold) in Fa2N-4 cells, whereas the expression of pregnane X-receptor (PXR) and aryl hydrocarbon receptor (AhR) was similar between Fa2N-4 cells and human hepatocytes. By using an optimized induction assay for Fa2N-4 cells, CYP3A4 induction by rifampicin, the prototypical PXR activator, increased from 1.5- to 7-fold at the level of functional activity. With nine selected compounds, which are known inducers of CYP3A4 either via activation of PXR, CAR, or both, we evaluated CYP3A4 and CYP2B6 mRNA induction using Fa2N-4 cells and human hepatocytes. No response was observed in Fa2N-4 cells treated with the selective CAR activators 6-(4-chlorophenyl)imidazo[2,1-b][1,3]-thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and artemisinin. CYP3A4 and CYP2B6 induction in Fa2N-4 cells were also low for phenytoin, phenobarbital, and efavirenz, which are dual activators of PXR/CAR. This finding was in agreement with the lack of expression of CAR. The EC(50) value for rifampicin-mediated CYP3A4 induction was 10-fold higher than that in human hepatocytes. This result could be attributed to the low expression of hepatic organic anion-transporting polypeptides OATP1B1 and OATP1B3 in Fa2N-4 cells. In summary, our findings identify limitations of Fa2N-4 cells as a predictive induction model.
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http://dx.doi.org/10.1124/dmd.108.020677DOI Listing
June 2008

Cytochrome P450 enzymes and transporters induced by anti-human immunodeficiency virus protease inhibitors in human hepatocytes: implications for predicting clinical drug interactions.

Drug Metab Dispos 2007 Oct 16;35(10):1853-9. Epub 2007 Jul 16.

Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, USA.

Although many of the clinically significant drug interactions of the anti-human immunodeficiency virus (HIV) protease inhibitors (PIs) can be explained by their propensity to inactivate CYP3A enzymes, paradoxically these drugs cause (or lack) interactions with CYP3A substrates that cannot be explained by this mechanism (e.g., alprazolam). To better understand these paradoxical interactions (or lack thereof), we determined the cytochromes P450 and transporters induced by various concentrations (0-25 microM) of two PIs, ritonavir and nelfinavir, and rifampin (positive control) in primary human hepatocytes. At 10 microM, ritonavir and nelfinavir suppressed CYP3A4 activity but induced its transcripts and protein expression (19- and 12- and 12- and 6-fold, respectively; a >2-fold change over control was interpreted as induction). At 10 microM, rifampin induced CYP3A4 transcripts, CYP3A protein, and activity by 23-, 12-, and 13-fold, respectively. The induction by rifampin of CYP3A activity was significantly correlated with its induction of CYP3A4 transcripts (r = 0.96, p < 0.05) and CYP3A protein (r = 0.89, p < 0.05). All three drugs (10 microM) induced CYP2B6 activity by 2- to 4-fold, CYP2C8 and 2C9 activity by 2- to 4-fold and the transcripts of CYP2B6, 2C8, and 2C9 by >3-, 5-, and 3-fold, respectively. CYP2C19 and 1A2 activity and transcripts were modestly induced (2-fold), whereas, as expected, CYP2D6 was not induced by any of the drugs. Of the transporters studied, protease inhibitors moderately induced multidrug resistance 1 (ABCB1) and multidrug resistance-associated protein (ABCC1) transcripts but had no or minimal effect on the transcripts of breast cancer resistance protein (ABCG2), organic anion-transporting peptide (OATP) 1B1 (SLCO1B1), or OATP1B3 (SLCO1B3). On the basis of these data, we concluded that many of the paradoxical drug interactions (or lack thereof) with the PIs are metabolismrather than transporter-based and are due to induction of CYP2B6 and 2C enzymes.
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http://dx.doi.org/10.1124/dmd.107.016089DOI Listing
October 2007

In vitro LC-MS cocktail assays to simultaneously determine human cytochrome P450 activities.

Biopharm Drug Dispos 2007 Jul;28(5):257-62

Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, USA.

Using liquid chromatography-mass spectrometry (LC-MS), two sensitive cocktail assays were developed, one to simultaneously determine activities of the cytochrome P450 enzymes, CYP1A2 (phenacitin), 2B6 (bupropion), 2C8 (amodiaquine) and 2C19 (omperazole), and the other to determine simultaneously activities of CYP3A4/5 (testosterone), 2C9 (tolbutamide) and 2D6 (dextromethorphan). These cocktail assays are sensitive, require only a small amount of microsomal protein, employ selective and high turnover CYP substrates and do not require post-incubation extraction. In each of these cocktails, no interactions were observed between the substrates. Combining the two cocktails into a single cocktail resulted in significant inhibition of CYP2D6 by amiodiaquine. These assays were used successfully to determine induction of CYP enzymes in microsomes isolated from human hepatocytes treated (72 h) with or without the prototypic inducer, rifampin.
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http://dx.doi.org/10.1002/bdd.552DOI Listing
July 2007

Induction of CYP3A4 by efavirenz in primary human hepatocytes: comparison with rifampin and phenobarbital.

J Clin Pharmacol 2004 Nov;44(11):1273-81

College of Pharmacy, University of Cincinnati Medical Center, 3223 Eden Avenue, Mail Location #0004, Cincinnati, OH 45267, USA.

The antiretroviral agent efavirenz enhances the systemic clearance of coadministered drugs that are cytochrome P450 (CYP) 3A4 substrates. The mechanism of the apparent increase in CYP3A4 activity by efavirenz and the magnitude of change relative to other known inducers are not known. The authors tested the hypothesis that increased enzymatic activity by efavirenz entails CYP3A4 induction and activation of the human pregnane X receptor (hPXR), a key transcriptional regulator of CYP3A4. Employing primary cultures of human hepatocytes, they compared the CYP3A4 inductive effects of efavirenz (1-10 microM) to rifampin (10 microM) and phenobarbital (2 mM). A cell-based reporter assay was employed to assess hPXR activation. The authors observed that efavirenz caused a concentration-dependent CYP3A4 induction and hPXR activation. Based on the CYP3A4 activity assay, the average magnitude of induction by efavirenz (5-10 microM) was approximately 3- to 4-fold. In comparison, phenobarbital (2 mM) and rifampin (10 microM) caused a 5- and 6-fold induction, respectively.
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http://dx.doi.org/10.1177/0091270004269142DOI Listing
November 2004

Dose-dependent induction of cytochrome P450 (CYP) 3A4 and activation of pregnane X receptor by topiramate.

Epilepsia 2003 Dec;44(12):1521-8

College of Pharmacy, University of Cincinnati Medical Center, 3223 Eden Avenue, Cincinnati, OH 45267, U.S.A.

Purpose: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription.

Methods: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay.

Results: Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation.

Conclusions: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.
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http://dx.doi.org/10.1111/j.0013-9580.2003.06203.xDOI Listing
December 2003
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