Publications by authors named "Jairam Palamanda"

26 Publications

  • Page 1 of 1

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

Application of a Rat Liver Drug Bioactivation Transcriptional Response Assay Early in Drug Development That Informs Chemically Reactive Metabolite Formation and Potential for Drug-induced Liver Injury.

Toxicol Sci 2020 09;177(1):281-299

Safety Assessment & Laboratory Animal Resources.

Drug-induced liver injury is a major reason for drug candidate attrition from development, denied commercialization, market withdrawal, and restricted prescribing of pharmaceuticals. The metabolic bioactivation of drugs to chemically reactive metabolites (CRMs) contribute to liver-associated adverse drug reactions in humans that often goes undetected in conventional animal toxicology studies. A challenge for pharmaceutical drug discovery has been reliably selecting drug candidates with a low liability of forming CRM and reduced drug-induced liver injury potential, at projected therapeutic doses, without falsely restricting the development of safe drugs. We have developed an in vivo rat liver transcriptional signature biomarker reflecting the cellular response to drug bioactivation. Measurement of transcriptional activation of integrated nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1) electrophilic stress, and nuclear factor erythroid 2-related factor 1 (NRF1) proteasomal endoplasmic reticulum (ER) stress responses, is described for discerning estimated clinical doses of drugs with potential for bioactivation-mediated hepatotoxicity. The approach was established using well benchmarked CRM forming test agents from our company. This was subsequently tested using curated lists of commercial drugs and internal compounds, anchored in the clinical experience with human hepatotoxicity, while agnostic to mechanism. Based on results with 116 compounds in short-term rat studies, with consideration of the maximum recommended daily clinical dose, this CRM mechanism-based approach yielded 32% sensitivity and 92% specificity for discriminating safe from hepatotoxic drugs. The approach adds new information for guiding early candidate selection and informs structure activity relationships (SAR) thus enabling lead optimization and mechanistic problem solving. Additional refinement of the model is ongoing. Case examples are provided describing the strengths and limitations of the approach.
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http://dx.doi.org/10.1093/toxsci/kfaa088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553701PMC
September 2020

Evaluation of the Drug Interaction Potential of Doravirine.

Antimicrob Agents Chemother 2019 04 27;63(4). Epub 2019 Mar 27.

Merck & Co., Inc., Kenilworth, New Jersey, USA

Doravirine is a novel nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1 infection. studies were conducted to assess the potential for drug interactions with doravirine via major drug-metabolizing enzymes and transporters. Kinetic studies confirmed that cytochrome P450 3A (CYP3A) plays a major role in the metabolism of doravirine, with ∼20-fold-higher catalytic efficiency for CYP3A4 versus CYP3A5. Doravirine was not a substrate of breast cancer resistance protein (BCRP) and likely not a substrate of organic anion transporting polypeptide 1B1 (OATP1B1) or OATP1B3. Doravirine was not a reversible inhibitor of major CYP enzymes (CYP1A2, -2B6, -2C8, -2C9, -2C19, -2D6, and -3A4) or of UGT1A1, nor was it a time-dependent inhibitor of CYP3A4. No induction of CYP1A2 or -2B6 was observed in cultured human hepatocytes; small increases in CYP3A4 mRNA (≤20%) were reported at doravirine concentrations of ≥10 μM but with no corresponding increase in enzyme activity. transport studies indicated a low potential for interactions with substrates of BCRP, P-glycoprotein, OATP1B1 and OATP1B3, the bile salt extrusion pump (BSEP), organic anion transporter 1 (OAT1) and OAT3, organic cation transporter 2 (OCT2), and multidrug and toxin extrusion 1 (MATE1) and MATE2K proteins. In summary, these findings indicate that CYP3A4 and CYP3A5 mediate the metabolism of doravirine, although with different catalytic efficiencies. Clinical trials reported elsewhere confirm that doravirine is subject to drug-drug interactions (DDIs) via CYP3A inhibitors and inducers, but they support the notion that DDIs (either direction) are unlikely via other major drug-metabolizing enzymes and transporters.
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http://dx.doi.org/10.1128/AAC.02492-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437506PMC
April 2019

Overcoming Time-Dependent Inhibition (TDI) of Cytochrome P450 3A4 (CYP3A4) Resulting from Bioactivation of a Fluoropyrimidine Moiety.

J Med Chem 2018 12 15;61(23):10700-10708. Epub 2018 Nov 15.

Herein we describe structure-activity relationship (SAR) and metabolite identification (Met-ID) studies that provided insight into the origin of time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) by compound 1. Collectively, these efforts revealed that bioactivation of the fluoropyrimidine moiety of 1 led to reactive metabolite formation via oxidative defluorination and was responsible for the observed TDI. We discovered that substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of 1 was necessary to ameliorate this TDI as exemplified by compound 19.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01326DOI Listing
December 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

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans.

Drug Metab Dispos 2015 Jun 26;43(6):851-63. Epub 2015 Mar 26.

Merck Sharp & Dohme Corporation, Kenilworth, New Jersey (X.C., G.H.C., K.O., S.W., X.C., D.N., J.C.P., G.S., J.P., A.L., M.J.S., R.E.); Translational Medicine Research Centre, Singapore (S.J.S., R.S., H.H., A.F., C.K.N.).

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.
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http://dx.doi.org/10.1124/dmd.115.063347DOI Listing
June 2015

Prednisone has no effect on the pharmacokinetics of CYP3A4 metabolized drugs - midazolam and odanacatib.

J Clin Pharmacol 2014 Nov 25;54(11):1280-9. Epub 2014 Jun 25.

Merck Sharp & Dohme, Whitehouse Station, NJ.

We evaluated the effect of prednisone on midazolam and odanacatib pharmacokinetics. In this open-label, 2-period crossover study in healthy male subjects, midazolam 2 mg was administered (Day -1) followed by odanacatib 50 mg (Day 1) during Part 1. In Period 2, prednisone 10 mg once daily (qd) was administered on Days 1-28; odanacatib was co-administered on Day 14 and midazolam 2 mg was co-administered on Days 1 and 28. Subjects were administered midazolam 2 mg on Days 42 and 56. Safety and tolerability were assessed throughout the study. A physiologically-based pharmacokinetic (PBPK) model was also built. There were 15 subjects enrolled; mean age was 31 years. The odanacatib AUC(0- ∞) GMR (90% CI) [odanacatib + prednisone (Day 14, Period 2)/odanacatib alone (Day 1, Period 1] was 1.06 (0.96, 1.17). AUC(0-∞) GMR (90%CI) [midazolam + prednisone (Day 28, Period 2)/midazolam alone (Day -1, Period 1] was 1.08 (0.93,1.26). There were no serious AEs or AEs leading to discontinuation. PBPK modeling showed that prednisone does not cause significant effects on the exposure of sensitive CYP3A4 substrates in vivo at therapeutic doses. Co-administration of prednisone 10 mg qd had no effect on pharmacokinetics of either odanacatib 10 mg or midazolam 2 mg.
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http://dx.doi.org/10.1002/jcph.338DOI Listing
November 2014

Pharmacological evaluation of selective α2c-adrenergic agonists in experimental animal models of nasal congestion.

J Pharmacol Exp Ther 2014 Apr 3;349(1):75-84. Epub 2014 Feb 3.

Departments of In Vivo Pharmacology (G.G.M., J.C.H., G.B.L., M.C., J.A.H., R.L.M.) and Immunology (Y.J.), Merck Research Laboratories, Boston, Massachusetts; Departments of Pharmacokinetics (J.R.P., H.M.) and Chemistry (C.W.B.), Merck Research Laboratories, Rahway, New Jersey; and Department of Cell Biology, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma (M.C.K., Y.Y.).

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1-3.0 mg/kg), compound B (0.3-5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC(80)) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion.
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http://dx.doi.org/10.1124/jpet.113.210666DOI Listing
April 2014

In vitro assessment of drug-drug interaction potential of boceprevir associated with drug metabolizing enzymes and transporters.

Drug Metab Dispos 2013 Mar 4;41(3):668-81. Epub 2013 Jan 4.

Merck Sharp & Dohme Corporation, Whitehouse Station, New Jersey, USA.

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.
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http://dx.doi.org/10.1124/dmd.112.049668DOI Listing
March 2013

Substituted imidazole of 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates cytochrome P450 2D6 by protein adduction.

Drug Metab Dispos 2011 Jun 21;39(6):974-83. Epub 2011 Mar 21.

Department of Chemistry, Kalamazoo College, 1200 Academy Street, Kalamazoo, MI 49006.

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 μM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.
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http://dx.doi.org/10.1124/dmd.110.037630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100908PMC
June 2011

Pharmacokinetics of pseudoephedrine in rats, dogs, monkeys and its pharmacokinetic-pharmacodynamic relationship in a feline model of nasal congestion.

Drug Metab Lett 2010 Apr;4(2):56-61

Exploratory Drug Metabolism and Pharmacokinetics, K-15-3, Mail Stop: 3700, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

The objectives of these studies were to characterize the pharmacokinetics (PK) of the nasal decongestant pseudoephedrine (PSE) in rats, dogs, and monkeys, and to evaluate its lower gastrointestinal tract regional bioavailability in rats. An LC-MS/MS assay with a lower limit of quantification (LLOQ) of 0.4 ng/mL of plasma was developed for the analysis of PSE in animal plasma. The total body clearance (CL) was the highest in rats (78 mL/min/kg), lowest in monkeys (15 mL/min/kg) and the dog averaged in between (33 mL/min/kg). The volume of distribution at steady state (Vdss) ranged from 3-5 L/kg in all species. In rats and dogs, the mean half-lives (t1/2) was ≈1.5 hr, while in monkeys the mean t1/2 was 4.6 hr, comparable to that observed in adult humans (4-8 hr). The oral bioavailability was 38, 58 and 78% in rats, dogs and monkeys. The bioavailability following intra-ileum or intra-colonic administration in rats was superior to that following oral dosing (66% and 78%, respectively) suggesting that colonic absorption may be compensating for the short half-life, thus enabling successful QD sustained release formulations of PSE. The pharmacokinetic/pharmacodynamic relationship (PK/PD) of PSE was also investigated in a feline model of nasal congestion to establish efficacious trough concentrations in cats for a comparison with that in humans. The PK/PD in the cat model followed a sigmoid Emax model with an EC50 (plasma concentration that elicits 50% of the maximum response) of 0.32 ±0.05 (SD) µM consistent with human plasma concentrations required for efficacy.
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http://dx.doi.org/10.2174/187231210791292762DOI Listing
April 2010

Loratadine and montelukast administered in combination produce decongestion in an experimental feline model of nasal congestion.

Am J Rhinol Allergy 2009 Nov-Dec;23(6):e17-22. Epub 2009 Sep 18.

Neurobiology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.

Background: Histamine and leukotrienes act to exert numerous local and systemic effects that contribute to the pathophysiology of allergic rhinitis. The aim of these experiments was to evaluate the nasal decongestant effects of loratadine and montelukast alone and in combination in a feline model of nasal congestion. We also studied the decongestant actions of the alpha-agonist adrenergic agonist D-pseudoephedrine with and without desloratadine.

Methods: Acoustic rhinometry was used to determine nasal cavity dimensions after intranasal compound 48/80. Cats were given D-pseudoephedrine (0.3 mg/kg) alone or in combination with desloratadine (5 mg/kg) 1 hour before nasal provocation with compound 48/80 (1%, 75 microliters) to either the left or right nasal passageway. Using a similar design, the nasal decongestant effects of montelukast (1 mg/kg) and loratadine (10 mg/kg) were studied alone and in combination.

Results: The addition of desloratadine to D-pseudoephedrine did not improve decongestant efficacy compared with each drug given individually. In contrast, when montelukast (1 mg/kg) was given in combination with loratadine (10 mg/kg), the decongestant activity was greater than when these drugs were administered separately. Sixty minutes after compound 48/80 provocation the nasal cavity volume ratio (volume ratio of the compound 48/80 treated/untreated nasal passageway) for the control, montelukast alone, loratadine alone, and the montelukast plus loratadine-treated groups were 0.20 +/- 0.03, 0.24 +/- 0.01, 0.28 +/- 0.03, and 0.50 +/- 0.03.

Conclusion: Concomitant montelukast plus loratadine produces a greater degree of nasal decongestion compared with montelukast or loratadine alone in an experimental model of nasal congestion.
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http://dx.doi.org/10.2500/ajra.2009.23.3365DOI Listing
June 2010

Evaluation of CYP1A1 and CYP2B1/2 m-RNA induction in rat liver slices using the NanoString technology: a novel tool for drug discovery lead optimization.

Drug Metab Lett 2009 Aug 1;3(3):171-5. Epub 2009 Aug 1.

Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

Cytochrome P450 (CYP) induction in rodents and humans is considered a liability for new chemical entities (NCEs) in drug discovery. In particular, CYP1A1 and CYP2B1/2 have been associated with the induction of liver tumors in oncogenicity studies during safety evaluation studies of potential drugs. In our laboratory, real time PCR (Taqman) has been used to quantify the induction of rat hepatic CYP1A1 and CYP2B1/2 in precision -cut rat liver slices. A novel technology that does not require m-RNA isolation or RT-PCR, (developed by NanoString Technologies) has been investigated to quantify CYP1A1 and CYP2B1/2 induction in rat liver slices. Seventeen commercially available compounds were evaluated using both Taqman and NanoString technologies. Precision-cut rat liver slices were incubated with individual compounds for 24 hr at 37 degrees C in a humidified CO(2) incubator and CYP1A1 and CYP2B1/2 m-RNA were quantified. The results from the NanoString technology were similar to those of the Taqman(R) with a high degree of correlation for both CYP isoforms (r(2)>0.85). Therefore, NanoString provides an additional new technology to evaluate the induction of CYP1A1 and 2B1/2, as well as potentially other enzymes or transporters in rat liver slices.
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http://dx.doi.org/10.2174/187231209789352094DOI Listing
August 2009

High-throughput evaluation of CYP1A1 and 2B1 induction in rat liver slices using a semi-automated system.

Drug Metab Lett 2009 Apr;3(2):108-14

Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

Drug candidates with the propensity to induce rat CYP1A1 or 2B1 isoforms are believed to possess a greater tendency to induce hepatic tumors in oncogenicity studies. We have previously published on a manual rat liver slice assay that showed a satisfactory relationship between in vitro CYP2B1 m-RNA induction using real time PCR and the ex vivo pentoxyresorufin O-dealkylase (PROD) activity in liver microsomes prepared from rats treated daily via the oral route for 14 consecutive days with inducers or non-inducers. We now describe this automated in vitro high throughput liver slice technique to screen out drug candidates that are potent rodent CYP1A1 and/or CYP2B1 inducers. A good concordance between in vitro and in vivo data was observed for both CYP1A1 (100 %) and CYP2B1 (90%) isoforms. Automation of key steps has enabled us to increase the annual screening throughput from 200 (manual) to 1500 compounds. The increase in throughput allowed the quick development of structure-induction relationships (SIR's) for multiple drug discovery programs in a facile manner.
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http://dx.doi.org/10.2174/187231209788654108DOI Listing
April 2009

Co-induction of CYP3A12 and 3A26 in dog liver slices by xenobiotics: species difference between human and dog CYP3A induction.

Drug Metab Lett 2009 Jan;3(1):61-6

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.

The induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds, suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity of ligands.
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http://dx.doi.org/10.2174/187231209787176399DOI Listing
January 2009

Temporal evaluation of CYP mRNA in mice administered with prototypical P450 inducers: comparison with conventional protein/enzyme methods.

Drug Chem Toxicol 2008 ;31(4):501-13

Department of Genetic and Molecular Toxicology, Schering-Plough Corporation, Summit, New Jersey 07901, USA.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.
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http://dx.doi.org/10.1080/01480540802390825DOI Listing
January 2009

Discovery of a novel, orally active himbacine-based thrombin receptor antagonist (SCH 530348) with potent antiplatelet activity.

J Med Chem 2008 Jun 1;51(11):3061-4. Epub 2008 May 1.

Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.

The discovery of an exceptionally potent series of thrombin receptor (PAR-1) antagonists based on the natural product himbacine is described. Optimization of this series has led to the discovery of 4 (SCH 530348), a potent, oral antiplatelet agent that is currently undergoing Phase-III clinical trials for acute coronary syndrome (unstable angina/non-ST segment elevation myocardial infarction) and secondary prevention of cardiovascular events in high-risk patients.
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http://dx.doi.org/10.1021/jm800180eDOI Listing
June 2008

Heterotricyclic himbacine analogs as potent, orally active thrombin receptor (protease activated receptor-1) antagonists.

J Med Chem 2007 Oct 14;50(21):5147-60. Epub 2007 Sep 14.

Central Nervous System and Cardiovascular Chemical Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA.

Pursuing our earlier efforts in the himbacine-based thrombin receptor antagonist area, we have synthesized a series of compounds that incorporate heteroatoms in the C-ring of the tricyclic motif. This effort has resulted in the identification of several potent heterocyclic analogs with excellent affinity for the thrombin receptor. Several of these compounds demonstrated robust inhibition of platelet aggregation in an ex vivo model in cynomolgus monkeys following oral administration. A detailed profile of 28b, a benchmark compound in this series, with a Ki of 4.3 nM, is presented.
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http://dx.doi.org/10.1021/jm070704kDOI Listing
October 2007

Metabolism-based identification of a potent thrombin receptor antagonist.

J Med Chem 2007 Jan;50(1):129-38

Central Nervous System and Cardiovascular Chemical Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

The metabolism of our prototypical thrombin receptor antagonist 1, Ki = 2.7 nM, was studied and three major metabolites (2, 4, and 5) were found. The structures of the metabolites were verified independently by synthesis. Compound 4 was shown to be a potent antagonist of the thrombin receptor with a Ki = 11 nM. Additionally, compound 4 showed a 3-fold improvement in potency with respect to 1 in an agonist-induced ex-vivo platelet aggregation assay in cynomolgus monkeys after oral administration; this activity was sustained with 60% inhibition observed at 24 h post-dose. Compound 4 was highly active in functional assays and showed excellent oral bioavailability in rats and monkeys. Compound 4 showed a superior rat enzyme induction profile relative to compound 1, allowing it to replace compound 1 as a development candidate.
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http://dx.doi.org/10.1021/jm061043eDOI Listing
January 2007

Quantitative PCR assay for cytochromes P450 2B and 3A induction in rat precision-cut liver slices: correlation study with induction in vivo.

J Pharmacol Toxicol Methods 2005 Sep-Oct;52(2):234-43. Epub 2005 Apr 1.

Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, K15-D209, 2015 Galloping Hill Rd., Kenilworth, NJ 07033, USA.

Introduction: In drug development, new chemical entities that cause cytochrome P450 induction are considered to be undesirable since P450 induction is linked to tumor formation and may compromise the evaluation of drug safety when autoinduction results in poor drug exposure.

Methods: We evaluated the use of the precision-cut liver slice as a model for measuring induction of cytochrome P450 in rats. Quantitative real-time reverse-transcription polymerase chain reaction was used to analyze the induction of selected forms of cytochrome P450 at the mRNA level. Firstly, the system was validated against known inducers of CYP2B and 3A. Subsequently, 26 proprietary compounds were tested in rat liver slices and rats in vivo for CYP2B and 3A induction.

Results: Exposure of liver slices to the known CYP2B inducers phenobarbital, benzoyl-pyridine, cabarmazepine, metyrapone, RU486 and dexamethasone caused elevation of CYP2B1/2 expression 10- to 40-fold compared to the control values. The CYP3A inducers PCN, dexamethasone, nicardipine, nifedipine, clotrimazole and RU486 induced a 4- to 50-fold expression of CYP3A14. For 26 proprietary compounds, a correlation with an R(2) value of 0.74 was established between the induction of CYP2B expression in liver slices and that in rats in vivo. When liver slice results were used to predict the induction of CYP2B in rats in vivo, the success rate was 91%. The induction of CYP3A in rats in vivo was analyzed by Western blot, then quantified by densitometry. There was a good correlation between CYP3A induction in liver slices and induction in vivo as assessed by Western blot, with an 86% positive prediction rate.

Discussion: The use of liver slices in combination with TaqMan technology provides a good model for predicting CYP induction in the rat. This method is useful for identifying compounds with CYP2B and 3A induction liability in the early phase of drug discovery.
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http://dx.doi.org/10.1016/j.vascn.2005.02.001DOI Listing
June 2006

Optimization of purine based PDE1/PDE5 inhibitors to a potent and selective PDE5 inhibitor for the treatment of male ED.

Bioorg Med Chem Lett 2005 May;15(9):2365-9

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.

In search of a PDE5 inhibitor for erectile dysfunction, an SAR was developed from a PDE1/PDE5 purine series of leads, which had modest PDE5 potency and poor isozyme selectivity. A compound (41) with PDE5 inhibition and in vivo activity similar to sildenafil was discovered from this effort. In addition, purine 41 demonstrated superior overall PDE isozyme selectivity when compared to the approved PDE5 inhibitors sildenafil, vardenafil, and tadalafil, which may result in a more favorable side-effect profile.
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http://dx.doi.org/10.1016/j.bmcl.2005.02.083DOI Listing
May 2005

SAR development of polycyclic guanine derivatives targeted to the discovery of a selective PDE5 inhibitor for treatment of erectile dysfunction.

Bioorg Med Chem Lett 2004 Mar;14(5):1291-4

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.

Development of structure-activity relationship of cyclic guanines I lead us to discovery of a potent and selective series of phosphodiesterase 5 inhibitors 52-59 (IC50=1.3-11.0 nM, PDE6/5=116-600).
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http://dx.doi.org/10.1016/j.bmcl.2003.12.027DOI Listing
March 2004

Assessment of temporal biochemical and gene transcription changes in rat liver cytochrome P450: utility of real-time quantitative RT-PCR.

Pharm Res 2003 Sep;20(9):1373-80

Department of Genetic and Molecular Toxicology, Schering-Plough Research Institute, PO Box 32, Lafayette, New Jersey 07848, USA.

Purpose: A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model.

Methods: Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A. Liver P450 levels were measured after 0.5, 1, 2, 4, and 8 days of dosing by qRT-PCR, Western blot, and enzyme activity.

Results: CYP1A, CYP3A, and CYP4A genes were maximally induced very rapidly (0.5-1 day), whereas the CYP2B gene was maximally induced after a lag time of 4 days. In all cases, fold changes in induction detected by qRT-PCR were greater than fold changes in protein levels and enzyme activities.

Conclusions: Maximal persistent and larger fold changes observed by qRT-PCR either preceded or occurred simultaneously with maximal sustained fold changes in protein levels as measured by Western blots and enzyme activity assays. Our data show that qRT-PCR provides increased sensitivity and specificity over conventional assays and may be key information for reliable assessment of drug-related changes in CYP induction during the transition from discovery to toxicology studies.
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http://dx.doi.org/10.1023/a:1025793707794DOI Listing
September 2003

A high-throughput cell-based reporter gene system for measurement of CYP1A1 induction.

J Pharmacol Toxicol Methods 2002 May-Jun;47(3):143-51

Department of Exploratory Drug Discovery, Schering-Plough Research Institute, D209, K15-2700, Kenilworth, NJ 07033, USA.

Introduction: Enzyme induction is undesirable in new drug discovery process, with consequences spanning from auto-induction to toxicity. Cytochrome P450 (CYP) 1A1 has long been known to be one of the metabolic enzymes involved in activating many procarcinogens, the first step toward tumor formation during chemical carcinogenesis. Induction of CYP1A1 during drug treatment may predispose the patients to some risk of chemical carcinogenesis.

Methods: Based on the signal-transduction mechanism of CYP1A1 induction, a high-throughput reporter-gene system was established by stable transformation of H4IIE cells to incorporate the luciferase gene under control of CYP1A1 promoter. This stable cell line was validated with known CYP1A1 inducers, such as 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF), alpha-naphthoflavone (alpha-NF) and 3-indocarbinol. Thirty in-house new chemical entities (NCEs) were then screened with this reporter-gene system, and also administered to rats to evaluate in vivo CYP1A1 induction.

Results: CYP1A1 reporter gene system can be used to identify strong inducers, such as 3-MC, beta-NF and alpha-NF, and weak inducers, such as 3-indocarbinol. In vitro induction of 30 in-house compounds in reporter gene system did not correlate with in vivo induction in rat liver microsome measured by ethoxyresorufin-O-dealkylation (EROD) activity, but had a reasonable correlation with Western blot signals.

Discussion: This reporter-gene system may be useful in eliminating compounds that can cause CYP1A1 induction at an early stage of drug discovery.
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http://dx.doi.org/10.1016/S1056-8719(02)00222-8DOI Listing
October 2003

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors.

Bioorg Med Chem Lett 2002 Nov;12(21):3149-52

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).
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http://dx.doi.org/10.1016/s0960-894x(02)00646-7DOI Listing
November 2002
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