Publications by authors named "Syed Muzeeb"

18 Publications

  • Page 1 of 1

Pharmacokinetics and Determination of Tumor Interstitial Distribution of a Therapeutic Monoclonal Antibody Using Large-Pore Microdialysis.

J Pharm Sci 2021 08 2;110(8):3061-3068. Epub 2021 Apr 2.

Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL, United States.

R7072 is a fully human monoclonal antibody (mAb) exerting anti-tumor activity via blockade of insulin like growth factor 1 receptor. The tumoral interstitial concentrations are anticipated to be better surrogates of active site concentrations than commonly used serum concentrations for pharmacokinetic-pharmacodynamic correlation of anti-tumor mAbs. Previously, a large-pore microdialysis technique for measuring tissue interstitial concentrations of R7072 in non-tumor bearing mice was established. In the current studies, the serum pharmacokinetics of R7072 were assessed and tissue interstitial concentrations were measured by large-pore microdialysis following intravenous and intraperitoneal administration of R7072 in tumor bearing mice. R7072 exhibited nonlinear pharmacokinetics in the studied dose range. Tumor and subcutaneous interstitial concentration data suggested some delay in tissue distribution after dosing. A dose-dependent increase in the ratio of tumor interstitial to serum concentration was observed indicating target-mediated drug disposition in tumor tissue. However, subcutaneous interstitial to serum concentration ratios were similar across the doses as observed previously in non-tumor bearing mice. A two-compartment population pharmacokinetic model with subcutaneous and tumor as open-loop compartments comprising of parallel linear and non-linear elimination from serum, linear disposition from subcutaneous interstitium and non-linear disposition from tumor interstitium was developed to simultaneously describe the pharmacokinetic data from all matrices.
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http://dx.doi.org/10.1016/j.xphs.2021.03.022DOI Listing
August 2021

Area under the curve predictions of dalbavancin, a new lipoglycopeptide agent, using the end of intravenous infusion concentration data point by regression analyses such as linear, log-linear and power models.

Xenobiotica 2018 Feb 14;48(2):148-156. Epub 2017 Mar 14.

c Suramus Bio, Drug Development , Bangalore , Karnataka , India.

1. Dalbavancin, a lipoglycopeptide, is approved for treating gram-positive bacterial infections. Area under plasma concentration versus time curve (AUC) of dalbavancin is a key parameter and AUC/MIC ratio is a critical pharmacodynamic marker. 2. Using end of intravenous infusion concentration (i.e. C) C versus AUC relationship for dalbavancin was established by regression analyses (i.e. linear, log-log, log-linear and power models) using 21 pairs of subject data. 3. The predictions of the AUC were performed using published C data by application of regression equations. The quotient of observed/predicted values rendered fold difference. The mean absolute error (MAE)/root mean square error (RMSE) and correlation coefficient (r) were used in the assessment. 4. MAE and RMSE values for the various models were comparable. The C versus AUC exhibited excellent correlation (r > 0.9488). The internal data evaluation showed narrow confinement (0.84-1.14-fold difference) with a RMSE < 10.3%. The external data evaluation showed that the models predicted AUC with a RMSE of 3.02-27.46% with fold difference largely contained within 0.64-1.48. 5. Regardless of the regression models, a single time point strategy of using C (i.e. end of 30-min infusion) is amenable as a prospective tool for predicting AUC of dalbavancin in patients.
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http://dx.doi.org/10.1080/00498254.2017.1294278DOI Listing
February 2018

Review of the bioanalytical methods for the determination of methotrexate and its metabolites in in vitro, preclinical and clinical studies: Case studies and perspectives.

Biomed Chromatogr 2017 Jan 17;31(1). Epub 2016 Oct 17.

Zydus Research Center, Ahmedabad, India.

Methotrexate is an old drug that has found use in several therapeutic areas, such as cancer to treat various malignancies, rheumatoid arthtritis and inflammatory bowel disease. Owing to its structural properties of possessing two carboxylic acid groups and having low native fluorescence, it has provided technical challenges for development of bioanalytical methods. Also, in vivo metabolism leading to circulatory metabolites such as 7-hydroxymethotrexate and 2,4-diamino N -methylpteroic acid, as well as the formation of polyglutamate metabolites intracellularly have added further complexity for the assays in terms of the analytes that need to be quantified in addition to methotrexate. The present review is aimed at providing a concise tabular summary of chromatographic assays with respect to method nuances including assay/chromatographic conditions, key validation parameters and applicable remarks. Several case studies are reviewed under various subheadings to provide the challenges involved in the method development for methotrexate and metabolites. Finally, a discussion section is devoted to overall perspectives obtained from this review.
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http://dx.doi.org/10.1002/bmc.3849DOI Listing
January 2017

Clinical Drug-Drug Pharmacokinetic Interaction Potential of Sucralfate with Other Drugs: Review and Perspectives.

Eur J Drug Metab Pharmacokinet 2016 Oct;41(5):469-503

Suramus Bio, Drug Development, I Phase, J.P. Nagar, Bangalore, 560 078, India.

Sucralfate, a complex of aluminium hydroxide with sulfated sucrose, forms a strong gastrointestinal tract (GIT) mucosal barrier with excellent anti-ulcer property. Because sucralfate does not undergo any significant oral absorption, sucralfate resides in the GIT for a considerable length of time. The unabsorbed sucralfate may alter the pharmacokinetics of the oral drugs by impeding its absorption and reducing the oral bioavailability. Because of the increased use of sucralfate, it was important to provide a reappraisal of the published clinical drug-drug interaction studies of sucralfate with scores of drugs. This review covers several category of drugs such as non-steroidal anti-inflammatory drugs, fluoroquinolones, histamine H2-receptor blockers, macrolides, anti-fungals, anti-diabetics, salicylic acid derivatives, steroidal anti-inflammatory drugs and provides pharmacokinetic data summary along with study design, objectives and key remarks. While the loss of oral bioavailability was significant for the fluoroquinolone class, it generally varied for other classes of drugs, suggesting that impact of the co-administration of sucralfate is manageable in clinical situations. Given the technology advancement in formulation development, it may be in order feasible to develop appropriate formulation strategies to either avoid or minimize the absorption-related issues when co-administered with sucralfate. It is recommended that consideration of both in vitro and preclinical studies may be in order to gauge the level of interaction of a drug with sucralfate. Such data may aid in the development of appropriate strategies to navigate the co-administration of sucralfate with other drugs in this age of polypharmacy.
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http://dx.doi.org/10.1007/s13318-016-0335-4DOI Listing
October 2016

Intranasal Pharmacokinetic Data for Triptans Such as Sumatriptan and Zolmitriptan Can Render Area Under the Curve (AUC) Predictions for the Oral Route: Strategy Development and Application.

J Pain Palliat Care Pharmacother 2016 11;30(1):13-24. Epub 2016 Feb 11.

Limited pharmacokinetic sampling strategy may be useful for predicting the area under the curve (AUC) for triptans and may have clinical utility as a prospective tool for prediction. Using appropriate intranasal pharmacokinetic data, a Cmax vs. AUC relationship was established by linear regression models for sumatriptan and zolmitriptan. The predictions of the AUC values were performed using published mean/median Cmax data and appropriate regression lines. The quotient of observed and predicted values rendered fold-difference calculation. The mean absolute error (MAE), mean positive error (MPE), mean negative error (MNE), root mean square error (RMSE), correlation coefficient (r), and the goodness of the AUC fold prediction were used to evaluate the two triptans. Also, data from the mean concentration profiles at time points of 1 hour (sumatriptan) and 3 hours (zolmitriptan) were used for the AUC prediction. The Cmax vs. AUC models displayed excellent correlation for both sumatriptan (r = .9997; P < .001) and zolmitriptan (r = .9999; P < .001). Irrespective of the two triptans, the majority of the predicted AUCs (83%-85%) were within 0.76-1.25-fold difference using the regression model. The prediction of AUC values for sumatriptan or zolmitriptan using the concentration data that reflected the Tmax occurrence were in the proximity of the reported values. In summary, the Cmax vs. AUC models exhibited strong correlations for sumatriptan and zolmitriptan. The usefulness of the prediction of the AUC values was established by a rigorous statistical approach.
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http://dx.doi.org/10.3109/15360288.2015.1124165DOI Listing
January 2017

A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids.

Biomed Chromatogr 2016 May 17;30(5):721-48. Epub 2016 Feb 17.

Suramus Bio, Drug Development, J.P. Nagar I Phase, Bangalore, 560078, Karnataka, India.

Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the analysis of MPA alone or with MPAG/AcMPAG in various biological specimens including plasma/serum, urine, ultrafiltrate, saliva, PBMC, dried blood spots, tissue extract, tumor biopsies and vitreous humor. Based on the need for experimental work, a proper choice of the assay and internal standard may be made using the choices in the literature. While the chemical methods involving high-performance liquid chromatography (HPLC) or LC coupled with triple quadrupole mass spectrometry (LC-MS/MS) are popular, enzymatic assays, in spite of their higher bias, have been used for the routine drug monitoring of MPA. The objectives of the present review are: (a) to provide a focused systematic compilation of the HPLC or LC-MS/MS methods for MPA, MPAG and/or AcMPAG published in the last decade (2005 to current) to enable visual comparison of the methods; (b) to compare and contrast a few enzymatic assays with those of the chemical methods; and (c) to discuss relevant issues/limitations and perspectives on select assays under various subheadings.
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http://dx.doi.org/10.1002/bmc.3682DOI Listing
May 2016

Applicability of a Single Time Point Strategy for the Prediction of Area Under the Concentration Curve of Linezolid in Patients: Superiority of Ctrough- over Cmax-Derived Linear Regression Models.

Drugs R D 2016 Mar;16(1):69-79

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Background And Objectives: Linezolid, a oxazolidinone, was the first in class to be approved for the treatment of bacterial infections arising from both susceptible and resistant strains of Gram-positive bacteria. Since overt exposure of linezolid may precipitate serious toxicity issues, therapeutic drug monitoring (TDM) may be required in certain situations, especially in patients who are prescribed other co-medications.

Methods: Using appropriate oral pharmacokinetic data (single dose and steady state) for linezolid, both maximum plasma drug concentration (Cmax) versus area under the plasma concentration-time curve (AUC) and minimum plasma drug concentration (Cmin) versus AUC relationship was established by linear regression models. The predictions of the AUC values were performed using published mean/median Cmax or Cmin data and appropriate regression lines. The quotient of observed and predicted values rendered fold difference calculation. The mean absolute error (MAE), root mean square error (RMSE), correlation coefficient (r), and the goodness of the AUC fold prediction were used to evaluate the two models.

Results: The Cmax versus AUC and trough plasma concentration (Ctrough) versus AUC models displayed excellent correlation, with r values of >0.9760. However, linezolid AUC values were predicted to be within the narrower boundary of 0.76 to 1.5-fold by a higher percentage by the Ctrough (78.3%) versus Cmax model (48.2%). The Ctrough model showed superior correlation of predicted versus observed values and RMSE (r = 0.9031; 28.54%, respectively) compared with the Cmax model (r = 0.5824; 61.34%, respectively).

Conclusions: A single time point strategy of using Ctrough level is possible as a prospective tool to measure the AUC of linezolid in the patient population.
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http://dx.doi.org/10.1007/s40268-015-0117-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767722PMC
March 2016

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria.

Toxicol In Vitro 2016 Apr 9;32:26-40. Epub 2015 Dec 9.

Section of Analytical Biosciences, Department of Pharmacy, School of Pharmaceutical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 μM)>celecoxib (IC50: 14.92 ± 6.40 μM)>valdecoxib (IC50: 161.4 ± 28.6 μM)>rofecoxib (IC50: 238.4 ± 79.2 μM)>etoricoxib (IC50: 405.1 ± 116.3 μM). Mechanism based inhibition of ATP synthesis (Kinact 0.078 min(-1) and KI 21.46 μM and Kinact/KI ratio 0.0036 min(-1)μM(-1)) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs and these metabolites did not alter the inhibition profile of ATP synthesis of the parent compound. The results suggest that coxibs themselves could be involved in the hepatotoxic action through inhibition of ATP synthesis.
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http://dx.doi.org/10.1016/j.tiv.2015.12.003DOI Listing
April 2016

Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI).

Toxicol In Vitro 2016 Mar 25;31:93-102. Epub 2015 Nov 25.

Section of Analytical Biosciences, Department of Pharmacy, School of Pharmaceutical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half maximal rate of inactivation (KI) of 7.69 ± 2.48 μM with Kinact/KI ratio of 0.343 min(-1) μM(-1). Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis.
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http://dx.doi.org/10.1016/j.tiv.2015.11.020DOI Listing
March 2016

Inhibition of ATP synthesis by fenbufen and its conjugated metabolites in rat liver mitochondria.

Toxicol In Vitro 2016 Mar 21;31:23-9. Epub 2015 Nov 21.

Section of Analytical Biosciences, Department of Pharmacy, School of Pharmaceutical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen-S-glutathione thioester (F-SG) were found to be more potent inhibitors compared to parent fenbufen (F), whereas fenbufen-O-carnitine (F-carn), fenbufen-glycine (F-gly) and fenbufen-N-acetyl lysine amide (F-NAL) were less potent compared to fenbufen. Fenbufen-CoA thioester (F-CoA) was equally potent as fenbufen in inhibiting ATP synthesis. Fenbufen showed time and concentration dependent inhibition of ATP synthesis with Kinact of 4.4 min(-1) and KI of 0.88 μM and Kinact/KI ratio of 5.01 min(-1) μM(-1). Data show that fenbufen did not act through opening MPT pore, nor did incubation of mitochondria with reduced GSH and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria. The results from the present experiments provide evidence that fenbufen and its metabolites could be involved in mitochondrial toxicity through inhibition of ATP synthesis.
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http://dx.doi.org/10.1016/j.tiv.2015.11.013DOI Listing
March 2016

Effect of some organic solvents on oxidative phosphorylation in rat liver mitochondria: Choice of organic solvents.

Toxicol In Vitro 2013 Dec 19;27(8):2135-41. Epub 2013 Sep 19.

Section of Analytical Biosciences, Department of Pharmacy, School of Pharmaceutical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences in potencies. Among the tested organic solvents, acetonitrile and acetone were more potent than ethanol, methanol, and DMSO. There was no significant difference in oxidative phosphorylation, compared to controls, when the concentrations of acetone was below 1% (v/v), of acetonitrile below 2% (v/v), of DMSO below 10% (v/v), of ethanol below 5% or of methanol below 2%, respectively. There was complete inhibition of oxidative phosphorylation at 50% (v/v) of acetone, acetonitrile and ethanol. But in the case of DMSO and methanol there were some residual activities observed at the 50% concentration level. DMSO showed least effect on oxidative phosphorylation with an IC50 value of 13.3±1.1% (v/v), followed by methanol (IC50 value 8.3±1.0), ethanol (IC50 value 4.6±1.1), acetone (IC50 value 4.3±1.0) and finally acetonitrile (IC50 value 2.1±1.0). All the organic solvents showed modulatory effects on 2,4-dinitrophenol (DNP) mediated inhibition of oxidative phosphorylation with potentiation of the action of DNP. Acetonitrile showed the highest potentiation effect followed by acetone, ethanol, methanol, and DMSO in presence of DNP. The use of organic solvents for investigation of the effects of compounds on oxidative phosphorylation in mitochondria should therefore include the use of relevant concentrations of the organic solvent in order to validate the contribution.
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http://dx.doi.org/10.1016/j.tiv.2013.09.010DOI Listing
December 2013

Design and optimization of quinazoline derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

Bioorg Med Chem Lett 2012 May 23;22(9):3157-62. Epub 2012 Mar 23.

Discovery Research, Dr. Reddy's Laboratories Ltd, Bollaram Road, Miyapur, Hyderabad 500049, India.

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays a role in metabolic and CNS disorders. The modeling-supported design, synthesis and multi-parameter optimization (biological activity, solubility, metabolic stability, hERG) of novel quinazoline derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified. Clusters of refined hMCHR1 homology models derived from the X-ray structure of the β2-adrenergic receptor, including extracellular loops, were developed and used to guide the design.
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http://dx.doi.org/10.1016/j.bmcl.2012.03.050DOI Listing
May 2012

Species difference in the in vitro and in vivo metabolism of amtolmetin guacil.

Arzneimittelforschung 2010 ;60(11):667-74

Bioanalytical Department, Integrated Product Development, Dr. Reddy's Laboratories Ltd, Bachupalli, Hyderabad, India.

Tolmetin (TMT, CAS 26171-23-3) is a non-steroidal anti-inflammatory drug (NSAID) indicated for the relief of signs and symptoms of osteoarthritis, rheumatoid arthritis and juvenile rheumatoid arthritis. As TMT causes gastro-intestinal side effects like other NSAIDs, its nonacidic prodrug amtolmetin guacil (AMG, CAS 87344-06-7) was synthesized. AMG has similar NSAID properties like TMT with additional gastroprotective property. The aim of this study was to investigate whether TMT and AMG are differentially metabolised in rat and human plasma (fresh and acidified) and liver microsomes. TMT was found to be stable in all the matrices tested viz., rat and human plasma (fresh and acidified) and liver microsomes. AMG was found to be stable only in acidified rat and human plasma. On the contrary, in fresh human plasma and human liver microsomes AMG was rapidly converted to two metabolites, which were subsequently identified as MED5 and MED5 methyl ester, without yielding any intact TMT. However, in rat fresh plasma and liver microsomes, AMG formed MED5 (predominant) and TMT. To corroborate the in vitro findings, in vivo pharmacokinetics (PK) studies were done following separate dosing of AMG in both rats and humans. In rats, the PK data substantiated that following oral administration of AMG it will be converted to TMT resulting in similar PK parameters observed for TMT when it was administered alone. In humans, however, AMG yields low levels of TMT which substantes the in vitro results. Levels of AMG were not detectable in the plasma. These results confirm the species differences in the in vitro and in vivo metabolism and disposition of AMG. More research work to further explore and understand AMG metabolism in humans is required.
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http://dx.doi.org/10.1055/s-0031-1296345DOI Listing
January 2011

Discovery of novel, orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

Bioorg Med Chem Lett 2010 Sep 29;20(18):5443-8. Epub 2010 Jul 29.

Discovery Research, Dr. Reddy's Laboratories Ltd, Bollaram Road, Miyapur, Hyderabad 500 049, India.

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.
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http://dx.doi.org/10.1016/j.bmcl.2010.07.086DOI Listing
September 2010

Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile.

J Chromatogr B Analyt Technol Biomed Life Sci 2007 Jun 15;853(1-2):88-96. Epub 2007 Mar 15.

Drug Metabolism and Pharmacokinetics, Discovery Research, Dr. Reddy's Laboratories Ltd., Miyapur, Hyderabad 500 049, India.

Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved deproteinization of 500 microL of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 microL acetonitrile containing 25% perchloric acid. An aliquot of 100 microL supernatant was injected onto a C18 column. The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid:acetonitrile:methanol:water at a flow rate of 1.0 mL/min. The detection of analyte peaks were achieved by monitoring the eluate using an UV detector set at 250 nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide (EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56 min, respectively. Average extraction efficiencies of EZM, EZM-G and IS was >75-80% and for EZM-K was >50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02 microg/mL. Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies.
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http://dx.doi.org/10.1016/j.jchromb.2007.02.053DOI Listing
June 2007

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

Eur J Drug Metab Pharmacokinet 2006 Oct-Dec;31(4):299-309

Drug Metabolism and Pharmacokinetics, Discovery Research, Dr. Reddy's Laboratories Ltd., Miyapur, Hyderabad, India.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.
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http://dx.doi.org/10.1007/BF03190471DOI Listing
April 2007

Analysis of five HMG-CoA reductase inhibitors-- atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin: pharmacological, pharmacokinetic and analytical overview and development of a new method for use in pharmaceutical formulations analysis and in vitro metabolism studies.

Biomed Chromatogr 2006 Mar;20(3):282-93

Drug Metabolism and Pharmacokinetics, Discovery Research, Dr. Reddy's Laboratories Ltd, Miyapur, Hyderabad, India.

A specific, accurate, precise and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of five 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, viz. atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin, in pharmaceutical formulations and extended the application to in vitro metabolism studies of these statins. Ternary gradient elution at a flow rate of 1 mL/min was employed on an Intertisl ODS 3V column (4.6 x 250 mm, 5 microm) at ambient temperature. The mobile phase consisted of 0.01 m ammonium acetate (pH 5.0), acetonitrile and methanol. Theophylline was used as an internal standard (IS). The HMG-CoA reductase inhibitors and their metabolites were monitored at a wavelength of 237 nm. Drugs were found to be 89.6-105.6% of their label's claim in the pharmaceutical formulations. For in vitro metabolism studies the reaction mixtures were extracted with simple liquid-liquid extraction using ethyl acetate. Baseline separation of statins and their metabolites along with IS free from endogenous interferences was achieved. Nominal retention times of IS, atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were 7.5, 17.2, 21.6, 28.5, 33.5 and 35.5 min, respectively. The proposed method is simple, selective and could be applicable for routine analysis of HMG-CoA reductase inhibitors in pharmaceutical preparations as well as in vitro metabolism studies.
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http://dx.doi.org/10.1002/bmc.561DOI Listing
March 2006

Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes--in vitro study.

Mol Cell Biochem 2005 Sep;277(1-2):49-53

Owaisi Hospital and Research Centre, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad, 500 058, India.

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m2 which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0-1250 Joules/m2, whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m2. Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m2.
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http://dx.doi.org/10.1007/s11010-005-4881-3DOI Listing
September 2005
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