W Griffith Humphreys

Bristol-Myers Squibb, New York City, New York, United States

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Publications (80)277.74 Total impact

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    ABSTRACT: Abstract The study described here investigated the impact of intestinal excretion (IE, excretion of drug directly from circulation to intestinal lumen), enteroenteric recirculation (EER), and renal tubule recirculation (RTR) on apixaban pharmacokinetics and disposition. The experimental approaches involve integrating apixaban elimination pathways with pharmacokinetic profiles obtained from bile duct-cannulated (BDC) rats and dogs receiving intravenous (IV) doses together with orally administration of activated charcoal (AC). Additionally, the role of P-gp and BCRP in apixaban disposition was evaluated in experiments using transporter inhibitors and transporter knockout (KO) rats. Approximately 20-50% of an apixaban IV dose was found in feces of BDC rats and dogs, suggesting IE leading to fecal elimination and intestinal clearance (IC). The fecal elimination, IC, and systemic clearance of apixaban were increased upon AC administration in both BDC rats and dogs, and were decreased in BDC rats dosed with GF-120918 (a BCRP and P-gp inhibitor). BCRP appeared to play a more important role for absorption, intestinal and renal elimination of apixaban than P-gp in transporter-KO rats after oral and IV dosing, which led to a higher level of active renal excretion in rat than other species. These data demonstrate that apixaban undergoes IE, EER, and RTR that are facilitated by efflux transporters. Intestinal re-absorption of apixaban could be interrupted by AC even at 3 h post drug dose in dogs (late charcoal effect). This study demonstrates that the intestine is an organ for direct clearance and redistribution of apixaban. The IE, EER, and RTR contribute to overall pharmacokinetic profiles of apixaban. IE as a clearance pathway, balanced with metabolism and renal excretion, helps decrease the impacts of intrinsic (renal or hepatic impairment) and extrinsic (drug-drug interactions) factors on apixaban disposition.
    Drug metabolism and disposition: the biological fate of chemicals 02/2013; · 3.74 Impact Factor
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    ABSTRACT: The studies reported here were conducted to investigate the transport characteristics of apixaban and to understand the impact of transporters on apixaban distribution and disposition. In human P-gp and BCRP-cDNA transfected cell monolayers as well as Caco-2 cell monolayers, the apparent efflux ratio of basolateral to apical (Pc(B-A)) versus apical to basolateral permeability (Pc(A-B)) of apixaban was >10. The P-gp and BCRP-facilitated transport of apixaban was concentration- and time-dependent and did not show saturation over a wide range of concentrations (1-100 μM). The efflux transport of apixaban was also demonstrated by the lower mucosal to serosal permeability than that of the serosal to mucosal direction in the isolated rat jejunum segments. Apixaban did not inhibit digoxin transport in Caco-2 cells. Ketoconazole decreased the P-gp-mediated apixaban efflux in Caco-2 and the P-gp-cDNA transfected cell monolayers, but did not affect the apixaban efflux to a meaningful extent in the BCRP-cDNA transfected cell monolayers. Co-incubation of a P-gp inhibitor (ketoconazole or cyclosporin A) and a BCRP inhibitor (Ko134) provided more complete inhibition of apixaban efflux in Caco-2 cells than separate inhibition by individual inhibitors. Naproxen inhibited apixaban efflux in Caco-2 cells, but showed only a minimal effect on apixaban transport in the BCRP-transfected cells. Naproxen was the first NSAID that was demonstrated as a weak P-gp inhibitor. These results demonstrate that apixaban is a substrate for efflux transporters P-gp and BCRP, which can help explain its low brain penetration, low fetus exposures, and milk excretion in rats.
    Drug metabolism and disposition: the biological fate of chemicals 02/2013; · 3.74 Impact Factor
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    ABSTRACT: AIM(S): This study assessed the effect of differences in renal function on the pharmacokinetics and pharmacodynamics of dapagliflozin, an SGLT2 inhibitor for treatment of type 2 diabetes mellitus (T2DM). METHODS: A single 50-mg dose of dapagliflozin was used to assess pharmacokinetics and pharmacodynamics in five groups: healthy nondiabetic subjects; patients with T2DM and normal kidney function; and patients with T2DM and mild, moderate or severe renal impairment based on estimated creatinine clearance. Subsequently, 20-mg once-daily multiple doses were evaluated in the patients with T2DM. Formation rates of dapagliflozin 3-O-glucuronide (D3OG), an inactive metabolite, were evaluated using isolated human kidney and liver microsomes. RESULTS: Plasma concentrations of dapagliflozin and D3OG were incrementally increased with declining kidney function. Steady-state C(max) for dapagliflozin were 4%, 6% and 9% higher and for D3OG were 20%, 37% and 52% higher in patients with mild, moderate, and severe renal impairment, respectively, compared to normal function. AUC(0-τ) was likewise higher. D3OG formation in kidney microsomes was 3-fold higher than liver and 109-fold higher than intestine. Compared to patients with normal renal function, pharmacodynamic effects were attenuated with renal impairment. Steady-state renal glucose clearance was reduced by 42%, 83%, and 84% in patients with mild, moderate, or severe renal impairment, respectively. CONCLUSIONS: These results indicate that both kidney and liver significantly contribute to dapagliflozin metabolism, resulting in higher systemic exposure with declining kidney function. Dapagliflozin pharmacodynamics in diabetic subjects with moderate to severe renal impairment are consistent with the observation of reduced efficacy in this patient population.
    British Journal of Clinical Pharmacology 12/2012; · 3.58 Impact Factor
  • Yanou Yang, Feng Qiu, Jonathan L Josephs, William Griffith Humphreys, Yue-Zhong Shu
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    ABSTRACT: Certain functional groups/structural motifs are known to generate chemically reactive metabolites that can covalently modify essential cellular macromolecules, and therefore have the potential to disrupt biological function and elicit idiosyncratic adverse drug reactions. In this report, we describe the bioactivation of 5-substituted 2-(alkylthio)-1, 3, 4 thiadiazoles and 2-(alkylthio)-1, 3-benzothiazoles, which can be added to the growing list of structural alerts. When 5-substituted 2-(methylthio)-1, 3, 4 thiadiazoles and 2-(methylthio)-1, 3-benzothiazole were incubated with pooled human liver microsomes (HLM) in the presence of NADPH and GSH, unusual GSH adducts were formed. Characterization of these GSH adducts by high resolution mass spectrometry indicated the replacement of the methylthio- group by GSH, and NMR experiments ascertained the proposed structures. Based on the metabolic profile change in incubation samples with/without GSH, we proposed that the GSH adduct formation involves two steps: 1) enzymatic oxidation of the alkylthio- group to form sulfoxide and sulfone; 2) nucleophilic displacement of the formed sulfoxide and sulfone by GSH. The proposed mechanism was confirmed by the formation of the same GSH adduct from the incubation of synthetically prepared sulfoxide and sulfone compounds in buffer. We found the sulfur oxidation step was significantly inhibited (80-100%) by preincubation with 1-aminobenzotriazole (1-ABT) but was much less affected by thermo-inactivation (0-45%), suggesting that the sulfoxidation step is primarily catalyzed by cytochrome P450s and not by flavin monooxygenases (FMO). We also investigated the presence of this bioactivation pathway in more than a dozen compounds containing 2-(alkylthio)-1, 3, 4 thiadiazole and 2-(alkylthio)-1, 3-benzothiazoles. The common GSH adduct formation pathway demonstrated by current studies raises a new structural alert and potential liability when 2-alkylthio derivatives of 1, 3-benzothiazoles and 1, 3, 4-thiadiazoles are incorporated in drug design.
    Chemical Research in Toxicology 11/2012; · 3.67 Impact Factor
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    ABSTRACT: An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.
    Bioanalysis 08/2012; 4(15):1855-70. · 3.25 Impact Factor
  • Mary F Grubb, William G Humphreys, Jonathan L Josephs
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    ABSTRACT: An integrated method that provides rates of both parent disappearance and metabolite formation was developed. Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 µM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 µM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 µM samples to UV areas in order to compare relative metabolite concentrations. The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.
    Bioanalysis 07/2012; 4(14):1747-61. · 3.25 Impact Factor
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    ABSTRACT: Saxagliptin is a potent dipeptidyl peptidase-4 inhibitor approved for the treatment of type 2 diabetes mellitus. The pharmacokinetics and disposition of [(14)C]saxagliptin were investigated in healthy male subjects after a single 50-mg (91.5 μCi) oral dose. Saxagliptin was rapidly absorbed (T(max), 0.5 h). Unchanged saxagliptin and 5-hydroxy saxagliptin (M2), a major, active metabolite, were the prominent drug-related components in the plasma, together accounting for most of the circulating radioactivity. Approximately 97% of the administered radioactivity was recovered in the excreta within 7 days postdose, of which 74.9% was eliminated in the urine and 22.1% was excreted in the feces. The parent compound and M2 represented 24.0 and 44.1%, respectively, of the radioactivity recovered in the urine and feces combined. Taken together, the excretion data suggest that saxagliptin was well absorbed and was subsequently cleared by both urinary excretion and metabolism; the formation of M2 was the major metabolic pathway. Additional minor metabolic pathways included hydroxylation at other positions and glucuronide or sulfate conjugation. Cytochrome P450 (P450) enzymes CYP3A4 and CYP3A5 metabolized saxagliptin and formed M2. Kinetic experiments indicated that the catalytic efficiency (V(max)/K(m)) for CYP3A4 was approximately 4-fold higher than that for CYP3A5. Therefore, it is unlikely that variability in expression levels of CYP3A5 due to genetic polymorphism will impact clearance of saxagliptin. Saxagliptin and M2 each showed little potential to inhibit or induce important P450 enzymes, suggesting that saxagliptin is unlikely to affect the metabolic clearance of coadministered drugs that are substrates for these enzymes.
    Drug metabolism and disposition: the biological fate of chemicals 04/2012; 40(7):1345-56. · 3.74 Impact Factor
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    ABSTRACT: A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.
    Chemical Research in Toxicology 03/2012; 25(3):556-71. · 3.67 Impact Factor
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    ABSTRACT: UHPLC coupled with orthogonal acceleration hybrid quadrupole-TOF (Q-TOF)-MS is an emerging technique offering new strategies for the efficient screening of new chemical entities and related molecules at the early discovery stage within the pharmaceutical industry. In the first part of this article, we examine the main instrumental parameters that are critical for the integration of UHPLC-Q-TOF technology to existing bioanalytical workflows, in order to provide simultaneous quantitative and qualitative bioanalysis of samples generated following in vivo studies. Three modern Q-TOF mass spectrometers, including Bruker maXis™, Agilent 6540 and Sciex TripleTOF™ 5600, all interfaced with UHPLC systems, are evaluated in the second part of the article. The scope of this work is to demonstrate the potential of Q-TOF for the analysis of typical small molecules, therapeutic peptides (molecular weight <6000 Da), and enzymatically (i.e., trypsin, chymotrypsin and pepsin) cleaved peptides from larger proteins. This work focuses mainly on full-scan TOF data obtained under ESI conditions, the major mode of TOF operation in discovery bioanalytical research, where the compounds are selected based on their pharmacokinetic/pharmacodynamic behaviors using animal models prior to selecting a few desirable candidates for further development. Finally, important emerging TOF technologies that could potentially benefit bioanalytical research in the semi-quantification of metabolites without synthesized standards are discussed. Particularly, the utility of captive spray ionization coupled with TripleTOF 5600 was evaluated for improving sensitivity and providing normalized MS response for drugs and their metabolites. The workflow proposed compromises neither the efficiency, nor the quality of pharmacokinetic data in support of early drug discovery programs.
    Bioanalysis 03/2012; 4(5):511-28. · 3.25 Impact Factor
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    ABSTRACT: A liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1-50 ng/mL for saxagliptin and 0.2-100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis(®) dC18 column (50 mm × 2.1 mm, 5 μm) for LC-MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at -20°C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 02/2012; 889-890:77-86. · 2.78 Impact Factor
  • Qian Ruan, Qin C Ji, Mark E Arnold, W Griffith Humphreys, Mingshe Zhu
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    ABSTRACT: Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.
    Analytical Chemistry 12/2011; 83(23):8937-44. · 5.70 Impact Factor
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    ABSTRACT: An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.
    Rapid Communications in Mass Spectrometry 11/2011; 25(21):3245-51. · 2.51 Impact Factor
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    ABSTRACT: The development of compounds with the potential for genotoxicity poses significant safety risks as well as risks of attrition. Although genotoxicity evaluation of the parent molecule is routine and reasonably predictive, assessing the risk of commercialization when release of a genotoxic degradant and/or metabolite from a nongenotoxic parent molecule is suspected is much more challenging and resource intensive. Much of the risk of the formation of a genotoxic degradant/metabolite can be discharged with the conduct of carcinogenicity studies in models where the compound is formed, but this approach requires a great deal of time and resources. In this manuscript, we investigated the contribution of various factors (pH, serum instability, and hepatic metabolism) to the formation of a mutagenic aromatic amine from a potent and highly selective thyromimetic compound ([3-(3,5-dibromo-4-(4-hydroxy-3-isopropyl-5-methylphenoxy)-2-methylphenylamino)-3-oxopropanoic acid], compound 1), under in vitro conditions. The kinetic parameters obtained from in vitro experiments combined with the pharmacokinetics of 1in vivo (e.g., plasma concentration-time profile and clearance) were used to estimate the extent of in vivo formation of [4-(4-amino-2,6-dibromo-3-methylphenoxy)-2-isopropyl-6-methylphenol] (compound 2), in rats upon administration of a single oral dose of 1. The agreement between the predicted values (1.9% conversion of total administered dose) with the observed levels of 2 in rats (0.2%-2.2% of the 10 mg/kg dose, 10 mg/kg) further prompted the utilization of this approach to predict the extent of release of this mutagen in humans upon administration of 1. The projection of 0.13% conversion to 2 from an efficacious daily dose of 15 mg of 1 translated to the generation of 20 μg of 2 and provided the basis for the decision to terminate the development of 1.
    Chemical Research in Toxicology 06/2011; 24(6):905-12. · 3.67 Impact Factor
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    ABSTRACT: (3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of ErbB human epidermal growth factor receptors (HER1, 2, and 4) and vascular endothelial growth factor receptors 1 to 3 that has been under clinical development for solid tumor malignancies. BMS-690514 is primarily cleared by metabolism with the primary metabolic pathways being direct glucuronidation (M6), hydroxylation (M1, M2, and M37), and O-demethylation (M3). In the current investigation, the metabolic drug-drug interaction potential of BMS-690514 was evaluated in a series of in vitro studies. Reaction phenotyping experiments with cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes and human liver microsomes (HLM) in the presence of P450 or UGT inhibitors suggested that CYP3A4, CYP2D6, and CYP2C9 were the major enzymes responsible for the oxidative metabolism of BMS-690514, whereas both UGT2B4 and UGT2B7 were responsible for the formation of M6. BMS-690514 did not cause direct or time-dependent inhibition of P450 enzymes (IC(50) values ≥40 μM) in incubations with HLM and probe substrates of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4. The compound also did not substantially induce CYP1A1, CYP1A2, CYP2B6, CYP3A4, or UGT1A1 at concentrations up to 10 μM in cultured human hepatocytes. Considering the submicromolar plasma C(max) concentration at the anticipated clinical dose of 200 mg, BMS-690514 is unlikely to cause clinically relevant drug-drug interactions when coadministered with other medications. In addition, because multiple enzymatic clearance pathways are available for the compound, inhibition of an individual metabolic pathway either via coadministered drugs or gene polymorphisms is not expected to cause pronounced (>2-fold) increases in BMS-690514 exposure.
    Drug metabolism and disposition: the biological fate of chemicals 06/2011; 39(9):1658-67. · 3.74 Impact Factor
  • Mingshe Zhu, Haiying Zhang, W Griffith Humphreys
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    ABSTRACT: Mass spectrometry plays a key role in drug metabolite identification, an integral part of drug discovery and development. The development of high-resolution (HR) MS instrumentation with improved accuracy and stability, along with new data processing techniques, has improved the quality and productivity of metabolite identification processes. In this minireview, HR-MS-based targeted and non-targeted acquisition methods and data mining techniques (e.g. mass defect, product ion, and isotope pattern filters and background subtraction) that facilitate metabolite identification are examined. Methods are presented that enable multiple metabolite identification tasks with a single LC/HR-MS platform and/or analysis. Also, application of HR-MS-based strategies to key metabolite identification activities and future developments in the field are discussed.
    Journal of Biological Chemistry 06/2011; 286(29):25419-25. · 4.65 Impact Factor
  • Jinping Gan, Peggy Liu-Kreyche, W Griffith Humphreys
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    ABSTRACT: To assess the inhibition and induction potential of tanespimycin and its major metabolite, 17-amino-17-demethoxygeldanamycin (17-AG) on cytochrome P450 (CYP) enzymes. The inhibitory effect of tanespimycin and 17-AG on various CYP enzymes was determined in human liver microsomes. The inductive effects of tanespimycin and 17-AG on CYP1A2, CYP2B6, and CYP3A4/5 were determined in cultured primary human hepatocytes. Tanespimycin did not inhibit the activities of CYP1A2, 2A6, 2B6, and 2E1 up to a concentration of 60 μM, while it moderately inhibited CYP3A4/5 and 2C19, and weakly inhibited CYP2C8, 2C9, and 2D6. In addition, its inhibition on CYP3A4/5 was time-dependent. 17-AG moderately inhibited the activities of CYP3A4/5 and CYP2C19, but did not inhibit other CYPs up to a concentration of 30 μM. The inhibition of CYP3A4/5 by 17-AG was not time-dependent. Tanespimycin and 17-AG did not significantly induce the activities of CYP1A2, CYP2B6, or CYP3A4/5 in cultured human hepatocytes at concentrations up to 40 and 20 μM for tanespimycin and 17-AG, respectively. Tanespimycin together with its active metabolite, 17-AG are moderate inhibitors of CYP3A4/5 and CYP2C19, but not inducers of CYPs. Therefore, co-administration of tanespimycin has the potential to increase the exposure of substrates of CYP2C19 and CYP3A4/5.
    Cancer Chemotherapy and Pharmacology 05/2011; 69(1):51-6. · 2.80 Impact Factor
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    ABSTRACT: The thiophene moiety has been recognized as a toxicophore because of the potential of oxidative bioactivation leading to electrophilic species. The introduction of bulky or electron-withdrawing groups at the α-carbon to the sulfur atom has the potential to reduce or eliminate bioactivation. In this article, we describe the bioactivation of a variety of substituted thiophenes. These compounds were incubated in NADPH-fortified human liver microsomes with or without the addition of reduced glutathione (GSH) as a trapping agent. The resulting GSH adducts were characterized by high performance liquid chromatography/high resolution mass spectrometry with the aid of a background subtraction methodology. Four of the five α-chlorothiophenes tested formed NADPH-dependent GSH adducts. Most adducts had masses consistent with the nominal substitution of chlorine by GSH. LC/MS/MS and proton NMR of the major GSH adduct of 1-(5-chlorothiophen-2-yl)ethanone (1a) confirmed that GSH displaced chlorine. To further explore the effect of different substitutions on the bioactivation potential, a series of 2-acetylthiophenes substituted at the C4 or C5 positions were tested in a quantitative thiol-trapping assay using dansyl glutathione. Substitutions at the C4 or C5 positions gave adduct levels that decreased in the following order: 4-H, 5-H (no substitution) > 4-Br ∼ 4-Cl > 5-Cl > 5-CN > 4-CH(3) > 5-Br > 5-CH(3) (no adduct detected). In conclusion, bioactivation was detected in a series of substituted thiophenes. Although substitutions on the thiophene ring can reduce the formation of reactive metabolites, the degree of reduction is dependent on the substitution position and substituent.
    Chemical Research in Toxicology 03/2011; 24(5):663-9. · 3.67 Impact Factor
  • W Griffith Humphreys
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    ABSTRACT: The sensitive and specific detection of adducts derived from reactive intermediates during discovery metabolite profiling has been made feasible by advances in LC-MS/MS instrumentation. Many companies employ screens with nucleophilic trapping agents as a routine part of early screening efforts. Although certainly not as straightforward as initial adduct detection, the positives in the profiling experiment can be followed-up with determination of exact adduct structure. This information feeds naturally into drug design efforts as the structural motifs responsible for reactive metabolite formation can be altered to reduce the property. While the process of generation of reactive metabolite data has become more straightforward, the conversion of that data into an optimization paradigm remains challenging. Recent studies have shown a very loose correlation between extent of reactive metabolite formation and observed toxicity, so setting stringent criteria likely leads to discarding compounds that would not have problems. On the other hand, the central role of reactive metabolites in most accepted mechanisms of drug-induced toxicity points to the fact that there is value in minimizing the property. Decision making based on information on reactive metabolite formation remains a difficult process in all phases of drug discovery and development. Decisions on compounds in discovery can be made based on a fixed threshold value or relative to a reference point within a chemical series, but should be made with a firm understanding of the limitation of the data.
    Chemico-biological interactions 01/2011; 192(1-2):56-9. · 2.46 Impact Factor
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    ABSTRACT: Mass spectrometry-based assays are used in drug discovery and development to detect, characterize and quantify drugs, metabolites, impurities and degradants. Recently, high resolution-based mass spectrometers have begun to emerge as a platform with potential for performing integrated qualitative and quantitative assays in order to streamline the drug discovery and development process. However, the widely different LC–MS response observed for a drug and its metabolites limit the direct use of LC–MS responses for relative quantitative determination of metabolites. This in turn limits the use of conventional LC–ESI-MS methods, in the absence of reference standards, as an integrated technique for detection, characterization and quantification of drugs and metabolites. The goal of this study was to explore the use of LC–captive spray ionization (CSI)-mass spectrometry for detection, characterization and quantification of drugs and metabolites. CSI allows the use of conventional HPLC or uHPLC columns and flow rates of 0.35–0.6 mL/min (before post-column flow splitting) and can be considered as a technique which can function as a nanospray or microspray. Also, in comparison to conventional nanospray ionization (NSI) techniques, setup and maintenance of CSI do not require: (1) X, Y, and Z positioning or cameras to guide the spray positioning, (2) difficult to control splitters to deliver nano-flow ratios and difficult to maintain nanospray nozzles. Evaluations using equimolar mixture of buspirone and four monoxy metabolites present in human plasma show that LC–CSI-MS is a highly sensitive technique that gives a near equimolar response for the compounds used in this example. Comparisons of LC–ESI-MS data with that obtained using LC–CSI-MS show that reasonable quantification of metabolites may be achievable without using reference standards or administration of radiolabeled drugs.Graphical abstractView high quality image (158K)Research highlights▶ LC-Captive Spray Ionization (CSI)-MS allows integration of quantitative and qualitative bioanalysis. ▶ LC-CSI-MS provides near equimolar response for drugs and metabolites. ▶ LC-CSI-MS provides opportunities to quantify drugs and metabolites without reference standards.
    International Journal of Mass Spectrometry. 01/2011; 301:127-135.
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    ABSTRACT: To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [³H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil.
    British Journal of Clinical Pharmacology 12/2010; 70(6):870-80. · 3.58 Impact Factor

Publication Stats

1k Citations
277.74 Total Impact Points

Institutions

  • 2003–2013
    • Bristol-Myers Squibb
      • • Department of Biotransformation
      • • Department of Pharmaceutical Candidate Optimization
      • • Department of Discovery Chemistry
      • • Department of Metabolism and Pharmacokinetics
      New York City, New York, United States
  • 2009
    • University at Albany, The State University of New York
      • School of Public Health
      New York City, New York, United States