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ABSTRACT: The purpose of this study was to perform a comparative analysis of various in vitro--in vivo extrapolation (IVIVE) methods used for predicting hepatic metabolic clearance (CL) of drugs on the basis of intrinsic CL data determined in microsomes. Five IVIVE methods were evaluated: the "conventional and conventional bias-corrected methods" using the unbound fraction in plasma (fu(p) ), the "Berezhkovskiy method" in which the fu(p) is adjusted for drug ionization, the "Poulin et al. method" using the unbound fraction in liver (fu(liver) ), and the "direct scaling method," which does not consider any binding corrections. We investigated the effects of the following scenarios on the prediction of CL: the use of preclinical or human datasets, the extent of plasma protein binding, the magnitude of CL in vivo, and the extent of drug disposition based on biopharmaceutics drug disposition classification system (BDDCS) categorization. A large and diverse dataset of 139 compounds was collected, including those from the literature and in house from Genentech. The results of this study confirm that the Poulin et al. method is robust and showed the greatest accuracy as compared with the other IVIVE methods in the majority of prediction scenarios studied here. The difference across the prediction methods is most pronounced for (a) albumin-bound drugs, (b) highly bound drugs, and (c) low CL drugs. Predictions of CL showed relevant interspecies differences for BDDCS class 2 compounds; the direct scaling method showed the greatest predictivity for these compounds, particularly for a reduced dataset in rat that have unexpectedly high CL in vivo. This result is a reflection of the direct scaling method's natural tendency to overpredict the true metabolic CL. Overall, this study should facilitate the use of IVIVE correlation methods in physiologically based pharmacokinetics (PBPK) model. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:4308-4326, 2012.
Journal of Pharmaceutical Sciences 08/2012; 101(11):4308-26. · 3.06 Impact Factor
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ABSTRACT: INTRODUCTION: The traditional in vitro approach for assessing potential CYP induction has been to simply compare changes in CYP activities using known CYP-specific probe substrates following exposure to the test compound to that of vehicle and/or positive controls in primary cultured human hepatocytes. The objective of these current studies was to develop and implement a highly efficient 96-well CYP induction assay in which mRNA levels, protein levels, and the conventional enzyme activities of CYP1A2, CYP2B6, and CYP3A4/5 are all measured in the same well after 48h. Cytotoxicity is also assessed in the same well after 24 and 48h of incubation. Since enzymatic activity data alone often 'misses' CYP induction due to compounding factors, such as CYP mechanism-based inactivation, this 'all-inclusive' approach efficiently maximizes the generation of additional useful and comprehensive data. This data can more readily identify potential CYP induction liabilities in the drug discovery process and, therefore, avoid potential drug-drug interactions in the clinic. METHODS: One 96-well plate with cryopreserved human hepatocytes accommodated up to nine test compounds at three clinically relevant concentrations, positive and negative controls for CYP1A2, CYP2B6, and CYP3A4/5, and a vehicle control (0.1% DMSO) in three different lots of cryopreserved human hepatocytes. Ritonavir, a positive control for CYP3A inactivation/induction, and staurosporine, a positive control for cytotoxicity, were included. The compounds 3-methylcholanthrene (a CYP1A2 inducer), phenobarbital (a CYP2B6 inducer), and rifampicin (a CYP3A4/5 inducer) served as positive controls. RESULTS: Data showed a strong correlation between the fold-increases in CYP activity, mRNA level, and protein level after incubation of the CYP isoforms with positive controls compared to the vehicle control. Ritonavir resulted in a decrease in CYP3A/5 activity, yet a concomitant increase in mRNA and protein levels of CYP3A4. Cytotoxicity was positive for staurosporine but negative for the other compounds. DISCUSSION: An 'all-inclusive' 96-well method for identifying potential drug-drug interactions in vitro was successfully developed and implemented. This is timely, as the recent FDA draft guidance on such studies now recommends using mRNA levels as an important endpoint.
Journal of pharmacological and toxicological methods 07/2012; · 2.32 Impact Factor
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ABSTRACT: Early in the drug discovery process, the identification of cytochrome P450 (CYP) time-dependent inhibition (TDI) is an important step for compound optimization. Here we describe a high-throughput, automated method for the evaluation of TDI utilizing human liver microsomes and conventional CYP-specific mass spectrometer-based probes in a 384-well format. One of the key differences from other published TDI assays is the use of a shift in area the under curve of the percent activity remaining versus inhibitor concentration plot (AUC shift) rather than the traditional fold-shift in IC50, to determine the magnitude of TDI. An AUC shift of < 15% suggests negative TDI and > 15% suggests potential TDI. This AUC shift was used to achieve quantitative data reporting, even in the case of weak inhibitors for which IC50 values cannot be quantified. An Agilent Technologies BioCel 1200 System was programmed such that the TDI liability of up to 77 test compounds, incubated at four test concentrations, with and without NADPH in the pre-incubation, can be analyzed in a single run. The detailed automated methodology, assay validation, data reporting and the novel TDI AUC shift approach to describe magnitude of TDI are presented.
Drug metabolism letters. 02/2012; 6(1):43-53.
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ABSTRACT: Intrinsic clearances of seven diverse compounds in rat liver microsomes were measured at intracellular pH 7.0 and extracellular pH 7.4. The obtained values were quite close for each compound. These results confirm the validity of the recently published novel equations for calculation of hepatic clearance and drug time course in liver that account for pH differences in extracellular water and hepatocytes.
Journal of Pharmaceutical Sciences 11/2011; 101(2):516-8. · 3.06 Impact Factor
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ABSTRACT: The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers. GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies. Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells. The clearance of GDC-0941 was high in mouse (63.7 mL/min/kg), rat (49.3 mL/min/kg) and cynomolgus monkey (58.6 mL/min/kg), and moderate in dog (11.9 mL/min/kg). The volume of distribution ranged from 2.52 L/kg in rat to 2.94 L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse. Predicted human clearance and volume of distribution using allometry were 6 mL/min/kg and 2.9 L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models. GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.
Xenobiotica 08/2011; 41(12):1088-99. · 1.79 Impact Factor
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ABSTRACT: Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.
Drug metabolism letters. 08/2011; 5(3):220-30.
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ABSTRACT: The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.
European Journal of Drug Metabolism and Pharmacokinetics 02/2011; 36(1):1-16. · 0.36 Impact Factor
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ABSTRACT: Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.
Chemical Research in Toxicology 04/2010; 23(5):861-3. · 3.78 Impact Factor
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ABSTRACT: In the early stages of drug discovery, the formation of reactive metabolites is often assessed by co-incubating the drug in liver microsomes with a trapping agent in the presence of NADPH. Our group assessed the capability of commonly used trapping agents to reversibly inhibit major cytochrome P450 (CYP) isoforms. Glutathione and cyanide did not inhibit the enzymes at concentrations up to 10 mM; however methoxylamine did show inhibition, with IC(50) values of 0.53 mM for CYP1A2, 4.12 mM for CYP2C9, 2.04 mM for CYP2C19, 9.72 mM for CYP2D6, and 1.26 and >10 mM for CYP3A4/5 (for testosterone and midazolam, respectively, as substrates).
Drug metabolism letters. 04/2009; 3(2):125-9.
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ABSTRACT: It was suggested that in vivo hepatic clearance, CL(h), may be predicted rather accurately with the in vitro values of intrinsic clearance, CL(int), obtained using the microsomal incubation mix containing diluted plasma, and consequently calculated by the well-stirred model equation. Conceivably the improvement could be due to the direct account of plasma protein binding in the measured values of CL(int). It is shown in this article that the prediction of CL(h) done in this manner may not yield accurate results, both substantial underestimation or overestimation of the true value is possible. The procedure may be useful to reduce the overestimation of CL(h) for highly protein bound drugs, though the obtained value of CL(h) may be far off from the correctly calculated one. The accurate way of calculating CL(h), based on the value of CL(int) obtained in diluted plasma, is presented. It takes into account both the drug protein binding in diluted plasma and microsomal binding, as well as blood-plasma concentration ratio. The prediction of CL(h) by the suggested calculation using the experimental data on CL(int), measured at different plasma dilutions for several drugs, yields consistent (dilution independent) values of hepatic clearance. It does not seem possible to avoid the measurement of plasma protein binding, microsomal binding and blood-plasma concentration ratio for an accurate and consistent prediction of CL(h), even if the value of CL(int) were obtained in the pure (undiluted) plasma. In an early stage screening using plasma in the microsomal incubation mix may be beneficial for fast metabolizing drugs with relatively high protein binding. This would reduce a possible overestimation CL(h), and also lead to the increase of the half-life in the microsomal incubation, so that it could be measured more accurately.
Journal of Pharmaceutical Sciences 11/2008; 98(6):1922-7. · 3.06 Impact Factor
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ABSTRACT: In vitro metabolic stability assays are used to screen compounds for stability in the presence of various drug metabolizing enzymes, usually cytochrome P450 in liver preparations (e.g., liver microsomes). High-throughput metabolic stability assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME optimization stage. In our laboratory, we have improved the metabolic stability assay using the cassette analysis method, column switching, and incorporated time saving techniques in method development to yield a robust method which reduces data turnaround time, increases compound throughput, and maximizes mass spectrometer usage. This method can determine metabolic stability using microsomes or hepatocytes from any species. We describe our findings following incubation of 40 different compounds with human liver microsomes and analysis by the cassette and discrete analysis methods. Similar metabolic stability results were obtained using the cassette analysis and discrete analysis method. An overall 70% time savings was achieved by pooling four new compounds into one sample for method development/MS optimization, cassetting four samples into one sample to minimize the number of injections on LC/MS/MS analysis, and using a column switching system to analyze the samples, which results in a two-fold decrease in the LC/MS/MS analysis time.
Drug metabolism letters. 02/2007; 1(1):67-72.
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ABSTRACT: White oils or waxes [mineral hydrocarbons (MHCs)] with substantial levels of saturated hydrocarbons in the range of C18 to C32 have produced hepatic microgranulomas and lymph node microgranulomas (also referred to as histiocytosis) after repeated administration to female Fischer-344 (F-344) rats. Female Sprague-Dawley (S-D) rats are less sensitive to these MHC-induced hepatic and lymph node effects. Studies reported herein characterized the pharmacokinetics and disposition of a representative C-26 MHC, [1-(14)C]1-eicosanylcyclohexane ([(14)C]EICO), in these two rat strains. Female F-344 and S-D rats were administered by oral gavage either a high (1.80 g/kg) or a low (0.18 g/kg) dose of MHC in olive oil (1:4, v/v) containing [(14)C]EICO as a tracer. Blood, urine, feces, liver, and mesenteric lymph nodes (MLNs) were analyzed for [(14)C]EICO and (14)C-metabolites. After the high dose, F-344 rats had a higher blood C(max) of [(14)C]EICO, a longer time to C(max), and a greater area under the systemic blood concentration-time curve from zero to time infinity compared with S-D rats. After the low dose, F-344 rats displayed a unique triphasic blood concentration-time profile, meaning two distinct C(max) values were observed. Fecal excretion was the major route of [(14)C]EICO elimination for both rat strains (70-92% of the dose). S-D rats eliminated the majority of [(14)C]EICO metabolites recovered in the urine by 16 h (8-17% of the dose), whereas F-344 rats did not excrete the same amount until 72 to 96 h. Beyond 24 h, a greater level of [(14)C]EICO was recovered in livers of F-344 rats; at 96 h, 3 and 0.1% of the dose was retained in livers of F-344 and S-D rats, respectively. The major urinary metabolites of EICO in both rat strains were identified as 12-cyclohexyldodecanoic acid and 10-cyclohexyldecanoic acid. Based on the pharmacokinetic parameters and disposition profiles, the data indicate inherent strain differences in the total systemic exposure, rate of metabolism, and hepatic and lymph node retention of [(14)C]EICO, which may be associated with the different strain sensitivities to the formation of liver granulomas and MLN histiocytosis.
Drug Metabolism and Disposition 01/2003; 30(12):1470-7. · 3.73 Impact Factor
