Gary L. Skiles

Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data.

An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of high resolution liquid chromatography/mass spectrometry (LC/MS) data for the detection and structural characterization of oxidative metabolites with mass defects similar to or significantly different from those of the parent drugs. The effectiveness of this approach was investigated using nefazodone as a model compound, which is known to undergo multiple common and uncommon oxidative reactions. Through the selective removal of all ions that fall outside of the preset filter windows, the MDF process facilitated the detection of all 14 nefazodone metabolites presented in human liver microsomes in the MDF-filtered chromatograms. The capability of the MDF approach to remove endogenous interferences from more complex biological matrices was examined by analyzing omeprazole metabolites in human plasma. The unprocessed mass chromatogram showed no distinct indication of metabolite peaks; however, after MDF processing, the metabolite peaks were easily identified in the chromatogram. Compared with precursor ion scan and neutral loss scan techniques, the MDF approach was shown to be more effective for the detection of metabolites in a complex matrix. The comprehensive metabolite detection capability of the MDF approach, together with accurate mass determination, makes high resolution LC/MS a useful tool for the screening and identification of both common and uncommon drug metabolites.

Modeling, prediction, and in vitro in vivo correlation of CYP3A4 induction.

CYP3A4 induction is not generally considered to be a concern for safety; however, serious therapeutic failures can occur with drugs whose exposure is lower as a result of more rapid metabolic clearance due to induction. Despite the potential therapeutic consequences of induction, little progress has been made in quantitative predictions of CYP3A4 induction-mediated drug-drug interactions (DDIs) from in vitro data. In the present study, predictive models have been developed to facilitate extrapolation of CYP3A4 induction measured in vitro to human clinical DDIs. The following parameters were incorporated into the DDI predictions: 1) EC50 and Emax of CYP3A4 induction in primary hepatocytes; 2) fractions unbound of the inducers in human plasma (fu, p) and hepatocytes (fu, hept); 3) relevant clinical in vivo concentrations of the inducers ([Ind]max, ss); and 4) fractions of the victim drugs cleared by CYP3A4 (fm, CYP3A4). The values for [Ind]max, ss and fm, CYP3A4 were obtained from clinical reports of CYP3A4 induction and inhibition, respectively. Exposure differences of the affected drugs in the presence and absence of the six individual inducers (bosentan, carbamazepine, dexamethasone, efavirenz, phenobarbital, and rifampicin) were predicted from the in vitro data and then correlated with those reported clinically (n = 103). The best correlation was observed (R2 = 0.624 and 0.578 from two hepatocyte donors) when fu, p and fu, hept were included in the predictions. Factors that could cause over- or underpredictions (potential outliers) of the DDIs were also analyzed. Collectively, these predictive models could add value to the assessment of risks associated with CYP3A4 induction-based DDIs by enabling their determination in the early stages of drug development.

Prediction of Human Drug-Drug Interactions from Time-Dependent Inactivation of CYP3A4 in Primary Hepatocytes Using a Population-Based Simulator

Time-dependent inactivation (TDI) of human cytochromes P450 3A4 (CYP3A4) is a major cause of clinical drug-drug interactions (DDIs). Human liver microsomes (HLM) are commonly used as an enzyme source for evaluating the inhibition of CYP3A4 by new chemical entities. The inhibition data can then be extrapolated to assess the risk of human DDIs. Using this approach, under- and overpredictions of in vivo DDIs have been observed. In the present study, human hepatocytes were used as an alternative to HLM. Hepatocytes incorporate the effects of other mechanisms of drug metabolism and disposition (i.e., phase II enzymes and transporters) that may modulate the effects of TDI on clinical DDIs. The in vitro potency (KI and kinact) of five known CYP3A4 TDI drugs (clarithromycin, diltiazem, erythromycin, verapamil, and troleandomycin) was determined in HLM (pooled, n = 20) and hepatocytes from two donors (D1 and D2), and the results were extrapolated to predict in vivo DDIs using a Simcyp population trial-based simulator. Compared with observed DDIs, the predictions derived from HLM appeared to be overestimated. The predictions based on TDI measured in hepatocytes were better correlated with the DDIs (n = 37) observed in vivo (R2 = 0.601 for D1 and 0.740 for D2) than those from HLM (R2 = 0.451). In addition, with the use of hepatocytes a greater proportion of the predictions were within a 2-fold range of the clinical DDIs compared with using HLM. These results suggest that DDI predictions from CYP3A4 TDI kinetics in hepatocytes could provide an alternative approach to balance HLM-based predictions that can sometimes substantially overestimate DDIs and possibly lead to erroneous conclusions about clinical risks.

Biosynthesis of Drug Metabolites Using Microbes in Hollow Fiber Cartridge Reactors: Case Study of Diclofenac Metabolism by Actinoplanes Species

Fungal and bacterial microbes are known to mimic mammalian cytochrome P450 metabolism. Traditionally, microbial biotransformation screening and small scale-ups (<1 liter) are performed in shake-flask reactors. An alternative approach is the use of hollow fiber cartridge (HFC) reactors. The performance of HFC reactors is compared with shake-flask reactors using diclofenac as a model substrate. Actinoplanes sp. (American Type Culture Collection 53771) in a shake-flask reactor hydroxylated diclofenac (50 μM) with 100% turnover in less than 5 h. A scaled-up production resulted in the formation of 4′-hydroxy (169 mg, 54% yield), 5-hydroxy (42 mg, 13% yield), and 4′,5-dihydroxy (25 mg, 7.7% yield) metabolites. HFC reactors with Teflon, polysulfone, and cellulose membranes were screened for nonspecific binding of diclofenac. Concentration-time profiles for turnover of 50 to 2000 μM diclofenac by Actinoplanes sp. were then determined at 22 and 30°C in an HFC reactor. Cellulose-based HFC reactors exhibited the lowest nonspecific binding (87% of 50 μM diclofenac remaining after 5 h) and offered the best conditions for its biotransformation (100% conversion; < 5 h at 30°C at 50 μM; 25 h at 500 μM). The time profile for substrate turnover was equivalent in both a cellulose membrane HFC reactor and shake-flask reactor. Two cellulose membrane HFC reactors were also tested to evaluate the reusability of the cartridges for diclofenac metabolism (50 μM, 22°C, 15 h; 500 μM, 30°C, 36 h). Up to seven reaction cycles with intermediate wash cycles were tested. At least 98% conversion was observed in each reaction cycle at both diclofenac concentrations.

Simulation of Clinical Drug-Drug Interactions from Hepatocyte CYP3A4 Induction Data and Its Potential Utility in Trial Designs

Rifampin and carbamazepine have been recommended in the U.S. Food and Drug Administration draft drug interaction guidance as CYP3A4 inducers for clinical drug-drug interaction (DDI) studies. To optimize the dose regimens of these inducers for use in DDI studies, their effect at various doses and dosing durations on the area under the curve (AUC) of multiple probe substrates was simulated using a population-based simulator. A similar assessment of the inducer phenobarbital was also conducted. CYP3A4 induction by all three inducers was previously determined in hepatocytes, and the results were incorporated into simulations. The pharmacokinetics of the three inducers and their associated CYP3A4 drug interactions were predicted and compared with in vivo observations. The predicted Cmax and AUC of all the inducers and substrates correlated closely with those observed clinically. The predicted magnitudes of the DDIs caused by CYP3A4 induction were also in good agreement with the observed clinical results. Comparison of the maximal CYP3A4 induction potential among the three inducers indicated that rifampin is the most potent inducer and is the best choice for clinical CYP3A4 induction DDI studies. Moreover, a near-maximal CYP3A4 DDI was predicted to result from administration of rifampin for approximately 7 days at 450 to 600 mg q.d. or 200 to 300 mg b.i.d. These results suggest optimal dose regimens for clinical trials that maximize the probability of detecting a DDI caused by CYP3A4 induction. The simulation strategy provides the means to predict the induction profiles of compounds in development.

Reductive Isoxazole Ring Opening of the Anticoagulant Razaxaban Is the Major Metabolic Clearance Pathway in Rats and Dogs

Razaxaban is a selective, potent, and orally bioavailable inhibitor of coagulation factor Xa. The molecule contains a 1,2-benzisoxazole structure. After oral administration of [14C]razaxaban to intact and bile duct-cannulated rats (300 mg/kg) and dogs (20 mg/kg), metabolism followed by biliary excretion was the major elimination pathway in both species, accounting for 34 to 44% of the dose, whereas urinary excretion accounted for 3 to 13% of the dose. Chromatographic separation of radioactivity in urine, bile, and feces of rats and dogs showed that razaxaban was extensively metabolized in both species. Metabolites were identified on the basis of liquid chromatography/tandem mass spectrometry and comparison with synthetic standards. Among the 12 metabolites identified, formation of an isoxazole-ring opened benzamidine metabolite (M1) represented a major metabolic pathway of razaxaban in rats and dogs. However, razaxaban was the major circulating drug-related component (>70%) in both species, and M1, M4, and M7 were minor circulating components. In addition to the in vivo observations, M1 was formed as the primary metabolite in rat and dog hepatocytes and in the rat liver cytosolic fraction. The formation of M1 in the rat liver fraction required the presence of NADH. Theses results suggest that isoxazole ring reduction, forming a stable benzamidine metabolite (M1), represents the primary metabolic pathway of razaxaban in vivo and in vitro. The reduction reaction was catalyzed by NADH-dependent reductase(s) in the liver and possibly by intestinal microflora on the basis of the recovery of M1 in feces of bile duct-cannulated rats.

Cytochrome P450-Mediated Epoxidation of 2-Aminothiazole-Based AKT Inhibitors: Identification of Novel GSH Adducts and Reduction of Metabolic Activation through Structural Changes Guided by in Silico and in Vitro Screening

A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT, a serine/threonine kinase. When incubated in rat and human liver microsomes in the presence of NADPH, 1 underwent significant metabolic activation on its 2-aminothiazole ring, leading to substantial covalent protein binding. Upon addition of glutathione, covalent binding was reduced significantly, and multiple glutathione adducts were detected. Novel metabolites from the in vitro incubates were characterized by LC-MS and NMR to discern the mechanism of bioactivation. An in silico model was developed based on the proposed mechanism and was employed to predict bioactivation in 23 structural analogues. The predictions were confirmed empirically for the bioactivation liability, in vitro, by LC-MS methods screening for glutathione incorporation. New compounds were identified with a low propensity for bioactivation.

Metabolite Generation via Microbial Biotransformations with Actinomycetes: Rapid Screening for Active Strains and Biosynthesis of Important Human Metabolites of Two Development-Stage Compounds, 5-[(5S,9R)-9-(4-Cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic Acid (BMS-587101) and Dasatinib

The enzymes present in many microbial strains are capable of carrying out a variety of biotransformations when presented with drug-like molecules. Although the enzymes responsible for the biotransformations are not well characterized, microbial strains can often be found that produce metabolites identical to those found in mammalian systems. However, traditional screening for microbial strains that produce metabolites of interest is done with many labor intensive steps that include multiple shake flasks and many manual manipulations, which hinder the application of these techniques in drug metabolite preparation. A 24-well microtiter plate screening system was developed for rapid screening of actinomycetes strains for their ability to selectively produce metabolites of interest. The utility of this system was first demonstrated with the well characterized cytochrome P450 substrate diclofenac. Subsequently, the use of this system allowed the rapid identification of several actinomycetes strains that were capable of converting two drug candidates under development, 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic acid and N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, Sprycel, BMS-345825), to mammalian metabolites of interest. Milligram quantities of the metabolites were then prepared by scaling-up the microbial biotransformation reactions. These quantities were sufficient for initial characterization, such as testing for pharmacological activity and use as analytical standards, prior to the availability of authentic chemically synthesized compounds.

Chapter 9 Quantification and structural elucidation of low quantities of radiolabeled metabolites using microplate scintillation counting (msc) techniques in conjunction with lc-ms

Publisher Summary This chapter presents the quantification and structural elucidation of low quantities of radiolabeled metabolites using microplate scintillation counting (MSC) techniques in conjunction with liquid chromatography-mass spectrometry (LC-MS). Radioactive isotopes, mainly 14C, are routinely used to trace the fate of drugs in disposition and metabolism studies conducted during their development. Information derived from these studies is a critical component of developing safe and effective new drugs and is also a regulatory requirement for registration approval. MSC is an off-line liquid chromatographic detector that has been available for only the past few years; however, in that short time it has shown great promise as a powerful and versatile tool for detection and quantitative analysis of low levels of radiolabeled metabolites. Compared to conventional HPLC-LSC methods, HPLC-MSC not only has comparable precision, better sensitivity and increased analytical throughput, it also significantly reduces radioactive waste and requires minimal manual operation. HPLC-MSC coupled with an electrospray ionization mass spectrometer is an ideal choice for structural elucidation of low abundance radiolabeled metabolites. Additionally, LC-MSC has been used in conjunction with high mass-resolution MS-based mass defect filtering technique for selective detection of metabolite ions in complex biological matrices. Finally, micro flow LC-MS/MS analysis of radioactive metabolites recovered from microplates has shown excellent sensitivity for identification/characterization of very small quantities of metabolites.

Novel cytochrome p450 bioactivation of a terminal phenyl acetylene group: formation of a one-carbon loss benzaldehyde and other oxidative products in the presence of N-acetyl cysteine or glutathione.

Compounds 1 (N1-(3-ethynylphenyl)-6-methyl-N5-(3-(6-(methylamino)pyrimidin-4-yl)pyridin-2-yl) isoquinoline-1,5-diamine) and 2 (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine; Erlotinib/Tarceva) are kinase inhibitors that contain a terminal phenyl acetylene moiety. When incubated in the presence of P450 and NADPH, the anticipated phenyl acetic acid metabolite was formed. When 10 mM of N-acetyl-l-cysteine was added to the incubation mixtures, the phenyl acetic acid product was reduced and at 25 mM or higher concentration of NAC, formation of the phenyl acetic acid was abolished. Instead, the phenyl acetylene moiety lost a carbon and formed a benzaldehyde product. Other oxidation products incorporating one or more equivalents of NAC were also observed. The identities of the metabolites were characterized by MS and NMR. Addition of deferoxamine or ascorbic acid diminished the formation of the NAC influenced products. Similar products were also observed when 1 or 2 were incubated in P450 reactions supplemented with GSH, in Fenton reactions supplemented with NAC or GSH, and in peroxidase reactions supplemented with NAC. We propose the thiols act as a pro-oxidant readily undergoing a one-electron oxidation to form thiyl radicals which in turn initiates the formation of other peroxy radicals that drive the reaction to the observed products. These in vitro findings suggest that one-electron oxidation of thiols may promote the cooxidation of xenobiotic substrates.