Evan Smith

Assessment of Three Human in Vitro Systems in the Generation of Major Human Excretory and Circulating Metabolites

An early understanding of key metabolites of drugs is crucial in drug discovery and development. As a result, several in vitro models typically derived from liver are frequently used to study drug metabolism. It is presumed that these in vitro systems provide an accurate view of the potential in vivo metabolites and metabolic pathways. However, no formal analysis has been conducted to validate their use. The goal of the present study was to conduct a comprehensive analysis to assess if the three commonly used in vitro systems, pooled human liver microsomes, liver S-9 fraction, and hepatocytes, adequately predict in vivo metabolic profiles for drugs. The second objective was to compare the overall capabilities of these three systems to generate in vivo metabolic profiles. Twenty-seven compounds in the Pfizer database and 21 additional commercially available compounds of diverse structure and routes of metabolism for which the human ADME data was available were analyzed in this study to assess the performance of the in vitro systems. The results suggested that all three systems reliably predicted human excretory and circulating metabolite profiles. Furthermore, the success in predicting primary metabolites and metabolic pathways was high (>70%), but the predictability of secondary metabolites was less reliable in the three systems. Thus, the analysis provides sufficient confidence in using in vitro systems to reliably produce primary in vivo human metabolites and supports their application in early discovery to identify metabolic spots for optimization of metabolic liabilities anticipated in humans in vivo. However, the in vitro systems cannot solely mitigate the risk of disproportionate circulating metabolites in humans and may need to be supplemented with metabolic profiling of plasma samples from first-in-human studies or early human radiolabeled studies.

Identifying a Selective Substrate and Inhibitor Pair for the Evaluation of CYP2J2 Activity

CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC50 = 77 nM) and O-demethylation of astemizole (Ki = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC50 values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues.

Validation of isolated metabolites from drug metabolism studies as analytical standards by quantitative NMR.

In discovery and development, having a qualified metabolite standard is advantageous. Chemical synthesis of metabolite standards is often difficult and expensive. As an alternative, biological generation and isolation of metabolites in the nanomole range are readily feasible. However, without an accurately defined concentration, these isolates have limited utility as standards. There is a significant history of NMR as both a qualitative and a quantitative technique, and these concepts have been merged recently to provide both structural and quantitative information on biologically generated isolates from drug metabolism studies. Previous methodologies relied on either specialized equipment or the use of an internal standard to the isolate. We have developed a technique in which a mathematically generated signal can be inserted into a spectrum postacquisition and used as a quantitative reference: artificial signal insertion for calculation of concentration observed (aSICCO). This technique has several advantages over previous methodologies. Any region in the analyte spectra, free from interference, can be chosen for the reference signal. In addition, the magnitude of the inserted signal can be modified to appropriately match the intensity of the sample resonances. Because this is postacquisition quantification, no special equipment or pulse sequence is needed. Compared with quantitation via the addition of an internal standard (10 mM maleic acid), the signal insertion method produced similar results. For each method, precision and accuracy were within ±5%, stability of signal response over 8 days was ±5%, and the dynamic range was more than 3 orders of magnitude: 10 to 0.01 mM.

Genotoxicity of 2-(3-Chlorobenzyloxy)-6-(piperazinyl)pyrazine, a Novel 5-Hydroxytryptamine2c Receptor Agonist for the Treatment of Obesity: Role of Metabolic Activation

2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT2C agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA after incubations with [14C]3 in the presence of rat S9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S9/NADPH in the presence of methoxylamine and cyanide led to the detection of amine and cyano conjugates of 3. The mass spectrum of the amine conjugate was consistent with condensation of amine with an aldehyde metabolite derived from hydroxylation of the secondary piperazine nitrogen-α-carbon bond. The mass spectrum of the cyano conjugate suggested a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen followed by two-electron oxidation to generate an electrophilic nitrone, which reacted with cyanide. The 3-chlorobenzyl motif in 3 was also bioactivated via initial aromatic ring hydroxylation followed by elimination to a quinone-methide species that reacted with glutathione or with the secondary piperazine ring nitrogen in 3 and its monohydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.

Identification of a Novel Glutathione Conjugate of Flutamide in Incubations with Human Liver Microsomes

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

Bioactivation of flutamide metabolites by human liver microsomes.

Flutamide, a widely used nonsteroidal antiandrogen drug for the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. A novel N-S glutathione adduct has been identified in a previous bioactivation study of flutamide (Kang et al., 2007). Due to the extensive first pass metabolism, flutamide metabolites such as 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1) have achieved plasma concentrations higher than the parent in prostate cancer patients. In vitro studies in human liver microsomes were conducted to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide metabolites and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. Several GSH adducts (G1, Flu-1-G1, Flu-1-G2, Flu-6-Gs) derived from the metabolites of flutamide were identified and characterized. A comprehensive bioactivation mechanism was proposed to account for the formation of the observed GSH adducts. Of interest were the formation of a reactive intermediate by the desaturation of the isopropyl group of M5 and the unusual bioactivation of Flu-1. Studies using recombinant P450s suggested that the major P450 isozymes involved in the bioactivation of flutamide and its metabolites were CYP1A2, CYP3A4, and CYP2C19. These findings suggested that, in addition to the direct bioactivation of flutamide, the metabolites of flutamide could also be bioactivated and contribute to flutamide-induced hepatotoxicity.

Optimization of potent, selective, and orally bioavailable pyrrolodinopyrimidine-containing inhibitors of heat shock protein 90. Identification of development candidate 2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide.

A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.

Metabolism, excretion and pharmacokinetics of [14C]crizotinib following oral administration to healthy subjects

Abstract 1. Crizotinib (XALKORI®), an oral inhibitor of anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition factor kinase (c-Met), is currently approved for the treatment of patients with non-small cell lung cancer that is ALK-positive. 2. The metabolism, excretion and pharmacokinetics of crizotinib were investigated following administration of a single oral dose of 250 mg/100 µCi [14C]crizotinib to six healthy male subjects. 3. Mean recovery of [14C]crizotinib-related radioactivity in excreta samples was 85% of the dose (63% in feces and 22% in urine). 4. Crizotinib and its metabolite, crizotinib lactam, were the major components circulating in plasma, accounting for 33% and 10%, respectively, of the 0–96 h plasma radioactivity. Unchanged crizotinib was the major excreted component in feces (∼53% of the dose). In urine, crizotinib and O-desalkyl crizotinib lactam accounted for ∼2% and 5% of the dose, respectively. Collectively, these data indicate that the primary clearance pathway for crizotinib in humans is oxidative metabolism/hepatic elimination. 5. Based on plasma exposure in healthy subjects following a single dose of crizotinib and in vitro potency against ALK and c-Met, the crizotinib lactam diastereomers are not anticipated to contribute significantly to in vivo activity; however, additional assessment in cancer patients is warranted.

IN VITRO METABOLIC ACTIVATION OF THIABENDAZOLE VIA 5-HYDROXYTHIABENDAZOLE: IDENTIFICATION OF A GLUTATHIONE CONJUGATE OF 5-HYDROXYTHIABENDAZOLE

Thiabendazole (TBZ) is a broad-spectrum antihelmintic used for treatment of parasitic infections in animals and humans and as an agricultural fungicide for postharvest treatment of fruits and vegetables. It is teratogenic and nephrotoxic in mice, and cases of hepatotoxicity have been observed in humans. Recent reports have demonstrated a correlation between 5-hydroxythiabendazole (5-OHTBZ) formation, a major metabolite of TBZ, and covalent binding of [14C]TBZ to hepatocytes, suggesting another pathway of activation of TBZ. Current in vitro studies were undertaken to probe the bioactivation of TBZ via 5-OHTBZ by cytochrome P450 (P450) and peroxidases and identify the reactive species by trapping with reduced glutathione (GSH). Microsomal incubation of TBZ or 5-OHTBZ supplemented with NADPH and GSH afforded a GSH adduct of 5-OHTBZ and was consistent with a bioactivation pathway that involved a P450-catalyzed two-electron oxidation of 5-OHTBZ to a quinone imine. The same adduct was detected in GSH-fortified incubations of 5-OHTBZ with peroxidases. The identity of the GSH conjugate suggested that the same reactive intermediate was formed by both these enzyme systems. Characterization of the conjugate by mass spectrometry and NMR revealed the addition of GSH at the 4-position of 5-OHTBZ. In addition, the formation of a dimer of 5-OHTBZ was discernible in peroxidase-mediated incubations. These results were consistent with a one-electron oxidation of 5-OHTBZ to a radical species that could undergo disproportionation or an additional one-electron oxidation to form a quinone imine. Overall, these studies suggest that 5-OHTBZ can also play a role in TBZ-induced toxicity via its bioactivation by P450 and peroxidases.

Myeloperoxidase-mediated bioactivation of 5-hydroxythiabendazole: A possible mechanism of thiabendazole toxicity

Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity. Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been proposed to yield a reactive intermediate. Here we show that this reactive intermediate can be catalyzed by myeloperoxidase (MPO), a neutrophil-bourne peroxidase. Using a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPO-generated metabolites in cell-based models including primary rat proximal tubule epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells. Timecourse experiments with MPO showed complete turnover of 5OH-TBZ within 15 min and a dramatic leftward shift in dose-response curves after 12h. After a 24h exposure in vitro, the LC(50) of this reactive intermediate was 23.3 ± 0.2 μM reduced from greater than 200 μM from 5OH-TBZ alone, an approximately 10-fold decrease. LC(50) values were equal in all cell types used. Comparison of lactate dehydrogenase leakage and caspase 3/7 activity revealed that cell death caused by the reactive intermediate is primarily associated with necrosis rather than apoptosis. This toxicity can be completely rescued via incubation with rutin, an inhibitor of MPO. These results suggest that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate which may play a role in TBZ-induced toxicity.