Characterization of 1-Aminobenzotriazole and Ketoconazole as Novel Inhibitors of Monoamine Oxidase (MAO): An In Vitro Investigation.

BACKGROUND AND OBJECTIVES: 1-Aminobenzotriazole, a known time-dependent inhibitor of cytochrome P450 (CYP) enzymes, and ketoconazole, a strong inhibitor of the human CYP3A4 isozyme, are used as standard probe inhibitors to characterize the CYP and/or non-CYP-mediated metabolism of xenobiotics. In the present investigation, 1-Aminobenzotriazole and ketoconazole are characterized as potent monoamine oxidase (MAO) inhibitors in vitro using mouse, rat and human liver microsomes and S9 fractions. METHODS: Inhibition potential of 1-aminobenzotriazole and ketoconazole was studied in mice, rat and human liver microsomes, S9 fractions, MAO-A and MAO-B expressed enzymes by monitoring the formation of 4-hydroxyquinoline (4-HQ) from kynuramine, a specific substrate of MAO by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mechanism of MAO inhibition was studied by incubating varying concentration of kynuramine with mouse, rat and human S9 fractions at varying concentration of 1-aminobenzatriazole and ketoconazole and monitoring the formation of 4-HQ. RESULTS: 1-aminobenzotriazole and ketoconazole inhibited both MAO isozymes (MAO-A and MAO-B) with more specificity towards MAO-B. Kynuramine substrate kinetics in mouse, rat and human S9 fractions with varying 1-aminobenzotriazole and ketoconazole concentrations showed decreased maximum rate (V max) for 4-HQ formation without affecting the Michaelis-Menten constant (K m). A non-competitive inhibition model was constructed and inhibition constants (K i) for 1-aminobenzotriazole (7.87 ± 0.61, 8.61 ± 0.92, 65.2 ± 1.61 µM for mice, rat and humans, respectively) and ketoconazole (0.12 ± 0.01, 2.04 ± 0.08, 5.52 ± 0.47 µM for mice, rat and humans, respectively) were determined. CONCLUSIONS: 1-Aminobenzotriazole and ketoconazole are characterized as non-competitive inhibitors of mice, rat and human MAO in vitro and the extent of their MAO inhibition potential is species specific. 1-Aminobenzotriazole or ketoconazole can be used as a probe inhibitor in vitro for screening the involvement of MAO-dependent metabolism of new chemical entities (NCE) in early drug discovery.

Mechanism of Drug-Drug Interactions Between Warfarin and Statins.

The anticoagulant drug warfarin and the lipid-lowering statin drugs are commonly co-administered to patients with cardiovascular diseases. Clinically significant drug-drug interactions (DDIs) between these drugs have been recognized through case studies for many years, but the biochemical mechanisms causing these interactions have not been explained fully. Previous theories include kinetic alterations in cytochrome P-450-mediated drug metabolism or disturbances of drug-protein binding, leading to anticoagulant activity of warfarin; however, neither the enantioselective effects on warfarin metabolism nor the potential disruption of drug transporter function have been well investigated. This study investigated the etiology of the DDIs between warfarin and statins. Liquid chromatography-mass spectrometry methods were developed and validated to quantify racemic warfarin, 6 of its hydroxylated metabolites, and pure enantiomers of warfarin; these methods were applied to study the role of different absorption, distribution, metabolism, and excretion properties, leading to DDIs. Plasma protein binding displacement of warfarin was performed in the presence of statins using equilibrium dialysis method. Substrate kinetics of warfarin and pure enantiomers were performed with human liver microsomes to determine the kinetic parameters (Km and Vmax) for the formation of all 6 hydroxywarfarin metabolites, inhibition of warfarin metabolism in the presence of statins, was determined. Uptake transport studies of warfarin were performed using overexpressing HEK cell lines and efflux transport using human adenocarcinoma colonic cell line cells. Fluvastatin significantly displaced plasma protein binding of warfarin and pure enantiomers; no other statin resulted in significant displacement of warfarin. All the statins that inhibited the formation of 10-hydroxywarfarin, atorvastatin, pitavastatin, and simvastatin were highly potent compared to other statins; in contrast, only fluvastatin was found to be a potent inhibitor of formation of 7-hydroxy warfarin. Uptake and efflux drug transporters do not play any role in these DDIs. The results showed that DDIs between warfarin and statins are primarily caused by cytochrome P-450 inhibition.

Comparison of enzyme kinetics of warfarin analyzed by LC-MS/MS QTrap and differential mobility spectrometry.

Warfarin is an anticoagulant used in the treatment of thrombosis and thromboembolism. It is given as a racemic mixture of R and S enantiomers. These two enantiomers show differences in metabolism by CYPs: S-warfarin undergoes 7 hydroxylation by CYP2C9 and R-warfarin by CYP3A4 to form 10 hydroxy warfarin. In addition, warfarin is acted upon by different CYPs to form the minor metabolites 3'-hydroxy, 4'-hydroxy, 6-hydroxy, and 8-hydroxy warfarin. For analysis, separation of these metabolites is necessary since all have the same m/z ratio and similar fragmentation pattern. Enzyme kinetics for the formation of all of the six hydroxylated metabolites of warfarin from human liver microsomes were determined using an LC-MS/MS QTrap and LC-MS/MS with a differential mobility spectrometry (DMS) (SelexION™) interface to compare the kinetic parameters. These two methods were chosen to compare their selectivity and sensitivity. Substrate curves for 3'-OH, 4'-OH, 6-OH, 7-OH, 8-OH and 10-OH warfarin formation were generated to determine the kinetic parameters (Km and Vmax) in human liver microsomal preparations. The limit of quantitation (LOQ) for all the six hydroxylated metabolites of warfarin were in the range of 1-3nM using an LC-MS/MS QTrap method which had a run time of 22min. In contrast, the LOQ for all the six hydroxylated metabolites using DMS interface technology was 100nM with a run time of 2.8min. We compare these two MS methods and discuss the kinetics of metabolite formation for the metabolites generated from racemic warfarin. In addition, we show inhibition of major metabolic pathways of warfarin by sulfaphenazole and ketoconazole which are known specific inhibitors of CYP2C9 and CYP3A4 respectively.

Effect of excipients on acetaminophen metabolism and its implications for prevention of liver injury.

Acetaminophen poisoning is the most frequent cause of acute hepatic failure in the US. Toxicity requires reductive metabolism of acetaminophen, primarily via CYP2E1. Liquid acetaminophen preparations contain propylene glycol, a common excipient that has been shown to reduce hepatocellular injury in vitro and in rodents. Children are less susceptible to acetaminophen toxicity for unclear reasons. We conducted a pharmacokinetic single-blinded crossover study of 15 healthy adult volunteers comparing the CYP2E1 and conjugative metabolism of a 15 mg/kg dose of liquid versus solid preparations of acetaminophen. Measured AUC's for the CYP2E1 metabolites were 16-17% lower and extrapolated AUC's were 25-28% lower in the liquid formulation arm while there was no difference in conjugative metabolite production. The formation rate constants for reductive metabolites were equivalent between solid and liquid formulations indicating that enzyme inhibition was competitive. Propylene glycol, an established CYP2E1 competitive antagonist, was detected in the liquid formulation but not solid formulation arm. Since children tend to ingest liquid preparations, the protective effect of this excipient could explain their decreased susceptibility to acetaminophen toxicity. A less hepatotoxic formulation of acetaminophen could potentially be developed if co-formulated with a CYP2E1 inhibitor.

Stability of vitamin B complex in multivitamin and multimineral supplement tablets after space flight.

The effect of storage in space on the stability of vitamin B complex in two commercial vitamin tablets was examined. Multiple vitamin samples returned after storage on the space shuttle and International Space Station (ISS) along with two ground control and three positive control groups were included in the study. Content of vitamin B(3) in the tablets and in vitro dissolution rate were determined using a modified high performance liquid chromatographic assay from USP/NF 2010. Results indicate that vitamin B(3) in one of the brands tested (#2) may be subject to marginal degradation after storage on ISS for 4 months as indicated by the chromatograms for all six tablets showing a split peak appearing as a notch at the peak tip. Chromatograms were not different for ground and flight samples for Brand #1 suggesting that this may be more suitable for use in space.

In vitro metabolism of oxymetazoline: evidence for bioactivation to a reactive metabolite.

Oxymetazoline (6-tert-butyl-3-(2-imidazolin-2-ylmethyl)-2,4-dimethylphenol) has been widely used as a nonprescription nasal vasoconstrictor for >40 years; however, its metabolic pathway has not been investigated. This study describes the in vitro metabolism of oxymetazoline in human, rat, and rabbit liver postmitochondrial supernatant fraction from homogenized tissue (S9) fractions and their microsomes supplemented with NADPH. The metabolites of oxymetazoline identified by liquid chromatography (LC)/UV/tandem mass spectrometry (MS/MS), included M1 (monohydroxylation of the t-butyl group), M2 (oxidative dehydrogenation of the imidazoline to an imidazole moiety), M3 (monohydroxylation of M2), M4 (dihydroxylation of oxymetazoline), and M5 (dihydroxylation of M2). Screening with nine human expressed cytochromes P450 (P450s) identified CYP2C19 as the single P450 isoform catalyzing the formation of M1, M2, and M3. Glutathione conjugates of oxymetazoline (M6) and M2 (M7) were identified in the liver S9 fractions, indicating the capability of oxymetazoline to undergo bioactivation to reactive intermediate species. M6 and M7 were not detected in those liver S9 incubations without NADPH. Cysteine conjugates (M8 and M9) derived from glutathione conjugates and hydroxylated glutathione conjugates (M10 and M11) were also identified. The reactive intermediate of oxymetazoline was trapped with glutathione and N-acetyl cysteine and identified by LC/MS/MS. M6 was isolated and identified by one-dimensional or two-dimensional NMR as the glutathione conjugate of a p-quinone methide. We have shown the tendency of oxymetazoline to form p-quinone methide species via a bioactivation mechanism involving a CYP2C19-catalyzed two-electron oxidation. Nevertheless, we conclude that the formation of this reactive species might not be a safety concern for oxymetazoline nasal products because of the typical low-dose and brief dosage regimen limited to nasal delivery.

Identification and characterization of oxymetazoline glucuronidation in human liver microsomes: evidence for the involvement of UGT1A9.

The incubation of oxymetazoline, a nonprescription nasal decongestant, with human liver microsomes (HLMs) supplemented with uridine-5-diphosphoglucuronic acid (UDPGA) generated glucuronide metabolite as observed by LC/MS/MS. The uridine glucuronosyltransferases (UGTs) responsible for the O-glucuronidation of oxymetazoline remain thus far unidentified. The glucuronide formed in HLMs was identified by LC/MS/MS and characterized by one- and two-dimensional NMR to be the ß-O-glucuronide of oxymetazoline. UGT screening with expressed UGTs identified UGT1A9 as the single UGT isoform catalyzing O-glucuronidation of oxymetazoline. Oxymetazoline O-glucuronidation by using HLMs was best fitted to the allosteric sigmoidal model. The derived S(50) and V(max) values were 2.42 ± 0.40 mM and 8.69 ± 0.58 pmole/(min mg of protein), respectively, and maximum clearance (CL(max)) was 3.61 L/min/mg. Oxymetazoline O-glucuronidation by using expressed UGT1A9 was best fitted to the substrate inhibition model. The derived K(m) and V(max) values were 2.53 ± 1.03 mM and 54.18 ± 16.92 pmole/(min mg of protein), respectively, and intrinsic clearance (CL(int)) was 21.41 L/(min mg). Our studies indicate that oxymetazoline is not glucuronidated at its nanomolar intranasal dose and thus is eliminated unchanged, because UGT1A9 would only contribute to its elimination at the toxic plasma concentrations.

Identification of human UGT2B7 as the major isoform involved in the O-glucuronidation of chloramphenicol.

Chloramphenicol (CP), a broad spectrum antibiotic, is eliminated in humans by glucuronidation. The primary UGT enzymes responsible for CP O-glucuronidation remain unidentified. We have previously identified the 3-O-CP (major) and 1-O-CP (minor) glucuronides by beta-glucuronidase hydrolysis, liquid chromatography-tandem mass spectrometry, and 1D/2D H NMR. Reaction phenotyping for the glucuronidation of CP with 12 expressed human liver UGT isoforms has identified UGT2B7 as having the highest activity for 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. The kinetics of CP 3-O-glucuronidation by pooled human liver microsomes (HLMs) exhibited biphasic Michaelis-Menten kinetics with the apparent high-affinity K(m1) and low-affinity K(m2) values of 46.0 and 1027 microM, whereas expressed UGT2B7 exhibited Michaelis-Menten kinetics with the apparent K(m) value of 109.1 microM. The formation of 1-O-CP glucuronide by pooled HLM and expressed UGT2B7 exhibited substrate inhibition kinetics with apparent K(m) values of 408.2 and 115.0 microM, respectively. Azidothymidine (AZT) and hyodeoxycholic acid (substrates of UGT2B7) inhibited 3-O- and 1-O-CP glucuronidation in pooled HLMs. In 10 donor HLM preparations, both CP 3-O- and CP 1-O-glucuronidation showed a significant correlation with AZT glucuronidation (UGT2B7) (r(s) = 0.85 and r(s) = 0.83, respectively) at 30 microM CP, whereas no significant correlation was observed between CP 3-O-glucuronidation and serotonin glucuronidation (UGT1A6) or propofol glucuronidation (UGT1A9) at this CP concentration. These results suggest that UGT2B7 is the primary human hepatic UDP-glucuronosyltransferase isoform catalyzing 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9.

Effect of potassium channel and cytochrome P450 inhibition on transient hypotension and survival during lipopolysaccharide-induced endotoxic shock in the rat.

The purpose of this study was to determine whether inhibition of potassium channels or cytochrome P450 attenuates the transient phase of hypotension during endotoxic shock in vivo, and to determine whether these interventions improve the rate of survival. Male Sprague-Dawley rats were pretreated with saline (0.2 ml, i.v.), tetraethylammonium chloride (TEA 30 mg/kg; 0.2 ml, i.v.), proadifen (SKF-525 A; 50 mg/kg, i.p.) or ketoconazole (50 mg/kg, i.p.) and challenged with lipopolysaccharide (LPS; 20 mg/kg, i.p.). Changes in heart rate, mean (MAP), systolic (SP) and diastolic (DP) arterial pressures as well as survival rate were then monitored for 45 min. Potassium channel inhibition with TEA had no effect on LPS-induced hypotension at any time point compared with saline (maximal fall in MAP of 79 +/- 18 and 80 +/- 13 mm Hg, respectively). Pretreatment with proadifen or ketoconazole, inhibitors of cytochrome P450, significantly attenuated LPS-induced hypotension compared with saline (maximal fall in MAP of 34, 26 and 63% below baseline, respectively). This effect was evident in all arterial pressures measured, MAP, SP and DP. At 45 min, the survival rate in the saline group was 66%. Pretreatment with TEA significantly reduced survival rate to 50% and pretreatment with proadifen or ketoconazole improved survival to 100% (p < 0.05). These results suggest that an arachidonic acid metabolite produced by a cytochrome P450-catalyzed reaction may contribute to the transient phase of LPS-induced hypotension. However, these effects do not appear to be mediated through potassium channel activation.

Glucuronidation of haloperidol by rat liver microsomes: involvement of family 2 UDP-glucuronosyltransferases.

The purposes of this study were to develop a HPLC method to assay for haloperidol glucuronide (HALG); to apply this assay method to the in vitro determination of haloperidol (HAL) UDP-glucuronosyltransferase (UGT) enzyme kinetics in rat liver microsomes (RLM); and to identify the UGT isoforms catalyzing glucuronidation of HAL in rats. Incubation of Brij-activated RLM with HAL and UDP-glucuronic acid (UDPGA) in TRIS pH 7.4 buffer resulted in the formation of a single peak in the HPLC chromatogram at 270 nm. The identity of this peak was confirmed to be that of HALG by 1) beta-glucuronidase hydrolysis; 2) incubation without UDPGA; 3) UV spectral analysis; and 4) LC/MS/MS to yield the expected mass of 552.1. Enzyme kinetic studies using single enzyme Michaelis-Menton model showed an apparent Vmax = 271.9 +/- 10.1 pmoles min(-1) mg protein(-1) and Km = 61 +/- 7.2 microM. Glucuronidation activity in homozygous Gunn (j/j) rats was approximately 80% as compared to Sprague-Dawley RLM. HALG formation was approximately doubled in PB-induced RLM. There was no increase in glucuronidation activities in 3MC-induced RLM. The Gunn rat and the PB-induced RLM data suggest predominant but not exclusive involvement of the UGT2B family in the formation of HALG. Because the UGTs exhibit overlapping substrate specificities and most substrates are glucuronidated by more than one isoform, inhibition studies with UGT2B1 substrate probe testosterone and the UGT2B12 substrate probe borneol were conducted. UGT2B1 and UGT2B12 exhibited 40% and 90% inhibition of HAL glucuronidation, respectively. Thus, UGT2B12 and UGT 2B1 isoforms are responsible for catalyzing HAL glucuronidation in rats. Our HPLC assay provides a specific and sensitive technique for the measurement of in vitro HAL-UGT activity.

Time course of recovery of cytochrome p450 3A function after single doses of grapefruit juice.

BACKGROUND: Components of grapefruit juice may impair the activity of intestinal cytochrome P450 (CYP) 3A enzymes, sometimes resulting in clinically important drug interactions. The time course of recovery from CYP3A inhibition after a single exposure to grapefruit juice is not clearly established. METHODS: Healthy volunteer subjects (N = 25) received a single 6-mg oral dose of the CYP3A substrate midazolam in the control condition without exposure to grapefruit juice. Two days later, midazolam was administered 2 hours after 300 mL of regular-strength grapefruit juice. Subjects were then randomly assigned to 3 different groups, receiving a third midazolam challenge at 26, 50, or 74 hours after exposure to grapefruit juice. The capacity of 6'7'-dihydroxybergamottin and bergamottin to inhibit human CYP3A was studied in vitro using human liver microsomes. RESULTS: The area under the plasma concentration curve (AUC) for midazolam increased by a factor of 1.65 (ratio compared with control) when midazolam was given 2 hours after grapefruit juice. At 26, 50, and 74 hours after grapefruit juice, the AUC ratios (mean AUC value at the indicated time divided by the mean control AUC on day 1) were 1.29, 1.29, and 1.06, respectively. The relationship of time after grapefruit juice exposure versus AUC increase over control indicated a recovery half-life estimated at 23 hours. The midazolam elimination half-life did not change significantly from the control value at any time after grapefruit juice exposure. 6'7'-Dihydroxybergamottin inhibited midazolam alpha-hydroxylation in vitro, with a mean 50% inhibitory concentration of 4.7 micro mol/L; preincubation of microsomes with 6'7'-dihydroxybergamottin greatly reduced the 50% inhibitory concentration to 0.31 micro mol/L, consistent with mechanism-based inhibition. Bergamottin itself had much weaker inhibitory potency compared to 6'7'-dihydroxybergamottin. CONCLUSIONS: A usual single exposure to grapefruit juice appears to impair the enteric, but not the hepatic, component of presystemic extraction of oral midazolam. Recovery is largely complete within 3 days, consistent with enzyme regeneration after mechanism-based inhibition. 6'7'-Dihydroxybergamottin was verified as a potent mechanism-based inhibitor of midazolam alpha-hydroxylation by CYP3A in vitro.