A personalized stereotactic fixture for implantation of depth electrodes in stereoelectroencephalography.

BACKGROUND: The stereoelectroencephalographic (SEEG) implantation procedures still represent a challenge due to the intrinsic complexity of the method and the number of depth electrodes required. OBJECTIVES: We aim at designing and evaluating the accuracy of a custom stereotactic fixture based on the StarFix™ technology (FHC Inc., Bowdoin, ME) that significantly simplifies and optimizes the implantation of depth electrodes used in presurgical evaluation of patients with drug-resistant epilepsy. METHODS: Fiducial markers that also serve as anchors for the fixture are implanted into the patient's skull prior to surgery. A 3D fixture model is designed within the surgical planning software, with the planned trajectories incorporated in its design, aligned with the patient's anatomy. The stereotactic fixture is built using 3D laser sintering technology based on the computer-generated model. Bilateral rectangular grids of guide holes orthogonal to the midsagittal plane and centered on the midcommissural point are incorporated in the fixture design, allowing a wide selection of orthogonal trajectories. Up to two additional grids can be accommodated for targeting structures where oblique trajectories are required. The frame has no adjustable parts, this feature reducing the risk of inaccurate coordinate settings while simultaneously reducing procedure time significantly. RESULTS: We have used the fixture for the implantation of depth electrodes for presurgical evaluation of 4 patients with drug-resistant focal epilepsy, with nearly 2-fold reduction in the duration of the implantation procedure. We have obtained a high accuracy with a submillimetric mean positioning error of 0.68 mm for the anchor bolts placed at the trajectory entry point and 1.64 mm at target. CONCLUSIONS: The custom stereotactic fixture design greatly simplifies the planning procedure and significantly reduces the time in the operating room, while maintaining a high accuracy.

Preclinical development of an EP2 antagonist for post-seizure cognitive deficits.

Cognitive comorbidities can substantially reduce quality of life in people with epilepsy. Inflammation is a component of all chronic diseases including epilepsy, as well as acute events like status epilepticus (SE). Neuroinflammation is the consequence of several broad signaling cascades including cyclooxygenase-2 (COX-2)-associated pathways. Activation of the EP2 receptor for prostaglandin E2 appears responsible for blood-brain barrier leakage and much of the inflammatory reaction, neuronal injury and cognitive deficit that follows seizure-provoked COX-2 induction in brain. Here we show that brief exposure of mice to TG11-77, a potent, selective, orally available and brain permeant EP2 antagonist, eliminates the profound cognitive deficit in Y-maze performance after SE and reduces delayed mortality and microgliosis, with a minimum effective i.p. dose (as free base) of 8.8 mg/kg. All in vitro studies required to submit an investigational new drug (IND) application for TG11-77 have been completed, and non-GLP dose range-finding toxicology in the rat identified no overt, organ or histopathology signs of toxicity after 7 days of oral administration at 1000 mg/kg/day. Plasma exposure in the rat was dose-linear between 15 and 1000 mg/kg dosing. TG11-77 thus appears poised to continue development towards the initial clinical test of the hypothesis that EP2 receptor modulation after SE can provide the first preventive treatment for one of the chief comorbidities of epilepsy.

Second-Generation Prostaglandin Receptor EP2 Antagonist, TG8-260, with High Potency, Selectivity, Oral Bioavailability, and Anti-Inflammatory Properties.

EP2, a G-protein-coupled prostaglandin-E2 receptor, has emerged as a seminal biological target for drug discovery. EP2 receptor activation is typically proinflammatory; therefore, the development of EP2 antagonists to mitigate the severity and disease pathology in a variety of inflammation-driven central nervous system and peripheral disorders would be a novel strategy. We have recently developed a second-generation EP2 antagonist TG8-260 and shown that it reduces hippocampal neuroinflammation and gliosis after pilocarpine-induced status epilepticus in rats. Here, we present details of synthesis, lead optimization on earlier leads that resulted in TG8-260, potency and selectivity evaluations using cAMP-driven time-resolved fluorescence resonance energy-transfer (TR-FRET) assays and [H3]-PGE2-binding assays, absorption, distribution, metabolism, and excretion (ADME), and pharmacokinetics. TG8-260 (2f) showed Schild K B = 13.2 nM (3.6-fold more potent than the previous lead TG8-69 (1c)) and 500-fold selectivity to EP2 against other prostanoid receptors. Pharmacokinetic data indicated that TG8-260 has a plasma half-life of 2.14 h (PO) and excellent oral bioavailability (77.3%). Extensive ADME tests indicated that TG8-260 is a potent inhibitor of CYP450 enzymes. Further, we show that TG8-260 displays antagonistic activity on the induction of EP2 receptor-mediated inflammatory gene expression in microglia BV2-hEP2 cells; therefore, it can serve as a tool for investigating anti-inflammatory pathways in peripheral inflammatory disease animal models.

Characterization of 1'-hydroxymidazolam glucuronidation in human liver microsomes.

Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1'-hydroxymidazolam, which is equipotent to midazolam. 1'-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1'-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1'-hydroxymidazolam in human liver microsomes enriched with uridine 5'-diphosphoglucuronic acid (UDPGA). The structure of the N-glucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1'-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n = 1.2-1.5). The apparent S(50) values for the formation of O- and N-glucuronides were 43 and 18 microM, respectively, and the corresponding apparent V(max) values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1'-hydroxymidazolam in human liver microsomes.

Clinical Accuracy of Customized Stereotactic Fixtures for Stereoelectroencephalography.

BACKGROUND: Stereoelectroencephalograpy (SEEG) is a diagnostic method involving 3-dimensional exploration of brain structures using depth electrodes for locating epileptogenic foci in patients with drug-resistant epilepsy. A variety of frame-based, frameless, and robotic stereotactic systems have been designed for the accurate placement of depth electrodes. OBJECTIVES: Using the FHC microTargeting platform as a model, we introduce a fully customized design that has all the constructive elements positioned by a computer algorithm, according to the planned trajectories, anchoring points, and anatomic constraints. All the constructive elements form a single-body fixture, which allows for the efficient implantation of multiple depth electrodes following trajectories having a wide range of orientations. We aim at evaluating the safety and accuracy of this stereotactic system in a clinical setting. METHODS: A total of 173 depth electrodes were implanted in 21 patients with drug-resistant epilepsy. Matlab and DEETO software packages were used to postoperatively evaluate the targeting accuracy. Automatic detection of electrode locations eliminated any subjectivity in calculating the targeting errors. RESULTS: As a result of using custom geometry of the stereotactic platform, the new design is optimized for each patient and streamlines the surgical procedures. The most important results characterizing the platform's accuracy are the values of 1.22 mm for the median lateral target point localization error and 1.17 mm for the median lateral entry point localization error. CONCLUSIONS: The patient-customized platforms are comparable in terms of safety, accuracy, and simplicity of use to the existing robotic devices for implantation of depth electrodes in patients undergoing SEEG investigations.

Identification of a new source of interference leached from polypropylene tubes in mass-selective analysis.

An interference leached from polypropylene tubes was identified to be a sulfoxide oxidative product of didodecyl 3,3'-thiodipropionate (DDTDP) that is used to prevent oxidative degradation of synthetic polymers. A sulfone oxidative product of DDTDP leached from the polypropylene tubes was also observed. The interfering compounds were isolated by LC and characterized using time-of-flight mass spectrometry and NMR. Authentic sulfoxide and sulfone products of DDTDP were also prepared by reacting DDTDP with hydrogen peroxide reaching an unequivocal structural assignment. In conclusion, when analytes of interest are solubilized in predominantly organic solvents and kept in polypropylene containers, the possibility of contamination from leached chemicals should be taken into account.

In vitro metabolism of a new oxazolidinedione hypoglycemic agent utilizing liver microsomes and recombinant human cytochrome P450 enzymes.

The compound, 5-{4-[3-(4-cyclohexyl-2-propylphenoxy)propoxy]phenyl}-1,3-oxazolidine-2,4-dione (compound A) is a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist. PPARgamma agonists have proven useful in the treatment of type 2 diabetes, which is characterized by hyperglycemia, insulin resistance and/or abnormal insulin secretion. The metabolism of this oxazolidinedione (OZD) was investigated in male rat, dog, monkey and human liver microsomes, and recombinant human cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in the presence of NADPH. Routes of metabolism included monohydroxylation of the cyclohexane ring at multiple positions, monohydroxylation of the n-propyl side chain or the tether linkage, and OZD ring opening, giving rise to the keto amide and alcohol amide entities. Liver microsomes showed subtle qualitative and quantitative metabolic differences among rat, dog, monkey and human preparations. Further, CYP2C8 and CYP2C19 did not display different regioselectivity for hydroxylation on the cyclohexane ring with both of them giving rise to C-3 and C-4 hydroxy metabolites, but they did display different stereoselectivity with CYP2C8 preferring cyclohexane hydroxylation in equatorial positions and CYP2C19 in axial positions.

(2R)-2-ethylchromane-2-carboxylic acids: discovery of novel PPARalpha/gamma dual agonists as antihyperglycemic and hypolipidemic agents.

A series of chromane-2-carboxylic acid derivatives was synthesized and evaluated for PPAR agonist activities. A structure-activity relationship was developed toward PPARalpha/gamma dual agonism. As a result, (2R)-7-(3-[2-chloro-4-(4-fluorophenoxy)phenoxy]propoxy)-2-ethylchromane-2-carboxylic acid (48) was identified as a potent, structurally novel, selective PPARalpha/gamma dual agonist. Compound 48 exhibited substantial antihyperglycemic and hypolipidemic activities when orally administered in three different animal models: the db/db mouse type 2 diabetes model, a Syrian hamster lipid model, and a dog lipid model.

Automated online dual-column extraction coupled with teicoplanin stationary phase for simultaneous determination of (R)- and (S)-propranolol in rat plasma using liquid chromatography-tandem mass spectrometry.

An automated online sample extraction method for rat plasma was developed and validated for the quantification of (R)- and (S)-propranolol following the intravenous administration of either the racemate or the individual enantiomers at 5 mg/kg. A dual-column extraction system coupled to a chiral stationary phase (CSP) was used in conjunction with liquid chromatography-tandem mass spectrometry. In this method, two Oasis HLB extraction columns (50x1.0 mm) in parallel were used for online plasma sample purification and teicoplanin CSP (Chirobiotic T) was used for the enantiomeric separation. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for re-conditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a relatively shorter run time and higher throughput. The lower limit of detection was 0.5 ng/ml and the lower limit of quantification was 2 ng/ml for each enantiomer using 25 microl of rat plasma. The method was validated with a linear calibration curve between 2 and 2000 ng/ml for (R)- and (S)-propranolol, respectively. The intra- and inter-day precision (C.V.) was no more than 7.6% and the accuracy of the assay was between 92 and 103%. The teicoplanin CSP proved to be rugged with excellent reproducibility of chromatographic parameters.

An in vitro, high throughput, seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS.

A validated method for the simultaneous characterization of xenobiotic compound-mediated inhibition of seven major cytochrome P450 (CYP) enzymes in pooled human liver microsomes through the use of specific CYP probe substrates (cocktail assay) with low protein content, and a rapid, three minute LC-MS/MS analytical method is described. The specific CYP substrates used in this cocktail assay included phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4/5). The LC-MS method incorporated the aforementioned seven CYP substrates along with their respective major metabolites, and one internal standard, labetalol. In a cross-validation analysis, the concentrations of each CYP probe substrate in the assay had minimal effect (i.e., inhibition or activation) on the other CYP activities. Furthermore, the assay conditions for the multiple probe substrate, ie., cocktail assay, were validated against the single probe substrate assay using 18 compounds with known CYP inhibition liabilities and 10 proprietary compounds. The inhibitory constant (Ki) determined with this cocktail assay was highly correlated (R(2) ≥ 0.77 for each individual probe substrate) with that of the single probe substrate assay for all 27 CYP inhibitors. This seven CYP inhibition cocktail assay has increased the efficiency to assess compounds for inhibition of the major CYP isoforms in a high throughput, drug discovery setting.

Simultaneous assessment of cytochrome P450 activity in cultured human hepatocytes for compound-mediated induction of CYP3A4, CYP2B6, and CYP1A2.

INTRODUCTION: The human nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are known to regulate gene expression of the cytochrome P450 (CYP) enzymes, 3A4, 2B6, and 1A2, respectively. In conventional CYP induction studies, the activity of each CYP enzyme is assessed in a separate incubation with the appropriate marker substrate. The objective of this study was to assess, simultaneously, the induction of CYP3A4, CYP2B6, and CYP1A2 activity in cultured human hepatocytes treated with various prototypical ligands of PXR, CAR, and AhR by utilizing an optimized substrate cocktail, as well as a rapid, sensitive liquid chromatography-mass spectrometry method. METHODS: To evaluate the xenobiotic-mediated induction of hepatocellular gene expression, the prototypical inducers rifampicin (10 µM) and phenobarbital (1 mM) were used for CYP3A4, CITCO (1 µM) and artemisinin (50 µM) were used for CYP2B6, and 3-methylcholanthrene (1 µM) and omeprazole (50 µM) were utilized for induction of CYP1A2. Primary human hepatocytes were treated with each compound for 48h, followed by a 30-min incubation of the hepatocyte culture along with the addition of three marker substrates for specific CYP activity: midazolam (CYP3A4; 5 µM), bupropion (CYP2B6; 50 µM), and phenacetin (CYP1A2; 100µM). The assessment of CYP activity was performed with a rapid, sensitive liquid chromatography-tandem mass spectrometry method which simultaneously assessed activity of CYP3A4, CYP2B6, and CYP1A2 in a single 3-min method by examining the formation of the probe substrate metabolites, 1'-hydroxymidazolam, hydroxybupropion, and acetaminophen, respectively. RESULTS: The average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable between the cocktail and the conventional assay. DISCUSSION: The combination of three marker substrates in a single 30-min incubation, in addition to a rapid, sensitive LC-MS/MS method, resulted in an efficient and robust method for assessing cytochrome P450 induction as compared to the conventional methodology.

Metabolism of MK-0524, a prostaglandin D2 receptor 1 antagonist, in microsomes and hepatocytes from preclinical species and humans.

(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D(2) receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (K(m) and V(max)) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of K(m) values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat approximately monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.

Absorption, metabolism, and excretion of [14C]MK-0767 (2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N-[[4-(trifluoromethyl)phenyl] methyl]benzamide) in humans.

MK-0767 (KRP-297; 2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N-[[4-(trifluoromethyl)phenyl] methyl]benzamide) is a thiazolidinedione (TZD)-containing dual agonist of the peroxisome proliferator-activated receptors alpha and gamma that has been studied as a potential treatment for patients with type 2 diabetes. The metabolism and excretion of [14C]MK-0767 were evaluated in six human volunteers after a 5-mg (200 microCi) oral dose. Excretion of 14C radioactivity was found to be nearly equal into the urine (approximately 50%) and feces (approximately 40%). Elimination of [14C]MK-0767 was primarily by metabolism, with minimal excretion of parent compound into the urine (<0.5% of dose) and feces (approximately 14% of the dose). [14C]MK-0767 was the major circulating compound-related entity (>96% of radioactivity) through 48 h postdose. It was also found that approximately 91% of the total radioactivity area under the curve was due to intact MK-0767. Several minor metabolites were detected in plasma (<1% of radioactivity, each), formed by cleavage of the TZD ring and subsequent S-methylation and oxidation. All the metabolites excreted into urine were formed by TZD cleavage, whereas the major metabolite in feces was the O-demethylated derivative of MK-0767.

Species differences in the elimination of a peroxisome proliferator-activated receptor agonist highlighted by oxidative metabolism of its acyl glucuronide.

A species difference was observed in the excretion pathway of 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C), an alpha-weighted dual peroxisome proliferator-activated receptor alpha/gamma agonist. After intravenous or oral administration of [14C]MRL-C to rats and dogs, radioactivity was excreted mainly into the bile as the acyl glucuronide metabolite of the parent compound. In contrast, when [14C]MRL-C was administered to monkeys, radioactivity was excreted into both the bile and the urine as the acyl glucuronide metabolite, together with several oxidative metabolites and their ether or acyl glucuronides. Incubations in hepatocytes from rats, dogs, monkeys, and humans showed the formation of the acyl glucuronide of the parent compound as the major metabolite in all species. The acyl glucuronide and several hydroxylated products, some which were glucuronidated at the carboxylic acid moiety, were observed in incubations of MRL-C with NADPH- and uridine 5'-diphosphoglucuronic acid-fortified liver microsomes. However, metabolism was more extensive in the monkey microsomes than in those from the other species. When the acyl glucuronide metabolite of MRL-C was incubated with NADPH-fortified liver microsomes, in the presence of saccharo-1,4-lactone, it underwent extensive oxidative metabolism in the monkey but considerably less in the rat, dog, and human liver microsomes. Collectively, these data suggested that the oxidative metabolism of the acyl glucuronide might have contributed to the observed in vivo species differences in the metabolism and excretion of MRL-C.

Enantiomer ratio of MK-0767 in humans and nonclinical species.

MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione-containing dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK-0767 contains a chiral center at the C-5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)-(R) to (-)-(S) enantiomers of MK-0767 were determined in plasma from humans (in vitro only) and nonclinical species used in the toxicological evaluation of rac-MK-0767, namely CD-1 mice, Sprague-Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (approximately 1.5-1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo. In mice, the ratio was higher in vitro (approximately 1) than in vivo (approximately 0.6), while in rabbits it was higher in vivo (approximately 1) than in vitro (approximately 0.5). These results suggested that differential binding of the MK-0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo, since in protein-free systems MK-0767 exists as the racemate.

Mechanistic studies on the metabolic scission of thiazolidinedione derivatives to acyclic thiols.

Thiazolidinedione (TZD) derivatives have been reported to undergo metabolic activation of the TZD ring to produce reactive intermediates. In the case of troglitazone, it was proposed that a P450-mediated S-oxidation leads to TZD ring scission and the formation of a sulfenic acid intermediate, which may be trapped as a GSH conjugate. In the present study, we employed a model compound {denoted MRL-A, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethoxy)phenyl]methyl]benzamide} to investigate the mechanism of TZD ring scission. When MRL-A was incubated with monkey liver microsomes (or recombinant P450 3A4 and NADPH-P450 reductase) in the presence of NADPH and oxygen, the major products of TZD ring scission were the free thiol metabolite (M2) and its dimer (M3). Furthermore, a GSH conjugate of M2 (M4) also was formed when the incubation mixture was supplemented with GSH. Experiments with isolated M2 suggested that this metabolite was unstable and underwent spontaneous autooxidation to M3. A qualitatively similar metabolite profile was observed when MRL-A was incubated with recombinant P450 3A4 and cumene hydroperoxide. Because an oxygen atom is transferred to MRL-A under these conditions, these data suggested that S-oxidation alone may result in TZD ring scission and formation of M2 via a sulfenic acid intermediate. Also, because the latter incubation mixture did not contain any reducing agents, the formation of M2 may have occurred due to disproportionation of the sulfenic acid. When NADPH was added to the incubation mixture containing P450 3A4 and cumene hydroperoxide, the formation of M3 increased, suggesting that the sulfenic acid was reduced to M2 by NADPH and subsequently underwent dimerization to yield M3 (vide supra). When NADPH was replaced by GSH, the formation of M4 increased, consistent with reduction of the sulfenic acid by GSH. In summary, these results suggest that the TZD ring in MRL-A is activated by an initial P450-mediated S-oxidation step followed by spontaneous scission of the TZD ring to a putative sulfenic acid intermediate; the latter species then undergoes reduction to the free thiol by GSH, NADPH, and/or disproportionation. Finally, the thiol may dimerize to the corresponding disulfide or, in the presence of S-adenosylmethionine, form the stable S-methyl derivative.

Study of the fragmentation mechanism of protonated 6-hydroxychlorzoxazone: application in simultaneous analysis of CYP2E1 activity with major human cytochrome P450s.

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186-->130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 microM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within +/- 15% of the spiked concentration at all levels.

Integration of knowledge-based metabolic predictions with liquid chromatography data-dependent tandem mass spectrometry for drug metabolism studies: application to studies on the biotransformation of indinavir.

Despite recent advances in the application of data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) to the identification of drug metabolites in complex biological matrixes, a prior knowledge of the likely routes of biotransformation of the therapeutic agent of interest greatly facilitates the detection and structural characterization of its metabolites. Thus, prediction of the [M + H]+ m/z values of expected metabolites allows for the construction of user-defined MS(n) protocols that frequently reveal the presence of minor drug metabolites, even in the presence of a vast excess of coeluting endogenous constituents. However, this approach suffers from inherent user bias, as a result of which additional "survey scans" (e.g., precursor ion and constant neutral loss scans) are required to ensure detection of as many drug-related components in the sample as possible. In the present study, a novel approach to this problem has been evaluated, in which knowledge-based predictions of metabolic pathways are first derived from a commercial database, the output from which is used to formulate a list-dependent LC/MS(n) data acquisition protocol. Using indinavir as a model drug, a substructure similarity search on the MDL metabolism database with a similarity index of 60% yielded 188 "hits", pointing to the possible operation of two hydrolytic, two N-dealkylation, three N-glucuronidation, one N-methylation, and several aromatic and aliphatic oxidation pathways. Integration of this information with data-dependent LC/MS(n) analysis using an ion trap mass spectrometer led to the identification of 18 metabolites of indinavir following incubation of the drug with human hepatic postmitochondrial preparations. This result was accomplished with only a single LC/MS(n) run, representing significant savings in instrument use and operator time, and afforded an accurate view of the complex in vitro metabolic profile of this drug.

Simultaneous determination of MK-0767 and seven metabolites in rat urine using liquid chromatography/tandem mass spectrometry.

MK-0767, 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, Table 1), is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor --> product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8-800 ng/mL, and 8-8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra-day and inter-day variation using this method were < or = 13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0-24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague-Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 microg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6- to 45-fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 microg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S-methylation and oxidation of the sulfide intermediate.

Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis.

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP trade mark LC/MS/MS system (Q TRAP trade mark ), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma. With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS(2)) and MS(3) experiments to obtain structural information of drug metabolites 'on-the-fly'. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage.