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.

EP2, a G-protein-coupled prostaglandin-E2 recep-tor, 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 neuroinflamma-tion 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 KB = 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.

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.

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.

While studying the metabolism of unique MEK inhibitors, including AR00340509 (4‐((4‐bromo‐2‐fluorophenyl)amino)‐N‐(2‐hydroxyethoxy)‐1,5‐dimethyl‐6‐oxo‐1,6‐dihydropyridine‐3‐carboxamide), we identified a novel O‐glucuronide linked through the carbonyl oxygen of the hydroxamate moiety. This glucuronide metabolite was observed in UDPGA‐supplemented hepatic microsomes and hepatocytes from a number of animal species including humans and was formed by rUGT1A1 and rUGT1A3. The terminal O‐linked glucuronide on the hydroxamate terminal OH side chain and N‐glucuronide of the amide metabolite were observed. The identity of the O‐linked carbonyl glucuronide metabolite was supported by the synthesis of the peracetate derivative of the glucuronide. Within this structural class of MEK inhibitors, some but not all, of the O‐linked carbonyl glucuronides were unstable and identification of the pathway was carried out via the more stable peracetate. Accurate mass determination of the peracetylated glucuronide of AR00340509 was carried with LC‐QTOF instrumentation, which yielded an M+H ion of 730.1258 and a structural formula of C29 H34 Br F N3 O13. The structural class of MEK inhibitors studied lend themselves to enhanced tautomer enolization, thereby facilitating the formation of these unique O‐glucuronides. We are investigating factors affecting the formation of carbonyl glucuronides, including substitutions with both electron withdrawing and electron donating entities.

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.

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.

There is great appeal, scientifically, financially and temporally, for the use of predictions in the drug metabolism and toxicology evaluations used, and in many instances, required, in chemical compound design and development. Indeed, there have been great strides in hardware and software development, as well as many scientific advances that have made predictions not only feasible but in many instances, accurate, when compared with data acquired from lengthy and costly human or animal studies. However, despite the wide array of available software capable of making predictions in the fields of ADME/Tox, the user needs to err on the side of caution when attempting to fit their data from a new structural entity into a database where the learning set may be somewhat or even grossly different from the new entity. There must be a great deal of thought given to what programs are to be utilized as well as to the robustness of the data being entered-there is still no replacement for the expression "garbage in, garbage out". Further, decisions must be made as to whether the predictions made by the software are superior to other ways of gaining human predictions from in vitro and in vivo ADME/Tox information-if acquiring in vitro data is easier (high throughput) and more reliable, then maybe a new predictive software program is not the best answer, but it if is, we need to choose wisely. Acceptance of predictive software is difficult but it will, eventually, be well worth the effort. Until that time, the end-user needs to go into the prediction business with his or her eyes wide open as to the potential pitfalls and ramifications of an over exuberance of predictions.

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.

(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 D2 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 (Km and Vmax) 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 Km 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 ∼ 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.

MK-0524 is a potent, selective and orally active Prostaglandin D-2 receptor 1 (DP1) antagonist currently under clinical development for the treatment of niacin-induced flushing. Experiments to study the pharmacokinetics, metabolism and excretion of MK-0524 were conducted in rats, dogs and monkeys. MK-0524 displayed linear kinetics and rapid absorption following an oral dose. Following intravenous (i.v.) administration of MK-0524 to rats and dogs (1 and 5 mg/kg), the mean Cl-p was similar to 2 and similar to 6 ml/min/kg, the T-1/2 was similar to 7 and similar to 13 h and the Vd(ss) was similar to 1 and similar to 5 L/kg, respectively. In monkeys dosed i.v. at 3 mg/kg, the corresponding values were 8 ml/min/kg, 3 h and 1 L/kg, respectively. Following oral dosing of MK-0524 to rats (5, 25 and 100 mg/kg), dogs (5 mg/kg) and monkeys (3 mg/kg), the absorption was rapid with the mean C-max occurring between 1 and 4 h. Absolute oral bioavailability values in rats, dogs and monkeys were 50, 70 and 8%, respectively. The major circulating metabolite was the acyl glucuronide of MK-0524 (M2), with ratios of glucuronide to the parent aglycone being highest in the monkey followed by dog and rat. In bile duct-cannulated rats and dogs, MK-0524 was eliminated primarily via acyl glucuronidation followed by biliary excretion of the acyl glucuronide, M2, the major drug-related entity in bile.

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.