Validation of diacyl glycerolacyltransferase I as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor.

A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1(-/-) mice.

Rapid hit to lead evaluation of pyrazolo[3,4-d]pyrimidin-4-one as selective and orally bioavailable mGluR1 antagonists.

Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties. These improvements were paralleled by rat PK study characterized by low clearance and quantitative bioavailability. Compound 2f represented a true lead-like molecule that is amenable for further lead optimization (LO) evaluation.

Solid phase extraction as a faster alternative to HPLC: application to MS analysis of metabolic stability samples.

Due to recent advances in high throughput organic synthesis, discovery teams now need to profile increased numbers of analogs in vitro for their absorption, distribution, metabolism, and excretion (ADME) properties. Consequently, pharmaceutical companies are developing lower cost and higher throughput methods for ADME testing. As demands for metabolic stability testing have increased in our laboratory, the time required to analyze samples using high-pressure liquid chromatography-mass spectrometry (HPLC-MS) has grown rapidly and ultimately limited our data output. In this study we show that solid phase extraction-mass spectrometry (SPE-MS) is a viable alternative to HPLC-MS for monitoring small molecule stability in liver microsomes. Using the SPE-MS approach, samples can be analyzed in 24 s compared to 2.5 min on the HPLC-MS without compromising data quality, thereby alleviating the analytical bottleneck.

Automatic mass spectrometry method development for drug discovery: application in metabolic stability assays.

High-throughput metabolic screening has been requested routinely to keep pace with high-throughput organic synthesis. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a fast gradient has become the method of choice for the task due to its sensitivity and selectivity. We have developed an automated system that consists of a robotic system for in vitro incubation and a commercially available software package for automatic MS/MS method development. A short, generic LC gradient and MS conditions that are applicable to most compounds have been developed to minimize the method development time and data analysis. This system has been used to support a number of in vitro screening assays in early drug discovery phase including microsomal stability and protein binding.

Evaluation of a novel in vitro Caco-2 hepatocyte hybrid system for predicting in vivo oral bioavailability.

A novel in vitro Caco-2 hepatocyte hybrid system was set up and tested for its ability to predict the oral bioavailability (F) in humans of 24 randomly chosen marketed drugs. Caco-2 cells were cultured on the transwell filters to form tight junctions. Pooled cryopreserved human hepatocytes were placed in the basolateral receiver compartment. To evaluate the permeability and hepatic first pass in one experiment, compounds were dissolved in medium and added to the apical donor compartment of the transwell apparatus, and the amount of the parent compound appearing in the basolateral receiver compartment was determined over a 3-h time course. The area under the concentration versus time curve (AUC) of the parent compound was determined. The predictive usefulness of this Caco-2 hepatocyte model was tested by comparing the AUC with the in vivo oral bioavailability reported in the literature. Linear regression analysis shows a reasonable correlation (R(2) = 0.86) between the in vitro AUC and oral bioavailability reported in the literature. Based on the literature data, the compounds were classified into low (F < 20%), medium (20 < F < 50%), and high (F > 50%) bioavailability categories. The oral bioavailability predicted from the experimental Caco-2 hepatocyte system successfully matches the appropriate literature-based bioavailability category for 22 of 24 of the compounds. The results presented in this study suggest that it may be feasible to combine Caco-2 cells and hepatocytes into one system for the prediction of oral absorption and first-pass effect in humans.

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry.

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.

Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes.

Sixty-four compounds with diverse structures were used in evaluation of intrinsic clearance by various hepatocyte preparations from rats, dogs, monkeys, and humans. Intrinsic clearance (CL(int)) was calculated from the ratio of the initial amount of the test compound minus the amount remaining after 2 h of incubation and the corresponding area under the concentration versus time curve. The predictive potential of this in vitro model was tested by comparing the intrinsic clearance with in vivo clearance using linear regression analysis. In addition, the intrinsic clearance values obtained from three different types of hepatocytes (cryopreserved, fresh, and sandwich-cultured) from the same species were compared to determine the influence of preservation and culture conditions. It seems that intrinsic clearance determined from human cryopreserved hepatocyte (R(2) = 0.87) was the best predictor for the corresponding human in vivo clearance. Dog and rat hepatocyte clearances were also demonstrated to be reasonable predictors (R(2) ranged 0.68-0.85 in dogs and 0.65-0.72 in rats) for their corresponding in vivo clearances. Monkey hepatocyte clearance seems to be the worst predictor for the corresponding in vivo clearance (R(2) = 0.35-0.67). Comparison of intrinsic clearance generated from cryopreserved and fresh hepatocytes demonstrated a very good correlation in dog (R(2) = 0.82) followed by rat (R(2) = 0.77), and then by monkey (R(2) = 0.70). A similar correlation profile was shown between cryopreserved hepatocytes and sandwich-cultured hepatocytes. These results demonstrated the predictive potential of intrinsic clearance for rat, dog, and human in vivo clearance, but also showed some limitation of the approach for monkey.

A high-throughput cell-based reporter gene system for measurement of CYP1A1 induction.

INTRODUCTION: Enzyme induction is undesirable in new drug discovery process, with consequences spanning from auto-induction to toxicity. Cytochrome P450 (CYP) 1A1 has long been known to be one of the metabolic enzymes involved in activating many procarcinogens, the first step toward tumor formation during chemical carcinogenesis. Induction of CYP1A1 during drug treatment may predispose the patients to some risk of chemical carcinogenesis. METHODS: Based on the signal-transduction mechanism of CYP1A1 induction, a high-throughput reporter-gene system was established by stable transformation of H4IIE cells to incorporate the luciferase gene under control of CYP1A1 promoter. This stable cell line was validated with known CYP1A1 inducers, such as 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF), alpha-naphthoflavone (alpha-NF) and 3-indocarbinol. Thirty in-house new chemical entities (NCEs) were then screened with this reporter-gene system, and also administered to rats to evaluate in vivo CYP1A1 induction. RESULTS: CYP1A1 reporter gene system can be used to identify strong inducers, such as 3-MC, beta-NF and alpha-NF, and weak inducers, such as 3-indocarbinol. In vitro induction of 30 in-house compounds in reporter gene system did not correlate with in vivo induction in rat liver microsome measured by ethoxyresorufin-O-dealkylation (EROD) activity, but had a reasonable correlation with Western blot signals. DISCUSSION: This reporter-gene system may be useful in eliminating compounds that can cause CYP1A1 induction at an early stage of drug discovery.