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Matrix-assisted laser desorption/ionization hyphenated with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI-QTOF following a direct infusion of (3)H-clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedures.

Tween 80 (Polysorbate 80) is a hydrophilic nonionic surfactant commonly used as an ingredient in dosing vehicles for pre-clinical in vivo studies (e.g., pharmacokinetic studies, etc.). Tween 80 increased apical to basolateral permeability of digoxin in Caco-2 cells suggesting that Tween 80 is an in vitro inhibitor of P-gp. The overall objective of the present study was to investigate whether an inhibition of P-gp by Tween 80 can potentially influence in vivo absorption of P-gp substrates by evaluating the effect of Tween 80 on the disposition of digoxin (a model P-gp substrate with minimum metabolism) after oral administration in rats. Rats were dosed orally with digoxin (0.2 mg/kg) formulated in ethanol (40%, v/v) and saline mixture with and without Tween 80 (1 or 10%, v/v). Digoxin oral AUC increased 30 and 61% when dosed in 1% and 10% Tween 80, respectively, compared to control (P < 0.05). To further examine whether the increase in digoxin AUC after oral administration of Tween 80 is due, in part, to a systemic inhibition of digoxin excretion in addition to an inhibition of P-gp in the GI tract, a separate group of rats received digoxin intravenously (0.2 mg/kg) and Tween 80 (10% v/v) orally. No significant changes in digoxin IV AUC was noted when Tween 80 was administered orally. In conclusion, Tween 80 significantly increased digoxin AUC and Cmax after oral administration, and the increased AUC is likely to be due to an inhibition of P-gp in the gut (i.e., improved absorption). Therefore, Tween 80 is likely to improve systemic exposure of P-gp substrates after oral administration. Comparing AUC after oral administration with and without Tween 80 may be a viable strategy in evaluating whether oral absorption of P-gp substrates is potentially limited by P-gp in the gut.

The ET(A) receptor antagonist (2) (N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)-[1,1'-biphenyl]-2-sulfonamide, BMS-193884) shares the same biphenyl core as a large number of AT(1) receptor antagonists, including irbesartan (3). Thus, it was hypothesized that merging the structural elements of 2 with those of the biphenyl AT(1) antagonists (e.g., irbesartan) would yield a compound with dual activity for both receptors. This strategy led to the design, synthesis, and discovery of (15) (4'-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2'-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1'-biphenyl]-2-sulfonamide, BMS-248360) as a potent and orally active dual antagonist of both AT(1) and ET(A) receptors. Compound 15 represents a new approach to treating hypertension.

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.KEYWORDS Hepatitis C virus NS3 serine protease inhibitor, R-ketoamide, narlaprevir, SCH 900518 H epatitis C virus (HCV) infection is a global health crisis leading to liver cirrhosis, hepatocellular carcinoma and liver failure in humans. 1 An estimated 3% of the human population worldwide is chronically infected with HCV. 2 Currently the only available treatment regimens are subcutaneous R-interferon or long-acting pegylated-interferon, alone or in combination with oral antiviral agent ribavirin. 3 The approved therapy is still far from ideal for the hard to treat genotype-1 patients 4 and is frequently accompanied by adverse side effects. There is an unmet medical need to discover new, more effective and tolerable regimens for the treatment of HCV infection. Advances in the understanding of molecular pathways of HCV replication have resulted in several small molecule direct-acting antivirals entering clinical trials in the past few years.Since identification of this virus, the NS3 serine protease contained within the N-terminal region of the NS3 protein has been studied extensively. 5 This chymotrypsin-like serine protease plays a pivotal role in viral replication and, therefore, is an attractive target for HCV antiviral therapeutics. 6,7 Intense efforts were focused in the past decade to discover novel small molecule agents that inhibit NS3 serine protease. 8 Proof of concept studies in humans with BILN 2061, a noncovalent P1-P3 macrocyclic inhibitor, validated this hypothesis. 9 Since then, several NS3 protease inhibitors have progressed to human clinical trials. Currently the most advanced among those are boceprevir (SCH 503034), 1, 10,11 and telaprevir (VX950), 12,13 from the slow-binding reversible R-ketoamide class, in phase III human evaluation. Inhibitors in phase II studies, from the structurally distinct noncovalent macrocyclic class, include 14 15 and MK-7009 (P2-P4 macrocycle). 16 Other NS3 protease inhibitors currently in clinical evaluation (structure not yet disclosed) include BI-201335, ABT-450, PHX-1766, ACH-1625 and VX-813. 8 Inhibitor 1 exhibited K i * = 14 nM in the enzyme binding assay, 17 EC 90 = 350 nM in the cell-based replicon assay, 18 and acceptable pharmacokinetic profile in rats and dogs (Figure 1). In our efforts to discover a second generation HCV protease inhibitor, we focused mainly on improving the in vitro potency and preclinical pharmacokinetic profile of the inhibitor, specifically exposure in monkeys. Further...