Development and validation of an LC-MS/MS method for the quantitative analysis of the adenosine A2a receptor antagonist NIR178 and its monohydroxy metabolite in human plasma: Application to clinical pharmacokinetics.

We report a selective LC-MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 ml plasma. Separation was achieved in 10 min on an Acquity UPLC BEH C18 1.7 µm, 2.1 × 50 mm column heated at 60°C with a gradient elution at 0.6 ml/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring. The linear response range was 1.00-1,000 ng/ml using a 1/x2 weighting factor. The intra- and inter-day accuracies (bias %) and intra- and inter-day precisions (CV, %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor calculated from six lots of normal, lipemic and hemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 compared with their internal standards were consistent and reproducible with a CV ≤8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.

Effects of Rifampin on the Pharmacokinetics of a Single Dose of Istradefylline in Healthy Subjects.

Istradefylline, a selective adenosine A2A inhibitor, is under development for the treatment of Parkinson's disease. The effect of oral steady-state rifampin 600 mg/day, a potent cytochrome P450 (CYP) 3A4 inducer, on the disposition of a single oral dose of istradefylline 40 mg was determined in a crossover study in 20 healthy subjects by measuring plasma concentrations of istradefylline and its M1 and M8 metabolites and their derived pharmacokinetic parameters. Based on the geometric mean ratio of log-transformed data, rifampin reduced istradefylline exposure: Cmax , 0.55 (90%CI, 0.49-0.62); AUClast , 0.21 (90%CI, 0.19-0.22); and AUCinf , 0.19 (90%CI, 0.18-0.20), indicating nonequivalence. These changes were primarily because of the effect of rifampin on the elimination parameters of istradefylline; mean CL/F was increased from 4.0 to 20.6 L/h, and mean t1/2 was reduced from 94.8 to 31.5 hours. The effect of rifampin coadministration on the disposition of the istradefylline M1 and M8 metabolites was inconsistent and variable. Furthermore, as exposure of the istradefylline M1 and M8 metabolites in plasma was generally <9% of total drug exposure, it would be expected to have a negligible impact on the pharmacodynamic effect of istradefylline. Caution should be exercised when istradefylline is administered concurrently with strong CYP3A4 inducers and dose adjustment considered.

Continuous adenosine A2A receptor antagonism after focal cerebral ischemia in spontaneously hypertensive rats.

Antagonism of the adenosine A2A receptor (A2AR) has been shown to elicit substantial neuroprotective properties when given immediately after cerebral ischemia. We asked whether the continuous application of a selective A2AR antagonist within a clinically relevant time window will be a feasible and effective approach to treat focal cerebral ischemia. To answer this question, we subjected 20 male spontaneously hypertensive rats to permanent middle cerebral artery occlusion and randomized them equally to a verum and a control group. Two hours after stroke onset, the animals received a subcutaneous implantation of an osmotic minipump filled with 5 mg kg(-1) day(-1) 8-(3-chlorostyryl) caffeine (CSC) or vehicle solution. The serum level of CSC was measured twice a day for three consecutive days. The infarct volume was determined at days 1 and 3 using magnetic resonance imaging. We found the serum level of CSC showing a bell-shaped curve with its maximum at 36 h. The infarct volume was not affected by continuous CSC treatment. These results suggest that delayed and continuous CSC application was not sufficient to treat acute ischemic stroke, potentially due to unfavorable hepatic elimination and metabolization of the pharmaceutical.

Evaluation of the effects of a high-fat meal on the oral bioavailability of a single dose of preladenant in healthy subjects.

The aim of this study was to evaluate the effect of food on the oral bioavailability of preladenant, a novel adenosine A(2A) receptor antagonist. This open-label, randomized, single-dose, 2-way crossover study evaluated the effects of a high-fat, high-calorie meal on the pharmacokinetics of preladenant and its metabolite (SCH434748) following oral administration of a single 25-mg preladenant capsule to 24 healthy subjects. When administered with food, the time of maximum concentration (T(max)) of preladenant was prolonged compared with administration in the fasting state. Whereas T(max) was increased from 0.9 hours to 2.6 hours and maximum concentration (C(max)) was decreased (from 212 ng/mL to 128 ng/mL), the extent of absorption (area under the plasma concentration-time curve from time 0 to time of final quantifiable sample, or AUC([tf])) was unaffected by the meal. Similarly, SCH434748 T(max) was prolonged in the fed state, and C(max) decreased from 43.7 ng/mL to 28.6 ng/mL. The assessment of AUC([tf]) and area under the plasma concentration-time curve from time 0 to infinity [AUC([I])] together suggests that the AUC for the metabolite remained unchanged. No serious, significant, or unexpected adverse events occurred. In summary, food delays absorption and reduces peak exposure (C(max)) but does not alter the extent of preladenant exposure (AUC). These small changes are unlikely to be of clinical importance. A single 25-mg dose of preladenant is safe and well tolerated in healthy subjects under both fed and fasting conditions.

The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated with severe motor impairments caused by the loss of dopaminergic innervation of the striatum. Previous studies have demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGlu4), including N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide, can produce antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu4 PAMsexhibited unsuitable physiochemical properties for systemic dosing, requiring intracerebroventricular administration and limiting their broader utility as in vivo tools to further understand the role of mGlu4 in the modulation of basal ganglia function relevant to PD. In the present study, we describe the pharmacologic characterization of a systemically active mGlu4 PAM, N-(3-chlorophenyl)picolinamide (VU0364770), in several rodent PD models. VU0364770 showed efficacy alone or when administered in combination with L-DOPA or an adenosine 2A (A2A) receptor antagonist currently in clinical development (preladenant). When administered alone, VU0364770 exhibited efficacy in reversing haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, and attentional deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced catalepsy when given in combination. The effects of VU0364770 to reverse forelimb asymmetry were also potentiated when the compound was coadministered with an inactive dose of L-DOPA, suggesting that mGlu4 PAMs may provide L-DOPA-sparing activity. The present findings provide exciting support for the potential role of selective mGlu4 PAMs as a novel approach for the symptomatic treatment of PD and a possible augmentation strategy with either L-DOPA or A2A antagonists.