Capillary microsampling in mice: effective way to move from sparse sampling to serial sampling in pharmacokinetics profiling.

Pharmacokinetic studies are an integral part of drug discovery and development. Mice are the commonly used species for pharmacokinetics studies during early discovery studies. Conventionally, composite PK profiles are obtained from mice studies due to the physiological limitations of the total blood volume that can be drawn over a certain period.With advancements in bioanalytical instrumentation and in blood sampling techniques, analysis with small volume (<50 µL) became feasible enabling serial blood sampling from the mouse for PK studies. The objective of the current study was to develop and establish a serial blood sampling technique in mouse and compare it with the conventional sparse sampling method (composite PK) following oral administration of widely used NSAIDs, diclofenac, celecoxib and tenoxicam, into Swiss Albino mice.The pharmacokinetic parameters of all three probe drugs by serial blood sampling were comparable with that of sparse sampling method. There was no significant difference between the whole blood concentration time profiles of all three drugs between serial sampling and sparse sampling suggesting serial blood sampling method can be easily implemented for mice PK studies.Serial blood sampling technique requires use of fewer number of animals, less quantity of test compound and reduces the possible dosing errors as fewer number of animals need to be dosed resulting in quality PK data and enabling comparison of inter-animal differences in PK profile.

Discovery and pharmacological evaluation of indole derivatives as potent and selective RORγt inverse agonist for multiple autoimmune conditions.

Targeting nuclear receptor RORγ is recognized to be beneficial in multiple autoimmune disorders. We disclosed new indole analogues as potent RORγ inverse agonists. RO-2 as one of the potent and orally bioavailable compounds was evaluated in various models of autoimmune disorder. It showed potent suppression of downstream markers of RORγt activity in murine and human primary cells, ex vivo PD assay and in multiple animal models of autoimmune diseases. The results indicate the potential of these indole analogues as orally bioavailable small molecule inverse agonists of RORγt, efficacious in various Th17 driven models of autoimmune disorders.

Development and validation of a chiral LC-ESI-MS/MS method for simultaneous determination of the enantiomeric metabolites isosorbide 2-mononitrate and isosorbide 5-mononitrate of isosorbide dinitrate in rat and human plasma.

A specific liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and validated for simultaneous determination of two active metabolites of isosorbide dinitrate (ISDN), namely, isosorbide 2-mononitrate (IS 2-MN) and isosorbide 5-mononitrate (IS 5-MN). A simple protein precipitation extraction technique was employed using 13C6 isosorbide 5-mononitrate as the internal standard. The two isomers were separated on a chiral column and mass detection was carried out by electrospray ionization (ESI) in negative multiple reaction monitoring (MRM) mode (ESI -ve). As neutral organic nitrates do not ionize well in ESI ion source, adduct formation of IS 2-MN and IS 5-MN were evaluated. Acetate adduct ions of IS 2-MN and IS 5-MN were well ionized and fragmentable in the negative mode by liquid chromatography electrospray ionization/tandem mass spectrometry. These acetates adduct ions, of IS 2-MN and IS 5-MN were selected as parent mass for quantitation. The method was developed and validated in rat and human plasma with K2EDTA as an anticoagulant. This simultaneous quantitation method was shown to be linear over a working range of 25.0 ng/mL to 5050 ng/mL and 12.4 ng/mL to 2500 ng/mL for IS 2-MN (r2 > 0.99) and IS 5-MN (r2 > 0.99), respectively, in rat and human plasma. Sensitivity was determined as 25.0 ng/mL for IS 2-MN and 12.4 ng/mL for IS 5-MN in rat and human plasma. Inter- and intra-day accuracy and precision were within ±15% in both method validations. This validated method was subsequently applied to a pharmacokinetic (PK) study of ISDN in rat after oral administration.

A simple and sensitive LC-MS/MS method for quantification of Bepridil in rat plasma and its application to pharmacokinetic studies.

Bepridil is potent inhibitor of Na+, K+ and Ca+ channel in cardiomyocytes. It has demonstrated strong antianginal effect with type I antiarrhythmic and with minimum antihypertensive therapeutic effect. Till date, a specific LC-MS/MS method to quantify Bepridil concentrations in biological matrix have not been reported yet. In current study, a highly sensitive, specific and simple LC-MS/MS method for quantification of antianginal drug Bepridil in rat plasma is presented. The LC-MS/MS method was validated in terms of selectivity, specificity, sensitivity, accuracy and precision, matrix effect, extraction recovery and stability as per USFDA's bioanalytical method validation guideline. The validated assay was applied for quantification of Bepridil from pharmacokinetic study in rats following oral and intravenous administration. The lower limit of quantification (LLOQ) of Bepridil was 1 ng/mL. The calibration curve ranges from 1 ng/mL to 1000 ng/mL with desirable linearity and r2 > 0.99. The method exhibited 10-fold dilution integrity. The intra-day and inter-day accuracy were within 101.32-96.80% and 102.87-95.35% with coefficient of variation 10.11-2.89% and 10.45-3.97% respectively. No significant interference observed by endogenous peak at the retention time of Bepridil and IS. The assay was free from any matrix effect, precise recovery across the calibration curve range and samples were stable under all experimental conditions. The validated assay was successfully applied to analyze plasma samples of pharmacokinetic study in rat to determine concentrations of Bepridil. In summary, a novel method for analyzing Bepridil in rat plasma has been successfully validated and is now being utilized for quantification of Bepridil from pre-clinical studies.

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model.

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3-30 mg/kg po for 7 days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and Tramadol (40 mg/kg).

Evaluation of separate role of intestine and liver in first pass metabolism of budesonide in rat.

1. Budesonide, a potent topical corticosteroid, reported to have low oral bioavailability in mice, rat, dog and human due to rapid first pass metabolism. However, there is insufficient information available in literature regarding the role of intestine and or liver responsible for the first pass metabolism of budesonide. 2. Current study in rats investigates the role of intestine and liver in first pass metabolism of budesonide using two in vivo models. Additionally, budesonide was also evaluated in in vitro assays such as thermodynamic solubility, permeability in Caco-2 cells and stability in simulated gastric (SGF), intestinal fluids (SIF) to understand the underlaying cause for low oral bioavailability. 3. Budesonide showed low oral, intra-duodenal and high intra-portal bioavailability in rat. In a dual vein cannulated rat model, intestinal and hepatic extraction ratios calculated based upon intestinal availability (Fa·Fg) and hepatic availability (Fh), suggests hepatic extraction of budesonide is minimal compared to intestinal. 4. In vitro results suggest, solubility and permeability may not be a barrier for the observed low oral bioavailability in rats. 5. Correlating the in vitro and in vivo data together, it can be concluded that, intestine might be playing major role in first pass metabolism of budesonide.

Modifications of flexible nonyl chain and nucleobase head group of (+)-erythro-9-(2's-hydroxy-3's-nonyl)adenine [(+)-EHNA] as adenosine deaminase inhibitors.

A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e wherein 5'C of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-EHNA. Docking of the representative compounds into the active site of ADA was performed to understand structure-activity relationships. Compounds 7a (Ki: 1.1nM) 7b (Ki: 5.2nM) and 26a (Ki: 5.9nM) showed suitable balance of potency, microsomal stability and demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may have therapeutic potential for various inflammatory diseases, hypertension and cancer.

Discovery of Potent and Selective A2A Antagonists with Efficacy in Animal Models of Parkinson's Disease and Depression.

Adenosine A2A receptor (A2AAdoR) antagonism is a nondopaminergic approach to Parkinson's disease treatment that is under development. Earlier we had reported the therapeutic potential of 7-methoxy-4-morpholino-benzothiazole derivatives as A2AAdoR antagonists. We herein described a novel series of [1,2,4]triazolo[5,1-f]purin-2-one derivatives that displays functional antagonism of the A2A receptor with a high degree of selectivity over A1, A2B, and A3 receptors. Compounds from this new scaffold resulted in the discovery of highly potent, selective, stable, and moderate brain penetrating compound 33. Compound 33 endowed with satisfactory in vitro and in vivo pharmacokinetics properties. Compound 33 demonstrated robust oral efficacies in two commonly used models of Parkinson's disease (haloperidol-induced catalepsy and 6-OHDA lesioned rat models) and depression (TST and FST mice models).

Discovery of liver-directed glucokinase activator having anti-hyperglycemic effect without hypoglycemia.

Glucokinase activators (GKAs) are among the emerging drug candidates for the treatment of type 2 diabetes (T2D). Despite effective blood glucose lowering in clinical trials, many pan-GKAs "acting both in pancreas and liver" have been discontinued from clinical development mainly because of their potential to cause hypoglycemia. Pan-GKAs over sensitize pancreatic GK, resulting in insulin secretion even at sub-normoglycemic level which might be a possible explanation for hypoglycemia. An alternative approach to minimize the risk of hypoglycemia is to use liver-directed GKAs, which are reported to be advancing well in clinical development. Here, we report the discovery and structure-activity relationship (SAR) studies on a novel 2-phenoxy-acetamide series with the aim of identifying a liver-directed GKA. Incorporation of a carboxylic acid moiety as an active hepatocyte uptake recognizing element at appropriate position of 2-phenoxy-acetamide core led to the identification of 26, a potent GKA with predominant liver-directed pharmacokinetics in mice. Compound 26 on oral administration significantly reduced blood glucose levels during an oral glucose tolerance test (oGTT) performed in diet-induced obese (DIO) mice, while showing no sign of hypoglycemia in normal C57 mice over a 10-fold dose range, even when dosed at fasted condition. Together, these data demonstrate a liver-directed GKA has beneficial effect on glucose homeostasis with reduced risk of hypoglycemia.

Discovery and evaluation of 1H-pyrrolo[2,3-b]pyridine based selective and reversible small molecule BTK inhibitors for the treatment of rheumatoid arthritis.

In a pursuit to identify reversible and selective BTK inhibitors, two series based on 7H-pyrrolo[2,3-d]pyrimidine and 1H-pyrrolo[2,3-b]pyridine as the hinge binding core, have been identified. Structure activity relationship (SAR) exploration led to identification of two advanced lead molecules, 11 and 13, which demonstrated desired BTK inhibitory potency in different cellular assays, excellent selectivity in a panel of 50 diverse kinases, favorable in vivo PK properties in mice and anti-arthritic effect in a mouse model of CIA.