Synthesis and evaluation of dual antiplatelet activity of bispidine derivatives of N-substituted pyroglutamic acids.

N-aralkylpyroglutamides of substituted bispidine were prepared and evaluated for their ability to inhibit collagen induced platelet aggregation, both in vivo and in vitro. Some compounds showed high anti-platelet efficacy (in vitro) of which six inhibited both collagen as well as U46619 induced platelet aggregation with concentration dependent anti-platelet efficacy through dual mechanism. In particular, the compound 4j offered significant protection against collagen epinephrine induced pulmonary thromboembolism as well as ferric chloride induced arterial thrombosis, without affecting bleeding tendency in mice. Therefore, the present study suggests that the compound 4j displays a remarkable antithrombotic efficacy much better than aspirin and clopidogrel.

Assessement of the pharmacokinetics, tissue distribution and excretion studies of a novel antiplatelet agent S007-867, following administration to rats.

S007-867 is a promising novel antiplatelet agent with better efficacy and lesser bleeding risk than existing agents. The present study investigated the absorption, tissue distribution, and excretion of S007-867 in rat model for further advancement of the molecule. A simple and robust ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) bioanalytical method was used to determine S007-867 in various matrices. Following oral administration, the compound was quickly dispersed in the various tissues and peak concentration levels were achieved within 0.5-1 h. Overall, exposure of drug, i.e., AUC in different tissues was found in the order of small intestine > liver > heart > spleen > lungs > kidney > brain. The total recoveries of the S007-867 within 96 h were 3.36% in urine and faeces. This might be due to a first-pass effect by the liver and intestine as most of the drug was eliminated in metabolite form. These findings provide a crucial information about further development of S007-867 as antithrombotic agent. Copyright © 2015 John Wiley & Sons, Ltd.

Pharmacokinetics and tissue distribution study of novel potent antiplatelet agent S007-867 in mice using HPLC-MS/MS.

1. S007-867 is a novel antiplatelet agent that shows promising in vitro and in vivo efficacy. For further development and better pharmacological elucidation, we characterized pharmacokinetics and tissue distribution of S007-867 in a mouse model. 2. A sensitive, selective and robust LC-MS/MS method was developed and validated in the mouse plasma and tissue for quantification of S007-867. The chromatographic separation was performed on Waters Symmetry Shield C18 column (150 × 4.6 mm, 5 µm) using methanol and ammonium acetate buffer. 3. S007-867 was rapidly absorbed and distributed to various tissues. Following single oral administration of S007-867 in the mouse, the concentration was in the order of C intestine > C liver > C kidney > C heart > C spleen > C lungs > C brain. Tissue to plasma area under the plasma curve ratio suggested that the maximum amount of drug was found in the intestine and liver. Half life of S007-867 was found longer in the heart (8.08 h), spleen (∼ 7.94 h) and kidney (∼ 15.41 h) as compared with other tissues. 4. The preclinical pharmacokinetics and tissue distribution data obtained using this LC-MS/MS method are expected to assist the future clinical investigations of S007-867 as a promising antiplatelet agent.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Synthesis and identification of chiral aminomethylpiperidine carboxamides as inhibitor of collagen induced platelet activation.

A series of chiral lactam carboxamides of aminomethylpiperidine were synthesized and investigated for the collagen induced in vitro anti-platelet efficacy and collagen plus epinephrine induced in vivo pulmonary thromboembolism. The compound 31a (30 µM/kg) displayed a remarkable antithrombotic efficacy (60% protection) which was sustained for more than 24 h and points to its excellent bioavailability. The compounds 31a (IC50 = 6.6 µM) and 32a (IC50 = 37 µM), as well as their racemic mixture 28i (IC50 = 16 µM) significantly inhibited collagen-induced human platelet aggregation in vitro. Compound 34c displayed dual mechanism of action against both collagen (IC50 = 3.3 µM) and U46619 (IC50 = 2.7 µM) induced platelet aggregation. The pharmacokinetic study of 31a indicated very faster absorption, prolonged and constant systemic exposure and thereby exhibiting better therapeutic response.

alpha-Amino acid derivatives as proton pump inhibitors and potent anti-ulcer agents.

In a program to identify new anti-ulcer compounds, a series of N-acyl derivatives of alpha-amino acids were screened for their in vitro H(+)/K(+) ATPase inhibitory activity, and in vivo efficacy in Pylorus ligation model. 3D-QSAR studies were carried out and a representative compound 13 was studied for the nature of its proton pump inhibition.

Progress in the design of low molecular weight thrombin inhibitors.

Intravascular thrombosis and its complication, embolism, is a leading cause of morbidity and mortality throughout the world. Past few decades have seen a great deal of progress in the development of antithrombotic agents, though the current treatment options are limited to heparin, LMW heparins, and warfarin. Detailed understanding of the biochemical and biophysical mechanisms of activation and regulation of blood coagulation have helped in developing specific inhibitors of enzymes, especially thrombin, within the coagulation cascade. Thrombin plays a central role in the coagulation cascade and so has become the primary target for the development of antithrombotic drugs. The review covers the main pharmacological aspects of haemostasis and thrombosis and provides an update on low molecular weight thrombin inhibitors along with the limitations of the prevalent antithrombotic agents. Recent developments in small molecule inhibitors of Protease Activated Receptor-1 (PAR-1) which can be helpful for the treatment of thrombotic and vascular proliferative disorders, have also been discussed.

Metabolism of CDRI-85/92, a new potent anti-ulcer agent, involving cis-trans conversion.

CDRI 85/92, an anti-ulcer drug, is a new proton pump inhibitor, currently in an advanced stage of drug development. To know more about the drug it was our objective to delineate/identify the metabolic pathway as well as the enzymes responsible for the formation of metabolites. Metabolism of CDRI-85/92 (cis-5-styryl-2-oxazolidinone-4-carboxylic acid) was investigated in rat liver cellular fractions (S9, microsomes and cytosol) using reverse-phase HPLC and mass spectrometry techniques. Two major metabolites were produced by rat liver S9 fractions and reducing factor generating system from either untreated rats or phenobarbitone (PB)-pretreated rats. Incubation of CDRI-85/92 with postmitochondrial fraction (S9) for 24 h resulted in a cis to trans conversion (metabolite M2). Further cis-trans metabolizing capacity was measured separately in the cytosolic and microsomal fractions. Incubation with the cytosolic fraction resulted in an increased rate of cis-trans conversion, while the microsomal fraction showed no cis to trans conversion, thereby restricting the cis to trans conversion to Phase II enzymes, which are mainly located in the cytosol. Studies with PB-pretreated rat liver S9 fractions resulted in an increased rate of cis to trans conversion. Another metabolite was also present (M1) which was identified as an oxygenated metabolite by mass spectrometry. The major urinary metabolite from CDRI-85/92-treated Sprague-Dawley rats (20 mg/kg p.o.) was identified as M2. Studies using sulfobromophthalein and N-ethylmaleimide, as specific inhibitors of GST, showed a complete absence of metabolism, thus indicating the involvement of GST in the metabolism of CDRI-85/92. This study will be helpful in providing clues about factors influencing the bioavailability of CDRI-85/92 as well as drug-drug interactions.