Antiplatelet Activity, P2Y1 and P2Y12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5′,5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors

Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation

McCormick\u27s Evidence, 7th (Hornbook Series)

This single-volume treatise is largely free of citations to authority, but retains the most notable footnotes. Topics covered include preparing and presenting evidence, cross-examination, and the procedure for admitting and excluding evidence. Discusses privilege against self-incrimination, privilege concerning improperly obtained evidence, scientific evidence, and demonstrative evidence. Reviews authentication, the hearsay rule, burdens of proof, and presumptions. Text also identifies current issues. - From the Publisherhttps://elibrary.law.psu.edu/fac_books/1010/thumbnail.jp

McCormick\u27s Evidence, 7th (Hornbook Series)

This single-volume treatise is largely free of citations to authority, but retains the most notable footnotes. Topics covered include preparing and presenting evidence, cross-examination, and the procedure for admitting and excluding evidence. Discusses privilege against self-incrimination, privilege concerning improperly obtained evidence, scientific evidence, and demonstrative evidence. Reviews authentication, the hearsay rule, burdens of proof, and presumptions. Text also identifies current issues. - From the Publisherhttps://elibrary.law.psu.edu/fac_books/1010/thumbnail.jp

McCormick on Evidence, 7th (Practitioner Treatise Series)

Recognized as the foremost authority on evidence law today, McCormick on Evidence offers comprehensive and authoritative analysis of the rules and theory of evidence. When there are specific questions of evidence for which a jurisdiction has no precedent, this treatise provides both general theories that may be argued to suggest the answer and varying views from other jurisdictions. Like the Dean McCormick\u27s original text, the Seventh Edition continues to provide a pragmatic approach to the law of evidence. - From the Publisherhttps://elibrary.law.psu.edu/fac_books/1009/thumbnail.jp

McCormick on Evidence, 7th (Practitioner Treatise Series)

Recognized as the foremost authority on evidence law today, McCormick on Evidence offers comprehensive and authoritative analysis of the rules and theory of evidence. When there are specific questions of evidence for which a jurisdiction has no precedent, this treatise provides both general theories that may be argued to suggest the answer and varying views from other jurisdictions. Like the Dean McCormick\u27s original text, the Seventh Edition continues to provide a pragmatic approach to the law of evidence. - From the Publisherhttps://elibrary.law.psu.edu/fac_books/1009/thumbnail.jp

Antiplatelet activity, P2Y₁ and P2Y₁₂ inhibition, and metabolism in plasma of stereoisomers of diadenosine 5',5'″-P¹ ,P⁴-dithio-P²,P³-chloromethylenetetraphosphate.

Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5',5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma.We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma.The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers.The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors