Publisher Correction: GLS-409, an Antagonist of Both P2Y1 and P2Y12, Potently Inhibits Canine Coronary Artery Thrombosis and Reversibly Inhibits Human Platelet Activation.

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GLS-409, an Antagonist of Both P2Y1 and P2Y12, Potently Inhibits Canine Coronary Artery Thrombosis and Reversibly Inhibits Human Platelet Activation.

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12 ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes.

Self-neutralizing oligonucleotides with enhanced cellular uptake.

There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by phosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.

Synergistic Inhibition of Both P2Y1 and P2Y12 Adenosine Diphosphate Receptors As Novel Approach to Rapidly Attenuate Platelet-Mediated Thrombosis.

OBJECTIVE: Unlike currently approved adenosine diphosphate receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet adenosine diphosphate receptor P2Y1 and may, therefore, be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409. APPROACH AND RESULTS: We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited adenosine diphosphate- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, that is, platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a nonsignificant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake. CONCLUSIONS: Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy.

New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors.

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5') tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.

Prodrugs of herpes simplex thymidine kinase inhibitors.

BACKGROUND: Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. METHODS: Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. RESULTS: Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. CONCLUSIONS: Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo.

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.

A novel agent effective against Clostridium difficile infection.

N(2)-(3,4-Dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine (MorE-DCBG, 362E) is a synthetic purine that selectively inhibits the replication-specific DNA polymerase of Clostridium difficile. MorE-DCBG and its analogs strongly inhibited the growth of a wide variety of C. difficile strains. When administered orally in a hamster model of C. difficile-specific colitis, 362E was as effective as oral vancomycin, the current agent of choice for treating severe forms of the human disease.

P1,P2-diimidazolyl derivatives of pyrophosphate and bis-phosphonates--synthesis, properties, and use in preparation of dinucleoside tetraphosphates and analogs.

P(1),P(2)-Diimidazolyl derivatives of pyrophosphate and halomethylene-bis-phosphonates have been synthesized and characterized, and the mechanism of their formation was studied. These reagents enable synthesis of dinucleoside tetraphosphates and tetraphosphonates conveniently and in high yields.

L-valine ester of cyclopropavir: a new antiviral prodrug.

BACKGROUND: Following the example of L-valine prodrugs of antiviral nucleoside analogues, L-valine ester of cyclopropavir (valcyclopropavir) was synthesized. METHODS: The known tetrahydropyranylcyclopropavir was transformed to N-(tert-butoxycarbonyl)-L-valine ester, which was deprotected to valcyclopropavir. RESULTS: Stability of valcyclopropavir towards hydrolysis at pH 7.0 roughly corresponded to that of valganciclovir. Valcyclopropavir inhibited replication of human cytomegalovirus (HCMV, Towne and AD169 strains) to approximately the same extent as the parent drug cyclopropavir. Pharmacokinetic studies in mice established that the oral bioavailability of valcyclopropavir was 95%. CONCLUSIONS: The prodrug valcyclopropavir offers some improved therapeutic parameters over the parent compound cyclopropavir.

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody.

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.