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.

In vivo colocalization of antigen and CpG [corrected] within dendritic cells is associated with the efficacy of cancer immunotherapy.

Immunostimulatory cytidyl guanosyl (CpG) motifs are of great interest as cancer vaccine adjuvants. They act as potent inducers of Th1 responses, including the activation of cytotoxic CD8(+) T lymphocytes (CTL). Whereas animal models have provided clear evidence that CpG enhances antitumor immunity, clinical trials in humans have thus far been less successful. Applying cryosurgery as an instant in situ tumor destruction technique, we now show that timing of CpG administration crucially affects colocalization of antigen and CpG within EEA-1(+) and LAMP-1(+) compartments within dendritic cells in vivo. Moreover, antigen/CpG colocalization is directly correlated with antigen cross-presentation, the presence of CTL, and protective antitumor immunity. Thus, failure or success of CpG as a vaccine adjuvant may depend on colocalization of antigen/CpG inside DCs and hence on the timing of CpG administration. These data might aid in the design of future immunotherapeutic strategies for cancer patients.

Detection of inflamed atherosclerotic lesions with diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) and positron-emission tomography.

Diadenosine-5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) and its analog P(2),P(3)-monochloromethylene diadenosine-5',5'''-P(1),P(4)-tetraphosphate (AppCHClppA) are competitive inhibitors of adenosine diphosphate-induced platelet aggregation, which plays a central role in arterial thrombosis and plaque formation. In this study, we evaluate the imaging capabilities of positron-emission tomography (PET) with P(2),P(3)-[(18)F]monofluoromethylene diadenosine-5',5'''-P(1),P(4)-tetraphosphate ([(18)F]AppCHFppA) to detect atherosclerotic lesions in male New Zealand White rabbits. Three to six months after balloon injury to the aorta, the rabbits were injected with [(18)F]AppCHFppA, and microPET imaging showed rapid accumulation of this radiopharmaceutical in the atherosclerotic abdominal aorta, with lesions clearly visible 30 min after injection. Computed tomographic images were coregistered with PET images to improve delineation of aortoiliac tracer activity. Plaque macrophage density, quantified by immunostaining with RAM11 against rabbit macrophages, correlated with PET measurements of [(18)F]AppCHFppA uptake (r = 0.87, P < 0.0001), whereas smooth-muscle cell density, quantified by immunostaining with 1A4 against smooth muscle actin, did not. Biodistribution studies of [(18)F]AppCHFppA in normal rats indicated typical adenosine dinucleotide behavior with insignificant myocardial uptake and fast kidney clearance. The accumulation of [(18)F]AppCHFppA in macrophage-rich atherosclerotic plaques can be quantified noninvasively with PET. Hence, [(18)F]AppCHFppA holds promise for the noninvasive characterization of vascular inflammation.

A simple kinetic method for rapid mechanistic analysis of reversible enzyme inhibitors.

A simple diagnostic method for mechanistic analysis of reversible enzyme inhibitors is presented. The method involves simple experimentation to determine how the inhibition by a reversible inhibitor changes in response to the substrate concentration varied in the assay. Four types of inhibitors are categorized based on their kinetic characteristic: (1) competitive or mutually exclusive inhibitors that compete with the substrate for the enzyme; (2) noncompetitive inhibitors that are independent of the substrate for binding to the enzyme; (3) antagonistic inhibitors where the binding affinity of the inhibitor is partially reduced by the substrate binding to the enzyme; and (4) synergistic inhibitors where inhibitor binding is enhanced by substrate binding. An equation was derived for data fitting and subsequent determination of inhibitor binding mode. The method was evaluated in three model enzyme systems, i.e., PK, adenylate kinase, and LDH, with known inhibitors. The method was also used to characterize a large number of unknown Csp3 inhibitors identified from HTS of a compound library consisting of 120,000 distinct chemical entities, a field test that validated the utility of the method. Among 76 Csp3 inhibitors analyzed, 70 were found to be non-mutually exclusive inhibitors, suggesting the existence of an allosteric site(s) in Csp3 for effective inhibition. The implication of this observation is discussed.

Rapid noninvasive detection of experimental atherosclerotic lesions with novel 99mTc-labeled diadenosine tetraphosphates.

The development of a noninvasive imaging procedure for identifying atherosclerotic lesions is extremely important for the clinical management of patients with coronary artery and peripheral vascular disease. Although numerous radiopharmaceuticals have been proposed for this purpose, none has demonstrated the diagnostic accuracy required to replace invasive angiography. In this report, we used the radiolabeled purine analog, 99mTc diadenosine tetraphosphate (Ap4A; AppppA, P1,P4-di(adenosine-5')-tetraphosphate) and its analogue 99mTc AppCHClppA for imaging experimental atherosclerotic lesions in New Zealand White rabbits. Serial gamma camera images were obtained after intravenous injection of the radiolabeled dinucleotides. After acquiring the final images, the animals were sacrificed, ex vivo images of the aortas were recorded, and biodistribution was measured. 99mTc-Ap4A and 99mTc AppCHClppA accumulated rapidly in atherosclerotic abdominal aorta, and lesions were clearly visible within 30 min after injection in all animals that were studied. Both radiopharmaceuticals were retained in the lesions for 3 hr, and the peak lesion to normal vessel ratio was 7.4 to 1. Neither of the purine analogs showed significant accumulation in the abdominal aorta of normal (control) rabbits. The excised aortas showed lesion patterns that were highly correlated with the in vivo and ex vivo imaging results. The present study demonstrates that purine receptors are up-regulated in experimental atherosclerotic lesions and 99mTc-labeled purine analogs have potential for rapid noninvasive detection of plaque formation.

Characterization of diadenosine polyphosphate transport into chromaffin granules from adrenal medulla.

The transport of diadenosine polyphosphates into chromaffin granules from bovine adrenal medulla has been studied by using the radiolabeled substrate [3H]Ap5A and the fluorescent substrate analog di(1,N6-ethenoadenosine)polyphosphate, epsilon-(Ap(n)A) (n=3-5). The vesicular concentration increase was time dependent and the substrates were not metabolized to any extent during the transport experiments. The saturation curve indicates the existence of kinetic and allosteric cooperativity during Ap(n)A (diadenosine polyphosphates) transport and could be the result of the presence of various affinity states of the transporter with K values of 16 +/- 1 microM and 75 +/- 6 microM, and corresponding Hill numbers of 2 and 4, when epsilon-(Ap4A) was the substrate. The saturation studies for [3H]Ap5A were performed in a broader concentration range; in this case a three-step curve was obtained with K values of 16 +/- 2 microM, 125 +/- 9 microM, and 545 +/- 11 microM; the corresponding Hill numbers were 2, 4, and 6. This kinetic behavior can be explained on the basis of a mnemonic model, as already demonstrated for the vesicular transport of ATP. The nonhydrolyzable adenine nucleotide analogs, ATPgammaS and ADPbetaS, inhibited the diadenosine polyphosphate transport at concentrations in the millimolar range. Ap(n)A transport was also inhibited by the P2 receptor antagonist suramin, the mitochondrial ATP/ADP exchange inhibitor atractyloside, the proton translocator FCCP, and N-ethylmaleimide.

Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain.

The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [3H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation. The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system.

Disposition of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) in rats.

The disposition of diadenosine 5'5"'-P1,P4-tetraphosphate (Ap4A), an endogenous dinucleotide, was investigated in rats. The degradation of Ap4A in rat plasma was very rapid and could be explained by a Michaelis-Menten equation: Km and Vmax values were 1.69 micrograms/ml and 4.32 micrograms/min/ml, respectively. Ap4A was degraded in rat plasma to ATP and AMP, but not to 2 ADP molecules, and these nucleotides were further degraded through adenosine. The degradations kinetics were examined. After intravenous bolus injection, Ap4A in plasma declined rapidly and the rate of elimination was dose-dependent: the biological half-life was about 3s at the dose of 1 mg/kg and was longer at 3 mg/kg. When Ap4A was administered by intravenous infusion (0.5 or 1.0 mg/kg/min), the plasma level rapidly reached a steady-state, which then rapidly declined after stopping the infusion.

Ca2+ dependency of diadenosine 5',5"'-p1,p4-tetraphosphate degradation in dog plasma.

Diadenosine 5',5"'-p1,p4-tetraphosphate (Ap4A) degradation in dog plasma was inhibited by the addition of EDTA. The Ap4A degrading activity seemed to be recovered by the addition of metal ions, Ca2+, Co2+, Cu2+, Mg2+, Mn2+ or Zn2+ to plasma in which the activity was lost by the addition of EDTA. Especially, the degradation was accelerated by the addition of Ca2+ to the plasma, and the degradation rate was dependent on the concentration of Ca2+. The results of this study suggested that the enzymatic degradation of Ap4A in dog plasma was modulated by the concentration of Ca2+.