Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

ß-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum.

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to ß-nicotinamide adenine dinucleotide (ß-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, ß-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. ß-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of ß-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of ß-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for ß-NAD at intestinal neuromuscular junctions. The data suggest that ß-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of ß-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.

RNA-directed agent, cordycepin, induces cell death in multiple myeloma cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy that is slow-growing, and thus traditional DNA-replication directed chemotherapeutics are ineffective. We hypothesized that those agents that target RNA-directed processes would be successful in MM. To test this postulate, cordycepin, a polyadenylation inhibitor was used as a proof-of-principle towards MM cell lines. Cordycepin accumulated in MM.1S cells as its triphosphate metabolite, 3'dATP and subsequently inhibits RNA synthesis and cell growth. Cell death was via apoptosis induction and over 50% of treated cells were annexin-V positive after 48 h. As a consequence of RNA synthesis inhibition, we hypothesized that specific genes with short half-lives may be downregulated, leading to a reduction in protein. Indeed, a reduction in the transcript levels for MET, a survival gene for MM, was detected as early as 4 h and transcripts were reduced to c. 10% of control after 48 h. Interestingly, no significant change in protein levels was observed for Bcl-2, XIAP, Mcl-1 or survivin. Stabilization of p53 was not observed, and caspases-8, -9 and -3 showed activation following cordycepin treatment but were not required for cell death. Our results suggest that RNA-directed agents may be a new group of agents for the treatment of MM.

Similar dynamics of G-protein coupled receptors molecules in response to antagonist binding.

In this communication we would like to draw attention on surprisingly similar transition of G-protein coupled receptors (GPCR) molecules, observed in these kinetic studies made with different receptors and their antagonists.

Kinetics of [35S]dATPalphaS interaction with P2Y1 purinoceptor in rat brain membranes.

Kinetics of [35S]dATPalphaS (2'deoxyadenosine-5'-[alpha-35S]-thiotriphosphate) interaction with rat brain membrane fragments was studied at 25 degrees C and at radioligand concentrations from 2 to 250 nM. At least two different ways of [35S]dATPalphaS interaction with the membranes were distinguished on the basis of radioligand on-rate. Firstly, the binding sites characterized by 'fast' on-rate can be observed. Secondly, the 'slow' binding sites were kinetically identified and quantified. As in both cases the bound radioligand could be displaced by excess of ATP, all these binding sites can be defined as 'specific sites'. In the 'slow' binding sites isomerization of the receptor-ligand complex was observed, as is typical for interaction of antagonists with G-protein coupled receptors, and the kinetic parameters for this interaction were similar with the appropriate data for the hP2Y1 receptors expressed in 1321N1 astrocytoma cells Therefore these sites could be assigned to the same receptor subtype in brain membranes while the 'fast' binding sites belong to other membrane-bound proteins, also interacting with ATP and its analogues. The kinetic properties of the latter sites were not analysed in detail.

Repeated cocaine administration differentially affects NMDA receptor subunit (NR1, NR2A-C) mRNAs in rat brain.

We investigated the effects of intermittent intraperitoneal (i.p.) injections of cocaine (20 mg/kg) on subunit mRNAs of N-methyl-D-aspartate (NMDA) receptors (NR1/NR2A-2C) in the rat brain by in situ hybridization using phosphor screen analysis. The level of NR1 subunit mRNA significantly increased in hippocampal complexes 1 h after a single i.p. injection of cocaine. After repeated cocaine injection, the mean scores of stereotyped behavior were increased with the number of injections. The level of NR1 subunit mRNA was obviously decreased in the striatum and cortices 24 h (early withdrawal) after a final injection following 14 days of subchronic administration. During the early withdrawal period, the amount of the NR1 subunit decreased in the nucleus accumbens, globus pallidus, and subiculum. In the dentate gyrus, the NR1 mRNA level significantly increased during early withdrawal in rats subchronically treated with cocaine. Levels of NR2B subunit mRNA were reduced in the cortices and striatum. During late withdrawal from cocaine, the level of NR2C subunit mRNA in the cerebellum was also reduced. These findings suggest that the disruption of NR1, NR2B, and NR2C subunits in the discrete brain regions occurs under the cocaine-related behavioral abnormalities and would be closely implicated in the initiation and expression of behavioral sensitization induced by repeated cocaine administration. Further studies on the changes in non-NMDA receptors are required to elucidate the biological significance of glutamate receptors for the mechanisms underlying the development of behavioral sensitization.

Decay rates of anti-HIV dideoxynucleotides in tissue culture systems: a simple correction for the effect of cell replication.

Measurement of intracellular drug levels in cell culture systems can be of predictive value in establishing rational clinical dosage schedules. Such in vitro measurements carried out with anti-HIV agents of the 2',3'-dideoxynucleoside (ddN) class have shown that many of the pharmacologically active ddNTP metabolites of these agents have relatively long intracellular half-lives and little or no host-cell cytotoxicity. As a consequence, replication of drug-exposed cells continues at an unperturbed rate so that a systematic dilution error occurs in the measurement of ddNTP decay half-times. The aim of this study is to present a simple general formulation for the correction of measured t1/2-values for ddNTPs and for other agents with similar intracellular pharmacokinetic properties. Two factors of practical interest emerge: first, the error is greater for agents with slow intracellular clearance rates than for agents with rapid rates; and second, for cell lines with long doubling times, the measured t1/2-values approach more closely to the true t1/2-values, until with the extreme case (quiescent or "G(o)" cells), the observed and true decay times are identical. The greatest dilution errors are seen with adenodine-based agents such as ddATP and 2'-F-ddATP, while the smallest errors are seen with rapidly cleared agents of the dideoxythymidine class.

Anomalous accumulation and decay of 2',3'-dideoxyadenosine-5'-triphosphate in human T-cell cultures exposed to the anti-HIV drug 2',3'-dideoxyinosine.

The rates of accumulation and decay of 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) have been examined after incubation with the anti-human immunodeficiency virus (HIV) agents 2',3'-dideoxyinosine (ddIno) and 2',3'-dideoxyadenosine (ddAdo) in human T-cell systems frequently used for assay of anti-HIV agents (MOLT-4 and CEM). Formation of ddATP from ddIno or ddAdo was rapid and concentration-dependent, with no saturation of phosphorylation being observed up to extremely high levels (1 mM) of drug. Rates of removal of ddATP from MOLT-4 cells were slow (t1/2 = 25-40 hr) and appeared to be monophasic. These unusually long half-times for ddATP utilization are not a general property of purine dideoxypurine nucleosides: when the corresponding guanine analog (2',3'-dideoxyguanosine) was examined under the same conditions, the t1/2 of ddGTP removal was only 3-5 hr. Similar results were observed with the human T-cell line CCRF-CEM. Coadministration with ddIno of inosine monophosphate dehydrogenase inhibitors, such as ribavirin and tiazofurin, yielded higher levels of ddATP in MOLT-4 and CEM cells, but did not influence the slow removal of ddATP from T-cells. The long half-time for disappearance of ddATP from cells may permit the maintenance of pharmacologically effective levels of ddATP within cells with relatively infrequent administration of the parent drug (ddIno or ddAdo).