8-substituted adenosine and theophylline-7-riboside analogues as potential partial agonists for the adenosine A1 receptor.

A series of 8-substituted adenosine and theophylline-7-riboside analogues (28 and 9 compounds, respectively) was tested on adenosine A1 and A2A receptors as an extensive exploration of the adenosine C8-region. Alkylamino substituents at the 8-position cause an affinity decrease for adenosine analogues, but an affinity increase for theophylline-7-riboside derivatives. The affinity decrease is probably due to a direct steric hindrance between the C8-substituent and the binding site as well as to electronic effects, not to a steric influence on the ribose moiety to adopt the anti conformation. The 8-substituents increase the affinity of theophylline-7-riboside analogues probably by binding to a lipophilic binding site. The intrinsic activity was tested in vitro for some 8-substituted adenosine analogues, by determining the GTP shift in receptor binding studies and the inhibition of adenylate cyclase in a culture of rat thyroid FRTL-5 cells, and in vivo in the rat cardiovascular system for 8-butylaminoadenosine. Thus, it was shown that 8-ethyl-, 8-butyl-, and 8-pentylamino substituted analogues of adenosine may be partial agonists in vitro, and that 8-butylaminoadenosine is a partial agonist for the rat cardiovascular A1 receptor in vivo.

Characterization of ecto-ATPase on human blood cells. A physiological role in platelet aggregation?

Ecto-ATPase (EC 3.6.1.15) is a plasma membrane-bound enzyme which degrades extracellular triphosphate nucleotides. Although its physiological function is still unclear, the enzyme obscures the study of P2 purinoceptors (i.e. receptors for ATP and other di- and triphosphate nucleotides), since it is capable of metabolizing the pharmacological ligands, such as ATP, for these receptors. We characterized the ecto-ATPase activity on human blood cells with a [gamma 32P]ATP assay and HPLC measurements. We also determined whether ecto-ATPase activity could affect the anti-aggregatory role of ATP in whole human blood. The Km for ATP of the ecto-ATPase on human blood cells was 8.5 +/- 2.3 microM and the maximum degradation rate, at 37 degrees, was 2.7 +/- 1.1 nmol ATP/(min x mL whole blood). In whole blood the major part of ATP was broken down by the blood cells, predominantly by the leukocytes. ATP and UTP were broken down equally well, mainly yielding the corresponding di- and monophosphates. In search of inhibitors for the ecto-ATPase, we studied several analogs of ATP. 8-Bromo-ATP as well as 2'- and 3'-deoxy-ATP were substrates for the enzyme. In contrast, modification of the phosphate side chain yielded inhibitors. Subsequently, a possible role of the ecto-ATPase in platelet aggregation was verified. To assess the role of the plasma membrane-bound enzyme, platelet aggregation was determined in whole blood instead of platelet-rich plasma. In the presence of ATP alone, an antagonist of ADP-induced platelet aggregation, some aggregation was still observed. As breakdown of ATP by the ecto-ATPase leads to gradual formation of ADP, as mentioned above, we compared the effects of a stepwise versus bolus addition of ADP. Subsequent dosing of ADP (1.5, 2.5, 5 and 10 microM) resulted in platelet aggregation but to a much smaller extent, at most approximately 60%, compared to the amount of platelet aggregation obtained with a bolus addition of ADP (10 microM). In conclusion, human blood cells possess a high affinity ecto-ATPase which degrades ATP as well as ATP analogs with modified base and ribose moieties. ATP analogs with a modified phosphate chain are inhibitors of the ecto-ATPase. A direct role of the ecto-ATPase activity on platelet aggregation is probably small, as degradation of ATP to ADP proceeds slowly and cumulative addition of ADP to platelets in whole blood results in a modest amount of aggregation.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of nucleoside uptake in human erythrocytes by a new series of compounds related to lidoflazine and mioflazine.

The zero-trans influx of uridine in human erythrocytes is inhibited by lidoflazine and analogs thereof. The concentrations required for inhibition of nucleoside transport were higher when the compounds were simultaneously added with uridine than upon preincubation of the inhibitors with the erythrocytes. R70380 proved to be the most active compound in this respect, its IC50 value being 13 nM after preincubation. Even the reference compounds nitrobenzylthioinosine and dilazep were remarkably more potent with preincubation; dipyridamole, however, was not.

Influence of the molecular structure of N6-(omega-aminoalkyl)adenosines on adenosine receptor affinity and intrinsic activity.

The affinities of a series of N6-(omega-aminoalkyl)adenosines as probes for A1 and A2 adenosine receptors were determined in various radioligand binding assays and the intrinsic activities were measured in adenylate cyclase assays. Clear species differences were noticed for A1 receptor affinity of these adenosine receptor agonists, the compounds being more active in calf than in rat brain tissue. The affinity profile within the series was, however, rather similar in both membrane preparations, with N6-9-aminononyladenosine displaying highest affinity. The A2 receptor affinities were comparable to values measured for the A1 receptor in its low affinity state, as assessed with a radiolabelled antagonist in the presence of 1 mM GTP. Calculation of the intrinsic activities of the adenosine analogues from their modulating action on adenylate cyclase showed almost all the compounds to be equally effective to (-)-N6-(R-phenylisopropyl) adenosine, on either A1 or A2 adenosine receptors. N6-3-Aminopropyl- and N6-12-aminododecyladenosine, however, proved to be partial agonists, the first on A1 and the second on A2 adenosine receptors. The data are used as the basis for a discussion of adenosine receptor subtype selectivity and intrinsic activity in general.

The combined use of high-performance liquid chromatography and radioimmunoassay for the bioanalysis of nicomorphine and its metabolites.

Methods have been developed for the determination of nicomorphine using reversed-phase HPLC with UV detection; for the simultaneous assay of morphine and mononicotinoylmorphine by a coupled normal-phase HPLC-radioimmunoassay method; and for conjugates of morphine and mononicotinoylmorphine by radioimmunoassay. The methods have been evaluated and applied to a pharmacokinetic study of nicomorphine administered intramuscularly.