Characterization of the pharmacokinetics, brain distribution, and therapeutic efficacy of the adenosine A1 receptor partial agonist 2'-deoxy-N6-cyclopentyladenosine in sarin-poisoned rats

The objective of the present study was to determine (1) the influence of sarin poisoning (144 microg/kg s.c.) on the pharmacokinetics and brain distribution of the adenosine A1 receptor partial agonist 2'-deoxy-N6-cyclopentyladenosine (2'dCPA), and (2) the effect of 2'dCPA (20 mg/kg i.v.) on the central acetylcholine (ACh) release and protection against sarin toxicity. A five-compartment model successfully described the pharmacokinetic profile of 2'dCPA in blood and brain microdialysate. A covariate analysis revealed that the volume of distribution of 2'dCPA in blood was different in sarin-poisoned rats, 177 +/- 7 versus 148 +/- 8 ml in control rats. However, the transport of 2'dCPA from blood to the brain was unaffected as reflected by the values of the intercompartmental transport clearances, 0.21 +/- 0.02 and 0.21 +/- 0.04 microl/min in control and sarin-poisoned rats, respectively. Also the area-under-curve (AUC) ratios of brain microdialysate and blood were identical with values of 0.02 +/- 0.001 and 0.02 +/- 0.002, respectively, demonstrating the restricted transport of 2'dCPA into the brain in both treatment groups. Treatment of sarin-poisoned rats by 2'dCPA did not adequately prevent the accumulation of ACh in the central nervous system. 2'dCPA delayed the emergence of concomitant symptoms compared to untreated rats, but eventually only 29% of the animals survived 24 h. In conclusion, the pharmacokinetic profile of 2'dCPA in blood was slightly changed by sarin, but not the distribution of 2'dCPA into the brain. The therapeutic efficacy of 2'dCPA against sarin was limited, presumably due to insufficient quantities of 2'dCPA reaching the brain.     

Pharmacokinetic/pharmacodynamic modelling of the anti-hyperalgesic and anti-nociceptive effect of adenosine A1 receptor partial agonists in neuropathic pain

The objective of this investigation was to characterise the pharmacokinetic-pharmacodynamic correlation of adenosine A1 receptor partial agonists in the chronic constriction injury model of neuropathic pain. Following intravenous administration of 8-methylamino-N6-cyclopentyl-adenosine (MCPA; 10 mg/kg) and 2'deoxyribose-N6-cyclopentyl-adenosine (2'dCPA; 20 mg/kg), the time course of the effect on the mechanical paw pressure threshold was determined in conjunction with plasma concentrations. Population pharmacokinetic/pharmacodynamic analysis was applied to derive individual concentration-effect relationships. A composite model consisting of an E(max) model for the anti-hyperalgesic effect in combination with a linear model for the anti-nociceptive effect accurately described the concentration-effect relationship. For both compounds, a full anti-hyperalgesic effect was observed. The values of the EC50 for the anti-hyperalgesic effect were (mean+/-S.D.): 3170+/-1460 and 2660+/-1200 ng/ml for MCPA and 2'dCPA versus 178+/-51 ng/ml for the reference full agonist 5'deoxyribose-N6-cyclopentyl-adenosine (5'dCPA). The values of the slope for the anti-nociceptive effect were 1.9+/-0.30 and 1.2+/-0.20 g.microl/ng, respectively, versus 55+/-8 g microl/ng for 5'dCPA. Adenosine A1 receptor partial agonists behave as full agonists with regard to the anti-hyperalgesic effect in neuropathic pain, but the anti-nociceptive effect is diminished.     

Modelling of the pharmacodynamic interaction of an A1 adenosine receptor agonist and antagonist in vivo: N6-cyclopentyladenosine and 8-cyclopentyltheophylline.

1. The purpose of this investigation was to develop a pharmacokinetic-pharmacodynamic model for the interaction between an adenosine A1 receptor agonist and antagonist in vivo. The adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) and the antagonist, 8-cyclopentyltheophylline (CPT) were used as model drugs. The CPA-induced reduction in mean arterial pressure and heart rate were used as measurements of effect. 2. Four groups of eight rats each received 200 micrograms kg-1 of CPA i.v. in 5 min during a steady-state infusion of CPT at a rate of 0, 57, 114 or 228 micrograms kg-1 h-1. The haemodynamic parameters were continuously measured and frequent blood samples were taken to determine the pharmacokinetics of the drugs. 3. CPT had no influence on the pharmacokinetics of CPA and the baseline values of the haemodynamic variables. Furthermore, no clear antagonism by CPT was observed of the CPA-induced reduction in mean arterial pressure. However, CPT antagonized the effect on heart rate, and with increasing CPT concentrations, a parallel shift of the CPA concentration-effect relationship to the right was observed. 4. An agonist-antagonist interaction model was used to characterize the interaction quantitatively. On the basis of this model, the pharmacodynamic parameters of both CPA and CPT could be estimated. For CPA the values were (mean +/- s.e.): Emax = 198 +/- 11 b.p.m., EC50 = 2.1 +/- 0.7 ng ml-1, Hill factor = 2.3 +/- 0.6 and for CPT: EC50 = 3.7 +/- 0.3 ng ml-1 and Hill factor = 3.1 +/- 0.1. 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine deaminase inhibition enhances the inotropic response mediated by A1 adenosine receptor in hyperthyroid guinea pig atrium.

The aim of the present study was to test the hypothesis that inhibition of adenosine deaminase (ADA) enhances the efficiency of signal-transduction of myocardial A1 adenosine receptors in hyperthyroidism. The inotropic response to N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist resistant to ADA, was investigated in the absence or presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an ADA and cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2) inhibitor, or of pentostatin (2'-deoxycoformycin; DCF), an exclusive ADA inhibitor, in left atria isolated from eu- or hyperthyroid guinea pigs. Both ADA inhibitors enhanced the effect of CPA only in hyperthyroid atria. EHNA significantly increased the Emax (mean+/-S.E.M.) from 83.8+/-1.2% to 93.4+/-1.2%, while DCF significantly decreased the logEC50 from -7.5+/-0.07 to -7.83+/-0.07 in hyperthyroid samples. Conversely, EHNA also diminished the logEC50 (from -7.5+/-0.07 to -7.65+/-0.07) and DCF also raised the Emax (from 83.8+/-1.2% to 85.7+/-2%) in hyperthyroidism, but these changes were not significant. In conclusion, ADA inhibition moderately but significantly enhanced the efficiency of A(1) adenosine receptor signaling pathway in the hyperthyroid guinea pig atrium. This suggests that elevated intracellular adenosine level caused by ADA inhibition may improve the suppressed responsiveness to A1 adenosine receptor agonists associated with the hyperthyroid state. Alternatively or in addition, the role of decreased concentration of adenosine degradation products cannot be excluded. Furthermore, in the case of EHNA, inhibition of PDE2 also appears to contribute to the enhanced A1 adenosine receptor signaling in the hyperthyroid guinea pig atrium.     

Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists

1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery.     

Brain penetration of synthetic adenosine A1 receptor agonists in situ: role of the rENT1 nucleoside transporter and binding to blood constituents.

The blood-brain barrier (BBB) transport of synthetic A(1) receptor agonists was studied in an in situ brain perfusion model in the presence and absence of the selective nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI). For 8-methylamino-N(6)cyclopentyladenosine (MCPA), N(6)-cyclopentyladenosine (CPA), 2'deoxy-N(6)-cyclopentyladenosine (2'dCPA) and 5'deoxy-N(6)-cyclopentyl adenosine (5'dCPA) the brain uptake clearance was low with values of 0.0045+/-0.0012, 0.018+/-0.0020, 0.022+/-0.0028 and 0.12+/-0.054 ml min(-1)g(-1), respectively. In the presence of an average NBTI plasma concentration of 2.6+/-0.3 microg ml(-1) (NBTI dose: 3 mg kg(-1) i.v.) the values of the brain uptake clearance were 0.0062+/-0.0012, 0.013+/-0.0017, 0.014+/-0.0030 and 0.13+/-0.066 ml min(-1)g(-1), respectively and not significantly different from the values in the absence of NBTI. In a separate experiment the brain uptake of MCPA from phosphate buffered saline (PBS) and whole blood were compared. The brain uptake clearance from whole blood (0.0012+/-0.001 ml min(-1)g(-1)) was significantly lower than from PBS (0.0045+/-0.0012 ml min(-1)g(-1)). The results of these studies show that the rENT1 nucleoside transporter does not contribute significantly to the transport of synthetic A(1) receptor agonists across the BBB and that binding to blood constituents restricts the brain uptake.     

Clearance of cyclopentyladenosine and cyclohexyladenosine in rats following a single subcutaneous dose.

A(1)-adenosine receptor agonist N(6)-cyclopentyladenosine (CPA) (3.0 mg kg(-1) body weight), or N(6)-cyclohexyladenosine (CHA) (3.0 mg kg(-1) body weight) was subcutaneously (s.c.) administered as a single dose to rats. After that, the blood samples (150 microl) were collected from vena saphena into heparinized capillary tubes at the different time points. The concentrations of CPA and CHA were assayed by reversed-phase high performance liquid chromatography (HPLC). Based on the present study, the retention times for CPA and CHA were 3.029 +/- 0.017 min and 5.07 +/- 0.031 min, respectively. In addition, the maximum concentrations of CPA and CHA were measured 60 and 80 min after the administration, and the half-lives of these compounds in the circulation were approximately 48.5 +/- 4.4 min and 106.6 +/- 7.4 min, respectively. Presumably, the difference between these values due to the larger cycloalkyl substituent of CHA and different absorption properties of these compounds. Moreover, in the present study, the elimination half-life was approximately sevenfold longer than that previously reported for CPA (48.5 +/- 4.4 min versus 6.9 +/- 0.5] min). The discrepancy in the half-life is not surprising because the administration route was different. On the other hand, after the s.c. administration, the absorption rates of these adenosine analogues are known to be slow. Based on the results of this paper, the authors conclude that the half-life of CHA is approximately 2.2-fold compared to that of CPA.     

Allosteric modulation, thermodynamics and binding to wild-type and mutant (T277A) adenosine A1 receptors of LUF5831, a novel nonadenosine-like agonist

The interaction of a new nonribose ligand (LUF5831) with the human adenosine A1 receptor was investigated in the present study. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A1 receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N6-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A1 receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A1 receptor (Ki=122+/-22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively.The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A1 receptor showed that LUF5831 had a submaximal effect (37+/-1%) in comparison to CPA (66+/-5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A1 receptor.     

Lack of adenosine A1 and dopamine D2 receptor-mediated modulation of the cardiovascular effects of the adenosine A2A receptor agonist CGS 21680

Some behavioral and biochemical effects of the systemically administered adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) in rats are potentiated by adenosine A(1) receptor agonists and counteracted by dopamine D2 receptor agonists. In the present study we compared potentiating and antagonistic interactions between CGS 21680 and adenosine A(1) and dopamine D2 receptor agonists on motor activity and on cardiovascular responses (arterial blood pressure and heart rate). The motor-depressant effects produced by CGS 21680 (0.5 mg/kg, i.p.) were potentiated by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 0.3 mg/kg, i.p.) and counteracted by the dopamine D2 receptor agonist quinpirole (0.5 mg/kg, i.p.). In contrast, neither CPA nor quinpirole significantly modified the decrease in arterial pressure or the increase in heart rate induced by CGS 21680. However, the adenosine A(2A) receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3, 3 mg/kg, i.p.) counteracted both the motor-depressant and cardiovascular effects of CGS 21680. Therefore, the effects of the systemically administered adenosine A(2A) receptor agonist CGS 21680 on cardiovascular function, in contrast to its effects on motor behavior, appear to be independent of the effects of adenosine A(1) and dopamine D2 receptor activity.     

Partial A(1) adenosine receptor agonists from a molecular perspective and their potential use as chronic ventricular rate control agents during atrial fibrillation (AF)

This review provides a molecular perspective of partial agonism at the A(1) adenosine receptor. The structure-activity relationships (SAR) for affinity and intrinsic efficacy of analogues of the full agonist N6-cyclopentyladenosine (CPA) are emphasized. Both general models of activation of G protein-coupled receptors and specific molecular models of the A(1)-adenosine receptor are used to interpret the results of efforts to synthesize and assay effects of partial agonists. The SAR of affinity and intrinsic efficacy of the 2', 3', and especially the 5'-deoxy derivatives of CPA is presented. From this analysis, the nature of the interactions of specific atoms and substituents of the CPA molecule with the A(1)-adenosine receptor are deduced and presented pictorially. As an example of the therapeutic potential of partial agonists, the design and testing of analogues of CPA to provide chronic ventricular rate control during atrial fibrillation is described. The challenges associated with designing a partial A(1)-adenosine receptor agonist for providing chronic ventricular rate control during atrial fibrillation are many. To meet these challenges, further medicinal chemistry efforts in the area of partial A(1)-adenosine receptor agonism are still needed.     

Differences in intrinsic efficacy of benzodiazepines are reflected in their concentration-EEG effect relationship.

1. The relevance of EEG effect parameters as a measure of the central nervous system effects of benzodiazepines was evaluated. The concentration-EEG effect relationships of the benzodiazepine agonist midazolam, partial agonist bretazenil, antagonist flumazenil and inverse agonist Ro 19-4603 were quantified and compared with the intrinsic efficacy and affinity of these compounds at the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex. 2. The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous bolus administration of 5 mg kg-1 midazolam, 2.5 mg kg-1 bretazenil, 10 mg kg-1 flumazenil or 2.5 mg kg-1 Ro 19-4603 to male Wistar derived rats. In a separate experiment the distribution between blood, cerebrospinal fluid and brain concentrations of these compounds was determined. A sensitive assay was developed to measure bretazenil and Ro 19-4603 concentrations in small samples of biological fluids. 3. The benzodiazepine-induced changes in amplitudes in the 11.5-30 Hz frequency band, as determined by aperiodic analysis, was used as EEG effect measure. Concentration-EEG effect relationships were derived by a pharmacokinetic-pharmacodynamic modelling procedure and in the case of midazolam, bretazenil and Ro 19-4603 successfully quantified by the sigmoidal Emax model. Large differences in maximal effect of midazolam (Emax = 73 +/- 2 microVs-1), bretazenil (Emax = 19 +/- 1 microVs-1) and Ro 19-4603 (Emax = -6.5 +/- 0.4 microVs-1) were observed, reflecting their differences in intrinsic efficacy. A close correlation was found between the EC50 values based on free drug concentration and receptor affinity as determined by displacement of [3H]-flumazenil in a washed brain homogenate at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The antinociceptive effects of the systemic adenosine A1 receptor agonist CPA in the absence and in the presence of spinal cord sensitization

Adenosine A1 receptor agonists are effective antinociceptive agents in neuropathic and inflammatory pain, though they appear to be weak analgesics in acute nociception. Important discrepancies are observed on the effectiveness and potency of adenosine analogues when comparing different studies, probably due to the use of different ligands, models of antinociception, routes of administration and types of sensitization. We studied the systemic antinociceptive effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in spinal cord neuronal responses from adult male rats in acute nociception and in sensitization due to arthritis and neuropathy. The experiments showed that CPA was effective in the three experimental conditions, with a similar potency in reducing responses to noxious mechanical stimulation (ID50s: 20 +/- 1.2 microg/kg in acute nociception, 18 +/- 1.1 microg/kg in arthritis, 17.4 +/- 2 microg/kg in neuropathy). The phenomenon of wind-up was also dose-dependently reduced by CPA in the three experimental situations although the main action was seen in arthritis. Depression of blood pressure by CPA was not dose-dependent. We conclude that systemic CPA is a potent and effective analgesic in sensitization due to arthritis and neuropathy but also in acute nociception. The effect is independent of the cardiovascular activity and is centrally mediated since wind-up was inhibited.     

Study of the novel non-xanthine heterocyclic compound GU285 as a potent non-selective adenosine receptor antagonist in the rat

The novel pyrazolo[3,4-d]pyrimidine compound GU285 (4-amino-6-alpha-carbamoylethylthio-1- phenylpyrazolo[3,4-d]pyrimidine, CAS 134896-40-5) was examined for its ability (1) to inhibit binding of adenosine (ADO) receptor ligands in rat brain membranes, (2) to antagonise functional responses to ADO agonists in rat right and left atria and coronary resistance vessels, and (3) to reduce the fall in heart rate and arterial blood pressure produced by the ADO A1 agonist N6-cyclopentyladenosine (CPA) in the intact, anaesthetized rat. GU285 competitively inhibited binding of the ADO A1 agonist [3H]-R-N6-phenylisopropyladenosine (R-PIA) yielding a Ki value of 11 (7-18) nmol.l-1 (geometric mean +/- 95% Cl). When assayed against the ADO A2A selective agonist [3H]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamidoadenosine, (CGS21680), a Ki of 15 (10-24) nmol.l-1 was obtained. In spontaneously beating right atria, GU285 competitively antagonized negative chronotropic effects of R-PIA with a pA2 of 8.7 +/- 0.3 and in electrically paced left atria, GU285 competitively antagonized negative inotropic effects of R-PIA with a pA2 of 9.0 +/- 0.1. In the potassium-arrested, perfused rat heart GU285 (1 mumol.l-1) antagonized only the high sensitivity, ADO A2B mediated component of the biphasic relaxation of the coronary vasculature produced by NECA. The low sensitivity component was unchanged. GU285 (1 mumol.kg-1) antagonized the negative chronotropic and hypotensive effects of the adenosine A1 agonist CPA in anaesthetized rats, producing a 10-fold rightward shift in the dose-response relationship. These data demonstrate that in the rat, GU285 is a potent, non-selective adenosine receptor antagonist that maintains its activity in vivo.     

Adenosine receptor agonists: synthesis and biological evaluation of 1-deaza analogues of adenosine derivatives

In a search for more selective A1 adenosine receptor agonists, N6-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N6-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N6-cyclohexyl-1-deazaadenosine (1-deazaCHA, 3c), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-beta-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine. On the other hand, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-beta-D-ribofuranu ronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-beta-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine, in an attempt to find a more selective A2 agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase and in radioligand binding studies. 1-DeazaNECA proved to be a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N6-substituted 1-deazaadenosines largely retain the A1 agonist activity of their parent compounds, but lose some of their A2 agonist activity. This results in A1-selective compounds, of which N6-cyclopentyl-2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A1 adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A1 receptor mediated adenosine actions.     

Failure of CGS15943A to block the hypotensive action of agonists acting at the adenosine A3 receptor.

1. Adenosine receptor agonists were evaluated for their activity at the putative adenosine A3 receptor which mediates a 'xanthine-resistant' hypotensive response in the anaesthetized rat. The compounds tested were: the A1/A3 receptor agonist, N-[2-(4-aminophenyl)ethyl]adenosine (APNEA), the non-selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), the adenosine A1 receptor-selective agonists, N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR79236) and N6-cyclopentyl adenosine (CPA), the A2a receptor-selective agonists, 2-[[2-[4-(2-carboxyethyl) phenyl] ethyl] amino]-N- ethylcarboxamidoadenosine (CGS21680) and 2-phenylaminoadenosine (CV1808), and the moderately A2b selective agonist, N-[(2-methylphenyl)methyl]adenosine (metrifudil). 2. In confirmation of literature findings, APNEA (1-1000 nmol kg-1) induced hypotension and bradycardia; the hypotension was not blocked by pretreatment with the xanthine antagonist, 8-P-sulphophenyltheophylline (8-sPT; 40 mg kg-1, i.v.), whereas the bradycardia was attenuated. The non-xanthine antagonist, 9-fluoro-2-(2-furyl)-5,6-dihydro [1,2,4]triazolo[1,5-c]- quinazin-5-imine (CGS15943A; 3 mg kg-1 i.v.), also attenuated the bradycardia without affecting the hypotension. 3. The adenosine A1 receptor-selective agonists, GR79236 and CPA, both produced dose-dependent falls in blood pressure and heart rate which were antagonized by 8-sPT (40 mg kg-1) and CGS15943A (3 mg kg-1). 4. The adenosine A2a receptor-selective agonists, CGS21680 and CV1808, produced only a hypotensive response which was antagonized by 8-sPT (40 mg kg-1) and to a much greater extent by CGS15943A (3 mg kg-1), consistent with the response being mediated solely by A2a receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of the adenosine receptor mediating inhibition of peristalsis in the rat jejunum.

1. The non-selective adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal diarrhoea in rats. More recently we found that NECA exerts its antidiarrhoeal effect by inhibiting secretion in both the jejunum and ileum and also by inhibiting peristalsis in the ileum. The specific aim of this study was to characterize the receptor in the rat jejunum mediating inhibition of peristalsis via functional studies using a range of metabolically stable adenosine analogues based on the pharmacological criteria of relative agonist and antagonist potencies. 2. Peristalsis in the rat isolated jejunum was achieved by raising the pressure to between 7-11 cmH2O for 3 min followed by a 3 min rest period (pressure at zero). The mean rate of peristalsis during inflation was 7.3 +/- 0.1 peristaltic waves per 3 min and this rate remained consistent for up to 30 min, in 5 separate tissues. The inhibitory effects of the adenosine analogues were quantified by expressing their effects as a % reduction in the mean number of peristaltic contractions derived from the control tissues. 3. The rank order of agonist potency to reduce the rate of peristalsis was: N6-cyclopentyladenosine (CPA) > NECA > R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) > chloroadenosine (2-CADO) > S-PIA > 2-phenylaminoadenosine (CV-1808). This order complies well with the rank order of agonist potency that represents the activation of the A1 receptor subtype (CPA > R-PIA = CHA = > NECA > 2-CADO > S-PIA > CV-1808).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anxiolytic activity of adenosine receptor activation in mice.

1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine.     

The plasma protein extravasation induced by adenosine and its analogues in the rat dorsal skin: evidence for the involvement of capsaicin sensitive primary afferent neurones and mast cells

1. The contribution of sensory neurons and mast cells to the oedema evoked by adenosine A1 (N(6)-cyclopentyladenosine, CPA, 3 - 30 nmol site(-1)), A2 (5'N-ethylcarboxamidoadenosine, NECA, 1 - 10 nmol site(-1)) and A3 receptor agonists (N6-[3-iodobenzyl]-N-methyl-5'-carboxiamidoadenosine, IB-MECA, 0.01 - 3 nmol site(-1)) was investigated in the rat skin microvasculature, by the extravascular accumulation of intravenously-injected (i.v.) 125I-albumin. 2. Intradermal (i.d.) injection of adenosine and analogues induced increased microvascular permeability in a dose-dependent manner (IB-MECA > NECA > CPA > adenosine). The non-selective adenosine receptor antagonist theophylline (5 - 50 nmol site(-1)) markedly inhibited adenosine, CPA or NECA but not IB-MECA-induced plasma extravasation. The A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.3 - 3 micromol kg(-1), i.v.) significantly reduced CPA-induced plasma extravasation whereas responses to adenosine, NECA or IB-MECA were unchanged. The A2 receptor antagonist 3,7-dymethyl-1-proprargylxanthine (DMPX, 0.5 - 50 nmol site(-1)) significantly reduced NECA-induced plasma extravasation without affecting responses to adenosine, CPA and IB-MECA. 3. The tachykinin NK1 receptor antagonist (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane chloride (SR140333), but not the NK2 receptor antagonist (S)-N-methyl-N[4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl]-benzamide (SR48968), significantly inhibited the plasma extravasation evoked by higher doses of adenosine (100 nmol site(-1)), CPA (100 nmol site(-1)), NECA (1 nmol site(-1)) and IB-MECA (0.1 - 1 nmol site(-1)). In rats treated with capsaicin to destroy sensory neurons, the response to higher doses of adenosine, CPA and NECA, but not IB-MECA, was significantly inhibited. 4. The effects of adenosine and analogues were largely inhibited by histamine and 5-hydroxytryptamine (5-HT) antagonists and by compound 48/80 pretreatment. 5. In conclusion, our results provide evidence that adenosine A1 and A2, but not A3, receptor agonists may function as cutaneous neurogenic pro-inflammatory mediators; acting via microvascular permeability-increasing mechanisms that can, depending on dose of agonist and purine receptor under study, involve the tachykinin NK1 receptor and mast cell amines.     

Ischaemia/reperfusion selectively attenuates coronary vasodilatation to an adenosine A2- but not to an A1-agonist in the dog.

1. The effects of myocardial ischaemia/reperfusion were tested on the coronary vasorelaxant responses to agonists selective for the A1 and A2 adenosine receptor subtypes in the dog. The left anterior descending (LAD) coronary artery was occluded distal to the first diagonal branch. The occlusion was maintained for 1 h, followed by 1 h of reperfusion. 2. In the first series of experiments, LAD and circumflex arteries were excised and contracted with prostaglandin F2 alpha (PGF2 alpha). Ischaemia/reperfusion did not significantly alter the vasorelaxation produced by either sodium nitroprusside (endothelium-independent) or acetylcholine (endothelium-dependent). The A1 selective agonist, cyclopentyladenosine (CPA), produced coronary vasorelaxation in both normally perfused vessels and vessels subjected to ischaemia/reperfusion. In contrast, the relaxation produced by the A2-selective agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl) ethyl] adenosine (DPMA) was significantly attenuated by ischaemia/reperfusion (14 fold shift in EC50). 3. In the second series of experiments, coronary blood flow was increased by administration of the A1 and A2 agonists before and after ischaemia/reperfusion of the LAD in anaesthetized dogs. Both compounds dose-dependently increased coronary blood flow. The slopes of the dose-response functions to CPA or DPMA were not significantly altered in the normally perfused circumflex vascular bed. Similarly, the CPA dose-response function in the LAD was unaltered by ischaemia/reperfusion. However, the slope of the coronary vasodilator response to the A2 agonist was significantly reduced following ischaemia/reperfusion of the LAD. 4. We conclude that ischaemia/reperfusion reduces responsiveness to an adenosine A2 receptor subtype agonist, but not an A1 receptor subtype agonist. These data confirm the independent nature of A1- and A2-mediated coronary vasodilatation.     

N6,5'-Disubstituted adenosine derivatives as partial agonists for the human adenosine A3 receptor

5'-(Alkylthio)-substituted analogues of N6-benzyl- and N6-(3-iodobenzyl)adenosine were synthesized in 37-61% overall yields. The affinities of these compounds for the adenosine A1, A2A, and A3 receptors were determined using rat brain cortex, rat brain striata, and stably transfected human A3 receptors in HEK 293 cells, respectively. The compounds proved to be selective for the adenosine A3 receptor and displayed affinities in the nanomolar range. Compounds 8, 10, and 11 had the highest affinities for the A3 receptor with Ki values ranging from 8.8 to 27.7 nM. In the N6-benzyl series, compound 4 (LUF 5403), with a 5'-methylthio group, maintained a reasonable affinity and had the highest selectivity for the A3 receptor. Compound 12 (LUF 5411), with an N6-(3-iodobenzyl) group and a 5'-(n-propylthio) substituent, had the highest A3 selectivity of all of the compounds and also displayed high affinity for this receptor (Ki = 44.3 nM). The compounds were also evaluated for their ability to stimulate [35S]GTPgamma[S] binding in cell membranes expressing the human adenosine A3 receptor. It appeared that the N6,5'-disubstituted adenosine derivatives behaved as partial agonists. Compounds 2, 4, 8, and 10 had the highest intrinsic activities. Additionally, when tested in a cAMP assay, these compounds also behaved as partial agonists.