R.A.A. Mathôt

Deoxyribose analogues of N6-cyclopentyladenosine (CPA): partial agonists at the adenosine A1 receptor in vivo

1 The purpose of the present study was to quantify the cardiovascular effects of the 2′‐, 3′‐ and 5′‐deoxyribose analogues of the selective adenosine A1 receptor agonist, N6‐cyclopentyladenosine (CPA) in vivo. The blood concentration‐effect relationships of the compounds were assessed in individual rats and correlated to their receptor binding characteristics. 2 The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous infusion of 0.80 mg kg−1 (2.5 μmol kg−1) of 5′dCPA, 1.2 mg kg−1 (3.8 μmol kg−1) of 3′dCPA or 20 mg kg−1 (63 μmol kg−1) of 2′ dCPA. The heart rate (HR) and mean arterial blood pressure (MAP) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration. 3 The relationship between blood concentrations and the reductions in both heart rate and blood pressure were described according to the sigmoidal Emax model. For the bradycardiac effect, the potencies based on free drug concentrations (EC50, u) of 5′dCPA, 3′dCPA and 2′dCPA in blood were 5.9 ± 1.7, 18 ± 4 and 260 ± 70 ng ml−1 (19 ± 6, 56 ± 11 and 830 ± 210 nM), respectively, and correlated well with the adenosine A1 receptor affinity in vitro. The Emax value of 2′dCPA was significantly less than those of the other compounds, suggesting that this compound may be regarded as a partial agonist when compared to the other analogues. The rank order of the maximal reduction in heart rate of the compounds corresponded well with the order of the GTP‐shifts, as determined in vitro. 4 It is concluded that deoxyribose derivatives of CPA may be partial agonists for the adenosine A1 receptor and may serve as tools for further investigation of adenosine receptor partial agonism in vivo.

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.

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 μg kg−1 of CPA i.v. in 5min during a steady‐state infusion of CPT at a rate of 0, 57, 114 or 228 μg 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 ± 0.7ng 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 It is concluded that the competitive agonist‐antagonist interaction model may be of value to characterize quantitatively the pharmacodynamic interactions between adenosine A1 receptor ligands in vivo.

Pharmacokinetic modelling of the haemodynamic effects of the A2a adenosine receptor agonist CGS 21680C in conscious normotensive rats

1 The aim of the present investigation was to determine the relationship between the blood concentration and haemodynamic effects of the adenosine A2a receptor agonist, CGS 21680C (the sodium salt of 2‐p‐(2‐carboxyethyl)phenylethylamino‐5′‐N‐ethylcarboxamidoadenosine) in conscious normotensive rats. 2 Chronically cannulated rats were randomly assigned to three groups which received 300, 1000 or 3000 μg kg−1 (0.56, 1.9 or 5.6 μmol kg−1) of CGS 21680C intravenously over 15min. The mean arterial blood pressure (MAP) and heart rate (HR) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur. 3 For each individual rat the reduction in mean arterial pressure was related to the blood concentration according to the sigmoidal Emax model. The concentration‐effect relationships were consistent for the different treatment groups. The potency based on free drug concentrations (EC50,u) was 5.8 ng ml−1 (11 nm) (mean ± s.e.; n = 19) and correlated well with the reported adenosine A2a receptor affinity (Ki 19 nm). In comparison with the reduction in blood pressure, CGS 21680C exhibited a greater potency for the reduction of the ratio MAP/HR. 4 It is concluded that estimates can be obtained for the potency and intrinsic activity of adenosine A2a receptor agonists in vivo by pharmacokinetic‐pharmacodynamic analysis of mean arterial pressure data in a rat model. In future studies, total peripheral resistance may also be useful as a pharmacodynamic parameter for A2a activation, provided that possible changes of the stroke volume are also assessed.

Pharmacokinetic‐haemodynamic relationships of 2‐chloroadenosine at adenosine A1 and A2a receptors in vivo

1 The purpose of the present study was to develop an experimental strategy for the quantification of the cardiovascular effects of non‐selective adenosine receptor ligands at the adenosine and A1 and A2a receptor in vivo. 2‐Chloroadenosine (CADO) was used as a model compound. 2 Three groups of normotensive conscious rats received an short intravenous infusion of 1.4 mg kg−1 CADO during constant infusions of the A1‐selective antagonist, 8‐cyclopentyltheophylline (CPT; 20 μm min−1 kg−1), the A2a‐selective antagonist, 8‐(3‐chlorostyryl)caffeine (CSC; 32 μm min−1 kg−1) or the vehicle. The heart rate (HR) and mean arterial blood pressure (MAP) were recorded continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentrations. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur. 3 During the infusion of CPT, CADO produced a reduction in both blood pressure and MAP/HR by activation of the A2a receptor. The concentration‐effect relationships were described according to the sigmoidal Emax model, yielding potencies based on free drug concentrations (EC50,u) of 61 and 68 ng ml−1 (202 and 225 nM) for the reduction of blood pressure and MAP/HR, respectively. During the infusion of CSC, an EC50,u value of 41 ng ml−1 (136 nM) was observed for the A1 receptor‐mediated reduction in heart rate. The in vivo potencies correlated with reported receptor affinities (Ki(A1)=300 nM and Ki(A2a)=80 nM). The maximal reductions in MAP/HR and heart rate were comparable to those of full agonists, with the values of −12 ± 1 × 1O−2 mmHg b.p.m.−1 and −205 b.p.m. respectively. 4 It is concluded that this integrated pharmacokinetic‐pharmacodynamic approach can be used to obtain quantitative information on the potency and intrinsic activity of new non‐selective adenosine receptor agonists at different receptor subtypes in vivo.

Time course of action of three adenosine A1 receptor agonists with differing lipophilicity in rats: comparison of pharmacokinetic, haemodynamic and EEG effects.

In this study we investigated the relationship between the pharmacokinetics and the cardiovascular and electroencephalogram (EEG) effects of three adenosine agonists with differing lipophilicity. Conscious normotensive rats received either 600 μg/kg N6-(p-sulphophenyl) adenosine (SPA), 200 μg/kg N6-cyclopentyladenosine (CPA) or 600 μg/kg 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCA) in a 5-min intravenous infusion. Changes in haemodynamics and EEG were monitored in conjunction with arterial blood sampling to determine blood concentrations of the compounds. The three adenosine agonists showed large differences in pharmacokinetic properties, resulting in terminal half-lives of 66 ± 10, 8.2 ± 0.4 and 24 ± 1 min (mean ± SEM) for SPA, CPA, and DCCA respectively. SPA had a significantly lower blood clearance relative to CPA and DCCA, whereas DCCA had the largest volume of distribution and degree of plasma protein binding. The relationship between concentration and heart rate could be described adequately by the sigmoidal Emax model. For SPA, CPA, and DCCA the EC50 values based on free drug concentrations were 423 ± 92, 1.8 ± 0.4 and 9.5 ± 1.1 nM respectively. These in vivo values correlated closely with the affinity of the compounds for the adenosine A1 receptor as determined in radioligand binding studies, with corresponding Ki values of 1410 ± 220, 4.7 ± 0.6 and 102 ± 74 nM (mean ± SEM) respectively. In the EEG, only CPA produced a small decrease in the amplitude of beta waves. This study demonstrates that the three adenosine analogues have large differences in pharmacokinetics, which complicates comparison of their cardiovascular and central responses simply on the basis of dose. The application of an integrated PK/PD approach permits estimates of potency and activity which are independent of underlying dose and pharmacokinetics.

Partial agonists for adenosine receptors

Publisher Summary This chapter discusses the partial agonists for adenosine receptors. Adenosine is generally considered as a “local hormone” with profound physiological activity. It is thought to mediate a large variety of effects, as diverse as vasodilation in the cardiovascular system, inhibition of lipolysis in fat cells, and depression of neuronal activity in the central nervous system (CNS). Most of its effects are mediated by membrane-bound receptors, called P i -purinoceptors, of which currently three subclasses are defined: A l , A 2 and A 3 . All three classes have been cloned, and are coupled to the enzyme adenylate cyclase, A 1 and A 3 adenosine receptors in an inhibitory, and A 2 receptors, of which two further subtypes A 2a and A 2b exist, in a stimulatory fashion. The chapter discusses some methods for screening partial agonists as well as application of these screening methods to adenosine receptors. There is also a description of how partial agonists for adenosine receptors were obtained for the purpose of this chapter. In view of the overwhelming number of therapeutic strategies already in clinical practice, the hypotensive effects of adenosine receptor agonists are probably of limited use. On the contrary, the potentially beneficial metabolic, antiarrhythmic, and CNS effects elicited by these compounds are confounded by the cardiovascular actions.

Assessment of the enantiomeric purity of R- and S-N6-phenylisopropyladenosine (PIA): Implications for adenosine receptor subclassification

A chiral column high-performance liquid chromatographic method was developed for the assessment of the enantiomeric purity of the stereoisomers of N6-phenylisopropyladenosine (PIA). The observed chiral purity of R-PIA was greater than 99.90%, whereas S-PIA was found to contain 4.4% of the R-enantiomer. In radioligand binding studies, the observed affinity of S-PIA for the adenosine A1 receptor (IC50 240 nM) could entirely be attributed to its content of R-PIA (IC50 7.8 nM). Calculation of a theoretical IC50 of pure S-PIA for the A2 receptor yielded a value of 6700 nM, which was 35-fold higher than for R-PIA (190 nM). Concludingly, the utilization of enantiomeric impure S-PIA in the definition of adenosine receptor subclasses is questionable.

Liquid chromatographic determination of the adenosine receptor agonist CGS 21680 in blood using on-line solid-phase extraction on a phenylboronic acid support and fluorescence detection.

An analytical method is described for the selective determination of A1 or A2 adenosine receptor agonists in blood. By implementing solid-phase extraction using immobilized-phenylboronic acid (PBA) in sample pretreatment, all adenosine derivatives are retained via their intact cis-diol group. On-line desorption of the analytes from the PBA support to the C18 analytical column is performed by injection of a small plug of perchloric acid. Fluorescence and UV detection are employed for the different adenosine derivatives. The method is applied to the determination of 2-[p-(2-carboxyethyl)phenylethylamino]-5-N- ethylcarboxyamidoadenosine (CGS 21680, I) in blood using fluorescence detection. The only off-line sample handling step is the extraction of blood with ethyl acetate and subsequent evaporation of the extraction solvent. The detection limit of the method was 0.25 ng (signal-to-noise ratio 3:1) and the determination limit for I in blood (pretreatment of 100 microliters) was 5 ng/ml. The method was validated and used to study the pharmacokinetics of I in rats.