Adenosine Ai Receptor Agonists

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The endogenous nucleoside adenosine exerts a variety of physiological effects via interactions with a family of G protein-coupled receptors consisting of four subtypes: the adenosine A^ A2A, A2B, and A3 receptors.49 The A1 receptor is the most comprehensively studied adenosine receptor subtype and has been cloned from a number of species, including humans. Ai receptors are believed to act mainly via the Gi/o family of G proteins and are widely expressed in the brain, spinal cord and a variety of peripheral tissues such as adipose tissue, heart, eye, adrenal gland, liver, kidney, salivary glands, and GI tract.49 Despite this widespread distribution, A1 knockout mice develop normally and display relatively subtle differences compared to wild-type animals, suggesting that adenosine A1 receptors are particularly important under pathophysiological conditions.50

There has been considerable interest in the adenosine A1 receptor as a potential target for therapeutic intervention for a variety of indications, such as cardiac arrhythmias, metabolic diseases, neurodegenerative diseases, neuropathic pain, and renal disorders.51'52 Stimulation of the A1 receptors in the heart produces negative dromo-, chrono-, and inotropic effects and adenosine itself is used clinically to terminate paroxysmal supraventricular arrhythmias and for myocardial perfusion imaging.51,52 In addition, an N6-substituted derivative of adenosine, tecadenoson (CVT-510) is a selective A1 receptor agonist in phase III clinical trials for the treatment of supraventricular tachycardia.138 On the other hand, the pronounced cardiodepressant effects induced by stimulating the A1 receptor have been a major impediment to the research into the development of selective A1 receptor ligands for other potential indications. One potential strategy to overcome this issue is based on the concept that low-efficacy (partial) agonists may display greater tissue selectivity compared to high-efficacy (full) agonists,53 and during the last decade significant efforts have been put into medicinal chemistry programs targeting the development of selective, partial agonists for the adenosine A1 receptor.52,54,55

An example of how such tools have been used to develop novel, mechanism-based PK/PD models is a project exploring the potential of a series of deoxribose and 8-alkylamino analogs of N6-cyclopentyladenosine (CPA), which were identified as adenosine A1 receptor agonists with reduced intrinsic efficacy54,55 as antilipolytic agents for the treatment of non insulin-dependent diabetes mellitus. Increased levels of non-esterified fatty acids (NEFAs) are a characteristic of non insulin-dependent diabetes and are believed to exacerbate insulin resistance and hyperglycemia. Therefore, selective A1 receptor agonists may provide a novel therapeutic strategy for the treatment of diabetes, since stimulation of adenosine A1 receptors in adipose tissue has been shown to decrease NEFA levels in rat and human.51 A key first step in this project was the development of an efficient and quality animal model and sensitive analytical assays for simultaneous and detailed characterization of the pharmacokinetics and time course of effect of efficacy and safety biomarkers. For this purpose, conscious, freely moving rats were used with several arterial and venous cannulae which were implanted under anesthesia several days prior to the experiment, which in a single animal allowed for continuous hemodynamic recordings and repeated arterial blood sampling for measurement of drug and NEFA levels following controlled intravenous drug infusion.56,57

The CPA analogs that were studied in this in vivo model had previously been demonstrated to display lower 'GTP shifts' in an in vitro binding assay of the adenosine A1 receptor than CPA itself. The guanosine triphosphate (GTP) shift is defined as the ratio between the apparent affinity in the presence and absence of GTP and is considered to be an in vitro measure of efficacy of A1 receptor ligands.54 By applying an integrated PK/PD modeling approach, it was found that the CPA analogs with a lower in vitro GTP shift displayed a reduced cardiovascular response and did indeed behave as partial agonists in vivo as evidenced by a significantly lower estimate of intrinsic activity (a) for the effect on heart rate.56,57 Subsequently, the cardiovascular data for 10 analogs were analyzed simultaneously using the operational model of agonism (eqn [5]) and the 'comparative method' (see Section, assuming a direct link between drug exposure and pharmacodynamic effect. The key finding of this analysis, which provided the first 'proof of principle' for this mechanism-based PK/PD approach, was that the estimates of in vivo affinity (KA) and efficacy (t) were highly


Figure 3 Relationship between in vitro GTP shift at the adenosine A-, receptor and intrinsic activity (a) for the in vivo effect on NEFAs and heart rate in rats.

GTP shift

Figure 3 Relationship between in vitro GTP shift at the adenosine A-, receptor and intrinsic activity (a) for the in vivo effect on NEFAs and heart rate in rats.

consistent with the results obtained from in vitro studies, i.e., in vivo estimates of KA correlated very well with the in vitro estimates of Ki and the in vivo estimates of t with the in vitro estimates of GTP shift.22 A main utility of this approach compared to more empirical modeling strategies is that it provides a more robust basis for prediction of drug effects between different systems, since it explicitly separates drug and system properties in the model. This is exemplified in Figure 3, which shows the nonlinear relationship between the in vitro GTP shift and maximum effect on heart rate in vivo predicted by the mechanistic model.22 Information like this not only provides novel mechanistic insights but can also be used to design effective and predictive in vitro and in silico screening strategies in drug discovery programs.

In order to validate the hypothesis that reducing intrinsic efficacy within the CPA series could enhance the cardiovascular safety window, the next step in this program was to compare the effects on heart rate with those on NEFA levels. It was indeed found that several analogs displayed profoundly reduced cardiovascular effects but still produced near-maximal inhibition of NEFAs in the rat model.58 Once again, the operational model of agonism (eqn [5]) was employed to simultaneously analyze the NEFA effects of all ligands, this time incorporated into an indirect effect model (eqn [10]) to account for hysteresis between drug concentration and pharmacodynamic response. This approach yielded estimates of in vivo efficacy and affinity for the adenosine A1 receptor-mediated effect on NEFAs that could be compared directly with those obtained for the effect on heart rate. The main outcome of this analysis was that on average all compounds displayed an ~38-fold higher efficacy (t in eqn [5]) for the NEFA effect compared to the cardiovascular effect, consistent with findings that the density of adenosine A1 receptors is ~ 25-fold higher in adipocytes compared to cardiac tissue.23 Finally, this integrated PK/PD model for efficacy and safety was linked back to the in vitro data as described above and Figure 3 shows the final model which can predict the therapeutic window in vivo for any compound from in vitro GTP shift data. An important conclusion from the model in Figure 3 is that it indicates that even compounds with GTP shift values close to unity (i.e., ligands that appear to behave as antagonists in vitro) may still produce significant inhibition of lipolysis in vivo, whereas they are expected to be without cardiodepressant side effects. This illustrates our view that mechanism-based PK/PD modeling can yield insights that can provide new directions for drug discovery program in the search for compounds with improved efficacy and/or safety profiles.

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