The effect of two selective A1‐receptor agonists and the bitopic ligand VCP746 on heart rate and regional vascular conductance in conscious rats

Background and Purpose Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A1, A2A, A2B, and A3). We have investigated the effect of two A1‐receptor‐selective agonists and the novel A1‐receptor bitopic ligand VCP746 on the rat cardiovascular system. Experimental Approach The regional haemodynamic responses of these agonist was investigated in conscious rats. Male Sprague–Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes on the renal, mesenteric arteries and the descending abdominal aorta and the jugular vein and caudal artery catheterized. Cardiovascular responses were measured following intravenous infusion (3 min each dose) of CCPA (120, 400, and 1,200 ng·kg−1·min−1), capadenoson or adenosine (30, 100, and 300 μg·kg−1·min−1), or VCP746 (6, 20, and 60 μg·kg−1·min−1) following pre‐dosing with DPCPX (0.1 mg·kg−1, i.v.) or vehicle. Key Results CCPA produced a significant A1‐receptor‐mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A1‐receptor‐mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A1‐receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A2A‐ and A2B‐receptors. Conclusions and Implications These results suggest VCP746 mediates its cardiovascular effects via activation of A2 rather than A1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A1 allosteric ligand moieties.

Background and Purpose: Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A 1 , A 2A , A 2B , and A 3 ). We have investigated the effect of two A 1 -receptor-selective agonists and the novel A 1receptor bitopic ligand VCP746 on the rat cardiovascular system.
Key Results: CCPA produced a significant A 1 -receptor-mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A 1 -receptor-mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A 1 -receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A 2A -and A 2B -receptors.
Conclusions and Implications: These results suggest VCP746 mediates its cardiovascular effects via activation of A 2 rather than A 1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A 1 allosteric ligand moieties.
In addition, it has been shown that selective A 2A adenosine receptor agonists are potent vasodilators in the rat that reduce BP and induce marked increases in heart rate and plasma renin activity (Alberti et al., 1997). The A 2A effect on heart rate appears to be secondary to an increase in reflex sympathetic nervous activity (Alberti et al., 1997).
Previous work in conscious rats has also shown that an A 1 -selective agonist can cause significant decreases in heart rate, a fall in mean arterial pressure and vasoconstriction in both the renal and mesenteric vascular beds, but vasodilatation in the hindquarters (Jolly, March, Kemp, Bennett, & Gardiner, 2008).
A number of selective agonists and antagonists are now available for the A 1 -receptor (Borea et al., 2018;Fredholm et al., 2011;. Some A 1 -receptor-selective agonists and partial agonists have undergone evaluation for cardiovascular disease indications such as paroxysmal supraventricular tachycardia, atrial fibrillation, angina pectoris and heat failure (Meibom et al., 2017;. However, the ubiquitous distribution of adenosine receptors in the body can often limit therapeutic application because of the effects of adenosine receptor ligands on the same receptor in a different tissue or cell type . One way in which the activity of endogenous adenosine can be subtly regulated at the level of its target receptor is via drugs that bind to an allosteric site on the receptor protein of interest. These allosteric modulators act to enhance or inhibit the binding of adenosine to its receptor binding site (the orthosteric site) and/or change the resulting functional response (Keov, Sexton, & Christopoulos, 2010;Hill et al., 2014;Göblyös & IJzerman, 2011;Kimatrai-Salvador, Baraldi, & Romagnoli, 2012;Cooper et al., 2019). VCP171, (2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone, has been described as a novel positive allosteric modulator for the adenosine A 1 -receptor (Aurelio et al., 2009Imlach, Bhola, May, Christopoulos, & Christie, 2015;Valant et al., 2010;Vincenzi et al., 2014). Recently, a hybrid molecule (or bitopic ligand) has been described that comprises adenosine attached via a linker to this positive allosteric modulator (Valant et al., 2014). The resulting hybrid ligand (VCP746) has been suggested to exhibit signalling bias towards Gi-mediated signalling and furthermore was also able to protect against ischaemic damage in cardiomyocytes expressing native A 1receptors (Valant et al., 2014). It did not, however, alter heart rate in an isolated rat atrial preparation (Valant et al., 2014). VCP746 has also been shown to reduce cardiac myocyte hypertrophy and remodelling .

What is already known
• Adenosine is a local mediator that regulates physiological processes via stimulation of the adenosine A 1 -receptor.
• VCP746 is adenosine linked to an A 1 -allosteric enhancer (VCP171), reported to be a biased A 1 -agonist.

What this study adds
• VCP746 causes increases in heart rate and vascular conductance that are not mediated by A 1 -receptors.
• Reporter gene studies confirm that VCP746 is a potent agonist of both A 2A-and A 2B -receptors.
What is the clinical significance reporter gene and the human adenosine A 2A -receptor or human adenosine A 2B -receptor were generated and grown to confluence in clear 96-well plates as described previously (Stoddart, Vernall, Briddon, Kellam, & Hill, 2015). On the day prior to analysis, normal growth medium was removed and replaced with serum-free medium (SFM; DMEM/F12 supplemented with 2-mM L-glutamine). On the day of the experiment, fresh SFM was added to the cells with increasing concentrations of the required test compounds. CRE-SPAP cells were then incubated for 5 hr at 37 C in humidified air containing 5% CO 2 .
After 5-hr incubation, all medium was removed from the cells and replaced with 40 μl of SFM and incubated for a further 1 hr.

| Catheter implantation
Catheter implantation commenced at least 10 days after the Doppler flow probe implantation surgery and after a satisfactory inspection from the Named Veterinary Surgeon. Under anaesthesia (as described above) catheters filled with heparinised saline (15 UÁml −1 ) were implanted into the distal abdominal aorta, via the ventral caudal artery (for the measurement of arterial BP and heart rate) and implanted into the right jugular vein (for drug administration; Carter et al., 2017). Three separate intravenous catheters were placed in the jugular vein to enable independent administration of different substances. At this stage, the probe wires were soldered into a miniature plug (Omnetic Connector Corporation, USA), which was mounted onto a custom-designed harness worn by the rat. The catheters emerged from the same point as the probe wires and were fed through a protective spring secured to the harness and attached to a counterbalanced pivot system. Reversal of anaesthetic and analgesia was administered (as described above) and the animals were single housed with free access to food and water. The arterial catheter was connected to a fluid-filled swivel for overnight infusion of heparinised (15 UÁml −1 ) saline to maintain potency.
Experiments began 24 hr after surgery for catheter implantation, with animals fully conscious and unrestrained in home cages and with free access to food and water.

| Cardiovascular recordings
Haemodynamic variables (heart rate, arterial BP, renal, mesenteric, and hindquarters Doppler shifts) were recorded using a customized, μgÁkg −1 Ámin −1 ) on day 1 and day 3. Each dose of adenosine was given as a 3-min infusion. Cardiovascular recordings were continued for a further 4 hr after administration of the adenosine.

| Study 5
Seven animals were used to assess the cardiovascular responses to VCP171 (Aurelio et al., 2009) in the presence or absence of DPCPX.
Following a period of baseline, rats were randomized into two groups.
where E max is the maximal response, [A] is the concentration of agonist, and the EC 50 is the molar concentration of agonist required to generate 50% of the E max . Statistical analysis of concentration-

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMA-COLOGY (Harding et al., 2018) and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos, et al., 2017;Alexander, Fabbro, et al., 2017).

| RESULTS
Baseline cardiovascular variables before the administration of  The effect of DPCPX alone was evaluated for 5 min (relative to a vehicle control) prior to agonist administration in all five of the studies involving DPCPX described under Section 2. Figure 3 shows the combined data for DPCPX and vehicle from all of the studies.
Administration of DPCPX produced a small increase in HR and MAP consistent with antagonism of a small contribution to basal activity from an A 1 -receptor-mediated response to endogenous adenosine ( Figure 3). There was also a small direct effect of DPCPX on MVC ( Figure 3). A similar effect on MVC has been previously reported by

| Effect of the A 1 -receptor-selective partial agonist capadenoson
Capadenoson is a selective A 1 -receptor partial agonist (Albrecht-

| Effect of the bitopic A 1 -receptor ligand VCP746 on cardiovascular responses in the rat
VCP746 is a hybrid molecule containing the adenosine moiety linked to the positive A 1 -receptor allosteric modulator VCP171 (Figure 1). It has been previously reported to act as a biased A 1 -receptor agonist that has no direct negative chronotropic effect in isolated rat atria (Valant et al., 2014). Application of VCP746 (6, 20 and 60 μgÁkg −1 min −1 , i.v.; 3-min infusion), however, produced a significant (P < .05) increase in HR in conscious freely moving rats that occurred in parallel with marked increases in renal and mesenteric vascular conductance (Figure 5a,b). These effects of VCP746 were not significantly antagonized by the A 1 -selective antagonist DPCPX (Figure 5a,b).

| Effect of adenosine, VCP171 and both in combination
To investigate the extent to which the cardiovascular responses to VCP746 observed above are due to the component molecules (adenosine and VCP171) contained within VCP746, we have studied their effect on the cardiovascular system alone and in combination.
The endogenous ligand adenosine (30, 100 and 300 μgÁkg −1 Ámin −1 ; 3-min intravenous infusion) produced a similar cardiovascular profile, in terms of a significant (P < .05) increase in HR, and associated increases in renal and mesenteric VC, to that observed with VCP746 ( Figure 6a,b). In addition, adenosine also produced a significant fall in MAP (P < .05) and a large increase in HVC (P < .05; Figure 6a The effect of VCP171 alone was investigated at equivalent equimolar doses to those at which the parent hybrid molecule VCP746 elicited significant effects on heart rate and renal/mesenteric VC. The doses chosen were based on the fraction of the MW of VCP746 that was contributed by the VCP171 component (Figure 1). Infusion of VCP171 (2.6, 8.5 and 25.6 μgÁkg −1 Ámin −1 ; 3-min intravenous infusions) produced a small decrease in VC in the mesenteric vascular bed (P < .05) but no consistent effect on heart rate or renal and hindquarters VC (Figure 7a,b). The reduction in mesenteric VC was not antagonized by DPCPX (Figure 7a,b). In combination, adenosine and VCP171 produced essentially similar cardiovascular responses to those obtained with adenosine alone (Figure 8a,b). In the presence of VCP171, there was a hint that the increase in heart rate produced by adenosine was somewhat blunted, perhaps consistent with an enhanced contribution of a minor A 1receptor-mediated bradycardia (Figure 8a,b).

| Agonist effect of VCP746 on adenosine A 2Aand A 2B -receptor-mediated CRE-SPAP responses
The lack of effect of the A 1 -antagonist DPCPX on the cardiovascular responses to VCP746, and their similarity with those obtained with adenosine, suggests that VCP746 is able to stimulate other adenosine receptors in the cardiovascular system. It has previously been shown that VCP746 lacks agonist efficacy at adenosine A 3 -receptors (Valant et al., 2014). Vecchio et al. (2016) have recently shown that VCP746 is a high affinity and potent agonist of A 2B -receptors. However, the potent vasodilator and tachycardia effects of VCP746 observed in the present study were more consistent with an effect on A 2A -receptors (Alberti et al., 1997;Borea et al., 2018;Headrick, Ashton, Meyer, & Peart, 2013). As a consequence, we have evaluated the agonist effect of VCP746 on CRE-mediated reporter gene responses in CHO-K1 cells stably expressing either the human A 2A -receptor or the human A 2B -receptor ( Figure 9). Consistent with previous reports (Vecchio et al., 2016), VCP746 was a potent and efficacious agonist of A 2Breceptor-mediated CRE-gene expression (pEC 50 = 8.7 ± 0.1, E max = 103.0 ± 4.6% of response to 1-μM NECA, n = 5; Figure 9; Table 2). VCP746 was, however, also a potent and high efficacy agonist of A 2A -receptor-mediated CREP-SPAP responses (pEC 50 = 7.4 ± 0.1, E max = 104.0 ± 15.0% of response to 1-μM NECA, n = 5; Figure 9; Table 2).

| DISCUSSION
In the present study, we have investigated the effect of a novel bitopic ligand VCP746, which has been reported to be a biased A 1agonist (Valant et al., 2014), on cardiovascular responses in conscious, freely moving rats. The aim was to establish whether a hybrid molecule containing both adenosine and the positive allosteric enhancer, VCP171, could provide a mechanism to activate a subset of A 1 -receptor-mediated responses in particular vascular beds. To determine whether differences in signalling efficiency or preferential activation of particular signalling pathways (i.e., via biasedagonism) can be exploited by a bitopic ligand, such as VCP746, we have investigated the effect of this ligand in conscious rats subject to normal autonomic reflex control. Previous work in isolated rat atria showed that VCP746 did not produce the normal A 1 -receptormediated negative chronotropic effect, but it was able to reduce ischaemic damage, hypertrophy and remodelling in cardiomyocytes Valant et al., 2014). Application of VCP746, however, produced a significant increase in HR in conscious, freely moving rats that occurred in parallel with marked increases in renal and mesenteric vascular conductance. These effects were the complete opposite of those expected of an A 1 -receptor agonist and were not inhibited by the selective A 1 -antagonist DPCPX.
The responses to VCP746 were in many respects similar to those elicited by adenosine, which produced tachycardia, increased vascular conductance in all three vascular beds and a substantial fall in mean arterial BP. All of these effects of adenosine were unaffected by DPCPX treatment and were very similar to those reported by Jolly et al. (2008) for adenosine using a similar conscious rat model. VCP171, at doses that were the equimolar equivalent of those found in pharmacologically active doses of VCP746, did not produce any major changes in the responses to adenosine. VCP171 certainly did not markedly enhance the contribution of A 1 -receptors in the final response, with the possible exception of a small effect on heart rate.
VCP171 does not produce a large enhancement of the affinity of adenosine at the rat A 1 -receptor (Cooper et al., 2019) and it is likely that the positive A 1 -receptor allosteric action of the VCP171 entity within the larger VCP746 molecule is not sufficient to overcome the apparently more potent effect of the adenosine moiety on adenosine A 2A -or A 2B -receptors in the cardiovascular system. This finding points to the need swap adenosine for a more A 1 -selective agonist within any future bitopic ligands and to match carefully the concentration range over which the orthosteric and allosteric components operate.
It is also worth pointing out that the probe dependence of allosteric modulators also needs to be considered (Cooper et al., 2019). For example, VCP171 does not produce a significant positive allosteric effect on the binding of capadenoson to the rat A 1 -receptor (Cooper et al., 2019).
Cardiovascular responses to adenosine in the heart (via both A 1and A 2A -receptors) are complex with an A 1 -mediated reduction in heart rate balanced by a A 2A -mediated increase in heart rate (Evoniuk, Jacobson, Shamim, Daly, & Wurtman, 1987;Jolly et al., 2008). It is worth noting that A 2A -receptors are also found in the cardiovascular regulatory regions of the brain (Thomas, St Lambert, Dashwood, & Spyer, 2000). Thus, an action at adenosine receptors in both the brain and the periphery is likely to contribute to the overall effect of systemically administered adenosine (Schindler et al., 2005). Furthermore, A 2A -receptor effects on the heart have been proposed to be partly not only due to a baroreceptor reflex response to the A 2Areceptor-mediated vasodilatation (Alberti et al., 1997;Barraco, Janusz, Polasek, Parizon, & Roberts, 1988;Ohnishi, Biaggioni, Deray, Branch, & Jackson, 1986;Thomas et al., 2000) but also due to a direct activation of A 2A -receptors in the heart (Lappe, Sheldon, & Cox, 1992) and the CNS (Schindler et al., 2005). Lappe et al. (1992) have also shown that systemic administration of an A 2A agonist produces larger increases in heart rate in conscious animals than anaesthetized animals. It is therefore likely that in conscious animals, the A 1 -mediated effects on heart rate are masked by a greater contribution from A 2Areceptors via central A 2A -receptors and via baroreceptive mechanisms. This balance of A 1 -and A 2A -responses (and the role of reflex mechanisms) in conscious animals may also explain the lack of effect of VCP746 on heart rate in isolated rat atria (Valant et al., 2014).
It has been shown previously that VCP746 is a potent and high efficacy agonist at adenosine A 2B -receptors (Vecchio et al., 2016).
However, the potent vasodilator and tachycardia effects of both VCP746 and adenosine observed in the present study seem more consistent with an effect on A 2A -receptors (Alberti et al., 1997;Borea et al., 2018;Headrick et al., 2013). As mentioned above, A 2A -receptor effects on heart rate have been proposed to be due to both baroreceptor reflex responses to A 2A -receptor-mediated vasodilatation (Barraco et al., 1988;Ohnishi et al., 1986;Thomas et al., 2000) and direct activation of A 2A -receptors in the heart (Lappe et al., 1992) and brain (Schindler et al., 2005). However, in the case of activation by VCP746, there was no change in mean arterial BP, suggesting that its effects are due to direct activation of A 2A -receptors in the heart and CNS or alternatively a consequence of A 2B -receptor activation.
To establish whether VCP746 has agonist activity at A 2A -receptors, in addition to its reported A 2B -effect, we investigated its ability to stimulate cAMP response element reporter gene responses in CHO-K1 cells expressing either the human A 2A -receptor or the human A 2B -receptor. In keeping with previous published work (Vecchio et al., 2016), VCP746 was nearly two orders of magnitude more potent than NECA at activating A 2B -receptor. However, as predicted from the in vivo cardiovascular data obtained in the present manuscript, VCP746 was also a potent and high efficacy agonist of A 2A -mediated CRE-SPAP responses. An agonist effect of VCP746 on cAMP formation at A 2A -receptors has also been reported recently in CHO cells (Aurelio et al., 2018).
In summary, the present study has shown that the bitopic ligand, VCP746, does not stimulate cardiovascular responses that are mediated by the adenosine A 1 -receptor. In contrast, it produces a marked increase in heart rate and vascular conductance in both the renal and mesenteric circulation that were opposite to those expected of an A 1agonist. Studies of cAMP-mediated gene transcription responses in CHO-K1 cells have confirmed that VCP746 is a potent and efficacious agonist of the adenosine A 2A -receptor, in addition to its known actions at the A 1 -and A 2B -receptors. Taken together, these data suggest that the cardiovascular responses to VCP746 are mediated by stimulation of cardiovascular A 2A -or A 2B -receptors rather than A 1receptors. This has implications for the design of future bitopic ligands that incorporate A 1 allosteric ligand moieties to overcome on-target cardiovascular side effects of drugs designed as A 1 -agonists for the treatment of ischaemic heart disease.