Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure

Figures

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  FIGURE 1. Multi-pathway profiling at the A1R. Adenosine receptor agonists were analyzed in CHO-hA1R cells in assays of calcium mobilization (A; n = 4), inhibition of cAMP accumulation (B; n = 6), cell survival (D; n = 3) or phosphorylation of ERK1/2 (E; n = 5) and Akt1/2/3 (F; n = 3). These data were used to calculate bias, where Log(τ/KA) are normalized to NECA (ΔLog(τ/KA)) and cAMP accumulation (ΔΔLog(τ/KA); C). Statistical significance of bias is reported in Supplementary Figure S1. Concentration response data are expressed as mean ± SEM.

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  FIGURE 2. Multi-pathway profiling at the A2AR, A2BR and A3R. Adenosine receptor agonism was analyzed in CHO-hA2AR (left panels), CHO-hA2BR (center panels) and CHO-hA3R cells (right panels). Cells were analyzed in cAMP (top row), calcium (second row), and phosphorylation of ERK1/2 (third row) and Akt1/2/3 (fourth row), with calculated bias shown in the lower row. As bias data are normalized to NECA and the cAMP pathway, where there is no response to NECA at a pathway, ligand-induced cAMP in a cell line, bias cannot be calculated. Statistical analyses of bias are shown in Supplementary Figure S1. Concentration response data are expressed as mean ± SEM.

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  FIGURE 3. Tissue-specific expression of adenosine receptors may influence side-effect profile of compounds used in vivo. VCP746 and neladenoson were studied further in the indicated ex vivo and in vivo assays (boxed) to determine expected side-effect profiles. Known sites of primary functional adenosine receptor expression are indicated by blue (A1R), orange (A2AR) and pink (A2BR) receptors.

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  FIGURE 4. VCP746 and neladenoson reduce cardiomyocyte hypertrophy. Rat primary neonatal ventricular cardiomyocytes were exposed to hypertrophic stimuli TNFα (10 ng/ml; A; n = 9), IL1β (10 ng/ml; B; n = 10) or AngII (100 nM; C; n = 9) for 72 h after 2 h pretreatment with the indicated adenosine receptor agonists. Hypertrophy was assessed by incorporation of 3H-leucine as an indicator of protein synthesis. Statistical significance was assessed by repeated measures one-way ANOVA with Dunnett’s multiple comparisons, compared with hypertrophic treatment (black bar) alone (*p < 0.05, **p < 0.01, ***p < 0.001).

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  FIGURE 5. Adenosinergic agonists show some anti-fibrotic activity in rat primary neonatal cardiac fibroblasts. Fibrosis was measured by the incorporation of 3H-proline as an indicator of collagen formation. After 72 h 10 nM AngII (A) or 10 ng/ml TGFβ (B) treatment, VCP746 and neladenoson pre-treatment (2 h) inhibited the AngII-fibrotic effect only, at 300 nM (repeated measures one-way ANOVA, Dunnett’s multiple comparisons, in comparison to fibrotic treatment alone; *(p < 0.05)).

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  FIGURE 6. Ex vivo and in vivo A1R-mediated chronotropic effects. In isolated neonatal ventricular cardiomyocytes, VCP746 (n = 7) had no effect on the spontaneous contraction rate of cells (A), while neladenoson (30 nM: n = 7; 300 nM: n = 8) showed a significant depressive effect, which is completely abrogated by pre-treatment with the A1R antagonist SLV320 (statistics were performed on raw data; paired Student’s t-test, comparison of VCP746/neladenoson-treated cells to the same cells prior to treatment; *(p < 0.05)). Data are mean ± SEM. In isolated atria (B), NECA produced a concentration-dependent effect (n = 8), while only minor effects of VCP746/neladenoson (n = 4–9) were observed at high concentrations (1 or 10 μM; one-way ANOVA compared with vehicle alone; ***(p < 0.01)). (C) Adenosinergic compounds were also tested in vivo. NECA (n = 6) decreased heart rate relative to vehicle A (n = 2). Neladenoson (n = 4) significantly reduced heart rate at 1µM relative to vehicle B (n = 7), while VCP746 (n = 5) increased heart rate at high concentrations in vivo (two-way ANOVA, Sidak’s multiple comparisons, compared with vehicle B). Data for (B) and (C) are expressed as mean ± SD.

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  FIGURE 7. VCP746 and neladenoson induce A2BR-mediated renal vasodilation but no A1R-mediated vasoconstriction. (A) Renal vasodilation was measured by administering VCP746 or the A2AR/A2BR agonist BAY 60-6583 to methoxamine-stimulated rat kidney and measuring resulting perfusion pressure (n = 3). The concentration-dependent vasodilation produced, with order of potency VCP467 > BAY 60-6583 > neladenson suggest an A2BR-mediated mechanism of action, which was confirmed using selective antagonists (Supplementary Figure S5). (B) In order to measure renal vasoconstriction, kidneys were stimulated with methoxamine/forskolin and then NECA (n = 6), neladenoson (n = 6) or VCP746 (n = 5) was applied. NECA induced a potent constrictor activity, with no response observed to neladenoson/VCP746. This was confirmed to be an A1R-mediated effect (Supplementary Figure S5). Data are expressed as mean ± SEM; where error bars are not visible, they are contained within the dimensions of the symbol.

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  FIGURE 8. NECA, but not VCP746 or neladenoson induces endothelium-dependent relaxation of rat thoracic aorta. NECA concentration-dependently inhibited phenylephrine-mediated contraction (pEC50 6.6 ± 0.2) while VCP746 and neladenoson displayed negligible effects (n = 3). Antagonist studies suggest this is a predominately A2AR-mediated mechanism (Supplementary Figure S6). Data are expressed as mean ± SEM; where error bars are not visible, they are contained within the dimensions of the symbol. Statistical significance treatment was assessed by repeated measures mixed effects model with Dunnett’s multiple comparisons, compared with vehicle (*p < 0.05, **p < 0.01, ***p < 0.001).