Linda Merkel

Restenosis Following Angioplasty in the Swine Coronary Artery Is Inhibited By an Orally Active PDGF-Receptor Tyrosine Kinase Inhibitor, RPR101511A

BACKGROUND Platelet-derived growth factor (PDGF), a purported mediator of arterial response to injury, stimulates proliferation, chemotaxis, and matrix production by activation of its membrane receptor tyrosine kinase. Because these activities underlie restenosis, inhibition of the PDGF-receptor tyrosine kinase (PDGFr-TK) is postulated to decrease restenosis. METHODS AND RESULTS RPR101511A is a novel compound which selectively and potently inhibits the cell-free and in situ PDGFr-TK and PDGFr-dependent proliferation and chemotaxis in vascular smooth muscle cells (VSMC). To evaluate the effect of RPR101511A (30 mg. kg-1. d-1 BID for 28 days following PTCA) on coronary restenosis, PTCA was performed in hypercholesterolemic minipigs whose left anterior descending (LAD) coronary artery had been injured by overdilation and denudation, yielding a previously existing lesion. Angiographically determined prePTCA minimal lumen diameters (MLD) were similar in vehicle and RPR101511A-treated pigs (1.98+/-0.09 versus 2.01+/-0.08 mm) and increased to the same extent in the 2 groups following successful PTCA (2.30+/-0.06 versus 2.52+/-0.13). At termination, there was an average 50% loss of gain in the vehicle-treated group but no loss of gain with RPR101511A (2.16+/-0. 05 versus 2.59+/-0.11, P<0.001). Morphometric analysis of the LAD showed that RPR101511A caused a significant decrease in total intimal/medial ratio (0.96+/-0.58 versus 0.67+/-0.09, P<0.05). CONCLUSIONS RPR101511A, which acts by inhibition of the PDGFr-TK, completely prevented angiographic loss of gain following PTCA and significantly reduced histological intimal hyperplasia.

Cardiovascular and Antilipolytic Effects of the Adenosine Agonist GR79236

Adenosine is known to produce cardiovascular effects such as bradycardia and hypotension via activation of myocardial (A1) and vascular (A2) receptors and antilipolytic effects through activation of adipocyte (A1) receptors. We established the cardiovascular and antilipolytic profile of the adenosine A1 agonist GR79236 (N6-[(1S,trans)-2-hydroxycyclopentyl]-adenosine) and compared it with CPA (N6-cyclopentyl-adenosine). GR79236 was approximately 3-fold less potent than CPA in inhibiting in vitro lipolysis. In conscious rats, both agents were shown to have antilipolytic and glucose-lowering properties. In rats instrumented with telemetry transmitters, orally administered CPA was one log unit more potent than GR79236 as a hypotensive and bradycardiac agent. In summary, GR79236 is an A1-selective adenosine agonist which reduces heart rate and mean arterial pressure and produces decreased plasma lipids and glucose levels.

Enzyme inhibition during the conversion of squalene to cholesterol

Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in cholesterol biosynthesis. One assay detects inhibition of delta 5.7-sterol delta 7-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. Delta 5.7-Sterol delta 7-reductase was inhibited by both RPR 101821, a protonated cyclohexylamine, and BM 15.766, a piperazine derivative, with IC50 values of 1 microM. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmosterol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing reactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a conversion of squalene to cholesterol in the order of 50%. The time required to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible values of Ki, kon, and koff for the reaction between enzymes and inhibitor to form enzyme-inhibitor complexes, it was predicted that in the presence of inhibitors, intermediate accumulation could still be observed after 6 h incubation. The experimental results were in agreement with this prediction.

Modulation of vascular reactivity by vasoactive peptides in aortic rings from hypercholesterolemic rabbits

The effect of moderate elevation in serum cholesterol on vascular reactivity to epidermal growth factor (EGF), endothelin (ET-1) and thrombin, vasoactive peptides present at sites of vascular injury, was examined in isolated aortic rings from rabbits fed either a casein-rich or a control diet for 10-12 weeks. In rings from hypercholesterolemic rabbits, development of maximal isometric tension to all peptide agonists was increased 22 +/- 0.6% while the EC50 for contraction was decreased. Vasorelaxant responses to nitroprusside, an endothelium-independent dilator, were largely intact, while those to A231897, an endothelium-dependent agent, were attenuated. These data suggest that elevation in serum cholesterol in the absence of atherosclerotic lesions is sufficient to increase vascular reactivity to peptide vasoactive mediators, an effect which may predispose arteries to vasospasm.

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells

Abstract This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by Δ12PGJ2 was first observed at 10 μM, rose to 4000–5000 ng/mL within one log unit and a maximum response was not observed; concentrations of Δ12PGJ2 higher than 30 μM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 μM and maintained to 1 μM without toxicity. Both, Δ12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 μM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with Δ12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of Δ12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3–4 hours of exposure to Δ12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or Δ12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Pharmacological characterization of AMP 579, a novel adenosine A1/A2 receptor agonist and cardioprotective

AMP 579 1S‐[1α,2β,3β,4α(S*)]‐4‐[7‐[[1‐[(3‐chloro‐2‐thienyl)methyl]propylamino]‐3H‐imidazo[4,5‐b]pyridin‐3‐yl]‐N‐ethyl‐2,3‐dihydroxy cyclopentanecarboxamide) is a novel cardioprotective adenosine agonist with the following order of affinity at adenosine receptors: A1 > A2A > A3. Agonism at A1 receptors was demonstrated in vitro in three different systems: 1) inhibition of lipolysis in rat and human isolated adipocytes, 2) restoration of insulin‐dependent glucose transport in rat adipocytes, and 3) reduction of heart rate in spontaneously beating rat right atria. Agonism at A2A receptors was reflected in vasorelaxation of porcine coronary arterial rings (IC50 = 0.3 μM); in comparison, agonism at A2B receptors was ∼100‐fold weaker, as reflected in relaxation of guinea pig aorta (IC50 = 28 μM). When given iv to conscious Sprague‐Dawley (SD) rats, AMP 579 dose‐dependently lowered free fatty acids (FFA), heart rate (HR), and mean arterial pressure (MAP), but was 25‐fold more potent at reducing FFA than at decreasing HR and MAP. In anesthetized rats undergoing myocardial ischemia‐reperfusion injury, AMP 579 (3 μg/kg + 0.3 μg/kg/min iv and 10 μg/kg + 1 μg/kg/min iv) was able to reduce infarct size by 55% and 63%, respectively, compared to control animals, when given 10 min prior to and throughout the first hour of reperfusion. These cardioprotective doses of AMP 579 caused no significant change in blood pressure or coronary blood flow. In summary, AMP 579 is a novel adenosine A1/A2A receptor agonist which causes long‐lasting reductions in FFA in vivo and has cardioprotective effects in a rat model of myocardial ischemia‐reperfusion injury at doses which have minimal hemodynamic effects. Thus, AMP 579 has significant potential for the therapy of acute myocardial infarction. Drug Dev. Res. 45:30–43, 1998.© 1998 Wiley‐Liss, Inc.

Differential effects of the adenosine A1 receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A(1) receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [3H]cyclohexyladenosine ([3H]CHA) to A(1) receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81, 723 (0.3-100 microM) produced a concentration-dependent enhancement of [3H]CHA binding to rat brain A(1) receptors. These effects were also species-dependent with larger enhancements (150-200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [3H]CHA binding to A(1) receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 microM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A(1) receptor selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 microM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [3H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 microM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A(1) receptor in a high-affinity state and that the relative proportions of high-affinity A(1) receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A(1) receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A(1) receptor.

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 F2α (PGF2α). 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.

Synthesis of a Potent A1 Selective Adenosine Agonist: N6-[1-R-[(3-Chloro-2-thienyl)methyl]propyl]adenosine, RG 14718(-)

Abstract This report describes the preparation of (R)-(3-chloro-2-thienyl)-2-butylamine and its conversion to the titled N6-substituted adenosine agonist, RG 14718(−). RG 14718(−) was found to have exceptional affinity and selectivity for the adenosine A1 receptor, IC50 = 5.7 + /− 1.6 pM.