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Antiplatelet and Antiproliferative Effects of SCH 51866, a Novel Type 1 and Type 5 Phosphodiesterase Inhibitor

Vemulapalli, Subbarao; Watkins, Robert; Chintala, Madhu; Davis, Harry; Ahn, Ho-Sam; Fawzi, Ahmad; Tulshian, Deen; Chiu, Peter; Chatterjee, Meeta; Lin, Chin-Chung; Sybertz, Edmund

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Journal of Cardiovascular Pharmacology: December 1996 - Volume 28 - Issue 6 - p 862-869
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Abstract

It is well established that guanosine 3′,5′-cyclic monophosphate (cGMP) and or agents that increase intracellular cGMP modulate vascular tone (1), inhibit vascular smooth-muscle cell proliferation (2), and prevent platelet aggregation (3,4) and adhesion (5-7). The intracellular levels of cGMP are regulated by cyclases or by cGMP phosphodiesterase (PDE) or both. Multiple isozymes of PDEs with different substrate specificity, tissue distribution, and sensitivity to inhibitors have been reported (8-10). Thus, PDEs have been divided into seven major families. PDE3 inhibitors augment adenosine 3′,5′-cyclic monophosphate (cAMP) in target tissues and are used clinically as positive inotropic agents (11,12). Dual inhibitors of PDE4/3, such as ORG 20241, are under preclinical investigation as antiasthmatic agents (13). Inhibition of PDE1 and PDE5 (8,10) augments cGMP in vascular smooth muscle and platelets, which mediates the vascular and antiplatelet (3,4) effects of cGMP PDE inhibitors. Although dipyridamole and zaprinast enhance cGMP by inhibiting PDE5, these compounds did not gain widespread clinical use because of lack of selectivity and potency.

We describe the PDE inhibitory properties of SCH 51866 {(+)-cis-5,6a,7,8,9,9a-hexahydro-2-[4-[trifluro-methyl] phenylmethyl]-5-methyl-cylopent[4,5]imidazo [2,1-b]purin-4(3H)one} (Fig. 1), a novel and potent type 1 and type 5 cGMP PDE inhibitor. We also compared the antiplatelet and vascular protective effects of SCH 51866 with E4021 (Fig. 1), a selective type 5 cGMP PDE inhibitor (14) in rats.

METHODS

All animal experiments were conducted in accordance with the recommendations set forth in the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and the Animal Welfare Act in a program accredited by the American Association for the Accreditation of Laboratory Animal Care under an approved institutional animal protocol.

In vitro studies

In vitro PDE-inhibitory activity of SCH 51866. PDE-inhibitory activity was measured in duplicate based on a radioenzymatic assay, as previously described (15). The PDE1 and PDE5 inhibitory activity was determined by using bovine aorta PDE1 and bovine lung PDE5 (kindly supplied by Dr. J. Corbin) isozymes as the enzyme sources. The PDE2, PDE3, and PDE4 activities were measured by using recombinant bovine adrenal PDE, bovine heart (kindly supplied by Dr. B. Mutas) and dog lung PDE isozymes (kindly supplied by Dr. J. Thompson) as the enzyme sources. The enzymes were incubated at 30°C for 20 min with an appropriate unlabeled and tritium-labeled (80,000 cpm) substrates and, where necessary, with an allosteric activator in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2. The reaction mixture in PDE1 through PDE5 assays contained 1 μM [3H cGMP], 0.5 μM calmodulin plus 0.5 mM CaCl2; 1 μM [3H cAMP] plus 5 μM cGMP; 1 μM [3H cAMP]; 0.25 μM [3H cAMP] and 1 μM [3H cGMP], respectively.

Effects of SCH 51866 on washed human platelet aggregation. Washed human platelets were prepared according to the methods described by Radomski and Moncada (16). In brief, blood (60 ml) was obtained from healthy human volunteers (who had not ingested any platelet-altering drugs for 2 weeks before donation) by venous puncture into Vacutainers containing 4.0% acid citrate dextrose (acid citrate dextrose 1 ml for 10 ml whole blood). Prostaglandin E1 (PGE1; 2 μg/ml whole blood) was added, and the blood was centrifuged at 250 g for 15 min at 15°C. The resulting platelet-rich plasma was then removed and 0.3 μml PGE1 was added and centrifuged at 900 g for 7 min at 15°C. The supernatant was decanted, and 10 ml of wash buffer containing in millimoles per liter; NaCl, 130; KCl, 4.74; and glucose, 11.5. Bovine serum albumin (BSA; 0.2%), HEPES, 10 mM; EGTA, 0.02 mM; and PGE1, 0.3 μg/ml was added, and the platelets were resuspended and centrifuged at 900 g for 7 min at 15°C. The wash buffer was then decanted, and the platelets were resuspended in a suspension buffer containing (in millimoles per liter; NaCl, 130; KCl, 4.74; KH2PO4, 1.2; NaHCO3, 4; glucose, 11.5; HEPES, 10; MgCl2 6 H2O; and CaCl2. H2O,) with BSA (0.2%) without PGE1 to yield a final concentration of 3 × 108 platelets/ml. Human fibrinogen (400 μg/ml) was then added to the platelet suspension and stored at 4°C until required. Platelet aggregation was performed in a dual-channel Chrono-log aggregometer (Model 440, Chrono-log Corp., Havertown, PA, U.S.A.) 1 h after the final resuspension. SCH 51866, E4021, or vehicle (20 μl) was added to the platelet suspension (475 μl) and incubated at 37°C for 5 min. Platelet aggregation was then induced by adding 1 μg/ml collagen, and platelet aggregation was monitored for an additional 5 min. The results were expressed as the drug concentration required to produce 50% inhibition of platelet aggregation (IC50).

In vivo studies

Effects of SCH 51866 on platelet adhesion in nylon filament-injured rat aorta. The method was described in detail previously by Reidy et al. (17). In brief, male Charles River CD rats (300-325 g) were anesthetized with inactin 100 mg/kg, i.p. The trachea was cannulated to facilitate spontaneous breathing. The left jugular vein and left femoral artery were cannulated to infuse drugs and to record blood pressure, respectively. Blood pressure was recorded with Gould pressure transducers connected to a Grass polygraph. The rectal temperature was maintained at 37°C. A monofilament (Cortland Line Co, Inc., Cortland, NY, U.S.A.) with a 45° angle was inserted into the left femoral artery and advanced to the iliac bifurcation. The rats were allowed to stabilize for a period of 30 min. The rats were infused with vehicle, SCH 51866 (0.03 and 0.1 mg/kg/min), E4021 (0.03 and 0.1 mg/kg/min), or zaprinast (1.0 mg/kg/min) throughout the study. Fifteen minutes after the start of drug infusion, each rat was injected with 0.5 ml of 111In-labeled platelets (1.50 × 109). Thirty minutes later, vascular injury was produced by advancing the nylon filament to the level of left renal artery. This procedure was repeated two additional times. Fifteen minutes after vascular injury, a 20-μl blood sample was taken to count for radioactivity. The injured abdominal aorta and the noninjured thoracic aorta were removed, cut open to drain the blood, rinsed in normal saline, weighed, and counted in a LKB gamma counter (Wallac Oy, Turku, Finland). The results were expressed as the number of platelets adhered to 1 mg wet weight of the injured abdominal aorta, according to the following formula: eqn. (1)

Labeling of platelets with 111In oxine. Platelets were labeled with 111In oxine as described by Reidy et al. (17). In brief, trunk blood (9 ml) from inactin-anesthetized rats was collected into sodium citrate Vacutainer tubes (Becton Dickinson, Rutherford, NJ, U.S.A.). The whole blood was centrifuged at 130 g at room temperature for 15 min to obtain platelet-rich plasma (PRP). The PRP was diluted to 10 ml with calcium-free Tyrode's solution and recentrifuged at 750 g for 7.5 min to obtain a platelet pellet, which was washed again with 10 ml of Tyrode's solution. The platelet pellet was resuspended in 3 ml of Tyrode's solution and incubated at 37°C for 5 min. 111In oxine (25 μCi/rat) was added to the platelet pellet and incubated for an additional 5 min. The labeled platelet suspension was centrifuged at 750 g for 7.5 min, and the supernatant was discarded. The labeled pellet was resuspended and washed twice. The final suspension was made with 0.9% normal saline, and the volume adjusted according to the number of rats used in each day's experiment (0.5 ml/rat). Of the final suspension, 5 μl was counted for radioactivity to determine the labeling efficiency according to the following formula:eqn. (2)

Effects of SCH 51866 on platelet cyclic nucleotides in rats. Male Charles River CD rats (300-325 g) were anesthetized as described and instrumented with arterial and venous catheters. After an equilibration period of 30 min, the rats were infused with saline vehicle (40 μl/min), SCH 51866, E4021 (0.03 and 0.10 mg/kg/min), or zaprinast (1.0 mg/kg/min) for 60 min. At the end of drug infusion, aortic blood samples were collected into 5 ml K3EDTA Vacutainers. PRP was obtained by centrifugation for 15 min at 130 g. One hundred microliters of 10 mM papaverine was added to a 1-ml aliquot of the PRP. The platelet pellet was resuspended in 1.0 ml of modified Tyrode's solution containing 0.35% BSA and 5 mM HEPES (pH 7.35) as described by Watanabe et al. (18). The platelet suspensions were kept frozen at -80°C until assayed for cyclic nucleotides. After removing 0.1 ml of the platelet suspension for protein determination by using a protein-assay kit (BIO-RAD, Richmond, CA, U.S.A.), the remaining 0.9 ml was brought to 6% trichloroacetic acid by adding 0.45 ml of 18% trichloroacetic acid. The samples were centrifuged at 2,500 g for 20 min at 40°C. A 1-ml aliquot of the supernatant was transferred to 15-ml polypropylene tubes and extracted 3 times with water-saturated ethyl ether. The residual ether was removed by heating at 50°C for 15 min. The platelet samples were assayed for cyclic nucleotides with radioimmunoassay kits purchased from Biomedical Technologies Inc. (Stoughton, MA, U.S.A.).

Balloon-injury model. The method was described in detail previously by Clowes et al. (19). In brief, male spontaneously hypertensive rats (SHRs) weighing (300-350 g) were anesthetized with methoxyflurane vapors (metofane), and a 2F Fogarty embolectomy catheter (Baxter, McGraw Park, IL, U.S.A.), was inserted into the left external carotid artery. The catheter was advanced 5 cm and inflated and retracted 3 times. The rats were dosed orally with vehicle, spirapril (30 mg/kg, once daily) SCH 51866 (1, 3, and 10 mg/kg, twice daily), or E4021 (3, 10, and 30 mg/kg, twice daily) a day before and 14 days after injury. Two hours after the last dose, the rats were killed by exsangunation under anesthesia with ketamine (50 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.). Five-milliliter blood samples were collected by heart puncture, and serum was obtained by centrifugation to measure serum concentration of drugs by high-performance liquid chromatography (HPLC). The left and right carotid arteries (5 mm) of aortic origin were removed, and the DNA content was measured as described by LaBarca and Paigen (20). The DNA measurements were expressed as the ratio of injured left carotid artery to the uninjured right carotid artery. Carotid artery specimens were fixed in 4% paraformaldehyde containing 10% sucrose (wt/vol) for histologic examination. Paraffin-embedded sections were stained with the Gomori trichrome-aldehyde fuchsin stain, and computer-assisted morphometric analyses were performed by using Bioquant System IV image analyzer.

Antihypertensive activity of SCH 51866 in SHRs. Male SHRs (Taconic Farms, Germantown, NY, U.S.A.; 275-300 g) were anaesthetized with ether, and the abdominal aorta was cannulated via the caudal artery with PE-50 tubing and connected to Viggo-Spectramed (Oxnard, CA, U.S.A.) pressure transducers to record blood pressure. The rats were placed in plastic restrainers and allowed to recover from ether anesthesia and surgery for ≥90 min. The rats were dosed orally with 0.4% aqueous methylcellulose vehicle, SCH 51866 (0.3-10 mg/kg), or E4021 (3-30 mg/kg), and blood pressure was recorded continuously. Analog blood pressure signals were recorded on Beckman oscillographs. A cardiovascular monitoring system (Buxco Electronics, Inc., Sharon, CT, U.S.A.) and digital computers were used to derive mean blood pressures and heart rates at 30-min intervals. In parallel experiments, SHRs were dosed orally with SCH 51866 or E4021 and trunk blood was obtained at different times to measure plasma levels of drugs.

Determination of plasma concentration of SCH 51866 and E4021. Five hundred μl of plasma from rats dosed with SCH 51866 was mixed with 1 ml of internal standard (SCH 51866 2.5 μg/ml in water). To this mixture, 5 ml of 30% methylene chloride in hexane was added, and the sample was shaken for 15 min in an Ebenbach shaker. The sample was centrifuged and placed in crushed ice. The solvent was decanted and evaporated to dryness at 40-45°C. The residue was dissolved in 0.3 ml of HPLC mobile phase (50% acetonitrile in ammonium phosphate, pH 5.5). The sample (100 μl) was injected into the Waters HPLC system with an S5 CN column and read in a spectrophotometer set at 290 mm.

Plasma from rats dosed with E4021 (100 μl) was added to 2 ml of acetonitrile, vortexed for 30 s, centrifuged, and the acetonitrile was decanted. The residual acetonitrile was evaporated to dryness under nitrogen and reconstituted in 0.3 ml of HPLC mobile phase (60% acetonitrile, 30% distilled water, and 10% 0.2 M ammonium phosphate buffer, pH 5.5). Two hundred microliters of the sample was injected into the Waters HPLC system (Pump model 6000A, programmable model 490 detector and model 712 WISP injector) with a zorbax column and a spectrophotometer set at 240-nm wavelength.

Materials

SCH 51866, E4021, zaprinast, and spirapril were synthesized at Schering-Plough Research Institute (Kenilworth, NJ, U.S.A.). 111In oxine was obtained from Amersham (Arlington Heights, IL, U.S.A.). PGE1 was obtained from Sigma (St. Louis, MO, U.S.A.).

Statistical analysis

All values are presented as mean ± SEM. The data were analyzed by analysis of variance; a p value <0.05 was considered significant.

RESULTS

Evaluation of PDE-inhibitory properties of SCH 51866

The ability of SCH 51866 to inhibit purified PDE types 1, 2, 3, 4, and 5 was assessed in vitro (Table 1). SCH 51866 inhibited the purified PDE1 and 5 isozymes with an IC50 of 70 and 60 nM, respectively. The inhibition of PDE1 by SCH 51866 was competitive with respect to cGMP (H.-S. Ahn, personal communication). SCH 51866 inhibited PDE2, PDE3, and PDE4 with an IC50 of 2.1, 5, and 30 μM, respectively (Table 1). In comparison, E4021 inhibited PDE5 with an IC50 of 4 nM(Table 1).

Effects of SCH 51866 on collagen-induced washed human platelet aggregation

The inhibitory effects of SCH 51866, E4021, aspirin, and sodium nitroprusside on collagen-induced washed human platelet aggregation are summarized in Table 2. SCH 51866 and E4021 prevented washed human platelet aggregation with IC50s of 10 and 4 μM, respectively. Sodium nitroprusside and aspirin inhibited washed human platelet aggregation with IC50s of 0.3 and 42 μM, respectively.

Effects of SCH 51866 on platelet adhesion in the nylon filament-injured rat aorta

The platelet-labeling efficiency was 85.4 ± 1.2% (n = 8), and the mean platelet count was 9.6 × 108/ml (n = 18). The mean platelet count was used to calculate the number of platelets deposited on the injured vessel.

Platelet adhesion to the nylon filament-injured rat aorta in the sham, experimental, SCH 51866-, and E4021-treated groups is shown in Fig. 2. Vascular injury caused by the nylon filament significantly increased the adherence of platelets to the injured subendothelium (1.1 × 106 ± 1.2 × 103 in the untreated experimental group vs. 9.1 × 103 ± 0.9 × 102; p < 0.05 in the sham group). In the sham group, the nylon filament was inserted into the femoral artery but was not advanced into the abdominal aorta. Neither SCH 51866 nor E4021 infusion at 0.03 mg/kg/min significantly affected platelet adhesion to the injured abdominal aorta, but SCH 51866 and E4021 at 0.10 mg/kg/min infusion significantly attenuated the adhesion of platelets to the nylon filament-injured abdominal aorta. SCH 51866, E4021, and zaprinast reduced platelet adhesion by 55 ± 5, 62 ± 4, and 53 ± 6%, respectively. SCH 51866 at 0.03 and 0.10 mg/kg/min decreased blood pressure by 11 ± 4 and 37 ± 4 mm Hg, respectively. In comparison, E4021 reduced blood pressure by 5 ± 2 and 23 ± 3 mm Hg at 0.03 and 0.10 mg/kg/min, respectively. Zaprinast decreased blood pressure by 32 ± 3 mm Hg.

Effects of SCH 51866 and E4021 cyclic nucleotide levels in anesthetized rats

The effects of SCH 51866, E4021, and zaprinast on platelet cGMP and cAMP levels in anesthetized rats are summarized in Table 3. Intravenous infusion of SCH 51866 and E4021 (0.03 mg/kg/min × 60) did not significantly (p > 0.05) alter platelet cGMP or cAMP. However, SCH 51866 and E4021 (0.10 mg/kg/min × 60) caused significant (p < 0.05) increases in platelet cGMP without affecting platelet cAMP. Similarly, zaprinast significantly increased platelet cGMP without influencing platelet cAMP. However, there is a discrepancy between the ability of zaprinast to increase platelet cGMP and its ability to attenuate platelet adhesion. The reasons for this discrepancy are not clear.

Effects of SCH 51866 and E4021 on myointimal proliferation in SHRs

The effects of SCH 51866 and E4021 on myointimal proliferation in carotid arteries of SHRs subjected to balloon angioplasty are shown in Fig. 3. Spirapril, an angiotensin-converting enzyme inhibitor, was used as a positive control. Balloon angioplasty significantly increased the intimal/medical ratio of left carotid artery of vehicle-treated SHRs, indicating marked intimal thickening. The contralateral right carotid arteries that were not subjected to balloon angioplasty appeared normal (i.e., no intimal thickening was observed; data not shown). Spirapril, as expected, significantly inhibited the neointima formation. SCH 51866 at 1, 3, or 10 mg/kg p.o. significantly attenuated myointimal proliferation. SCH 51866 inhibited intimal cross-sectional area by 30, 35, and 50% at 1, 3, and 10 mg/kg p.o., respectively, and caused a comparable reduction in intimal/medical ratio. The plasma concentration of SCH 51866 at 1, 3, and 10 mg/kg p.o. reached 0.074 ± 0.001, 0.345 ± 0.026, and 0.854 ± 0.081 μg/ml, respectively. In contrast, E4021, a highly selective cGMP PDE5 inhibitor, failed to affect myointimal proliferation at 3-30 mg/kg p.o. (Fig. 3). However, the plasma concentration of E4021 at 3, 10, and 30 mg/kg p.o. was 1.97 ± 0.2, 7.06 ± 0.6, and 11.99 ± 1.0 μg/ml, respectively.

Antihypertensive effects of SCH 51866 in conscious SHRs

The effects of SCH 51866 on blood pressure and heart rate in conscious SHRs are summarized in Fig. 4. The baseline blood pressure among the different treatment groups was similar. SCH 51866 at doses of 1, 3, and 10 mg/kg p.o. reduced blood pressure significantly by 16 ± 2, 34 ± 9, and 39 ± 3 mm Hg, respectively. The blood pressure started to decrease soon after oral dosing with SCH 51866, and the maximal reduction in blood pressure was usually observed 1 h after dosing; the reduction in blood pressure was sustained throughout the study. The antihypertensive activity of SCH 51866 was accompanied by significant tachycardia. The antihypertensive doses of SCH 51866 (1-10 mg/kg p.o.) caused dose-dependent increases in plasma levels of SCH 51866 (Fig. 5). In contrast, E4021 at 3-30 mg/kg p.o. did not affect either blood pressure or heart rate in conscious SHRs (data not shown). The inability of E4021 to modify blood pressure was not the result of poor oral absorption of E4021 because E4021 at 10 mg/kg achieved a plasma concentration of 2.37-3.81 μg/ml at 0.5-4 h after oral dosing. Higher doses of E4021 were not tested.

DISCUSSION

The results of our study indicate that SCH 51866 is a highly selective type 1 and 5 cGMP PDE inhibitor. SCH 51866 inhibited platelet aggregation and adhesion and prevented myointimal proliferation in carotid arteries of SHRs subjected to balloon angioplasty and decreased blood pressure in SHRs.

SCH 51866 inhibited PDE1 and 5 isolated from bovine aorta and bovine lungs at low nanomolar range. The inhibition of PDE1 by SCH 51866 was competitive with respect to cGMP (H.- S. Ahn, personal communication). SCH 51866 was significantly less effective in inhibiting PDE2, 3, and 4 isozymes. SCH 51866 is ≈10-fold less active in inhibiting PDE5 than is E4021, but 1,000-fold more potent against PDE1. A comparison of the published data indicates that SCH 51866 is approximately fivefold more potent than zaprinast in inhibiting PDE5 (14).

The ability of SCH 51866 to prevent collagen-induced washed human platelet aggregation was assessed in vitro. SCH 51866 and E4021 inhibited collagen-induced platelet aggregation. However, E4021 was slightly more potent than SCH 51866 in inhibiting collagen-induced human platelet aggregation. A review of the literature suggests that SCH 51866 is ≈10-fold more potent than zaprinast in inhibiting platelet aggregation (21). The IC50 of aspirin to inhibit collagen-induced platelet aggregation reported in this study was similar to that reported by Dohi et al. (22).

The platelet antiadhesive effects of SCH 51866 were assessed in the nylon filament-injured rat abdominal aorta. In the vehicle-treated experimental control, a great number of indium-labled platelets adhered to the injured subendothelium. Intravenous administration of SCH 51866 significantly reduced the number of platelets adhered to the injured subendothelium. Similarly, E4021 and zaprinast also prevented the adhesion of indium-labeled platelets at sites of vascular injury. The doses of SCH 51866, zaprinast, and E4021 that inhibited platelet adhesion caused significant increases in platelet cGMP, indicating that cGMP mediates the platelet antiadhesive effects of SCH 51866, zaprinast, and E4021. These results support our previous findings and those of others that agents that increase intracellular cGMP but not cAMP prevent accumulation of platelets after balloon angioplasty in rats (7), pigs (23), and ex vivo in perfused rat lungs (5). Similarly, Chintala et al. (24) reported that intracellular increase of cGMP but not cAMP prevented accumulation of platelets in the kidney after ischemia-reperfusion in rats. However, the mechanism(s) by which cGMP prevents platelet adhesion is(are) not clearly understood. cGMP had been reported to inhibit human platelet secretion (25) and binding of fibrinogen to human platelets (26). The mechanism by which cGMP prevents these intracellular events remains to be clarified.

Lines of evidence suggest that platelets adhere to the damaged subendothelium and release growth factors that may play a role in restenosis. Restenosis occurs in 30-40% of patients within 6 months after percutaneous transluminal angioplasty (PTCA) and clinically is a major problem. Antiplatelet drugs such as aspirin and dipyridamole (27,28) are used to reduce ischemic complications after PTCA. More recently glycoprotein (GP) IIb/IIIa antagonists have been administered during and or shortly after angioplasty (29,30) to prevent ischemic episodes. Although the results of using GP IIb/IIIa antagonists in PTCA studies are encouraging, bleeding is a major side effect with these drugs. Moreover, the antiplatelet drugs are directed at thrombotic complications but not at restenosis. In our study, SCH 51866 exhibited significant antiproliferative effects. Similarly, 8-Br-cGMP (31); cGMP-increasing vasodilators (2), and SPM-5185, a nitric oxide donor (32,33), prevented neointima formation in several experimental models. The mechanism by which SCH 51866 affords protection against balloon catheter-induced neointima formation remains to be clarified. However, it is probable that selective inhibition of cGMP PDE1 and 5 isozymes by SCH 51866 and the resultant increases in smooth-muscle cell and platelet cGMP contributed to its antiproliferative effects in SHR. SCH 51866 also prevented neointima formation in minipigs by 53% (34), suggesting inhibition of neointima formation by SCH 51866 is not species specific. In this respect, SCH 51866 differs from other vasoactive agents such as angiotensin-converting enzyme inhibitors that prevent restenosis in rat models but not in pigs (34,35). It is important to mention that the vasodepressor effects of SCH 51866 did not contribute to its antiproliferative effects, because potent hypotensive agents such as verapamil and minoxidil failed to inhibit restenosis in rat models (36). Moreover, nifedipine had no effect on myointimal proliferation in our rat model of restenosis (37). In contrast to SCH 51866, E4021, a highly selective PDE5 inhibitor (14), failed to demonstrate antiproliferative effects in balloon catheter-injured carotid arteries of SHRs. The reasons for this disparate finding are not clear. However, it is reasonable to speculate that it is essential to inhibit vascular PDE1 to prevent neointima formation in injured carotid arteries of SHRs.

In summary, SCH 51866 selectively inhibited PDE1 and 5 isozymes and inhibited washed human platelet aggregation and platelet adhesion in the nylon filament-injured rat aorta. SCH 51866 prevented neointima formation in balloon catheter-injured carotid arteries of SHRs and decreased blood pressure in SHRs. The antiplatelet and antiproliferative properties of SCH 51866 make it an ideal candidate to assess the role of cGMP in restenosis.

Acknowledgment: We gratefully acknowledge the expert technical assistance of the following people: George Boykow, Arthur Brown, Rene Cleven, John Cook, Lizbeth Hoos, Stanley Kurowski, Daniel McGregor, Kathyrn Pula, Richard Tedesco, and Hongtao Zhang. Dr. Andrew Stamford synthesized E4021.

FIG. 1.
FIG. 1.:
Chemical structures of SCH 51866 and E4021.
FIG. 2.
FIG. 2.:
Effects of SCH 51866, zaprinast, and E4021 on estimated number of platelets adhered to the abdominal aorta injured with a nylon filament. SCH 51866 and E4021 (0.03 and 0.1 mg/kg/min) and zaprinast (1.0 mg/kg/min) were infused for 60 min (see Methods for details). N = 6-10 per group. *p < 0.05 versus Exper-Vehicle.
FIG. 3.
FIG. 3.:
Inhibition of neointimal formation by SCH 51866 but not by E4021 in spontaneously hypertensive rats (SHRs). SHRs were dosed with SCH 51866 (1-10 mg/kg, twice daily) or E4021 (3-30 mg/kg, twice daily) a day before and 14 days after balloon injury. Values were derived from the overall number of lesions analyzed and represent the ratio of total intimal area to total medial area. Columns and bars represent mean ± SEM, respectively, n = 6-13 per group. *p < 0.05 versus vehicle.
FIG. 4.
FIG. 4.:
Effects of SCH 51866 on blood pressure (top) and heart rate (bottom) in conscious spontaneously hypertensive rats (SHRs). Conscious SHRs were dosed orally with SCH 51866 (0.3-10 mg/kg), and blood pressure was monitored continuously for 4 h. The values to the right of each curve denote the baseline blood pressure (mm Hg) and heart rate (beats/min). *p < 0.05 versus vehicle.
FIG. 5.
FIG. 5.:
Plasma levels of SCH 51866 in spontaneously hypertensive rats (SHRs) dosed orally with SCH 51866 (1-10 mg/kg). Arterial blood samples (1.0 ml) were obtained at different times for the determination of plasma concentration of SCH 51866 by high-performance liquid chromatography (HPLC).

REFERENCES

1. Murad F. Cyclic guanosine monophosphate as a mediator of vasodilation. J Clin Invest 1986;78:1-5.
2. Kariya K-I, Kawahara Y, Araki S-I, Fukuzaki H, Takai Y. Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic muscle cells. Atherosclerosis 1989;80:143-7.
3. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987;2:1057-8.
4. Siney L, Lewis MJ. Endothelium-derived relaxing factor inhibits platelet adhesion to cultured porcine endocardial endothelium. Eur J Pharmacol 1990;229:223-6.
5. Venturini CM, Weston LK, Kaplan JE. Platelet cGMP, but not cAMP, inhibits thrombin-induced platelet adhesion to pulmonary vascular endothelium. Am J Physiol 1992;262:H606-12.
6. Radomski MW, Palmer RMJ, Moncada S. Characterization of the L-arginine:nitric oxide pathway in human platelets. Br J Pharmacol 1990;101:325-8.
7. Vemulapalli S, Chiu PJS, Kurowski S, Brown A, Hartman BA, Leach MW. In vivo inhibition of platelet adhesion by a cGMP-mediated mechanism in balloon catheter injured rat carotid artery. Pharmacology 1996;52:235-42.
8. Beavo JA, Reifsnyder DH. Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors. Trends Pharmacol Sci 1990;11:150-5.
9. Beavo JA, Conti M, Heaslip R. Multiple cyclic nucleotide phosphodiesterases. Mol Pharmacol 1994;46:399-405.
10. Nicholson CD, Challiss RAJ, Shahid M. Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci 1991;12:19-27.
11. Silver PJ, Pagani ED. Biochemical and preclinical pharmacology of selective inhibitors of cardiovascular phosphodiesterase isozymes. In: Gwathmey JK, Briggs GM, Allen PD, eds. Heart failure: basic science and clinical aspects. New York: Marcel Dekker, 1993: 367-86.
12. Goldstein RA, Fleming RM. Clinical aspects of phosphodiesterase inhibitors. In: Gwathmey JK, Briggs GM, Allen PD, eds. Heart failure: basic science and clinical aspects. New York: Marcel Dekker, 1993:387-98.
13. Nicholson CD, Shahid M, Bruin J, et al. Characterization of ORG 20241, a combined phosphodiesterase IV/III cyclic nucleotide phosphodiesterase inhibitor in asthma. J Pharmacol Exp Ther 1995;274:678-87.
14. Saeki T, Adachi H, Takase Y, Yoshitake S, Souda S, Saito I. A selective type V phosphodiesterase inhibitor, E4021, dilates porcine large coronary artery. J Pharmacol Exp Ther 1995;272:825-31.
15. Ahn HS, Crim W, Romano M, Sybertz E, Pitts B. Effects of selective inhibitors on cyclic nucleotide phosphodiesterases of rabbit aorta. Biochem Pharmacol 1989;38:3331-9.
16. Radomski M, Moncada S. An improved method for washing of human platelets with prostacyclin. Thromb Res 1983;30:383-9.
17. Reidy MA, Yoshida K, Harker LA, Schwartz SM. Vascular injury: quantification of experimental focal endothelial denudation in rats using indium-111-labeled platelets. Arterioscelerosis 1986;6:305-11.
18. Watanabe H, Kakihana M, Ohtsuka S, et al. Platelet cGMP: a potentially useful indicator to evaluate the effects of nitroglycerin and nitrate tolerance. Circulation 1993;88:29-36.
19. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983;49:208-15.
20. Labarca C, Paigen KA. A simple, rapid and sensitive DNA assay procedure. Anal Biochem 1980;102:344-52.
21. Anderson TLG, Vinge E. Interactions between isoprenaline, sodium nitroprusside, and isozyme-selective phosphodiesterase inhibitors on ADP-induced aggregation and cyclic nucleotide levels in human platelets. J Cardiovasc Pharmacol 1991;18:237-42.
22. Dohi M, Sakata Y, Seki J, et al. The anti-platelet actions of FR 122047, a novel cyclooxygenase inhibitor. Eur J Pharmacol 1993;243:179-84.
23. Groves PH, Penny WH, Cheadle HA, Lewis MA. Exogenous nitric oxide inhibits in vivo platelet adhesion following balloon angioplasty. Cardiovasc Res 1992;26:65-8.
24. Chintala MS, Bernardino V, Chiu PJS. Cyclic GMP but not cyclic AMP prevents renal platelet accumulation after ischemia-reperfusion in anesthetized rats. J Pharmacol Exp Ther 1994;271:1203-8.
25. Lieberman EH, O'Neil S, Mendelsohn ME. S-nitrosocystein inhibition of human platelet secretion is correlated with increases in platelet cGMP levels. Circ Res 1991;68:1722-8.
26. Mendelsohn ME, O'Neil S, George D, et al. Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine. J Biol Chem 1990;265:19028-34.
27. Barnathan ES, Schwartz JS, Taylor L, et al. Aspirin and dipyridamole in the prevention of acute coronary thrombosis complicating coronary angioplasty. Circulation 1987;76:125-34.
28. Lembo NJ, Black AJ, Roubin GS, et al. Effect of pretreatment with aspirin versus aspirin plus dipyridamole on frequency and type of acute complications of percutaneous transluminal coronary angioplasty. Am J Cardiol 1990;65:422-6.
29. Frishman WH, Burns B, Atac B, et al. Novel antiplatelet therapies for treatment of patients with ischemic heart disease: inhibitors of the platelet glycoprotein IIb/IIIa integrin receptor. Am Heart J 1995;130:877-92.
30. Tcheng JE. Enhancing safety and outcomes with the newer antithrombotic and antiplatelet agents. Am Heart J 1995;130:673-9.
31. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-Br-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 1989;13:1874-84.
32. Guo J, Milhoan KA, Tuan RS, Lefer AM. Beneficial effect of SPM-5185, a cysteine-containing nitric oxide donor, in rat carotid artery intimal injury. Circ Res 1994;75:77-84.
33. De Meyer GRY, Bult H, Ustunes L, et al. Effect of nitric oxide donors on neointima formation and vascular reactivity in the collared carotid artery of rabbits. J Cardiovasc Pharmacol 1995;26:272-9.
34. Watkins RW, Davis HR, Fawzi A, et al. Antihypertensive, hemodynamic and vascular protective effects of SCH 51866, an inhibitor of cGMP hydrolysis [Abstract]. Fed Am Soc Exp Biol 1995;9:A342.
35. Lam JYT, Lacoste L, Bourassa M. Cilazapril and early atherosclerotic changes after balloon injury of porcine carotid arteries. Circulation 1992;85:1542-7.
36. Powell JS, Muller RKM, Baumgartner HR. Suppression of the vascular response to injury: the role of angiotensin-converting enzyme inhibitors. J Am Coll Cardiol 1991;17:137-42b.
37. Chatterjee M, Davis HR, Watkins RW, et al. The inhibition of myointimal proliferation by selective inhibition of cGMP phosphodiesterases (PDEs) [Abstract]. Circulation 1994;90:I-627.
Keywords:

Phosphodiesterase inhibition; SCH 51866; E4021; Neointima; Antiplatelet

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