Synergistic Effects on Thromboresistance in Man

Br. J. clin. Pharmac. (1992), 33, 289-292

Prostaglandin '2 and the nitric oxide donor molsidomine have synergistic effects on thromboresistance in man

H. SINZINGER', F. RAUSCHA2, J. O'GRADY* & P. FITSCHA3 1Department of Nuclear Medicine, University of Vienna, Ludwig Boltzmann-Institute for Nuclear Medicine, Vienna, Wilhelm Auerswald-Atherosclerosis Research Group (ASF) Vienna, Atherosclerosis Research Group of the Austrian Academy of Sciences (ATK) Vienna, 2Department of Cardiology, University of Vienna and 32nd Department of Internal Medicine, Policlinic Vienna, Austria

1 In vitro synergistic effects of nitric oxide and prostaglandin I2 (PGI2) have been shown. Consequently we examined any potentiating effect of the nitric oxide donor molsidomine on the reduction in thrombogenicity produced by PGI2 in patients with peripheral vascular disease. 2 Thirty-six patients all with peripheral and also coronary artery disease were randomly allocated to receive PGI2 5 ng kg-1 min-' for 6 h daily, 5 days a week for 5 weeks, alone (12 patients), with molsidomine 12 mg daily (12 patients) or molsidomine 12 mg daily alone (12 patients). 3 The effect of each treatment regimen was measured in terms of femoral artery platelet uptake and platelet survival after autologous ll'Indium-oxine labelling. Molsidomine alone had no effect on platelet uptake or survival but in combination with PGI2 it significantly potentiated the decreased platelet uptake and prolonged platelet survival observed with PGI2 alone.

Keywords prostaglandin '2 (PGI2) endothelium derived relaxing factor (EDRF) nitric oxide (NO) molsidomine thromboresistance

Introduction Methods The antiaggregatory prostaglandins (PG) 12 and E1 act Subjects and treatments by increasing intracellular platelet cyclic AMP (Gorman et al., 1977; Tateson et al., 1977). Endothelium derived Thirty-six patients all suffering from both clinically relaxing factor (EDRF; Furchgott & Zawadzki, 1980) manifest coronary artery disease and peripheral vascular recently identified as nitric oxide (Moncada et al., disease for at least 1 year with a platelet uptake ratio of 1988a) inhibits platelet aggregation by activation of at least 1.25 over one of the femoral arteries were cellular cytosolic guanylate cyclase resulting in enhanced involved in this open study. [The platelet uptake ratio platelet cGMP (Bohme et al., 1983; Moncada et al., is derived from the division of the actual radioactive 1988b). PGI2 and nitric oxide act synergistically to counts over a lesion site by those over the contralateral inhibit platelet aggregation (Radomski et al., 1987a) in side after background subtraction (Sinzinger & Fitscha vitro as does the stable PGI2-'analogue' iloprost for 1984)]. Patients were randomly assigned to one of three example with sodium nitroprusside (Lidbury et al., 1919). treatment groups: Group 1: 12 patients (54-71 years; A similar in vitro (Katzenschlager et al., 1991) synergism nine male, three female) received 3 x 4 mg molsidomine of PGE1 and nitric oxide in reducing platelet aggregation (Albert Roussel, Wiesbaden, Germany) daily, Group 2: was reproduced in vivo by the co-administration of 12 patients, (52-73 years; nine male, three female) isosorbide dinitrate and PGE1, resulting in a decreased received PGI2 (kindly provided by Salvador Moncada, platelet uptake on active human atherosclerotic lesions Wellcome Research Labs, Beckenham, Kent, UK) at (Sinzinger et al., 1990). We have applied this technique a rate of 5 ng kg-1 min- 1 intravenously (using a portable in order to assess whether molsidomine via its in vivo infusion pump, Pharmacia, Sweden) for 6 h daily for 5 metabolite SIN-1 A shows a similar synergistic effect days a week and Group 3: 12 patients (55-73 years; in vivo with PGI2. SIN-1 A is formed from SIN-1 eight male, four female) received a combination of (Noack et al., 1989). molsidomine and PGI2. The treatments were admini-

Correspondence: Professor Helmut Sinzinger, Wilhelm Auerswald-Atherosclerosis Research Group (ASF), Nadlergasse, A-1090 Vienna, Austria *Dr John O'Grady is visiting professor in the Department of Pharmacology, University of Vienna, Austria 289 290 H. Sinzinger et al.

Table 1 Patients characteristics days. The actual radioactive counts over lesion sites Group divided by the counts over the contralateral unaffected 1 2 3 Total side per pixel after background subtraction resulted in a platelet uptake ratio. A minimum of 2000 counts was Sex (m/f) 9/3 9/3 8/4 26/10 acquired. Smokers (yes/no) 3/9 4/8 4/8 11/25 HYP (yes/no) 2/10 3/9 3/9 8/28 Statistical methods HLP (yes/no) 4/8 5/7 6/6 15/21 FA (yes/no) 5/7 5/7 5/7 15/21 MOD (yes/no) 2/10 1/11 1/11 4/32 Values are presented as mean ± s.e. mean; calculations CHD (duration) 4.33 4.08 4.42 4.28 of significance were performed using Student's t-test PVD (duration) 2.17 1.96 2.01 2.08 for paired data and analysis of variance. MI (yes/no) 3/9 3/9 4/8 10/26 Groups: 1 molsidomine, 2 PGI2, 3 combination 1 + 2 HYP ... hypertension; HLP ... hyperlipoproteinemia; FA . .. positive familial Results anamnesis for cardiovascular events; MOD ... maturity-onset diabetes; CHD . . . coronary Molsidomine alone had no significant effect on either heart disease; PVD . . . peripheral vascular platelet uptake ratio (Figure 1) or platelet survival (Figure disease; MI . .. myocardial infarction; values for 2). PGI2 in contrast significantly (P < 0.001) diminished CHD and PVD are mean durations in years since platelet uptake ratio and significantly (P < 0.001) pro- first clinical diagnosis. longed platelet survival. Administration of both treatments induced a significant further reduction of platelet stered for 5 weeks on an outpatient base in all three uptake ratio and increase in survival time. The labelling groups. Any co-medication known to influence either characteristics were unaffected by the treatments (Table platelet function or the prostaglandin system was 2). The treatment did not cause a change in blood excluded. Patients with cerebrovascular disease were pressure. Patients in group 1 and 3 showed a significant excluded. The demographic data are summarized in (P < 0.01) improvement in number and severity of Table 1. The study population included patients with anginal attacks (detailed data not shown) as compared hypertension and with hyperlipoproteinemia. The study with group 2 (PGI2 alone). was performed in accordance with the Declaration of Helsinki. All patients had given written informed consent. Discussion Radiolabelling ofplatelets The action of sydnonimines, a group of antianginal sub- One diagnostic radiolabelling of platelets was performed stances (Bohn & Schonafinger, 1989), is believed to be 1 to 2 weeks prior to treatment. A second labelling was due to nitric oxide release from inactive prodrugs. After performed at the beginning of the 5th week of treatment. Autologous platelets were labelled using a radiolabelling kit (Karmed, Kosterneuburg, Austria) as described 1.351 earlier (Sinzinger & Fitscha, 1984). Briefly, 16 ml of blood were anticoagulated with 4 ml acid citrate dextrose. Platelet rich plasma was isolated by a 10 min 1.30 - centrifugation at 100 g. This platelet rich plasma was further centrifuged at 500 g for 5 min to obtain a platelet 0 pellet. This was resuspended in Tyrode buffer (final 1.25 - volume 100 ,tl; pH 6.2). 100 ,uCi '11-Indium-oxine (SGAE were Seibersdorf, Austria) used for radiolabelling. Before a) reinjection of autologous radiolabelled platelets an 1 .201- aliquot was drawn for the estimation of labelling Cua) efficiency. Thereafter, blood samples (2 ml) were drawn 4) 3 times daily for 1 week. The 1 h blood sample was used to calculate the recovery. For the calculation of platelet 1.15 - survival we used the multiple hit model computer program kindly provided by Professor A. duP.Heyns, SA Blood Transfusion Service, Johannesburg, South- 1.10 Africa. Gamma-camera images Pre Post Figure 1 Synergism of molsidomine and PGI2 on platelet Gamma-camera images were obtained using a large uptake ratio (mean ± s.e. mean, n = 12). The combined field of view gamma-camera (Siemens, Erlangen, application was most effective, while molsidomine alone was Germany) and a matrix of 64 x 64 in AP-view. Exposure not effective. *P < 0.01. 0 molsidomine, * PGI2, time was 4-5 min. Images were generated daily for 5 V molsidomine + PGI2. Prostaglandin I2 and molsidomine 291

180- antiplatelet agents examined in the same test system reduce thrombogenicity and prolong platelet survival to a much lesser extent. However, if this assumption is true, it confirms that exogenous nitric oxide-supplemen- 170 tation results in an additional in vivo benefit. As compared with earlier studies using isosorbide dinitrate (in combination with PGE1), the effects of molsidomine 160 (together with PGI2) are ofsimilar magnitude (Sinzinger et al., 1990). This is surprising, considering that SIN- 1 A shows the maximal nitric oxide liberation at pH 7.4 > 150- (Gryglewski et al., 1990), whereas other nitric oxide donors do so at lower pH. While SIN-1 increases cGMP (Gerzer et al., 1988), other nitrates do not. The role of 140 other mechanisms, such as the decrease in leukotriene B4-formation (Ney et al., 1990) and inhibition of mono- cyte adhesion (via cGMP-increase, (R. J. Gryglewski, personal communication) alone does not seem to bene- ficially affect thromboresistance. A synergism between PGI2 and nitric oxide or between Pre Post iloprost and sodium nitroprusside in decreasing platelet Figure 2 Synergistic prolongation in platelet survival (h) by activity in vitro is probably mediated via enhanced molsidomine and prostaglandin 12 (mean ± s.e. mean, n = 12). platelet cAMP (Gorman et al.,1977) and cGMP. An *P < 0.01. 0 molsidomine, * PGI2, V molsidomine + PGI2. equivalent synergism with glyceryl trinitrate has not been examined so far. Mechanical factors such as stress Table 2 Labelling characteristics and pulsatile flow are likely to be major stimuli for nitric oxide and PGI2 release. The nitric oxide activity is Group highest in large arterioles where the hydraulic resistance 1 2 3 and shear stress are also highest. A variety of risk factors such as hypertension (Lockette et al., 1986), Labelling hyperlipoproteinemia (Shimokawa & Vanhoutte, 1989), 89 ± 4 91 ± 3 88 ± 5 before diabetes (Kamata et al., 1989), and atherosclerosis efficiency (%) 91 ± 4 90 ± 4 90 ± 4 after (Ludmer et al., 1986) affect PGI2 and EDRF-release 60±6 57±8 61±7 before (Gryglewski et al., 1989). Hence, both the difference in recovery (%) 62 ± 7 59 ± 7 60 ± 8 after the synthesis of local autacoids as well as the stimuli the endothelium is subjected to, are likely to act as Groups: 1 molsidomine, 2 PGI2, 3 combination 1 + 2 important regulating factors. Activated platelets and normal values: labelling efficiency > 80%, recovery also leucocytes play an important role in the pathogenesis > 50%. of critical ischaemia. A locally reduced availability of nitric oxide (Vallance et al., 1989) could lead to enhanced adhesion of platelets and monocytes to the vessel wall administration of molsidomine the initial step is the (Radomski et al., 1987b). Although various EDRFs hydroxyl ion-dependent conversion, the transformation have been described, at least one shares the properties of SIN-1 to SIN-1 A which occurs immediately at of nitric oxide. In view of these findings the role of the neutral pH. The subsequent mechanism of nitric oxide- clinical use of cyclooxygenase inhibitors has to be release, however, is not completely understood (Bohn addressed. From the pharmacological point of view a & Schonafinger, 1989). dose of 40 mg daily or even lower seems to be preferable These results extend the finding of in vivo synergism (Patrono et al., 1980; Sinzinger et al., 1988). The cell- between PGI2 and molsidomine transformation products selective synergism of nitric oxide and PGI2 in main- (Bult et al., 1989). Decreased thrombogenicity has also taining separation between platelets and leucocytes and been shown after molsidomine and PGI2 in cultured the vessel wall offers a rationale for the treatment of porcine femoral artery endothelial cells (Berenger- acute events such as unstable angina, while avoiding Bahnet & Rolland, 1989) as well as in a tubular segment hypotensive side effects. of rabbit aorta (Gryglewski et al., 1989). Hence endogenous EDRF may contribute to the beneficial local The authors gratefully acknowledge the valuable help of Dr haemostatic effects of PGI2 reported earlier (Sinzinger K. Weiss, in performing the statistical analysis and of Eva et al., 1984). This is supported by the finding that other Unger in preparing and typing the manuscript.

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