Published ahead of print on October 2, 2003, doi:10.1164/rccm.200303-346OC
© 2004 American Thoracic Society A Combination of Oral Sildenafil and Beraprost Ameliorates Pulmonary Hypertension in RatsDepartment of Internal Medicine, National Cardiovascular Center; Departments of Cardiac Physiology, Biochemistry, and Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, Osaka; and Second Department of Internal Medicine, Nara Medical University, Nara, Japan Correspondence and requests for reprints should be addressed to Noritoshi Nagaya, M.D., Department of Internal Medicine, National Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: nagayann{at}hsp.ncvc.go.jp
Sildenafil, an oral phosphodiesterase type-5 inhibitor, has vasodilatory effects through a cyclic guanosine 3', 5'-monophosphate–dependent mechanism, whereas beraprost, an oral prostacyclin analog, induces vasorelaxation through a cAMP-dependent mechanism. We investigated whether the combination of oral sildenafil and beraprost is superior to each drug alone in the treatment of pulmonary hypertension. Rats were randomized to receive repeated administration of saline, sildenafil, beraprost, or both of these drugs twice a day for 3 weeks. Three weeks after monocrotaline (MCT) injection, there was significant development of pulmonary hypertension. The increases in right ventricular systolic pressure and ratio of right ventricular weight to body weight were significantly attenuated in the Sildenafil and Beraprost groups. Combination therapy with sildenafil and beraprost had additive effects on increases in plasma cAMP and cyclic guanosine 3', 5'-monophosphate levels, resulting in further improvement in pulmonary hemodynamics compared with treatment with each drug alone. Unlike MCT rats given saline, sildenafil, or beraprost alone, all rats treated with both drugs remained alive during 6-week follow-up. These results suggest that combination therapy with oral sildenafil and beraprost attenuates the development of MCT-induced pulmonary hypertension compared with treatment with each drug alone.
Key Words: pulmonary hypertension • sildenafil • beraprost • monocrotaline Primary pulmonary hypertension is a rare but life-threatening disease characterized by progressive pulmonary hypertension that leads to right ventricular (RV) failure and death (1). The median survival is considered to be 2.8 years from the time of diagnosis. Therefore, a novel therapeutic strategy for primary pulmonary hypertension is desirable. Sildenafil is a phosphodiesterase type-5 inhibitor, which has been used for the treatment of erectile dysfunction in men (2, 3). Interestingly, phosphodiesterase type-5 is abundantly expressed not only in the corpus cavernosum but also in the lungs (4), especially in vascular smooth muscle cells (5). Thus, sildenafil serves as a pulmonary vasodilator through a cyclic guanosine 3', 5'-monophosphate (cGMP)–dependent mechanism (6–10). Prostacyclin produces strong vasodilation and inhibition of platelet aggregation. Beraprost is an orally active prostacyclin analog whose biological effects are believed to be mediated by cAMP (11). In humans, beraprost has been reported to be partially effective for pulmonary arterial hypertension (12–14). Currently, continuous intravenous infusion of epoprostenol is considered to be a therapeutic breakthrough for the treatment of primary pulmonary hypertension. In the clinical setting, however, oral ingestion may be more simple, noninvasive, and comfortable than continuous intravenous infusion therapy. Considering that sildenafil and beraprost dilate pulmonary vessels through different mechanisms, combination therapy with both drugs may have additive or synergistic effects on pulmonary hemodynamics. Thus, the purpose of this study was to investigate whether the combination of oral sildenafil and beraprost ameliorates monocrotaline (MCT)-induced pulmonary hypertension compared with each drug alone.
Animals Male Wistar rats weighing 80 to 100 g were used in this study. Rats were randomly given a subcutaneous injection of either 60 mg/kg MCT or 0.9% saline and assigned to receive oral administration of 0.9% saline, sildenafil, beraprost, or both of these drugs. This protocol resulted in the creation of five groups: normal rats given saline (Sham group, n = 10), MCT rats given saline (Control group, n = 10), MCT rats treated with oral sildenafil (Sildenafil group, n = 10), MCT rats treated with oral beraprost (Beraprost group, n = 10), and MCT rats treated with both of these drugs (Combination group, n = 10). Hemodynamic measurements and histologic analyses were performed 3 weeks after MCT injection. Additionally, 40 rats were studied to evaluate the effects of these treatments on survival in MCT rats (Control, Sildenafil, Beraprost, and Combination groups, n = 10 each). Finally, 20 rats were studied to examine the effects of sildenafil and beraprost on plasma cAMP and cGMP (Control, Sildenafil, Beraprost, and Combination groups, n = 5 each).
Experimental Protocol Hemodynamic studies were performed on Day 22. Rats were anesthetized with intraperitoneal pentobarbital (30 mg/kg) and placed on a heating pad to maintain body temperature at 37 to 38°C throughout the study. A polyethylene catheter (PE-50) was inserted into the right carotid artery to measure heart rate and mean arterial pressure. A polyethylene catheter (PE-50) was inserted through the right jugular vein into the RV for measurement of RV pressure. These hemodynamic variables were measured with a pressure transducer (model P 23 ID; Gould, Cleveland, OH) connected to a polygraph and recorded with a thermal recorder (7758 B System; Hewlett-Packard, Waltham, MA). Finally, cardiac arrest was induced by injection of 2 mmol potassium chloride through the catheter. The ventricles and lungs were excised, dissected free, and weighed. The measurement of the RV weight excluded the intraventricular septum. The ratio of RV weight to body weight (RV/BW), the ratio of left ventricular plus septum (LV + S) weight to body weight (LV + S/BW), and the ratio of RV weight to LV + S weight (RV/LV + S) were calculated as indexes of ventricular hypertrophy. To evaluate the effects of combination therapy on plasma cAMP and cGMP, normal rats were assigned to receive a single administration of saline, sildenafil (25 mg/kg), beraprost (100 µg/kg), or both drugs. Blood was drawn from the right carotid artery for measurements of cAMP and cGMP at 0, 30, 60, 120, and 180 minutes after administration.
Morphometric Analysis of Pulmonary Arteries
Assay for Plasma cAMP and cGMP Levels
Survival Analysis
Statistical Analysis
Effects of Combination Therapy on Physiologic Profile Body weight did not significantly differ among the four MCT groups (Table 1) . RV/BW was significantly increased after MCT injection (Figure 1) . However, the increase was significantly attenuated in the Sildenafil, Beraprost, and Combination groups compared with that in the Control group. Importantly, RV/BW in the Combination group was significantly lower than that in the Sildenafil and Beraprost groups.
Effects of Combination Therapy on Hemodynamics RV systolic pressure was significantly increased after MCT injection (Figure 2) . However, the increase was significantly attenuated in the Sildenafil and Combination groups compared with the Control group. Combination therapy caused further improvement in RV systolic pressure after MCT injection. There were no significant differences in heart rate or mean arterial pressure among the five groups (Table 1).
Morphometric Analysis of Pulmonary Arteries Representative photomicrographs showed that hypertrophy of the pulmonary vessel wall was attenuated in the Combination group compared with that in the Control, Sildenafil, and Beraprost groups (Figure 3) . Quantitative analysis of peripheral pulmonary arteries demonstrated that the increase in %wall thickness after MCT injection was significantly attenuated in the Sildenafil, Beraprost, and Combination groups (Figure 4) . Expectedly, hypertrophy of the pulmonary vessel wall was significantly attenuated in the Combination group compared with that in the Sildenafil and Beraprost groups.
Effects of Combination Therapy on Plasma cAMP and cGMP Levels Plasma cAMP level was significantly higher in the Beraprost and Combination groups than in the Control and Sildenafil groups 30 minutes after administration (Figure 5) . Interestingly, the increase in plasma cAMP level lasted longer than 180 minutes in the Combination group, whereas it reached maximum at 30 minutes and substantially decreased in the Beraprost group. As a result, the plasma cAMP level from 60 to 180 minutes was significantly higher in the Combination group than in the Beraprost group. The plasma cGMP level was significantly higher in the Sildenafil than in the Control and Beraprost groups. Combination therapy induced a further increase in plasma cGMP level compared with sildenafil therapy alone.
Survival Analysis Kaplan–Meier survival curves demonstrated that the Sildenafil, Beraprost, and Combination groups had a significantly higher survival rate than the Control group (Figure 6) . Unlike MCT rats given saline, sildenafil, or beraprost alone, all rats in the Combination group remained alive during 6-week follow-up (Control group, 30%; Sildenafil group, 90%; Beraprost group, 80%; Combination group, 100%).
In the present study, we demonstrated that (1) the combination of oral sildenafil and beraprost ameliorated the development of MCT-induced pulmonary hypertension and pulmonary vascular remodeling compared with each drug alone, and that (2) unlike MCT rats given saline, sildenafil, or beraprost alone, all rats in the Combination group remained alive during 6-week follow-up. We also demonstrated that (3) combination therapy induced additive effects on increases in plasma cAMP and cGMP levels. Currently, intravenous infusion of prostacyclin is established as treatment for primary pulmonary hypertension. The dramatic success of long-term intravenous prostacyclin is now leading to the development of prostacyclin analogs using new drug delivery systems (oral beraprost, aerosolized iloprost, and subcutaneous treprostinil) (17–21). Sildenafil, a therapeutic agent for erectile dysfunction, has recently been shown to attenuate pulmonary hypertension (6–10). Sildenafil and beraprost allow oral administration, which is attractive for the patient and doctor. Unfortunately, however, each drug is not effective enough to inhibit pulmonary hypertension. Sildenafil causes pulmonary vasorelaxation through a cGMP-dependent mechanism, whereas beraprost dilates pulmonary vessels through a cAMP-dependent mechanism. As a general pharmacologic principle, these drugs that produce similar effects through different mechanisms may have additive or synergistic effects when combined. In fact, the combination of oral sildenafil and beraprost significantly attenuated the increases in RV systolic pressure and RV/BW compared with treatment with each drug alone. These findings suggest that the combination of oral sildenafil and beraprost is superior to each drug alone in ameliorating the development of MCT-induced pulmonary hypertension. Interestingly, no significant decrease in systemic arterial pressure was observed during combination therapy as well as treatment with each drug alone, suggesting that oral administration of these drugs causes relatively selective pulmonary vasodilation. MCT-induced endothelial cell injury has been shown to activate platelets and vasoconstrictive factors, resulting in pulmonary hypertension and pulmonary vascular remodeling (22). Sildenafil has an inhibitory effect on pulmonary hypertension and vascular remodeling in rats through the endothelial nitric oxide synthase–nitric oxide–cGMP pathway (6). Prostacyclin not only has an antiplatelet aggregation but also inhibits smooth muscle cell proliferation (23, 24). In the present study, histologic examination of MCT rats revealed that combination therapy significantly inhibited an increase in medial wall thickness of peripheral pulmonary arteries compared with treatment with each drug alone. These findings suggest that the combination of oral sildenafil and beraprost is superior to each drug alone in inhibiting the development of pulmonary vascular remodeling after MCT injection. Interestingly, the combination of oral sildenafil and beraprost increased plasma cAMP level compared with treatment with beraprost alone. This may be explained by a recent finding that sildenafil inhibits not only cGMP breakdown by phosphodiesterase type-5 but also cAMP breakdown by phosphodiesterase type-3 in vivo (25). On the other hand, Koide and coworkers (26) showed that an elevation of intracellular cAMP positively regulates nitric oxide production, resulting in increased cGMP. In fact, combination therapy induced a further increase in plasma cGMP level compared with sildenafil therapy alone. These results suggest that combination therapy with sildenafil and beraprost has additive effects on increases in plasma cAMP and cGMP levels. It is interesting to speculate that the additive effects of sildenafil and beraprost may be responsible for marked improvement in pulmonary hemodynamics in MCT rats. Oral administration of sildenafil or beraprost improved survival in MCT rats, which is consistent with earlier studies (10, 27, 28). Surprisingly, all rats treated with both drugs remained alive during 6-week follow-up in the present study. The increased survival rates in MCT rats are considered to be associated with the amelioration of pulmonary hypertension (27). Thus, this combination therapy may be an alternative approach for severe pulmonary hypertension that is refractory to conventional therapy. Sildenafil and beraprost can be given by oral administration. Given the potential risks and high medical costs of an invasive method, oral combination therapy may be worth trying before intravenous infusion therapy is considered. However, the therapeutic potential of this combination therapy in patients with pulmonary arterial hypertension should be confirmed by long-term, large-scale clinical studies. This study includes some study limitations. The present study demonstrated that combination therapy attenuated the development of MCT-induced pulmonary hypertension. Further studies are necessary to investigate whether this therapy is effective for the treatment of established pulmonary hypertension. MCT injection induces a liver toxicity (29), which may influence pharmacokinetics of drugs. Nevertheless, both sildenafil and beraprost have been reported to attenuate pulmonary hypertension in humans (8, 12–14). Clinical trials are necessary to confirm the efficacy of the combination therapy for treatment of pulmonary hypertension. In conclusion, the combination of oral sildenafil and beraprost attenuated the development of MCT-induced pulmonary hypertension compared with treatment with each drug alone. This combination therapy may be a new therapeutic strategy for the treatment of pulmonary arterial hypertension.
Supported by RHGTEFB-genome-005 and the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan. Conflict of Interest Statement: T.I. has no declared conflict of interest; N.N. has no declared conflict of interest; T.F. has no declared conflict of interest; T.I. has no declared conflict of interest; N.N. has no declared conflict of interest; K.H. has no declared conflict of interest; K.K. has no declared conflict of interest; H.K. has no declared conflict of interest. Received in original form March 9, 2003; accepted in final form September 26, 2003
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ABSTRACT
METHODS
p < 0.05 versus Sildenafil group; 
p < 0.05 versus Beraprost group.
