A Long-Acting Prostacyclin Agonist with Thromboxane Inhibitory Activity for Pulmonary Hypertension

Pulmonary arterial hypertension is a rare but life-threatening disease (1, 2). The pathogenesis includes pulmonary vasoconstriction, endothelial cell proliferation, smooth muscle cell proliferation, and in situ thrombosis (3, 4). Prostacyclin, a metabolite of arachidonic acid, has vasoprotective effects, including vasodilation, antiplatelet aggregation, and inhibition of smooth muscle cell proliferation (5-8). Thus, continuous intravenous infusion of prostacyclin (epoprostenol) has become recognized as a therapeutic breakthrough for pulmonary arterial hypertension (9-16). The dramatic success of long-term intravenous prostacyclin has led to the development of prostacyclin analogs (oral beraprost, aerosolized iloprost, and subcutaneous treprostinil; Figure 1) (17-20). Nevertheless, treatment with prostacyclin or its analogs has some problems in the clinical setting. Their duration of acting is so short that they need to be continuously infused or frequently administered (9-20). In addition, these compounds failed to inhibit thromboxane synthesis during treatment (21).

We developed a new type of prostacyclin agonist, ONO-1301, which has long-lasting prostacyclin activity and thromboxane synthase inhibitory activity. Prostacyclin and its analogs are unstable because 15-hydroxyprostaglandin dehydrogenase metabolizes their prostanoid structures, including a five-membered ring and allylic alcohol. In contrast, ONO-1301 is chemically and biologically stable because of the absence of prostanoid structures. Interestingly, ONO-1301 has thromboxane synthase inhibitory activity because of the presence of a 3-pyridine radical. It has been reported that augmented release of thromboxane A^sub 2^, which is both a potent pulmonary vasoconstrictor and a procoagulant (22), occurs in patients with pulmonary hypertension (23, 24). The imbalance of thromboxane and prostacyclin is considered to contribute to the development of pulmonary arterial hypertension. These findings raise the possibility that administration of ONO-1301 may have beneficial effects on pulmonary hemodynamics.

Thus, the purposes of this study were (1) to investigate whether a single subcutaneous administration of ONO-1301 has long-lasting prostacyclin activity in rats, (2) to investigate whether subcutaneous administration of ONO-1301 inhibits thromboxane synthesis in monocrotaline (MCT)-induced pulmonary hypertension in rats, (3) to examine whether intermittent subcutaneous ONO-1301 improves pulmonary hemodynamics and survival in MCT rats, and (4) to elucidate the underlying mechanisms responsible for the beneficial effects of this compound.

METHODS

Animals

We used 95 male Wistar rats weighing 100 to 120 g. The rats were randomly given a subcutaneous injection of either 60 mg/kg MCT (MCT rats) or 0.9% saline (vehicle) and assigned to receive repeated subcutaneous injection of ONO-1301 (Ono Pharmaceutical Co., Ltd., Osaka, Japan) or vehicle. This protocol resulted in the creation of three groups: sham rats given vehicle (n = 10), MCT rats given vehicle (n = 10), and MCT rats treated with ONO-1301 (n = 13). In addition, 20 rats were studied to evaluate the effect of ONO-1301 on survival in MCT rats. Furthermore, 42 rats were studied to evaluate the effect of ONO1301 on plasma cAMP level (n = 12) and 11-dehydro-thromboxane B^sub 2^ (TXB^sub 2^) level (n = 30).

In Vivo Experimental Protocol

After rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg), they were given a subcutaneous injection of either 60 mg/kg MCT or vehicle. Then, ONO-1301 (20 mg/kg/d) or vehicle was injected subcutaneously twice per day for 3 wk after MCT injection. Animals were maintained on standard rat chow. Hemodynamic studies were performed on Day 22. 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 right ventricle (RV) for measurement of RV pressure. Finally, cardiac arrest was induced by injection of 2 mmol/L potassium chloride through the catheter. The ventricles and lungs were excised, dissected free, and weighed. The ratio of RV weight to body weight (RV/BW), the ratio of left ventricular plus septal weight to body weight (LV + S/BW), and the ratio of RV weight to left ventricular plus septal weight (RV/LV + S) were calculated as indexes of ventricular hypertrophy, as reported previously (25). All protocols were performed in accordance with guidelines of the Animal Care Ethics Committee of the National Cardiovascular Center Research Institute.

Morphometric Analysis of Pulmonary Arteries

Paraffin sections 4-

Assay for Plasma ONO-1301 Concentration

To estimate the half-life of ONO-1301, we measured plasma ONO-1301 concentration in rats after a subcutaneous injection (n = 4). Blood was drawn at 0.25, 0.5, 1, 2, 4, 8, and 24 h after a single subcutaneous administration of ONO-1301 (10 mg/kg). Plasma ONO-1301 concentration was measured by liquid chromatography tandem mass spectrometry assay.

Assay for Plasma cAMP Level

To investigate whether a single subcutaneous administration of ONO-1301 has long-lasting prostacyclin activity in rats, we measured plasma cAMP levels after ONO-1301 injection. Twelve rats were assigned to receive a single subcutaneous injection of ONO-1301 (10 mg/kg) or vehicle (n = 6 each). Blood was drawn from the right carotid artery at baseline and 1, 2, 4, 6, and 8 h after ONO-1301 injection. Blood was immediately transferred into a chilled glass tube containing disodium ethylenediaminetetraacetic acid (1 mg/ml) and aprotinin (500 U/ml) and centrifuged immediately. Plasma cAMP levels was measured with a radioimmunoassay kit (cAMP assay kit; Yamasa Shoyu, Chiba, Japan), as reported previously (27).

Assay for Plasma 11-dehydro-TXB^sub 2^ Level

To investigate the acute effect of ONO-1301 or prostacyclin (epoprostenol) on thromboxane synthesis in MCT rats, we measured plasma 11-dehydro-TXB^sub 2^, a metabolite of thromboxane A^sub 2^, after administration of ONO-1301 (10 mg/kg), epoprostenol, or vehicle (n = 5 each). Epoprostenol was infused via a polyethylene catheter (PE-50) inserted into the right jugular vein. Infusion of epoprostenol was begun at 10 ng/kg/min, increased gradually to 150 ng/kg/min over 30 min, escalated to 300 ng/kg/min over the next 30 min, and held at this dose for 1 h, as reported previously (21, 28, 29).

To investigate the chronic effect of ONO-1301 on thromboxane synthesis in MCT rats, we measured plasma 11-dehydro-TXB^sub 2^ after repeated subcutaneous injection of ONO-1301 (20 mg/kg/d) or vehicle twice per day for 3 wk (n = 5 each). Blood was drawn from the right carotid artery and plasma 11-dehydro-TXB^sub 2^ level was measured with an enzyme immunoassay kit (11-dehydro-TXB^sub 2^ assay kit; Cayman Chemical Co., Ann Arbor, MI), as reported previously (30).

Survival Analysis

To evaluate the effect of intermittent subcutaneous administration of ONO-1301 on survival in MCT rats, 20 rats received repeated injection of ONO-1301 or vehicle twice per day (n = 10 each). Survival was estimated from the date of MCT injection to the death of the rat or 6 wk after injection.

Statistical Analysis

All data were expressed as mean ± SEM. Comparisons of parameters among the three groups were made by one-way analysis of variance, followed by Newman-Keuls' test. Comparisons of the time course of parameters between the two groups were made by two-way analysis of variance for repeated measures, followed by Newman-Keuls' test. Survival curves were derived by the Kaplan-Meier method and compared by log-rank test. A value of p < 0.05 was considered statistically significant.

RESULTS

Effects of ONO-1301 on Pulmonary Hemodynamics and Vascular Remodeling

RV systolic pressure was significantly increased 3 wk after MCT injection (Figure 2A). However, the increase was significantly attenuated by subcutaneous administration of ONO-1301 (10 mg/kg twice per day). Similarly, the increases in RV/BW and RV/LV + S in MCT rats were significantly attenuated by treatment with ONO-1301 (Figures 2B and 2C). There were no significant differences in heart rate or mean arterial pressure among the three groups (Table 1).

Representative photomicrographs showed that hypertrophy of the pulmonary vessel wall after MCT injection was attenuated in MCT rats treated with ONO-1301 compared with those given vehicle (Figure 3A). Quantitative analysis demonstrated a significant increase in percent wall thickness after MCT injection, but this change was ameliorated by ONO-1301 (Figure 3B).

Long-Lasting Activity of ONO-1301

We measured plasma ONO-1301 concentrations after a single subcutaneous administration of ONO-1301. The increase in plasma ONO-1301 concentration reached a peak at 4 h, and the half-life of plasma ONO-1301 concentration was approximately 5.6 h (Figure 4). In addition, a single subcutaneous administration of ONO-1301 significantly increased plasma cAMP level in rats (Figure 5). The increase in plasma cAMP level reached a peak at 6 h and lasted at least up to 8 h after ONO-1301 injection. These results suggest that subcutaneous administration of ONO-1301 has long-lasting activity in rats.

Inhibitory Effect of ONO-1301 on Thromboxane Synthase

Although administration of prostacyclin (epoprostenol) markedly increased plasma 11-dehydro-TXB^sub 2^ level in MCT rats with established pulmonary hypertension, ONO-1301 did not significantly increase plasma 11-dehydro-TXB^sub 2^ level, even after bolus injection (Figure 6A). Plasma 11-dehydro-TXB^sub 2^ level was markedly elevated 3 wk after MCT injection (Figure 6B). However, 3-wk treatment with ONO-1301 significantly attenuated the increase in plasma 11-dehydro-TXB^sub 2^ level in MCT rats.

Survival Analysis

Kaplan-Meier survival curves demonstrated that MCT rats treated with ONO-1301 had a significantly higher survival rate than MCT rats given vehicle (80 vs. 30% in 6-wk survival; Figure 7).

DISCUSSION

In the present study, we demonstrated that (7) a novel prostacyclin agonist (ONO-1301) ameliorated the development of MCT-induced pulmonary hypertension and improved survival in MCT rats; (2) ONO-1301 had a long half-life of approximately 5.6 h, and a single administration of ONO-1301 caused a longlasting increase in plasma cAMP level; and (3) ONO-1301 attenuated the increase in plasma 11-dehydro-TXB^sub 2^ level in MCT rats.

Conventional prostacyclin and its analogs need continuous infusion or frequent administration because of their short duration of acting. Epoprostenol has a very short half-life (< 6 min), iloprost has a serum half-life of 20 to 25 min, and the elimination half-life of beraprost is 35 to 40 min after oral administration (31). Treprostinil sodium, a stable prostacyclin analog, has been reported to have a half-life of 4.6 h after cessation of continuous subcutaneous infusion (32). With regard to cAMP, a second messenger of prostacyclin and its analogs, it has been reported that plasma cAMP levels remained increased at 4 h and normalized at 6 h after inhalation of iloprost (33), and that plasma cAMP levels reached a peak at 30 min and subsequently returned to baseline levels at 2 h after administration of oral beraprost (27). In our results, the half-life of plasma ONO-1301 concentration was approximately 5.6 h, and a single subcutaneous administration of ONO-1301 increased plasma cAMP level at least up to 8 h. Because the method for administration was different between ONO-1301 and conventional prostacyclin analogs (compare a subcutaneous single shot of ONO-1301, continuous intravenous infusion of epoprostenol, continuous subcutaneous infusion of treprostinil, inhalation of iloprost, and oral administration of beraprost), it is difficult to directly compare the lasting effects of prostacyclin activity of ONO-1301 with that of conventional prostacyclin analogs. Nevertheless, the long half-life of ONO-1301 and long-lasting increases in plasma cAMP levels indicate that ONO-1301 exhibits chemical and biologic stability comparable to that of conventional prostacyclin and its analogs. ONO-1301 does not contain prostanoid structures such as a five-membered ring and allylic alcohol, which are subject to metabolism by 15-hydroxyprostaglandin dehydrogenase. These may be the reason for the long-lasting activity of ONO-1301. The present study also demonstrated that repeated administration of ONO-1301 twice per day markedly attenuated the development of MCT-induced pulmonary hypertension, as indicated by significant decreases in RV systolic pressure and RV weight. Thus, intermittent subcutaneous administration of ONO-1301 may be sufficient for the treatment of pulmonary hypertension.

Thromboxane, produced by endothelial cells and platelets, has a potent vasoconstrictor effect, smooth muscle mitogenic property, and platelet aggregation effect (22). Earlier studies have demonstrated impaired prostacyclin synthesis and increased thromboxane production in patients with pulmonary arterial hypertension, suggesting that imbalance of the release of thromboxane and prostacyclin plays an important role in the development of pulmonary hypertension (23, 24). Furthermore, thromboxane-receptor density is increased in the RV of patients with pulmonary hypertension (34). Rich and colleagues have shown that inhibition of thromboxane synthase modestly improves pulmonary hemodynamics in patients with pulmonary arterial hypertension (35). ONO-1301 has a 3-pyridine radical, which is known to inhibit thromboxane synthase through interaction with carboxylic acid via a hydrogen bond. In the present study, plasma 11-dehydro-TXB^sub 2^ level was markedly elevated in MCT rats. However, treatment with ONO-1301 greatly diminished its level. Furthermore, in the acute phase, plasma 11-dehydro-TXB^sub 2^ level in MCT rats did not significantly increase after administration of ONO-1301, although the plasma level markedly increased after epoprostenol infusion, which is consistent with the earlier study of Cuiper and colleagues (21). Therefore, it is possible that ONO-1301 attenuates MCT-induced pulmonary hypertension partly via improvement of prostacyclin/thromboxane imbalance.

In the present study, ONO-1301 also attenuated the increase in medial wall thickness of peripheral pulmonary arteries. Activation of prostacyclin receptors has been shown to suppress the growth of vascular smooth muscle cells through a cAMPdependent pathway. Thus, ONO-1301 may attenuate the development of pulmonary vascular remodeling at least in part via a cAMP-dependent pathway. Importantly, repeated administration of ONO-1301 improved survival in MCT rats compared with vehicle administration. The increased survival rate in MCT rats is considered to be associated with the amelioration of pulmonary hypertension. Thus, intermittent subcutaneous administration of ONO-1301 may be an alternative approach for severe pulmonary hypertension refractory to conventional therapy.

In conclusion, subcutaneous administration of a novel prostacyclin agonist (ONO-1301) markedly attenuated MCT-induced pulmonary hypertension and improved survival in rats. The beneficial effects of ONO-1301 may occur through its long-lasting stimulation of cAMP and inhibition of thromboxane synthase. Thus, administration of this compound may be a promising therapeutic strategy for the treatment of pulmonary arterial hypertension.

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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