help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on November 1, 2007, doi:10.1164/rccm.200703-349OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200703-349OCv1
177/2/195    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Obata, H.
Right arrow Articles by Nagaya, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Obata, H.
Right arrow Articles by Nagaya, N.
American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 195-201, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200703-349OC

Original Article

Single Injection of a Sustained-release Prostacyclin Analog Improves Pulmonary Hypertension in Rats

Hiroaki Obata1,2, Yoshiki Sakai3, Shunsuke Ohnishi1, Satoshi Takeshita4, Hidezo Mori5, Makoto Kodama2, Kenji Kangawa6, Yoshifusa Aizawa2 and Noritoshi Nagaya1,4

1 Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, Osaka, Japan; 2 Division of Cardiology, Niigata University Graduate School of Medical and Dental Science, Niigata, Japan; 3 Ono Pharmaceutical Co. Ltd., Research Headquarters, Osaka, Japan; 4 Department of Internal Medicine, National Cardiovascular Center, Osaka, Japan; 5 Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Osaka, Japan; and 6 Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan

Correspondence and requests for reprints should be addressed to Noritoshi Nagaya, M.D., Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: nnagaya{at}ri.ncvc.go.jp


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Although prostacyclin is recognized as a therapeutic breakthrough for pulmonary hypertension, it needs continuous infusion because of its short action. Therefore, we developed a new drug delivery system for prostacyclin. We prepared ONO-1301MS, a novel sustained-release prostacyclin analog polymerized with poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres.

Objectives: We examined whether ONO-1301MS attenuates monocrotaline (MCT)-induced pulmonary hypertension in rats, and attempted to elucidate the underlying mechanisms responsible for the beneficial effects of ONO-1301MS.

Methods: After MCT injection, rats were randomized to receive a single subcutaneous injection of 100 mg/kg ONO-1301MS or vehicle.

Measurements and Main Results: We prepared ONO-1301MS, which was polymerized with PLGA to release ONO-1301 for 3 weeks. A single administration of ONO-1301MS achieved sustained elevation of its circulating level and plasma cyclic adenosine 3',5'-monophosphate level for 3 weeks, and attenuated an increase in a metabolite of thromboxane A2 level. Rats had developed pulmonary hypertension 3 weeks after MCT injection; however, treatment with ONO-1301MS significantly attenuated the increases in right ventricular systolic pressure and right ventricular weight to body weight ratio. ONO-1301MS significantly inhibited hypertrophy of pulmonary arteries. Phosphorylation of extracellular signal-regulated protein kinase (ERK) in the lung was significantly increased in the control group, whereas this increase was markedly attenuated by treatment.

Conclusions: We developed a new drug delivery system for prostacyclin using PLGA and ONO-1301. A single injection of ONO-1301MS resulted in sustained activity for 3 weeks, and attenuated pulmonary hypertension, partly through its antiproliferative effect on vascular smooth muscle cells via inhibition of ERK phosphorylation.

Key Words: pulmonary hypertension • prostacyclin analog • sustained-release preparation • extracellular signal regulated kinase • poly(lactic-co-glycolic acid)



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Although prostacyclin is recognized as a therapeutic breakthrough for pulmonary hypertension, it needs continuous infusion because of its short action. For patients with pulmonary hypertension, development of a sustained-release prostacyclin would be beneficial in terms of stable hemodynamics and quality of life.

What This Study Adds to the Field
A single injection of ONO-1301MS resulted in sustained activity for 3 weeks, and attenuated pulmonary hypertension in rats.

 
Pulmonary arterial hypertension is a rare but life-threatening disease characterized by progressive pulmonary hypertension that leads to right ventricular (RV) failure and death (1). Prostacyclin, a metabolite of arachidonic acid, has vasoprotective effects, including vasodilation, antiplatelet aggregation, and inhibition of smooth muscle cell (SMC) proliferation (2–4). Thus, continuous intravenous infusion of prostacyclin (epoprostenol) has become recognized as a therapeutic breakthrough for pulmonary arterial hypertension (5–7). The dramatic success of long-term intravenous prostacyclin has led to the development of prostacyclin analogs (8–11). Nevertheless, treatment with prostacyclin or its analogs has some problems in the clinical setting. Epoprostenol therapy requires a continuous intravenous infusion device, and is therefore more invasive and uncomfortable than taking prostacyclin analogs. On the other hand, prostacyclin analogs, such as subcutaneously infused treprostinil, inhaled iloprost, and oral beraprost, need continuous infusion or frequent administration because of their short duration of action (5–11). In fact, epoprostenol has a very short half-life (<6 min) (12), treprostinil has been reported to have a half-life of 4.6 hours after cessation of continuous subcutaneous infusion (13), iloprost has a serum half-life of 20 to 25 minutes, and the elimination half-life of beraprost is 35 to 40 minutes after oral administration (12).

Recently, we developed a new type of prostacyclin agonist, ONO-1301 (Figure 1), which has long-lasting prostacyclin activity and an inhibitory effect on thromboxane synthase (14). ONO-1301 does not contain prostanoid structures, such as a five-membered ring or allylic alcohol, which are digested by 15-hydroxyprostaglandin dehydrogenase (Figure 1). These structures are considered to be crucial for the stable activity of ONO-1301. This agent is metabolized by cytochrome P450, and the half-life was about 5.6 hours in our previous study (14). In addition, ONO-1301 has a 3-pyridine radical, which is known to inhibit thromboxane synthase through interaction with carboxylic acid via a hydrogen bond (Figure 1). Repeated administration of ONO-1301 attenuated monocrotaline (MCT)-induced pulmonary hypertension and improved survival in rats. Although the half-life of plasma ONO-1301 concentration is longer than that of any other prostacyclin analogs, ONO-1301 still needs to be administered twice a day subcutaneously to achieve a significant improvement in pulmonary hypertension. For patients with pulmonary hypertension, development of a long-acting, sustained-release prostacyclin analog would be beneficial in terms of stable hemodynamics and quality of life. To overcome these problems, we developed a new drug delivery system for prostacyclin. We prepared a novel sustained-release prostacyclin analog polymerized with poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres (ONO-1301MS). PLGA microspheres, which are biodegradable and biocompatible compounds, have been used as a controlled delivery system for proteins and drugs (15–20). The release of drug from PLGA microspheres occurs through degradation of the polymeric matrix. Here, we showed that a single subcutaneous administration of ONO-1301MS achieved sustained elevation of its circulating level for 3 weeks.


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		Figure 1. Chemical structures of ONO-1301, epoprostenol (prostacyclin analog), and ozagrel (thromboxane synthase inhibitor). Epoprostenol shares common characteristics with prostanoid structures, including a five-membered ring and an allylic alcohol (blue and yellow circles, respectively). In contrast, ONO-1301 has a carboxylic acid and a lipid-soluble functional group that activates the prostacyclin receptor (green circles), but does not have prostanoid structures, which allow long-lasting prostacyclin activity. Unlike epoprostenol, ONO-1301 has thromboxane synthase inhibitory activity because of a 3-pyridine radical and carboxylic acid within its molecule (red circles), similar to ozagrel.

 
Thus, the purposes of this study were as follows: (1) to investigate whether a single subcutaneous administration of ONO-1301MS attenuates MCT-induced pulmonary hypertension in rats and (2) to elucidate the underlying mechanisms responsible for the beneficial effects of this compound.


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of ONO-1301MS
ONO-1301MS is polymerized ONO-1301 with PLGA microspheres. ONO-1301 and PLGA (polylactic acid to glycolic acid ratio of 50:50) were dissolved in dichloromethane. The dissolved polymer was added to polyvinyl alcohol aqueous solution to form an oil-in-water emulsion. Then, dichloromethane was evaporated by stirring. After centrifugation and washing, ONO-1301MS was isolated by lyophilization.

Morphologic Studies by Scanning Electron Microscopy
To evaluate the shape and surface morphology of ONO-1301MS, we used a scanning electron microscope (model S-2460N; Hitachi, Tokyo, Japan). After lyophilization, the microspheres were mounted on an aluminum stub and coated with a thin layer (200 Å) of gold by an ion sputter (model E-1010; Hitachi). The surface morphology of the microsphere samples was then visualized under a scanning electron microscope.

Particle Diameter
ONO-1301MS was suspended in distilled water and dispersed by sonication. The particle diameter was measured by a laser diffraction particle size analyzer (model SALD-2100; Shimadzu, Kyoto, Japan).

Encapsulation Efficiency
Acetonitrile containing n-propyl 4-hydroxybenzoate served as an internal control to obtain the encapsulation efficiency, and this solution was homogenized by a sonicator. The concentration of ONO-1301 in this solution was analyzed by high-performance liquid chromatography (HPLC). The encapsulation efficiency was calculated as follows:

Formula

In Vitro Release of ONO-1301 from PLGA Microspheres
ONO-1301MS was suspended in phosphate-buffered saline (0.067 mol/L salt concentration, pH 6.8) containing 0.2% Tween-80 to adjust the concentration of ONO-1301 to 100 µg/ml. This solution was aliquoted into 1 ml and incubated at 37°C. At various time intervals, one of the aliquots was centrifuged for 5 minutes at 12,000 rpm. The supernatant was discarded, the pellet was dissolved in acetonitrile, and the remaining amount of ONO-1301 was analyzed by HPLC.

Animal Models
We used 5-week-old male Wistar rats weighing 95 to 110 g. The rats were randomly given a subcutaneous injection of either 60 mg/kg MCT or 0.9% saline, and assigned to receive a subcutaneous injection of 100 mg/kg ONO-1301MS or 0.9% saline. This protocol resulted in the creation of three groups: normal rats given 0.9% saline (sham group, n = 10), MCT rats given 0.9% saline (control group, n = 11), and MCT rats treated with ONO-1301 MS (treated group, n = 11). We chose the maximum dose that did not induce significant hypotension (see Figure E1 in the online supplement).

In Vivo Experimental Protocol
After anesthetization by an intraperitoneal injection of 30 mg/kg pentobarbital, rats were given a subcutaneous injection of either 60 mg/kg MCT or 0.9% saline. Subsequently, rats received a single subcutaneous injection of 100 mg/kg ONO-1301MS or 0.9% saline. ONO-1301MS was suspended with 0.9% saline containing 0.2% Tween-80. Hemodynamic measurements and histologic analyses were performed on Day 21. For hemodynamic measurements, rats were anesthetized by intraperitoneal injection of 20 mg/kg pentobarbital, and the following indexes were recorded after an equilibration period. A polyethylene catheter (model PE-50; BD Biosciences, San Jose, CA) was inserted into the right carotid artery to measure heart rate and mean arterial pressure. The catheter was inserted through the right jugular vein into the right ventricle for the measurement of RV pressure. The values of heart rate, mean arterial pressure, and systolic RV pressure were calculated from a series of 20 consecutive heart beats in each rat. 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 RV weight to body weight ratio (RV/BW), left ventricular plus septal weight to body weight ratio (LV + S/BW), and RV weight to left ventricular plus septal weight ratio (RV/LV + S) were calculated as indexes of ventricular hypertrophy, as reported previously (21). All protocols were performed in accordance with the guidelines of the Animal Care Ethics Committee of the National Cardiovascular Center Research Institute (Osaka, Japan).

Morphometric Analysis of Pulmonary Arteries
Paraffin sections of 4-µm thickness were obtained from the lower region of the right lung and stained with hematoxylin and eosin. Analysis of the medial wall thickness of the pulmonary arteries was performed as described previously (22). In brief, the external diameter and the medial wall thickness were measured in 20 muscular arteries (25–100-µm external diameter) per lung section. For each artery, the medial wall thickness was expressed as follows:

Formula
A lung section was obtained from individual rats for comparison among the three groups (n = 5 in each group).

Assay of Plasma Levels of ONO-1301 and Cyclic AMP
To investigate whether a single subcutaneous administration of ONO-1301MS produces long-lasting prostacyclin activity in rats, we measured plasma levels of ONO-1301 and cyclic AMP (cAMP) after ONO-1301MS injection. Fourteen rats were assigned to receive a single subcutaneous injection of 100 mg/kg ONO-1301MS or 0.9% saline (n = 7 in each group), and blood was drawn from the tail vein on Days 0, 7, 14, and 21. Blood was immediately transferred to a chilled glass tube containing 1 mg/ml disodium ethylenediaminetetraacetic acid and 500 U/ml aprotinin, and centrifuged immediately. Plasma ONO-1301 level was measured by liquid chromatography tandem mass spectrometry assay. Plasma cAMP level was measured with a radioimmunoassay kit (cAMP assay kit; Yamasa Co., Chiba, Japan), as reported previously (23).

Assay of Urinary Level of 11-Dehydro Thromboxane B2
To investigate the effect of ONO-1301MS on thromboxane synthesis in rats, we measured urinary level of 11-dehydro thromboxane B2 (11-DTXB2), a metabolite of thromboxane A2 (TXA2), after single subcutaneous injection of ONO-1301MS (100 mg/kg) or vehicle (n = 8 in each group). Urine samples were collected for 24 hours on Day 14 by using metabolic cages, and urinary concentration of 11-DTXB2 was measured with an enzyme immunoassay kit (11-DTXB2 assay kit; Cayman Chemical Co., Ann Arbor, MI). The urinary level of 11-DTXB2 was expressed as the ratio of urinary 11-DTXB2 concentration to that of creatinine, as reported previously (24).

Western Blot Analysis
To investigate the effect of ONO-1301MS on proliferative signaling pathways in homogenized lung tissue, the protein expression of extracellular signal-regulated protein kinase (ERK) 1/2 and phospho-ERK1/2 was determined by Western blotting. Western blotting was performed using rabbit monoclonal antibodies raised against ERK1/2 and phospho-ERK1/2 (Cell Signaling Technology, Danvers, MA). Peripheral samples of lung tissue were obtained on Day 21 from individual rats for comparison among the three groups (n = 6 in each group). Positive protein bands were visualized by means of chemiluminescence (enhanced chemiluminescence kit; Amersham Biosciences, Little Chalfont, UK). Western blot analysis using a mouse polyclonal antibody raised against β-actin (Sigma Chemical Corp., St. Louis, MO) was used as a protein loading control. The resultant bands were quantified using Image J 1.36 imaging software (National Institutes of Health; http://rsb.info.nih.gov/ij/).

Statistical Analysis
All data were expressed as mean ± SEM. Comparisons of parameters among the three groups were made by one-way analysis of variance (ANOVA), followed by Newman-Keuls test. Comparisons of the time course of parameters between the two groups were made by two-way ANOVA for repeated measures, followed by Newman-Keuls test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of ONO-1301MS
We prepared three kinds of ONO-1301MS (samples 1, 2, and 3). The external surface morphology of ONO-1301MS (sample 2 as a representative sample) exhibited a spherical shape with a smooth and uniform surface (Figure 2A). The particle size in samples 1, 2, and 3 was 21.2, 42.0, and 71.1 µm, respectively (Figure 2B; sample 2 as a representative sample). Encapsulation efficiency in each sample was 5.1, 21.8, and 17.4%, respectively. In vitro, each sample had different time periods of ONO-1301 release at 2, 3, and 4 weeks, respectively (Figure 2C). These data suggest that we were able to vary the release period of ONO-1301.


Figure 2
View larger version (43K):
[in this window]
[in a new window]

  		Figure 2. Physicochemical characteristics and in vitro release of ONO-1301. (A) Morphology of ONO-1301MS (sample 2) studied by scanning electron microscopy. Scale bar = 50 µm. (B) Particle diameter of ONO-1301MS (sample 2) obtained by a laser diffraction particle size analyzer. (C) Release profiles of ONO-1301MS in each sample.

 
Long-lasting Activity of ONO-1301MS
To investigate the pharmacokinetics in vivo, we measured plasma ONO-1301 level after a single subcutaneous administration of ONO-1301MS, which was designed to release ONO-1301 for 3 weeks (sample 2). ONO-1301 was detected in plasma for 3 weeks, whereas plasma ONO-1301 level at baseline in the ONO-1301MS group and at all times in the vehicle group was below the detection limit (Figure 3A). In addition, plasma cAMP level after a single subcutaneous administration of ONO-1301MS was significantly higher than that in the control group (Figure 3B). Interestingly, the increase in plasma cAMP level lasted for over 2 weeks in parallel with the change in plasma ONO-1301MS level (Figure 3). These results suggest that subcutaneous administration of ONO-1301MS achieves long-lasting activity in rats.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Figure 3. Time course changes in plasma ONO-1301 and cAMP. (A) Plasma ONO-1301 concentration after a single subcutaneous administration of ONO-1301MS or vehicle. The shaded area indicates below the lower limit of quantification (0.025 ng/ml) and is treated as 0 in the statistical analysis. (B) Changes in plasma cAMP level after a single subcutaneous administration of ONO-1301 MS or vehicle. Data are mean ± SEM. *P < 0.05 versus vehicle.

 
Inhibitory Effect of ONO-1301MS on Thromboxane Synthase
Urinary level of 11-DTXB2 was markedly elevated 14 days after MCT injection (Figure 4). However, treatment with ONO-1301MS significantly decreased urinary level of 11-DTXB2 in MCT rats. These results suggest that ONO-1301MS has a sustained inhibitory effect on thromboxane synthase activity.


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Figure 4. Effect of ONO-1301MS on thromboxane synthesis. Changes in urinary 11-dehydro thromboxane B2 (11-DTXB2) level on Day 14. Sham = sham rats given vehicle; control = monocrotaline (MCT)-treated rats given vehicle; treated = MCT rats treated with ONO-1301MS. Data are mean ± SEM. *P < 0.05 versus sham; {dagger}P < 0.05 versus control.

 
Effects of ONO-1301MS on Pulmonary Hemodynamics and Vascular Remodeling
Three weeks after MCT injection, RV systolic pressure was markedly increased (Figure 5). However, the increase in RV systolic pressure was significantly attenuated in the treated group. Similarly, the increases in RV/BW and RV/LV + S in MCT rats were significantly attenuated by treatment with ONO-1301MS (Figure 5). There were no significant differences in heart rate or mean arterial pressure among the three groups (Table 1). Histologic examination demonstrated that hypertrophy of the pulmonary vascular wall was attenuated in the treated group compared with that in the control group (Figure 6).


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		Figure 5. Effects of ONO-1301MS on pulmonary hemodynamics. (A) Effects of ONO-1301MS on right ventricular (RV) systolic pressure, (B) RV weight to body weight (RV/BW), and (C) RV weight to left ventricular plus septal weight (RV/LV + S). Sham = sham rats given vehicle; control = monocrotaline (MCT)-treated rats given vehicle; treated = MCT rats treated with ONO-1301MS. Data are mean ± SEM. *P < 0.05 versus sham; {dagger}P < 0.05 versus control.

 

Figure 6
View larger version (72K):
[in this window]
[in a new window]

  		Figure 6. Effect of ONO-1301MS on vascular remodeling. (A) Representative photomicrographs of peripheral pulmonary arteries on Day 21. Scale bars = 50 µm. (B) Quantitative analysis of percentage of wall thickness in peripheral pulmonary arteries. Data are mean ± SEM. *P < 0.05 versus sham; {dagger}P < 0.05 versus control.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. PHYSIOLOGIC PROFILES OF THREE EXPERIMENTAL GROUPS

 
No adverse reactions, such as flushing, diarrhea, or hypotension, were observed in the treated group, and there were no significant differences in blood biochemical markers of liver and renal function among the three groups (mean value ± SEM in sham, control, and treated group were, respectively: 139 ± 14, 145 ± 20, and 102 ± 12 IU/L in aspartate aminotransferase; 53 ± 3, 54 ± 5, and 45 ± 3 IU/L in alanine aminotransferase; 0.1 ± 0, 0.1 ± 0, and 0.1 ± 0 mg/dl in total bilirubin; 16.2 ± 1.1, 16.2 ± 0.4, and 15.4 ± 1.2 mg/dl in urea nitrogen; 0.22 ± 0.01, 0.21 ± 0.01, and 0.21 ± 0.01 mg/dl in creatinine; n = 5 in each group.). Moreover, no abnormality was observed at the injection site.

Inhibitory Effect of ONO1301-MS on Proliferative Signals
To investigate the effect of ONO1301-MS on proliferative signals in the lung, Western blot analyses were performed. There were no significant differences in the expression of ERK1 and ERK2 among the three groups (Figure 7). However, phosphorylation of ERK1 and ERK2 was significantly increased in the control group, whereas these increases were markedly attenuated in the treatment group (Figure 7).


Figure 7
View larger version (28K):
[in this window]
[in a new window]

  		Figure 7. Effect of ONO-1301MS on extracellular signal-regulated protein kinase (ERK) phosphorylation. (A) Representative Western blotting for ERK, phospho-ERK (P-ERK) and β-actin (protein loading control) in lungs on Day 21 (n = 6 in each group). (B and C) Scatter plot of quantitative analysis of P-ERK expression in lung tissue. Horizontal lines in this figure show the mean value. *P < 0.05 versus sham; {dagger}P < 0.05 versus control.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that (1) a novel sustained-release prostacyclin analog polymerized with PLGA microspheres (ONO-1301MS) allowed a 3-week elevation of its circulating level, (2) ONO-1301MS had a sustained inhibitory effect on thromboxane synthase activity, and (3) a single subcutaneous administration of ONO-1301MS attenuated MCT-induced pulmonary hypertension in rats.

Conventional prostacyclin and its analogs need continuous infusion or frequent administration because of their short duration of action. Previously, we reported a new type of prostacyclin agonist, ONO-1301, which has long-lasting prostacyclin activity and an inhibitory effect on thromboxane synthase (14). Although ONO-1301 has such interesting features, it still needs to be administered twice a day to achieve a significant improvement in pulmonary hypertension. To overcome this problem, we developed a new drug delivery system for prostacyclin. We polymerized ONO-1301 with PLGA microspheres to develop a novel sustained-release prostacyclin analog.

PLGA microspheres have been used as a controlled delivery system for bioactive agents (25). The release of bioactive agents from PLGA microspheres occurs through hydrolytic degradation of the polymeric matrix. Importantly, PLGA has already been used in humans. PLGA microspheres containing leuprorelin, a potent luteinizing hormone–releasing hormone analog, have been administered to patients with prostate and breast cancer by subcutaneous injection (26, 27). The rate of release of contents of PLGA microspheres can be changed by varying the factors affecting the hydrolytic degradation behavior of PLGA, such as lactate acid to glycolic acid ratio, average molecular weight of PLGA, and particle size (25). In the present study, we could control the degradation rate of ONO-1301MS. ONO-1301MS was designed to release ONO-1301 for 3 weeks, because it takes 3 weeks to induce pulmonary hypertension in rats after MCT injection. The present study demonstrated that the contained ONO-1301 was released for 3 weeks in vitro, producing a 3-week elevation of its circulating level after a single administration in vivo. It should be noted that only a single subcutaneous administration of ONO-1301MS attenuated MCT-induced pulmonary hypertension in rats. Thus, it might be possible to extend the administration interval for ONO-1301MS considerably longer than that with current prostacyclin analogs, and this could improve the quality of life in patients with pulmonary hypertension.

With regard to cAMP, which is a second messenger of prostacyclin and its analogs, it has been reported that plasma cAMP level remained increased after administration of prostacyclin analogs (23, 28). In our results, administration of ONO-1301MS increased the plasma cAMP level for over 2 weeks. This increase in plasma cAMP level was parallel to the change in plasma ONO-1301 level. In addition, ONO-1301MS attenuated the increase in urinary 11-DTXB2 level in MCT rats 14 days after single administration. These results support that a single administration of ONO-1301MS produced a sustained beneficial effect for 3 weeks.

In the present study, we chose the maximum dose that did not induce significant hypotension. We did dose–response studies using ONO-1301MS (30, 100, and 300 mg/kg, respectively) (see Figure E1). ONO-1301MS at 300 mg/kg has induced significant hypotension. In addition, ONO-1301MS at 30 mg/kg did not significantly decrease RV systolic pressure (see Figure E3). On the other hand, ONO-1301MS at 100 mg/kg significantly decreased RV systolic pressure without significant hypotension. Furthermore, a single injection of 100 mg/kg ONO-1301 without PLGA or PLGA without ONO-1301 to MCT rats did not influence hemodynamics and vascular remodeling (see Figures E1–E3). These results suggest that a single injection of ONO-1301MS ameliorates MCT-induced pulmonary hypertension. Consistent with these hemodynamic data, RV/BW and medial wall thickness of pulmonary arteries, as indexes of RV hypertrophy and elevation of pulmonary arteriolar resistance, respectively, were significantly attenuated by the treatment with ONO-1301MS.

In histologic analysis, hypertrophy of pulmonary vessels after MCT injection was significantly attenuated by treatment with ONO-1301MS. An earlier clinical trial has shown that long-term therapy with epoprostenol significantly reduces pulmonary vascular resistance in patients who have no short-term response to vasodilators (5). It is speculated that such a beneficial effect of epoprostenol is caused not only by vasodilatation and antiplatelet aggregation but also by an antiproliferative effect on SMCs and reverse remodeling of pulmonary arteries. In the present study, phosphorylated ERK1/2 in the lung tissue was significantly increased after MCT injection. However, this increase was markedly attenuated by treatment with ONO-1301MS. ERK is the final component of the mitogen-activated protein kinase cascade. Prostacyclin has been shown to inhibit phosphorylation of ERK1/2 through the activation of cAMP (29). With respect to this signaling, protein kinase A (PKA), an intracellular effector of cAMP, has been shown to negatively regulate the Ras–ERK cascade by phosphorylating Raf and by preventing its association with active Ras (30). Furthermore, we previously reported that ONO-1301 inhibited pulmonary fibroblast proliferation through activation of the cAMP/PKA pathway (31). Therefore, it is interesting to speculate that ONO-1301MS may have antiproliferative effects on pulmonary vascular SMCs at least in part through inhibition of ERK via a cAMP-dependent pathway, although the precise mechanism remains to be elucidated.

ONO-1301MS significantly decreased urinary level of 11-DTXB2, a metabolite of TXA2. TXA2 is a vasoconstrictor and a potent stimulator of platelet aggregation (32, 33). Moreover, it has been demonstrated that TXA2 induces mitosis in vascular SMCs through activation of ERK (34, 35). It has been suggested that imbalance of thromboxane and prostacyclin plays an important role in the development of pulmonary hypertension (36). Previous reports showed that administration of thromboxane synthase inhibitor modestly attenuated pulmonary hypertension (37, 38). Thus, an inhibitory effect of ONO-1301MS on thromboxane synthase may also contribute to improvement in pulmonary hypertension.

In the present study, no adverse reactions, such as flushing, diarrhea, hypotension, renal dysfunction, or hepatic dysfunction, were observed in the treated group. However, further preclinical studies are necessary to confirm the safety and efficacy of ONO-1301MS before clinical trials start in patients with pulmonary arterial hypertension.

We did not measure cardiac output because of technical and mechanical problems. To support our hemodynamic data, we evaluated a variety of indexes, such as RV/BW and medial wall thickness of pulmonary arteries. These physiologic and histologic findings have been consistent with data on RV systolic pressure. Therefore, it is unlikely that the reduction in RV systolic pressure observed in the present study was related to the reduced cardiac output.

In conclusion, we developed a novel sustained-release prostacyclin analog polymerized with PLGA microspheres (ONO-1301MS), which achieved a 3-week elevation of its circulating level and simultaneously increased plasma cAMP levels for over 2 weeks, and had an inhibitory effect on thromboxane synthase. A single subcutaneous administration of ONO-1301MS attenuated MCT-induced pulmonary hypertension in rats. ONO-1301MS may have an antiproliferative effect through inhibition of ERK phosphorylation. This drug delivery system for a prostacyclin analog may be a new therapeutic strategy for the treatment of pulmonary arterial hypertension.


   FOOTNOTES
 
Supported by research grants from Ono Pharmaceutical Co., Ltd. (no. 526); Human Genome Tissue Engineering 009 from the Ministry of Health, Labor, and Welfare; the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO); and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology.

This article contains an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200703-349OC on November 1, 2007

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

Received in original form March 1, 2007; accepted in final form October 26, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. McLaughlin VV, McGoon MD. Pulmonary arterial hypertension. Circulation 2006;114:1417–1431.[Free Full Text]
  2. Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 1976;263:663–665.[CrossRef][Medline]
  3. Moncada S, Vane JR. Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. N Engl J Med 1979;300:1142–1147.[Medline]
  4. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 2004;351:1425–1436.[Free Full Text]
  5. McLaughlin VV, Genthner DE, Panella MM, Rich S. Reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension. N Engl J Med 1998;338:273–277.[Abstract/Free Full Text]
  6. McLaughlin VV, Shillington A, Rich S. Survival in primary pulmonary hypertension: the impact of epoprostenol therapy. Circulation 2002;106:1477–1482.[Abstract/Free Full Text]
  7. Sitbon O, Humbert M, Nunes H, Parent F, Garcia G, Herve P, Rainisio M, Simonneau G. Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol 2002;40:780–788.[Abstract/Free Full Text]
  8. Okano Y, Yoshioka T, Shimouchi A, Satoh T, Kunieda T. Orally active prostacyclin analogue in primary pulmonary hypertension. Lancet 1997;349:1365.[CrossRef][Medline]
  9. Nagaya N, Uematsu M, Okano Y, Satoh T, Kyotani S, Sakamaki F, Nakanishi N, Miyatake K, Kunieda T. Effect of orally active prostacyclin analogue on survival of outpatients with primary pulmonary hypertension. J Am Coll Cardiol 1999;34:1188–1192.[Abstract/Free Full Text]
  10. Olschewski H, Simonneau G, Galie N, Higenbottam T, Naeije R, Rubin LJ, Nikkho S, Speich R, Hoeper MM, Behr J, et al. Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 2002;347:322–329.[Abstract/Free Full Text]
  11. Simonneau G, Barst RJ, Galie N, Naeije R, Rich S, Bourge RC, Keogh A, Oudiz R, Frost A, Blackburn SD, et al. Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 2002;165:800–804.[Abstract/Free Full Text]
  12. Badesch DB, McLaughlin VV, Delcroix M, Vizza CD, Olschewski H, Sitbon O, Barst RJ. Prostanoid therapy for pulmonary arterial hypertension. J Am Coll Cardiol 2004;43:56S–61S.[Abstract/Free Full Text]
  13. Laliberte K, Arneson C, Jeffs R, Hunt T, Wade M. Pharmacokinetics and steady-state bioequivalence of treprostinil sodium (Remodulin) administered by the intravenous and subcutaneous route to normal volunteers. J Cardiovasc Pharmacol 2004;44:209–214.[Medline]
  14. Kataoka M, Nagaya N, Satoh T, Itoh T, Murakami S, Iwase T, Miyahara Y, Kyotani S, Sakai Y, Kangawa K, et al. A long-acting prostacyclin agonist with thromboxane inhibitory activity for pulmonary hypertension. Am J Respir Crit Care Med 2005;172:1575–1580.[Abstract/Free Full Text]
  15. Alonso MJ, Gupta RK, Min C, Siber GR, Langer R. Biodegradable microspheres as controlled-release tetanus toxoid delivery systems. Vaccine 1994;12:299–306.[CrossRef][Medline]
  16. Seagal J, Edry E, Keren Z, Leider N, Benny O, Machluf M, Melamed D. A fail-safe mechanism for negative selection of isotype-switched B cell precursors is regulated by the Fas/FasL pathway. J Exp Med 2003;198:1609–1619.[Abstract/Free Full Text]
  17. Mullerad J, Cohen S, Benharroch D, Apte RN. Local delivery of IL-1 alpha polymeric microspheres for the immunotherapy of an experimental fibrosarcoma. Cancer Invest 2003;21:720–728.[CrossRef][Medline]
  18. Mullerad J, Cohen S, Voronov E, Apte RN. Macrophage activation for the production of immunostimulatory cytokines by delivering interleukin 1 via biodegradable microspheres. Cytokine 2000;12:1683–1690.[CrossRef][Medline]
  19. Sanchez A, Gupta RK, Alonso MJ, Siber GR, Langer R. Pulsed controlled-released system for potential use in vaccine delivery. J Pharm Sci 1996;85:547–552.[CrossRef][Medline]
  20. Roullin VG, Lemaire L, Venier-Julienne MC, Faisant N, Franconi F, Benoit JP. Release kinetics of 5-fluorouracil-loaded microspheres on an experimental rat glioma. Anticancer Res 2003;23:21–25.[Medline]
  21. Kimura H, Kasahara Y, Kurosu K, Sugito K, Takiguchi Y, Terai M, Mikata A, Natsume M, Mukaida N, Matsushima K, et al. Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. Lab Invest 1998;78:571–581.[Medline]
  22. Ono S, Voelkel NF. PAF antagonists inhibit monocrotaline-induced lung injury and pulmonary hypertension. J Appl Physiol 1991;71:2483–2492.[Abstract/Free Full Text]
  23. Itoh T, Nagaya N, Fujii T, Iwase T, Nakanishi N, Hamada K, Kangawa K, Kimura H. A combination of oral sildenafil and beraprost ameliorates pulmonary hypertension in rats. Am J Respir Crit Care Med 2004;169:34–38.[Abstract/Free Full Text]
  24. Fiorucci S, Mencarelli A, Meneguzzi A, Lechi A, Morelli A, del Soldato P, Minuz P. NCX-4016 (NO-aspirin) inhibits lipopolysaccharide-induced tissue factor expression in vivo: role of nitric oxide. Circulation 2002;106:3120–3125.[Abstract/Free Full Text]
  25. Shive MS, Anderson JM. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 1997;28:5–24.[CrossRef][Medline]
  26. Fornara P, Jocham D. Clinical study results of the new formulation leuprorelin acetate three-month depot for the treatment of advanced prostate carcinoma. Urol Int 1996;56:18–22.[CrossRef][Medline]
  27. Schmid P, Untch M, Kosse V, Bondar G, Vassiljev L, Tarutinov V, Lehmann U, Maubach L, Meurer J, Wallwiener D, et al. Leuprorelin acetate every-3-months depot versus cyclophosphamide, methotrexate, and fluorouracil as adjuvant treatment in premenopausal patients with node-positive breast cancer: the TABLE study. J Clin Oncol 2007;25:2509–2515.[Abstract/Free Full Text]
  28. Beghetti M, Reber G, de Moerloose P, Vadas L, Chiappe A, Spahr-Schopfer I, Rimensberger PC. Aerosolized iloprost induces a mild but sustained inhibition of platelet aggregation. Eur Respir J 2002;19:518–524.[Abstract/Free Full Text]
  29. Li RC, Cindrova-Davies T, Skepper JN, Sellers LA. Prostacyclin induces apoptosis of vascular smooth muscle cells by a cAMP-mediated inhibition of extracellular signal-regulated kinase activity and can counteract the mitogenic activity of endothelin-1 or basic fibroblast growth factor. Circ Res 2004;94:759–767.[Abstract/Free Full Text]
  30. Cook SJ, McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science 1993;262:1069–1072.[Abstract/Free Full Text]
  31. Murakami S, Nagaya N, Itoh T, Kataoka M, Iwase T, Horio T, Miyahara Y, Sakai Y, Kangawa K, Kimura H. Prostacyclin agonist with thromboxane synthase inhibitory activity (ONO-1301) attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2006;290:L59–L65.[Abstract/Free Full Text]
  32. Hamberg M, Svensson J, Samuelsson B. Prostaglandin endoperoxides: a new concept concerning the mode of action and release of prostaglandins. Proc Natl Acad Sci USA 1974;71:3824–3828.[Abstract/Free Full Text]
  33. Svenssen J, Strandberg K, Tuvemo T, Hamberg M. Thromboxane A2: effects on airway and vascular smooth muscle. Prostaglandins 1977;14:425–436.[CrossRef][Medline]
  34. Sachinidis A, Flesch M, Ko Y, Schror K, Bohm M, Dusing R, Vetter H. Thromboxane A2 and vascular smooth muscle cell proliferation. Hypertension 1995;26:771–780.[Abstract/Free Full Text]
  35. Morinelli TA, Tempel GE, Jaffa AA, Silva RH, Naka M, Folger W, Halushka PV. Thromboxane A2/prostaglandin H2 receptors in streptozotocin-induced diabetes: effects of insulin therapy in the rat. Prostaglandins 1993;45:427–438.[CrossRef][Medline]
  36. Christman BW, McPherson CD, Newman JH, King GA, Bernard GR, Groves BM, Loyd JE. An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 1992;327:70–75.[Abstract]
  37. Nagata T, Uehara Y, Hara K, Igarashi K, Hazama H, Hisada T, Kimura K, Goto A, Omata M. Thromboxane inhibition and monocrotaline-induced pulmonary hypertension in rats. Respirology 1997;2:283–289.[Medline]
  38. Rich S, Hart K, Kieras K, Brundage BH. Thromboxane synthetase inhibition in primary pulmonary hypertension. Chest 1987;91:356–360.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200703-349OCv1
177/2/195    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Obata, H.
Right arrow Articles by Nagaya, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Obata, H.
Right arrow Articles by Nagaya, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2008 American Thoracic Society