INTRODUCTION

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Keywords: dipyridamole; iloprost; pentoxifylline; phosphodiesterase inhibitor; pulmonary hypertension; tolafentrine; U46619

Continuous intravenous prostacyclin has been demonstrated to be beneficial in the long-term treatment of patients with primary pulmonary hypertension (1) and has also been suggested for secondary pulmonary hypertension (4). Because of the nonselective route of application, however, the pulmonary vasodilatory effect is accompanied by major systemic vasodilation. Moreover, ventilation-perfusion mismatch with possible impairment of arterial oxygenation may occur, particularly in patients with pre-existing lung disease. In patients with acute pulmonary hypertension due to acute respiratory distress syndrome (ARDS), inhaled vasodilators (nitric oxide and prostanoids) have been shown to cause selective pulmonary vasodilation, without affecting systemic blood pressure (5, 6), accompanied by improved ventilation-perfusion matching. Moreover, inhalation of aerosolized iloprost has been demonstrated to cause largely selective pulmonary vasodilation in severe pulmonary hypertension without affecting gas exchange to a disadvantage (7). Iloprost is a stable prostacyclin analog with strong vasodilatory (8) and antithrombotic (9) properties. Inhaled iloprost decreases pulmonary artery pressure and increases cardiac output without affecting mean systemic arterial pressure in severe primary and secondary pulmonary hypertension, and evidence of long-term beneficial effects of daily repetitive iloprost inhalations has been reported (7, 10). In comparison with the duration of inhaled prostacyclin (10-20 min), the pulmonary vasodilatory response to nebulized iloprost lasts significantly longer (45-90 min). Nevertheless, patients need multiple daily inhalation maneuvers to achieve substantial alleviation of pulmonary hypertension when employing inhaled iloprost for long-term treatment. One approach to prolong and possibly increase the vasorelaxant effect of inhaled iloprost might be the concomitant use of phosphodiesterase (PDE) inhibitors, for stabilization of the second-messenger cyclic AMP. In previous studies addressing this issue, mono- and dual-selective PDE inhibitors were, indeed, shown to prolong the lung vasodilatory efficacy of inhaled prostacyclin in perfused rabbit lungs and intact rabbits (14, 15). Combined inhibition of PDE3 and PDE4 was found to be particularly effective in these investigations. We now extended this approach by asking whether coadministration of PDE inhibitors might also open an additional window of efficacy when employing the per se longer-acting agents iloprost, and whether clinically available nonselective PDE inhibitors might be suitable for this purpose.

	METHODS

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Materials

Iloprost (Ilomedin) was from Schering (Berlin, Germany) and the nonspecific PDE inhibitor pentoxifylline (Trental) was from Hoechst (Hoechst Marion Roussel, Bad Soden, Germany). The thromboxane A₂ mimetic U46619 was supplied by Sigma (Deishofen, Germany). Dipyridamole (Persantin) was from Boehringer (Ingelheim, Germany), and tolafentrine was from Byk Gulden Pharmaceuticals (Konstanz, Germany). All other chemicals and drug supplies were from standard commercial sources.

Surgical Preparation

As described previously (14), rabbits of either sex were initially anesthetized with a mixture of xylazine and ketamine and anticoagulated with heparin 200 U/kg. Anesthesia was maintained by a constant intravenous infusion of xylazine and ketamine through the right peripheral ear vein. After tracheostomy the animals were ventilated with a frequency of 40 breaths/min and a tidal volume of 8 ml/kg, using a volume-controlled respirator (cat ventilator; Hugo Sachs Elektronik, March Hugstetten, Germany). The fraction of inspired oxygen (FI_O2) was set at 0.5 and a positive end-expiratory pressure of 0.5 mm Hg was used throughout. A catheter was inserted into the right femoral vein for infusion of saline and PDE inhibitors and the left carotid artery was cannulated for arterial pressure monitoring. A pulmonary artery catheter (4F; Braun, Melsungen, Germany) was inserted into the pulmonary artery through the right external jugular vein.

Hemodynamics and Blood Gases

Setting the level of the left atrium to zero, mean pulmonary artery pressure () and mean systemic arterial pressure () were continuously recorded with fluid-filled force transducers (Combitrans; Braun). Pulmonary and systemic vascular resistances were calculated by standard formulas. Cardiac output () was calculated by using the Fick principle as described (14). Hemoglobin and oxygen saturation of arterial and venous blood samples (1 ml) was measured with an OSM2 hemoximeter (Radiometer; Copenhagen, Denmark). Oxygen uptake of the animals was measured online (O₂ controller; Labotect, Goettingen, Germany).

Nebulization

For nebulization of iloprost and the phosphodiesterase inhibitors, an ultrasonic nebulizer (Pulmo Sonic 5500; DeVilbiss Medizinische Produkte, Langen, Germany) was used throughout. This device produces an aerosol with a mass median aerodynamic diameter (MMAD) of 4.5 µm and a geometric standard deviation (GSD) of 2.3, as measured with a laser difractometer (HELOS; Sympatec, Clausthal-Zellerfeld, Germany). As described previously, the nebulizer was placed in the inspiratory limb of the ventilation system (16). For measurement of bronchoalveolar deposition of the nebulized materials, a laser photometer was located at the inlet of the tracheal tube, allowing breath-by-breath assessment of inhaled and exhaled aerosol mass (17).

Experimental Protocols

U46619 was infused at a dosage between 0.5 and 2 µg/kg · min to induce an increase in from ~ 13 to ~ 26 mm Hg within 20 min. As described previously, stable pulmonary hypertension is established by this approach (14). Dose-effect curves of the PDE inhibitors were created after reaching a stable U46619-induced pressure plateau, employing randomized doses for short-term infusions (10 min) or inhalations (10 min) of the various PDE inhibitors. Hemodynamics and blood gases were measured at the end of the 10-min infusion or inhalation period. Concerning aerosolization of the stable prostaglandin I₂ (PGI₂) analog, nebulized iloprost at a dosage of 40 ± 8 ng/kg · min was found to achieve selective pulmonary vasodilation in all animals, without affecting systemic arterial pressure. This dose was used throughout all studies. In the group with sole iloprost inhalation the aerosolization maneuver was performed after reaching a stable pressure plateau, and the hemodynamic effects were monitored. In the combination experiments, the PDE inhibitor was either infused as a short-term infusion (10 min) or nebulized (10 min) after establishing stable pulmonary hypertension, and iloprost was aerosolized subsequently. Hemodynamics and blood gases were measured at the end of each intervention period. For the intravenous route of application, tolafentrine (50 µg/kg · min), dipyridamole (1 µg/kg · min), and pentoxifylline (50 µg/ kg · min) were applied. The sequential nebulization of the PDE inhibitors and iloprost was performed with tolafentrine (30 µg/kg · min), dipyridamole (50 ng/kg · min), and pentoxifylline (100 µg/kg · min). Further experiments were performed with higher doses of PDE inhibitors, which per se caused hemodynamic effects, and which were also employed for sequential nebulization with iloprost. The following doses were applied for this approach: tolafentrine (600 µg/kg · min), dipyridamole (10 µg/kg · min) and pentoxifylline (600 µg/kg · min).

Data Analysis

Data are given as means ± SEM. Differences between the various groups were assessed by use of analysis of variance and the Student- Newman-Keuls test for multiple comparisons, with a p value < 0.05 regarded to be significant. Differences between means of two groups were analyzed with an unpaired t test.

	RESULTS

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Baseline and U46619-induced Pulmonary Hypertension

All baseline hemodynamic data and blood gases were in physiologic ranges. After a stable steady state period, U46619 infusion at a mean of 1.5 ± 0.8 µg/kg · min resulted in a significant increase in pulmonary artery pressure to ~ 26 mm Hg as compared with ~ 15 mm Hg before U46619 (Tables 1 and 2). Mean systemic pressure () and cardiac output did not change significantly. There were no significant changes in blood gases as compared with control animals and with the baseline period preceding U46619 administration.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Influence of iloprost nebulization and its combination with subthreshold doses of inhaled PDE inhibitors on U46619-elicted pulmonary hypertension. Mean pulmonary artery pressure (Ppa [PPA], as a percentage of U46619- induced increase) is given (mean ± SEM of eight independent experiments each; SEM bars are not included when smaller than symbol). Iloprost nebulization (40 ng/kg · min) is indicated by the horizontal bar. The PDE inhibitors were prenebulized as follows: 30 µg/kg · min tolafentrine, 50 ng/kg · min dipyridamole, and 100 µg/kg · min pentoxifylline (10-min aerosolization maneuvers). Compared with sole iloprost inhalation, all time points from 28 to 34 min were significantly different for iloprost/dipyridamole (p < 0.05) and all time points from 26 to 36 min were significantly different for iloprost/tolafentrine (p < 0.05).

View larger version (33K): [in this window] [in a new window]   Figure 4.   Influence of iloprost nebulization and its combination with effective doses of inhaled PDE inhibitors on U46619-elicted pulmonary hypertension. Mean pulmonary artery pressure (Ppa [PPA], as a percentage of U46619-induced increase) is given (mean ± SEM of eight independent experiments each; SEM bars are not included when smaller than symbol). Iloprost nebulization (40 ng/kg · min) is indicated by the horizontal bar. The PDE inhibitors were prenebulized as follows: 600 µg/kg · min tolafentrine, 10 µg/kg · min dipyridamole, and 600 µg/kg · min pentoxifylline (10-min aerosolization maneuvers). Compared with sole iloprost inhalation, all time points from 12 to 36 min were significantly different for iloprost/pentoxifylline (p < 0.05), all time points from 10 to 48 min for iloprost/dipyridamole were significantly different (p < 0.05), and all time points from 14 to 50 min for iloprost/tolafentrine were significantly different (p < 0.05).

View larger version (32K): [in this window] [in a new window]   Figure 2.   Influence of iloprost nebulization and its combination with subthreshold doses of intravenous PDE inhibitors on U46619-elicted pulmonary hypertension. Mean pulmonary artery pressure (Ppa [PPA], as a percentage of U46619-induced increase) is given (mean ± SEM of eight independent experiments each; SEM bars are not included when smaller than symbol). Iloprost nebulization (40 ng/kg · min) is indicated by the horizontal bar. The PDE inhibitors were preapplied as short-term infusion as follows: 50 µg/kg · min pentoxifylline, 1 µg/kg · min dipyridamole, and 50 µg/kg · min tolafentrine. Compared with sole iloprost inhalation, all time points from 24 to 34 min were significantly different for iloprost/pentoxifylline (p < 0.05). The same was true for all time points from 26 to 34 min for iloprost/dipyridamole (p < 0.05) and all time points from 14 to 46 min for iloprost/tolafentrine (p < 0.05).

Dose-Effect Curves of PDE Inhibitors

As shown in Figure 1, all PDE blockers reduced pulmonary artery pressure in a dose-dependent manner via both the intravenous and inhalative routes. Dipyridamole was the most efficient vasodilator (dose range, 1-20 µg/kg · min for 10-min infusion and 0.05-10 µg/kg · min for 10-min aerosolization), followed by tolafentrine (dose range, 50-500 µg/kg · min for intravenous route and 30-600 µg/kg · min for aerosolization) and pentoxifylline (dose range, 50-1,000 µg/kg · min for intravenous route and 100-600 µg/kg · min for aerosolization).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Dose-effect curves of intravenous (a) and inhaled (b) PDE inhibitors on U46619-elicted pulmonary hypertension. Mean pulmonary artery pressure drop (Ppa [PPA], in mm Hg) is given (mean ± SEM of six independent experiments each). PDE inhibitors were applied at various doses as short-term infusion or inhalation (10 min each) in randomized order.

Nebulization of Iloprost

In pilot experiments, dose-response curves of nebulized iloprost were established (data not shown). Inhalation of nebulized iloprost at 40 ng/kg · min resulted in a significant decrease in U46619-induced pulmonary hypertension (%) by 25.0 ± 3.4% (Figures 2-4). This was accompanied by a moderate increase in cardiac output (from 422 to 436 ml/min, NS) and a concomitant change in pulmonary vascular resistance (Rpv) (Figure 5). Mean systemic arterial pressure (; Figure 6) and blood gases did not change significantly (Tables 1 and 2), within ~ 18 min, 95% of the U46619-induced PPA plateau was reached again, and the iloprost effect completely leveled off within ~ 30 min. The calculated area under the curve (AUC) was 468 ± 76% · min (Figure 7).

View larger version (50K): [in this window] [in a new window]   Figure 5.   Pulmonary vascular resistance (RL [PVR]) in response to inhaled iloprost in the presence and absence of PDE inhibitors. Measurements were performed at the end of the iloprost nebulization period (40 ng/kg · min) in the presence and absence of the various PDE inhibitors, which were either preinfused or prenebulized, and the decrease in PVR (as a percentage) is given (mean ± SEM of eight independent experiments each). The following doses were applied: Pent iv, 50 µg/ kg · min; Pent aer, 100 µg/kg · min; Pent aer hd, 600 µg/kg · min; Dip iv, 1 µg/kg · min; Dip aer, 50 ng/kg · min; Dip aer hd, 10 µg/kg · min; Tola iv, 50 µg/kg · min; Tola aer, 30 µg/kg · min; Tola aer hd, 600 µg/ kg · min. Ilo = iloprost; Pent = pentoxifylline; Dip = dipyridamole; Tola = tolafentrine; iv = intravenous; aer = aerosolized; hd = high dose. *p < 0.05 as compared with sole iloprost nebulization.

View larger version (15K): [in this window] [in a new window]   Figure 6.   Mean systemic arterial pressure (Psa [PSA]) in response to inhaled iloprost in the presence and absence of PDE inhibitors. Measurements were performed at the end of the iloprost nebulization period (40 ng/kg · min) in the presence and absence of various PDE inhibitors, which were either preinfused or prenebulized, and the decrease in Psa (as a percentage) is given (mean ± SEM of eight independent experiments each). The following doses were applied: Pent iv, 50 µg/kg · min; Pent aer, 100 µg/kg · min; Pent aer hd, 600 µg/kg · min; Dip iv, 1 µg/kg · min; Dip aer, 50 ng/kg · min; Dip aer hd, 10 µg/kg · min; Tola iv, 50 µg/kg · min; Tola aer, 30 µg/kg · min; Tola aer hd, 600 µg/ kg · min. Ilo = iloprost; Pent = pentoxifylline; Dip = dipyridamole; Tola = tolafentrine; iv = intravenous; aer = aerosolized; hd = high dose. *p < 0.05 as compared with sole iloprost nebulization.

View larger version (65K): [in this window] [in a new window]   Figure 7.   Influence of combined PDE inhibitors on the area under the curve (AUC) of iloprost-induced decrease in mean pulmonary artery pressure (Ppa [PPA]). Measurements were performed from the onset of iloprost nebulization until 68 min postaerosolization. The AUC was calculated by standard techniques and is given as %PPA · min. The maneuvers correspond to those depicted in Figures 2-4. The following doses were applied: Pent iv, 50 µg/kg · min; Pent aer, 100 µg/kg · min; Pent aer hd, 600 µg/kg · min; Dip iv, 1 µg/kg · min; Dip aer, 50 ng/kg · min; Dip aer hd, 10 µg/kg · min; Tola iv, 50 µg/kg · min; Tola aer, 30 µg/kg · min; Tola aer hd, 600 µg/kg · min. Ilo = iloprost; Pent = pentoxifylline; Dip = dipyridamole; Tola = tolafentrine; iv = intravenous; aer = aerosolization; hd = high dose. *p < 0.05 as compared with sole iloprost nebulization.

Combined Subthreshold Administration of Intravenous PDE Inhibitors and Iloprost Nebulization

In the presence of subthreshold intravenous pentoxifylline (50 µg/kg · min), the iloprost-induced drop tended to increase (34.7 ± 2.8 versus 25.0 ± 3.4%, NS), and the vasodilatory effect, defined by values below 95% of the U46619- induced pressure plateau, was significantly prolonged, from 18 to 40 min (Figure 2). This prolongation was also reflected in the calculated AUC, which more than doubled (958 ± 118% · min for iloprost/pentoxifylline; Figure 7). Comparable efficacy was noted for intravenous subthreshold dipyridamole (1 µg/kg · min), which prolonged the postnebulization vasodilatory effect to 42 min and increased the AUC from 468 ± 76 to 872 ± 126% · min. Tolafentrine (50 µg/kg · min) both enhanced the maximum decrease in response to iloprost aerosolization (51.3 ± 6.2 instead of 25.0 ± 3.4% of the U46619-induced pressure elevation; p < 0.01) as well as prolonged the effect to 52 min. The AUC was significantly increased (from 468 ± 76 to 1,555 ± 225% · min; p < 0.01).

Combined Subthreshold Administration of Inhaled PDE Inhibitors and Iloprost

Conebulization of a subthreshold dose of pentoxifylline (100 µg/kg · min) and iloprost neither amplified nor prolonged the iloprost-induced drop (Figure 3). Correspondingly, no significant changes in blood gases, hemodynamics, and AUC were measured. In contrast, the vasodilatory effect of iloprost in the presence of nebulized subthreshold doses of dipyridamole and tolafentrine was significantly prolonged, from 18 to 36 min for both agents (Figure 3). The calculated AUC was increased from 468 ± 76 to 873 ± 177% · min (combination with dipyridamole, NS) and 901 ± 206% · min (combination with tolafentrine, NS).

Combined Inhalation of Effective Doses of PDE Inhibitors and Iloprost

When aerosolized in doses effecting per se a moderate decrease in without affecting , both nonspecific PDE inhibitors pentoxifylline (600 µg/kg · min) and dipyridamole (10 µg/kg · min) significantly amplified the iloprost-induced drop to 46.4 ± 1.8% (p < 0.01) and 48.1 ± 4.4% (p < 0.01), respectively (Figure 4). In addition, the vasodilatory effect was markedly prolonged to 38 min (pentoxifylline) and 48 min (dipyridamole). This was also true for the dual-selective PDE inhibitor tolafentrine (600 µg/kg · min), which amplified the iloprost-induced drop to 43.9 ± 1.6% (p < 0.05) and prolonged the pressure drop to 52 min. Correspondingly, the calculated AUC was markedly increased for conebulized pentoxifylline (945 ± 109% · min), dipyridamole (1,261 ± 166% · min), and tolafentrine (1,908 ± 180% · min). This was also true for pulmonary vascular resistance values, which significantly decreased for all combinations of high-dose aerosolized PDE inhibitors and iloprost (Figure 5).

	DISCUSSION

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Infusion of the thromboxane A₂ mimetic U46619 has repeatedly been employed for induction of stable pulmonary hypertension in animals, suitable for testing the effects of vasodilators on pulmonary hemodynamics (14, 18). When establishing dose- effect curves for the currently investigated phosphodiesterase inhibitors, comparable efficacy was noted for tolafentrine and pentoxifylline for both the intravascular and inhalative routes of administration. In contrast, dipyridamole was effective in a lower concentration range (by three to four orders of magnitude). This difference in efficacy may not be easily explained by currently available pharmacologic data, characterizing pentoxifylline as a nonselective PDE1-5 inhibitor with IC₅₀ values ranging between 50 and 200 µM, tolafentrine as a selective PDE3/4 inhibitor with IC₅₀ values of 60 and 100 nM, respectively (14), and dipyridamole as a nonselective PDE inhibitor (PDE5/6/8/10) with IC₅₀ values ranging between 1 and 5 µM (21). The potent pulmonary vasodilatory efficacy of dipyridamole compared with tolafentrine and pentoxifylline may indicate a dominant role of PDE5 and cGMP in the lung vasculature. Alternatively, a particular role of PDE8 and PDE10 may be considered; however, the presence of these newly characterized PDE families in lung vascular cells is currently not settled (21).

The pulmonary vasodilatory efficacy of various PDE inhibitors has been demonstrated in different animal models. However, the suitability of effective doses of PDE inhibitors for treatment of pulmonary hypertension is hampered by severe systemic side effects of the agents. The combination of low-dose PDE inhibitors with short-term-acting agonists such as nitric oxide or prostacyclin has previously been reported as an effective treatment protocol in experimental models of pulmonary hypertension by this group and by others (14, 15, 18, 22). Following this line, a combination of subthreshold doses of pentoxifylline, dipyridamole, and tolafentrine with the longer-acting prostacyclin analog iloprost was undertaken in the present investigation. Subthreshold doses were characterized by the dose-effect curves when employing the PDE inhibitors as sole agents. Indeed, some amplification (infused tolafentrine) and prolongation of the pulmonary vasodilatory effect of inhaled iloprost were noted for subthreshold doses of all agents either for the intravenous or for the inhalative route; however, the efficacy was less impressive compared with the combination of subthreshold PDE inhibitors with PGI₂. This finding is most probably explained by the fact that iloprost does per se possess longer efficacy, with minor extra benefit being achievable via additional cAMP stabilization by low-dose PDE inhibition. A second approach presently investigated was based on the fact that PDE inhibitors may be nebulized in doses that cause some direct lung vasorelaxation but that are still too low to provoke systemic vasodilatory effects. When combining this approach with iloprost aerosolization, marked augmentation of the pulmonary vasodilatory response to the prostanoid was noted. For all agents (aerosolized tolafentrine, pentoxifylline, and dipyridamole), the maximum decline in response to iloprost approximately doubled, and the area under the curve of decrease over time was 2- to 4-fold increased. Notably, this marked enhancement of iloprost-induced pulmonary vasodilation again occurred in the absence of any systemic pressure decline, and without any deterioration of gas exchange.

Strongest potency was noted for coaerosolized tolafentrine, a dual-selective PDE3 and PDE4 inhibitor. This underscores the major role of PDE3 and PDE4 as cAMP-hydrolyzing pathways in pulmonary smooth muscle cells (23). It is also in line with the previous observation that intravenous tolafentrine markedly enhances the prostacyclin-induced pulmonary vasodilatory effect in perfused rabbit lungs and intact animals (14, 15).

Interestingly, the combination of aerosolized dipyridamole and iloprost was also noted to enhance and markedly prolong the postiloprost vasodilation. Dipyridamole had been noted to amplify pulmonary vasodilation of inhaled nitric oxide (24) and to attenuate the nitric oxide-induced rebound pulmonary hypertension in congenital heart disease (25). There are three possible explanations for the current observation that dipyridamole augmented the vasodilative efficacy of iloprost without causing systemic side effects. First, dipyridamole acts as a nonselective PDE inhibitor, thereby inhibiting cAMP-hydrolyzing isozymes in the lung vasculature, for example, PDE8 or PDE10, as discussed above. Second, the dipyridamole-linked inhibition of PDE5 with related cGMP increase and the iloprost-induced increase in cAMP may closely interact to forward the lung vasodilatory effects. This may be partially mediated by PDE3, which is inhibited by cGMP with an IC₅₀ of 0.1-1 µM (26). Increased intracellular cGMP levels induced by dipyridamole may result in a significant inhibition of PDE3 and therefore prolong the iloprost-induced vasodilation, as has been shown in studies that addressed the combination of the PDE5 inhibitor zaprinast and inhaled prostacyclin (14), as well as investigations of isolated vascular strips in which the NO/cGMP pathway interacts with cAMP-mediated vasodilation (27). And third, additional pharmacologic effects of dipyridamole such as inhibition of the adenosine uptake of endothelial cells (28) may amplify the vasodilatory profile of inhaled prostanoids.

Pentoxifylline was somewhat less effective then dipyridamole and, in particular, tolafentrine in augmenting iloprost-induced pulmonary vasodilation in the present study, again without causing any systemic side effects. Considering the profile of pentoxifylline as a nonselective PDE1-5 inhibitor, its effect on the response to iloprost may largely be attributable to some stabilization of cAMP due to PDE3 and PDE4 inhibition. However, its impact on PDE5 and cGMP levels may also be relevant, as discussed for dipyridamole.

In conclusion, subthreshold doses of the PDE inhibitors tolafentrine, pentoxifylline, and dipyridamole, whether aerosolized or infused, prolonged the pulmonary vasodilatory effect of nebulized iloprost in a model of experimental pulmonary hypertension. Even stronger efficacy for amplification of the iloprost response was noted, when the aerosolization route was employed for codelivery of PDE inhibitor doses that caused per se some pulmonary vasodilation, but no systemic effect or gas exchange deterioration. Conebulization, even of nonselective clinically available PDE inhibitors, might thus be considered for enhancement and in particular prolongation of the lung vasorelaxant response to inhaled iloprost.