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to nitroglycerin (NTG; IC50 49 nM), but much more potent than isosorbide dinitrate (ISD; ... avoided by co-administration of a nitrate and a β-blocker. Hence, the ...
Cardiovascular Drug Reviews Vol. 23, No. 2, pp. 149–160 © 2005 Neva Press, Branford, Connecticut

Preclinical Profile of PF9404C, a Nitric Oxide Donor with â Receptor Blocking Properties 1Mercedes

Villarroya, 1Manuela G. López, 1Ricardo de Pascual and 1,2Antonio G. García 1

Instituto de Farmacología Teófilo Hernando, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain; 2 Servicio de Farmacología Clínica e Instituto de Gerontología, Hospital de la Princesa, Madrid, Spain

Keywords: â-Blocker — NO donor — PF9404C.

ABSTRACT PF9404C ((2¢S),(2S)-3-isopropylamine, 1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2¢-propranol) is the S-S diesteroisomer of a novel blocker of â-adrenergic receptors with vasorelaxing properties. It causes a concentration-dependent relaxation of rat aorta helical strips precontracted with 10–6 M norepinephrine (NE; IC50 33 nM). It is equipotent to nitroglycerin (NTG; IC50 49 nM), but much more potent than isosorbide dinitrate (ISD; IC50 15,000 nM). In rat aorta smooth muscle cells, at 10 ìM, PF9404C increased the formation of cGMP from 3 pmol/mg protein in basal conditions to 53 pmol/mg protein, suggesting that the mechanism of its vasorelaxing effects involves the slow generation of NO. This is supported by the facts that (i) ODQ (a blocker of guanylate cyclase) inhibited the vasodilatory effects of PF9404C; and (ii) PF9404C generates NO, as indirectly measured by the Griess reaction. In the electrically driven guinea pig left atrium, PF9404C blocks the inotropic effects of isoproterenol in a concentration-dependent manner. Its IC50 (30 nM) was similar to that of S-propranolol (22.4 nM) and lower than that of metoprolol (120 nM) or atenolol (192 nM). The â adrenergic ligand (–)-[3H]-CGP12177 (4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride) (0.2 nM) is displaced from its binding sites in rat brain membranes with a Ki of 7, 17, 170, and 1200 nM for PF9404C, S-(–)propranolol, metoprolol, and atenolol, respectively. PF9404C blocks 45Ca2+ entry into bovine adrenal chromaffin cells induced by direct depolarization with 70 mM K+ or by the nicotinic agonist dimethylphenylpiperazinium (DMPP). Address correspondence and reprint requests to: Dr. Mercedes Villarroya, Instituto de Farmacologia Teófilo Hernando, Facultad de Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo 4, 28029, Madrid, Spain. Tel.: +34 914975387; Fax: +34 914975397; E-mail: [email protected]

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PF9404C exhibits about 3-fold higher potency than NTG to relax the majority of the vessels studied, especially when they were contracted with K+, and shows a certain selectivity of action for the renal artery. It produces auto-tolerance that is ca. 20-fold less pronounced than that observed with NTG. Cross-tolerance in preparations pre-exposed to PF9404C and later relaxed with NTG, was much greater than auto-tolerance. This makes PF9404C a useful pharmacological tool for the development of novel NO-donor compounds with a lesser degree of vascular tolerance than those currently available.

INTRODUCTION â-Adrenoceptor antagonists, or â-blockers, have been used in the treatment of hypertension, chronic cardiac failure, angina pectoris, arrhythmias or glaucoma for a long time. Their use was initiated with the discovery of propranolol (4,5) and continued with the development of new and more selective molecules, like metoprolol, atenolol, acebutolol or esmolol. The administration of a â-blocker alone can lead to an initial rise of peripheral vascular resistance, followed by a gradual decrease, but remaining higher than in normotensive subjects, whereas cardiac output is reduced (21,26). Organic nitrates are vasodilators; they are commonly used in the treatment of coronary heart disease and congestive cardiac failure. The administration of a vasodilator alone leads to a fall of peripheral resistance and arterial blood pressure which, in a counter-regulatory response, induces an increase in heart rate and catecholamine levels as well as activation of the renin-angiotensin-aldosterone system. This problem has been usually avoided by co-administration of a nitrate and a â-blocker. Hence, the search for a hybrid molecule having these two different mechanisms of action, i.e., â-adrenoceptor blockade and vasodilation, has been pursued for a number of years. This was, for example, the idea in the case of carvedilol, a combined á-â blocker that improves cardiac function at rest and lessens symptoms in patients with cardiac failure (11,30,35). One of the most commonly used â-blockers is metoprolol, a â-receptor blocker with preferential selectivity for â1 receptors. This drug has high hepatic metabolism and, consequently, a low bioavailability; its half-life ranges from 3 to 7 h. In the search for new antihypertensive compounds endowed with the above mentioned double mechanism of action, we synthesized a derivative of metoprolol with NO2 groups, in collaboration with chemists of Laboratorios Almirall Prodesfarma. This compound named PF9404C [((2¢S),(2S)-3-isopropylamine, 1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2¢-propanol] (Fig. 1), is capable of generating nitric oxide (NO) when in contact with tissues or cells. In this article we describe its principal pharmacological properties.

CHEMISTRY PF9404C is derived from PF9104, a molecule obtained from metoprolol which incorporates two functional NO2 groups in the aliphatic chain. It has two asymmetric carbon centers and, therefore, four different enantiomers. The S-S enantiomer, (2¢S),(2S)-3-isopropylamine, 1-[4–(2,3-dinitroxy)propoxymethyl]-phenoxy-2¢-propanol exhibiting the

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highest potency as a â-blocker and vasodilator, was named PF9404C and selected for further development (46).

N H

S

O H

OH

O

S

CH3

PHARMACOLOGY In Vitro Pharmacology Vasorelaxation PF9404C causes a concentration-dependent, slowly developing and endothelium-independent relaxation of rat aorta strips precontracted with norepinephrine; its IC50 is 33 ± 12 nM. Compared with various known NO donors, PF9404C is among the most potent nitrovasodilators (SNP, NOR3, NTG, and NOR4) and is substantially more potent than SNAP, NOR2, NOR1, or ISD (Table 1).

O2 NO

ONO2 H

FIG. 1. Chemical structure of PF9404C.

NO production The fact that PF9404C behaves as other well known NO donors does not prove that the mechanism of action of PF9404C involves NO release. However, several other pharmacological experiments suggest that this is so. For instance, oxyhemoglobin (a NO sequestering agent) displaced the concentration-response response curve of PF9404C to the right; thus, the IC50 of 19 nM obtained under control conditions in these experiments was increased up to 53 nM in the presence of oxyhemoglobin. A similar shift was observed for NTG, showing an IC50 of 14 nM before oxyhemoglobin, and of 61 nM in its presence. In both cases, the effects of oxyhemoglobin were significant over the concentration range of

TABLE 1. IC50s and time constants (ô) for the relaxation of rat aorta strips induced by PF9404C and different NO donors Compound PF9404C SNP NOR3 NTG NOR4 NOR2 SNAP NOR1 ISD

IC50 (nM) 33 ± 12 1.9 ± 1 5.8 ± 0.5 49 ± 22 58 ± 24 110 ± 17 190 ± 80 240 ± 71 15,000 ± 5900

n 9 8 3 14 3 3 6 3 11

Time constant (ô) for relaxation (min) 3.23 — 2.32 1.70 5.88 1.92 — 1.21 —

See addendum for abbreviations. IC50 data are means ± S.E.M. of the number of strips shown in n, from at least 3 different animals. Time constants are means of the values calculated from individual concentration-response relaxation curves. Adapted from ref. 46 with permission.

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PF9404C and NTG. Furthermore, cysteine that is known to accelerate NO formation (13,23), accelerated the reversal of the vasorelaxant effects of the compound. NO relaxes the vessels by activating a soluble guanylate cyclase to form cGMP in smooth muscle cells. Two well known inhibitors of this enzyme, methylene blue (MB) (20) and 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) (17,32) have been shown to reduce PF9404C- and NTG-induced relaxation. This finding indicated that relaxation could have been dependent on the production of NO. MB (1 ìM) reduced PF9404C-induced relaxation from 80–90% to 40%; similarly ODQ (1 ìM) reduced relaxation to 20%. At 1 ìM, NTG relaxed the aorta by 90% under control conditions; the relaxation decreased to 30% in the presence of MB and to 15% in the presence of ODQ. In these experiments an indirect method to measure release of NO to the medium, the colorimetric Griess reaction that produces a purple “azo” compound, has been used. Rat aorta smooth muscle cells in culture were incubated in the presence of different NO donors at the concentration of 100 ìM for 15 min and the concentration of NO -2 in the medium was estimated (Table 2). No statistical significant differences were observed in the concentrations of NO -2 produced by PF9404C, NTG or SNP. SNAP induced the highest accumulation of NO -2 , that was significantly different from that caused by other compounds (p < 0.001). Another indirect measure of NO production involves cGMP formation in rat aorta smooth muscle cells. Table 3 demonstrates generation of cGMP by PF9404C and NTG. There was no statistically significant difference between the effects of the two compounds.

Calcium channel blocking effects Some studies support the view that nitric oxide inhibits the entry of Ca2+ into cells by blocking voltage-dependent calcium channels (VDCCs) (8,39). To verify this hypothesis for a NO donor like PF9404C, we used bovine chromaffin cells as a model, since four different types of VDCCs (L, N, P/Q, R) (2,25) and nicotinic acetylcholine receptors (nAChRs) (15,24) are expressed in these cells. It was assumed that, after activation of nAChRs, Na+ enters the cell through the ionophore associated with this receptor, cell depolarizes, VDCC opens and calcium enters the cell. We studied the effect of PF9404C, measuring 45Ca2+ entry induced by direct activation of VDCC with depolarizing concentrations of KCl and by activation of nAChRs with the nicotinic agonist dimethylphenylpi-

TABLE 2. Concentration of NO2- (ìM) in the culture medium after incubation of rat aorta smooth muscle cells with different NO donors at the concentration of 100 ìM for 15 min Compound PF9404C NTG SNAP SNP

[NO-2 ] (ìM) 7.4 ± 0.3 5.7 ± 0.1 18.3 ± 0.3 6.3 ± 0.2

n 8 8 8 4

n, number of wells of at least two different cultures. Data are means ± S.E.M. for the number of wells shown in n. See addendum for abbreviations.

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perazinium (DMPP). The effect of PF9404C was compared with those of NTG and other NO donors (Table 4). When depolarization was induced by 70 ìM K+ there was no statistically significant difference in the effects of PF9404C and NTG on 45Ca2+ entry. However, PF9404C was more effective than NTG in blocking 45Ca2+ entry induced by DMPP (p < 0.001). Most of the other NO donors tested blocked 45Ca2+ entry to a lesser extent, even though some of them (like SNAP) released more NO than PF9404C (Table 2). These results suggest that PF9404C may possibly have a calcium channel blocking effect at concentrations much higher than required for its activity as a NO donor. Additional experiments will be necessary to determine whether this effect is due to the presence of NO groups in the molecule or to some other intrinsic characteristic of PF9404C.

Blockade of â-adrenoceptors The rationale behind the development of PF9404C was to obtain a hybrid molecule with vasodilator and â-adrenoceptor blocking effects. To test the â-adrenoceptor blocking activity two experimental protocols were used: (i) antagonism of the inotropic response to isoproterenol on the isolated left atria of guinea pigs and (ii) biochemical studies to determine the displacement of the â-adrenoceptor ligand CGP12177 by PF9404C. The an-

TABLE 3. Generation of cyclic GMP (pmol/mg protein) in rat aorta smooth muscle cells incubated for 5 min with different concentrations of PF9404C and nitroglycerin (NTG) Drug (ìM) PF9404C NTG

0 3.6 3.6

0.3 21.8 ± 0.9 11.8 ± 5.7

1 28.2 ± 1.9 23.6 ± 3.8

3 31.8 ± 5.2 16.4 ± 8.5

10 50.9 ± 1.4 49.1 ± 2.8

30 50.9 ± 1.4 76.4 ± 8.5

Data are means ± S.E.M. for four wells from the same cell culture. Adapted from ref. 46 with permission.

TABLE 4. Effects of PF9404C and other NO donors on 45Ca2+ uptake induced by 70 mM K+ or by the nicotinic agonist DMPP (100 ìM) into bovine chromaffin cells % Inhibition 70 ìM K+ Compound PF9404C 30 ìM NTG 30 ìM SNAP 30 ìM SNP 30 ìM NOR-1 10 ìM NOR-2 10 ìM NOR-3 10 ìM NOR-4 10 ìM

63 ± 2.2 60 ± 3.1 25 ± 8.2 39 ± 4.5 62 ± 4.4 29 ± 3.5 34 ± 4.4 22 ± 6.5

100 ìM DMPP n 9 20 9 14 12 12 12 12

92 ± 1.5 55 ± 4.2 20 ± 5.2 57 ± 6.7 60 ± 4 50 ± 3.5 36 ± 5.3 40 ± 6.5

n 9 17 9 11 12 12 12 12

n, number of wells from at least three different cell cultures. Data are means ± S.E.M. of the number of wells shown in n. See addendum for abbreviations.

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tagonism of the inotropic actions of isoproterenol in the guinea pig left atrium indicated that PF9404C blocks cardiac â-adrenoceptors. Its â-blocking potency is close to that of S(–)propranolol, 4-fold higher than that of metoprolol, and 6-fold higher than that of atenolol. This finding was confirmed by radioligand binding experiments in rat brain membranes, where the binding of (–)-[3H]-CGP12177 (0.2 nM) has been displaced with the following order of potencies: PF9404C > S-(–)propranolol > metoprolol > atenolol (Table 5).

Vascular selectivity To explore the potential cardiovascular indications for PF9404C, its vascular selectivity for different rabbit arteries and veins (aorta, renal, femoral and central ear arteries and portal and saphenous veins) was studied. Parallel experiments were conducted with NTG for comparison. Two different types of stimuli were used to pre-contract the arteries and veins prior to relaxation with PF9404C or NTG: i) phenylephrine, which induces á-adrenoceptor-mediated mobilization of calcium from the intracellular stores with the consequent contraction of the vessels (42); and ii) high K+, that opens L-type calcium channels through membrane depolarization, permitting the entry of calcium and subsequent contraction of vascular strips (7,45). In general, both drugs were more potent as inhibitors of phenylephrine- than of K+-induced contractions in most of the vessels studied, with the exception of the central ear artery (Table 6). PF9404C exhibited a slightly higher potency (about 3-fold) than NTG. In addition, it is worth noting that PF9404C always exerts complete relaxation of all vessels, contracted by either phenylephrine or K+. In contrast, NTG does not fully relax vessels contracted by K+. It is conceivable that PF9404C has some calcium-antagonist property, thus explaining the greater blockade of K+-evoked vascular contractions (14). A second aspect to consider was the possible selectivity of the two drugs for the different arteries and veins. NTG shows a certain selectivity for the aorta and renal artery with respect to the femoral artery (IC50 to inhibit the phenylephrine-induced contraction around 0.03 and 0.3 ìM, respectively), and greater selectivity for these three vessels in comparison to the central ear artery (IC50 = 4.6 ìM). We found a clearer vasoselectivity for PF9404C. The differences in relaxation for the two compounds are statistically significant at all concentrations from 1 to 100 nM in aorta and at 3 to 30 ìM for portal and saphenous veins. The order of potencies to inhibit the phenylephrine-induced contraction of the arteries were (Table 6): aorta (IC50 = 0.007 ìM)

TABLE 5. Effects of PF9404C and different â-receptor blockers on the inotropic effects of isoproterenol in the guinea pig atria and [3H]CGP12177 binding to rat brain membranes Compound PF9404C S-propranolol Metoprolol Atenolol

IC50 (nM) to block the inotropic effect of 30 nM isoproterenol 30 ± 9.9 22.4 ± 3.8 120 ± 20.6 192 ± 53

IC50 (nM) for the displacement of [3H]CGP12177 binding 23 ± 4 53 ± 4 530 ± 110 3700 ± 1600

n 8 5 5 5

Data are means ± S.E.M. of the number of experiments shown in n. Adapted from ref. 46 with permission.

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> renal artery (IC50 = 0.015 ìM) > femoral artery (IC50 = 0.098 ìM) > central ear artery (IC50 = 3.7 ìM). The differences were even greater when the contraction of the arteries was evoked with high K+. While NTG never inhibits by 100% the contraction, PF9404C is a very efficient inhibitor, that is capable of completely relaxing all arteries, with the following order of potencies: aorta (IC50 = 0.12 ìM) > renal artery (IC50 = 1.3 ìM) > central ear artery (IC50 = 1.9 ìM) > femoral artery (IC50 = 2.8 ìM). In this case, the differences in relaxation between NTG and PF9404C were statistically significant at all concentrations from 3 nM to 30 ìM for aorta and from 3 to 30 ìM for portal vein. There was no differences in the relaxant effects of the two drugs in saphenous vein preparations. NTG, as most of the organic nitrates, is considered to be preferentially venoselective (3,29,40). Surprisingly, we did not find a clear venoselectivity for either of the two drugs. As mentioned above the IC50s for the relaxant effects in portal or saphenous veins were higher than those for aorta, renal, or femoral arteries (Table 6). The fact that PF9404C shows certain selectivity for the renal in comparison to the femoral and the central ear arteries, suggests a possible beneficial effect on renal blood flow in situations with a deficit in renal perfusion, such as congestive cardiac failure or certain types of hypertension. Additionally, the drug could have a nephroprotective effect in different nephropathies.

Development of tolerance There is a trend for the beneficial effects of nitrates to diminish during long-term therapy. This phenomenon, termed tolerance, is well documented in patients with congestive cardiac failure (12,36,43). After continuous administration of the drug, the efficacy of the treatment is lost, leaving the patients vulnerable to ischemia. The cause of the tolerance is still controversial, although many studies have been conducted on the origin

TABLE 6. IC50s for vasorelaxant effects of PF9404C and nitroglycerin (NTG) in rabbit vessels precontracted with 35 mM K+ or 1 ìM phenylephrine

Potassium

Phenylephrine

Vessel Aorta Renal artery Femoral artery Central ear artery Portal vein Saphenous vein Aorta Renal artery Femoral artery Central ear artery Portal vein Saphenous vein

Maximum contraction (mN) 29.8 ± 0.9 48 ± 6 41 ± 5 20.4 ± 2 7.2 ± 1.3 26 ± 3 28.2 ± 0.9 36 ± 4 40 ± 5 26.5 ± 1.8 6.5 ± 0.7 29 ± 3

PF9404C IC50 (ìM) 0.12 1.34 2.86 1.95 4.60 0.20 0.01 0.015 0.1 3.7 2.1 0.6

NTG IC50 (ìM) NC 4.1 NC 2.2 NC 0.5 0.025 0.04 0.3 4.5 NC 0.5

Ratio NTG/PF9404C NC 3 NC 1.1 NC 2.5 3.6 2.5 3 1.2 NC 0.8

n 16 13 15 21 13 16 13 17 22 17 18 26

NC, not calculable. The maximum contraction data are means ± S.E.M. of the number of preparations shown in n. Adapted from ref. 41 with permission.

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and mechanisms underlying this phenomenon (1,6,9,10,33,34,38). We studied the development of tolerance to PF9404C and compared it to the tolerance that develops to NTG (Table 7). At 30 ìM PF9404C completely relaxed preparations, contracted with either phenylephrine or K+, while NTG, at the same concentration, relaxed aortas by only 60 or 40%, respectively. It is of interest that there was significantly less tolerance to PF9404C than to NTG. Two major mechanisms have been proposed to explain tolerance to organic nitrates: 1) impaired enzymatic release of NO (“impaired bioconversion”) and 2) increased endothelial generation of superoxide (18,26). The first mechanism is currently more widely accepted. In the case of NTG, its activation of guanylate cyclase is sulfhydryl-dependent in vitro (23) and N-acetylcysteine is known to potentiate the effect of organic nitrates (22,26). This is consistent with our observation that in the presence of N-acetylcysteine, the tolerance to NTG did not develop. However, in the case of PF9404C, after co-incubation with N-acetylcysteine, tolerance was slightly increased. This suggests that the mechanism of action of PF9404C in relaxing the vessels must be different from that of NTG. One possible cause would be the slower release of NO by PF9404C, as compared to NTG (46). We studied cross-tolerance for NTG or PF9404C in aortic strips pretreated with these drugs and contracted with 1 ìM phenylephrine. In this study IC50 for NTG (5.9 ìM) was higher, while IC50 for PF9404C (0.41 ìM) was lower than in the autotolerance study. Given the importance of the development of tolerance with nitrates in the treatment of patients with ischemic heart disease or cardiac failure, PF9404C or similar NO-donors much less capable of developing tolerance will offer a significant therapeutic advantage.

Antagonism of endothelin-1-induced increase in renal perfusion pressure Arterial hypertension, cardiac failure and shock are often associated with a renal hypoperfusion. The effects of PF9404C on renal perfusion pressure were studied in isolated rat kidneys perfused with 10 nM endothelin-1. No significant reduction of renal perfusion pressure was observed for NTG or metoprolol at any of the concentrations used. PF9404C or carvedilol elicited, however, a concentration-dependent reduction in the endothelin-1induced increase in the perfusion pressure. This effect was statistically significant only at 100 ìM of PF9404C or carvedilol (Table 8).

Therapeutic potential The rationale for the combination of a â-adrenoceptor antagonist with a vasodilator in the same molecule is clearly established (44). When propranolol or a similar â-blocker

TABLE 7. Auto-tolerance to PF9404C and nitroglycerin (NTG) in rat aorta contracted with 1 ìM phenylephrine or 35 mM K+, expressed as IC50 for the relaxation of the vessel (ìM)

PF9404C NTG

Phenylephrine control pre-expossed 0.018 (14) 0.82 (22) 0.0035 (11) 2.90 (13)

ratio 45 829

Potassium control pre-expossed 0.011 (11) 1.5 (11) 0.0043 (11) NC (11)

ratio 138 —-

NC, not calculable. Data are the means of the number of preparations shown in parentheses. Data from ref. 41 with permission.

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alone is administered to hypertensive patients, the arterial pressure remains to a large extent unaltered at the start of treatment and decreases gradually during chronic treatment, whereas cardiac output is consistently reduced. However, even during chronic treatment the total peripheral resistance remains higher than in normotensive subjects (21). Thus, pure â-blockers are normally not able to correct the hemodynamic disturbance. A vasodilator alone leads to a fall of peripheral resistance and of the arterial blood pressure. However, heart rate and plasma catecholamine levels increase and renin-angiotensin-aldosterone system is activated. These counterregulatory responses can be prevented by simultaneous â-blockade. It is known that NO exerts potent vasodilating actions (37), inhibits leukocyte-endothelial cell interaction (28,31), platelet adherence and aggregation (19), as well as vascular smooth muscle cell proliferation (16). These actions constitute important protective mechanisms against atherogenesis and myocardial ischemia-reperfusion injury. Hence, the reduction in NO activity observed in hypercholesterolemia may promote atherogenesis and aggravate reperfusion injury. Conversely, the preservation of NO activity by a slow NO donor, such as PF9404C, may exert beneficial effects in hypercholesterolemia and myocardial ischemia. On the other hand, the fact that PF9404C shows a certain selectivity for the renal artery in relation to the femoral and the central ear arteries, give us basis to predict a possible beneficial effect improving renal blood flow in conditions associated with diminished renal perfusion, such as in congestive cardiac failure or in certain types of hypertension. Additionally, it could have a nephroprotective effect in different nephropathies. Finally, it is interesting that the tolerance (cross-tolerance and auto-tolerance) to the vasorelaxing effect of PF9404C is 20-fold lower than that to NG and apparently involves a different mechanism. Given the importance of the development of tolerance to nitrates in the treatment of ischemic heart disease or cardiac failure, PF9404C or similar compounds with much less potential for developing tolerance may represent a new approach to the treatment of these diseases.

Conclusions and perspectives PF9404C is a potent vasorelaxing agent, as well as a blocker of cardiac â adrenergic receptors. The mechanism of its vasorelaxing effect involves, at least in part, slow generation of NO. This compound can, therefore, exhibit antihypertensive and cardiopro-

TABLE 8. Antagonism of endothelin-1 (10 nM)-induced reduction of perfusion pressure in isolated rat kidneys by PF9404C and other drugs

PF9404C NTG Metoprolol Carvedilol

Reduction of retrograde perfusion at 100 ìM % control n 81 ± 7.2*** 8 15 ± 6.3 5 26 ± 8.1 4 68 ± 12.6** 5

Data are means ± S.E.M. for the number of experiments shown in n. **p < 0.005, ***p < 0.001, with respect to control flow.

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tective actions through a dual mechanism of action i.e., NO donation and blockade of â-adrenoceptors. Although PF9404C and NTG are both NO donors, the profiles of their vasodilator actions are different. Furthermore, the tolerance to the vasodilator effect is less pronounced with PF9404C and seems to be mediated by a mechanism different from that of nitrates. This makes PF9404C a useful pharmacological tool for further development of novel NO-donors with a lesser tolerance than those now available. Acknowledgments. This work was supported in part within a collaborative research program between Laboratorios Almirall Prodesfarma (Barcelona, Spain) and Instituto Teófilo Hernando (Universidad Autónoma de Madrid, Spain).

Addendum. Abbreviations ISD, isosorbide dinitrate; ISO, isoproterenol; DMPP, 1,4-dimethylphenyl-piperazinium; MB, methylene blue; NE, norepinephrine; nAChR, nicotinic acetylcholine receptor; NO, nitric oxide; NOR1, (+)-(E)-methyl-2-[(E)-hydroxyminol]-5-nitro-6-methoxy-3-hexenamide]; NOR2, [(+)-(E)-Methyl-2-[(E)-hydroxyminol]-5-nitro-3-hexenamide]; NOR3, [(+)-(E)-ethyl-2-[(E)-hidroxyminol-5-nitro-3-hexenamide]; NOR4, [3-((+)-(E)-ethyl-2¢-[(e)-hydroxyminol]-5-nitro-3-hexenecarbamoyl)-pyridine]; NTG, nitroglycerin; ODQ, (1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one); SNAP, S-nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside; VDCCs, voltage-dependent calcium channels.

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