Edward D. Pagani

Zaprinast increases cyclic GMP levels in plasma and in aortic tissue of rats

The purpose of this study was to determine if significant relationships exist between plasma and aortic cyclic GMP (cGMP) levels and pharmacodynamic effect after the i.v. administration of the cGMP-selective phosphodiesterase inhibitor zaprinast to conscious, spontaneously hypertensive rats. Zaprinast dose-dependently increased plasma and aortic cGMP levels at 10, 18 and 30 mg/kg and decreased mean arterial blood pressure (MAP) at 18 and 30 mg/kg. The concentrations of cGMP in the plasma and in the aorta were significantly correlated (r = 0.765, P < 0.0001). The changes in MAP were significantly correlated to aortic (r = -0.750, P < 0.0001) and plasma (r = -0.762, P < 0.0001) cGMP levels. We conclude that plasma cGMP may be an index of cGMP-selective phosphodiesterase inhibition in vivo.

Reversal of nitroglycerin tolerance in vitro by the cGMP-phosphodiesterase inhibitor zaprinast

Following in vitro exposure of rat aortic rings to 550 microM nitroglycerin for 1 h, tolerance was demonstrated by a significant increase in EC50 values for nitroglycerin-induced relaxation. However, cross-tolerance to sodium nitroprusside was not observed. Co-incubation of aortic rings with the cGMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast (10 microM), during incubation with 550 microM nitroglycerin, did not prevent the development of tolerance. However, the addition of 0.30 or 10 microM zaprinast to tolerant aortic rings did restore responsiveness to nitroglycerin. The increase in cGMP in tolerant aortic rings in response to 300 nM nitroglycerin (2-4 fmol/micrograms) was significantly less than that observed for non-tolerant rings (6.6-12 fmol/micrograms), but cGMP levels were restored in tolerant rings by zaprinast (7-12 fmol/micrograms). These data suggest that inhibition of vascular cGMP-PDE activity does not prevent the development of tolerance in vitro, but does reverse the loss of vasorelaxant potency to nitroglycerin via restoration of intracellular cGMP levels.

Inhibition of low Km cyclic GMP phosphodiesterases and potentiation of guanylate cyclase activators by cicletanine.

Cicletanine is an antihypertensive/vasorelaxant/natriuretic agent of unknown mechanism. We wished (a) to determine if cicletanine interacts with guanylate cyclase activators that modulate vasomotor tone and sodium balance [i.e., atriopeptin II (AP II), endothelium-derived relaxing factor (EDRF), and sodium nitroprusside (SNP)], and (b) to define the subcellular basis for this interaction by quantitating the effects of cicletanine on low Km cyclic GMP phosphodiesterase (PDE) activity. In phenylephrine-contracted rat aortic smooth muscle, the vasorelaxant potency of cicletanine was increased twofold in the presence of a threshold-relaxant concentration of AP II, and functional cyclic GMP PDE inhibition was also evident from the three- to sixfold potentiation by cicletanine of AP II- or SNP-induced vasorelaxation. Vasorelaxation produced by cicletanine was not endothelium dependent, however. In further studies, intravenous (i.v.) administration of cicletanine or the low Km cyclic GMP PDE inhibitor, zaprinast, decreased blood pressure (BP) ≤20% in conscious spontaneously hypertensive rats (SHR). These results are consistent with the additional finding that cicletanine inhibited Ca2+ -calmodulin (CaM) cyclic GMP PDE and zaprinast-sensitive cyclic GMP specific PDE over a concentration range (10–600 μM) similar to that for vasorelaxation. Thus, inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant effect of cicletanine as well as the potentiation by cicletanine of the vasorelaxant actions of guanylate cyclase activators. The extent to which this mechanism contributes to the antihypertensive efficacy of cicletanine has not yet been fully determined.

Reversal or nitroglycerin tolerance by the cGMP phosphodiesterase inhibitor zaprinast

In in vitro experiments, aortic rings (2-3mm) from Sprague-Dawley rats (250-350g) were incubated for 1 h with 550 μM nitroglycerin or vehicle (1.65% PEG-400), extensively rinsed, transferred to naive tissue baths, and subsequently tested for concentration-related vasorelaxation to nitroglycerin in the presence and absence of zaprinast. In in vivo experiments, spontaneously hypertensive rats (17 weeks) were injected with 100 mg nitroglycerin/kg body weight s.c., 3 times/day for 3 days. Decreases in mean arterial blood pressure (MAP) in response to nitroglycerin in the presence or absence of zaprinast were quantitated prior to the initiation of nitroglycerin injection (day 0), and after 3 days of nitroglycerin injection (fig. 1, bottom)

Enrichment and analysis of desmosine and isodesmosine in biological fluids

A method has been developed for the enrichment and analysis of the elastin crosslinks, desmosine and isodesmosine, in biological fluids and tissues. It is adapted from published methods, offering improved recovery, sensitivity, resolution, and speed of analysis. Samples were hydrolyzed in 6 M HCl, after which the desmosines were enriched by CF1 cellulose chromatography and analyzed by HPLC with a C18 column. Isodesmosine and desmosine were quantitated based on absorbance at 275 nm, with a limit of detection of approximately 30 pmol and recovery of approximately 66% in urine. Their tR values on our HPLC system were approximately 9 and 12 min, respectively. This method was used to evaluate the daily and weekly variation in the concentrations of desmosine and isodesmosine in human urine. The results suggest that this method can be used to process large numbers of biological samples for analysis of desmosine and isodesmosine.

Zaprinast, but not dipyridamole, reverses hemodynamic tolerance to nitroglycerin in vivo

Abstract Hemodynamic tolerance to nitroglycerin was developed in spontaneously hypertensive rats following 2–3 days of pretreatment with 100 mg/kg of nitroglycerin administered s.c. 3 times/day. Tolerance was evaluated both in vivo, by administering ascending bolus doses of nitroglycerin of 1–300 μg/kg i.v., and ex vivo in isolated, denuded aortic vascular rings by exposure to ascending concentrations of nitroglycerin of 0.0003–100 μM. Tolerance was observed as a significant blunting of the hypotensive and vasorelaxant effect of nitroglycerin. Co-incubation of tolerant aortic rings and pretreatment of tolerant SHR with 10 μM and 0.1–10 mg/kg zaprinast, respectively, resulted in full restoration of the vasorelaxant and hypotensive effect of nitroglycerin. Zaprinast partially reversed hemodynamic tolerance at 0.01 mg/kg. Conversely, dipyridamole (10 μM) reversed tolerance ex vivo, but was ineffective in reversing tolerance in vivo at pretreatment doses of 30 and 60 mg/kg. Following a 100-μg/kg i.v. challenge dose of nitroglycerin, aortic cyclic guanosine monophosphate (cGMP) levels were lower in nitroglycerin tolerant SHR when compared to non-tolerant SHR. Pretreatment of tolerant SHR with 10 mg/kg zaprinast restored the increase in cGMP levels to nitroglycerin to that seen in non-tolerant SHR. Conversely, dipyridamole (30 mg/kg) pretreatment was not effective in restoring cGMP levels. These data therefore suggest that reversal of hemodynamic tolerance in vivo is related to restoration of changes in vascular cGMP levels. Zaprinast, a selective cGMP phosphodiesterase inhibitor, effectively reverses tolerance and dipyridamole, a rather non-selective inhibitor, does not.

Nω-Nitro-L-arginine attenuates the accumulation of aortic cyclic GMP and the hypotension produced by zaprinast

To determine if N omega-nitro-L-arginine (NNA), an inhibitor of the synthesis and/or release of endothelium-derived relaxing factor (EDRF), alters the response to zaprinast, a selective inhibitor of cyclic GMP (cGMP) phosphodiesterase, zaprinast (3-30 mg/kg) or vehicle (1 ml/kg) was given to conscious, spontaneously hypertensive rats (SHR) in a cumulative i.v. dose-response manner 30 min after pretreatment with NNA (1 or 3 mg/kg) or saline (1 ml/kg). Mean arterial pressure (MAP) was measured 5 min after each dose of zaprinast. Five minutes after the last dose of zaprinast (30 mg/kg), the rats were anesthetized with pentobarbital (25 mg i.v.). A segment of the abdominal aorta was freeze-clamped in situ and removed for the determination of cGMP levels. NNA (3 mg/kg) decreased basal aortic cGMP levels by 54% and increased MAP by 37 +/- 2 mm Hg. Zaprinast (30 mg/kg) increased aortic cGMP by 187% and decreased MAP by 49 +/- 4 mm Hg. NNA (3 mg/kg) reduced the accumulation of cGMP in aortic tissue (from 4.1 +/- 0.4 to 1.3 +/- 0.1 fmol/microgram protein) and attenuated the depressor response (from -49 +/- 4 to -31 +/- 4 mm Hg) produced by zaprinast. These data are consistent with the hypothesis that NNA inhibits the tonic release of EDRF and that the depressor effects of zaprinast are due, at least in part, to the potentiation of the vasodilator effects of EDRF in vivo. Moreover, since the changes in MAP produced by NNA and zaprinast were significantly correlated with cGMP levels in aortic tissue, the concentration of cGMP in vascular tissue may be a determinant of blood pressure in SHR.

Cardiovascular activity of WIN 65579, a novel inhibitor of cyclic GMP phosphodiesterase 5.

This study describes the phosphodiesterase inhibitory potency and cardiovascular actions of WIN 65579 (1-cyclopentyl-3-ethyl-6-(3-ethoxy-4-pyrridyl)-1H-pyrazolo[3,4-d]p yrimidin-4-one), a potent, new cGMP phosphodiesterase 5 inhibitor. WIN 65579 is a competitive inhibitor of phosphodiesterase 5, with IC50 values of 2-3 nM for phosphodiesterase 5 from human or canine vascular sources. WIN 65579 has low affinity for phosphodiesterases 1, 2 and 3 (IC50 > 3-10 microM), and is somewhat selective for phosphodiesterase 4 (IC50 approximately 100 nM). WIN 65579 is an endothelial-dependent relaxant of rat aortic smooth muscle (EC50 = 60 nM) and lowers mean arterial blood pressure in conscious spontaneous hypertensive rats following intravenous or oral dosing. WIN 65579 also increases plasma cGMP levels, and reinstates vascular responsiveness to nitroglycerin in conscious rats that are nitroglycerin-tolerant. These data show that WIN 65579 is one of the more potent phosphodiesterase 5 inhibitors, and that WIN 65579 possesses cardiovascular activities consistent with vascular phosphodiesterase 5 inhibition in vivo.

Chapter 20. Human Leukocyte Elastase Inhibitors

Publisher Summary Human leukocyte elastase (HLE) is a serine protease and a basic glycoprotein, with isoforms of 25-30 kD. The active site of HLE is a channel on the protein surface that has extended substrate binding sites. The catalytic triad of Serlg5, Hisr7, and Asplo2 that resides in the active site channel catalyzes amide bond hydrolysis of various proteins, including elastin, the connective tissue of the lung. The S1 specificity pocket of serine proteases is largely responsible for the selectivity of the class of enzymes to cleave proteins at specific sites. HLE has a relatively small S1 pocket that is lined, with hydrophobic residues, thus HLE preferentially cleaves proteins at sites, with small lipophilic residues, such as alanine and valine. The specificity has been exploited in the design of HLE inhibitors. The isoforms of HLE have identical amino acid sequences and catalytic properties, but differ based on the nature of the carbohydrate content. The different carbohydrate content of the E-1 and E-3 isoforms of elastase is proposed to direct these isoforms to secretory and lysosomal functions, respectively. Human elastase, from polymorphonuclear neutrophils (PMN) and from purulent sputum, display identical kinetics, with various substrates and inhibitors, suggesting that elastase, from sputum, is from polymorphonuclear leukocytes (PMNs). In the chapter, the term HLE will be used to indicate elastase, from either PMNs or purulent sputum.

Imidazotriazinone inhibitors of the Ca2+-calmodulin sensitive phosphodiesterase (PDE I)

Abstract Hybrid structural analogs 1 of the PDE V and PDE III inhibitors, zaprinast, milrinone, and CI-930 were prepared to identify dual PDE inhibitors. The SAR study led unexpectedly to the identification of WIN 61691 (8d), a potent inhibitor of PDE I (IC50 = 85 nM). A potent and selective inhibitor of PDE I would be a useful tool to elucidate the physiologic function of PDE I and other PDE isozymes in biological systems.