Manlio Bolla

RELEASE OF LEUKOTRIENE A4 VERSUS LEUKOTRIENE B4 FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES

The reactive intermediate formed by 5-lipoxygenase metabolism of arachidonic acid, leukotriene A4, is known to be released from cells and subsequently taken up by other cells for biochemical processing. The objective of this study was to determine the relative amount of leukotriene A4 synthesized by human polymorphonuclear leukocytes (PMNL) that is available for transcellular biosynthetic processes. This was accomplished by diluting cell suspensions and measuring the relative amounts of enzymatic versus nonenzymatic leukotriene A4-derived metabolites after challenge with the Ca2+ ionophore A23187. Nonenzymatic leukotriene A4-derived metabolites were used as a quantitative index of the amount of leukotriene A4 released into the extracellular milieu. The results obtained demonstrated that in human PMNL, the relative amounts of nonenzymatic versus enzymatic leukotriene A4-derived metabolites increased with decreasing cell concentrations. After a 20-fold dilution of PMNL in cell preparations, a doubling in the amount of nonenzymatic leukotriene A4-derived metabolites was observed following challenge (from 53.9 ± 1.3 to 110.4 ± 8.9 pmol/106 PMNL, p < 0.01). Reduction of possible cell-cell interactions by dilution suggested that over 50% of leukotriene A4 synthesized is released from the PMNL. These data provide evidence that, in human PMNL preparations, transfer of leukotriene A4 to neighboring PMNL is taking place, resulting in additional formation of leukotriene B4 and its ω-oxidized metabolites 20-hydroxy- and 20-carboxy-leukotriene B4. Neutrophil reuptake of extracellular leukotriene A4 leads to an underestimation of the fraction of leukotriene A4 that is in fact available for transcellular metabolism when tight cell-cell interactions occur, such as during PMNL adhesion to the microvascular endothelium and diapedesis.

Gastrointestinal-Sparing Effects of Novel NSAIDs in Rats with Compromised Mucosal Defence

Nonsteroidal anti-inflammatory drugs are among the most commonly used prescription and over-the-counter medications, but they often produce significant gastrointestinal ulceration and bleeding, particularly in elderly patients and patients with certain co-morbidities. Novel anti-inflammatory drugs are seldom tested in animal models that mimic the high risk human users, leading to an underestimate of the true toxicity of the drugs. In the present study we examined the effects of two novel NSAIDs and two commonly used NSAIDs in models in which mucosal defence was expected to be impaired. Naproxen, celecoxib, ATB-346 (a hydrogen sulfide- and naproxen-releasing compound) and NCX 429 (a nitric oxide- and naproxen-releasing compound) were evaluated in healthy, arthritic, obese, and hypertensive rats and in rats of advanced age (19 months) and rats co-administered low-dose aspirin and/or omeprazole. In all models except hypertension, greater gastric and/or intestinal damage was observed when naproxen was administered in these models than in healthy rats. Celecoxib-induced damage was significantly increased when co-administered with low-dose aspirin and/or omeprazole. In contrast, ATB-346 and NCX 429, when tested at doses that were as effective as naproxen and celecoxib in reducing inflammation and inhibiting cyclooxygenase activity, did not produce significant gastric or intestinal damage in any of the models. These results demonstrate that animal models of human co-morbidities display the same increased susceptibility to NSAID-induced gastrointestinal damage as observed in humans. Moreover, two novel NSAIDs that release mediators of mucosal defence (hydrogen sulfide and nitric oxide) do not induce significant gastrointestinal damage in these models of impaired mucosal defence.

Cyclooxygenase-inhibiting nitric oxide donators for osteoarthritis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) remain the most commonly used medications for the treatment of the symptoms of many chronic inflammatory diseases, including osteoarthritis. Unfortunately, the toxicity of NSAIDs substantially limits their long-term use. Some newer NSAIDs, namely selective cyclooxygenase (COX)-2 inhibitors, exhibit greater gastrointestinal safety, and concomitant use of anti-secretory drugs can also reduce NSAID-induced gastropathy. However, NSAIDs also adversely affect the cardiovascular system. A new class of anti-inflammatory drugs, COX-inhibiting nitric oxide donators (CINODs), has been designed to exert similar anti-inflammatory effects as NSAIDs, but with an improved safety profile. CINODs release nitric oxide, providing protective effects in the gastrointestinal tract and attenuating the detrimental effects on blood pressure normally associated with NSAIDs. We provide an outline of the rationale for CINODs and their activity, in addition to an overview of the pre-clinical and clinical profile of the most advanced CINOD, naproxcinod.

Delapril slows the progression of atherosclerosis and maintains endothelial function in cholesterol-fed rabbits

The renin-angiotensin system is an important modulator of arterial blood pressure and inhibitors of the angiotensin-converting enzyme (ACE-Is) and are currently used in the treatment of hypertension. The pleiotropic actions exerted by angiotensin II (AngII) on the functionality of the vessel wall may have pro-atherosclerotic outcomes; evidence for an anti-atherosclerotic effect of ACE-Is has been presented and an antioxidant effect has been attributed to thiol-containing ACE-Is, like Captopril. The present study has been undertaken to investigate the effect of Delapril, a lipophilic ACE-I, on the development of atherosclerosis in cholesterol-fed rabbits. While it did not correct hyperlipidemia, Delapril dose dependently inhibited the development of atherosclerosis, expressed as aortic area covered by lesions (23.3+/-4.1, 21.3+/-2.4 and 18.5+/-3.3% with Delapril at the daily dose of 5, 10 and 20 mg/kg, respectively, versus 38.2%+/-6.4 for control animals) and its effect was similar to that of Captopril (14.5+/-5.1% at the daily dose of 25 mg/kg). Furthermore, Delapril partially and dose dependently restored endothelium-dependent relaxation, which is impaired in vessels from hypercholesterolemic animals (51.80+/-12.18, 59.74+/-5.16, 69.13+/-8.70 maximal percent relaxation versus 48.26+/-3.05% for the untreated control and 67.67+/-6.72% for Captopril-treated animals). An antioxidant mechanism is unlikely to explain this data, since Delapril does not contain thiol groups. These observations suggest that Delapril may represent an effective pharmacological approach for the treatment of atherosclerosis during its early phases.

A topical nitric oxide-releasing dexamethasone derivative : effects on intraocular pressure and ocular haemodynamics in a rabbit glaucoma model

Background: Topical nitric oxide-releasing dexamethasone (NCX1021) may avoid the negative effects of dexamethasone phosphate. Aims: To obtain more information on the role of nitric oxide in glaucoma and to compare a nitric oxide-releasing dexamethasone with dexamethasone phosphate with regard to intraocular pressure (IOP) and ocular haemodynamics in an experimental rabbit model. Methods: Six rabbits were treated with dexamethasone phosphate 0.1% in the right eye and with NCX1021 in the left eye for 5 weeks. The parameters considered were IOP, nitric oxide marker levels in aqueous humour, ocular haemodynamics of ophthalmic artery (by means of colour Doppler imaging), expression of endothelial nitric oxide synthase (eNOS)in ciliary processes and histology of ciliary bodies. Results: Dexamethasone increased IOP levels, NCX1021 did not. Nitrite and cyclic guanosine monophosphate levels in aqueous humour were lowered by dexamethasone and increased by NCX1021. Resistivity index of the ophthalmic artery was increased, eNOS expression was reduced and ciliary bodies showed histological lesions in dexamethasone-treated eyes, not in NCX1021-treated ones. Conclusions: NCX1021 may avoid the IOP increase, impairment of ocular blood flow and the morphological changes in the ciliary bodies possibly induced by corticosteroid treatment.

Pharmacological characterization of the cysteinyl-leukotriene antagonists CGP 45715A (iralukast) and CGP 57698 in human airways in vitro

Cysteinyl‐leukotrienes (cysteinyl‐LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. We investigated the pharmacological profile of the cysteinyl‐LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. Competition curves vs [3H]‐LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]‐LTD4; (b) as in all the G‐protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2=16.6 nM±36% CV and Ki1= Ki2=5.7 nM±19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. In functional studies: (a) iralukast and CGP 57698 antagonized LTD4‐induced contraction of human bronchi, with pA2 values of 7.77±4.3% CV and 8.51±1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. Both antagonists significantly inhibited antigen‐induced contraction of human isolated bronchial strips in a concentration‐dependent manner, lowering the upper plateau of the anti‐IgE curves. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases.

Prevention of Bleomycin-Induced Lung Fibrosis in Mice by a Novel Approach of Parallel Inhibition of Cyclooxygenase and Nitric-Oxide Donation Using NCX 466, a Prototype Cyclooxygenase Inhibitor and Nitric-Oxide Donor

Cyclooxygenase (COX)-inhibiting nitric oxide (NO) donors (CINODs) are designed to inhibit COX-1 and COX-2 while releasing NO. COX inhibition is responsible for anti-inflammatory and pain-relieving effects, whereas NO donation can improve microcirculation and exert anti-inflammatory and antioxidant actions. In an in vivo mouse model of bleomycin-induced lung fibrosis, we evaluated whether a prototype CINOD compound, (S)-(5S)-5,6-bis(nitrooxy)hexyl)2-(6-methoxynaphthalen-2-yl)propanoate (NCX 466), may show an advantage over naproxen, its congener drug not releasing NO. Bleomycin (0.05 IU) was instilled intratracheally to C57BL/6 mice, which were then treated orally with vehicle, NCX 466 (1.9 or 19 mg/kg), or an equimolar dose of naproxen (1 or 10 mg/kg) once daily for 14 days. Afterward, airway resistance, assumed as lung stiffness index, was assayed, and lung specimens were collected for analysis of lung inflammation and fibrosis. NCX 466 and naproxen dose-dependently prevented bleomycin-induced airway stiffness and collagen accumulation. NCX 466, at the highest dose, was significantly more effective than naproxen in reducing the levels of the profibrotic cytokine transforming growth factor-β and the oxidative stress markers thiobarbituric acid reactive substance and 8-hydroxy-2′-deoxyguanosine. NCX 466 also decreased myeloperoxidase activity, a leukocyte recruitment index, to a greater extent than naproxen. A similar inhibition of prostaglandin E2 was achieved by both compounds. In conclusion, NCX 466 has shown a significantly higher efficacy than naproxen in reducing lung inflammation and preventing collagen accumulation. These findings suggest that COX inhibition along with NO donation may possess a therapeutic potential in lung inflammatory diseases with fibrotic outcome.

Comparative analysis of isolated human bronchi contraction and biosynthesis of cysteinyl leukotrienes using a direct 5-lipoxygenase inhibitor

Quantitation of cysteinyl leukotriene production and smooth muscle contraction upon immunological challenge of isolated human bronchi was evaluated. Analysis of picomole amounts of leukotriene C4, D4, and E4 was achieved using HPLC separation and enzyme immunoassay quantitative determination. The aim of the study was to correlate the contraction of airway smooth muscle and cysteinyl leukotriene production with and without 5-lipoxygenase inhibition. In human isolated bronchial tissue treated with indomethacin and pyrilamine to make their contractile responses leukotriene dependent only, the novel 5-lipoxygenase inhibitor 5,6-Dihydroxy-2-(N,N-Dimethylhydrazino)-1,2,3,4-tetrahydro-naph talene bromide (CHF 1909) caused a concentration-dependent inhibition of the immunologically induced contraction, showing an IC50 value of 13 +/- 2.2 microM (mean +/- CV). At the concentration of 30 microM, this compound caused more than 90% inhibition of the maximal bronchoconstriction in vitro, and inhibited cysteinyl leukotriene production by 90% as well. Contemporary measurement of immunologically induced contraction and production of cysteinyl leukotrienes in isolated human bronchi provided a direct correlation between smooth muscle contraction and synthesis of leukotriene C4, D4, and E4.

Synthesis and biological evaluation of 14,15-dehydro-LTA4 analog

Abstract New stable leukotriene A 4 analog with acetylenic function in positions 14,15 has been synthesized. Conversion of this analog to the corresponding LTC 4 derivative and the structural and biological characterization are presented.

Evaluation of the Pharmacological Activity of the Pure Cysteinyl-Leukotriene Receptor Antagonists CGP 45715A (Iralukast) and CGP 57698 in Human Airways

Cysteinyl-containing leukotrienes (cysteinyl-LTs) produce bronchoconstriction, mucus hypersecretion, and pro-inflammatory effects (Dahlen et al., 1980; Dahlen et al., 1981; Foster C von Sprecher et al., 1991; von Sprecher et al., 1996).