WO2002085376A2 - A method for decreasing superoxide anion production and for the treatment of diseases associated with oxidative stress - Google Patents

Abstract

The present invention relates to a new method for reducing vascular, cardiac and colonic tissue O2- generation by lowering the NAD(P)H oxidase activity of these tissues in normal and hypertensive subjects using ASA, nimesulide and indomethacin. Although ASA did not show any acute effect in vitro, chronic oral treatment or chronic incubation with ASA significantly lowered the O¿2?- basal or NAD(P)H activated production in aorta and smooth muscle cells from normotensive and hypertensive rats. These effects were dose-dependent and needed more than 3 days to onset in vivo condition. ASA treatment significantly improved the impaired aortic relaxation response to acetylcholine in SHR and significantly attenuated the age-dependent development of hypertension in young SHR. In another model of hypertension and insulin resistance induced by high glucose feeding, which was also found to be associated with a higher production of superoxide anion in tissues from the cardiovascular system, chronic ASA treatment was found to prevent simultaneously the development of hypertension, insulin resistance and the production of superoxide anion. Finally, in another hypertension model induced by the chronic administration of angiotensin II which has the property to activate NAD(P)H oxidase and to enhance the superoxide production in vessels, the concomitant treatment with ASA was also found to simultaneously prevent the development of hypertension and the enhanced superoxide anion production.

Description

A METHOD FOR DECREASING SUPEROXIDE ANIONS PRODUCTION AND TREATMENT OF OXIDATIVE STRESS

BACKGROUND OF THE INVENTION (a) Field of the Invention

The invention relates to a method for treating oxidative stress by decreasing superoxide anions production, (b) Description of Prior Art

Recently, numerous major clinical trials have demonstrated the efficacy of acetylsalicylic acid (ASA, ASPIRIN™) for reducing cardiovascular events (ischaemic strokes and myocardial infarction) and total mortality in patients with coronary artery disease (Mehta JL. Clin Cardiol. 21:879-84, 1998), with hypertension (Thun MJ. Namboodiri MM. Heath CW Jr. N Engl J Med. 325:1593-1596, 1991) or at cardiovascular risk (Chisolm GM. Steinberg D., Free Radio Biol Med. 28: 1815-1826, 2000). These beneficial effects associated to ASA treatment cannot be explained completely by its platelet inhibitory effects (Mehta JL. Clin Cardiol.l . :879-84, 1998) as other platelet inhibitory agents have not been found to be as effective as ASA. Furthermore, other epidemiological studies have indicated that ASA therapy may also reduce the incidence of cancers, especially colon cancer (Mehta JL. Clin Carc /o/.21:879-84, 1998).

Accumulating evidences indicate that oxidative stress may play an important role in the pathogenesis and/or progression of cardiovascular diseases, especially in atherosclerosis (Chisolm GM. Steinberg D., Free Radio Biol Med. 28: 1815-1826, 2000) and hypertension (Dhalla NS. Temsah RM. Netticadan T., J Hypertension, 18(6):655-673, June 2000). Highly reactive oxidative agents, especially superoxide anion (0₂ ^"") and hydrogen peroxide (H₂0₂), can trigger platelet aggregation, induce vessel constriction (or spasm), impair endothelium-dependent vasodilatory functions, stimulate proliferation of SMC or cause neutrophil infiltration. Most of those effects facilitate the progression of atherosclerosis, favor the occlusion of coronary vessels, and stimulate arterial constriction and hypertrophy. Moreover, recent studies suggest that oxidative DNA damage is important in the etiology of many human cancers (Marnett LJ., Carcinogenesis, 21 :361 -370, 2000). Experimental and clinical studies have provided substantial evidence supporting the pathogenic role of inflammation in the development of atherosclerosis and hypertension (Dhalla NS. Temsah RM. Netticadan T., J Hypertension, 18(6):655-673, June 2000; and Ross R. N Engl J Med. 340(2):115-126, 1999). Indeed, circulating levels of C- reactive protein (CRP), an inflammation marker, have been shown to constitute an independent risk factor for cardiovascular disease (Munzel T, Sayegh H, Freeman BA, et al., J Clin Invest. 95(1): 187-194, 1995). It is known that inflammation can enhance tissue superoxide level through numerous mechanisms, including the release of superoxide by infiltrating leukocytes (polymorphonuclear leukocytes and monocytes) and the stimulating effects of the pro-inflammatory cytokines (such as IL-1 , and TNF-α) on the superoxide production by local tissue cells (fibroblasts, endothelium and smooth muscle cells) (Barbacanne MA, Souchard JP, Darblade B, lliou JP, Nepveu F, Pipy B, Bayard F and Arnal JF. Free Radical Biol Med. 29:388-396, 2000).

It is also known that the association of diabetes and hypertension potentiates the degree of cardiovascular risk, but the exact mechanisms underlying this potentiation are not understood at present. Previous studies have suggested that reactive oxygen species including superoxide anion (0₂ ^") may be involved in the pathogenesis and complications of both conditions (Giugliano D., et al., Diabetes Care, 19:257-267, 1996).

It is well known that ASA has a potent anti-inflammatory effect. It is also well known that there are two forms of cyclooxygenase (COX), COX-1 and COX-2, involved in the synthesis of prostaglandins in mammalian cells and NSAIDs strongly inhibit these COX activity. Among the NSAIDs, ASA and indomethacin inhibit both the COX-1 and COX-2 although ASA is considered sometimes as a preferential COX-1 inhibitor, while nimesulide selectively inhibits COX-2 (Laudanno OM, Cesolari JA, Esnarriaga J, San Miguel P, Bedini OA. Dig Dis Sci. 45:1359-1365, 2000). In a clinical study, Ridker et al (Ridker PM. Cushman M. Stampfer MJ. et al. N Engl J Med. 336:973-979, 1997) have suggested that ASA treatment decreases circulating CRP level and significantly reduces the risk of myocardial infarction in 'healthy' men. In an in vitro study, Leo et al (Leo R. Pratico D. luliano L. et al. Circulation. 95(4):885-891 , 1997) have demonstrated that platelets exposed to the process of anoxia- reoxygenation showed spontaneous aggregation and increased superoxide production. Both phenomenons were inhibited or attenuated by pre-incubation with superoxide dismutase and ASA. Recently, nimesulide was reported to inhibit the dextran sodium sulfate stimulated superoxide. generation and mutagenic oxidative lesion in rat colonic mucosa (Tardieu D, Jaeg JP, Deloly A, Corpet DE, Cadet J, Petit CR. Carcinogenesis 21:973-976, 2000). Nimesulide was also reported to inhibit the stimulated superoxide production in activated polymorphonuclear leukocytes (Bevilacqua M, Vago T, Baldi G, Renesto E, Dallegri F, Norbiato G., Eur J Pharmacol. 268:415-423, 1994).

It would be highly desirable to be provided with a new method for the treatment of oxidative stress by reducing production of superoxide anions.

SUMMARY OF THE INVENTION

One object of the present invention is to evaluate the effect of ASA on basal 0 ^~ production and on NAD(P)H oxidase in aortic rings and in aortic cultured smooth muscle cells (SMC).

Another object of the present invention was to investigate the possible beneficial properties of ASA on the improvement of aortic endothelium- dependent relaxation functions and on the prevention of the development of hypertension, as well as the potential relationship between the present object and the one above.

Still a further object of the present invention to evaluate the effect of ASA, nimesulide and indomethacin treatment on basal 0₂ ^~ production and on NAD(P)H oxidase in aortic, cardiac and colonic tissues, as well as to clarify the possible molecular mechanism of these anti- oxidative effects.

In accordance with the present invention there is provided a method for preventing and/or treating the development of hypertension in a patient, using acetylsalicylic acid. Still in accordance with the present invention, there is provided a method for reducing superoxide anion production in a patient, using acetylsalicylic acid.

Further in accordance with the present invention, there is provided a method for reducing NAD(P)H oxidase activity causing reduction of superoxide anion production, using acetylsalicylic acid, preferably as multiple doses, and more preferably at doses containing each at least 80 mg/day of acetylsalicylic acid.

In accordance with the present invention, there is also provided a method for restoring impaired aortic vasodilatory response in a hypertensive patient, using acetylsalicylic acid.

Still in accordance with the present invention, there is provided a method for reducing superoxide anion production in cardiac and colonic tissues of a patient, using acetylsalicylic acid, nimesulide and indomethacin.

The present invention also provides a method for treating cardiovascular diseases associated to an increased oxidative stress, using acetylsalicylic acid.

There is still provided in accordance with the present invention a method for reducing insulin resistance in a patient, using acetylsalicylic acid.

In accordance with the present invention, acetylsalicylic acid may also be used in a method for treating a patient suffering from hyperglycemia. Of course, acetylsalicylic acid may also be used in accordance with the present invention for the manufacture of a medicament for the various therapeutic uses described herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A to 1 D illustrate Basal superoxide production (Figs. 1A and 1C) and DPI-inhibitable superoxide production (Figs. 1B and 1 D) were evaluated by lucigenin chemiluminescence in aortic rings (Figs. 1A and 1 B) and ventricular slices (Figs. 1C and 1 D) from control (n=6) and ASA (n=6), Ang II (n=5) or Ang ll+ASA (n=5) treated rats; Fig. 2 illustrates inhibitory effects of ASA on Ang ll-stimulated superoxide production evaluated in aortic rings from control, Ang II and Ang ll+ASA treated rats;

Figs 3A and 3B illustrate the effect of ASA on chronic Ang II infusion-induced hypertension;

Fig. 4 illustrates the effect of 12-day ASA, Ang II or Ang ll+ASA treatment on heart (mg)/body (g) weight ratio;

Fig. 5 illustrates a linear regression analysis of aortic superoxide level and blood pressure from control, Ang II and Ang ll+ASA treated rats; Fig. 6 illustrates the effect of ASA, losartan and PD 123 319 on

Ang ll-induced superoxide production in cultured aortic SMC;

Fig. 7 illustrates inhibitory effects of ASA compared with losartan on Ang ll-stimulated [³H]leucine incorporation in cultured aortic SMCs;

Fig. 8 illustrates a time course of the effect of ASA treatment (100 mg/kg/day) on basal aortic 0₂ ^~ production in Sprague-Dawley rats;

Figs. 9A and 9B illustrate dose-response curves of chronic ASA treatment (12 days) on basal (Fig. 9A) and NADH (100 μmol/L) activated (Fig. 9B) superoxide (0₂ ^~) production by aortic rings from Sprague-Dawley rats; Figs. 10A and 10B illustrate the effects of NSAIDs treatment on basal 0₂ ^~ production (Fig. 10A) and on NAD(P)H oxidase activity (Fig. 10B) in aortic, cardiac and colonic tissues;

Figs. 11 A to 11 D illustrate the effects of ASA treatment on basal

(Figs. 11A and 11C) and NADH (100 μmol/L) activated (Figs. 11 B and 11 D) 0₂ ^~ production in aortic rings (ASA 100 mg/kg/day for 12 days) and cultured aortic smooth muscle cells (ASA 10-4 mol/L for 48 hours) in WKY and SHR;

Fig. 12 illustrates the effects of pre-treatment with ASA (100 mg/kg/day, 12 days) on vascular dose-response vasodilatory effects of acetylcholine in aortic rings from 12-week old WKY and SHR rats;

Fig. 13 illustrates the effects of chronic ASA treatment (100mg/kg/day) on the evolution of blood pressure in WKY and SHR rats;

Fig. 14 illustrates the evolution of systolic arterial pressure recorded in rats during three weeks in control and in 10% glucose-fed (glucose-fed) combined or not with aspirin treatment; Figs. 15A to 15D illustrate the effects of chronic glucose feeding combined or not with aspirin treatment on plasma level expressed in mmol/L (Fig. 15A), on plasma insulin levels expressed in pmol/L (Fig. 15B), on the index of insulin resistance (plasma glucose x insulin / 22.5) (HOMA) (Fig. 15C), and on anion superoxide production in aorta expressed in cpm/min/mg of aorta (Fig. 15D); and

Figs. 16A to 16C illustrate the correlations between the basal aortic superoxide production expressed in cpm/min/mg of aorta and the systolic blood pressure expressed in mmHg (Fig. 16A), between insulin resistance index and the basal aortic superoxide production (Fig. 16B), and between the systolic blood pressure and insulin resistance index in control (D), glucose-fed (o), and glucose-fed treated with aspirin (Δ) rats.

DETAILED DESCRIPTION OF THE INVENTION Angiotensin II (Ang II) induced oxidative stress has been suspected to play an important role in the pathogenesis of many cardiovascular diseases such as atherosclerosis, hypertension, congestive heart failure, and cardiovascular remodeling. It is hereby demonstrated that acetylsalicylic acid (ASA, aspirin) possesses potent antioxidative properties through reducing vascular superoxide anion (0₂ ^") production and inhibiting the vascular NAD(P)H oxidase activity in normal and hypertensive rats.

As it is now recognized that oxidative stress and inflammation play an important role in the development and evolution of cardiovascular diseases as well as colon cancer, the present invention thus provides a new method for treating oxidative stress by reducing superoxide anions (0₂ ^~) production.

In the present invention, It was found that ASA treatment could reduce vascular tissue 0₂ ^~ generation in aortic ring and in cultured aortic smooth muscle cells from normotensive (WKY) and hypertensive (SHR) rats by means of the lucigenin-enhanced chemiluminescence method. The effects of ASA, indomethacin and nimesulide treatment on 0₂ ^_ generation were also investigated in aortic, cardiac and colonic tissue in normotensive Sprague-Dawley rats by means of the lucigenin-enhanced chemiluminescence method. Although ASA did not show any acute effect on 0₂ ^"~ production in vitro, chronic oral treatment (100 mg/kg/day, 12 days) significantly (p<0.01) lowered the basal aortic tissue 0₂ ^~ production from control level of 4.7 + 0.1 to 2.5 + 0.2 cpmχ10³/mg tissue in normotensive rats and from the higher initial levels of 7.1 ± 0.3 to 3.9 + 0.2 cpm^χ103/mg tissue in hypertensive rats while simultaneously decreasing the NAD(P)H activated 0₂ ^~ production in both groups. These effects were dose- dependant from 10 to 100 mg/kg/day and became detectable more than 3 days after the onset of treatment. A similar reduction of 0₂ ^~ production was also observed following a 48 hours ASA (10-4 mol/L) treatment in cultured aortic SMC and this effect was greater in SMC from SHR. Moreover, ASA treatment significantly improved the impaired aortic relaxation response to acetylcholine in 12-week old SHR rats and significantly attenuated the age- dependent development of hypertension in young SHR treated from the age of 6 weeks, whereas no effects on the vascular reactivity or blood pressure were observed in age-matched WKY rats. Chronic treatment with all three non-steroidal anti-inflammatory drugs (NSAIDs) significantly decreased the aortic, cardiac and colonic tissue 0₂ ^~ production through reducing the tissue NAD(P)H oxidase activity in¹ normotensive rats.

Accordingly, NSAIDs treatment in vivo importantly reduced vascular, cardiac and colonic 0₂ ^~ production through lowering the tissue NAD(P)H oxidase activity mainly via inhibiting the prostaglandin synthesis enzyme cyclooxygenase (COX) type 2. The results also indicated that ASA treatment also reduces aortic 0₂ ^~ production in hypertensive rats, which are involved in restoring aortic vasorelaxation mechanism and in attenuating the development of hypertension in SHR. These findings provide a completely new and effective strategy for reducing tissue 0₂ ^~ production as well as a new insight into the understanding of the beneficial cardiovascular effects of ASA and the potential anticarcinogenic effects of NSAIDs in colon. The results provided in the present application also suggest that there exists a close relationship between the COX and the NAD(P)H oxidase activity.

The major findings in this invention are as follows: (1) chronic ASA, indomethacin and nimesulide oral treatment significantly reduced the 0₂ ^~ production in aortic, cardiac and colonic tissue through the inhibition of the NAD(P)H oxidase activity in these tissues; (2) the inhibition of COX-2 seems to play an important role in the mediation of antioxidative effects of those NSAIDs; (3) the ASA treatment produced a significant dose dependent (10-100 mg/kg/day) inhibition of 0₂ ^~ production in aorta of both normotensive and hypertensive rats, but this effect was more important in hypertensive rats; (4) ASA treatment (for 48 hours) similarly reduced 0₂ ^" production in cultured aortic SMC; (5) chronic ASA treatment improved significantly the impaired aortic vasodilatory response to Ach in 12-week old hypertensive rats, but did not alter the aortic reactivity in age-matched normotensive WKY rats; (6) a chronic ASA treatment initiated in young (6- week old) still normotensive SHR attenuated significantly the age- dependent development of hypertension; but the ASA treatment did not modify either the blood pressure of age-matched WKY rats or the blood pressure of 12-week old SHR once the hypertension is established.

The present invention are the first direct evidence that ASA, indomethacin and nimesulide treatment decrease aortic, cardiac and colonic tissue 0₂ ^" production through inhibiting local tissue NAD(P)H oxidase activity. The results presented herein indicate that ASA does not directly inhibit the NAD(P)H oxidase since neither the basal 0₂ ^" production nor the NAD(P)H oxidase activity were modified in aortic rings incubated acutely in vitro with ASA. Furthermore, the time-effect curve showed that the ASA treatment needed to last more than 3 days to produce a detectable and significant effect on 0₂ ^" production. These results indicate that ASA decreased the 0₂ ^" production and reduced the NAD(P)H oxidase activity through an indirect mechanism. Our results from the COX-2 selective inhibitor, nimesulide, as well as from the nonselective COX enzyme inhibitors, ASA and indomethacin, suggest that the antioxidative effects of those NSAIDs are mediated by their properties of inhibiting the COX-2 enzyme.

Inflammatory reactions, particularly those that are chronic, can constitute a significant source of oxidative stress and damage. Inflammation is associated with a marked rise in the number of polymorphonuclear leukocytes and monocytes in the affected tissues. These activated leukocytes at the inflammatory sites release a large quantity of reactive oxygen species including 0₂ ^". Moreover, the pro- inflammatory cytokines such as interleukin-1β and TNF-α stimulate local tissue cells including fibroblasts, kidney mesangial cells, endothelial cells, and smooth muscle cells to produce 0₂ ^" by activating the NAD(P)H oxidase pathway. Moreover, the anti-inflammatory effects of ASA mediated through inhibition and acetylation of cyclooxygenase isoform 2 (COX-2) shows a dose-effect relationship similar to the present dose-relationship for the anti-oxidative effects of ASA. So far, no direct evidence has demonstrated that the inhibition of COX-2 can decrease the NAD(P)H oxidase activity and 0₂ ^" production in cardiovascular tissues. Moreover, the effects of ASA on 0₂ ^" generation in cultured aortic SMCs suggest that ASA can directly exert its anti-oxidative effects on those cells independent of leukocytes infiltration. In addition, the effects of ASA on 0₂ ^~ generation in cultured aortic SMCs and the effectiveness of ASA in reducing 0₂ ^~ production in normal Sprague-Dawley rats suggest that ASA can directly exert its anti-oxidative effects independent of leukocytes infiltration and independent of the presence of inflammatory state, respectively. The similar degree of inhibition on 0₂ ^" production induced by ASA treatment observed in SMC and in aortic rings also suggests that the anti-oxidative effect of ASA on SMC seems to account for most of the effects of ASA on the whole artery. In pathological conditions, the excessive production of 0₂ ^" reacts with nitric oxide (NO) and reduces the level of that vasodilator while simultaneously producing the tissue damaging peroxynitrite in local vascular tissue. These effects could contribute to the impairment of the vascular relaxation functions observed in hypertension and other cardiovascular disease states. The present invention showed for the first time that ASA treatment significantly restored the impaired aortic relaxation function in SHR rats. Moreover, the beneficial effects of ASA on aortic relaxation was observed only in SHR rats characterized by high level of 0₂ ^~ production in aorta, but not in healthy normotensive WKY rats. These data indicate that ASA treatment improved the relaxation mainly through its antioxidant properties. The possible explanations for the observation of only a partial restoration of the endothelium-dependent relaxation despite the normalization of the high level of 0₂ ^"production in aorta from SHR, could be that: a) only part of the oxidative stress induced vascular damages are reversible; and/or b) oxidative stress is only one of the multipathogenic factors implicated in the relaxation alteration. Irreversible arterial damages induced by oxidative stress could be related to the remodeling, to SMC hypertrophy, to apoptotic mechanisms and fibrosis of the arterial wall. In normal condition, the influence of local tissue 0₂ ^" level on NO availability and on vessel relaxation properties are still unknown. The present results showed that, although ASA treatment also reduced vascular 0₂ ^" level in aorta of normotensive rats, this treatment did not change the aortic vasorelaxant response to Ach. These data suggest that in health and in the absence of an increased oxidative stress, further reduction in 0₂ ^" level does not modify the availability of NO or the endothelium-dependent relaxation mechanisms.

Another interesting finding in the present invention is that ASA treatment administered to young still normotensive SHR attenuated the development of their hypertension and decreased significantly their blood pressure level compared to age-matched untreated SHR. This effect of ASA was observed only in SHR rats and not in age-matched normotensive WKY rats, although the aortic tissue 0₂ ^" level was equally reduced by ASA treatment in both groups of rat. In contrast, the ASA treatment in 12-week old hypertensive SHR rats did not modify their blood pressure. These findings indicate that ASA does not exert a direct hypotensive action since ASA did not correct the already established hypertension in older SHR. Results have shown that there is a positive linear relationship between the arterial 0₂ ^" level and the blood pressure levels in SHR and that the hypertension developed in parallel with the rise in 0₂ ^" production in that model. Several other studies have also suggested that an enhanced 0₂ ^" production plays an important role in the development of hypertension. Taken together, the above-mentioned data suggest that ASA attenuated the development of hypertension in young SHR rats through its anti- oxidative properties. It is noticeable that, although the higher production of 0₂ ^" in SHR rats was completely normalized by the ASA treatment, only partial prevention of hypertension was achieved. This observation suggests that the oxidative stress although important, is not the unique etiological factor in the development of hypertension in that model. ln the present invention, ASA produced a significant decrease of 19% (p<0.01 ) in aortic basal 0₂ ^" production at a dose as low as 10 mg/kg/day. Depending on the interspecies dosage conversion factors based on equal body surface (7:1 for conversion from rat to man), the dose of 10 mg/kg for rats can be converted to 1.43 mg/kg for man or 100 mg for a man of 70 kg. Preferably, at least 80 mg/day can be used in accordance with the present invention. This dose is within the range of 'low dose' of ASA used for the prevention of cardiovascular diseases. The concentration of 10^"4 mol/L of ASA used in the present in vitro and in cultured cells studies is within the drug's normal pharmacological concentration, as clinical pharmacokinetic data indicate that, after single oral administration of 650 mg of ASA in human, the drug plasma concentration could reach 4χ10^"4 mol/L in 1-2 hours.

The effects of ASA on Ang ll-induced cardiac and vascular tissues 0₂ ^" production (lucigenin-enhanced chemiluminescence method), hypertension and cardiac hypertrophy has further been investigated. To investigate the molecular mechanism, the effects of ASA and Ang II on 0₂ ^" production and cellular protein synthesis were also studied in cultured aortic smooth muscle cells. Chronic Ang II infusion (200 ng/kg/min, for 12 days) increased the aortic and cardiac tissue 0₂ ^" production by 77 and 35% from the basal values of 1627 ± 112 and 231 ± 21 cpm/mg tissue to 2873 ± 444 and 312 ± 23 cpm/mg tissue, and also increased the aortic and cardiac tissues DPI (diphenylene iodonium)-inhibitable 0₂ ^" production (representing intrinsic NAD(P)H oxidase activity) by 46% and 39% from control level of 1031 ± 60 and 155 ± 12 cpm/mg tissue to 1504 ± 160 and 216 ± 18 cpm/mg tissue, respectively (p<0.05) (Figs. 1A to 1D). The progressive development of hypertension following angiotensin infusion appears to be closely related to the progressive increase in the production of 0₂ ^" whereas the combined treatment with ASA reverted within 5 days the initial-increased production of 0₂ ^" thus preventing the blood pressure rise (Fig. 2). These Ang ll-induced oxidative effects were accompanied with a significant and progressive increases in systolic blood pressure (from 135 to 194 mmHg) (Figs. 3A and 3B) and heart/body weight ratio (from 2.25 to 2.69, parameter of cardiac hypertrophy) (Fig. 4). ASA treatment for 12 days in control animals significantly reduced the aortic and cardiac tissue basal 0₂ ^" production by 31% and 33% to 1127 ± 71 and 156 ± 7 cpm/mg tissue, respectively, through reducing the NAD(P)H oxidase activity (36% and 33% reduction of DPI-inhibitable 0₂ ^" production, respectively) (Figs. 1A to 1 D). However this treatment did not modify the blood pressure and heart/body weight ratio in those rats. In contrast, the simultaneous ASA treatment in Ang ll-infused rats completely prevented not only the Ang ll-induced 0₂ ^" production (aortic 1776 ± 110, cardiac 230 ± 17 cpm/mg tissue, p<0.05 Vs Ang II alone) but also the hypertension (blood pressure 139 ± 4 mmHg) and cardiac hypertrophy (heart/body weight ratio 2.32 ± 0.05) associated to the chronic Ang II infusion (Figs. 1A to 1D, Figs. 3A and 3B, and Fig. 4). In Fig. 3A, rats were treated with ASA, Ang II, or simultaneous ASA plus Ang II for 12 days. In Fig. 3B, rats were treated with Ang II alone for the first 7 days, then, ASA treatment was added in Ang ll+ASA group for simultaneous treatment for the last 7 days while the Ang II group continue receiving Ang II during the last 7 days. Correlation analysis of the changes in aortic 0₂ ^" production and in blood pressure levels from control, Ang II and Ang ll+ASA treated rats showed a highly significant linear relationship between those two parameters with r² = 0.83 (pθ.001) (Fig. 5). In cultured aortic SMCs, the incubation for 48 hours with ASA

(10^"4 M) significantly reduced the 0₂ ^" production by 56% from a control level of 34 ± 3 to 15 ± 2 cpm/μg protein (Fig. 6), but did not modify the [³H]leucine incorporation (92 + 2 cpmχ10³/well), a cell growth parameter (Fig. 7), while the incubation with Ang II (10^"6 mol/L) increased by the 0₂ ^" production 109 ± 6 cpm/μg protein and increased [³H]leucine incorporation to 126 ± 3 cpmχ10³/well (p<0.01 Vs control). The simultaneous treatment of the Ang ll-treated SMC with ASA (10^"4 M) and losartan (10^"5 M), an ATι receptor selective antagonist, significantly reduced the 0₂ ^" production to 48 ± 4 and 59 ± 11 cpm/μg protein, and decreased also the [³H]leucine incorporation to 93 ± 2 and 99 ± 6 cpmχ10³/well, respectively. In contract, the simultaneous treatment with the AT₂ selective antagonist, PD 123 319

(10^"5 M) did not significantly modify the Ang ll-induced 0₂ ^" production (93 ±

10 cpm/μg protein) and [³H]leucine incorporation (112 ± 2 cpmχ10³/well).

In conclusion, the present results indicate that the NSAIDs treatment in vivo importantly reduced vascular, cardiac and colonic 0₂ ^~ production through lowering the tissue NAD(P)H oxidase activity in normal rats. The results also indicate that ASA treatment can also reduce 0₂ ^~ production in hypertensive rats, and the antioxidative properties of ASA are implicated in the effects of the drug to restore the impaired endothelium- dependent vascular relaxation in SHR rats and to attenuate the development of hypertension in young SHR rats. These antioxidative effects of the NSAIDs seem to be mediated by an indirect mechanism via inhibiting the prostaglandin synthesis enzyme COX-2. These findings provide us with a completely new and effective strategy for reducing tissue 0₂ ^~ production and also bring us a new insight into the understanding of the beneficial cardiovascular effects of ASA and the potential anticarcinogenic effects of NSAIDs in colon. The results also suggest that there exists a close relationship between the COX and the NAD(P)H oxidase activity. Chronic Ang II infusion significantly increases both cardiac and vascular tissue 0₂ ^" production through enhancing the NAD(P)H oxidase activity. This oxidative stress plays a major role in chronic Ang ll-induced hypertension and cardiovascular hypertrophy. The simultaneous ASA treatment totally prevented those Ang ll-induced changes through its anti- oxidative properties. These results provide a new insight into understanding the cardiovascular benefits of ASA observed in many cardiovascular diseases. In addition to demonstrating antioxidative properties, ASA was also shown herein to exert a protective effect against angiotensin II on the cardiovascular system. The results reported herein point out a new therapeutic use of ASA in the treatment of the multitude of cardiovascular diseases in which the pathology is associated in major part to an increased oxidative stress.

The present invention will be more readily understood by referring to the following examples, which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I ASA treatment for lowering the superoxide production Methods

Animals

Studies were performed in male rats of Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats(SHR). The animals were given free access to drinking water. For control rats, drinking water was free of any drug, whereas ASA (Aspirin™, Bayer) was added in the drinking water of treated rats. At the end of the treatment, the rats were killed by decapitation after light anesthesia with C0₂ and the thoracic aorta, the whole heart and about 5 cm of distal colon was quickly excised and immersed in ice cold Krebs-Hepes buffer solution containing (mmol/L): NaCI 99.01 , KCI 4.69, CaCI₂ 1.87, MgS0₄ 1.20, K₂HP0₄ 1.03, NaHC0₃ 25.0, Na-Hepes 20.0, glucose 11.1 (saturated with 95% 0₂ and 5 % C0₂, pH 7.4). The aortic periadventitial tissue was carefully removed and the aorta was cut into either 2 mm ring segments for 0₂ ^~ measurement or 4 mm ring for isometric tension studies. The luminal content of the colon was gently removed by irrigation with buffer solution and the colon was cut into 3^χ10 mm strip. The left ventricle of the heart was sliced into 1 mm thickness with a custom-made instrument and a slice of about 30 mg (5^χ5 mm) was used for 0₂ ^" measurement. Superoxide anion measurement

The superoxide anion production was measured using the lucigenin-enhanced chemiluminescence method. Briefly, after vessel preparation, a 2 mm ring segment (about 2-4 mg) was placed into the Krebs-Hepes buffer (saturated with 95% 0₂ and 5% C0₂, at room temperature). After 10 min equilibration, the aortic ring segment was transferred to a scintillation vial containing 250 μmol/L lucigenin in a total volume of 2 ml of Krebs-Hepes buffer for determining the basal 0₂ ^~ level. NADH (100 μmol/L) was added into the vial to evaluate the NADH- activated 0₂ ^~ generation. For cultured SMCs study, about 10⁶ cells were added into the counting vials. The chemiluminescence was recorded every minute for 15 minutes by a liquid scintillation counter (Wallac 1409, Turku, Finland) switched to the out-of-coincidence mode. The respective background was subtracted from total count. At the end of the chemiluminescence measurement, the fresh aortic ring was weighed and the total SMCs protein was determined with the Lowry's method. The superoxide generation was expressed as counts x 10³/min/mg fresh tissue (cpmχ10³/mg tissue) for aortic ring or counts/min/μg protein for cultured SMC. Recently, the chemiluminescence technique using high concentration of lucigenin has been challenged because reduced lucigenin can itself generate 0₂ ^~. To validate data obtained with high concentration of lucigenin, low concentration of lucigenin (5 μmol/L) was used in some experiments with aortic rings and in all experiments with cardiac and colonic tissues from Sprague-Dawley rats. The 0₂ ^~ production was also quantified by the cytochrome c (20 μmol/L) reduction technique in intact aortic rings. Similar inhibiting effect of ASA on 0₂ ^~ production was proven in all three approaches.

To measure NAD(P)H oxidase activity, NADH (100 μmol/L ) was added into the scintillation vial to evaluate the NADH-activated 0₂ ^~ production in intact aortic rings with 250 μmol/L of lucigenin. This approach was also validated by using an NAD(P)H oxidase selective inhibitor, diphenylene iodonium (DPI, 100 μmol/L). Aortic rings were incubated with DPI for 10 min at room temperature before the basal 0₂ ^" production was evaluated by chemiluminescence technique with 5 μmol/L of lucigenin. The DPI-inhibitable 0₂ ^~ production, which represents the intrinsic NAD(P)H oxidase activity, was expressed as the difference of aortic basal 0₂ ^~ production in the presence and in the absence of DPI. Protocols 1. Studies in Sprague-Dawley rats

Direct effect of ASA on superoxide production was evaluated. Sprague-Dawley rats (225-250 g, n=4) without any drug treatment were sacrificed and the thoracic aorta was excised and cut into 2 mm ring. The aortic rings were incubated in vitro with 10^"4 mol/L of ASA at room temperature (~ 22° C) for 10 min in Krebs-Hepes buffer solution. At the end of incubation, the aortic rings were washed twice with Krebs-Hepes buffer solution and were transferred into the counting vial for O₂ ^~ measurement. Dose-effect of ASA on aortic superoxide production

In 27 Sprague-Dawley rats (225-250 g), the dose-effect of ASA was evaluated by comparing animals treated either with water (control, n=9), or with one of 4 doses of ASA (10, 25, 50 and 100 mg/kg/day added in drinking water) for 12 days in 4, 5, 4 and 5 rats respectively. At the end of treatment, the animals were sacrificed and the aortic rings were prepared for immediate measure of 0₂ ^~ production. Time-effect of ASA on aortic superoxide production ASA treatment (100 mg/kg/day) was performed in 12, 4, 4, 4 and

5 Sprague-Dawley rats for 0, 3, 6, 9 and 12 days, respectively. At the end of treatment, the animals were sacrificed and the aortic rings were prepared for immediate measure of 0₂ ^~ production .

Effects of ASA, indomethacin and nimesulide treatment on superoxide production and NADH oxidase activity in aortic, cardiac and colonic tissue

Twenty four Sprague-Dawley rats were separated into 4 groups of 6 rats receiving water (control), ASA (100 mg/kg/day), indomethacin (5 mg/kg/day) or nimesulide (5 mg/kg/day), respectively, for 12 days. The drugs were added in the drinking water. At the end of treatment, the aortic rings, the ventricular slices and the colonic strips were prepared for the evaluation of basal and DPI-inhibitable 0₂ ^~ production with lucigenin (δμmol/L) enhanced chemiluminescence method. 2. Studies in WKY and SHR rats

To evaluate the effect of ASA on aortic superoxide production and vasorelaxation to acetylcholine, 12-week-old WKY and SHR rats were separated into ASA treated and control group. In ASA treated groups, rats were treated orally with 100 mg/kg/day ASA for 12 days in 7 WKY and 10 SHR rats, while the control groups (8 WKY and 10 SHR) received only water. At the end of treatment, the animals were sacrificed and the aortic rings were prepared for immediate measure of 0₂ ^~ production and for vasorelaxation studies to various concentration of acetylcholine.

Tests to evaluate the preventive effect of ASA on hypertension development were performed on 6 WKY and 6 SHR rats of age of 6 weeks. Either species of rats were separated into ASA treated (n=3) and control (n=3) groups. The ASA treated rats received ASA (100 mg/kg/day) while control rats received only water, and the treatment lasted for 53 days. During the treatment, the systolic blood pressure was measured regularly (at least twice/week) by a method of tail cuff plethysmography (Harvard Apparatus Ltd). At the end of 53 days of treatment, systolic blood pressure was also measured directly through a femoral artery catheter in rats anesthetized with pentobarbital sodium (40 mg/kg). 3. Studies in cultured aortic smooth muscle cells

The effect of ASA on 0₂ ^~ production was studied in cultured SMC from 12-week-old WKY (n=3) or SHR (n=3) rats. ASA was dissolved in culture medium and the pH of the drug solution was adjusted to 7.4 with NaOH. The drug solution (final concentration 10^"4 mol/L) or equal volume of buffer (control) was added into cell culture medium and cultured with SMC for 48 hours. At the end of the culture, cells were washed twice in situ with Krebs-Hepes buffer solution and then were mechanically scraped. These cells were washed twice and were purified by centrifugation (1500 rpm, 5 min) to remove cell debris. The concentration of the cells was estimated by a hemacytometer. These cells were immediately used for 0₂ ^~ measurement. Aortic smooth muscle cell culture

Aortic SMCs from 12 week old WKY and SHR rats were isolated and cultured. Briefly, rat aortas were isolated and connective tissues as well as endothelium were removed. The vessel was cut and enzymatically digested with collagenase-dispase, elastase, and collagenase in a stepwise manner. The dispersed cells were plated in the tissue culture flasks and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a C0₂ incubator at 37° C. Cultured SMCs were passed once a week by harvesting with trypsin treatment and splitting at a ratio of 1 :4. The medium was changed twice weekly. Cultured cells between passages 3 and 5 were used in the present study. Isometric tension studies Isometric tension studies were performed in 4 mm aortic rings from 6 control and 6 ASA treated WKY and SHR rats, respectively. The aortic rings were suspended in individual organ chambers filled with Krebs buffer solution containing (mmol/L): NaCI 118, KCI 4, CaCI₂ 2.5, MgCI₂ 1.2, KH₂P0₄ 1.2, NaHC0₃ 24, glucose 11.1. After an equilibration period of 45 min, the resting tension was gradually increased to about 4.0 g and then the ring segment was exposed to 70 mmol/L KCI to determine the maximal contraction. Rings were thereafter thoroughly washed and allowed to equilibrate for an additional 45 min. The aortic rings were then submaximally precontracted with 10^"7 mol/L of phenylephrine. After a stable contraction plateau was reached, the rings were exposed to cumulative acetylcholine concentrations (Ach 10^"9 - 10^"5 mol/L) to determine the endothelium-dependent relaxation. Responses to Ach were expressed as percent of the precontracted tension of phenylephrine. To prevent the synthesis of prostaglandins and their influence on the yasorelaxant response, all studies were performed in the presence of 10 μmol/L of indomethacin. Data analysis

Data are expressed as mean ± SEM. Statistical comparisons were made by Student's t-test for paired groups data or one way ANOVA followed by Tukey HSD analysis for multi-variance. The vasorelaxation responses and the levels of blood pressure were analyzed by repeated measures ANOVA. The critical level of significance was set at p<0.05. Results Direct effect of ASA on aortic superoxide production in vitro Aortic rings from Sprague-Dawley rats (n=4) were incubated in vitro without or with 10^"4 mol/L of ASA at room temperature for 10 min and the basal aortic superoxide production was evaluated. The 0₂ ^~ production by untreated aortic rings was 4.5 ± 0.1 cpmχ10³/mg tissue and the acute incubation with ASA did not modify the basal 0₂ ^~ production (4.3 ± 0.2 cpmχ10³/mg tissue).

Time-effect relationship of ASA treatment on aortic superoxide production in normotensive rats

Sprague-Dawley rats were treated orally with the dose of 100 mg/kg/day of ASA for 0 (control), 3, 6, 9 or 12 days and the basal 0₂ ^~ production was determined in aorta at the end of each treatment period. Fig. 8 showed that after 3 days of treatment, ASA did not produce a significant inhibitory effect on basal 0₂ ^~ production. However, subsequently, the 0₂ ^~ inhibitory effect of ASA accrued in a time dependent manner to reach a reduction of 48.5% at the end of the 12-days treatment. In Fig. 8, ** represents p<0.01 vs. untreated rats.

Dose-effect relationship of chronic in vivo ASA treatment on aortic superoxide production in normotensive rats

Twenty seven Sprague-Dawley rats were treated orally with incremental doses (0-100 mg/kg/day) of ASA for 12 days. ASA treatment produced a dose-dependent decrease in the basal aortic °₂ ^_ production

(Fig. 9A). The basal °_^~ level in rats without ASA treatment was 4.7 ± 0.1 cpmχ10³/mg tissue and the production was progressively and significantly decreased by 19.3%, 36.7%, 43.3% and 46.1 % with 10, 25, 50 and 100 mg/kg/day chronic ASA treatments, respectively (p<0.01 for all doses compared to untreated control). A similar dose related inhibitory effect of ASA treatment was also observed on the NADH-activated °₂ ^~ production reaching an inhibition of 53% at the highest dose (Fig. 9B) (In Figs. 9A and 9B, ** represents p<0.01 for all doses vs. untreated rats). The pattern of those dose-effect curves indicated that a maximum effect was reached at about the dose of 100 mg/kg/day, which was chosen for all following studies.

Effects of chronic ASA, indomethacin and nimesulide treatment on aortic, cardiac and colonic superoxide production in normotensive rats

The basal 0₂ ^~ production by aortic rings, cardiac slices and colonic strips in control rats were 1987 ± 60, 318 ± 6 and 396 + 10 cpm/mg tissue, respectively. ASA (100 mg/kg/day), indomethacin (5mg/kg/day) and nimesulide (5mg/kg/day) treatment reduced significantly those basal superoxide production by 33%, 17% and 40% (p<0.01 Vs control) in aortic rings, by 32%, 13% and 37% (p<0.01 Vs control) in cardiac tissue, and by 46%, 28% and 51% (p<0.001 Vs control) in colonic strips, respectively (Fig. 10A). When high concentration of DPI (100 μmol/L), an NAD(P)H oxidase selective inhibitor, were pre-incubated with these tissues, the 0₂ ^~ production by the NAD(P)H oxidase pathway was completely inhibited. Therefore, the DPI-inhibitable 0₂ ^~ production represents the intrinsic NAD(P)H oxidase activity in these tissues. In control rats, the DPI- inhibitable 0₂ ^~ production was 1429 + 36, 195 + 10 and 237 ± 25 cpmχ10³/mg tissue in aortic rings, in cardiac slice and in colonic stripes, respectively. ASA, indomethacin and nimesulide treatment inhibited significantly the DPI-inhibitable 0₂ ^~ production by 35%, 24% and 43% (p<0.01 Vs control) in aortic rings, by 32%, 13% and 33% (p<0.01 ASA and Nimesulide Vs control) in cardiac tissue, and by 34%, 27% and 48% (p<0.05 Vs control) in colonic strips, respectively (Fig. 10B). However, the inhibiting effects of indomethacin on both basal and DPI-inhibitable 0₂ ^~ production were significantly less than those of ASA and nimesulide treatment (p<0.05). In Figs. 10A and 10B, the effects of NSAIDs treatment on basal 0₂ ^~ production (Fig. 10A) and on NAD(P)H oxidase activity (Fig. 10B) were evaluated using the chemiluminescence method with 5 μmol/L lucigenin in aortic rings, cardiac slice and colonic strips from normal Sprague-Dawley rats. DPI-inhibitable 0₂ ^~ production, representing the intrinsic NAD(P)H oxidase activity, was obtained by subtracting the residual 0₂ ^~ production in the presence of DPI from the basal 0₂ ^~ production. (* p<0.05, ** p<0.01 , NS p>0.05 Vs untreated rats).

Effects of chronic ASA treatment on aortic superoxide production in normotensive and spontaneously hypertensive rats

To evaluate the effects of ASA (1 OOmg/kg/day) in hypertensive rats, 12 weeks old SHR and their age-matched WKY rats were treated for 12 days. The basal °₂- production in WKY and SHR rats without ASA treatment was 3.8 ± 0.1 and 7.1 + 0.3 cpmχ10³/mg tissue, respectively (p < 0.01 , SHR vs. WKY) and the NADH activated 0₂ ^~ production was 136.7 + 8.8 and 186.6 + 7.0 cpmχ10³/mg tissue in WKY and SHR aorta (p<0.01 , SHR vs. WKY). ASA treatment decreased by 26.7% and 44.5% the basal °₂ production respectively in WKY and SHR rats (p<0.001 vs. untreated rats) (Fig. 11A). A similar decrease of 25.4% (p<0.01) and 51.1 % (p<0.001 ) in NADH-activated °2^~ production was also observed in ASA treated WKY and SHR rats, respectively (Fig. 11 B). It is noticeable that in SHR rats, ASA treatment completely restored to normal the higher basal and NAD(P)H activated °₂ ^~ levels. The 12-day treatment with ASA did not change the blood pressure of either WKY or SHR rats (WKY: 144 + 4.7 before treatment vs. 145 ± 3.3 mmHg at the end of treatment; SHR: 216 ± 5.6 vs. 205 ± 6.4 mmHg, p=NS). In Figs. 11 A to 11 D, ** represents p<0.01 vs. untreated rats and + p<0.01 vs. WKY rats. Effects of ASA on superoxide production in cultured aortic smooth muscle cells

Cultured aortic SMCs from 12 week old WKY and SHR were in cultured with 10^"4 mol/L of ASA for 48 hrs. The basal °_^~ production was

36.7 + 2.7 and 54.7 + 2.5 cpm/μg protein in untreated cells from WKY and SHR, respectively (p<0.001 , SHR vs. WKY). Those basal levels were respectively decreased by 60.2% and 73.1 % with the ASA treatment (p<0.01) (Fig. 11C). The NADH-activated °₂ ^~ production which was 41.8 ± 1.4 and 89.9 + 9.9 cpmχ10³/μg protein in control cells from WKY and SHR rats respectively(p<0.001 , SHR vs. WKY) was lowered by 54.4% and 53.3% in ASA treated cells respectively (p<0.01 vs. untreated) (Fig. 11 D). Like in the aorta, the higher °_7 basal and NADH induced °₂ ^~ productions were normalized by the ASA treatment in SHR.

Effects of chronic ASA on acetylcholine induced vasorelaxation in aorta from normotensive and spontaneously hypertensive rats The endothelium-dependent vasorelaxation was evaluated following chronic in vivo ASA treatment for 12 days from WKY and SHR rats. Acetylcholine induced a 26.9+0.9 % relaxation in aortic rings from untreated aorta from WKY (Fig. 12). The dose-response curves of the Ach induced-relaxation in aorta of untreated SHR showed a marked impairment with a maximal relaxation of only 14.4 + 0.8 % (a reduction of 47 % in the response compared to WKY, p<0.01 ), whereas the sensitivity, as reflected by the EC₅0, was not altered. The chronic ASA treatment restored partially and significantly the impaired relaxation of aorta from SHR by increasing the maximal relaxation to 20.6 + 0.8 % (p<0.01 vs. untreated SHR). However, no change was induced by ASA treatment in the aortic response of WKY rats (maximal relaxation: 28.9 + 1.0 %) (Fig. 12). In Fig. 12, relaxation was expressed as percent of the precontracted tension induced by phenylephrine (10^"7 mol/L). (*^* p<0.01 , n = 6 for each group). Preventive effect of chronic ASA treatment on the development of hypertension in spontaneously hypertensive rats

Since a close direct correlation between the level of aortic basal

°2^~ production and the development of hypertension (r= 0.683, p<0.01) in young SHR has recently been reported by the inventors, it was of interest to investigate whether the antioxidative properties of ASA could prevent or attenuate the development of hypertension in that model. Six-week old SHR and WKY rats were orally treated with ASA (100mg/day/kg) or water for 53 days and their blood pressure was measured during that period. At the beginning of the study (at 6 weeks), there was no difference in blood pressure between WKY and SHR rats. In untreated SHR, the blood pressure increased progressively from a basal value of 141 + 2.7 to 216 ± 2.0 mmHg (75 mmHg increase) at the end of study. In ASA treated SHR rats (SHR-ASA), the rise of the blood pressure was significantly attenuated with an increase of only 45 mmHg (from 141 + 6.0 to 186 + 3.5 mmHg) at the end of 53-day treatment (p<0.001 vs. untreated SHR). In contrast, ASA treatment did not have any effect on blood pressure in age-matched normotensive WKY rats (Fig. 13). Systolic BP was also measured .through direct femoral artery cannulation in rats anesthetized with pentobarbital sodium at the end of treatment. The BP in ASA-treated SHR rats was significantly lower than that of untreated SHR (183 + 4 vs. 217 ± 2 mmHg, p<0.001 ). Conversely, no differences in blood pressure between control and ASA-treated WKY rats (control 140 + 5, ASA-treated 138 + 7 mmHg) were observed. ASA treatment did not modify the body weight gain of the WKY and SHR rats. In Fig. 13, the drug treatment was started at 6 week of age (day 0) and lasted for 53 days. (** p<0.01 , n= 3 for each group).

EXAMPLE II ASA prevention of insulin resistance

Methods Animals

After a few days of acclimatization, male Sprague-Dawley (SD) rats weighing 230-260 g were randomly divided into three groups. One group of SD ( n=8) was given 10% D-glucose to drink in addition to a normal chow diet during three weeks, one group of SD (n=8) was given a combination of 10% glucose and aspirin™ (100 mg/kg/day) in their drinking water during three weeks while another group of age-matched control SD (n=8) was given only tap water and normal chow diet during three weeks. The body weight and blood pressure of all rats were recorded weekly. Systolic blood pressure was measured by tail cuff photoplethysmography (Harvard Apparatus Ltd) at least three times one day before the study. At the conclusion of the three-week treatment period, the animals were anesthetized with C0₂ and sacrificed by decapitation. The blood samples were collected early in the morning after 16 hours fast for the subsequent measurement of glucose, insulin, and antioxidant reserve. The thoracic aorta was quickly excised, immersed in ice-cold Krebs-Hepes buffer and cut into 2 mm ring segments for subsequent 0₂ measurement. Laboratory analysis

Plasma glucose concentrations were measured with a glucometer (Elite, Bayer Inc., Toronto, Canada). Insulin levels were determined by radioimmunoassay method (kit 07260102, ICN Pharmaceuticals, Costa Mesa, California, USA). Erythrocytes and plasma superoxide dismutase (SOD) activity was determined spectrophotometrically (kit, Randox Laboratories Canada Ltd, Mississauga, Ontario, Canada). Gluthatione peroxidase (GPx) activity in erythrocytes and plasma was measured as described in Daret et al., (Daret et al., Gluthatione peroxidase: activity and steady state level of mRNA. In: Punchard NA, Kelly FJ (Editors). Free Radicals, A Practical Approach. Oxford, New York; pp 227-231 , 1996). To evaluate the degree of insulin resistance, the Homeostasis

Model Assessment (HOMA) was calculated using formula 1.

HOMA = insulin (μg/ml) X glucose (mmol/L) / 22.5 Superoxide anion measurement

The superoxide anion production was measured using the lucigenin-enhanced chemiluminescence method as described in the art. Briefly, the aortic ring was placed into the Krebs-Hepes buffer (saturated with 95% and 5% C0₂ at room temperature during 30 minutes). After a 10 minute equilibration, the aortic ring segment was gently transferred to a glass scintillation vial containing 5μmol/l lucigenin for the determination of basal 0₂ levels. The chemiluminescence was recorded every minute for 15 minutes at room temperature by a liquid scintillation counter. Background counts were determined from vessel-free incubation media and subtracted from the readings obtained with vessels. Lucigenin counts were expressed as cpm/mg of dry weight of vessel. Statistics

Data are expressed as mean ± SEM (n). Statistical analysis was performed by one-way analysis of variance (ANOVA). The statistical significances of the differences between groups were further established by the Bonferroni/Dunn multiple comparison test. Significance was set at P<0.05, and values were interpreted with the Bonferroni's correction when appropriate (P<0.0167), with three pairwise comparisons considered of interest: control rats Vs the two other groups and glucose fed rats Vs glucose and ASA fed rats. Simple regression analysis were used to evaluate the correlations between systolic blood pressure, insulin resistance index and aortic superoxide anion production. Results

Blood Pressure and Body Weight

The chronic administration of glucose in drinking water resulted in a significant increase (p<0.01 ) in systolic blood pressure, which reached an average of 163 mmHg after 3 weeks (Fig. 14). The chronic treatment with aspirin prevented completely the increase of systolic blood pressure in glucose fed rats so that the blood pressure curve of those animals followed the same pattern than in control normotensive rats. Chronic glucose feeding resulted in a significant increase in water intake in glucose fed rats in comparison to control rats (99.3 ± 15.2 ml Vs 34.3 ±5.0 ml; P<0.01). The addition of aspirin tended to decrease, although not significantly, the water intake in glucose fed rats. In Fig. 14, the number of rats in each group are indicated in brackets. The symbol "*" represents P<0.05 and the symbol "**" represents P<0.01 Vs control. The symbol "+" represents P<0.05 and the symbol "++" represents P<0.01 Vs glucose group. Plasma Glucose and Insulin Concentrations

As shown in Figs. 15A and 15B, the plasma levels of glucose and insulin were significantly higher in glucose fed rats in comparison to control rats. The treatment with aspirin prevented the rise in glucose levels in glucose-fed rats so that the plasma glucose levels did not statistically differ from those in control rats. In glucose-fed rats, insulin levels increased by 276% and the treatment with aspirin reduced this increase to 211 % in glucose-fed rats but those levels remained higher (PO.05) than in control rats. The development of insulin resistance was indicated by a 398% increase in HOMA (P<0.05) in glucose-fed rats (Fig. 15C) and the treatment with aspirin reduced significantly (P<0.05) this increase to 190% in insulin resistance in glucose-fed rats but those levels remained nevertheless significantly higher (P<0.05) than the control rats. Basal Aortic Superoxide Anion Production As shown in Fig. 15D, the basal aortic superoxide anion production was increased by 52% in glucose-fed rats in comparison to control rats (P<0.05). The treatment with aspirin totally prevented the rise in basal superoxide anion production in aorta of glucose-fed rats (PO.01). In Figs. 15A to 15D, data are represented as means ±SE. The number of rats in each group was 8. The symbol "*" represents P<0.05 and the symbol "**" represents P<0.01 Vs control. The symbol "+" represents P<0.05 and the symbol "++" represents P<0.01 Vs glucose group.

Relationships Between The Aortic Superoxide Anion Production, Insulin Resistance Index And Systolic Blood Pressure

Highly significant positive correlations were found between the aortic superoxide anion production and systolic blood pressure (r-0.723; P<0.01 ; Fig. 16A), between the aortic superoxide anion production and insulin resistance (HOMA) (r=0.821 ; P<0.01 ; Fig. 16B) as well as between HOMA and systolic blood pressure (r=0.760; P<0.01 ; Fig. 16C). Antioxidant Reserve

As shown in Table 1 , the chronic administration of glucose combined or not with aspirin had no effect on the activity of GPx both in plasma and in red blood cells.

TABLE 1 Body Weight And Systolic Blood Pressure

Data are means ± SE. * P < 0.01 Vs control rats, ** P < 0.01 Vs glucose-fed rats

As shown in Table 2, the erythrocytes SOD activity was similar in all groups but an increase in the plasma SOD activity was observed in glucose-fed rats (P < 0.05) but those levels tended to be higher in aspirin treated glucose-fed rats compared to control. TABLE 2

Gluthatione Peroxidase And Superoxide Dismutase Activities in Erythrocytes And Plasma Of Control, Glucose-Fed and Glucose-Fed

Rats Treated With Aspirin

Data are means ± SE. * P < 0.05 Vs control rats

In accordance with the present invention, it was thus found that: (1 ) the chronic treatment with glucose for three weeks resulted in increases in blood pressure, in insulin resistance and in basal superoxide anion production in the aorta; (2) the concomitant chronic aspirin treatment of glucose-fed rats prevented the rise in blood pressure, in glucose levels and in aortic superoxide anion production, whereas it attenuated significantly the increase in insulin resistance; and (3) highly positive correlations between systolic blood pressure and aortic superoxide anion production or insulin resistance were observed in these groups of rats.

In the present application, it is thus shown for the first time that aspirin (ASA) prevented simultaneously the development of hypertension, hyperglycemia as well as the rise in aortic superoxide anion production and attenuated the increase in insulin resistance in glucose-fed rats. In conclusion, the present invention thus demonstrates that chronic Aspirin treatment in vivo prevents the development of hypertension and reduces significantly the insulin resistance in chronically glucose-fed rats. Aspirin produces these effects through its anti-oxydative properties since this treatment was found to inhibit the increase in vascular oxidative stress observed in aorta of chronically glucose-fed rats. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for preventing and/or treating the development of hypertension in a patient, said method comprising administering to a patient acetylsalicylic acid.

2. A method for reducing superoxide anion production in a patient, said method comprising administering acetylsalicylic acid to said patient.

3. The method of claim 2, wherein said acetylsalicylic acid reduces NAD(P)H oxidase activity causing reduction of superoxide anion production.

4. The method of claim 1 , wherein said acetylsalicylic acid is administered as multiple doses.

5. The method of claim 4, wherein each of said multiple doses comprises a dose of acetylsalicylic acid of at least 80 mg/day.

6. A method for restoring impaired aortic vasodilatory response in a hypertensive patient, said method comprising administering acetylsalicylic acid to a patient for a time and with a dose sufficient to restore aortic vasorelaxation mechanism.

7. A method for reducing superoxide anion production in cardiac and colonic tissues of a patient, said method comprising administering acetylsalicylic acid, nimesulide and indomethacin to said patient.

8. The method of claim 7, wherein said acetylsalicylic acid, nimesulide and indomethacin reduce NAD(P)D oxidase activity causing reduction of superoxide anions production.

9. A method for treating cardiovascular diseases associated to an increased oxidative stress, said method comprising the step of administering to a patient in need thereof a pharmaceutically acceptable therapeutically effective amount of acetylsalicylic acid to a patient.

10. A method for reducing insulin resistance in a patient, said method comprising the step of administering to said patient a pharmaceutically acceptable therapeutically effective amount of acetylsalicylic acid.

11. A method for treating a patient suffering from hyperglycemia said method comprising the step of administering to said patient a pharmaceutically acceptable therapeutically effective amount of acetylsalicylic acid.

12. Use of acetylsalicylic acid for preventing and/or treating the development of hypertension in a patient.

13. Use of acetylsalicylic acid for reducing superoxide anion production in a patient.

14. Use of acetylsalicylic acid for reducing NAD(P)H oxidase activity causing reduction of superoxide anion production.

15. The use of claim 12, wherein said acetylsalicylic acid is administered as multiple doses.

16. The use of claim 15, wherein each of said multiple doses comprises a dose of acetylsalicylic acid of at least 80 mg/day.

17. Use of acetylsalicylic acid for restoring impaired aortic vasodilatory response in a hypertensive patient.

18. Use of acetylsalicylic acid, nimesulide and indomethacin for reducing superoxide anion production in cardiac and colonic tissues of a patient.

19. The use of claim 18, wherein said acetylsalicylic acid, nimesulide and indomethacin reduce NAD(P)D oxidase activity cause reduction of superoxide anions production.

20. Use of acetylsalicylic acid for treating cardiovascular diseases associated to an increased oxidative stress.

21. Use of acetylsalicylic acid for reducing insulin resistance in a patient.

22. Use of acetylsalicylic acid for treating a patient suffering from hyperglycemia.

23. Use of acetylsalicylic acid for the manufacture of a medicament for preventing and/or treating the development of hypertension in a patient.

24. Use of acetylsalicylic acid for the manufacture of a medicament for reducing superoxide anion production in a patient.

25. Use of acetylsalicylic acid for the manufacture of a medicament for reducing NAD(P)H oxidase activity causing reduction of superoxide anion production.

26. The use of claim 23, wherein said acetylsalicylic acid is administered as multiple doses.

27. The use of claim 26, wherein each of said multiple doses comprises a dose of acetylsalicylic acid of at least 80 mg/day.

28. Use of acetylsalicylic acid for the manufacture of a medicament for restoring impaired aortic vasodilatory response in a hypertensive patient.

29. Use of acetylsalicylic acid, nimesulide and indomethacin for the manufacture of a medicament for reducing superoxide anion production in cardiac and colonic tissues of a patient.

30. The use of claim 29, wherein said acetylsalicylic acid, nimesulide and indomethacin reduce NAD(P)D oxidase activity cause reduction of superoxide anions production.

31. Use of acetylsalicylic acid for the manufacture of a medicament for treating cardiovascular diseases associated to an increased oxidative stress.

32. Use of acetylsalicylic acid for the manufacture of a medicament for reducing insulin resistance in a patient.

33. Use of acetylsalicylic acid for the manufacture of a medicament for treating a patient suffering from hyperglycemia.