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Lack of gastric toxicity of nitric oxide-releasing aspirin, NCX-4016, in the stomach of diabetic rats
Life Sciences 67 (2000) 1639Ð1652
Lack of gastric toxicity of nitric oxide-releasing aspirin, NCX-4016, in the stomach of diabetic rats Kimihito Tashima, Akinobu Fujita, Masakazu Umeda, Koji Takeuchi* Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607-8414, Japan
Abstract We compared the gastric toxic effect of aspirin (ASA) in both normal and diabetic rats, with that of NCX-4016, a derivative of ASA with nitric oxide (NO) releasing moiety. Animals were injected with streptozotocin and used after 5 weeks of diabetes with blood glucose levels of .350 mg/dl in the presence of omeprazole. Oral administration of ASA (with 150 mM HCl) did not produce damage at 30 mg/kg in the conscious rat but caused hemorrhagic gastric lesions in STZ-diabetic rats. By contrast, NCX-4016 even at 190 mg/kg (a dose equimolar to 100 mg/kg of ASA) did not cause damage in both normal and STZ-diabetic rat stomachs. Plasma salicylic acid levels were not different between normal and diabetic rats after administration of ASA or NCX-4016, though the latter gave signiÞcantly lower levels as compared to ASA. Intragastric application of ASA (80 mM in 50 mM HCl) for 30 min caused a reduction of transmucosal PD and increase of luminal H1 loss with a minimal effect on mucosal blood ßow (GMBF) in both normal and diabetic rats, yet resulting in much severe damage in the stomach of the latter group. Mucosal application of NCX-4016, however, did not cause PD reduction and luminal H1 loss, but produced a marked hyperemia, resulting in no damage in the stomach of both normal and STZ-diabetic rats. The increased gastric toxicity of ASA in STZ-diabetic rats was signiÞcantly mitigated by co-application of a NO donor FK-409 together with ASA, with an increase of GMBF, despite similar degrees of PD reduction and luminal H1 loss being observed. We conclude that NCX-4016 does not have a toxic effect in either normal or diabetic rat stomachs, although the diabetic rat stomach is more vulnerable to ASA-induced damage. NCX-4016, though absorbed more slowly than ASA, counteracts the injurious effect of aspirin on the gastric mucosa, probably by increasing GMBF mediated by NO. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Diabetic rat; Gastric mucosa; Aspirin; NO-releasing aspirin (NCX-4016)
Introduction Diabetes mellitus is a chronic disease characterized by hyperglycemia and by complications that include microvascular diseases and a variety of neuropathies [1]. Patients with pro* Corresponding author. Tel.: 81-75-595-4679; fax: 81-75-595-4774. E-mail address: [email protected] (K. Takeuchi) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 4 6 -3
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longed diabetes have an increased risk of microvascular complications, including myocardial infarction and stroke, which are mostly attributable to an increased platelet adhesiveness and aggregation, with enhanced production of thromboxane [2,3]. Anti-platelet therapy has been shown to reduce the risk of stroke in such patients. This gives a rational to the use of aspirin (ASA) in diabetic patients to prevent microvascular complications. Recent studies showed an increased mucosal susceptibility to various ulcerogenic stimuli in streptozotocin (STZ)-induced diabetic rats, an accepted model of insulin-dependent diabetes [4Ð 7]. Since the short-term or long-term administration of nonsteroidal antiinßammatory drugs (NSAIDs) such as ASA causes damage in the stomach as an unwanted side effect in human and in laboratory animals [8Ð11], it is assumed that the gastric toxicity of aspirin is enhanced in diabetic patients. Yet, no study has investigated the gastric toxic effect of ASA in diabetic conditions. A recent attempt to develop NSAIDs that spare the gastrointestinal tract of injury has produced nitric oxide (NO)-releasing NSAIDs [12]. It has been shown that the coupling of a NO-releasing moiety to standard NSAIDs markedly reduced their ulcerogenic properties without altering their effectiveness as anti-inßammatory drugs or cyclooxygenase inhibitors [12Ð15]. The NO-releasing ASA is a similar derivatization of ASA and has been shown to exhibit comparable anti-thrombotic or anti-inßammatory activity to that of ASA, with less damage in the gastric mucosa [16Ð18]. In the present study, we therefore compared the gastric toxic effect of a NO-releasing ASA derivative, NCX-4016, in normal and STZ-diabetic rats,with that of the parent compound ASA. In addition, we examined the effect of an exogenous NO donor, FK-409, on gastric ulcerogenic and functional responses to ASA in diabetic rats. Materials and methods Animals Male Sprague-Dawley rats (260Ð280 g: Charles River, Shizuoka, Japan) were used. The animals were fed standard rat chow and tap water ad libitum. One week after purchase, they were given streptozotocin (STZ: 70 mg/kg, i.p.) and fed normally thereafter. Control animals received an equal volume of saline. All experimental procedures described here were approved by the Experimental Animal Research Committee of the Kyoto Pharmaceutical University. General procedures The experiments were performed in normal and 5 week STZ-diabetic rats. Blood was sampled from the tail vein and blood glucose levels (BGL) were determined by GlucostarGlucostix (Miles-Sankyo Co. Ltd., Tokyo, Japan). The STZ-treated animals with BGL of less than 350 mg/dl under nonfasting conditions were excluded from the study. In these groups of rats, the effects of oral administration of ASA and NCX-4016 on the gastric mucosa under unanesthetized conditions, and those of their topical application on the ulcerogenic and functional responses in the chambered stomach under urethane anesthetized conditions,were examined. Since STZ-diabetic rats had some damage in the stomach after fasting [5], all animals were treated with i.p. omeprazole (60 mg/kg) immediately before fasting to prevent fasting-induced lesions. The doses of NCX-4016 and ASA used in all studies are the same on
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a molar basis and have been shown to cause an equipotent inhibition of prostaglandin (PG) biosynthesis in the rat gastric mucosa [18,19]. Gastric ulcerogenic effects in conscious rats The rats were given ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg; equivalent to 100 mg/kg of ASA), both suspended in 150 mM HCl, p.o., and killed 4 hr later. The stomachs were removed, inßated by injecting 10 ml of 2% formalin for 10 min to Þx the tissue walls, and opened along the greater curvature of the stomach. The area (mm2) of hemorrhagic lesions developed in the stomach was measured under a dissecting microscope with a square grid (310), summed per stomach, and used as a lesion score. This procedure was applied to the subsequent studies. Gastric ulcerogenic and functional effects in anesthetized rats Animals were anesthetized with urethane (1.25 g/kg, i.p.), and the trachea was cannulated to ensure a patent airway. Simultaneous measurement of transmucosal potential difference (PD), gastric mucosal blood ßow (GMBF), and luminal acid loss was performed in the chambered stomach as described previously [20]. Brießy, the abdomen was incised, and the stomach was exposed and mounted on an ex-vivo chamber (area exposed, 3.14 cm2). At the beginning of each experiment, the mucosa was rinsed several times with a solution of 50 mM HCl plus 100 mM NaCl. When the gastric exudate became clear, 2 ml of the acid solution was instilled in the chamber, and 15 min later the gastric contents were recovered from the chamber. This procedure was repeated every 15 min, two times before and 6 times after exposure of the mucosa to 80 mM ASA or NCX-4016 for 30 min. PD was determined using two agar bridges, one positioned in the chamber and the other in the abdominal cavity. GMBF was measured by a laser Doppler ßowmeter (Advance Model ALF 21, Tokyo, Japan), placing the probe gently on the corpus mucosa using a balance, and changes in GMBF were continuously monitored on a two-channel recorder (U-228, Tokai-Irika, Tokyo, Japan) simultaneously with those of PD [21]. On the other hand, luminal acid loss (loss of luminal titratable acidity) was determined from the analyses of the collected acid solution. Each sample was analyzed for volume and acid concentration, which was determined by titration of the aliquot against 100 mM NaOH to pH 7.0 (Autoburette, Comtite-7, Hiranuma, Tokyo, Japan). The amount of luminal acid loss was calculated as the difference between the product of the Þnal volume and concentration and the product of initial volume and concentration, and the results were expressed as mEq/15 min. Ninety minutes after exposure to ASA or NCX-4016, the mucosa was examined for hemorrhagic lesions under a dissecting microscope. To prevent bias, the observer measuring the lesion was unaware of the treatment. In some cases, the effect of FK-409 on gastric ulcerogenic and functional responses to ASA in STZ-diabetic rats was examined. FK-409, an orally active spontaneous NO releasing drug which can generate NO [22], has been shown to afford gastric cytoprotection, inhibition of acid secretion as well as increase of GMBF in rats when administered p.o. [18,23], FK409 (1 mg/ml) was given topically for 30 min, together with 80 mM ASA. Determination of plasma salicylic acid Plasma levels of salicylic acid were determined in both normal and STZ-diabetic rats following p.o. administration of ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg) with 150
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mM HCl, according to the modiÞed method of Urushidani et al [24]. Three hundred ml of blood was collected from the tail vein in the presence of heparin, at 30, 60, 120 and 240 min after administration of these drugs. Samples were centrifuged at 10,000 rpm for 5 min at 48C and stored at 2808C until the assay. For determination of salicylic acid, the samples (100 ml) were mixed with 20 ml of 6 N HCl, boiled at 1008C for 10 min, and shaken with 600 ml of ethylene dichloride for 5 min. After extracting, the organic phase was transferred to another tube. Then, 200 ml of 1.76% of ferric nitrate was added and the mixture was shaken for 5 min. The aqueous layer was collected, and the salicylic acid levels were measured by spectrophotoßuorometry at 545 nm. A standard curve was made using rat plasma containing various concentrations of salicylic acid. Preparation of drugs Drugs used were urethane (Tokyo Kasei, Tokyo, Japan), streptozotocin (Nacalai tesque, Kyoto, Japan), aspirin (ASA: Sigma Chemicals, St. Louis, Missouri, USA), NCX-4016 (NOreleasing ASA; NiCox, Paris, France), and FK-409 (Fujisawa, Osaka, Japan). ASA or NCX4016 was suspended in 50 mM or 150 mM HCl, while other agents were dissolved in saline. Each agent was prepared immediately before use and administered i.p. or p.o. in a volume of 0.5 ml per 100 g body weight, or applied topically to the chamber in a volume of 2 ml per rat. Statistics Data are presented as the mean6SE from 4z7 rats per group. Statistical analyses were performed using a two-tailed DunnettÕs multiple comparison test, and values of p,0.05 were regarded as signiÞcant. Results Effects of ASA and NCX-4016 on gastric mucosa of conscious rats Oral administration of ASA with 150 mM HCl at 30 mg/kg did not cause any damage in the gastric mucosa of normal rats but at 100 mg/kg produced hemorrhagic lesions, the lesion score being 16.163.0 mm2 (Fig. 1). The toxic action of acidiÞed ASA at the same doses was markedly potentiated in STZ-diabetic rat stomachs; the lesion score at 100 mg/kg was significantly increased to 36.469.0 mm2, which is about 2 times greater than that observed in normal rats. Even at 30 mg/kg, ASA provoked apparent gastric damage in STZ-diabetic rats. By contrast, NCX-4016 at 190 mg/kg did not produce any macroscopic damage in either normal or STZ-diabetic rat stomachs. Likewise, control animals received vehicle alone did not show any damage in the stomach under normal and diabetic conditions (not shown). Gastric functional responses and mucosal injury induced by mucosal application of ASA and NCX-4016 in anesthetized rats PD response Under chambered conditions in the presence of omeprazole (inhibition of acid secretion) and exogenous acid (50 mM HCl plus 100 mM NaCl), the normal rat stomachs generated a
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Fig. 1. Mucosal ulcerogenic effects of ASA and NCX-4016 in normal and STZ-diabetic rats. ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg), suspended in 150 mM HCl, was administered orally, and the animals were killed 4 hr later. Control animals received vehicle alone did not show any damage in the stomach, either normal or diabetic conditions (not shown). Data are presented as the mean6SE from 5z7 rats per group. SigniÞcant difference at P,0.05; * from normal rats given ASA; # from the animals given ASA (100 mg/kg) in the corresponding group.
PD of 245z50 mV (mucosa negative) and maintained relatively constant GMBF (180~200 mV: arbitrary unit) during a 2-hr test period (Fig. 2). Exposure of the normal rat mucosa to ASA (80 mM) for 30 min caused a marked reduction of PD from 247.663.1 mV to 28.062.4 mV. After the exposure the reduced PD normalized gradually, yet was still signiÞcantly low 90 min later; the degree of PD recovery being 31.565.6%. Although STZ-diabetic rats exhibited a lower PD (235z40 mV) in basal conditions than normal rats, the values in GMBF were almost equivalent to those in normal rats. The mucosal application of ASA caused a similar degree of PD reduction in STZ-diabetic rats; DPD was 29.062.3 mV. However, the recovery of PD after exposure to ASA was slightly delayed in the diabetic rats; the degree of the recovery at 90 min post-treatment being 20.262.4%. On the other hand, NCX4016 (80 mM) applied topically to the stomach did not produce a decrease of PD in either normal or STZ-diabetic rats, and the PD remained in similar ranges before and after the exposure in both groups of animals. GMBF response The GMBF was slightly increased immediately after exposure of the mucosa to 80 mM ASA, but returned to or dropped below baseline values during the exposure, the increase observed at the end of exposure being 20.268.3% (Fig. 3). Essentially similar changes in GMBF were observed in STZ-diabetic rat stomachs after exposure to ASA, but the increase in GMBF at the end of exposure was 4.465.8%, slightly less than that observed in normal rats. On the other hand, the GMBF was signiÞcantly elevated when NCX-4016 (80 mM) was
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Fig. 2. Changes in PD after exposure of the stomach to ASA or NCX-4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK-409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. Data are presented as the mean6SE of values determined every 15 min from 4z7 rats per group. SigniÞcant difference at P,0.05; * from basal values (time 0) in the corresponding group; # from values in STZ-diabetic rats exposed to ASA alone.
applied to the stomach in both normal and STZ-diabetic rats, reaching a peak increase of 59.365.3% and 62.467.9%, respectively, at the end of the exposure period, and in both cases remained signiÞcantly elevated for 30 min even after removal of NCX-4016 from the chamber. In these rats, the GMBF showed a moderate increase (25.269.3% and 6.364.8%) even 60 min after exposure to NCX-4016. Luminal acid loss When the gastric mucosa was exposed to 50 mM HCl in the absence of acid secretion induced by omeprazole, a small but signiÞcant loss of luminal titratable acidity was consistently observed in normal rats under basal conditions; DH1 was less than 25 mEq/15 min (Fig. 4). Following the mucosal application of 80 mM ASA, the loss of titratable acidity was signiÞcantly increased, reaching a maximal value immediately after the exposure, then gradually decreasing to the pre-exposure levels 90 min later. The maximal DH1 value was 49.763.7 mEq/15 min in normal rats. The loss of luminal titratable acidity in STZ-treated rats was signiÞcantly great in basal conditions as compared to normal rats, and the values (DH1: 35.163.2 mEq/15 min) were approximately 1.5 times the levels observed in normal animals. The luminal acid loss in STZ-diabetic rats was further increased in response to ASA, similar to normal rats; the magnitude of H1 loss observed immediately after ASA being 54.764.3 mEq/15 min, and not signiÞcantly different from that observed in normal
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Fig. 3. GMBF responses induced by ASA or NCX-4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK-409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. Data show the GMBF response (%increase from basal values) at the end of the exposure period in each group, and values are the mean6SE from 4z7 rats. SigniÞcant difference at P,0.05; * from normal rats treated ASA; # from STZ-diabetic rats treated with ASA alone.
rats. By contrast, the mucosal exposure to NCX-4016 did not alter the luminal acid loss in either normal or STZ-diabetic rats, and the values in DH1 loss remained unchanged before and after exposure to NCX-4016. The luminal acid loss observed immediately after exposure to NCX-4016 was 33.863.1 mEq/15 min and 39.868.3 mEq/15 min, respectively, in normal and diabetic rat stomachs, neither of which was not signiÞcantly different from the corresponding pre-exposure values. Mucosal injury Mucosal application of 80 mM ASA or acid solution (50 mM HCl) by itself did not induce gross damage in the gastric mucosa (not shown), but these treatments (ASA plus HCl) applied together produced hemorrhagic damage in the mucosa of normal rats, the lesion score being 23.263.1 mm2 (Fig. 5). These lesions were signiÞcantly worsened in STZ-diabetic rats, the lesion score being 64.4612.5 mm2. Histologically, the response in normal rat stomachs to ASA was a widespread disruption of epithelial cells without deep damage, but in diabetic rats the damage reached deep into the mucosa with severe hemorrhaging (not shown). The mucosal application of NCX-4016, however, did not induce gross damage in the gastric mucosa of either normal or STZ-diabetic rats. Effects of FK-409 on gastric functional and ulcerogenic responses induced by ASA in STZ-diabetic rats To further investigate the beneÞcial inßuence of NO on gastric functional and ulcerogenic responses to ASA, we examined the effects of the NO donor FK-409 on these responses in
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Fig. 4. Amount of luminal acid loss (loss of titratable acidity) after exposure of the stomach to ASA or NCX4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. Data are the mean6SE of values determined every 15 min from 4z7 rats. SigniÞcant difference at P,0.05; * from normal rats without ASA treatment; # from normal rats without NCX-4016 treatment, u from basal values (time 0) in the corresponding group.
STZ-diabetic rat stomachs. As shown in Figs. 2Ð4, FK-409 (1 mg/ml), given topically together with 80 mM ASA, did not prevent PD reduction or luminal acid loss caused by ASA, but signiÞcantly increased GMBF during and after the exposure. In these animals, the recovery of PD seen after exposure to ASA was expedited by co-application of FK-409, the degree of PD recovery at 60 min and 75 min post-treatment being 33.167.1% and 33.366.6%, respectively, both of which were signiÞcantly greater than the values for STZ-diabetic rats treated with ASA alone (Fig. 2). The increased gastric ulcerogenecity to ASA in STZ-diabetic rats was apparently mitigated by the combined application of FK-409, and the lesion score was decreased to 29.064.1 mm2, which was signiÞcantly lower than that (64.4612.5 mm2) observed in the control group with ASA alone (Fig. 5). Plasma levels of salicylic acid after administration of ASA and NCX-4016 Following p.o. administration of ASA with 150 mM HCl in normal rats, the plasma levels of salicylic acid were increased dose- and time-dependently. At 100 mg/kg, a maximal value was observed within 30 min in both normal (307.2613.5 mg/ml) and diabetic animals
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Fig. 5. Development of gastric mucosal lesions after exposure of the stomach to ASA or NCX-4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK-409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. The animals were killed 90 min after the treatment with ASA or NCX-4016. Data are the mean6SE from 4z7 rats. SigniÞcant difference at P,0.05; * from normal rats treated with ASA alone; # from STZ-diabetic rats treated with ASA alone.
(312.2611.2 mg/ml), remaining in these ranges for 4 hr thereafter (Fig. 6). Although NCX4016 (190 mg/kg) given p.o. with 150 mM HCl produced a gradual increase in the plasma salicylic acid levels in both normal and diabetic rats, reaching 4 hr later about half the values observed with ASA; the values were 162.6611.0 mg/ml and 160.4616.3 mg/ml, respectively. No difference was found in plasma salicylic acid levels after administration of NCX4016 between normal and diabetic animals. Discussion The present study showed that gastric toxicity of ASA was enhanced in STZ-induced diabetic rats, conÞrming our previous observation that the diabetes predisposes rats to gastric ulceration [5,20]. In addition, we also found that NCX-4016, an ASA derivative with a NOreleasing moiety, prevented such gastric toxicity in STZ-diabetic as well as normal rats. These results give rational support to the use of a NO-releasing ASA for anti-platelet therapy in diabetic patients. STZ is known to possess diabetogenic properties and cause selective destruction of pancreatic b-cells. As expected, all STZ-treated animals developed a persistent hyperglycemia, which was observed one week after STZ injection. Although ASA provoked severe damage in the gastric mucosa in both normal and STZ-diabetic rats, the severity was much greater in the latter. Because all experiments in the present study were done in the presence of exogenous HCl under inhibition of acid secretion by omeprazole pretreatment, it is unlikely that the exacerbation of damage in diabetic rats is due to acid secretory changes. Furthermore, no difference was observed in plasma levels of salicylic acid 2 hr after administration of ASA
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Fig. 6. Plasma salicylic acid levels after p.o. administration of ASA or NCX-4016 in normal and STZ-diabetic rats. ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg), suspended in 150 mM HCl, was administered orally, and the blood samples were collected from a tail vein at various time points after the administration. Data are presented as the mean6SE from 5z6 rats per group. * SigniÞcant difference from the animals given ASA (100 mg/ kg) in the corresponding group, at P,0.05.
(30 mg/kg) and NCX-4016 (190 mg/kg), yet apparent damage was observed only in the diabetic animals treated with the former. Thus, it is unlikely that the less toxic effect of the latter is simply due to a low bioavailability of the compound. Two major components, namely the topical irritant effect on the epithelium and the suppression of PG synthesis, have been proposed to explain the gastric toxicity of ASA [10,25]. However, since ASA produces gastric lesions when administered orally but not parenterally, the former effect is more crucial in causing gastric mucosal damage [26]. Indeed, the present study showed that topical application of ASA caused PD reduction and luminal acid loss in normal and STZ-diabetic rats, the phenomena being observed in the stomach after disruption of the mucosal barrier [26]. The luminal acid loss is due to both H1 back-diffusion and neutralization of H1 with bicarbonate or plasma diffused into the lumen. The occurrence of H1 back-diffusion is followed by an increase of GMBF, partly mediated by endogenous PG, to prevent damage in the mucosa by limiting local accumulation of H1 ion. However, ASA by inhibiting PG biosynthesis hampered gastric hyperemic response associated with H1 back-diffusion and thereby provoked hemorrhagic damage in the stomach. We previously reported that the GMBF response induced by H1 back-diffusion was mitigated in diabetic animals, partly due to dysfunction of capsaicin-sensitive afferent neurons [20]. The increase of GMBF after ASA treatment was only 30% in normal rat stomachs and was even less in STZ-diabetic animals, yet the difference was not great enough to explain the exacerbation of ASA-induced damage in diabetic rat stomachs. At present, although the mechanisms by which ASA-induced gastric damage was worsened in STZ-diabetic rats remains unexplored, they may include a decrease of mucus secre-
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tion [4], an impairment of the anti-oxidative system [7] or some other complication observed in diabetic conditions [5,20]. Because there was no difference in plasma salicylic acid levels between normal and diabetic animals after administration of ASA, it is unlikely that the increased ulcerogenic response is due to an enhancement of its absorption from the stomach. We noted in this study that STZ-diabetic rats showed characteristic mucosal changes under basal conditions without any treatment; a decrease in basal PD values and an increase in luminal acid loss, in agreement with the Þndings by Hung et al. [27]. The enhanced luminal acid loss may be largely due to an increase of spontaneous H1 back-diffusion. These results account for the lowered PD values and are consistent with the increased mucosal permeability in STZ-diabetic rats as determined from the luminal appearance of Evans blue [5]. The reason for the enhanced mucosal permeability in diabetic rat stomachs remains unknown, yet this may also be a factor contributing to the increased susceptibility of the mucosa to ASA. It has been shown that chemical ablation of capsaicin-sensitive sensory neurons exacerbated gastric mucosal lesions induced by a variety of noxious stimuli including aspirin [28,29]. These results may suggest a similarity of deleterious inßuences on the mucosal integrity between diabetes and sensory deafferentation. Lincoln et al. [30] showed that the density of calcitonin gene-related peptide (CGRP)-immunoreactive Þbres in the intestine was markedly decreased in diabetic rats. We also reported that the amount of CGRP released in the isolated stomach in response to capsaicin was signiÞcantly lower in STZ-diabetic animals than in normal rats [21]. Thus, it is likely that the increased gastric toxicity of ASA in STZ-diabetic rats is at least partly accounted for by dysfunction of capsaicin-sensitive sensory neurons. On the other hand, topical application of NCX-4016, a NO-releasing ASA derivative, did not cause gross damage in rat stomachs even in the presence of acid, irrespective of whether or not the rats were subjected to diabetes by STZ injection. These results conÞrmed our previous observation that intragastric administration of NCX-4016 did not produce any damage in the stomach, despite decreasing the mucosal PGE2 contents [18,19]. In addition, NCX4016 had no effect on PD but produced a sustained increase of GMBF, suggesting a lack of direct irritating action on the mucosa and a release of NO from the compound. These results are consistent with the Þndings by others [12Ð17], who showed that the addition of a nitroxybutylester group to NSAIDs markedly reduced its short-term ulcerogenic property without altering its effectiveness as a cyclooxygenase inhibitor. In the present study, NCX-4016 gave lower values of plasma salicylic acid levels than ASA, following p.o. administration. However, this does not mean that NCX-4016 is more slowly absorbed, because this comound inhibited gastric mucosal PGE2 synthesis and showed antiinßammatory action against carrageenan-induced paw edema,both as effectively as ASA [18,19]. The release of salicylate from NCX-4016 may well require a more complicated metabolism than the simple loss of an acetyl group in the case of aspirin. In any case, it is conceivable that the therapeutic concentrations of ASA could be attained following p.o. administration of NCX-4016. We have previously shown that NOx levels in both serum and gastric contents were increased following NCX-4016, administered either p.o. or s.c. [18]. The mechanism responsible for the reduced ulcerogenic property of the NO-releasing derivative of ASA has not been elucidated, yet it is conceivable that the NO released from this compound exerts beneÞcial effects on the gastric mucosa by enhancing the mucosal defensive ability. This contention is supported by previous studies using several NO-releasing derivatives of NSAIDs such as nitrofenac, NO-naproxen
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and NO-ßurbiprofen [12Ð15]. It should also remind us that NO-releasing ASA (NCX-4215) is more effective in inhibiting platelet aggregation than ASA, despite having less of an effect on thromboxane B2 synthetic activity [16], suggesting that NO released from the compound by itself has an inhibitory effect on platelet aggregation. Just how NO protects the gastric mucosa against damage is not entirely clear. It is now well accepted that NO plays an important role in maintaining the mucosal integrity of the stomach and modulating various gastric functions such as mucosal blood ßow and mucus secretion [31Ð33]. Since NCX-4016 applied topically to the gastric mucosa caused a persistent increase of GMBF, this effect may contribute to the protective action of this compound [18]. This contention was supported by the present Þnding that the increased gastric toxicity of ASA in STZ-diabetic rats was signiÞcantly attenuated by the combined administration of FK-409, a NO donor [22]. This treatment did not affect the PD reduction or luminal H1 loss observed in the stomach following ASA treatment, but produced a marked increase of GMBF. In addition, recent studies showed an important role for neutrophils in the pathogenesis of ASA-induced gastric lesions [34,35]. Since NO inhibits neutrophil activation and scavenges oxygen metabolites [36], it is possible that the molecule interferes with the neutrophilrelated process in tissue injury. Indeed, Wallace et al. [37] recently showed that this particular NO-releasing derivative of ASA, NCX-4016, inhibited leukocyte adherence to the vascular endothelium and prevented gastric damage induced by hemorrhagic shock. It should also be noted in the present study that FK-409, when applied together with ASA, signiÞcantly promoted the recovery of PD in the stomach after exposure to ASA. GMBF is considered to play a role in maintaining the gastric mucosal integrity by supplying oxygen and nutrients, in addition to buffering H1 ions, and contributes to mucosal repair as well as mucosal protection. It is possible to assume that the process of PD recovery after ASA was signiÞcantly promoted by a NO donor, resulting in less severe gastric damage. Thus, the present study conÞrmed our previous results in normal rats [18] and further showed that unlike ASA, NCX-4016, the NO-releasing ASA derivative, totally spares the stomach from topical irritating or ulcerogenic action in diabetic rats, although the gastric mucosa of diabetic rats is more vulnerable to ASA-induced damage. The lack of gastric toxicity of NCX-4016 may be accounted for by some beneÞcial effects (i.e., increase of GMBF) of NO released from this compound. Since the addition of a NO releasing moiety to ASA does not alter its effectiveness as an anti-thrombotic or anti-inßammatory agent or cyclooxygenase inhibitor [16Ð19], it is concluded that NCX-4016 can be used safely in diabetic patients as anti-platelet therapy for preventing microvascular complications.
References 1. Nathan DM. Long-term complications of diabetes mellitus. New Engl. J. Med. 1993; 328: 1676Ð85. 2. Morrish NJ, Strevens LK, Fuller JH, Jarrett RJ, Keen HA. A prospective study of mortality among middleaged diabetic patients (the London cohort of the WHO Multinational study of Vascular Disease in Diabetes) I: causes and death rates. Diabetologia 1990; 33: 538Ð41. 3. Ruggeri ZM. The role of von Willebrand factor and Þbrinogen in the initiation of platelet adhesion to thrombogenic surfaces. Thromb. Haemost. 1995; 74: 460Ð3. 4. OÕReilly D, Long RG. Diabetes and gastrointestinal tract. Dig. Dis. Sci. 1987; 5: 57Ð64.
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5. Takeuchi K, Ueshima K, Ohuchi T, Okabe S. Induction of gastric lesions and hypoglycemic response by food deprivation in streptozotocin-diabetic rats. Dig. Dis. Sci. 1994; 39: 626Ð34. 6. Takehara K, Tashima K, Takeuchi K. Alterations in duodenal bicarbonate secretion and mucosal susceptibility to acid in diabetic rats. Gastroenterology 1997; 112: 418Ð28. 7. Goldin E, Ardite E, Elizalde JI, Odriozola A, Panes J, Pique JM, Fernandez-Checa JC. Gastric mucosal damage in experimental diabetes in rats: Role of endogenous glutathione. Gastroenterology 1997; 112: 855Ð63. 8. Levy M. Aspirin use in patients with major upper gastrointestinal bleeding and peptic ulcer disease: A report from the Boston Collaboration Drug Surveillance Program. New Engl. J. Med. 1974; 290: 1159Ð62. 9. Lanza FJ. Endoscopic studies of gastric and duodenal injury after the use of ibuprofen, aspirin, and other nonsteroidal antiinßammatory agents. Am. J. Med. 1984; 13: 19Ð24. 10. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (Lond) 1971; 231: 232Ð5. 11. Okabe S, Takeuchi K, Nakamura K, Takagi K. Pathogenesis of gastric lesions induced by aspirin in pylorusligated rats. Jpn. J. Pharmacol. 1974; 24: 363Ð71. 12. Wallace JL, Reuter BK, Cicala C, Mcknight W, Grisham MB, Cirino G. Novel nonsteroidal anti-inßammatory drug derivatives with markedly reduced ulcerogenic properties in the rat. Gastroenterology 1994; 107: 173Ð9. 13. Wallace JL, Reuter BK, Cicala C, Mcknight W, Elliott SN. A diclofenac derivative without ulcerogenic properties. Eur. J. Pharmacol. 1994; 257: 249Ð55. 14. Wallace JL, Cirino G, Mcknight W, Elliott SN. Reduction of gastrointestinal injury in acute endotoxic shock by ßurbiprofen nitroxybutylester. Eur. J. Pharmacol. 1995; 280: 63Ð8. 15. Davies NM, Roseth AG, Appleyard CB, Mcknight W, Del. Soldato P, Calignano A, Cirino G, Wallace JL. NO-naproxen vs. naproxen: Ulcerogenic, analgesic and anti-inßammatory effects. Aliment. Pharmacol. Ther. 1997; 11: 69Ð79. 16. Wallace JL, Mcknight W, Del. Soldato P, Baydoun AR, Cirino G. Antithrombotic effects of a nitric oxidereleasing, gastric-sparing aspirin derivative. J. Clin. Invest. 1995; 96: 2711Ð18. 17. Lechi C, Andrioli G, Gaino S, Tommasoli R, Zuliani V, Ortolani R, Degan M, Benoni G, Bellavite P, Lechi A, Minuz P. The antiplatelet effects of a new nitroderivative of acetylsalicylic acid: An in vitro study of inhibition on the early phase of platelet activation and on TXA2 production. Thromb. Haemost. 1996; 76: 791Ð8. 18. Takeuchi K, Ukawa H, Konaka A, Kitamura M, Sugawa Y. Effect of nitric oxide-releasing aspirin on gastric functional and ulcerogenic responses in rats: Comparison with aspirin. J. Pharmacol. Exp. Ther. 1998; 286: 115Ð21. 19. Takeuchi K, Suzuki K, Yamamoto H, Araki H, Mizoguchi H, Ukawa H. Effects of COX-2 selective and NOreleasing NSAIDs on gastric ulcerogenic responses. J. Physiol. Pharmacol. 1998; 49: 501Ð13. 20. Tashima K, Korolkiewicz RP, Kubomi M, Takeuchi K. Increased susceptibility of gastric mucosa to ulcerogenic stimulation in diabetic rats: Role of capsaicin-sensitive sensory neurons. Br. J. Pharmacol. 1998; 124: 1395Ð402. 21. Matsumoto J, Takeuchi K, Ueshima K, Okabe S. Role of capsaicin-sensitive afferent neurons in mucosal blood ßow response of rat stomach induced by mild irritants. Dig. Dis. Sci. 1992; 37: 1336Ð44. 22. Kita Y, Hirasawa Y, Maeda K, Nishio M, Yoshida K. Spontaneous nitric oxide release account for the potent pharmacological actions of FK409. Eur. J. Pharmacol. 1994; 257: 123Ð30. 23. Kato S, Kitamura M, Korolkiewicz RP, Takeuchi K. Role of nitric oxide in regulation of gastric acid secretion in rats: Effects of NO donors and NO synthase inhibitor. Br. J. Pharmacol. 1998; 123: 839Ð46. 24. Urushidani T, Okabe S, Takeuchi K, Takagi K. Strain differences in aspirin-induced gastric ulceration in rats. Jpn. J. Pharmacol. 1978; 28: 569Ð78. 25. Davenport HW. Gastric mucosal hemorrhage in dogs: Effect of acid, aspirin and alcohol. Gastroenterology 1969; 56: 439Ð49. 26. Ligmusky M, Golanska EM, Hansen DG, Kauffman GL. Aspirin can inhibit gastric mucosal cyclooxygenase without causing lesions in rats. Gastroenterology 1983; 84: 756Ð61. 27. Hung CR. Role of acid back-diffusion in the formation of mucosal ulceration and its treatment with drugs in diabetic rats. Digestion (abstract) 1994; 55: 15. 28. Holzer P, Sametz W. Gastric mucosal protection against ulcerogenic factors in the rat mediated by capsaicinsensitive afferent neurons. Gastroenterology 1986; 91: 975Ð81.
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29. Takeuchi K, Ohuchi T, Narita M, Okabe S. Capsaicin-sensitive sensory nerves in recovery of gastric mucosal integrity after damage by sodium taurocholate in rats. Jpn. J. Pharmacol. 1993; 63: 479Ð86. 30. Lincoln J, Bokor JT, Crowe R, GrifÞth SG, Haven AJ, Burnstock G. Myenteric plexus in streptozotocintreated rats: Neurochemical and histochemical evidence for diabetic neuropathy in the gut. Gastroenterology 1984; 86: 654Ð61. 31. MacNaughton WK, Cirino G, Wallace JL. Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach. Life Sci. 1989; 45: 1869Ð76 . 32. Whittle BJR, Lopez-Belmote J, Moncada S. Regulation of gastric mucosal integrity by endogenous nitric oxide: Interaction with prostaglandins and sensory neuropeptides in the rat. Br. J. Pharmacol. 1990; 99: 607Ð11. 33. Moncada S, Palmer RMJ, Higgs EA. Nitric Oxide: Physiology, pathophysiology and pharmacology. Pharmacol. Rev. 1991; 43: 109Ð42. 34. Wallace JL, Keenan CM, Granger DN. Gastric ulceration induced by nonsteroidal anti-inßammatory drugs is a neutrophil-dependent process. Am. J. Physiol. 1990; 259: G462ÐG467. 35. Yoshida N, Takemura T, Granger DN, Anderson DC, Wolf RE, Mcintire LV, Kvietys PR. Molecular determinants of aspirin-induced neutrophil adherence to endothelial cells. Gastroenterology 1993; 105: 715Ð24. 36. Gaboury J, Woodman RC, Granger DN. Nitric oxide prevents leukocyte adherence: role of superoxide. Am. J. Physiol. 1993; 265: H862ÐH867. 37. Wallace JL, Mcknight W, Wilson TL, Del. Soldato P, Cirino G. Reduction of shock-induced gastric damage by a nitric oxide-releasing aspirin derivative; Role of neutrophils. Am. J. Physiol. 1997; 273: G1246ÐG1251.
Lack of gastric toxicity of nitric oxide-releasing aspirin, NCX-4016, in the stomach of diabetic rats Kimihito Tashima, Akinobu Fujita, Masakazu Umeda, Koji Takeuchi* Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607-8414, Japan
Abstract We compared the gastric toxic effect of aspirin (ASA) in both normal and diabetic rats, with that of NCX-4016, a derivative of ASA with nitric oxide (NO) releasing moiety. Animals were injected with streptozotocin and used after 5 weeks of diabetes with blood glucose levels of .350 mg/dl in the presence of omeprazole. Oral administration of ASA (with 150 mM HCl) did not produce damage at 30 mg/kg in the conscious rat but caused hemorrhagic gastric lesions in STZ-diabetic rats. By contrast, NCX-4016 even at 190 mg/kg (a dose equimolar to 100 mg/kg of ASA) did not cause damage in both normal and STZ-diabetic rat stomachs. Plasma salicylic acid levels were not different between normal and diabetic rats after administration of ASA or NCX-4016, though the latter gave signiÞcantly lower levels as compared to ASA. Intragastric application of ASA (80 mM in 50 mM HCl) for 30 min caused a reduction of transmucosal PD and increase of luminal H1 loss with a minimal effect on mucosal blood ßow (GMBF) in both normal and diabetic rats, yet resulting in much severe damage in the stomach of the latter group. Mucosal application of NCX-4016, however, did not cause PD reduction and luminal H1 loss, but produced a marked hyperemia, resulting in no damage in the stomach of both normal and STZ-diabetic rats. The increased gastric toxicity of ASA in STZ-diabetic rats was signiÞcantly mitigated by co-application of a NO donor FK-409 together with ASA, with an increase of GMBF, despite similar degrees of PD reduction and luminal H1 loss being observed. We conclude that NCX-4016 does not have a toxic effect in either normal or diabetic rat stomachs, although the diabetic rat stomach is more vulnerable to ASA-induced damage. NCX-4016, though absorbed more slowly than ASA, counteracts the injurious effect of aspirin on the gastric mucosa, probably by increasing GMBF mediated by NO. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Diabetic rat; Gastric mucosa; Aspirin; NO-releasing aspirin (NCX-4016)
Introduction Diabetes mellitus is a chronic disease characterized by hyperglycemia and by complications that include microvascular diseases and a variety of neuropathies [1]. Patients with pro* Corresponding author. Tel.: 81-75-595-4679; fax: 81-75-595-4774. E-mail address: [email protected] (K. Takeuchi) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 4 6 -3
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longed diabetes have an increased risk of microvascular complications, including myocardial infarction and stroke, which are mostly attributable to an increased platelet adhesiveness and aggregation, with enhanced production of thromboxane [2,3]. Anti-platelet therapy has been shown to reduce the risk of stroke in such patients. This gives a rational to the use of aspirin (ASA) in diabetic patients to prevent microvascular complications. Recent studies showed an increased mucosal susceptibility to various ulcerogenic stimuli in streptozotocin (STZ)-induced diabetic rats, an accepted model of insulin-dependent diabetes [4Ð 7]. Since the short-term or long-term administration of nonsteroidal antiinßammatory drugs (NSAIDs) such as ASA causes damage in the stomach as an unwanted side effect in human and in laboratory animals [8Ð11], it is assumed that the gastric toxicity of aspirin is enhanced in diabetic patients. Yet, no study has investigated the gastric toxic effect of ASA in diabetic conditions. A recent attempt to develop NSAIDs that spare the gastrointestinal tract of injury has produced nitric oxide (NO)-releasing NSAIDs [12]. It has been shown that the coupling of a NO-releasing moiety to standard NSAIDs markedly reduced their ulcerogenic properties without altering their effectiveness as anti-inßammatory drugs or cyclooxygenase inhibitors [12Ð15]. The NO-releasing ASA is a similar derivatization of ASA and has been shown to exhibit comparable anti-thrombotic or anti-inßammatory activity to that of ASA, with less damage in the gastric mucosa [16Ð18]. In the present study, we therefore compared the gastric toxic effect of a NO-releasing ASA derivative, NCX-4016, in normal and STZ-diabetic rats,with that of the parent compound ASA. In addition, we examined the effect of an exogenous NO donor, FK-409, on gastric ulcerogenic and functional responses to ASA in diabetic rats. Materials and methods Animals Male Sprague-Dawley rats (260Ð280 g: Charles River, Shizuoka, Japan) were used. The animals were fed standard rat chow and tap water ad libitum. One week after purchase, they were given streptozotocin (STZ: 70 mg/kg, i.p.) and fed normally thereafter. Control animals received an equal volume of saline. All experimental procedures described here were approved by the Experimental Animal Research Committee of the Kyoto Pharmaceutical University. General procedures The experiments were performed in normal and 5 week STZ-diabetic rats. Blood was sampled from the tail vein and blood glucose levels (BGL) were determined by GlucostarGlucostix (Miles-Sankyo Co. Ltd., Tokyo, Japan). The STZ-treated animals with BGL of less than 350 mg/dl under nonfasting conditions were excluded from the study. In these groups of rats, the effects of oral administration of ASA and NCX-4016 on the gastric mucosa under unanesthetized conditions, and those of their topical application on the ulcerogenic and functional responses in the chambered stomach under urethane anesthetized conditions,were examined. Since STZ-diabetic rats had some damage in the stomach after fasting [5], all animals were treated with i.p. omeprazole (60 mg/kg) immediately before fasting to prevent fasting-induced lesions. The doses of NCX-4016 and ASA used in all studies are the same on
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a molar basis and have been shown to cause an equipotent inhibition of prostaglandin (PG) biosynthesis in the rat gastric mucosa [18,19]. Gastric ulcerogenic effects in conscious rats The rats were given ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg; equivalent to 100 mg/kg of ASA), both suspended in 150 mM HCl, p.o., and killed 4 hr later. The stomachs were removed, inßated by injecting 10 ml of 2% formalin for 10 min to Þx the tissue walls, and opened along the greater curvature of the stomach. The area (mm2) of hemorrhagic lesions developed in the stomach was measured under a dissecting microscope with a square grid (310), summed per stomach, and used as a lesion score. This procedure was applied to the subsequent studies. Gastric ulcerogenic and functional effects in anesthetized rats Animals were anesthetized with urethane (1.25 g/kg, i.p.), and the trachea was cannulated to ensure a patent airway. Simultaneous measurement of transmucosal potential difference (PD), gastric mucosal blood ßow (GMBF), and luminal acid loss was performed in the chambered stomach as described previously [20]. Brießy, the abdomen was incised, and the stomach was exposed and mounted on an ex-vivo chamber (area exposed, 3.14 cm2). At the beginning of each experiment, the mucosa was rinsed several times with a solution of 50 mM HCl plus 100 mM NaCl. When the gastric exudate became clear, 2 ml of the acid solution was instilled in the chamber, and 15 min later the gastric contents were recovered from the chamber. This procedure was repeated every 15 min, two times before and 6 times after exposure of the mucosa to 80 mM ASA or NCX-4016 for 30 min. PD was determined using two agar bridges, one positioned in the chamber and the other in the abdominal cavity. GMBF was measured by a laser Doppler ßowmeter (Advance Model ALF 21, Tokyo, Japan), placing the probe gently on the corpus mucosa using a balance, and changes in GMBF were continuously monitored on a two-channel recorder (U-228, Tokai-Irika, Tokyo, Japan) simultaneously with those of PD [21]. On the other hand, luminal acid loss (loss of luminal titratable acidity) was determined from the analyses of the collected acid solution. Each sample was analyzed for volume and acid concentration, which was determined by titration of the aliquot against 100 mM NaOH to pH 7.0 (Autoburette, Comtite-7, Hiranuma, Tokyo, Japan). The amount of luminal acid loss was calculated as the difference between the product of the Þnal volume and concentration and the product of initial volume and concentration, and the results were expressed as mEq/15 min. Ninety minutes after exposure to ASA or NCX-4016, the mucosa was examined for hemorrhagic lesions under a dissecting microscope. To prevent bias, the observer measuring the lesion was unaware of the treatment. In some cases, the effect of FK-409 on gastric ulcerogenic and functional responses to ASA in STZ-diabetic rats was examined. FK-409, an orally active spontaneous NO releasing drug which can generate NO [22], has been shown to afford gastric cytoprotection, inhibition of acid secretion as well as increase of GMBF in rats when administered p.o. [18,23], FK409 (1 mg/ml) was given topically for 30 min, together with 80 mM ASA. Determination of plasma salicylic acid Plasma levels of salicylic acid were determined in both normal and STZ-diabetic rats following p.o. administration of ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg) with 150
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mM HCl, according to the modiÞed method of Urushidani et al [24]. Three hundred ml of blood was collected from the tail vein in the presence of heparin, at 30, 60, 120 and 240 min after administration of these drugs. Samples were centrifuged at 10,000 rpm for 5 min at 48C and stored at 2808C until the assay. For determination of salicylic acid, the samples (100 ml) were mixed with 20 ml of 6 N HCl, boiled at 1008C for 10 min, and shaken with 600 ml of ethylene dichloride for 5 min. After extracting, the organic phase was transferred to another tube. Then, 200 ml of 1.76% of ferric nitrate was added and the mixture was shaken for 5 min. The aqueous layer was collected, and the salicylic acid levels were measured by spectrophotoßuorometry at 545 nm. A standard curve was made using rat plasma containing various concentrations of salicylic acid. Preparation of drugs Drugs used were urethane (Tokyo Kasei, Tokyo, Japan), streptozotocin (Nacalai tesque, Kyoto, Japan), aspirin (ASA: Sigma Chemicals, St. Louis, Missouri, USA), NCX-4016 (NOreleasing ASA; NiCox, Paris, France), and FK-409 (Fujisawa, Osaka, Japan). ASA or NCX4016 was suspended in 50 mM or 150 mM HCl, while other agents were dissolved in saline. Each agent was prepared immediately before use and administered i.p. or p.o. in a volume of 0.5 ml per 100 g body weight, or applied topically to the chamber in a volume of 2 ml per rat. Statistics Data are presented as the mean6SE from 4z7 rats per group. Statistical analyses were performed using a two-tailed DunnettÕs multiple comparison test, and values of p,0.05 were regarded as signiÞcant. Results Effects of ASA and NCX-4016 on gastric mucosa of conscious rats Oral administration of ASA with 150 mM HCl at 30 mg/kg did not cause any damage in the gastric mucosa of normal rats but at 100 mg/kg produced hemorrhagic lesions, the lesion score being 16.163.0 mm2 (Fig. 1). The toxic action of acidiÞed ASA at the same doses was markedly potentiated in STZ-diabetic rat stomachs; the lesion score at 100 mg/kg was significantly increased to 36.469.0 mm2, which is about 2 times greater than that observed in normal rats. Even at 30 mg/kg, ASA provoked apparent gastric damage in STZ-diabetic rats. By contrast, NCX-4016 at 190 mg/kg did not produce any macroscopic damage in either normal or STZ-diabetic rat stomachs. Likewise, control animals received vehicle alone did not show any damage in the stomach under normal and diabetic conditions (not shown). Gastric functional responses and mucosal injury induced by mucosal application of ASA and NCX-4016 in anesthetized rats PD response Under chambered conditions in the presence of omeprazole (inhibition of acid secretion) and exogenous acid (50 mM HCl plus 100 mM NaCl), the normal rat stomachs generated a
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Fig. 1. Mucosal ulcerogenic effects of ASA and NCX-4016 in normal and STZ-diabetic rats. ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg), suspended in 150 mM HCl, was administered orally, and the animals were killed 4 hr later. Control animals received vehicle alone did not show any damage in the stomach, either normal or diabetic conditions (not shown). Data are presented as the mean6SE from 5z7 rats per group. SigniÞcant difference at P,0.05; * from normal rats given ASA; # from the animals given ASA (100 mg/kg) in the corresponding group.
PD of 245z50 mV (mucosa negative) and maintained relatively constant GMBF (180~200 mV: arbitrary unit) during a 2-hr test period (Fig. 2). Exposure of the normal rat mucosa to ASA (80 mM) for 30 min caused a marked reduction of PD from 247.663.1 mV to 28.062.4 mV. After the exposure the reduced PD normalized gradually, yet was still signiÞcantly low 90 min later; the degree of PD recovery being 31.565.6%. Although STZ-diabetic rats exhibited a lower PD (235z40 mV) in basal conditions than normal rats, the values in GMBF were almost equivalent to those in normal rats. The mucosal application of ASA caused a similar degree of PD reduction in STZ-diabetic rats; DPD was 29.062.3 mV. However, the recovery of PD after exposure to ASA was slightly delayed in the diabetic rats; the degree of the recovery at 90 min post-treatment being 20.262.4%. On the other hand, NCX4016 (80 mM) applied topically to the stomach did not produce a decrease of PD in either normal or STZ-diabetic rats, and the PD remained in similar ranges before and after the exposure in both groups of animals. GMBF response The GMBF was slightly increased immediately after exposure of the mucosa to 80 mM ASA, but returned to or dropped below baseline values during the exposure, the increase observed at the end of exposure being 20.268.3% (Fig. 3). Essentially similar changes in GMBF were observed in STZ-diabetic rat stomachs after exposure to ASA, but the increase in GMBF at the end of exposure was 4.465.8%, slightly less than that observed in normal rats. On the other hand, the GMBF was signiÞcantly elevated when NCX-4016 (80 mM) was
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Fig. 2. Changes in PD after exposure of the stomach to ASA or NCX-4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK-409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. Data are presented as the mean6SE of values determined every 15 min from 4z7 rats per group. SigniÞcant difference at P,0.05; * from basal values (time 0) in the corresponding group; # from values in STZ-diabetic rats exposed to ASA alone.
applied to the stomach in both normal and STZ-diabetic rats, reaching a peak increase of 59.365.3% and 62.467.9%, respectively, at the end of the exposure period, and in both cases remained signiÞcantly elevated for 30 min even after removal of NCX-4016 from the chamber. In these rats, the GMBF showed a moderate increase (25.269.3% and 6.364.8%) even 60 min after exposure to NCX-4016. Luminal acid loss When the gastric mucosa was exposed to 50 mM HCl in the absence of acid secretion induced by omeprazole, a small but signiÞcant loss of luminal titratable acidity was consistently observed in normal rats under basal conditions; DH1 was less than 25 mEq/15 min (Fig. 4). Following the mucosal application of 80 mM ASA, the loss of titratable acidity was signiÞcantly increased, reaching a maximal value immediately after the exposure, then gradually decreasing to the pre-exposure levels 90 min later. The maximal DH1 value was 49.763.7 mEq/15 min in normal rats. The loss of luminal titratable acidity in STZ-treated rats was signiÞcantly great in basal conditions as compared to normal rats, and the values (DH1: 35.163.2 mEq/15 min) were approximately 1.5 times the levels observed in normal animals. The luminal acid loss in STZ-diabetic rats was further increased in response to ASA, similar to normal rats; the magnitude of H1 loss observed immediately after ASA being 54.764.3 mEq/15 min, and not signiÞcantly different from that observed in normal
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Fig. 3. GMBF responses induced by ASA or NCX-4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK-409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. Data show the GMBF response (%increase from basal values) at the end of the exposure period in each group, and values are the mean6SE from 4z7 rats. SigniÞcant difference at P,0.05; * from normal rats treated ASA; # from STZ-diabetic rats treated with ASA alone.
rats. By contrast, the mucosal exposure to NCX-4016 did not alter the luminal acid loss in either normal or STZ-diabetic rats, and the values in DH1 loss remained unchanged before and after exposure to NCX-4016. The luminal acid loss observed immediately after exposure to NCX-4016 was 33.863.1 mEq/15 min and 39.868.3 mEq/15 min, respectively, in normal and diabetic rat stomachs, neither of which was not signiÞcantly different from the corresponding pre-exposure values. Mucosal injury Mucosal application of 80 mM ASA or acid solution (50 mM HCl) by itself did not induce gross damage in the gastric mucosa (not shown), but these treatments (ASA plus HCl) applied together produced hemorrhagic damage in the mucosa of normal rats, the lesion score being 23.263.1 mm2 (Fig. 5). These lesions were signiÞcantly worsened in STZ-diabetic rats, the lesion score being 64.4612.5 mm2. Histologically, the response in normal rat stomachs to ASA was a widespread disruption of epithelial cells without deep damage, but in diabetic rats the damage reached deep into the mucosa with severe hemorrhaging (not shown). The mucosal application of NCX-4016, however, did not induce gross damage in the gastric mucosa of either normal or STZ-diabetic rats. Effects of FK-409 on gastric functional and ulcerogenic responses induced by ASA in STZ-diabetic rats To further investigate the beneÞcial inßuence of NO on gastric functional and ulcerogenic responses to ASA, we examined the effects of the NO donor FK-409 on these responses in
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Fig. 4. Amount of luminal acid loss (loss of titratable acidity) after exposure of the stomach to ASA or NCX4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. Data are the mean6SE of values determined every 15 min from 4z7 rats. SigniÞcant difference at P,0.05; * from normal rats without ASA treatment; # from normal rats without NCX-4016 treatment, u from basal values (time 0) in the corresponding group.
STZ-diabetic rat stomachs. As shown in Figs. 2Ð4, FK-409 (1 mg/ml), given topically together with 80 mM ASA, did not prevent PD reduction or luminal acid loss caused by ASA, but signiÞcantly increased GMBF during and after the exposure. In these animals, the recovery of PD seen after exposure to ASA was expedited by co-application of FK-409, the degree of PD recovery at 60 min and 75 min post-treatment being 33.167.1% and 33.366.6%, respectively, both of which were signiÞcantly greater than the values for STZ-diabetic rats treated with ASA alone (Fig. 2). The increased gastric ulcerogenecity to ASA in STZ-diabetic rats was apparently mitigated by the combined application of FK-409, and the lesion score was decreased to 29.064.1 mm2, which was signiÞcantly lower than that (64.4612.5 mm2) observed in the control group with ASA alone (Fig. 5). Plasma levels of salicylic acid after administration of ASA and NCX-4016 Following p.o. administration of ASA with 150 mM HCl in normal rats, the plasma levels of salicylic acid were increased dose- and time-dependently. At 100 mg/kg, a maximal value was observed within 30 min in both normal (307.2613.5 mg/ml) and diabetic animals
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Fig. 5. Development of gastric mucosal lesions after exposure of the stomach to ASA or NCX-4016 in normal and STZ-diabetic rats. The stomach was exposed to 2 ml of 80 mM ASA or NCX-4016 with 50 mM HCl for 30 min, and was applied with 2 ml of 50 mM HCl every 15 min before and after the exposure. FK-409 (1 mg/ml) was applied to the stomach together with 80 mM ASA in STZ-diabetic rats. The animals were killed 90 min after the treatment with ASA or NCX-4016. Data are the mean6SE from 4z7 rats. SigniÞcant difference at P,0.05; * from normal rats treated with ASA alone; # from STZ-diabetic rats treated with ASA alone.
(312.2611.2 mg/ml), remaining in these ranges for 4 hr thereafter (Fig. 6). Although NCX4016 (190 mg/kg) given p.o. with 150 mM HCl produced a gradual increase in the plasma salicylic acid levels in both normal and diabetic rats, reaching 4 hr later about half the values observed with ASA; the values were 162.6611.0 mg/ml and 160.4616.3 mg/ml, respectively. No difference was found in plasma salicylic acid levels after administration of NCX4016 between normal and diabetic animals. Discussion The present study showed that gastric toxicity of ASA was enhanced in STZ-induced diabetic rats, conÞrming our previous observation that the diabetes predisposes rats to gastric ulceration [5,20]. In addition, we also found that NCX-4016, an ASA derivative with a NOreleasing moiety, prevented such gastric toxicity in STZ-diabetic as well as normal rats. These results give rational support to the use of a NO-releasing ASA for anti-platelet therapy in diabetic patients. STZ is known to possess diabetogenic properties and cause selective destruction of pancreatic b-cells. As expected, all STZ-treated animals developed a persistent hyperglycemia, which was observed one week after STZ injection. Although ASA provoked severe damage in the gastric mucosa in both normal and STZ-diabetic rats, the severity was much greater in the latter. Because all experiments in the present study were done in the presence of exogenous HCl under inhibition of acid secretion by omeprazole pretreatment, it is unlikely that the exacerbation of damage in diabetic rats is due to acid secretory changes. Furthermore, no difference was observed in plasma levels of salicylic acid 2 hr after administration of ASA
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Fig. 6. Plasma salicylic acid levels after p.o. administration of ASA or NCX-4016 in normal and STZ-diabetic rats. ASA (30 and 100 mg/kg) or NCX-4016 (190 mg/kg), suspended in 150 mM HCl, was administered orally, and the blood samples were collected from a tail vein at various time points after the administration. Data are presented as the mean6SE from 5z6 rats per group. * SigniÞcant difference from the animals given ASA (100 mg/ kg) in the corresponding group, at P,0.05.
(30 mg/kg) and NCX-4016 (190 mg/kg), yet apparent damage was observed only in the diabetic animals treated with the former. Thus, it is unlikely that the less toxic effect of the latter is simply due to a low bioavailability of the compound. Two major components, namely the topical irritant effect on the epithelium and the suppression of PG synthesis, have been proposed to explain the gastric toxicity of ASA [10,25]. However, since ASA produces gastric lesions when administered orally but not parenterally, the former effect is more crucial in causing gastric mucosal damage [26]. Indeed, the present study showed that topical application of ASA caused PD reduction and luminal acid loss in normal and STZ-diabetic rats, the phenomena being observed in the stomach after disruption of the mucosal barrier [26]. The luminal acid loss is due to both H1 back-diffusion and neutralization of H1 with bicarbonate or plasma diffused into the lumen. The occurrence of H1 back-diffusion is followed by an increase of GMBF, partly mediated by endogenous PG, to prevent damage in the mucosa by limiting local accumulation of H1 ion. However, ASA by inhibiting PG biosynthesis hampered gastric hyperemic response associated with H1 back-diffusion and thereby provoked hemorrhagic damage in the stomach. We previously reported that the GMBF response induced by H1 back-diffusion was mitigated in diabetic animals, partly due to dysfunction of capsaicin-sensitive afferent neurons [20]. The increase of GMBF after ASA treatment was only 30% in normal rat stomachs and was even less in STZ-diabetic animals, yet the difference was not great enough to explain the exacerbation of ASA-induced damage in diabetic rat stomachs. At present, although the mechanisms by which ASA-induced gastric damage was worsened in STZ-diabetic rats remains unexplored, they may include a decrease of mucus secre-
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tion [4], an impairment of the anti-oxidative system [7] or some other complication observed in diabetic conditions [5,20]. Because there was no difference in plasma salicylic acid levels between normal and diabetic animals after administration of ASA, it is unlikely that the increased ulcerogenic response is due to an enhancement of its absorption from the stomach. We noted in this study that STZ-diabetic rats showed characteristic mucosal changes under basal conditions without any treatment; a decrease in basal PD values and an increase in luminal acid loss, in agreement with the Þndings by Hung et al. [27]. The enhanced luminal acid loss may be largely due to an increase of spontaneous H1 back-diffusion. These results account for the lowered PD values and are consistent with the increased mucosal permeability in STZ-diabetic rats as determined from the luminal appearance of Evans blue [5]. The reason for the enhanced mucosal permeability in diabetic rat stomachs remains unknown, yet this may also be a factor contributing to the increased susceptibility of the mucosa to ASA. It has been shown that chemical ablation of capsaicin-sensitive sensory neurons exacerbated gastric mucosal lesions induced by a variety of noxious stimuli including aspirin [28,29]. These results may suggest a similarity of deleterious inßuences on the mucosal integrity between diabetes and sensory deafferentation. Lincoln et al. [30] showed that the density of calcitonin gene-related peptide (CGRP)-immunoreactive Þbres in the intestine was markedly decreased in diabetic rats. We also reported that the amount of CGRP released in the isolated stomach in response to capsaicin was signiÞcantly lower in STZ-diabetic animals than in normal rats [21]. Thus, it is likely that the increased gastric toxicity of ASA in STZ-diabetic rats is at least partly accounted for by dysfunction of capsaicin-sensitive sensory neurons. On the other hand, topical application of NCX-4016, a NO-releasing ASA derivative, did not cause gross damage in rat stomachs even in the presence of acid, irrespective of whether or not the rats were subjected to diabetes by STZ injection. These results conÞrmed our previous observation that intragastric administration of NCX-4016 did not produce any damage in the stomach, despite decreasing the mucosal PGE2 contents [18,19]. In addition, NCX4016 had no effect on PD but produced a sustained increase of GMBF, suggesting a lack of direct irritating action on the mucosa and a release of NO from the compound. These results are consistent with the Þndings by others [12Ð17], who showed that the addition of a nitroxybutylester group to NSAIDs markedly reduced its short-term ulcerogenic property without altering its effectiveness as a cyclooxygenase inhibitor. In the present study, NCX-4016 gave lower values of plasma salicylic acid levels than ASA, following p.o. administration. However, this does not mean that NCX-4016 is more slowly absorbed, because this comound inhibited gastric mucosal PGE2 synthesis and showed antiinßammatory action against carrageenan-induced paw edema,both as effectively as ASA [18,19]. The release of salicylate from NCX-4016 may well require a more complicated metabolism than the simple loss of an acetyl group in the case of aspirin. In any case, it is conceivable that the therapeutic concentrations of ASA could be attained following p.o. administration of NCX-4016. We have previously shown that NOx levels in both serum and gastric contents were increased following NCX-4016, administered either p.o. or s.c. [18]. The mechanism responsible for the reduced ulcerogenic property of the NO-releasing derivative of ASA has not been elucidated, yet it is conceivable that the NO released from this compound exerts beneÞcial effects on the gastric mucosa by enhancing the mucosal defensive ability. This contention is supported by previous studies using several NO-releasing derivatives of NSAIDs such as nitrofenac, NO-naproxen
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and NO-ßurbiprofen [12Ð15]. It should also remind us that NO-releasing ASA (NCX-4215) is more effective in inhibiting platelet aggregation than ASA, despite having less of an effect on thromboxane B2 synthetic activity [16], suggesting that NO released from the compound by itself has an inhibitory effect on platelet aggregation. Just how NO protects the gastric mucosa against damage is not entirely clear. It is now well accepted that NO plays an important role in maintaining the mucosal integrity of the stomach and modulating various gastric functions such as mucosal blood ßow and mucus secretion [31Ð33]. Since NCX-4016 applied topically to the gastric mucosa caused a persistent increase of GMBF, this effect may contribute to the protective action of this compound [18]. This contention was supported by the present Þnding that the increased gastric toxicity of ASA in STZ-diabetic rats was signiÞcantly attenuated by the combined administration of FK-409, a NO donor [22]. This treatment did not affect the PD reduction or luminal H1 loss observed in the stomach following ASA treatment, but produced a marked increase of GMBF. In addition, recent studies showed an important role for neutrophils in the pathogenesis of ASA-induced gastric lesions [34,35]. Since NO inhibits neutrophil activation and scavenges oxygen metabolites [36], it is possible that the molecule interferes with the neutrophilrelated process in tissue injury. Indeed, Wallace et al. [37] recently showed that this particular NO-releasing derivative of ASA, NCX-4016, inhibited leukocyte adherence to the vascular endothelium and prevented gastric damage induced by hemorrhagic shock. It should also be noted in the present study that FK-409, when applied together with ASA, signiÞcantly promoted the recovery of PD in the stomach after exposure to ASA. GMBF is considered to play a role in maintaining the gastric mucosal integrity by supplying oxygen and nutrients, in addition to buffering H1 ions, and contributes to mucosal repair as well as mucosal protection. It is possible to assume that the process of PD recovery after ASA was signiÞcantly promoted by a NO donor, resulting in less severe gastric damage. Thus, the present study conÞrmed our previous results in normal rats [18] and further showed that unlike ASA, NCX-4016, the NO-releasing ASA derivative, totally spares the stomach from topical irritating or ulcerogenic action in diabetic rats, although the gastric mucosa of diabetic rats is more vulnerable to ASA-induced damage. The lack of gastric toxicity of NCX-4016 may be accounted for by some beneÞcial effects (i.e., increase of GMBF) of NO released from this compound. Since the addition of a NO releasing moiety to ASA does not alter its effectiveness as an anti-thrombotic or anti-inßammatory agent or cyclooxygenase inhibitor [16Ð19], it is concluded that NCX-4016 can be used safely in diabetic patients as anti-platelet therapy for preventing microvascular complications.
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