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Characterization of naproxen induced gastric antral ulceration and adaptation in hamsters
GASTROENTEROLOGY Vol. 114, No. 4
A122 AGA ABSTRACTS • G0500
PREVENTION OF GASTRIC DAMAGE BY NO-RELEASING ASPIRIN (NCX-4016) IS MEDIATED BY INHIBITION OF ICE/-LIKE PROTEASES IN GASTRIC MUCOSA AND ENDOTHELIAL CELLS. Fiorucci S., E. Antonelli, O. Morelli, B. Federici and A. Morelli. G.I. Unit, University of Perugia, Italy. Background. Tumor necrosis factor-ct (TNFtx), a proinflammatory cytokine, is released in vivo and in vitro after exposure to non-steroidal anti-inflammatorydrugs (NSAIDs). TNFct receptor binding leads to activation of multiple interleukin-1 converting enzyme (ICE)-like proteases (caspases) and endothelial cell death. Endothelial cell dysfunction is an early step in the process of NSAID-gastropathy. Nilric oxide(NO)-releasing NSAIDs, are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. NO-releasing agents (sodium nitroprusside [SNP] and L-Arginine) prevent TNF-induced ICE/caspase 1 activation in human endothelial ceils (HUVECs). Aims. To assess whether NO-NSAIDs modulate ICE-like proteases in vivo and in vitro and protect endothelial cells from TNFct-induced apoptosis. Methods. Macrophages, prepared from mouse spleen, were used to investigate TNFet release in vitro. Gastric damage was induced in rat by oral administration of 150 mg/kg aspirin or an equimolar dose (249 mg/kg) of NCX-4016 (NicOx, Milan, Italy). ICE-like activity was measured in cell Iysates and gastric homogenates using YVAD.AMC as substrate in a fluorimetric assay. HUVEC apoptosis was assessed by FACS-scan analysis. Results. In vitro studies demonstrated that aspirin, but not NCX-4016, released TNFct from mouse macrophages. Co-incubation with 1-100 laM SNP resulted in a concentrationdependent inhibition of aspirin-induced TNFct-release. In viw) aspirin administration caused a time-dependent increase in gastric mucosal damage and gastric mucosa ICE/caspase activity. Pretreating rats with 0.1 mM L-Arginine, SNP and Z.VAD.fmk, a pan-ICE/caspase inhibitor, prevented both gastric mucosal damage and ICE/caspaseupregulation induced by aspirin. No gastric damage was detectable with any dose of NCX-4016 tested. Like aspirin NCX-4016 was largely metabolized to salicylate. In contrast to aspirin NCX-416 significantly increased plasma nitrite/nitrate concentrations and inhibited the aspirin-induced upregulation of ICE/caspase activity into the gastric mucosa. Although aspirin had no effect on HUVEC apoptosis induced by TNFcq NCX-4016 and SNP caused a concentrationdependent inhibition of apoptosis and prevented ICEqike pmteases activation as demonstrated by Westem blot analysis and fluorimetric assay. NCX-4016 inhibited IL-113 release from endotoxin-stimulated macrophages. Conclusions. 1) Activation of ICE/caspase pmteases is a key step in the process of NSAID-gastropathy; 2) NCX-4016 spares the gastric mucosa and protects endothelial cells from TNFetinduced apoptosis. The potential of NO-releasing NSAIDs to modulate ICE-like pmteases and IL-113release might help to explain the anti-inflammatory activity of these compounds. • G0501
HEAT SHOCK PROTEIN 70 (HSP 70) IN GASTRIC MUCOSAL DAMAGE AND ADAPTATION: EFFECTS OF HELICOBACTER PYLORI INFECTION. H. Fischer, P,C. Konturek, V. Huber, J. Neumann, E.G. Hahn, W. Schmitz, W. Domschke and J.W. Konturek. Departments of Medicine and Pharmacology, University of Miinster, MUnster, Germany and Department of Medicine, University of Erlangen, Erlangen, Germany. Gastric adaptation to repeated exposures to aspirin (ASA) is well documented, but the involvement of Helicobacter pylori (H. pylori) in ASA-induced gastropathy and adaptation to ASA and the biochemical events underlying this adaptation remain unclear. The heat shock protein 70 (HSP 70) is part of a very sensitive and phylogenetically highly preserved defense mechanism. To evaluate the influence of H. pylori on gastric adaptation to ASA and the role of HSP 70, eight volunteers with H. pylori infection were given ASA 2g/day for 14 days. Three months after successful eradication of H. pylori with triple therapy with lansoprazole+amoxicillin+clarithromycin and H. pylori status being confirmed by 13C urea breath test and histology, all subjects received the same ASA treatment again. Mucosal damage, evaluated by endoscopy using the Lanza score system, and the gastric microbleeding rate were determined on days 0, 3, 7 and 14. Protein expression of HSP 70 was determined by quantitative immunoblotting of mucosal biopsies and mucosal expression of mRNA for HSP 70 was analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). In all subjects ASA induced gastric damage reached a maximum on day 3. In H. pylori-positive subjects, the extent of this damage was maintained at similar level up to the 14th day, while in H. pylori-eradicated subjects the damage was significantly lessened in terms of endoscopy and microbleeding rate on day 14. The lISP 70 protein expression as well as mRNA expression in gastric mucosa significantly increased following eradication of H. pylori. During ASA administration, in H. pylori-infected subjects a decrease of lISP 70 content from 5.4-+ 0.8 on day 0 to 3.5-+ 0.5x104 Phosphor-Imager units (arbitrary density) on day 14 was observed while after eradication HSP 70 increased from 8.7 -+ 1.2 on day 0 to 12.8 -+ 1.5x104 Phosphor-Imager units on day 14. We conclude, that gastric adaptation to ASA is impaired in H. pylori-positive subjects, but eradication of this bacterium restores this adaptive process. This is accompanied by different expression of HSP 70 during ASA-induced damage before and after eradication of H. pylori. Decreased expression of HSP 70 may reflect decreased mucosal resistance to the injury induced by
H. pylori and might be partly responsible for the impaired adaptation to ASA in H. pylori-positive subjects. G0502
CHARACTERIZATION OF NAPROXEN INDUCED GASTRIC ANTRAL ULC'~ERATIONAND ADAPTATION IN HAMSTERS. L.R. Fitzpatrick, T. Le and K. Sakurai, Maryland Research Laboratories, Otsuka America Pharmaceutical Inc., Rockville, MD. Chronic naproxen administration can cause ulceration of the gastric antral mncosa. Such ulceration is clearly reduced by misoprostol, but not bY H2 receptor antagonists. Moreover, gastric mucosal adaptation to naproxen has recently been demonstrated in humans (Lipscomb et al., Dig. Dis. Sci, 1996). Therefore, our goal was to establish a clinically relevant animal model of naproxen induced gastric antral ulceration and adaptation. Methods: Male Golden Syrian hamsters (100 - 130 grams) were utilized. Initial studies indicated that an 80 mg/kg oro-gastric dose of naproxen, given bid, was optimal for inducing ulceration. In an oro-gastric drug treatment study, naproxen treated hamsters (n=10) were dosed concomitantly for 3 days, With vehicle (0.5% methyl-cellulose), misoprostol (1 o r 100 lag&g, tid), or famotidine (30 mg/kg, tid). Macroscopic damage to the gastric antrum was assessed on a blind basis, as the number of penetrating gastric ulcers, and as a total ulcer area (total length x width in mm2). In the gastric adaptation study, hamsters (n=5-17) received either methyl-cellulose or naproxen for a 21 day period. On days 1, 3, 7, 14 and 21, macrosopic gastric damage was assessed. On day 21, hamsters were re-challenged with naproxen (80 mg/kg), indomethacin (30 mg/kg), or 70% ethanol. Macroscopic and histologic damage were then determined on a blind basis, either 1 or 4 hours later. Histologic damage to the gastric mucosa was assessed, using an eyepiece micrometer, as the length of damaged mucosa + total mucosal length (ethanol induced damage), or as the area of gastric erosion + total examined area of antral mucosa (naproxen and indomethacin induced damage). Results: Naproxen typically induced 1 or 2 gastric antral ulcers, which were confirmed by histology. On day 3, misoprostol (100 lag&g) treatment caused significant reductions (80-97%) in the ulcer number, area, and incidence. At a 1 lag/kg dose, misoprostol was also protective (30-58% decrease in ulcer indices). Famotidine treatment did not reduce the ulcer number or incidence, but it did significantly attenuate (45%) the ulcer area. Naproxen induced ulcer areas (ram2) were: 9.5 -+ 2.3 (day 1), 16.0 _ 2.2 (day 3), 8.6 -+ 2.0 (day 7), 4.1 -+ 1.2 (day 14), and 0.4 +-0.3 (day 21). On day 21, acute naproxen, ethanol, and indomethacin induced gastric damage was significantly less (67-87%) in chronic naprnxen treated hamsters, as opposed to vehicle treated animals. Acute histologic damage also tended to be significantly less (p < 0.05 vs. vehicle) in animals pre-treated with naproxen. The mean percent decreases in the naproxen pre-treated hamsters were: 44% (vs. ethanol), 61% (vs. indomethacin), and 86% (vs. naproxen). Conclusions: Naproxen induced antral ulceration was substantially prevented by concomitant misoprostol treatment, but not by dosing with famotidine. Gastric mucosal adaptation to naproxen was associated with a decreased ulcer area, and protection from acute gastric damage by various noxious agents (i.e., cross-adaptation). Administration of naproxen to hamsters results in reproducible gastric antral ulceration and adaptation, with apparent clinical relevance. This research was funded by Otsuka America Pharmaceutical Inc., Rockville, MD. G0503 COMMUNITY DYSPEPSIA CLINICS REDUCE MAINTENANCE THERAPY FOR PEPTIC ULCERATION IN HELICOBACTER POSITIVE AND NEGATIVE PATIENTS. EH Forrest, J Granger, S Ticcioni, L Murray, E Graham, JF MacKenzie, R Stuart, AJ Morris. Departments of Gastroenterology and Surgery, Glasgow Royal Infirmary, Scotland, UK.
Helicobacter Pylori (Hp) eradication is recommended for patients with peptic ulcer disease (PUD). Most studies of Hp eradication are hospital-based. We studied the effectiveness of Community Dyspepsia Clinics for managing PUD. Methods: 230 patients receiving prescribed maintenance therapy for diagnosed PUD were seen at Community Dyspepsia Clinics. Those patients Helisal Rapid Blood Test (HRBT) positive patients received Omeprazole 40mg od, Amoxycillin 500mg tds and Metronidazole 400mg tds for one week. At 6 weeks a urea breath test (UBT) was performed, and after 6 months Health Centre prescription records were reviewed. Results: 184 patients received Hp eradication, of whom 147 (80%) reattended at 6 weeks. The per protocol eradication was 91% (73% ITT eradication). 6 patients did not complete treatment, and 56 (38%) had side effects. At 6 weeks 100 patients (68%) noted subjective improvement, and only 33 (22%) had restarted acid suppression therapy. Six months after eradication therapy, 60 patients (44%) had restarted acid suppression. The number of maintenance prescriptions fell amongst patients initially HRBT negative who had not received eradication therapy, although less so compared with patients who received treatment (26% v 56%; p < 0.01). After 6 months relatively more proton pump inhibitors were prescribed than H2 antagonists (24% v 42%; p < 0.01).
A122 AGA ABSTRACTS • G0500
PREVENTION OF GASTRIC DAMAGE BY NO-RELEASING ASPIRIN (NCX-4016) IS MEDIATED BY INHIBITION OF ICE/-LIKE PROTEASES IN GASTRIC MUCOSA AND ENDOTHELIAL CELLS. Fiorucci S., E. Antonelli, O. Morelli, B. Federici and A. Morelli. G.I. Unit, University of Perugia, Italy. Background. Tumor necrosis factor-ct (TNFtx), a proinflammatory cytokine, is released in vivo and in vitro after exposure to non-steroidal anti-inflammatorydrugs (NSAIDs). TNFct receptor binding leads to activation of multiple interleukin-1 converting enzyme (ICE)-like proteases (caspases) and endothelial cell death. Endothelial cell dysfunction is an early step in the process of NSAID-gastropathy. Nilric oxide(NO)-releasing NSAIDs, are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. NO-releasing agents (sodium nitroprusside [SNP] and L-Arginine) prevent TNF-induced ICE/caspase 1 activation in human endothelial ceils (HUVECs). Aims. To assess whether NO-NSAIDs modulate ICE-like proteases in vivo and in vitro and protect endothelial cells from TNFct-induced apoptosis. Methods. Macrophages, prepared from mouse spleen, were used to investigate TNFet release in vitro. Gastric damage was induced in rat by oral administration of 150 mg/kg aspirin or an equimolar dose (249 mg/kg) of NCX-4016 (NicOx, Milan, Italy). ICE-like activity was measured in cell Iysates and gastric homogenates using YVAD.AMC as substrate in a fluorimetric assay. HUVEC apoptosis was assessed by FACS-scan analysis. Results. In vitro studies demonstrated that aspirin, but not NCX-4016, released TNFct from mouse macrophages. Co-incubation with 1-100 laM SNP resulted in a concentrationdependent inhibition of aspirin-induced TNFct-release. In viw) aspirin administration caused a time-dependent increase in gastric mucosal damage and gastric mucosa ICE/caspase activity. Pretreating rats with 0.1 mM L-Arginine, SNP and Z.VAD.fmk, a pan-ICE/caspase inhibitor, prevented both gastric mucosal damage and ICE/caspaseupregulation induced by aspirin. No gastric damage was detectable with any dose of NCX-4016 tested. Like aspirin NCX-4016 was largely metabolized to salicylate. In contrast to aspirin NCX-416 significantly increased plasma nitrite/nitrate concentrations and inhibited the aspirin-induced upregulation of ICE/caspase activity into the gastric mucosa. Although aspirin had no effect on HUVEC apoptosis induced by TNFcq NCX-4016 and SNP caused a concentrationdependent inhibition of apoptosis and prevented ICEqike pmteases activation as demonstrated by Westem blot analysis and fluorimetric assay. NCX-4016 inhibited IL-113 release from endotoxin-stimulated macrophages. Conclusions. 1) Activation of ICE/caspase pmteases is a key step in the process of NSAID-gastropathy; 2) NCX-4016 spares the gastric mucosa and protects endothelial cells from TNFetinduced apoptosis. The potential of NO-releasing NSAIDs to modulate ICE-like pmteases and IL-113release might help to explain the anti-inflammatory activity of these compounds. • G0501
HEAT SHOCK PROTEIN 70 (HSP 70) IN GASTRIC MUCOSAL DAMAGE AND ADAPTATION: EFFECTS OF HELICOBACTER PYLORI INFECTION. H. Fischer, P,C. Konturek, V. Huber, J. Neumann, E.G. Hahn, W. Schmitz, W. Domschke and J.W. Konturek. Departments of Medicine and Pharmacology, University of Miinster, MUnster, Germany and Department of Medicine, University of Erlangen, Erlangen, Germany. Gastric adaptation to repeated exposures to aspirin (ASA) is well documented, but the involvement of Helicobacter pylori (H. pylori) in ASA-induced gastropathy and adaptation to ASA and the biochemical events underlying this adaptation remain unclear. The heat shock protein 70 (HSP 70) is part of a very sensitive and phylogenetically highly preserved defense mechanism. To evaluate the influence of H. pylori on gastric adaptation to ASA and the role of HSP 70, eight volunteers with H. pylori infection were given ASA 2g/day for 14 days. Three months after successful eradication of H. pylori with triple therapy with lansoprazole+amoxicillin+clarithromycin and H. pylori status being confirmed by 13C urea breath test and histology, all subjects received the same ASA treatment again. Mucosal damage, evaluated by endoscopy using the Lanza score system, and the gastric microbleeding rate were determined on days 0, 3, 7 and 14. Protein expression of HSP 70 was determined by quantitative immunoblotting of mucosal biopsies and mucosal expression of mRNA for HSP 70 was analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). In all subjects ASA induced gastric damage reached a maximum on day 3. In H. pylori-positive subjects, the extent of this damage was maintained at similar level up to the 14th day, while in H. pylori-eradicated subjects the damage was significantly lessened in terms of endoscopy and microbleeding rate on day 14. The lISP 70 protein expression as well as mRNA expression in gastric mucosa significantly increased following eradication of H. pylori. During ASA administration, in H. pylori-infected subjects a decrease of lISP 70 content from 5.4-+ 0.8 on day 0 to 3.5-+ 0.5x104 Phosphor-Imager units (arbitrary density) on day 14 was observed while after eradication HSP 70 increased from 8.7 -+ 1.2 on day 0 to 12.8 -+ 1.5x104 Phosphor-Imager units on day 14. We conclude, that gastric adaptation to ASA is impaired in H. pylori-positive subjects, but eradication of this bacterium restores this adaptive process. This is accompanied by different expression of HSP 70 during ASA-induced damage before and after eradication of H. pylori. Decreased expression of HSP 70 may reflect decreased mucosal resistance to the injury induced by
H. pylori and might be partly responsible for the impaired adaptation to ASA in H. pylori-positive subjects. G0502
CHARACTERIZATION OF NAPROXEN INDUCED GASTRIC ANTRAL ULC'~ERATIONAND ADAPTATION IN HAMSTERS. L.R. Fitzpatrick, T. Le and K. Sakurai, Maryland Research Laboratories, Otsuka America Pharmaceutical Inc., Rockville, MD. Chronic naproxen administration can cause ulceration of the gastric antral mncosa. Such ulceration is clearly reduced by misoprostol, but not bY H2 receptor antagonists. Moreover, gastric mucosal adaptation to naproxen has recently been demonstrated in humans (Lipscomb et al., Dig. Dis. Sci, 1996). Therefore, our goal was to establish a clinically relevant animal model of naproxen induced gastric antral ulceration and adaptation. Methods: Male Golden Syrian hamsters (100 - 130 grams) were utilized. Initial studies indicated that an 80 mg/kg oro-gastric dose of naproxen, given bid, was optimal for inducing ulceration. In an oro-gastric drug treatment study, naproxen treated hamsters (n=10) were dosed concomitantly for 3 days, With vehicle (0.5% methyl-cellulose), misoprostol (1 o r 100 lag&g, tid), or famotidine (30 mg/kg, tid). Macroscopic damage to the gastric antrum was assessed on a blind basis, as the number of penetrating gastric ulcers, and as a total ulcer area (total length x width in mm2). In the gastric adaptation study, hamsters (n=5-17) received either methyl-cellulose or naproxen for a 21 day period. On days 1, 3, 7, 14 and 21, macrosopic gastric damage was assessed. On day 21, hamsters were re-challenged with naproxen (80 mg/kg), indomethacin (30 mg/kg), or 70% ethanol. Macroscopic and histologic damage were then determined on a blind basis, either 1 or 4 hours later. Histologic damage to the gastric mucosa was assessed, using an eyepiece micrometer, as the length of damaged mucosa + total mucosal length (ethanol induced damage), or as the area of gastric erosion + total examined area of antral mucosa (naproxen and indomethacin induced damage). Results: Naproxen typically induced 1 or 2 gastric antral ulcers, which were confirmed by histology. On day 3, misoprostol (100 lag&g) treatment caused significant reductions (80-97%) in the ulcer number, area, and incidence. At a 1 lag/kg dose, misoprostol was also protective (30-58% decrease in ulcer indices). Famotidine treatment did not reduce the ulcer number or incidence, but it did significantly attenuate (45%) the ulcer area. Naproxen induced ulcer areas (ram2) were: 9.5 -+ 2.3 (day 1), 16.0 _ 2.2 (day 3), 8.6 -+ 2.0 (day 7), 4.1 -+ 1.2 (day 14), and 0.4 +-0.3 (day 21). On day 21, acute naproxen, ethanol, and indomethacin induced gastric damage was significantly less (67-87%) in chronic naprnxen treated hamsters, as opposed to vehicle treated animals. Acute histologic damage also tended to be significantly less (p < 0.05 vs. vehicle) in animals pre-treated with naproxen. The mean percent decreases in the naproxen pre-treated hamsters were: 44% (vs. ethanol), 61% (vs. indomethacin), and 86% (vs. naproxen). Conclusions: Naproxen induced antral ulceration was substantially prevented by concomitant misoprostol treatment, but not by dosing with famotidine. Gastric mucosal adaptation to naproxen was associated with a decreased ulcer area, and protection from acute gastric damage by various noxious agents (i.e., cross-adaptation). Administration of naproxen to hamsters results in reproducible gastric antral ulceration and adaptation, with apparent clinical relevance. This research was funded by Otsuka America Pharmaceutical Inc., Rockville, MD. G0503 COMMUNITY DYSPEPSIA CLINICS REDUCE MAINTENANCE THERAPY FOR PEPTIC ULCERATION IN HELICOBACTER POSITIVE AND NEGATIVE PATIENTS. EH Forrest, J Granger, S Ticcioni, L Murray, E Graham, JF MacKenzie, R Stuart, AJ Morris. Departments of Gastroenterology and Surgery, Glasgow Royal Infirmary, Scotland, UK.
Helicobacter Pylori (Hp) eradication is recommended for patients with peptic ulcer disease (PUD). Most studies of Hp eradication are hospital-based. We studied the effectiveness of Community Dyspepsia Clinics for managing PUD. Methods: 230 patients receiving prescribed maintenance therapy for diagnosed PUD were seen at Community Dyspepsia Clinics. Those patients Helisal Rapid Blood Test (HRBT) positive patients received Omeprazole 40mg od, Amoxycillin 500mg tds and Metronidazole 400mg tds for one week. At 6 weeks a urea breath test (UBT) was performed, and after 6 months Health Centre prescription records were reviewed. Results: 184 patients received Hp eradication, of whom 147 (80%) reattended at 6 weeks. The per protocol eradication was 91% (73% ITT eradication). 6 patients did not complete treatment, and 56 (38%) had side effects. At 6 weeks 100 patients (68%) noted subjective improvement, and only 33 (22%) had restarted acid suppression therapy. Six months after eradication therapy, 60 patients (44%) had restarted acid suppression. The number of maintenance prescriptions fell amongst patients initially HRBT negative who had not received eradication therapy, although less so compared with patients who received treatment (26% v 56%; p < 0.01). After 6 months relatively more proton pump inhibitors were prescribed than H2 antagonists (24% v 42%; p < 0.01).