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Aspirin is an effective analgesic and antiplatelet drug that in addition to its ability to reduce pain, inflammation and fever, appears to have efficacy in the prevention/treatment of a range of diseases including heart disease, numerous cancers and Alzheimer's. It is important to understand the bioavailability of aspirin and its major metabolite, salicylic acid, since dosage and route of administration can vary for treating differing diseases, and the major side-effects of aspirin, upper gastrointestinal ulceration and bleeding, are dose-dependent. We examined the time course for gastroduodenal uptake of aspirin and the appearance of its major metabolite salicylic acid in blood and lymph after intragastric (to simulate oral) and intraduodenal (to simulate enteric-coating) dosing in rats. Results show that after intragastric dosing, intact aspirin is absorbed primarily by the gastric mucosa and to a lesser extent by the duodenal mucosa. When aspirin is dosed intragastrically or intraduodenally, a much greater concentration of aspirin enters the lymph than the blood. In contrast, the concentration of salicylic acid was higher in blood than in lymph. Lymph levels of both aspirin and salicylic acid were sufficiently high so as to perform a pharmacologic function there, possibly as a chemopreventive agent against colon cancer and potentially the metastatic spread of non-gastrointestinal cancers.

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OBJECTIVES: Breast feeding protects infants from many diseases, including necrotizing enterocolitis, peptic ulceration and infectious diarrhea. Conversely, maternal separation stress and Non-Steroidal Anti-Inflammatory Drugs (NSAID's) can induce intestinal injury and bleeding. This study aimed to evaluate in suckling rats if maternal separation/formula feeding leads to increased intestinal sensitivity to indomethacin (indo)-induced intestinal injury and to look at potential mechanisms involved. METHODS: Nine-day-old rats were dam-fed or separated/trained to formula-feed for 6 days prior to indo administration (5 mg/kg/day) or saline (control) for 3 days. Intestinal bleeding and injury were assessed by measuring luminal and Fecal Hemoglobin (Hob) and jejunal histology. Maturation of the intestine was assessed by measuring luminal bile acids, jejunal sucrase, serum corticosterone, and mRNA expression of ileal Apical Sodium-Dependent Bile Acid Transporter (ASBT). RESULTS: At 17 days, formula-fed indo-treated pups had a 2-fold increase in luminal Hb compared to formula-fed control pups and had evidence of morphological injury to the small intestinal mucosa as observed at the light microscopic level, whereas indo had no effect on dam-fed littermates. In addition, formula-fed rats had significant increases in luminal bile acid, sucrase specific activity, serum corticosterone, and expression of ASBT mRNA compared to dam-fed rats. CONCLUSION: Maternal separation stress may cause early intestinal maturational changes induced by corticosteroid release, including increased epithelial exposure to bile acids. These maturational changes may have a sensitizing rather than protective effect against indo-induced injury in the new-born.

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BACKGROUND AND PURPOSE: Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) that is limited in its enteral or parenteral use by side effects of gastroduodenal bleeding and ulceration. We have investigated the ability of phosphatidylcholine associated with indomethacin to form a therapeutically effective drug (INDO-PC) with reduced gastrointestinal (GI) toxicity for parenteral use. EXPERIMENTAL APPROACH: Rats were treated acutely by intravenous or chronically with subcutaneous injection of vehicle, indomethacin or INDO-PC using three related protocols. We then evaluated the following properties of these parenterally administered test drugs: (i) GI toxicity (luminal and faecal haemoglobin; intestinal perforations and adhesions; and haematocrit); (ii) bioavailability (plasma indomethacin); and (iii) therapeutic efficacy (analgesia from sensitivity to pressure; anti-inflammatory from ankle thickness; cyclo-oxygenase (COX) inhibition from synovial fluid prostaglandin E(2) concentration) in rats with adjuvant-induced joint inflammation. KEY RESULTS: Acute and chronic dosing with INDO-PC produced less GI bleeding and intestinal injury than indomethacin alone, whereas the bioavailability, analgesic, anti-inflammatory and COX inhibitory activity of INDO-PC were comparable to indomethacin. CONCLUSIONS AND IMPLICATIONS: The chemical association of phosphatidylcholine with indomethacin appears to markedly reduce the GI toxicity of the NSAID while providing equivalent therapeutic efficacy in a parenteral INDO-PC formulation.

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UNLABELLED: We have been developing a family of phosphatidylcholine (PC)-associated NSAIDs, which appear to have improved GI safety and therapeutic efficacy in both rodent model systems and pilot clinical trials. As naproxen has been demonstrated to be associated with the lowest cardiovascular adverse events in comparison with both COX-2 selective inhibitors and conventional NSAIDs, we have been developing a Naproxen-PC formulation for evaluation in animal models and clinical trials. We have determined that an oil-based formulation of naproxen and triple strength soy lecithin provides excellent GI protection in both: 1) an acute NSAID-induced intestinal bleeding model in rats pretreated with L-NAME that are intragastrically administered a single dose of naproxen (at a dose of 50 mg/kg) vs the equivalent dose of Naproxen-PC; and 2) a more chronic model (at a naproxen dose of 25 mg/kg BID) in rats that have pre-existing hindpaw inflammation (induced with a intradermal injection of Complete Freund's Adjuvant/CFA). Both models demonstrate the superior GI safety of Naproxen-PC vs naproxen while this novel formulation had significant anti-inflammatory efficacy to reduce hindpaw edema and the generation of PGE(2) in the collected joint synovial fluid. CONCLUSION: Naproxen-PC appears to induce significantly less GI injury and bleeding in two rodent model systems while maintaining anti-inflammatory and COX-inhibitory activity.

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BACKGROUND AND PURPOSE: Clinical studies demonstrate that aspirin consumption reverses the gastrointestinal (GI) benefits of coxibs, by an undefined mechanism. EXPERIMENTAL APPROACH: Rodent models were employed to investigate the effects of combinations of celecoxib and aspirin on gastric ulcerogenesis, bleeding, surface hydrophobicity (by contact angle analysis) and ulcer healing. We also evaluated the effects of phosphatidylcholine (PC)-associated aspirin in these rodent models and confirmed its cyclooxygenase (COX)-inhibitory activity by measuring mucosal prostaglandin E(2) (PGE(2)) concentration. We present evidence that aspirin's ability to induce gastric injury and bleeding in rats, was exacerbated in the presence of a coxib and was dependent on its ability to reduce gastric surface hydrophobicity. In contrast, co-administration of phosphatidylcholine (PC)-associated aspirin and celecoxib induced little or no gastric injury/bleeding and maintained the stomach's hydrophobic properties. Interestingly, aspirin and aspirin/PC equally inhibited gastric mucosal PGE(2) concentration. Aspirin in combination with a coxib retarded the healing of experimentally induced gastric ulcers, whereas healing rates of rats treated with celecoxib in combination with aspirin/PC were comparable to controls. CONCLUSIONS AND IMPLICATIONS: Aspirin's gastric toxicity in combination with a coxib can be dissociated from its ability to inhibit COX-1 and appears to be dependent, in part, on its ability to attenuate the stomach's surface hydrophobic barrier. This adverse drug interaction between aspirin and coxibs, which impacts the treatment of osteoarthritic and cardiac patients requiring cardiovascular prophylaxis, can be circumvented by the administration of phosphatidylcholine (PC)-associated aspirin, to maintain the stomach's hydrophobic properties.

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Altered gastrin expression associated with Helicobacter pylori infection may contribute to the pathogenesis of peptic ulcer disease or gastric cancer in man, but gastrin has not been investigated in a murine model of Helicobacter infection. C57BL/6 mice were inoculated with Helicobacter felis and examined after 4-21 weeks for G and D cell numbers, antral gastrin and somatostatin mRNA, and luminal pH. In H. felis-infected mice, gastrin mRNA declined at four and six weeks after infection to 57% and 23%, respectively, of uninfected control values. Concurrently, somatostatin mRNA showed no change at four weeks and a modest 25% decrease at six weeks after infection. Similar reductions were noted in G and D cell numbers, resulting in a decrease in the G/D cell ratio after mice were infected with H. felis. Infected animals also showed a loss of parietal and chief cells, and an increased gastric pH. H. felis infection in C57BL/6 mice leads to an early suppression of G cell number and gastrin mRNA. These changes precede an alteration in somatostatin cell number and mRNA and, coupled with reductions in parietal and chief cells, may contribute both to severe impairment of gastric acid output and the potential for carcinogenic processes.

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Recombinant human lactoferrin possesses in-vitro antibiotic and anti-inflammatory activity similar to the native form. It was tested for in-vivo activity in mice infected with the gastritis-inducing bacterium Helicobacter felis. A two-week course of treatment with lactoferrin was sufficient to partially reverse both infection-induced gastritis and the infection rate, and fully reverse gastric surface hydrophobicity changes. A comparison of lactoferrin with amoxicillin and standard triple therapy revealed no differences in infection rate. These results show that recombinant human lactoferrin is effective in a mouse model of Helicobacter infection, and support further testing of this promising agent for this application.

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Helicobacter pylori infection has been linked to the development of gastritis which can then progress to a number of disease entities including peptic ulcer disease and gastric cancer. Since the pathogenic mechanism by which the bacteria causes gastritis is unresolved, we employed a model system, the H. felis-infected mouse to investigate the temporal relationship between bacterially-induced alterations in the hydrophobic phospholipid barrier of the stomach and the development of gastritis. In the present study, C57BL/6 mice were inoculated with 10(9) CFU of H. felis and the changes in gastric wet weight, histology, surface hydrophobicity, phospholipid/phosphatidylcholine concentration, phospholipase A2 activity, and the pH of collected gastric juice were measured 0.5-2 months postinoculation. In related experiments, we investigated the effects of treating H. felis infected mice with antibiotic/ bismuth therapy on the above gastric properties. It was determined that both gastric surface hydrophobicity and phospholipid composition were significantly attenuated as early as 2-4 weeks postinfection, preceding signs of mucosal inflammation and glandular atrophy as indicated by increases in gastric wet weight, pH and a disappearance in parietal cells. These early H. felis-induced changes in gastric surface hydrophobicity and phospholipid concentration were reversed by antibiotic/bismuth therapy. Based on these results we conclude that H. felis infection induces an early transformation of the stomach from a hydrophobic to an acid-sensitive hydrophilic state that may trigger the subsequent development of gastritis.

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To investigate a potential new treatment for gastric Helicobacter pylori infection, we have examined the use of the natural antibiotic lactoferrin, found in bovine milk, for activity against Helicobacter species both in vitro and in vivo. Lactoferrin was bacteriostatic to H. pylori when cultured at concentrations > or =0.5 mg/ml. Growth of H. pylori was not inhibited by another milk constituent, lysozyme, or by a metabolite of lactoferrin, lactoferricin B, but growth was inhibited by the iron chelator deferoxamine mesylate. Lactoferrin inhibition of growth could be reversed by addition of excess iron to the medium. Lactoferrin in retail dairy milk was found to be more stable intragastrically than unbuffered, purified lactoferrin. Treatment of H. felis-infected mice with lactoferrin partially reversed mucosal disease manifestations. It is concluded that bovine lactoferrin has significant antimicrobial activity against Helicobacter species in vitro and in vivo. Bovine lactoferrin should be further investigated for possible use in H. pylori infections in man.

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BACKGROUND: Treatment and prevention of non-steroidal anti-inflammatory drug-induced gastropathy involve the concurrent use of antisecretory drugs. Recently, we have shown that the ability of these drugs to increase the intragastric pH to values > > pKa of NSAIDs compromises their therapeutic activity. In the present study, we evaluated the potential of omeprazole to interfere with the bioavailability of aspirin administered to rats either alone or complexed with the zwitterionic phospholipid, dipalmitoylphosphatidylcholine (DPPC). METHODS: Aspirin or aspirin/DPPC was administered intragastrically to rats pre-dosed with either saline or omeprazole. Concentrations of aspirin and salicylic acid in the blood and the gastric mucosa were assessed by HPLC and the 6-keto-PGF1 alpha gastric mucosal concentration by radioimmunoassay. RESULTS: Gastric absorption of aspirin and its relative bioavailability were reduced by an antisecretory dose of omeprazole; its inhibitory effect on gastric prostaglandin synthesis was consequently attenuated. However, these effects could be partly overcome if aspirin was administered as a complex with DPPC. CONCLUSIONS: These observations suggest that: (i) DPPC increases the lipid solubility and gastric permeability of NSAIDs; and (ii) neutralization of the gastric pH results in a shift of aspirin absorption toward the intestine where it could be degraded to salicylic acid.

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BACKGROUND & AIMS: The gastrointestinal side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are reduced by antisecretory agents. The effects of combination therapy on the gastrointestinal toxicity and therapeutic activity of free and phospholipid-associated NSAIDs were investigated in rats. METHODS: Fasted rats, pretreated with either saline or an antisecretory dose of omeprazole, ranitidine, or cimetidine, were intragastrically administered saline, aspirin, or indomethacin. In ulcer models, gastric lesions in aspirin-treated rats and intestinal bleeding in indomethacin-treated rats were measured. For antipyretic and analgesic activity, rectal body temperature in febrile rats and the rats' pain sensitivity to pressure applied to an inflamed limb were measured, respectively. RESULTS: NSAID-induced gastrointestinal ulceration and bleeding were reduced in rats pretreated with antisecretory agents and abolished in rats administered phospholipid-associated NSAIDs in combination with inhibitors of acid secretion. The antipyretic and analgesic activity of both NSAIDs was attenuated in rats pretreated with an antisecretory agent. This pH-dependent block in therapeutic activity was overcome if the NSAID was preassociated with a phospholipid to enhance the drug's lipophilic characteristics. CONCLUSIONS: Combination therapy of antisecretory agents and NSAIDs, chemically associated with phospholipids, has distinct advantages with regard to both low gastrointestinal toxicity and restored therapeutic activity.

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We have recently reported that the Gl toxicity of aspirin is markedly reduced when the drug is preassociated with the zwitterionic phospholipid dipalmitoylphosphatidycholine (DPPC) before intragastric administration. The present study was designed to determine whether the biological availability and therapeutic activity of aspirin were affected by chemically associating the drug with DPPC. To evaluate this, we compared the kinetics of entry of labeled aspirin into the blood, after intragastric administration of the drug in the free and lipid-associated states; we also tested the ability of the above formulations to inhibit fever, inflammation and pain in appropriate rodent model systems. We found that although the Gl absorption of free aspirin and that of the aspirin/DPPC complex were similar, in all three rodent models the complex had significantly greater antipyretic, anti-inflammatory and analgesic efficacy than aspirin alone. Dose-response analyses employing the fever model demonstrated that potency of aspirin to reduce fever was increased 5 to 10-fold when the aspirin was intragastrically administered in the lipid-associated state. We conclude that the therapeutic activity of aspirin to inhibit fever, inflammation and pain is remarkably enhanced when the drug is intragastrically administered in chemical association with the zwitterionic phospholipid DPPC. A number of molecular mechanisms have been proposed to explain the observed phospholipid-dependent increase in aspirin's therapeutic activity.

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BACKGROUND & AIMS: Persons infected with Helicobacter pylori show an enhanced meal-stimulated gastrin release compared with uninfected controls. The aim of this study was to determine in animal models whether this gastrin release could be related to chronic gastric inflammation, elevated luminal ammonia level, or a combination of these factors. METHODS: Two rat models of mild gastric inflammation were studied. Rats given a long-term diet of 20 g/dL ammonium acetate (AmAc) in rat chow or 0.1% iodoacetamide in drinking water for 2-3 weeks underwent a short-term challenge with a normal or AmAc-supplemented meal. Serum gastrin and antral gastrin messenger RNA levels were measured. RESULTS: Compared with normal postprandial gastrin release, animals given the long-term AmAc feeding showed a normal response to rat chow but a greatly exaggerated response to rat chow plus 20 g/dL AmAc. Long-term feeding with iodoacetamide also resulted in enhanced gastrin release and antral gastrin messenger RNA in response to a meal supplemented with AmAc, but not to a normal meal or one supplemented with sodium acetate. CONCLUSIONS: Inflamed gastric mucosa is more sensitive to the effects of luminal ammonia and responds with an increase in both synthesis and release of gastrin. These animal models may provide insight into the pathogenesis of hypergastrinemia associated the H. pylori infection.

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We investigated the ability of three dairy foods to prevent stress-induced gastric lesions and bleeding in rats. Skim milk, whole milk, and cream were all significantly gastroprotective with the greatest protection seen with the highest fat dairy food. Pretreatment of rats with cream for up to 2 hr prior to stress maintained a portion of the protective effect. Lipid extracts of cream, but not skim milk or whole milk, were gastroprotective. Surface hydrophobicity of the gastric mucosa was reduced by stress, but was maintained at prestress levels by treatment with milk, cream, or their lipid extracts, although this effect was not sufficient for protection in stressed rats. Alterations in gastric pH or titratable acid could not explain the protective effects of dairy foods or their lipid extracts. Milk was more gastroprotective in stressed rats than another food of equal caloric value. We conclude that both the lipid and nonlipid fractions of dairy foods possess gastroprotective activity against stress-induced ulcerogenesis in rats, a property that may be of therapeutic value for man.

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BACKGROUND/AIMS: Chronic infection with Helicobacter pylori causes persistent elevations in gastric juice ammonia levels. Thus, we studied the effects of experimentally induced increases in gastric juice ammonia levels on gastric structure and function and gastrin homeostasis. METHODS: Rats were fed either normal chow or the diet supplemented (20 g/dL) with ammonium or sodium acetate. RESULTS: Long-term dietary ammonium loading for 2 weeks or longer resulted in a 1.5-2-fold increase in the weight and mucosal thickness of the stomach and proximal duodenum with evidence of mild gastritis and enterochromaffinlike cell hyperplasia. The ammonium-containing diet also induced a significant 2-3-fold increase in both circulating gastrin levels of fed rats and an increase in the postprandial gastrin responses over control values. Antral gastrin levels were also markedly elevated by long-term ingestion of the test diet, which was increased 3-4-fold over control values in fasted animals and less so after meal stimulation. Consistent with these findings, gastrin-specific messenger RNA was increased 2.5-3-fold in the antrum of ammonium fed rats, whereas actin-specific messenger RNA was not affected or decreased. Animals fed a diet supplemented with 20 g/dL sodium acetate sustained modest increases in mucosal thickness and serum and antral gastrin concentration, suggesting that nonspecific gastric injury and inflammation is also a factor that influences G-cell function. CONCLUSIONS: Long-term exposure of the antral mucosa to elevated levels of ammonia in the gastric juice in the presence of gastritis, conditions similar to that occurring in subjects infected with H. pylori, seem to be causative factors in the development of G-cell hyperfunction.

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In this study we analyzed by gas chromatographic headspace analysis the composition and concentration of gastrin-stimulatory volatile aliphatic amines in the gastric juice of healthy subjects and acute renal failure patients. We demonstrated that although these aliphatic amines are present in the gastric juice of normal subjects in trace amounts, they accumulate in the gastric juice of uremic subjects. This 30-40-fold elevation in gastric juice amine concentration agreed favorably with the 40-50-fold augmentation in serum gastrin levels in acute renal failure, with a significant association (r = 0.87) existing between these two parameters. It was also determined that a 2-hr hemodialysis procedure resulted in a modest nonparallel decline in both gastric amine and serum gastrin levels. These results support the hypothesis that the accumulation of volatile aliphatic amines in the gastric juice of uremic individuals may induce an activation of the antral G cells, resulting in hypergastrinemia.

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The hydrophobic property of crude samples of canine and porcine mucus was demonstrated by its binding and reactivity with two fluorescent dyes, 1-anilinonaphthalene-8-sulfonic acid (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The contribution of lipids to these hydrophobic binding sites was indicated by our observations that the DPH-induced fluorescence of both porcine and canine gastric mucus was reduced greater than 75% after lipid extraction. Fluorescence microscopy revealed an extracellular band of intense reactivity in association with the mucus gel layer overlying the rat gastric mucosa that was abolished if the frozen sections were pretreated with lipid solvents. When rats pretreated with indomethacin were injected with a cytoprotective dose of 16,16-dimethyl-PGE2, there was an increase in fluorescence of the gastric perfusate treated with ANS, suggesting that hydrophobic factors, perhaps lipids, were being secreted in association with mucus. These extracellular lipids may play an important role in conferring protective barrier properties to the mucus gel layer.

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Secretory granules from rat antral tissue were isolated by differential centrifugation in sucrose and were confirmed as intact by electron microscopy. Gastrin release from the isolated granules was measured in response to stimulation with amino acids or their decarboxylated amine metabolites. Nine of 13 amino acids tested were ineffective at inducing gastrin release, whereas all 13 of the amine metabolites were potent stimulants of gastrin release. A pH gradient across the granule fraction membranes was estimated by acridine orange fluorescence and indicated an acidic interior. Changes in acridine orange fluorescence as an indicator of pH gradient dissipation showed that all of the amines, but only one of the amino acids, reversed acridine orange fluorescence. Ammonium chloride, similar to amines, both reversed acridine orange fluorescence and induced release of gastrin. It is concluded that amines 1) may directly stimulate gastrin granules to release their contents and 2) tend to alkalinize the gastrin granule interior. Some amino acids, in contrast, appear to directly stimulate gastrin release and do not affect the granule pH gradient.

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We investigated the biosynthesis of phospholipid, neutral lipids, glycoproteins, and DNA in primary cultures of rat oxyntic mucosal cells. In addition, responses of these biosynthetic pathways to the gastric protective agent 16,16-dimethyl prostaglandin E2 (dmPGE2) were studied. Cultured gastric cells under control conditions synthesized glycoprotein in a linear manner over time. The cells responded to dmPGE2 with an increase in glycoprotein synthesis without an effect on DNA synthesis. Investigations of lipid synthesis showed that phospholipid was produced in a linear fashion by these cells, however, no effect of exogenously administered dmPGE2 on its rate of formation was discernible. In contrast, the incorporation of labeled palmitate into neutral lipids revealed that triglyceride biosynthesis was significantly increased by the addition of dmPGE2 to the culture medium, which could be further enhanced by the administration of the phosphodiesterase inhibitor, isobutyl methyl xanthine. Cyclic nucleotide involvement was further suggested by our finding that triglyceride synthesis in cultured gastric mucous cells could be increased a comparable amount by the addition of both dbcAMP and dbcGMP to the medium. The possible relationship between these biochemical alterations and the gastric protective action of dmPGE2 is discussed.

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Ulcerogenesis of the duodenal mucosa frequently involves an inflammatory reaction with infiltration of leukocytes. Measurement of neutrophil myeloperoxidase activity might thus be a sensitive indicator of damage, before visible lesions occur. To test this possibility, a rat model for duodenal injury was used where fasted animals were treated with indomethacin and histamine-diHCl. Twenty-four hours after indomethacin treatment, duodenal tissues were collected for histochemical staining and biochemical assay for myeloperoxidase activity. Indomethacin- and histamine-challenged rats had significantly elevated myeloperoxidase activity compared to unchallenged controls (P less than 0.05) for both histochemistry and biochemistry. There was also a significant correlation between these two parameters (r = 0.68, P less than 0.001). The duodenal injury model then was used to test the effectiveness of known gastric protective agents. Results indicated that milk and buttermilk did not aggravate or protect against duodenal injury, while antacid and prostaglandin did significantly protect against inflammation (P less than 0.02). We concluded that measurement of myeloperoxidase activity is a sensitive and potentially useful estimate of duodenal injury that can be valuable in assessing ulcerogenesis and healing.

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