Role of surface-active phospholipids in gastric cytoprotection.

Intragastric administration of a liposomal surfactant suspension markedly reduced acid-induced gastric ulcerogenesis and bleeding in rats. The concentration of surface-active molecules intrinsically present in the gastric mucosa was increased two to six times by administration of 16,16-dimethyl prostaglandin E2. Thus, local accumulation of surface-active phospholipids may be an integral component of the cytoprotective mechanism activated by prostaglandin treatment.

Naproxen-PC: a GI safe and highly effective anti-inflammatory.

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.

Surface phospholipids in gastric injury and protection when a selective cyclooxygenase-2 inhibitor (Coxib) is used in combination with aspirin.

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.

Bioavailability of aspirin in rats comparing the drug's uptake into gastrointestinal tissue and vascular and lymphatic systems: implications on aspirin's chemopreventive action.

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.

Formula Feeding Predisposes Gut to NSAID-Induced Small Intestinal Injury.

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.

Effect of omeprazole on the bioavailability of unmodified and phospholipid-complexed aspirin in rats.

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.

Rat and human cardiac monoamine oxidase: a comparison with other tissues.

Experiments were made to test the proposal that rat heart mitochondrial monoamine oxidase (MAO) differs from that in certain other organs. Using kynuramine as the substrate, cardiac MAO of rats was compared with that in the vas deferens and liver. While the relative proportions of type A and B MAO varied between the three tissues, the sensitivity of the MAO types to preferential inhibitors, mixed substrate interactions and apparent Km determinations failed to reveal differences. Human atria contained mostly type B MAO, but the properties of the A and B types were not radically different from those found in rat tissues. No clorgyline-resistant MAO activity was detected with kynuramine, either in the rat or human tissues. Differences in rat heart MAO seem to be exposed by some substrates but not others, such as kynuramine.

Recombinant human lactoferrin is effective in the treatment of Helicobacter felis-infected mice.

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.

Gastrointestinal safety and therapeutic efficacy of parenterally administered phosphatidylcholine-associated indomethacin in rodent model systems.

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.

Observations on the monamine oxidase activity of rat vasa deferentia, major blood vessels and human saphenous vein.

Monoamine oxidase (MAO) activity was characterized using whole tissue homogenates and kynuramine as the substrate. In rat vasa deferentia, kynuramine deamination was differentially inhibited by clorgyline, less so by deprenyl and not at all by pargyline. These studies, and mixed substrate experiments with tryptamine, proved that kynuramine is a substrate for MAO types A and B. Experiments made with rat abdominal aorta and inferior vena cava disclosed clorgyline-sensitive and resistant MAO activity, the latter being inhibited by semicarbazide but not by deprenyl or pargyline. No semicarbazide-sensitive species of MAO was found in human saphenous vein which also differed from the rat vasculature in that the predominant MAO activity was of the B type. It is concluded that kynuramine is also a good substrate for clorgyline-resistant enzymes and that rat vasculature may be a poor model for predicting deaminating mechanisms in human venous tissue.

Development of gastric mucosal protection against acid in the rat. Role of corticosteroids and prostaglandins.

The ability of 16,16-dimethyl prostaglandin E2 (dmPGE2) to protect the gastric mucosa from acid-induced damage was assessed in suckling and adult rats. Suckling rats 9-11 days of age did not show protection with dmPGE2 at doses up to 100 micrograms/kg, whereas adult rats had an ED50 of approximately 2 micrograms/kg. The onset of responsiveness to dmPGE2 (5 micrograms/kg) occurred in the suckling rat at an age between 14 and 21 days. The "intrinsic" ability of the gastric mucosa to protect itself against acid-induced damage increased gradually between 7 and 21 days. The appearance of dmPGE2 sensitivity was unaffected by administration of hydrocortisone on day 7, although hydrocortisone alone induced a precocious development of the intrinsic protection against luminal acid. These data suggest that this mucosal protective property of the stomach appears during the end of the second week of life in the rat, just before the increase in acid secretory activity. This may be attributable to both the ontogenic appearance of a responsiveness to prostaglandins and an increase in circulating levels of adrenocorticosteroid hormones. Although the protective mechanisms responding to prostaglandins and the corticosteroids could be different, our results suggest that the two factors may act in concert to assure optimal protection against luminal acid.

Milk protection against experimental ulcerogenesis in rats.

The antiulcer activity in pasteurized/homogenized bovine milk and a lipid extract of this milk was tested in an attempt to isolate and identify the active component. Using 0.6 N HCl as a damaging agent in pylorus-ligated rats, the protective property of milk appeared to be related to its phospholipid content and not its protein constituents. With intact (non-pylorus-ligated) rats, milk had demonstrable protective activity against 0.6 N HCl, as well as 100% ethanol and 160 mM taurocholic acid. The increasing phospholipid concentrations in skim, whole, and buttermilk paralleled their antiulcer efficacy. A lipid extract of whole milk showed significant protection against 0.6 N HCl when given alone or following indomethacin treatment. Measurements of the contact angle (hydrophobicity) of the gastric surface showed that it was maintained near control levels in the presence of 0.6 N HCl, if rats were first pretreated with milk. These results are consistent with the possibility that surface-active lipids in dairy milk, such as phospholipids, may account for a significant portion of milk's antiulcer activity by maintaining the hydrophobicity of the luminal surface of the gastric mucosa in the presence of a damaging agent.

Surface hydrophobicity of the gastric mucosa in the developing rat. Effects of corticosteroids, thyroxine, and prostaglandin E2.

Surface hydrophobicity of the gastric mucosa and its variation in response to treatments with corticosteroids, thyroxine, and 16,16-dimethyl prostaglandin E2 were measured in developing rats. A developmental increase in the hydrophobicity of the luminal surface of the gastric mucosa was recorded between the first and third weeks of life. The hydrophobicity of the stomach was not consistently influenced by an acute administration of 16,16-dimethyl prostaglandin E2 (5 micrograms/kg, 30 min before examination) until the end of the third week of life, at which time a significant 40% increase was recorded. Similarly, the decrease in surface hydrophobicity that resulted from luminal administration of an ulcerogenic dose of HCl (0.6 N, 6 ml/kg) was blocked by 16,16-dimethyl prostaglandin E2 only in 3-wk-old rats and not in rats 1 and 2 wk of age. Neither the normal developmental increase nor the 16,16-dimethyl prostaglandin E2-induced enhancement in gastric surface hydrophobicity was induced precociously by corticosterone or thyroxine. The possible importance of these findings on the development of gastric surface hydrophobicity to the ontogeny of both gastric barrier function and prostaglandin-induced gastric protection is discussed.

A role for milk phospholipids in protection against gastric acid. Studies in adult and suckling rats.

Animal milk was tested directly for antiulcer activity in a rat model system. The severe mucosal necrosis and intragastric bleeding induced by the luminal administration of supraphysiologic 0.6 N HCl was significantly reduced (50%-70%) if both suckling rats and adults were pretreated per os with raw rat or bovine milk. Pasteurized/homogenized bovine milk also gave protection, although to a lesser extent (30%-50% reduction in bleeding). Treatment of milk with cholestyramine removed 50%-80% of the surface-active phospholipids from milk and also eliminated milk's protective property, an effect that was reversed upon addition of phospholipids to the extracted milk. The antiulcer action of milk in rats could be related to its concentration of dipalmitoyl lecithin. These findings suggest that milk has a potent antiulcer activity that may be attributable to its phospholipid constituents.

Effects of 16,16-dimethyl prostaglandin E2 on glycoprotein and lipid synthesis of gastric epithelial cells grown in a primary culture.

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.

Amino acid- and amine-induced gastrin release from isolated rat endocrine granules.

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.

Gastroprotection by dairy foods against stress-induced ulcerogenesis in rats.

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.

Effects of milk, prostaglandin, and antacid on experimentally induced duodenitis in the rat. Use of myeloperoxidase as an index of inflammation.

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.

Monoamine oxidase: an important intracellular regulator of gastrin release in the rat.

The role of monoamine oxidase (MAO) in the meal-induced or amino acid-induced release of gastrin was investigated. Rats that were pretreated with the nonspecific MAO inhibitor nialamide (200 mg/kg) showed a greater rise in meal-induced serum gastrin than did untreated controls. In vitro experiments demonstrated that gastrin secretion from dispersed antral G cells in response to a stimulatory dose of phenylalanine or methylbenzylamine (10 mM) was markedly enhanced if the cells were treated with nialamide. Studies with the more specific MAO inhibitors clorgyline and deprenyl indicated that antral mucosa contained predominantly type A activity. Inhibition of MAO type A with clorgyline, both in vivo and in vitro, resulted in a greater release of gastrin after stimulation by a meal or phenylalanine. It is concluded that MAO may play an important role in the regulation of gastrin release from the G cell by partially controlling the level of amines within the cell.