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Figure S-1. Chemical structures of aspirin and the predominant phospholipid in soybean phosphatidylcholine (PC).Figure S-2. Evaluation of the effects of aspirin-PC and aspirin on tube-formation. Figure S-3. In vitro evidence that the growth inhibitory activity of aspirin-PC and aspirin of ovarian cancer cell lines.

Figure S1. Aspirin and Aspirin-PC does not block the release of TGF from platelets following co-culture with colon cancer cells. Figure S2. Aspirin and Aspirin-PC block platelet induced upregulation of the EMT marker SNAIL in MC-26 cells. Figure S3. Aspirin and Aspirin-PC decrease collagen-induced activation of platelets obtained from the AOM/DSS cancer model. Figure S4. Time course of aspirin (ASA) test drugs on body weight in AOM/DSS mouse colon cancer model. Figure S5. Aspirin and Aspirin-PC do not alter changes in body weight induced by AOM/DSS. Figure S6. Effect of aspirin (ASA) test drugs on fecal hemoglobin in AOM/DSS mouse cancer model. Figure S7. Effect of aspirin (ASA) test drugs on hematocrit in AOM/DSS mouse cancer model.

Figure legends for Supplementary Figures 1-7.

To determine the efficacy of a novel and safer (for gastrointestinal tract) aspirin (aspirin-PC) in preclinical models of ovarian cancer, in vitro dose–response studies were performed to compare the growth-inhibitory effect of aspirin-PC versus aspirin on three human (A2780, SKOV3ip1, and HeyA8) and a mouse (ID8) ovarian cancer cell line over an 8-day culture period. In the in vivo studies, the aspirin test drugs were studied alone and in the presence of a VEGF-A inhibitor (bevacizumab or B20), due to an emerging role for platelets in tumor growth following antiangiogenic therapy, and we examined their underlying mechanisms. Aspirin-PC was more potent (vs. aspirin) in blocking the growth of both human and mouse ovarian cancer cells in monolayer culture. Using in vivo model systems of ovarian cancer, we found that aspirin-PC significantly reduced ovarian cancer growth by 50% to 90% (depending on the ovarian cell line). The efficacy was further enhanced in combination with Bevacizumab or B20. The growth-inhibitory effect on ovarian tumor mass and number of tumor nodules was evident, but less pronounced for aspirin and the VEGF inhibitors alone. There was no detectable gastrointestinal toxicity. Both aspirin and aspirin-PC also inhibited cell proliferation, angiogenesis, and increased apoptosis of ovarian cancer cells. In conclusion, PC-associated aspirin markedly inhibits the growth of ovarian cancer cells, which exceeds that of the parent drug, in both cell culture and in mouse model systems. We also found that both aspirin-PC and aspirin have robust antineoplastic action in the presence of VEGF-blocking drugs. Mol Cancer Ther; 15(12); 2894–904. ©2016 AACR.

The chemopreventive activity of non-steroidal anti-inflammatory drugs (NSAIDs), particularly aspirin, has been well demonstrated in preclinical and clinical studies. However, the primary side effect from this class of drug is gastrointestinal (GI) bleeding, which has limited the widespread use of NSAIDs for the prevention of cancer. The development of GI-safer NSAIDs, which are associated with phosphatidylcholine (PC) may provide a solution to this therapeutic problem. In the present study, the efficacy of two NSAIDs, aspirin and indomethacin, were compared using murine colon cancer cell line MC-26. Each NSAID was assessed alone and in combination with PC, using in vitro and in vivo systems. The results reveal that the PC-associated NSAIDs had a significantly higher degree of protection against cancer cell growth compared with the unmodified NSAIDs. It was also observed that Aspirin-PC and Indomethacin-PC prevented the metastatic spread of cancer cells in a syngeneic mouse model. These results support the potential use of PC-NSAIDs for the chemoprevention of colorectal cancer.

The chemopreventive activity of non-steroidal anti-inflammatory drugs (NSAIDs), particularly aspirin, has been well demonstrated in preclinical and clinical studies. However, the primary side effect from this class of drug is gastrointestinal (GI) bleeding, which has limited the widespread use of NSAIDs for the prevention of cancer. The development of GI-safer NSAIDs, which are associated with phosphatidylcholine (PC) may provide a solution to this therapeutic problem. In the present study, the efficacy of two NSAIDs, aspirin and indomethacin, were compared using murine colon cancer cell line MC-26. Each NSAID was assessed alone and in combination with PC, using in vitro and in vivo systems. The results reveal that the PC-associated NSAIDs had a significantly higher degree of protection against cancer cell growth compared with the unmodified NSAIDs. It was also observed that Aspirin-PC and Indomethacin-PC prevented the metastatic spread of cancer cells in a syngeneic mouse model. These results support the potential use of PC-NSAIDs for the chemoprevention of colorectal cancer.

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID–BA or NSAID–BA–PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA–IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA–IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA–IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA–IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.

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.

Background/Aims: Proton pump inhibitors (PPIs) are widely used to prevent nonsteroidal anti-inflammatory drug (NSAID)-induced peptic ulcers. NSAIDs produce small intestinal injury and some PPIs have been reported to protect against NSAID-induced small bowel injury in rats. The aim of this study was to compare PPIs, revaprazan, and phosphatidylcholine-associated indomethacin (Indo-PC) for protection against indomethacin (Indo)-induced small bowel injury. Methods: Rat intestinal epithelial cells (IEC-6) were pretreated with omeprazole, lansoprazole, or revaprazan prior to exposure to Indo or Indo-PC. Cell viability was assessed by methyl thiazolyl tetrazolium assay. Omeprazole, lansoprazole, or revaprazan was administered orally to rats prior to the vehicle or Indo. Indo-PC was administered alone. After 24 h, small intestinal erosions were counted; intestinal bleeding was assessed as the hemoglobin concentration of small intestinal fluid. Results: Omeprazole, lansoprazole, and revaprazan did not protect against Indo-induced IEC-6 cell injury. Indo-PC was less damaging in vitro than Indo alone. In vivo, neither omeprazole nor lansoprazole protected against Indo-induced small bowel injury; however, revaprazan pretreatment and Indo-PC resulted in significantly fewer erosions (>50% reduction) or bleeding (>80% reduction). Conclusion: PPIs showed no small bowel protective effect in vitro or in vivo. Revaprazan showed a small bowel protective effect in vivo, whereas Indo-PC was protective both in vitro and in vivo.

Endotoxemia from sepsis can injure the gastrointestinal tract through mechanisms that have not been fully elucidated. We have shown that LPS induces an increase in gastric permeability in parallel with the luminal appearance of secretory phospholipase A2 (sPLA2) and its product, lysophosphatidylcholine (lyso-PC). We proposed that sPLA2 acted on the gastric hydrophobic barrier, composed primarily of phosphatidylcholine (PC), to degrade it and produce lyso-PC, an agent that is damaging to the mucosa. In the present study, we have tested whether lyso-PC and/or sPLA2 have direct damaging effects on the hydrophobic barriers of synthetic and mucosal surfaces. Rats were administered LPS (5 mg/kg, i.p.), and gastric contents were collected 5 h later for analysis of sPLA2 and lyso-PC content. Using these measured concentrations, direct effects of sPLA2 and lyso-PC were determined on (a) surface hydrophobicity as detected with an artificial PC surface and with intact gastric mucosa (contact angle analysis) and (b) cell membrane disruption of gastric epithelial cells (AGS). Both lyso-PC and sPLA2 increased significantly in the collected gastric juice of LPS-treated rats. Using similar concentrations to the levels in gastric juice, the contact angle of PC-coated slides declined after incubation with either pancreatic sPLA2 or lyso-PC. Similarly, gastric contact angles seen in control rats were significantly decreased in sPLA2 and lyso-PC-treated rats. In addition, we observed dose-dependent injurious effects of both lyso-PC and sPLA2 in gastric AGS cells. An LPS-induced increase in sPLA2 activity in the gastric lumen and its product, lyso-PC, are capable of directly disrupting the gastric hydrophobic layer and may contribute to gastric barrier disruption and subsequent inflammation.

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 PGE2 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.

Background and Aims: A mechanism for protection of gastrointestinal (GI) and hepatic cells from damaging detergent actions of bile acids appears to involve the bile component, phosphatidylcholine (PC). Non-steroidal anti-inflammatory drugs (NSAIDs) induce intestinal injury in direct proportion to their ability to be excreted into bile, and are known to chemically associate with PC. We investigated the role of bile acids and PC in the mechanism of indomethacin-induced epithelial injury. Methods: Rats were injected orally or intravenously with radiolabeled indomethacin and their bile was collected over time for determination of NSAID secretion. Bile from rats treated with or without indomethacin was used in studies of red blood cell (RBC) hemolysis as a measure of membrane cytotoxicity. The bile salt, sodium deoxycholate (SDC), and indomethacin were tested alone and in combination with PC on RBC and on hepatic HepG2 cells. Results: Intravenously or orally given indomethacin was quantitatively excreted (approximately 50%) into bile over a 2-h study period. Bile from a rat treated with indomethacin or bile with exogenous indomethacin was cytotoxic to RBC, and the injury was prevented by the addition of PC. Hepatocytes exposed to SDC showed injury that could be dose-dependently prevented by PC, and reversed by indomethacin. Conclusions: Biliary PC plays an important physiological role in protecting GI and hepatic epithelia from the cytotoxic actions of bile salts. The ability of NSAIDs excreted into the bile to associate with mixed bile salt micelles and reduce the protective action of the PC may be a critical component in the drugs' pathogenic mechanism.

Background and Aim: The mechanism which protects the biliary and intestinal mucosa from the detergent properties of bile acids is not fully understood. We employed three contrasting in vitro model systems (human red blood cells, polarized intestinal [Caco-2] cells, and synthetic liposomes), to compare the efficacy of saturated and unsaturated phosphatidylcholine (PC) to protect cells and membranes from bile salt injury. Methods: Hemolysis of red blood cells, electrical resistance across confluent monolayers of Caco-2 cells, and disruption of synthetic PC liposomes were assessed after incubation with varying concentrations of bile salt (sodium deoxycholate) alone or in the presence of saturated or unsaturated PC. Results: The hemolytic activity of deoxycholate on red blood cells was observed at ≥2 mM, and could be blocked by equimolar concentration or greater of both saturated or unsaturated PC. In contrast, exposure of Caco-2 cells to deoxycholate at ≥0.8 mM induced a maximal decrease in resistance, which was reversed by ≥0.8 mM unsaturated PC or 5 mM saturated PC. Similarly, synthetic liposomes were permeabilized by 0.8 mM deoxycholate and were protected by a lower concentration of unsaturated PC (2 mM) than saturated (5 mM). Conclusions: Cells can show variable resistance to bile salt toxicity. Extracellular PC, especially in the unsaturated state, can directly protect cell and artificial membranes from bile salt injury. These findings support a role for biliary PC in the formation of mixed micelles that have low cytotoxic properties.

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 E2 (PGE2) concentration. Key results: 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 PGE2 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. British Journal of Pharmacology (2007) 150, 913–919. doi:10.1038/sj.bjp.0707176

In this review, we have discussed our current understanding of the barrier properties that are in place to protect the upper gastrointestinal mucosa from luminal acid, and the pathogenic mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce injury to the gastrointestinal tract. The changes in our view of the importance of NSAID-induced cyclo-oxygenase (COX) inhibition on the pathogenesis and prevention of NSAID-induced gastrointestinal injury is presented. The focus of this paper has been placed on the effects of NSAIDs on the mucosal surface, and specifically the effect of these powerful drugs in inducing changes in the hydrophobicity, fluidity, biomechanical and permeability properties of extracellular and membrane phospholipids. Lastly, recent evidence is presented that salicylic acid and related NSAIDs may alter the stability of membranes, inducing the formation of unstable pores that may lead to back-diffusion of luminal acid and membrane rupture. This understanding of the interaction of NSAIDs with membrane phos-pholipids may prove valuable in the design of novel NSAID formulations with reduced gastrointestinal side-effects.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat a number of conditions, and proton pump inhibitors (PPIs) are often used to prevent NSAID-induced gastric mucosal damage; however, the effects of NSAIDs on intestinal motility are poorly understood. The purpose of the present study is to determine the effects of a prototypical NSAID, indomethacin, either alone or in conjunction with the PPI omeprazole, on intestinal motility. Rats were randomly divided into four groups treated with vehicle, omeprazole, indomethacin, or a combination of indomethacin and omeprazole. Intestinal motility and transit were measured along with inflammatory mediators in the intestinal smooth muscle, markers of mucosal damage, and bacterial counts in the intestinal wall. Indomethacin, but not omeprazole, caused mucosal injury indicated by lower gut bleeding; however, both omeprazole and indomethacin suppressed contractile activity and frequency in the distal part of the small intestine. Cotreatment with omeprazole did not reduce indomethacin-induced intestinal bleeding. Furthermore, although indomethacin caused increased inflammation as indicated by increased edema development and inflammatory mediators, cotreatment with omeprazole did not reduce inflammation in the intestinal smooth muscle or prevent the increased bacterial count in the intestinal wall induced by indomethacin. We conclude that both NSAID and PPI treatment suppressed contractile activity in the distal regions of the small intestine. The suppression of intestinal contractility was associated with increased inflammation in both cases; however, indomethacin and omeprazole appear to affect intestinal motility by different mechanisms.

Sepsis of gastrointestinal origin can lead to life-threatening complications in vital organs due to bacterial overgrowth and/or translocation from the lumen into the blood. In a rat model of endotoxemia, changes in surface hydrophobicity (associated with barrier integrity) of the gastrointestinal mucosa were examined. Rats were treated with Escherichia coli lipopolysaccharide (LPS), and gastric and ileal tissue were collected for determination of surface hydrophobicity by contact angle analysis. A role for bile salts in hydrophobicity changes was tested by quantifying bile salts in the lumen of both the stomach and ileum after LPS and by the administration of LPS to bile duct-ligated rats. A single intraperitoneal dose of LPS induced a dose- and time-dependent reduction in hydrophobicity of both the stomach and ileum, with the stomach showing greater sensitivity at an earlier time than the ileum. LPS also induced gastric bleeding, reflux of bile acid into the gastric lumen, and decreased levels of bile salt in the ileum. The LPS-induced reductions in surface hydrophobicity of the stomach were prevented by prior bile duct ligation. We conclude that LPS disrupts gastrointestinal barrier integrity, in part by mechanisms involving bile constituents and an attenuation in the mucosa's hydrophobic characteristics.

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 hydro-phobicity 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.

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.

Helicobacter pylori infection has been linked tothe development of gastritis which can then progress toa number of disease entities including peptic ulcerdisease and gastric cancer. Since the pathogenic mechanism by which the bacteria causesgastritis is unresolved, we employed a model system, theH. felis-infected mouse to investigate the temporalrelationship between bacterially-induced alterations in the hydrophobic phospholipid barrier of thestomach and the development of gastritis. In the presentstudy, C57BL/6 mice were inoculated with 109CFU of H. felis and the changes in gastric wet weight, histology, surface hydrophobicity,phospholipid/phosphatidylcholine concentration,phospholipase A2 activity, and the pH ofcollected gastric juice were measured 0.5-2 monthspostinoculation. In related experiments, we investigated the effects oftreating H. felis infected mice with antibiotic/bismuththerapy on the above gastric properties. It wasdetermined that both gastric surface hydrophobicity and phospholipid composition were significantlyattenuated as early as 2-4 weeks postinfection,preceding signs of mucosal inflammation and glandularatrophy as indicated by increases in gastric wet weight, pH and a disappearance in parietal cells. Theseearly H. felis-induced changes in gastric surfacehydrophobicity and phospholipid concentration werereversed by antibiotic/bismuth therapy. Based on these results we conclude that H. felis infectioninduces an early transformation of the stomach from ahydrophobic to an acid-sensitive hydrophilic state thatmay trigger the subsequent development ofgastritis.

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.

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-PGF1α 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.

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. (Gastroenterology 1996 Mar;110(3):801-8)

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.

Background The gastric mucosa is protected in part by a hydrophobic layer of phosphatidylcholine (PC) that overlies the mucus gel on the stomach. Endotoxin treatment (i.e., lipopolysaccharide [LPS]) results in an apparent disruption of this layer, as evidenced by a reduction in surface hydrophobicity and an increase in transmural permeability. The current studies compared PC and lyso-PC levels in mucus and gastric mucosa before and after LPS treatment, and examined potential mechanisms for surface phospholipid changes. Methods Rats were administered LPS (5 mg//kg, intraperitoneally) and samples were collected after 5 h for analysis of PC and its primary degradant, lyso-PC, in the loosely and firmly adherent mucus layers and the mucosa. The dependence of LPS-induced effects on gastric alkalinization, PC synthetic activity, and intestinal reflux material was assessed. Results The gastric contents after LPS, which also contained duodenal reflux material, had greatly increased amounts of PC and lyso-PC. The firmly adherent mucus layer was unchanged. The gastric mucosa after LPS revealed significant reductions of PC levels and no change in lyso-PC content. These phospholipid changes were not caused by alkalinization of the stomach or altered PC synthesis. Prevention of duodenogastric reflux by pylorus ligation blocked the LPS-induced increase in luminal lyso-PC and the reduction in mucosal PC. Conclusions LPS appears to induce a release of PC from gastric mucosa into the lumen, along with degradation of PC to lyso-PC, without an effect on PC synthesis. Component(s) of intestinal reflux material appear to be required for these effects. The lowered PC levels in gastric mucosa after LPS may contribute to reduced barrier properties of this tissue.

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.

Diarrhea is one of the most common infirmities affecting international travelers, occurring in 20 to 50% of persons from industrialized countries visiting developing regions. Enterotoxigenic Escherichia coli (ETEC) is the most common causative agent and is isolated from approximately half of the cases of traveler's diarrhea. Rifaximin, a largely water-insoluble, nonabsorbable (E. coli . However, the drug has minimal effect on the bacterial flora or the infecting E. coli strain in the aqueous environment of the colon. The purpose of the present study was to evaluate the antimicrobial effect and bioavailability of rifaximin in aqueous solution in the presence and absence of physiologic concentrations of bile acids. The methods used included growth measurement of ETEC (strain H10407), rifaximin solubility measurements, total bacterial protein determination, and assessment of the functional activity of rifaximin by monitoring inhibition of bacterial β-galactosidase expression. Solubility studies showed rifaximin to be 70- to 120-fold more soluble in bile acids (approximately 30% in 4 mM bile acids) than in aqueous solution. Addition of both purified bile acids and human bile to rifaximin at subinhibitory and inhibitory concentrations significantly improved the drug's anti-ETEC effect by 71% and 73%, respectively, after 4 h. This observation was confirmed by showing a decrease in the overall amount of total bacterial protein expressed during incubation of rifaximin plus bile acids. Rifaximin-treated samples containing bile acids inhibited the expression of ETEC β-galactosidase at a higher magnitude than samples that did not contain bile acids. The study provides data showing that bile acids solubilize rifaximin on a dose-response basis, increasing the drug's bioavailability and antimicrobial effect. These observations suggest that rifaximin may be more effective in the treatment of infections in the small intestine, due to the higher concentration of bile in this region of the gastrointestinal tract than in the colon. The water insolubility of rifaximin is the likely explanation for the drug's minimal effects on colonic flora and fecal pathogens, despite in vitro susceptibility.

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.

The hydrophobic surface layer of the gastrointestinal (GI) tract, which has been attributed to the presence of phosphatidylcholine (PC) in the mucus gel, protects the mucosa of the GI tract and is disrupted by parenteral LPS treatment. We investigated the potential for repletion of this layer as a means to prevent LPS-induced GI injury. Rats were treated orally with PC 1 h before LPS (i.p.). Gastric and ileal tissues were assessed for changes in permeability 5 h later, and gastric fluid was analyzed for signs of GI-related LPS effects (bile acid reflux, increased volume, and pH) and gastric injury (bleeding). Serum TNF-alpha was assessed as a measure of a non-GI, LPS response. Radiolabeled PC was tracked through the GI tract to verify the extent of luminal exposure during the time of the study. Pretreatment with oral PC significantly blocked permeability increases in gastric and ileal tissue due to LPS. A portion of orally administered PC gained access to the entire GI tract in 1 h. Exogenous PC did not prevent the increase in serum TNF-alpha or gastric fluid volume or pH induced by LPS, nor did it prevent the duodenogastric reflux of bile acid. There was a tendency for PC to reduce gastric bleeding after LPS. Orally administered PC seems to act directly on the mucosa to prevent GI permeability disturbances due to LPS. Under the conditions studied, oral PC does not block systemic effects of LPS. However, enteral formulations containing PC may be useful adjuncts in the prevention of GI injury from endotoxemia.

Background: The pathogenesis of multiple organ failure (MOF) in trauma patients may involve the gastrointestinal tract, but its exact origins remain elusive. In a prospective study, the gastric fluid of major torso trauma patients was examined for evidence of duodenogastric reflux and potential gastric injury, and was compared with patient outcomes regarding MOF. Methods: Patient samples were collected daily for 4 days by nasogastric tube and analyzed for pH, hemoglobin, and bile acid. Blood was collected for analysis of C-reactive protein (CRP). Outcomes were recorded for the presence or absence of MOF. Results: The results showed that most patients exhibited alkaline gastric contents (pH ≥ 4.9) and elevated levels of hemoglobin immediately after the trauma. Although non-MOF patients demonstrated a decline of both mean gastric pH and bleeding by day 4, MOF patients maintained significant elevations in pH during this time period. Mean total bile acid levels were increased in all patients, signifying the presence of duodenogastric reflux. However, there were no clear differences in mean bile acid concentrations between MOF and non-MOF patients over time, although MOF patients tended to exhibit higher levels. All patients showed a progressive rise in serum CRP during the first 24 hours after trauma, which was maintained for 4 days. The initial rise in serum CRP in MOF patients was delayed compared with that in non-MOF patients. Conclusions: We conclude that duodenogastric reflux occurs in trauma patients in the first few days after trauma and may contribute to elevated gastric pH and bleeding. Further study is needed to verify whether monitoring the gastric juice of trauma patients during the first several days of hospitalization, for alkaline pH and excessive blood in the gastric lumen, could lead to better assessments of patient status.

A hydrophobic layer of phosphatidylcholine (PC) overlies and protects the surface of the gastrointestinal (GI) tract, contributing to barrier integrity. During critical illness such as sepsis, gut barrier integrity is compromised, which could be related to degradation of PC. The purpose of this study was to investigate a role for luminal (secretory) phospholipase A2 (sPLA(2)) in LPS-induced GI injury. Rats were treated with LPS (5 mg/kg) or saline for 0.5, 1, 3, and 5 h. The gastric and ileal luminal contents were collected for determination of sPLA(2) activity, and the luminal lipids were analyzed using thin layer chromatography for lyso-PC content. The GI permeability was assessed in vivo with fluorescein-isothiocyanate dextran 4000 and rats were tested with or without a specific sPLA(2) inhibitor. LPS induced significant increases in sPLA(2) activity and lyso-PC content in the gastric and ileal lumens at 5 h. In addition, LPS treated rats showed a significant increase in GI permeability to fluorescein-isothiocyanate dextran in both the stomach and ileum at 5 h, which was prevented by pretreatment with the sPLA(2) inhibitor. In response to LPS, sPLA(2) activity increases in the GI tract lumen where it may degrade the extracellular protective phospholipid layer and membranes, producing injurious lyso-PC and increased GI permeability. Pretreatment with an orally active sPLA(2) inhibitor blocks the LPS-induced increase in GI permeability, and may suggest a new approach to fortify the GI mucosal barrier and prevent complications from endotoxin in trauma and other patients.

It has been shown that intragastric treatment of rats with a suspension of dipalmitoylphosphatidylcholine and tripalmitin at a 1:4 ratio (5 mg lipid/mL per rat) provided rats with highly efficaceous and consistent protection against a variety of ulcerogenic agents and conditions. The gastric protective activity of this mixture was of long duration (t 1/2 approximately 9 hours. In an attempt to understand the mechanism of protection, it was determined that the ulcerogen-induced reduction in gastric surface hydrophobicity was reversed in rats pretreated with the mixture. However, the lipid mixture did not affect the gastric emptying rate and maintained its cytoprotective activity in indomethacin-treated rats. These results indicate that the mixture's protective effect was not mediated by alterations in either gastrointestinal motility or the gastric accumulation of lipids or "cytoprotective" metabolites (prostaglandins). The mixture also appreciably reduced gastric lesion score in response to acid if one or both the lipids was substituted for a metabolically inert ether analogue, suggesting that lipid metabolism makes a negligible contribution to the protective response. Electron microscopic observation indicated that the predominent structure in the mixture is a microemulsion in which a dipalmitoylphosphatidylcholine monolayer encapsulates a tripalmitin core. Last, the improved gastric protective activity of the mixture in comparison to dipalmitoylphosphatidylcholine liposomes is discussed regarding marked differences in the physical structure of the two suspensions and the rate at which lipids in these states adsorb to a surface to enhance its hydrophobic properties.

Recombinant human lactoferrin (RHLF) was tested for its ability to prevent non-steroidal anti-inflammatory drug (NSAID)-induced intestinal injury in rats and mice. Acute and chronic models using indometacin, naproxen and diclofenac were used. Measurements were made of intestinal bleeding and inflammation. Orally administered RHLF was effective at preventing acute NSAID-induced increases in gut bleeding and myeloperoxidase activity. Oral RHLF was also effective at blocking some chronic manifestations of indometacin usage. Protection by RHLF of the intestinal tract from NSAIDs appears to be linked to attenuation of neutrophil migration to the intestine, and is independent of prostaglandins and nitric oxide. RHLF does not bind to the NSAID or interfere with the NSAID biological activity. We conclude that orally administered RHLF is effective at preventing NSAID-induced intestinal injury in rodents and should be investigated for this potential therapeutic use in man.

The use of NSAIDs or COX-2 inhibitors for chemoprevention of colorectal cancer has been suggested for patients at high risk for this disease. However, the gastrointestinal side effects of traditional NSAIDs which consist of bleeding and ulceration, and the cardiovascular effects of COX-2 inhibitors may limit their usefulness. In preclinical studies, our laboratory has shown that the addition of phosphatidylcholine (PC) to the NSAIDs aspirin (ASA) or ibuprofen (IBU) results in a NSAID–PC with fewer GI side effects and also maintained or enhanced analgesic, anti-pyretic and anti-inflammatory efficacy over the unmodified NSAID. Because NSAID–PCs have not been tested for anti-cancer activity, in the present study, ASA–PC and IBU–PC were tested on the SW-480 human colon cancer cell line. SW-480 cells were incubated in media containing 1–5 mM NSAID or NSAID–PC for 2 days. Measurements were made of cell number, cell proliferation (DNA synthesis), and manner of cell death (necrosis and apoptosis). ASA and IBU reduced cell number in a dose-dependent manner with IBU showing a greater potency than ASA. The association of PC to the NSAID resulted in greater reductions of cell number for both NSAIDs. Furthermore, the NSAID–PC formulation had significantly greater efficacy and potency to inhibit cellular DNA synthesis than the unmodified NSAID. PC alone at the doses and times used had no effect on cell number in this cell line, but did have a small effect to reduce DNA synthesis. None of the drugs had a clear effect on cell death by necrosis. Only IBU and IBU–PC caused cell death by apoptosis in SW-480 cells. We conclude that NSAID–PCs have activity to impede the growth of colon cancer cells in vitro, which is due, in major part, to a marked reduction in DNA synthetic activity of these cells. This growth inhibitory effect appears to be independent of COX-2 activity, since it is known that SW-480 cells do not have this inducible COX isoform. Due to its greater efficacy in this model system, IBU–PC should be further evaluated as a chemopreventive agent that is safer for the GI tract than unmodified NSAID.

Background & Aims: Insight into the role of the different cyclooxygenase isoforms in prostaglandin biosynthesis, surface hydrophobicity, and gastric mucosal barrier integrity can be gained by comparing the effects of luminal damaging agents in wild-type and cyclooxygenase knockout mice. Methods: Fasted wild-type, cyclooxygenase-1, and cyclooxygenase-2 knockout mice were intragastrically administered saline, 0.6N HCl, or aspirin (aspirin 20 mmol/L) in combination with 0.6N HCl and killed 1 hour later, at which time the gastric lesion score was assessed and biopsy samples were taken for surface, biochemical, and morphological analyses. Results: The gastric mucosa of cyclooxygenase-1 knockout mice was more severely injured by both HCl alone and aspirin/HCl than that of wild-type and cyclooxygenase-2 knockout mice. HCl alone and aspirin/HCl also induced a more profound decrease in surface hydrophobicity in cyclooxygenase-1 knockout mice than in wild-type mice, whereas this surface property was unaffected in cyclooxygenase-2 knockout mice. The gastric injury induced by aspirin/HCl in cyclooxygenase-1 knockout mice could be prevented if the animals were treated with phosphatidylcholine-associated aspirin. Aspirin/HCl, in comparison to saline or HCl alone, induced a 4 – 6-fold increase in gastric mucosal prostaglandin E2 concentration in the cyclooxygenase-1 knockout mice, whereas it decreased prostaglandin E2 levels in wild-type and cyclooxygenase-2 knockout mice. This paradoxical aspirin-induced increase in gastric prostaglandin E2 in cyclooxygenase-1 knockout mice seemed to correspond to an increase in cyclooxygenase-2 messenger RNA and protein expression. The gastric lesion score seemed to be significantly associated with alterations in surface hydrophobicity but not with mucosal prostaglandin E2 concentration. Conclusions: Our evidence on cyclooxygenase knockout mice suggests that aspirin predominantly causes gastric injury by a nonprostaglandin mechanism, perhaps by attenuating surface hydrophobicity, a possibility supported by the low gastric toxicity of phosphatidylcholine/aspirin. However, prostaglandins generated by cyclooxygenase-1 may play an important permissive role in maintaining gastric mucosal barrier integrity. Aspirin seems to paradoxically increase the gastric mucosal prostaglandin E2 concentration in cyclooxygenase-1 knockout mice, possibly by the induction of cyclooxygenase-2.

Lactoferrin possesses antibiotic, antiinflammatory, and immune-modulating properties that may be active against the gastritis-, ulcer- and cancer-inducing bacterium Helicobacter pylori. In vitro testing of bovine and human lactoferrin by several laboratories has shown significant bacteriostatic and bactericidal activity. Subsequent in vivo testing of bovine lactoferrin in animal models of H. pylori infection has shown beneficial effects of this agent. Our laboratory has utilized a mouse model that is infected with the feline strain of this bacterium, H. felis. The resulting gastritis that develops in this model and the effects of bovine lactoferrin and recombinant human lactoferrin (from Aspergillus niger var. awamori, Agennix Inc., Houston, Tex.) treatment were assessed by various measures. Infected animals treated with orally administered lactoferrin showed reversals in all parameters. In addition, when recombinant human lactoferrin was used in combination with low doses of amoxicillin or tetracycline, there was an enhancement in gastritis-reducing activity. Possible mechanisms for these effects of lactoferrin are discussed. Lactoferrin has significant, orally active in vivo actions and should be further investigated for clinical situations involving Helicobacter infections where it may have utility when administered alone and also when given in combination with established antibiotic agents.Key words: lactoferrin, Helicobacter, gastritis, surface hydrophobicity.

Background: The chronic use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, is associated with a reduction in risk for colorectal and other cancers. However, when used chronically, these drugs carry a significant side effect to induce GI bleeding. Our laboratory has developed a class of NSAIDs that are complexed with phosphatidylcholine (PC-NSAIDs) which have been shown in preclinical and clinical trials to reduce NSAID GI toxicity. To date these drugs have been shown to possess equal or better efficacy compared to traditional NSAIDs in regard to anti-inflammatory, analgesic and anti-pyretic activities. To evaluate the chemopreventive activity of PC-NSAIDs, an animal model of carcinogeninduced colonic aberrant crypts was tested. Methods: Rats were treated at two weekly intervals with azoxymethane (AOM, 15mg/kg), followed two weeks later by daily oral dosing with control or test drugs for 4weeks (indomethacin, 2mg/kg; indomethacin-PC, 2mgNSAID/ kg; ibuprofen, 20mg/kg; ibuprofen-PC, 20mg NSAID/kg; or aspirin-PC, 20mg NSAID/kg). Assessments were made of GI toxicity/bleeding by measurement of the hematocrit and of hemoglobin in the feces, and of chemopreventive efficacy by the number of aberrant crypts in the distal colon. Results: At the doses used, no GI toxicity was detected from any of the test agents. The number of colonic aberrant crypts were significantly reduced by treatment with indomethacin-PC, but not by indomethacin (AOM control=40±11; indomethacin= 46±9; indomethacin-PC=10±2). In a separate study colonic aberrant crypts were significantly reduced by treatment with ibuprofen-PC and aspirin-PC, but not by ibuprofen (AOM control=133±18; ibuprofen=189±32; ibuprofen-PC=76±8; aspirin-PC=86±24). Conclusions: At equivalent doses, a novel class of PC-NSAIDs exhibit enhanced chemopreventive activity over traditional NSAIDs in a rodent model of colon carcinogenesis. (Supported by NIH grants R41CA171408 and R03CA171613).

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. (Gastroenterology 1996 Oct;111(4):990-5)

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.

Background: An increasing amount of evidence suggests that consumption of non-aspirin NSAIDs is associated with injury to the lower gut in humans, as has been established in rodent model systems. NSAID-induced lower gut injury may in fact explain the linkage between NSAID consumption and the development of ileitis, diverticulitis and inflammatory bowel disease. Evidence from our and other labs indicates that NSAID-induced injury to the lower gut is dependent on the presence of bile, based upon In Vitro (synthetic membranes and GI cells in culture) and In Vivo (rodent) studies, and we have hypothesized that NSAIDs may form toxic mixed micelles with bile acids (BAs). Aim: To test this hypothesis that NSAIDs and BAs spontaneously form mixed micellar aggregates we used a combination of powerful computational and spectroscopic techniques to gain insights into atomic associations between cholic acid (CA) and ibuprofen (IBU). Methods & Results: Using Molecular Dynamics (MD) simulations in tandem with Nuclear Magnetic Resonance (NMR) spectroscopy, we found that CA and IBU form mixed micelles in a concentration dependent manner, so that multiple CA molecules form ‘pseudo-rings' into which IBUs insert their hydrophobic tails. This ‘β-face-to-tail' packing between the steroid rings of CA and the alkyl carbons of IBU promotes the hydrophobic interaction of these two classes of amphipathic molecules. We also found that the IBU-CA mixed micelles have much larger sizes and higher net surface charges than simple CA micelles, the total charge being the sum of the single negative charge arising from each of the CA and IBU molecules. Furthermore, the IBU-CA mixed micelles adopt amore spherical shape that differs from the oblate shape formed by the pure CA clusters. These results highlight the power of a combined NMR/MD approach for characterizing the molecular interactions in the binarymixtures of NSAIDs and BAs. Conclusions: Our combined NMR/MD analysis yielded compelling structural evidence that NSAIDs and BAs form mixed micelles at concentrations greater than or equal to the CMC of the two species, which likely occur in the small intestinal lumen. The significance of the altered micellar morphology and charge density of these BA/NSAID mixed micelles to the drugs' cytotoxicity to the epithelium of the lower gut is an important area for future investigation. (Supported by NIH grants RC1 DK086304 and P30 DK56338.)