Richard Jaszewski

Duodenal and Gastric Ulcer Prevention with Misoprostol in Arthritis Patients Taking NSAIDs

Nonsteroidal anti-inflammatory drugs (NSAIDs) remain the agents of choice in the treatment of many rheumatic diseases because of their analgesic and anti-inflammatory properties. However, use of NSAIDs is also associated with an increased frequency of peptic ulcers and ulcer complications, such as major upper gastrointestinal bleeding and perforation [1-8]. It has been estimated that in the United States, NSAID-induced gastrointestinal complications account for at least 2600 deaths and 20 000 hospitalizations each year in rheumatoid arthritis patients alone [9]. The concern about NSAID-induced gastroduodenal damage is heightened because complications often occur without preceding symptoms of ulcer disease [10, 11]. The prevalence of endoscopically confirmed upper gastrointestinal ulcers in NSAID users has been reported to range between 15% and 31% [12-17]. Despite the predominance of gastric ulcers in chronic NSAID users, ulcer complications in NSAID users are often associated with duodenal ulcers [7, 11, 18-20]. Duodenal ulcers are more common than gastric ulcers in patients not taking NSAIDs, suggesting that many duodenal ulcers associated with NSAID use may reflect NSAID exacerbation of preexisting peptic ulcer disease [21, 22]. Misoprostol, a synthetic prostaglandin E1 analog, has been shown to be effective in decreasing the incidence of gastric ulcers in chronic NSAID users [17, 23] without interfering with the antirheumatic effects of the NSAID. The H2-receptor antagonist, ranitidine, and the topical agent, sucralfate, have not been effective in preventing gastric ulcers in long-term NSAID users [23-25]. Ranitidine-NSAID cotherapy decreased the frequency of duodenal ulcers in NSAID-treated patients, suggesting that duodenal ulcers in NSAID users may have a different pathogenesis than gastric ulcers [24, 25]. Our study was designed to investigate the efficacy of misoprostol for the prevention of NSAID-induced duodenal ulcers in arthritis patients receiving long-term NSAID therapy. Methods Patients were recruited from private practice offices, Veterans Affairs clinics, health maintenance organizations, and academic institutions. The study was done between June 1989 and February 1991. Patients were eligible to enter the study if they had rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, or the Reiter syndrome. Upper gastrointestinal pain was not required for entry into the study, but patients were expected to require at least 3 additional months of daily NSAID therapy with either ibuprofen, piroxicam, naproxen, sulindac, tolmetin, indomethacin, or diclofenac. Women were required to be postmenopausal, surgically sterilized, or practicing adequate contraception. Other exclusion criteria were a history of peptic ulcer disease requiring treatment in the 30 days immediately before entry, upper gastrointestinal malignancy, pyloric obstruction, acute hepatitis, pancreatitis, bleeding diathesis, upper gastrointestinal surgery within 30 days, or severe renal impairment. In addition, patients taking antineoplastic drugs, anticoagulants, anti-ulcer drugs other than the study drug, or prednisone at dosages of more than 7.5 mg/d were excluded. Study Design The study was a randomized, double-blind, placebo-controlled, multicenter trial of misoprostol, 200 g given four times a day (with meals and at bedtime with food). Upper gastrointestinal symptoms were scored using a 0 to 3 grading scale; zero corresponded to no abdominal pain, 1 corresponded to mild pain, 2 corresponded to moderate pain, and 3 corresponded to severe abdominal pain. Patients with gastric or duodenal ulcers of any size as well as those with erosions 0.3 cm in diameter at baseline upper gastrointestinal endoscopy were excluded. Patients who were accepted were randomized (randomization was balanced within each center) and received misoprostol or placebo within 72 hours of the entry endoscopic examination. Patients continued to take NSAIDs at the same dosage administered before the study. Patients were allowed to take up to three aluminum hydroxide antacid tablets (Amphojel [600 mg], Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania) per day for the first 2 weeks of the study for relief of upper gastrointestinal pain. After 4 weeks ( 3 days), 8 weeks ( 5 days), and 12 weeks ( 5 days) of treatment with misoprostol or placebo, endoscopic examinations and upper gastrointestinal pain evaluations were repeated. Endoscopists remained blinded to the patients medication. Patients were instructed to take the study medication the night before the endoscopy and not to take the next dose until after the procedure was completed. Noncompliance was defined as failure to take at least 60% of prescribed medication during the study period, determined by pill count at 4, 8, and 12 weeks of therapy. The protocol was approved by an institutional review board, and each patient gave written, informed consent. Data Analysis Any patient who received at least one dose of study medication qualified for analysis of the frequency of adverse experiences. The primary analysis for efficacy was of the intention-to-treat group. In addition, any patient who took at least 60% of the doses of study medication during the time of study participation, who had not missed more than 3 consecutive days of study medication, who satisfied the inclusion or exclusion criteria, and who had an initial and final endoscopic examination was considered evaluable for efficacy. Figure 1. Percentage of evaluable and intent-to-treat patients with duodenal ulcers. P P The primary end point of the study was the development of a duodenal ulcer at follow-up endoscopic examination. An ulcer was defined as a circumscribed break in the duodenal or gastric mucosa of 0.5 cm in diameter (0.5 cm by 0.5 cm or larger) and with perceptible depth. The comparability of the misoprostol and placebo treatment groups with respect to demographic characteristics at admission was assessed for all patients enrolled in the study. These characteristics included age, sex, race, height, and weight. When expected cell sizes were adequate, nominal measures were compared using the Pearson chi-square test. Otherwise, the Fisher exact test was used. Height, weight, and age measures were compared in the two treatment groups using the Wilcoxon rank-sum test. Treatment group differences in the proportion of patients who completed the study and the proportion of evaluable patients were assessed using the Pearson chi-square test. Patients who discontinued study participation without an endoscopy at week 4, week 8, or week 12 were classified as unknown for each time period at which their endoscopy status was undetermined. A chi-square analysis of duodenal ulcer development during the 12-week period was then conducted. Secondary analyses using identical methods were done for gastric ulcer development and gastroduodenal ulcer development. The rates of developing ulcers presented above did not account for censoring (that is, patients who discontinued the study before endoscopic verification of outcome or before they finished the full 12 weeks of study participation). Therefore, rates of developing duodenal, gastric, and gastroduodenal ulcer were also compared in the two treatment groups using a life-table analysis, which provided actuarial method estimates of the rate of ulcer development. The grouping intervals used for the life-table were prestudy to week-4 endoscopy, week-4 to week-8 endoscopy, and week-8 to week-12 endoscopy. The actual number of days from the prestudy endoscopy to the week 4, week 8, and week 12 endoscopies was not used to construct the life-table intervals; consequently, they can only be considered nominal categories. The log-rank test was used to compare the survival distributions of the two treatment groups. Figure 2. Percentage of intention-to-treat patients developing ulcers during the 12-week study period. Additional analyses using a stepwise logistic regression model were done to assess the effect of selected demographic and medical history measures on ulceration. Age, sex, alcohol consumption, smoking, type of arthritis, baseline erosions, and treatment group were the independent variables for these models. A forward selection procedure was used, with step selection of variables based on the maximum likelihood ratio and resulting P values from the chi-square statistics. A variable entered the model if its P value for entry was less than 0.15 and was retained in the model if its P value for removal was 0.15. Treatment group was initially entered in each model before beginning the stepping process. For these analyses, type of arthritis was categorized as osteoarthritis versus rheumatoid arthritis; patients who had both osteoarthritis and ankylosing spondylitis, or both osteoarthritis and psoriatic arthritis, were categorized as having osteoarthritis. Patients who had the other types of arthritis (including both osteoarthritis and rheumatoid arthritis) were excluded from the logistic regression analyses. The qualitative investigator assessments (daytime and nighttime pain and upper gastrointestinal symptoms) were tabulated at each visit by treatment group for evaluable patients. The distributions of the investigators severity ratings for daytime and nighttime pain were each compared for the two treatment groups at 12 weeks using the Fisher exact test. For patients who reported daytime or nighttime pain at the prestudy visit, changes from prestudy to week 12 in daytime and nighttime pain were categorized as better, same, or worse and compared in the two treatment groups using the Pearson chi-square test or the Fisher exact test, depending on the distribution of responses. For patients who did not report daytime or nighttime pain at the prestudy visit, the proportion of patients who developed daytime or nighttime pain at week 12 was compared in the two treatment groups using the Pearson chi

Aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa.

Although the incidence of colon cancer increases with advancing age, reasons for this increase are not fully understood. Earlier studies have demonstrated that in Fischer-344 rats, aging is associated with increased crypt cell production in the colon, an event considered to be central to the initiation of carcinogenesis. Apoptosis also plays a critical role in the development and progression of colon cancer. Therefore, we have examined the age-related changes in proliferation and apoptosis in the colonic mucosa of 4-5, 12-14, and 22-24 month-old Fischer-344 rats. We have observed that proliferative activity in the colon, as assessed by proliferating cell nuclear antigen immunoreactivity, is higher (50-80%) in 12-14 and 22-24 month-old rats than in their 4-6 month-old counterparts. In contrast, the number of apoptotic cells, (as determined by TdT-mediated dUTP nick-end labeling assay) in the colonic mucosa of 12-14 and 22-24 month-old rats are considerably lower (50-60%) than in 4-6 month-old animals. These changes are accompanied by a concomitant reduction (75%) in pro-apoptotic Bak and stimulation (200%) of anti-apoptotic Bcl-xL levels. Since activation of caspases is associated with initiation and maintenance of apoptosis, we also analyzed the levels of pro and active forms of caspase-3, 8 and 9. The levels of active forms of caspase-3, 8 and 9 are found to be considerably (60-80%) lower in the colonic mucosa of 22-24 month-old rats, compared to their younger counterparts. This is accompanied by decreased cleavage of poly(ADP-ribose) polymerase, a substrate for caspases. In conclusion, our data show that aging enhances proliferation, but attenuates apoptosis in the colonic mucosa. These changes may partly be responsible for the age-related rise in colorectal cancer.

Increased colonic mucosal angiotensin I and II concentrations in Crohn's colitis

To define a potential role for the angiotensin system in Crohns colitis, the colonic mucosal levels of angiotensin I and II were measured in endoscopic biopsy samples from patients with active Crohns colitis (n = 20), ulcerative colitis (n = 13), other forms of colitis (n = 3), and normal controls (n = 17). Colonic mucosal levels of angiotensin I and II were greater in patients with Crohns colitis than in normal subjects (p less than 0.001 and p less than 0.001, respectively). Mucosal levels of angiotensin I and II were also higher in Crohns colitis than in ulcerative colitis (p less than 0.001 and p less than 0.001, respectively), and levels of angiotensin II were higher in Crohns than in other forms of colitis (p = 0.014). Mucosal levels of angiotensin I and II correlated well with the degree of macroscopic inflammation in Crohns colitis (r = 0.86, p less than 0.001 and r = 0.68, p less than 0.001, respectively). Mucosal levels of angiotensin I correlated fairly well with the Crohns Disease Activity Index (r = 0.46, p less than 0.05) while angiotensin II levels correlated poorly. These studies suggest that angiotensin I and II may have a role in the inflammation associated with Crohns colitis.

Folic acid inhibition of EGFR-mediated proliferation in human colon cancer cell lines

Although accumulating evidence suggests a chemopreventive role for folic acid in colon cancer, the regulation of this process in unknown. We hypothesize that supplemental folic acid exerts its chemopreventive role by inhibiting mucosal hyperproliferation, an event considered to be central to the initiation of carcinogenesis in the gastrointestinal tract. The present investigation examines the effect of supplemental folic acid on proliferation of Caco-2 and HCT-116 colon cancer cell lines. Furthermore, because certain tyrosine kinases, particularly epidermal growth factor receptor (EGFR), play a role in regulating cell proliferation, we also examined the folic acid-induced changes in tyrosine kinase activity and expression of EGFR. In Caco-2 and HCT-116 cells, maintained in RPMI 1640 medium containing 1 microg/ml folic acid, we observed that the supplemental folic acid inhibited proliferation in a dose-dependent manner. Pretreatment of HCT-116 and Caco-2 cell lines with supplemental folic acid (1.25 microg/ml) completely abrogated transforming growth factor-alpha (TGF-alpha)-induced proliferation in both cell lines. Tyrosine kinase activity and the relative concentration of EGFR were markedly diminished in both cell lines following a 24-h exposure to supplemental folic acid. The folic acid-induced inhibition of EGFR tyrosine kinase activity in colon cancer cell lines was also associated with a concomitant reduction in the relative concentration of the 14-kDa membrane-bound precursor form of TGF-alpha. In conclusion, our data suggest that supplemental folic acid is effective in reducing proliferation in two unrelated colon cancer cell lines and that EGFR tyrosine kinase appears to be involved in regulating this process.Although accumulating evidence suggests a chemopreventive role for folic acid in colon cancer, the regulation of this process in unknown. We hypothesize that supplemental folic acid exerts its chemopreventive role by inhibiting mucosal hyperproliferation, an event considered to be central to the initiation of carcinogenesis in the gastrointestinal tract. The present investigation examines the effect of supplemental folic acid on proliferation of Caco-2 and HCT-116 colon cancer cell lines. Furthermore, because certain tyrosine kinases, particularly epidermal growth factor receptor (EGFR), play a role in regulating cell proliferation, we also examined the folic acid-induced changes in tyrosine kinase activity and expression of EGFR. In Caco-2 and HCT-116 cells, maintained in RPMI 1640 medium containing 1 μg/ml folic acid, we observed that the supplemental folic acid inhibited proliferation in a dose-dependent manner. Pretreatment of HCT-116 and Caco-2 cell lines with supplemental folic acid (1.25 μg/ml) completely abrogated transforming growth factor-α (TGF-α)-induced proliferation in both cell lines. Tyrosine kinase activity and the relative concentration of EGFR were markedly diminished in both cell lines following a 24-h exposure to supplemental folic acid. The folic acid-induced inhibition of EGFR tyrosine kinase activity in colon cancer cell lines was also associated with a concomitant reduction in the relative concentration of the 14-kDa membrane-bound precursor form of TGF-α. In conclusion, our data suggest that supplemental folic acid is effective in reducing proliferation in two unrelated colon cancer cell lines and that EGFR tyrosine kinase appears to be involved in regulating this process.

Neutrophil chemotaxis in gastric mucosa - A signal-to-response comparison

This prospective study compares levels of neutrophil chemotactic activity (NCA) in gastric juice to the neutrophil count in gastric biopsies. Sixty-three male patients enrolled in the study and had antral biopsies following collection of gastric juice during esophagogastroduodenoscopy. Biopsies were examined for the magnitude of gastritis, tissue PMN count, and presence ofHelicobacter pylori. Secretions were assayed for neutrophil chemotactic activity. Results show an increase in NCA and in tissue PMN counts with increasing severity of gastritis.H. pylori-positive patients had higher levels of NCA and PMN thanH. pylori-negative patients. Wide variability in NCA levels preclude a direct correlation between NCA and PMN counts.

Inhibition of gastric mucosal regeneration by tyrphostin: Evaluation of the role of epidermal growth factor receptor tyrosine kinase

Although induction of mucosal cell proliferation is a crucial event in gastric mucosal regeneration after injury, intracellular regulatory processes have not been fully elucidated. We hypothesize that tyrosine kinases (Tyr-k)--specifically the enzyme associated with epidermal growth factor receptor (EGF-R)--play an important role in mucosal regeneration. Utilizing tyrphostin--a Tyr-k inhibitor with a greater specificity for EGF-R Tyr-k than for other Tyr-ks--we have examined the role of EGF-R Tyr-k in gastric mucosal regeneration after injury. Gastric mucosal injury in 3-to 4-month-old rats was induced by orogastric administration of 2 mol/L NaCl, whereas the control animals received an equivalent volume of water. The animals were killed 24 hours later. During this 24-hour experimental period (reparative phase), one of the groups was also injected (IP) with tyrphostin-51 (0.65 mg/kg in 30% dimethyl sulfoxide), whereas the control group received the vehicle. In the absence of tyrphostin, the gastric mucosa showed signs of extensive regeneration, whereas in its presence the degree of regeneration was greatly attenuated. These changes were accompanied by parallel alterations in the number of proliferating cell nuclear antigen-immunoreactive cells and the Tyr-k activity of EGF-R. In water-fed control animals, tyrphostin also caused a significant 30% reduction in proliferating cell nuclear antigen-immunoreactive cells. In these animals, the Tyr-k activity of EGF-R was also decreased by 30%. At 24 hours after injury, EGF-R mRNA levels were increased 36-fold over the water-fed controls, and this increase was not significantly affected by tyrphostin. Our current data suggest that activation of EGF-R is an important event in mucosal regeneration.

Differential Responsiveness of Proximal and Distal Colonic Mucosa to Gastrin

In vivo and in vitro experiments were performed to examine the responsiveness of the proximal and distal colonic mucosa to the growth-promoting action of gastrin. Infusion (osmotic minipump) of gastrin G-17-I (250 ng/kg/h) for 5 days to 4-month-old male Fischer-344 rats resulted in a significant (90-150%) increase in proliferative activity (as assessed by BrdU or PCNA immunoreactivity) in the distal colonic mucosa. In contrast, gastrin caused no apparent change in proliferative activity in the proximal colon. Because tyrosine kinases (Tyr-ks) are thought to be critically involved in regulating the trophic action of gastrin, responsiveness of isolated colonocytes from both segments of the colon to gastrin (1 x 10(-9) M) was also examined. Exposure of isolated colonocytes from the distal, but not from the proximal, colon to gastrin for 2 min resulted in a significant (73%) stimulation in Tyr-k activity. This was also accompanied by a marked rise in phosphorylation of at least six membrane proteins with M, of 55, 60, 70, 94, and 170 kDa. Tyr-k activity induced by gastrin in colonocytes from the distal colon was inhibited by tyrphostin (3.2 microM) but not by staurosporine (20 nM). In colonocytes from the distal colon, gastrin also stimulated phospholipase C (PLC) activity, which could also be inhibited by tyrphostin, but not by staurosporine. We conclude that mucosa of the distal, but not the proximal, colon responds to the trophic action of gastrin. Tyr-ks are thought to be involved in the regulation of this process.

Differential expression of EGFR during early reparative phase of the gastric mucosa between young and aged rats

Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-alpha. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-alpha were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-α (TGF-α) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-α. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-α were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.

Persistence of gastric ulcers caused by plain aspirin or nonsteroidal antiinflammatory agents in patients treated with a combination of cimetidine, antacids, and enteric-coated aspirin.

Twenty-three patients chronically ingesting plain aspirin or nonsteroidal antiinflammatory drugs, who had endoscopically proven solitary or multiple gastric ulcers, were treated for eight weeks with cimetidine and antacids. Plain aspirin and nonsteroidal antiinflammatory drugs were discontinued in all patients. Seven patients received enteric-coated aspirin throughout the treatment phase and continuously for the entire study period (2.5–12 months). The remainder of patients (N=16) did not receive enteric-coated aspirin. An endoscopy was performed to assess ulcer healing. None of seven patients receiving enteric-coated aspirin had complete healing of their ulcer(s) while 15 of 16 patients not receiving enteric-coated aspirin demonstrated complete healing of their ulcer(s) (P<0.001). An eight-week course of cimetidine and antacids is ineffective in completely healing gastric ulcers caused by plain aspirin or nonsteroidal antiinflammatory drugs while enteric-coated aspirin is continued.

Chemopreventive role of folic acid in colorectal cancer.

Mortality from colorectal cancer, a leading cause of death in the U.S.A. and other western countries, has remained unchanged over the past 45 years. Therefore, the search for strategies to prevent the development and progression of colorectal cancer has markedly intensified. Chemoprevention is one such strategy. Accumulating evidence suggests that folic acid, a water soluble vitamin, could be an effective chemopreventive agent for colorectal cancer. Results from several studies have demonstrated that a diet deficient in folic acid may be associated with an increased risk of colonic neoplasia, whereas dietary supplementation of this nutrient may be chemopreventive. Although the mechanisms by which folic acid exerts its chemopreventive role in colorectal carcinogenesis remain to be fully elucidated, supplemental folic acid has been shown to arrest the loss of heterozygosity (LOH) of the tumor suppressor gene DCC (deleted in colorectal cancer) and to stabilize its protein in normal appearing rectal mucosa of patients with colorectal adenomas. Data from in vitro studies utilizing colon cancer cell lines suggest that supplemental folic acid or its metabolite 5-methyltetrahydrofolate (5-MTF) attenuates the expression and activation of EGF-receptor (EGFR) as well as proliferation of cells. The folic acid mediated reduction of EGFR function could partly be the result of suppression of EGFR gene through increased methylation of CpG sequences within its promoter.