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862 867 ( 2002)

Long-Term Rebamipide Therapy Improves Helicobacter pylori-Associated Chronic Gastritis

K. HARUMA, MD,* M. ITO, MD, S. KIDO, MD, N. MANABE, MD, Y. KITADAI, MD, M. SUMII, MD, S. TANAKA, MD, M. YOSHIHARA, MD, and K. CHAYAMA, MD

We investigated an antiinammatory effect of rebamipide {2-(4-chlorobenzoylamino)-3[2(1H)-quinolinon-4-yl] propionic acid}, a gastroprotective agent, in H. pylori-associated gastritis. Eighty-six patients with H. pylori-positive chronic gastritis were enrolled: 53 were treated with rebamipide (300 mg daily for 12 months) and 33 served as controls. Signicant decreases in mononuclear cell inltration into the antrum and corpus were noted in the rebamipide treatment group (before vs after, 1.42 0.15 vs 1.02 0.15; P 0.01 and 1.60 0.15 vs 1.21 0.14; P 0.05, respectively). Levels of inltrating neutrophil were also decreased in the antrum (before vs after, 0.98 0.14 vs 0.70 0.13; P 0.05) and were associated with a decrease in iNOS production. Sera from patients treated with rebamipide showed a signicant decrease in gastrin (276.3 58.3 pg/ml vs 173.0 34.2 pg/ml; P 0.05), whereas no change was observed in the control group. These suggest that long-term rebamipide treatment improved histologic gastritis and decreased serum gastrin levels in H. pylori-associated gastritis.
KEY WORDS: rebamipide; H. pylori; gastritis; iNOS.

Helicobacter pylori plays an important role in the induction of chronic gastritis (1). It has been accepted that there is a strong association between H. pyloriassociated gastritis and gastric diseases including peptic ulcer and gastric cancer (2 4). Therefore, it is important to investigate the pathogenesis of chronic gastritis for developing methods to control the occurrence of gastric diseases. Rebamipide {2-(4-chlorobenzoylamino)-3-[2(1H)quinolinon-4-yl] propionic acid} is used in East Asian countries as a gastroprotective agent for the treatManuscript received June 28, 2001; revised manuscript received November 2, 2001; accepted November 23, 2001. From the *Division of Gastroenterology II, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan; and First Department of Internal Medicine and Health Service Center, Hiroshima University School of Medicine, Hiroshima, Japan. Address for reprint requests: Dr. Ken Haruma, Division of Gastroenterology II, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima Kurashiki, 701-0192, Japan.

ment of acute gastritis, including gastritis induced by nonsteroidal antiinammatory drugs, and peptic ulcers (5, 6). Recent in vitro studies have demonstrated the antiinammatory effect of rebamipide on gastric mucosal cells. Rebamipide suppresses activation of neutrophils induced by H. pylori and protects gastric epithelial cell (7). Production of free radicals by activated neutrophils is also inhibited by rebamipide (8). Increased mucosal levels of various cytokines including IL-1, -6, -8 and tumor necrosis factor (TNF)- play critical roles in the pathogenesis of H. pylori-associated gastritis (9, 10). Production of IL-8 by epithelial cells stimulated by H. pylori infection is a crucial factor in H. pylori-induced gastritis (1113). Rebamipide has been reported to decrease levels of various cytokines including IL-8 in vitro (14). In addition, we showed that expression of IL-8 is dramatDigestive Diseases and Sciences, Vol. 47, No. 4 (April 2002)

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EFFECT OF REBAMIPIDE ON GASTRITIS

TABLE 1. CLINICAL FEATURES
OF

REBAMIPIDE

AND

CONTROL GROUP Control group (N 33) 59.0 (3978) 17/16 223.4 78.1 3.11 0.45

Rebamipide group (N 53) Age [yr, mean (range)] Sex (men/women) Serum PG (pg/ml, mean PG I/II (mean SE) 60.9 (3682) 32/21 276.3 58.3 3.21 0.28

SE)

ically suppressed by short-term treatment with rebamipide in patients with H. pylori-associated gastritis (15). Many clinical studies have demonstrated that rebamipide accelerates ulcer healing and prevents ulcer recurrence (5, 6). No report, however, has discussed whether rebamipide administration can improve chronic gastritis. We studied the effect of rebamipide on chronic gastritis in humans. Severity of gastritis was assessed histologically before and after long-term treatment with rebamipide. Serum gastrin and pepsinogen levels were measured in the same subjects. Moreover, the production of inducible nitric oxide synthase (iNOS), which is induced by H. pylori infection and is closely associated with gastric carcinogenesis, was investigated. MATERIALS AND METHODS
Patients. This study was performed in a prospective and open-randomized manner. Eighty-six patients with dyspepsia were included, and all gave informed consent. All patients were positive for H. pylori as determined by CLO-test, Giemsa staining, [13C]urea breath test or serum IgG antibodies against H. pylori (Amrad). No patient who had undergone gastrectomy or was taking H2 blocker/proton pump inhibitors was included in the study. Endoscopic examination showed that no localized lesion was present in any patient. Patients were requested to visit the hospital every four weeks and were randomized to treatment. We excluded data of patients who dropped out of the study or who were lost to follow up. A few patients needed other treatment within 12 months and were also excluded from the study. After diagnosis of H. pylori infection, 53 patients (32 men/21 women, mean age 60.9 years) were treated with 300 mg rebamipide daily for 12 months, and 33 patients (17 men/16 women, mean age 59.0 years) were followed up without medication. We found no signicant side effects of rebamipide. Prior to and 12 months after treatment, routine endoscopic examination was performed on each patient; ve biopsy specimens were obtained: three from the antrum and two from the corpus. The degree of gastritis was determined from hematoxylin and eosin (HE)-stained sections and scored on a scale of 0 to 3 according to the updated Sydney system (16). Serum Gastrin and Pepsinogens. Fasting serum was collected from all patients. The samples were centrifuged immediately at 4C and stored at 20C until use. Serum concentrations of serum gastrin and pepsinogens were determined by radioimmunoassay (Dainabot, Tokyo, Japan).
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Immunohistochemistry. Immunohistochemical analysis was performed as we have described previously (17). Fourmicron sections of formalin-xed parafn-embedded tissues were deparafnized and hydrated. After treatment with pepsin (Biomeda, Foster City, California, USA) for 10 min, endogenous peroxidase was quenched with 3% H2O2 in methanol for 10 min followed by a phosphate-buffered saline (PBS) rinse. Nonspecic binding was blocked by a 20-min incubation with PBS containing 1.5% normal goat serum. Sections were then incubated with a polyclonal antibody against human iNOS (diluted 1/500 with PBS; Santa Cruz Biotechnology, California, USA) overnight at 4C. A biotin-labeled secondary antibody (anti-rabbit IgG sheep antibody) was applied and incubated for 30 min at room temperature and followed by ABC procedure (Dako). The sections were developed with DAB chromogen (Merck, Darmstadt, Germany), counterstained with Mayers hematoxylin, dehydrated, and mounted. Grading for iNOS. Immunoreactivity was graded on a scale of 0 to 3 (0, no stained cells; 1, a few stained cells; 2, a moderate number of stained cells; 3, most cells stained) as described previously (18). Two investigators independently scored the slides without information regarding the patients. Only clear cytoplasmic stainings in inammatory cells were judged as positive. Statistics. Results are reported as mean standard error (SE). Statistical analysis was performed by paired t-test with StatView software (SAS Institute Inc., Cary, North Carolina, USA). P 0.05 was considered statistically signicant.

RESULTS Changes in Histologic Features of Gastritis by Long-Term Treatment with Rebamipide. Clinical features of patients are given in Table 1; no difference was seen between the two groups. Changes in the histologic features of gastritis are summarized in Table 2. In the rebamipide-treatment group, inltration by mononuclear cells was decreased signicantly after 12 months of rebamipide therapy, but no signicant difference was found in the control group. Neutrophil inltration in the antrum also improved after rebamipide therapy. The grades of atrophy before and after the therapy, however, did not differ in either groups. Serum Gastrin and Pepsinogens. Fasting levels of serum gastrin decreased signicantly after long-term rebamipide therapy (before, 276.3 58.3 pg/ml; af-

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TABLE 2. CHANGES
IN

HISTOLOGIC STATUS

OF

GASTRITIS

WITH

LONG-TERM REBAMIPIDE THERAPY Control group (N 33) Before 1.52 0.21 1.92 0.16 1.03 0.17 1.13 0.14 1.52 0.18 1.89 0.14 After 1.48 0.21 1.74 0.19 1.29 0.19 1.11 0.17 1.48 0.18 1.64 0.14

Rebamipide group (N 53) Before Mononuclear cell inltration Corpus Antrum Neutrophil inltration Corpus Antrum Atrophy Corpus Antrum *P 0.05. P 0.01. 1.60 0.15* 1.42 0.15 1.18 0.17 0.98 0.14* 1.51 0.14 2.17 0.13 After 1.21 0.14* 1.02 0.15 0.98 0.15 0.70 0.13* 1.59 0.14 2.17 0.13

ter, 173.0 34.2 pg/ml; P 0.01; Figure 1), whereas no signicant difference was found in the control group (before, 223.4 78.1 pg/ml; after, 226.6 94.6 pg/ml). However, the serum pepsinogen I/II ratio was not altered in the rebamipide group (before, 3.21 0.28; after, 3.22 0.28; Figure 2) or the control group (before, 3.11 0.45; after, 2.96 0.44). Production of iNOS. A signicant decrease in the iNOS score was observed after long-term rebamipide therapy in both the antrum (before, 1.07 0.21; after, 0.37 0.15; P 0.05; Figure 3) and the corpus (before, 1.23 0.25; after, 0.32 0.15; P 0.01). No signicant difference was found in the control group in the antrum (before, 1.19 0.20; after, 1.09 0.24) or the corpus (before, 0.95 0.17; after, 0.84 0.19). The iNOS immunoreactivity was mainly detected primarily in the cytoplasm of inammatory cells including the neutrophils, lymphocytes and macrophages (Figure 4).

DISCUSSION H. pylori is involved in the pathogenesis of gastritis, peptic ulcer, and gastric cancer (1 4), and atrophic gastritis is found primarily in patients with H. pylori infection (1). This suggests that H. pylori plays a critical role in the promotion of atrophic gastritis, which is a risk factor for gastric cancer (2 4). Epidemiological studies have indicated that infection with H. pylori is a risk factor for gastric cancer, and the World Health Organization classied this infection as a denite biological carcinogen in 1994 (19, 20). Therefore, eradication of H. pylori should reduce the frequency of atrophic gastritis and contribute to cancer prevention. Rebamipide, a gastroprotective agent, has been used clinically for treatment of acute gastritis and peptic ulcer (5, 6). In vitro studies have demonstrated that rebamipide suppresses the production of several cytokines and free radicals that play an important role

Fig 1. Effect of rebamipide on serum levels of gastrin. Serum gastrin levels were measured by RIA in patients treated with rebamipide (A) and in controls (B). Sera were collected prior to the start of therapy (left) and after 12 months of therapy (right). **P 0.01. NS, not signicant.

Fig 2. Effect of rebamipide on pepsinogen I/II levels. Serum pepsinogen levels were measured by RIA in patients treated with rebamipide (A) and in controls (B). Sera were collected prior to the start of therapy (left) and after 12 months of therapy (right). NS, not signicant.
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Fig 3. Effect of rebamipide on iNOS production. Production of iNOS was investigated by immunohistochemical analysis. All specimens were scored as described in Materials and Methods section. The results are shown as mean SE in the patients treated with rebamipide (A) and in controls (B). Biopsy specimens were collected from the antrum and the corpus prior to the start of therapy (left) and after 12 months of therapy (right). *P 0.05. **P 0.01. NS, not signicant.

in H. pylori-associated gastritis (7, 8). However, no reported studies have investigated the effect of rebamipide on chronic gastritis in humans. In the present study, we demonstrated that rebamipide improved gastritis induced by H. pylori infection in vivo. Improvement of histologic gastritis might be one of the important mechanisms of rebamipide to protect human gastric mucosa. A recent study has demonstrated that H. pylori attaches to gastric epithelial cells and injects pathogenic proteins into the epithelial cells (21). Intracellular cascades are stimulated by these events, leading to expression of NF-B and IL-8 (21). IL-8 expression by epithelial cells is a critical event caused by CagApositive H. pylori infection (11). Cag A protein is commonly expressed in H. pylori strains in Japan (22). In a previous study, rebamipide therapy (300 mg/day for two weeks) dramatically decreased mucosal levels of IL-8 protein (15). In our 53 patients with long-term treatment of rebamipide, the status of H. pylori infection was unaffected by long-term rebamipide therapy (data not shown). Therefore, after H. pylori attaches to epithelial cells, rebamipide might block the events upstream of IL-8 production by the gastric mucosa and improve histologic gastritis. It has been believed that eradication of H. pylori may be necessary for improvement of H. pylori-associated gastritis. Longterm rebamipide therapy should be considered for patients in whom successful H. pylori eradication could not be obtained to improve gastritis.
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It is thought that the atrophic changes associated with chronic gastritis are reversible. We have previously reported improvement in histologic atrophy in some patients after H. pylori eradication (23). However, we did not observe improvement in atrophy after 12 months of treatment with rebamipide alone. Pepsinogen I/II ratios were also unaffected by rebamipide therapy. We have followed some patients treated with rebamipide for more than ve years, but we have found no obvious improvement in atrophy. Serum levels of gastrin were decreased by longterm administration of rebamipide. This suggests that rebamipide may improve not only histologic but also functional abnormalities caused by H. pylori infection. The reason for the alteration of gastrin level is not clear because we could not demonstrate an improvement in atrophy in the corpus histologically and serologically. Acid secretion is controlled by some lymphokines, including IL-1, which inhibits the acid secretion of parietal cells. We examined the level of IL-1 by enzyme-linked immunosorbent assay and conrmed that rebamipide does not affect the IL-1 level in gastric mucosa (data not shown). The most likely mechanism of normalizing gastrin levels is the escape from controls of some lymphokines (except for IL-1) by improvement of histologic gastritis. Next, we have to evaluate acid output in the present cases and conrm whether rebamipide can improve acid output or not. It has been proposed that reactive oxygen and

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(18). In addition, in the present patients, we have conrmed the simultaneous expression of iNOS and nitrotyrosine. The production of iNOS, primarily by inammatory cells, was strongly suppressed by longterm rebamipide therapy, indicating that rebamipide may be useful for the prevention of gastric cancer in high risk subjects when H. pylori eradication is not possible. In vitro studies have demonstrated that rebamipide decreases the oxidative stress increased by H. pylori infection, supporting our present result (29). In conclusion, long-term rebamipide therapy is an effective treatment for chronic gastritis associated with H. pylori infection, and it may improve gastric function. Further studies are needed to assess improvement of clinical symptoms and possible indications for use of rebamipide in patients with functional dyspepsia. REFERENCES
1. Kawaguchi H, Haruma K, Komoto K, Yoshihara M, Sumii K, Kajiyama G: Helicobacter pylori infection is the major risk factor for atrophic gastritis. Am J Gastroenterol 91:959 962, 1996 2. Correa P: Helicobacter pylori and gastric carcinogenesis. Am J Surg Path 19(suppl 1):S37S43, 1995 3. Kohmoto K, Haruma K, Kamada T, Tanaka S, Yoshihara M, Sumii K, Kajiyama G, Talley NJ: Helicobacter pylori infection and gastric neoplasia: correlations with histological gastritis and tumor histology. Am J Gastroenterol 93:12711276, 1998 4. Haruma K, Komoto K, Kamada T, Ito M, Kitadai Y, Yoshihara M, Sumii K, Kajiyama G: Helicobacter pylori is a major risk factor for gastric carcinoma in young patients. Scand J Gastroenterol 35:255259, 2000 5. Naito Y, Yoshikawa T, Iinuma S, Yagi N, Matsuyama K, Boku Y, Fujii T, Yoshida N, Kondo M, Sasaki E: Rebamipide protects against indomethacin-induced gastric mucosal injury in healthy volunteers in double-blind, placebo-controlled study. Dig Dis Sci 43:83S 89S, 1998 6. Nebiki H, Higuchi K, Arakawa T, Ando K, Uchida T, Ito H, Harihara S, Kuroki T, Kobayashi K: Effect of rebamipide on Helicobacter pylori infection in patients with peptic ulcer. Dig Dis Sci 43:203S206S, 1998 7. Han BG, Kim HS, Rhee KH, Han HS, Chung MH: Effects of rebamipide on gastric cell damage by Helicobacter pyloristimulated human neutrophils. Pharmacol Res 32:201207, 1995 8. Yoshikawa T, Naito Y, Tanigawa T, Kondo M: Free radical scavenging activity of the novel anti-ulcer agent rebamipide studied by electron spin resonanse. Arzneim-Forsch/Drug Res 43:363366, 1993 9. Moss SF, Legon S, Davies J, Calam J: Cytokine gene expression in Helicobacter pylori associated antral gastritis. Gut 35:15671570, 1994 10. Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN: Mucosal tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8 production in patients with Helicobacter pylori infection. Scand J Gastroenterol 29:425 429, 1994
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Fig 4. Immunohistochemical staining of iNOS. Expression of iNOS was determined by immunohistochemical staining. Parafnembedded sections were obtained from gastric biopsies of a 56year-old female before (A) and after administration (B) of rebamipide. Positive signals were found primarily in the cytoplasm of mononuclear cells in the section of pre-administration of rebamipide. (120).

nitrogen species play roles in carcinogenesis (24). Reactive nitrogen species derived from nitric oxide are generated by iNOS in a variety of cell types, including activated macrophages and neutrophils (25). Increased activity of iNOS has been observed in patients with chronic gastritis and gastric cancer (26, 27). Recent studies have shown that H. pylori infection leads to formation of nitrotyrosine, which may contribute to DNA damage and apoptosis in the gastric mucosa (28). It is likely that iNOS expression is associated closely with gastric carcinogenesis. As we have previously reported, iNOS expression does not simply correlate with the degree of inammation and H. pylori status (18). Expressions of iNOS and nitrotyrosine were signicantly higher in the gastric cancer patients than in H. pylori-positive noncancer patients

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