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No significant difference in neutrophil activation found among three H2RAs
Digestive and Liver Disease 39 (2007) 130–135
Alimentary Tract
No significant difference in neutrophil activation found among three H2RAs M. Fukuda, D. Shirasaka ∗ , N. Aoyama, I. Miki, M. Kachi, Y. Morita, T. Tamura, M. Kasuga Division of Digestive Disease, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe, Hyogo 650-0017, Japan Received 25 March 2006; accepted 19 September 2006 Available online 11 December 2006
Abstract Background. Even with the most effective treatment, Helicobacter pylori eradication is difficult in some patients. Therefore, patients sometimes require acid-suppressive therapy without H. pylori eradication. It has been reported that ranitidine inhibits neutrophil activation, whereas famotidine does not. However, few studies have been published concerning the activation of neutrophils before and after treatment using clinical doses of histamine-2 receptor antagonists in patients with H. pylori infection. Aim. To examine the effects of neutrophil activation after treatment with three different histamine-2 receptor antagonists. Patients. This prospective, open-label, randomised, parallel-group study was conducted. Thirty patients with H. pylori infection were enrolled. These subjects were randomly assigned to receive one of the following treatments: (a) 150 mg ranitidine, (b) 20 mg famotidine, or (c) 10 mg lafutidine b.d., for 4 weeks. Before and after histamine-2 receptor antagonist treatment, histological findings, myeloperoxidase activity, and interleukin-8 in the gastric mucosa were evaluated. Results. On the basis of the histological findings between before and after histamine-2 receptor antagonist treatment, no significant differences were found in any groups. Similarly, there were no significant differences in myeloperoxidase activity or interleukin-8 levels. Conclusion. In patients with H. pylori, when used at clinical doses, any histamine-2 receptor antagonists can be used without concerning about inhibition of neutrophil activation. © 2006 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: H. pylori; Histamine-2 receptor antagonist; Neutrophil activation
1. Introduction Eradication of H. pylori infection plays an important role in the treatment and prevention of peptic ulcer disease [1,2]. However, even with the current treatment regimens, about 10–20% of patients with gastro-duodenal ulcer will fail to eradicate H. pylori infection. For the treatment of functional dyspepsia (FD) patients with ulcer-like dyspepsia, proton pump inhibitors (PPI) or histamine-2 receptor antagonist (H2RA) are one of the most effective drugs [3]. Oftentimes, FD patients with H. pylori infection require acid-suppressive ∗
Corresponding author. Tel.: +81 78 382 5861; fax: +81 78 382 2080. E-mail address: [email protected] (D. Shirasaka).
therapy without H. pylori cure. Many recent studies have shown that acid-suppressive therapy aggravates corpus gastritis in patients with H. pylori infection [4], promoting the development of atrophic gastritis and increasing the risk of carcinogenesis [5]. However, the best H2RA for FD patients with H. pylori infection has not been determined. Several researchers have studied the association of H2RA with inhibition of neutrophil activation [6,7]. Ranitidine inhibits neutrophil activation in vitro, while cimetidine and famotidine do not [7]. Neutrophils play an important role in the development of inflammation and tissue injury by releasing a variety of inflammatory mediators [8]. Therefore, it is possible that in H. pylori-positive patients with neutrophil infiltration, some H2RAs do not inhibit neutrophil activation,
1590-8658/$30 © 2006 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016/j.dld.2006.09.019
M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135
and as a result, induce gastric mucosa injury. However, few studies have been reported concerning the infiltration and activation of neutrophils before and after treatment using clinical doses of H2RA in patients with H. pylori infection. The aim of this study was to examine the effects of inhibition of neutrophil activation after treatment with three H2RAs, ranitidine, famotidine and lafutidine, in patients with H. pylori infection. In addition, we aimed to determine which H2RA is suitable for patients with H. pylori infection.
2. Materials and methods 2.1. Subjects The Interstitial Review Board of Kobe University Hospital approved this study. Between October 2002 and September 2004, C13 -urea breath test (UBT) was performed in 212 patients, who requested the screening of H. pylori status. The cut-off value was set at 2.5‰ 20 min after taking 100 mg C13 -urea. Seventy-two patients with H. pylori infection were provided informed consent in accordance with the declaration of Helsinki and its later revision. Thirty consecutive patients (12 females and 18 males, mean age 28.6 ± 8.80, range 21–50) agreed to explanation and participated in this study. Written informed consent was obtained from all the subjects. This prospective, open-label, randomised, parallelgroup study was conducted at Kobe University Hospital in Japan. Exclusion criteria included subjects who had been taking other drugs, known drug allergy to the study drugs, gastric cancer, gastroduodenal ulcer, liver cirrhosis, renal failure, severe concomitant disease, pregnancy or lactation and previous gastric surgery. Serum pepsinogen (Dinabot, Tokyo, Japan) and gastrin (Kyowa medex, Tokyo, Japan) were assayed with a commercial radioimmunoassay kit according to the manufacturer’s protocol. Subjects were randomly assigned to receive one of the following treatments: (a) 20 mg famotidine b.d., for 4 weeks (famotidine group; n = 10), (b) 150 mg ranitidine b.d., for 4 weeks (ranitidine group; n = 10), or (c) 10 mg lafutidine b.d., for 4 weeks (lafutidine group; n = 10). Compliance and side effects were documented using a detailed questionnaire during treatment. Before and 4 weeks after treatments, subjects underwent upper gastrointestinal endoscopy and no gastric cancers or gastroduodenal ulcers were observed. Biopsy specimens were obtained from the greater curvatures of the gastric antrum and mid-corpus. Throughout the study, histological gastritis for each subject was examined on slides with two (4 m each) biopsy specimens stained with haematoxylin–eosin. Parameters assessed included neutrophil activity, chronic inflammation and glandular atrophy and were graded 0 (normal), 1 (mild), 2 (moderate) and 3 (markedly abnormal) according to the visual analogue scale of the updated Sydney System [9].
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2.2. Myeloperoxidase (MPO) activity and interleukin (IL)-8 in the gastric mucosa MPO activity in the gastric mucosa in the greater curvatures of antrum and mid-corpus were determined before and after H2RA treatments. MPO activity was determined with a commercial radioimmunoassay kit (Pharmacia, Tokyo, Japan) according to the manufacturer’s protocol and expressed as the degradation of H2 O2 mol/min/g tissue. IL8 levels in the gastric mucosa in the greater curvatures of antrum and mid-corpus were determined before and after H2RA treatments. The concentration of IL-8 was determined with an enzyme amplified sensitivity immunoassay (ELISA) kit (BioSource, Nivelles, Belgium) according to the manufacturer’s protocol and expressed in pg/ml protein. 2.3. Statistics Results are expressed as mean ± standard deviation. Grades for gastritis, MPO activity and IL-8 levels, before and after H2RA treatments were compared by the nonparametric Mann–Whitney U-test. A p-value of <0.05 was considered statistically significant.
3. Results 3.1. Clinical characteristics The clinical characteristics of the participants are given in Table 1. In age, sex, serum pepsinogen and serum gastrin, significant differences were not found among the three groups (Table 1). 3.2. Neutrophil activity, chronic inflammation and glandular atrophy In the famotidine group, there were no significant differences between before and after medication, in neutrophil activity, chronic inflammation and glandular atrophy in the greater curvatures of the gastric antrum and mid-corpus (Table 2). Similarly, in the ranitidine and lafutidine groups, histological gastritis scores for neutrophil activity, chronic inflammation and glandular atrophy had not changed by the completion of the study (Table 2). Although all subjects were subdivided according to neutrophil activity and chronic inflammation scores at baseline, histological gastritis score had not changed significantly at the completion of the study (data not shown). In spite of subdivision according to serum pepsinogen I/II ratio and gastrin at baseline, the results were the same. 3.3. MPO activity In MPO activity, there were no significant differences between before and after medication in the greater curvatures
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M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135
Table 1 Clinical characteristics of participants
n (M/F) Age Pepsinogen I (ng/ml) Pepsinogen II (ng/ml) Pepsinogen I/II Gastrin (pg/ml)
Famotidine group
Ranitidine group
Lafutidine group
p
10 (6/4) 28.2 ± 8.7 98.1 ± 95.8 19.5 ± 11.0 4.9 ± 2.8 108.4 ± 66.9
10 (7/3) 28.9 ± 8.9 62.3 ± 26.8 23.8 ± 11.1 3.0 ± 1.1 95.8 ± 56.2
10 (5/5) 28.7 ± 9.7 79.5 ± 33.9 23.4 ± 12.2 3.7 ± 1.4 139.7 ± 29.8
N.S. N.S. N.S. N.S. N.S. N.S.
Table 2 Results before and after medication in neutrophil activity, chronic inflammation, and glandular atrophy Antrum Before
p After
Mid-corpus
p
Before
After
N.S. N.S. N.S.
0.5 ± 0.71 1.3 ± 0.82 0.10 ± 0.32
0.4 ± 0.52 1.3 ± 0.82 0.10 ± −0.32
N.S. N.S. N.S.
0.9 ± 0.32 1.8 ± 0.92 0.70 ± 0.68
N.S. N.S. N.S.
1.1 ± 0.88 1.7 ± 1.1 0.30 ± 0.48
1.0 ± 0.67 1.57 ± 0.68 0.10 ± 0.32
N.S. N.S. N.S.
0.89 ± 0.60 1.9 ± 0.78 0.78 ± 0.67
N.S. N.S. N.S.
0.44 ± 0.53 1.8 ± 0.83 0.4 ± 0.53
0.56 ± 0.52 1.56 ± 0.88 0.33 ± 0.50
N.S. N.S. N.S.
Famotidine Activity Inflammation Atrophy
0.8 ± 0.92 1.9 ± 1.1 0.70 ± 0.68
0.5 ± 0.53 1.8 ± 0.92 0.6 ± 0.52
Ranitidine Activity Inflammation Atrophy
1.0 ± 0.67 2.3 ± 0.82 0.90 ± 0.57
Lafutidine Activity Inflammation Atrophy
0.56 ± 0.67 2.0 ± 0.87 1.1 ± 0.60
Fig. 1. Before and after medication for MPO activity in antrum (a) and mid-corpus (b). Significant differences were not found.
M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135
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Fig. 2. Before and after medication for IL-8 in antrum (a) and mid-corpus (b). Significant differences were not found.
of the gastric antrum (a) and mid-corpus (b) in the famotidine group (Fig. 1). Similarly, in the ranitidine and lafutidine groups, MPO activity was not changed at the completion of the study (Fig. 1). 3.4. IL-8 In IL-8, there were no significant differences between before and after medication in the greater curvatures of the gastric antrum (a) and mid-corpus (b) in the famotidine group (Fig. 2). Similarly, in the ranitidine and lafutidine group, IL-8 was not changed at the completion of the study (Fig. 2).
4. Discussion In this study, no significant differences between before and after H2RA treatment were found for histological findings, MPO activity and IL-8. To our knowledge, few reports have been published concerning neutrophil activation and infiltration in human gastric mucosa before and after continuous medication with H2RA in patients without peptic disease. Therefore, these results are important as clinical evidence concerning the choice of H2RA in patients with H. pylori infection.
MPO is sufficiently included in azurophil granules and MPO activity in the gastric mucosa reflects tissue-associated neutrophil accumulation [10,11]. IL-8 is a chemoattractant protein for neutrophils and attachment of H. pylori to gastric epithelial cells leads to the production of IL-8, which in turn activates and recruits neutrophils to the site of infection [12,13]. Thus, the concentration of IL-8 in the gastric mucosa indicates neutrophil infiltration in H. pylori gastritis. Okajima et al. [7] reported inhibition of neutrophil activation by ranitidine in vivo in rats with stress-induced gastric mucosa injury, whereas famotidine did not inhibit neutrophil activation. In vitro, Okajima et al. [14] reported that ranitidine inhibited an increase in intracellular concentration of ionized calcium ([Ca2+ ]i ), whereas famotidine did not. An increase in [Ca2+ ]i plays an important role in neutrophil elastase release, ranitidine inhibited the activation of neutrophils, but famotidine enhanced this activation. On the contrary, Mikawa et al. [6] demonstrated that ranitidine had no effect on the increase in [Ca2+ ]i , while famotidine did have an effect. Concerning the inhibition induced by H2RA on neutrophil activation, there is still no consensual agreement among investigators. If an H2RA inhibits neutrophil activation, then that H2RA would be suitable for patients with H. pylori infection. The reason is that in patients with H. pylori infection, neutrophil infiltration is induced, and therefore any H2RA that can
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inhibit neutrophil activation, would be useful to prevent gastric mucosa injury in H. pylori-positive patients. However, our study contradicted previous reports. We found no effect on inhibition of neutrophil activation in any of the H2RAs examined. The reasons for this contradiction may be attributed to the factors described below. First, Okajima et al. carried out their investigation in rats with stress-induced gastric mucosal injury, whereas our subjects were patients with mild to moderate neutrophil infiltration, with an average neutrophilic score in the mid-corpus of 0.7 ± 0.8. In rat models subjected to water-immersion restraint stress, neutrophil infiltration in the gastric mucosa was observed, and in this condition, inhibition of neutrophil activation induced by H2RA was found. In patients with severe neutrophil infiltration induced by acute stresses or inflammation, the H2RA should be selected according to not only gastric acid antisecretory effect, but also the inhibition on neutrophil activation. However, in this study, even in patients with severe neutrophil infiltration, inhibition of neutrophil activation was not observed (data not shown). Therefore, in H. pylori-positive FD patients with mild to moderate neutrophil infiltration, any H2RA may be used. Second, the doses of H2RA used in previous in vivo and in vitro studies were much higher than those used in clinical patients. Yuki et al. [15] reported that the effective dose of famotidine that inhibits basal gastric acid secretion in 50% (ED50) was 0.43 mg/kg, whereas the famotidine dose used in Okajima et al [14] study was much higher, 5 mg/kg. Echizen et al. [16] reported that after oral administration of 40 mg famotidine, the maximum drug concentration in the plasma was approximately 0.1 M, whereas the famotidine doses used in in vitro studies were from 1 to 1000 M [14]. The plasma level of ranitidine in humans after an oral dose of 150 mg of ranitidine has been reported to be about 1.5 M [17], whereas the inhibition of neutrophil elastase release required more than 100 M of ranitidine [14]. Therefore, in high doses of H2RA, significant differences may be found; however, in the doses used clinically, no significant differences among the H2RAs examined were found concerning the inhibition of neutrophil activation. It is well known that effect of a gastric acid secretion inhibitor on histological gastritis varies by distribution of gastritis before therapy. It has been reported that pangastritis (active gastritis on the antrum and corpus) is at a high-risk for the development of gastric cancer. Patients taking H2RA were not in high-risk groups. However, for the development of atrophy, 4 weeks were not enough. We need more investigation for long time.
5. Conclusion In patients with H. pylori, no significant differences among H2RAs were found concerning the inhibition of neutrophil activation. When used at clinical doses, any H2RA can be
used without concern about neutrophil activation and gastric cancer development.
Practice points • Oftentimes, FD patients with H. pylori infection require acid suppressive therapy without H. pylori cure. • The association of H2RA with inhibition of neutrophil activation has been studied. • No significant differences in histological findings, MPO activity, or IL-8 before and after H2RA treatment were found in this study. • Pangastritis, a high-risk for gastric cancer development, was not induced after taking any H2RAs for 4 weeks. • When used at clinical doses, any H2RA can be used without concern about neutrophil activation and gastric cancer development.
Research agenda • Four weeks were not enough for development of atrophy. • A longer investigation time is needed.
Conflict of interest statement None declared. References [1] Caselli M, Parente F, Palli D, Covacci A, Alvisi V, Gasbarrini G, et al. ‘Cervia Working Group Report’: guidelines on the diagnosis and treatment of Helicobacter pylori infection. Dig Liver Dis 2001;33:75–80. [2] Vaira U, Gatta L, Ricci C, D’Anna L, Iglioli MM. Helicobacter pylori: diseases, tests and treatment. Dig Liver Dis 2001;33:788–94. [3] Talley NJ, Lauritsen K. The potential role of acid suppression in functional dyspepsia: the BOND, OPERA, PILOT, and ENCORE studies. Gut 2002;50:iv36–41. [4] Kuipers EJ, Uyterlinde AM, Pena AS, Hazenberg HJ, Bloemena E, Lindeman J, et al. Increase of Helicobacter pylori-associated corpus gastritis during acid suppressive therapy: implications for long-term safety. Am J Gastroenterol 1995;90:1401–6. [5] Logan RP, Walker MM, Misiewicz JJ, Gummett PA, Karim QN, Baron JH. Changes in the intragastric distribution of Helicobacter pylori during treatment with omeprazole. Gut 1995;36:12–6. [6] Mikawa K, Akamatsu H, Nishina K, Shiga M, Maekawa N, Obara H, et al. The effects of cimetidine, ranitidine, and famotidine on human neutrophil functions. Anesth Analg 1999;89:218–24. [7] Okajima K, Murakami K, Liu W, Uchiba M. Inhibition of neutrophil activation by ranitidine contributes to prevent stress-induced gastric mucosal injury in rats. Crit Care Med 2000;28:2858–65. [8] Harlan JM. Leukocyte-endothelial interaction. Blood 1985;65:513–25.
M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135 [9] Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol 1996;20:1161–81. [10] Matheson NR, Wong PS, Travis J. Isolation and properties of human neutrophil myeloperoxidase. Biochemistry 1981;20:325–30. [11] Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. [12] Tummuru MK, Cover TL, Blaser MJ. Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun 1993;61:1799–809. [13] Crabtree JE, Farmery SM, Lindley IJ, Figura N, Peichl P, Tompkins DS. CagA/cytotoxic strains of Helicobacter pylori and interleukin-
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8 in gastric epithelial cell lines. J Clin Pathol 1994;47:945– 50. Okajima K, Harada N, Uchiba M. Ranitidine reduces ischemia/ reperfusion-induced liver injury in rats by inhibiting neutrophil activation. J Pharmacol Exp Ther 2002;301:1157–65. Yuki H, Kamato T, Nishida A, Fujihara A, Takeda M, Miyata K. Antisecretory effects of a novel and long-lasting histamine H2receptor antagonist, YM-14471, in rats and dogs. Jpn J Pharmacol 1993;63:345–51. Echizen H, Ishizaki T. Clinical pharmacokinetics of famotidine. Clin Pharmacokinet 1991;21:178–94. Castaneda-Hernandez G, Flores-Murrieta FJ, Granados-Soto V, Herrera-Abarca A, Perez-Urizar J, Herrera JE, et al. Pharmacokinetics of oral ranitidine in Mexicans. Arch Med Res 1996;27:349–52.
Alimentary Tract
No significant difference in neutrophil activation found among three H2RAs M. Fukuda, D. Shirasaka ∗ , N. Aoyama, I. Miki, M. Kachi, Y. Morita, T. Tamura, M. Kasuga Division of Digestive Disease, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe, Hyogo 650-0017, Japan Received 25 March 2006; accepted 19 September 2006 Available online 11 December 2006
Abstract Background. Even with the most effective treatment, Helicobacter pylori eradication is difficult in some patients. Therefore, patients sometimes require acid-suppressive therapy without H. pylori eradication. It has been reported that ranitidine inhibits neutrophil activation, whereas famotidine does not. However, few studies have been published concerning the activation of neutrophils before and after treatment using clinical doses of histamine-2 receptor antagonists in patients with H. pylori infection. Aim. To examine the effects of neutrophil activation after treatment with three different histamine-2 receptor antagonists. Patients. This prospective, open-label, randomised, parallel-group study was conducted. Thirty patients with H. pylori infection were enrolled. These subjects were randomly assigned to receive one of the following treatments: (a) 150 mg ranitidine, (b) 20 mg famotidine, or (c) 10 mg lafutidine b.d., for 4 weeks. Before and after histamine-2 receptor antagonist treatment, histological findings, myeloperoxidase activity, and interleukin-8 in the gastric mucosa were evaluated. Results. On the basis of the histological findings between before and after histamine-2 receptor antagonist treatment, no significant differences were found in any groups. Similarly, there were no significant differences in myeloperoxidase activity or interleukin-8 levels. Conclusion. In patients with H. pylori, when used at clinical doses, any histamine-2 receptor antagonists can be used without concerning about inhibition of neutrophil activation. © 2006 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: H. pylori; Histamine-2 receptor antagonist; Neutrophil activation
1. Introduction Eradication of H. pylori infection plays an important role in the treatment and prevention of peptic ulcer disease [1,2]. However, even with the current treatment regimens, about 10–20% of patients with gastro-duodenal ulcer will fail to eradicate H. pylori infection. For the treatment of functional dyspepsia (FD) patients with ulcer-like dyspepsia, proton pump inhibitors (PPI) or histamine-2 receptor antagonist (H2RA) are one of the most effective drugs [3]. Oftentimes, FD patients with H. pylori infection require acid-suppressive ∗
Corresponding author. Tel.: +81 78 382 5861; fax: +81 78 382 2080. E-mail address: [email protected] (D. Shirasaka).
therapy without H. pylori cure. Many recent studies have shown that acid-suppressive therapy aggravates corpus gastritis in patients with H. pylori infection [4], promoting the development of atrophic gastritis and increasing the risk of carcinogenesis [5]. However, the best H2RA for FD patients with H. pylori infection has not been determined. Several researchers have studied the association of H2RA with inhibition of neutrophil activation [6,7]. Ranitidine inhibits neutrophil activation in vitro, while cimetidine and famotidine do not [7]. Neutrophils play an important role in the development of inflammation and tissue injury by releasing a variety of inflammatory mediators [8]. Therefore, it is possible that in H. pylori-positive patients with neutrophil infiltration, some H2RAs do not inhibit neutrophil activation,
1590-8658/$30 © 2006 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016/j.dld.2006.09.019
M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135
and as a result, induce gastric mucosa injury. However, few studies have been reported concerning the infiltration and activation of neutrophils before and after treatment using clinical doses of H2RA in patients with H. pylori infection. The aim of this study was to examine the effects of inhibition of neutrophil activation after treatment with three H2RAs, ranitidine, famotidine and lafutidine, in patients with H. pylori infection. In addition, we aimed to determine which H2RA is suitable for patients with H. pylori infection.
2. Materials and methods 2.1. Subjects The Interstitial Review Board of Kobe University Hospital approved this study. Between October 2002 and September 2004, C13 -urea breath test (UBT) was performed in 212 patients, who requested the screening of H. pylori status. The cut-off value was set at 2.5‰ 20 min after taking 100 mg C13 -urea. Seventy-two patients with H. pylori infection were provided informed consent in accordance with the declaration of Helsinki and its later revision. Thirty consecutive patients (12 females and 18 males, mean age 28.6 ± 8.80, range 21–50) agreed to explanation and participated in this study. Written informed consent was obtained from all the subjects. This prospective, open-label, randomised, parallelgroup study was conducted at Kobe University Hospital in Japan. Exclusion criteria included subjects who had been taking other drugs, known drug allergy to the study drugs, gastric cancer, gastroduodenal ulcer, liver cirrhosis, renal failure, severe concomitant disease, pregnancy or lactation and previous gastric surgery. Serum pepsinogen (Dinabot, Tokyo, Japan) and gastrin (Kyowa medex, Tokyo, Japan) were assayed with a commercial radioimmunoassay kit according to the manufacturer’s protocol. Subjects were randomly assigned to receive one of the following treatments: (a) 20 mg famotidine b.d., for 4 weeks (famotidine group; n = 10), (b) 150 mg ranitidine b.d., for 4 weeks (ranitidine group; n = 10), or (c) 10 mg lafutidine b.d., for 4 weeks (lafutidine group; n = 10). Compliance and side effects were documented using a detailed questionnaire during treatment. Before and 4 weeks after treatments, subjects underwent upper gastrointestinal endoscopy and no gastric cancers or gastroduodenal ulcers were observed. Biopsy specimens were obtained from the greater curvatures of the gastric antrum and mid-corpus. Throughout the study, histological gastritis for each subject was examined on slides with two (4 m each) biopsy specimens stained with haematoxylin–eosin. Parameters assessed included neutrophil activity, chronic inflammation and glandular atrophy and were graded 0 (normal), 1 (mild), 2 (moderate) and 3 (markedly abnormal) according to the visual analogue scale of the updated Sydney System [9].
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2.2. Myeloperoxidase (MPO) activity and interleukin (IL)-8 in the gastric mucosa MPO activity in the gastric mucosa in the greater curvatures of antrum and mid-corpus were determined before and after H2RA treatments. MPO activity was determined with a commercial radioimmunoassay kit (Pharmacia, Tokyo, Japan) according to the manufacturer’s protocol and expressed as the degradation of H2 O2 mol/min/g tissue. IL8 levels in the gastric mucosa in the greater curvatures of antrum and mid-corpus were determined before and after H2RA treatments. The concentration of IL-8 was determined with an enzyme amplified sensitivity immunoassay (ELISA) kit (BioSource, Nivelles, Belgium) according to the manufacturer’s protocol and expressed in pg/ml protein. 2.3. Statistics Results are expressed as mean ± standard deviation. Grades for gastritis, MPO activity and IL-8 levels, before and after H2RA treatments were compared by the nonparametric Mann–Whitney U-test. A p-value of <0.05 was considered statistically significant.
3. Results 3.1. Clinical characteristics The clinical characteristics of the participants are given in Table 1. In age, sex, serum pepsinogen and serum gastrin, significant differences were not found among the three groups (Table 1). 3.2. Neutrophil activity, chronic inflammation and glandular atrophy In the famotidine group, there were no significant differences between before and after medication, in neutrophil activity, chronic inflammation and glandular atrophy in the greater curvatures of the gastric antrum and mid-corpus (Table 2). Similarly, in the ranitidine and lafutidine groups, histological gastritis scores for neutrophil activity, chronic inflammation and glandular atrophy had not changed by the completion of the study (Table 2). Although all subjects were subdivided according to neutrophil activity and chronic inflammation scores at baseline, histological gastritis score had not changed significantly at the completion of the study (data not shown). In spite of subdivision according to serum pepsinogen I/II ratio and gastrin at baseline, the results were the same. 3.3. MPO activity In MPO activity, there were no significant differences between before and after medication in the greater curvatures
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M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135
Table 1 Clinical characteristics of participants
n (M/F) Age Pepsinogen I (ng/ml) Pepsinogen II (ng/ml) Pepsinogen I/II Gastrin (pg/ml)
Famotidine group
Ranitidine group
Lafutidine group
p
10 (6/4) 28.2 ± 8.7 98.1 ± 95.8 19.5 ± 11.0 4.9 ± 2.8 108.4 ± 66.9
10 (7/3) 28.9 ± 8.9 62.3 ± 26.8 23.8 ± 11.1 3.0 ± 1.1 95.8 ± 56.2
10 (5/5) 28.7 ± 9.7 79.5 ± 33.9 23.4 ± 12.2 3.7 ± 1.4 139.7 ± 29.8
N.S. N.S. N.S. N.S. N.S. N.S.
Table 2 Results before and after medication in neutrophil activity, chronic inflammation, and glandular atrophy Antrum Before
p After
Mid-corpus
p
Before
After
N.S. N.S. N.S.
0.5 ± 0.71 1.3 ± 0.82 0.10 ± 0.32
0.4 ± 0.52 1.3 ± 0.82 0.10 ± −0.32
N.S. N.S. N.S.
0.9 ± 0.32 1.8 ± 0.92 0.70 ± 0.68
N.S. N.S. N.S.
1.1 ± 0.88 1.7 ± 1.1 0.30 ± 0.48
1.0 ± 0.67 1.57 ± 0.68 0.10 ± 0.32
N.S. N.S. N.S.
0.89 ± 0.60 1.9 ± 0.78 0.78 ± 0.67
N.S. N.S. N.S.
0.44 ± 0.53 1.8 ± 0.83 0.4 ± 0.53
0.56 ± 0.52 1.56 ± 0.88 0.33 ± 0.50
N.S. N.S. N.S.
Famotidine Activity Inflammation Atrophy
0.8 ± 0.92 1.9 ± 1.1 0.70 ± 0.68
0.5 ± 0.53 1.8 ± 0.92 0.6 ± 0.52
Ranitidine Activity Inflammation Atrophy
1.0 ± 0.67 2.3 ± 0.82 0.90 ± 0.57
Lafutidine Activity Inflammation Atrophy
0.56 ± 0.67 2.0 ± 0.87 1.1 ± 0.60
Fig. 1. Before and after medication for MPO activity in antrum (a) and mid-corpus (b). Significant differences were not found.
M. Fukuda et al. / Digestive and Liver Disease 39 (2007) 130–135
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Fig. 2. Before and after medication for IL-8 in antrum (a) and mid-corpus (b). Significant differences were not found.
of the gastric antrum (a) and mid-corpus (b) in the famotidine group (Fig. 1). Similarly, in the ranitidine and lafutidine groups, MPO activity was not changed at the completion of the study (Fig. 1). 3.4. IL-8 In IL-8, there were no significant differences between before and after medication in the greater curvatures of the gastric antrum (a) and mid-corpus (b) in the famotidine group (Fig. 2). Similarly, in the ranitidine and lafutidine group, IL-8 was not changed at the completion of the study (Fig. 2).
4. Discussion In this study, no significant differences between before and after H2RA treatment were found for histological findings, MPO activity and IL-8. To our knowledge, few reports have been published concerning neutrophil activation and infiltration in human gastric mucosa before and after continuous medication with H2RA in patients without peptic disease. Therefore, these results are important as clinical evidence concerning the choice of H2RA in patients with H. pylori infection.
MPO is sufficiently included in azurophil granules and MPO activity in the gastric mucosa reflects tissue-associated neutrophil accumulation [10,11]. IL-8 is a chemoattractant protein for neutrophils and attachment of H. pylori to gastric epithelial cells leads to the production of IL-8, which in turn activates and recruits neutrophils to the site of infection [12,13]. Thus, the concentration of IL-8 in the gastric mucosa indicates neutrophil infiltration in H. pylori gastritis. Okajima et al. [7] reported inhibition of neutrophil activation by ranitidine in vivo in rats with stress-induced gastric mucosa injury, whereas famotidine did not inhibit neutrophil activation. In vitro, Okajima et al. [14] reported that ranitidine inhibited an increase in intracellular concentration of ionized calcium ([Ca2+ ]i ), whereas famotidine did not. An increase in [Ca2+ ]i plays an important role in neutrophil elastase release, ranitidine inhibited the activation of neutrophils, but famotidine enhanced this activation. On the contrary, Mikawa et al. [6] demonstrated that ranitidine had no effect on the increase in [Ca2+ ]i , while famotidine did have an effect. Concerning the inhibition induced by H2RA on neutrophil activation, there is still no consensual agreement among investigators. If an H2RA inhibits neutrophil activation, then that H2RA would be suitable for patients with H. pylori infection. The reason is that in patients with H. pylori infection, neutrophil infiltration is induced, and therefore any H2RA that can
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inhibit neutrophil activation, would be useful to prevent gastric mucosa injury in H. pylori-positive patients. However, our study contradicted previous reports. We found no effect on inhibition of neutrophil activation in any of the H2RAs examined. The reasons for this contradiction may be attributed to the factors described below. First, Okajima et al. carried out their investigation in rats with stress-induced gastric mucosal injury, whereas our subjects were patients with mild to moderate neutrophil infiltration, with an average neutrophilic score in the mid-corpus of 0.7 ± 0.8. In rat models subjected to water-immersion restraint stress, neutrophil infiltration in the gastric mucosa was observed, and in this condition, inhibition of neutrophil activation induced by H2RA was found. In patients with severe neutrophil infiltration induced by acute stresses or inflammation, the H2RA should be selected according to not only gastric acid antisecretory effect, but also the inhibition on neutrophil activation. However, in this study, even in patients with severe neutrophil infiltration, inhibition of neutrophil activation was not observed (data not shown). Therefore, in H. pylori-positive FD patients with mild to moderate neutrophil infiltration, any H2RA may be used. Second, the doses of H2RA used in previous in vivo and in vitro studies were much higher than those used in clinical patients. Yuki et al. [15] reported that the effective dose of famotidine that inhibits basal gastric acid secretion in 50% (ED50) was 0.43 mg/kg, whereas the famotidine dose used in Okajima et al [14] study was much higher, 5 mg/kg. Echizen et al. [16] reported that after oral administration of 40 mg famotidine, the maximum drug concentration in the plasma was approximately 0.1 M, whereas the famotidine doses used in in vitro studies were from 1 to 1000 M [14]. The plasma level of ranitidine in humans after an oral dose of 150 mg of ranitidine has been reported to be about 1.5 M [17], whereas the inhibition of neutrophil elastase release required more than 100 M of ranitidine [14]. Therefore, in high doses of H2RA, significant differences may be found; however, in the doses used clinically, no significant differences among the H2RAs examined were found concerning the inhibition of neutrophil activation. It is well known that effect of a gastric acid secretion inhibitor on histological gastritis varies by distribution of gastritis before therapy. It has been reported that pangastritis (active gastritis on the antrum and corpus) is at a high-risk for the development of gastric cancer. Patients taking H2RA were not in high-risk groups. However, for the development of atrophy, 4 weeks were not enough. We need more investigation for long time.
5. Conclusion In patients with H. pylori, no significant differences among H2RAs were found concerning the inhibition of neutrophil activation. When used at clinical doses, any H2RA can be
used without concern about neutrophil activation and gastric cancer development.
Practice points • Oftentimes, FD patients with H. pylori infection require acid suppressive therapy without H. pylori cure. • The association of H2RA with inhibition of neutrophil activation has been studied. • No significant differences in histological findings, MPO activity, or IL-8 before and after H2RA treatment were found in this study. • Pangastritis, a high-risk for gastric cancer development, was not induced after taking any H2RAs for 4 weeks. • When used at clinical doses, any H2RA can be used without concern about neutrophil activation and gastric cancer development.
Research agenda • Four weeks were not enough for development of atrophy. • A longer investigation time is needed.
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