Bovine lactoferrin is effective to suppress Helicobacter pylori colonization in the human stomach: A randomized, double-blind, placebo-controlled study

J Infect Chemother (2005) 11:265–269 DOI 10.1007/s10156-005-0407-x

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2005

ORIGINAL ARTICLE Masumi Okuda · Teruko Nakazawa · Koji Yamauchi Eikichi Miyashiro · Reiko Koizumi · Mina Booka Susumu Teraguchi · Yoshitaka Tamura Norishige Yoshikawa · Yukihiko Adachi · Ichiro Imoto

Bovine lactoferrin is effective to suppress Helicobacter pylori colonization in the human stomach: a randomized, double-blind, placebo-controlled study

Received: April 28, 2005 / Accepted: August 5, 2005

Abstract Bovine lactoferrin (bLF) has antibacterial activity against Helicobacter pylori in vitro and is effective to suppress bacterial colonization in mice. The aim of our study was to evaluate the efficacy of orally administered bLF on H. pylori colonization in humans by a randomized, double-blind, placebo-controlled study. Fifty-nine healthy subjects positive for H. pylori infection were recruited. Subjects were randomized into two groups. The bLF group received bLF tablets at a dosage of 200 mg b.i.d. for a period of 12 weeks, and the control group received placebo tablets without bLF. The 13C-urea breath test (UBT) was performed before, during, and at the end of administration, and again 4 weeks after administration. Positive response was defined as more than 50% decrease of the UBT value at the end of administration. Positive response was observed in 10 of 31 bLF-treated subjects (32.3%) and 1 of 28 control subjects (3.6%), indicating that the rate of positive response in the bLF group was significantly higher than that in the control group (bLF vs. control, P < 0.01). These results suggested that bLF administration is effective to suppress H. pylori colonization.

M. Okuda (*) · E. Miyashiro Department of Pediatrics, Wakayama Rosai Hospital, Wakayama, Koya 435, Wakayama 460-8435, Japan Tel. +81-73-451-3181; Fax +81-73-452-7171 e-mail: [email protected] T. Nakazawa Department of Microbiology, Yamaguchi University School of Medicine, Ube, Japan K. Yamauchi · R. Koizumi · S. Teraguchi · Y. Tamura Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Japan M. Booka · N. Yoshikawa Department of Pediatrics, Wakayama Medical University, Wakayama, Japan Y. Adachi · I. Imoto Department of Endoscopic Medicine, Mie University School of Medicine, Tsu, Japan

Key words Bovine lactoferrin · Helicobacter pylori · UBT (urea breath test)

Introduction Lactoferrin (LF) is an 80-kDa iron-binding glycoprotein that is found in milk and to a lesser extent in saliva, tears, and other exocrine secretions, as well as in specific granules of polymorphonuclear leukocytes.1 Antibacterial properties of LF against various gram-negative bacteria have been reported. The bacteriostatic activity of LF is attributed to iron depletion under iron-limiting conditions,2 whereas bactericidal activity is attributed to membrane disruption by LF as well as by a pepsin-derived fragment of LF that has the capacity to bind the lipid A portion of LPS.3,4 After successful cultivation of Helicobacter pylori, a curved or spiral gram-negative microaerophilic bacterium,5 evidence has been accumulating to indicate that H. pylori infection plays a significant role in the development of chronic gastritis, peptic ulcer diseases, mucosa-associated lymphoid tissue lymphoma, and gastric cancer.6–8 Triple therapy with two antibiotics and a proton pump inhibitor is established for patients with gastric diseases caused by H. pylori, but alternative therapeutic approaches are required because of the emergence of antibiotic resistance.9,10 Bovine lactoferrin (bLF) has been demonstrated to have antibacterial activity to H. pylori in vitro.11 The administration of bLF to H. pylori-infected mice resulted in marked decrease of bacterial colonization, attachment, and inflammation irrespective of iron-free or partially iron-saturated preparations.12,13 In humans with H. pylori infection, Di Mario et al. showed that bLF combined in a triple eradication regimen (rabeprazole, clarithromycin, and tinidazole) significantly improved the eradication rate.14 In the present study, we investigated the efficacy of a single administration of bLF in a randomized, double-blind, placebo-controlled trial.

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Methods and subjects Study design and subjects This study enrolled 59 H. pylori-infected healthy volunteers without upper gastrointestinal symptoms or volunteers with minimal upper gastrointestinal symptoms who were not being treated (34 adults and 25 children). H. pylori infection was diagnosed when both the 13C-urea breath test (UBT)15 and serum-based (HM-CAP; Enteric Products, New York, NY, USA) or urine-based (URINELISA; Otsuka Pharmaceutical, Tokyo, Japan) enzyme-linked immunosorbent assay (ELISA) were positive. The volunteers were medical staff of Wakayama Rosai Hospital or children who were enrolled in a previous epidemiological study with some of their parents.16 Exclusion criteria were a history of milk intolerance and use of antimicrobial and/or antiacid drugs within 4 weeks before or during the study. All the adult volunteers and the parents of the children gave written informed consent before participation in this study. When the children were able to understand the study design, informed consent was also obtained from them. The research protocol was reviewed and approved by the Human Research Ethics Committee of Wakayama Rosai Hospital. Subjects were randomly divided to a bLF group or a control group who took two 100-mg bLF tablets (Morinaga Milk Industry, Tokyo, Japan) or placebo tablets, respectively, twice daily between meals for a period of 12 weeks. The placebo tablet contained dextrin as a substitute for bLF and a natural coloring agent (0.55%) to adjust the color to that of the bLF tablets. Both tablets had a yogurt flavor, and their taste was almost the same. Compliance with taking medications was confirmed by tablet count and questioning at every medical examination. Subjects and physicians were not informed about the treatments until the end of the study. Siblings and their parents were grouped together to prevent mixing of tablets.

C-urea breath test

Subjects were fasted for at least 4 h before UBT. An infrared spectrometer (UBiT-IR300; Otsuka Electronics, Hirakata, Japan) was applied. The dosage of 13C-urea was altered with the age of volunteers: 50 mg for those under 6 years, 75 mg between 7 and 12 years, and 100 mg over 13 years. An increase of the UBT value by more than 2.5% or 3.5% in adults and children, respectively, was considered positive.17 UBT was performed before (week 0), during (week 8), and at the end of administration (week 12), and again 4 weeks after the administration (week 16). Positive response was defined as more than 50% decrease of the UBT value in week 12 compared to that in week 0. Statistical analysis The rate of subjects who showed a positive response was compared between groups using Fisher’s exact test. Baseline characteristics (mean UBT values in week 0) of subjects between groups were compared with the use of the t test or chi-square test. All statistical tests were performed using Stat View software (version 8.2; SAS Institute, Cary, NC, USA), and P < 0.05 was considered statistically significant.

Results Fifty-nine subjects, including 34 adults and 25 children, were divided into a bLF-treated or a placebo-treated control group (Table 1). In week 0, the mean UBT values of the two adult groups were not statistically different, whereas those of the child groups were significantly different (P < 0.01). During and after bLF ingestion for a period of 12 weeks, no significant adverse effects were observed. The individual time course of UBT values is shown in Fig. 1. Six of 17 bLF-treated adults, 1 of 17 control adults, 4

Table 1. Baseline profiles of the subjects in each group and response to bovine lactoferrin (bLF) treatment Adults

UBT value (‰) Range Sex (male/female) Age (years) Range Respondersb (%) 13

Children

Total

bLF (n = 17)

Control (n = 17)

bLF (n = 14)

Control (n = 11)

bLF (n = 31)

Control (n = 28)

28.2 ± 21.7a (3.6–62.5) 5/12

34.2 ± 22.0 (8.4–77.3) 4/13

50.9* ± 17.9 (19.1–74.6) 8/6

22.6 ± 9.7 (10–42.9) 5/6

38.4 ± 22.8 (3.6–74.6) 13/18

29.7 ± 18.8 (8.4–77.3) 9/19

41 ± 8 (29–59) 6/17 (35)

42 ± 8 (28–57) 1/17 (6)

6±2 (3–10) 4/14 (29)

10 ± 3 (6–14) 0/11 (0)

–

–

10/31 (32)**

1/28 (4)

UBT, C-urea breath test a Mean ± SD b A positive response was defined as a UBT value in week 12 being less than 50% of that in week 0 * P < 0.05 (pediatric bLF group vs. pediatric control group) by t test ** P < 0.01 (bLF group vs. control group) by Fisher’s exact test

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Fig. 1. Time course of C-urea breath test (UBT) values in adults (a) and children (b) after oral administration of bovine lactoferrin (bLF; 200 mg b.i.d.). The UBT was performed before (week 0), during (week 8), at the end (week 12), and after the treatment (week 16). Solid lines and dotted lines represent the UBT values of responders and nonresponders, respectively; hatched boxes represent the treatment period with bLF (left) or placebo (right)

of 14 bLF-treated children, and none of 11 control children showed a positive response. Altogether, 10 of 31 subjects in the bLF group and 1 of 28 subjects in the control group were responders (see Table 1), indicating that the rate of responders in the bLF group was significantly higher than that in the control group (P < 0.01). In an adult patient from the bLF group, the UBT value became negative in week 12, but

increased to a positive value at 4 weeks after administration. In the other subjects, the UBT value remained positive during the study period (Fig. 1). The profiles of the responders and nonresponder from the bLF group are shown in Table 2. The age and sex as well as UBT values in week 0 did not differ significantly between the responders and nonresponders.

268 Table 2. Baseline profiles of responders and nonresponders from the bLF group Adults

UBT value (‰) Range Age (years) Range Sex (male/female) a b

Children

Total

Respondersa (n = 6)

Nonresponders (n = 11)

Responders (n = 4)

Nonresponders (n = 10)

Responders (n = 10)

Nonresponders (n = 21)

38.6 ± 25.2b (5.7–62.5) 43 ± 11 (29–59) 1/5

22.5 ± 18.3 (3.6–52.2) 41 ± 7 (32–55) 4/7

46.2 ± 19.5 (19.1–64.8) 7±3 (3–9) 3/1

52.7 ± 17.9 (30.5–81) 6±2 (4–10) 5/5

41.6 ± 22.3 (5.7–64.8) –

36.9 ± 23.4 (3.6–81) –

4/6

9/12

A positive response was defined a UBT value in week 12 being less than 50% of that in week 0 Mean ± SD

Discussion UBT values of H. pylori-infected subjects correlate with the gastric colonization density of H. pylori as well as with the severity of related gastritis.18,19 In the present study, 10 of 31 subjects (32.3%) in the bLF group and 1 of 28 subjects (4%) in the control group showed more than 50% decrease of the UBT value after administration of 400-mg bLF daily for 12 weeks. This observation suggests that oral administration of bLF is effective to reduce the colonization density of H. pylori, although it does not lead to complete eradication. In most of the responders from the LF group, the UBT returned to the baseline level by 4 weeks after the end of administration, suggesting that LF suppressed H. pylori during oral administration. In the human stomach, bLF is degraded by pepsin, resulting in the formation of various peptides including lactoferricin B, an N-terminal 3124-Da peptide.20 In contrast to bLF, lactoferricin B has bactericidal activity against H. pylori at a concentration of more than 0.1 mg/ml (3 ¥ 10-5 mol/l), which is equivalent to 2.6 mg/ml bLF.11 We also reported previously that bLF and its degraded products were recovered in gastric juice at a total concentration of 5.7 ¥ 10-5 mol/l after administration of 2 g, i.e., 200 ml of 10 mg/ ml bLF (1.2 ¥ 10-4 mol/l), and the gastric contents contained lactoferricin B at a concentration of 5.6 ¥ 10-6 mol/l, which is five times less than the bactericidal concentration under these conditions.20 Lactoferricin B, but not bLF, has the capacity to inhibit urease of H. pylori at a concentration of approximately 3 mg/ml (1 ¥ 10-3 mol/l).11 The inhibition, however, might not affect UBT results in the present study, because lactoferricin B in the stomach was assumed to be far below the inhibitory concentration to urease, and UBT was performed at least 4 h after administration of bLF. In the present study, the concentration of bLF in gastric juice was estimated to be 4 mg/ml (5 ¥ 10-5 mol/l), assuming that 200 mg was dissolved in 50 ml gastric juice, an average volume in adults. The estimated bLF concentration was slightly higher than the inhibitory concentration of bLF in vitro, i.e., 1.0 mg/ml.11 There have been two reports about the effect of oral administration of recombinant human LF against H. pylori

infection. Opekun et al. administrated 250 mg or 1 g recombinant human LF to 12 subjects five times in a 24-h period and found no significant change in the UBT value.21 Guttner et al. administrated 5 g recombinant human LF per day for 5 or 14 days, and found neither conversion of the UBT from positive to negative nor consistent decrease of UBT values with a possible suppression of H. pylori.22 In contrast to the inefficacy of recombinant human LF, oral administration of bLF (200 mg b.i.d) in combination with a 7-day triple eradication regime (rabeprazole, clarithromycin, and tinidazole) was successful to eradicate H. pylori in 100% of patients, whereas the triple therapy eradication regimen without bLF was only effective in 76.9%.14 Although there was no conversion of the UBT value from positive to negative (as in the two recombinant human LF studies), administration of the same dosage of bLF alone was effective for reducing the density of H. pylori colonizing the stomach in the present study. Intriguingly, partially iron-saturated human LF, but not bLF, supported full growth of H. pylori in a medium lacking other iron sources.23 A 70-kDa LF-binding protein highly specific for human LF was identified in outer membrane proteins of H. pylori grown under iron-limiting conditions, and this might constitute the iron uptake system of H. pylori.24 Because the human LF concentration is estimated to be less than 0.05 mg/ml in gastric juice,25,26 it is plausible to assume that a high dosage of orally administrated bLF might sequester iron from human LF in the stomach, resulting in inhibition of the growth of H. pylori. Also, recombinant human LF exerted an antibacterial action against 8 of 13 clinical isolates of H. pylori tested in vitro, but 5 of 13 strains were resistant to a high concentration (3 mg/ml) of recombinant human LF.27 In week 0, the mean UBT values of the pediatric bLF group and control group were significantly different. The cause of this bias may have been that siblings were grouped together to prevent mixing of tablets in the present study. As unpublished data, we observed that the mean UBT value was decreased by 40% in week 8 compared with week 0 when volunteers ingested yogurt containing 0.4 g LF every day. In the present study, a 50% decrease of the UBT, which was larger than that seen in the previous study, was defined as a positive response. There were no differences between adults and children in the response to bLF treat-

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ment, as shown in Table 1. Therefore, we combined adults with children and compared the bLF group with the control group. In the present study, 21 of 31 subjects from the bLF group were nonresponders. Background profiles such as age and sex as well as UBT values at week 0 did not differ significantly between the responders and nonresponders. The range of UBT values in the responders was wide (5.7%–64.8%), so the mean UBT value of the responders (including subjects with relatively low values) was similar to that of the nonresponders. Thus, the response of subjects to bLF might depend on the H. pylori strain and/or host factors rather than factors such as age, sex, and UBT value. Although the UBT values of the pediatric LF and control groups were significantly different in week 0, this was not thought to influence the UBT response. Administration of bLF to H. pylori-infected gerbils resulted in decreasing not only the bacterial density but also the proportion of pepsinogen I-negative pyloric glands that might reflect preneoplastic lesions in the stomach.28 In addition, it has been reported that oral administration of bLF or its peptides enhances host protection against infection with various microorganisms, possibly as a result of an immunomodulatory effect.29–31 It will be necessary to elucidate the immunomodulatory effects of bLF on H. pylori infection in the future. This study suggested that continuous administration of bLF at a daily dose of 400 mg could suppress H. pylori colonization. Further studies are needed to demonstrate that oral bLF is useful for preventing chronic gastritis and gastric carcinogenesis associated with H. pylori infection.

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