Use of bovine lactoferrin for Helicobacter pylori eradication

Digestive and Liver Disease 35 (2003) 706–710 www.elsevier.com / locate / dld

Alimentary Tract

Use of bovine lactoferrin for Helicobacter pylori eradication F. Di Mario,a , *, G. Aragona a , N. Dal Bo` b , G.M. Cavestro a , L. Cavallaro a , V. Iori a , G. Comparato a , G. Leandro c , A. Pilotto d , A. Franze` e a

Chair of Gastroenterology, University of Parma, Parma, Italy b Gastroenterology Unit, University of Treviso, Treviso, Italy c Gastroenterological Hospital ‘ S. De Bellis’ IRCCS, Castellana Grotte, Bari, Italy d Geriatric Unit ‘ Casa Sollievo della Sofferenza’, IRCCS San Giovanni Rotondo, Foggia, Italy e Gastroenterology and Endoscopy Unit, Az. Ospedaliera, Parma, Italy Received 21 January 2003; accepted 3 April 2003 Institution at which the work was carried out: Universita` degli Studi di Parma, Dipartimento di Scienze Cliniche, Sezione di Gastroenterologia, Via Gramsci 14, 43100 Parma, Italy.

See commentary on pages 691 – 693

Abstract Background. One-week triple therapy is the most frequently recommended treatment for Helicobacter pylori infection. Eradication rate is satisfactory, nevertheless is advisable to look for more effective therapies. Aim. To test the efficacy of a standard triple therapy plus bovine lactoferrin in the eradication of H. pylori infection. Patients and Methods. One hundred and fifty consecutive H. pylori positive patients, suffering from dyspeptic symptoms were recruited in a 7-day triple therapy open randomised single centre study with rabeprazole, clarithromycin, tinidazole, bovine lactoferrin (group A) or rabeprazole, clarithromycin, tinidazole (group B), or a 10-day therapy with rabeprazole, clarithromycin, tinidazole (group C). H. pylori status was assessed 8 weeks after the end of the treatment by means of a 13 C-urea breath test or a H. pylori stool antigen-test. Results. Eradication rates (intention to treat / per protocol) were: group A (92.2 / 95.9%), group B (71.2 / 72.5%) and group C (70.2 / 75%). The efficacy of triple therapy added with lactoferrin was significantly higher than other two regimens ( p50.01, intention to treat analysis; p50.005, per protocol analysis). Conclusion. These results suggest that lactoferrin tested in the present study was effective in curing H. pylori and could be a new agent to assist the antimicrobials in the eradication of the bacterium.  2003 Published by Elsevier Ltd. on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: Bovine lactoferrin; Eradication; Helicobacter

1. Introduction It is well known that a 7-day triple therapy achieves eradication rates of H. pylori ranging between 80 and 90% [1–4]. Up to 20% of patients enrolled in clinical trials may experience a treatment failure. However, the rate of failure in clinical practice appears to be even higher and, even within the frame of clinical trials, up to 10% of patients remain infected after first- and second-line treatment [5,6]. *Corresponding author. Tel.: 139-521-991-772; fax: 139-521-291582. E-mail address: [email protected] (F. Di Mario,).

The reasons for treatment failure are various and include low compliance, treatment duration, number and dosages of the prescribed drugs and bacterial resistance to antibiotics [7]. Moreover, culture and susceptibility tests are sensible in refractory patients, but the results observed in vivo by following in vitro susceptibility to standard antiHelicobacter antibiotics are often disappointing [8]. Continuous search for new approaches is performed by gastroenterologists and microbiologists due to the importance of an increasing number of subjects suitable for H. pylori and to the large burden of drugs for a second line of therapy [9,10]. Bovine lactoferrin (bLf) is a glycoprotein of the transferrin family constituted by a single poly-

1590-8658 / 03 / $30  2003 Published by Elsevier Ltd. on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016 / S1590-8658(03)00409-2

F. Di Mario, et al. / Digestive and Liver Disease 35 (2003) 706–710

peptidic chain, it weights between 75 000 and 80 000 Da and it transports two iron atoms with a high affinity, also in an acidic environment [11,12]. bLf is present in milk, saliva, tears, bile, blood plasma and mucosal and genital secretions [13]. Moreover, bLf was demonstrated to have a bacteriostatic and bactericide action against various infectious agents [14,15]: its actions have been attributed to its ability to bind to iron with great affinity preventing iron utilisation by microbial agents (i.e., H. pylori) [16,17]. In this view, bLf at the dosage of 200 mg b.i.d. (capsules of 100 mg) was added at the schedule of a 1-week triple therapy, based on a PPI (rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole 500 mg b.i.d.), as the first line attempt of eradication. In the present study, the 200-mg bid dosage was chosen according to the recommended dosage reported on technical card of the drug.

2. Patients and methods A sample size of 150 patients was enrolled to achieve an appropriate statistical power of at least 0.9 at the 0.05 level, at which the smallest difference between treatment arms considered clinically important was 15%, assuming eradication rates of 80% with a rate between non lactoferrin versus lactoferrin group of 2:1 and dropout rate |5%. A total of 150 consecutive H. pylori-positive patients suffering from dyspeptic symptoms, gastritis and peptic ulcer disease were included in an open randomised single centre study. Patients, after being informed by the physicians on the impact of H. pylori infection and its association with chronic gastritis, peptic ulcer and gastric cancer, wished to be treated for H. pylori. In all subjects, the presence of bacterium was assessed at baseline by means of histology plus 13 C-urea breath test ( 13 C-UBT) or histology plus Helicobacter pylori stool antigen-test (HpSA) (Meridian Diagnostics, Milan, Italy). The 13 CUBT was considered positive if the d value over baseline at 309 (that is, the algebraic difference between d value at 309 and d value at baseline) was $5‰. For histopatological examination five biopsies (two at antrum, one from the incision and two from the body) were taken. Biopsies were oriented and embedded in paraffin. Sections were stained with hematoxylin and eosin for the histological evaluation ¨ of gastritis and with May Grunwald–Giemsa for identification of H. pylori. According to the recommendations of the Maastricht 2 consensus for monitoring H. pylori eradication treatment [18], 8–10 weeks after completion of the treatment, all subjects had voluntarily undergone H. pylori (Hp) testing by means of 13 C-UBT or HpSA indifferently. Patients were not taking acid inhibitors or antibiotics in the 3 weeks before testing for Hp status. Failure of eradication therapy was defined as a positive breath test or HpSA. According to a randomisation list, all patients received:

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rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d., tinidazole 500 mg b.i.d. and bLf 200 mg b.i.d. (Dicofarm– Rome, Italy) for 7 days (Group A) or rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole 500 mg b.i.d. for 7 days (Group B) or rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole 500 mg b.i.d. for 10 days (Group C). The content of bLf per capsule was standardised in the manufacturing process, 100 mg of bovine lactoferrin each capsule. Exclusion criteria were: previous attempt at H. pylori eradication, history of definitive acid-lowering surgery, reflux esophagitis .A, previous oesophageal surgery, treatment with proton pump inhibitors within the last 2 weeks or any antibiotics within the last 4 weeks before the study enrollment. Patients with a proved allergy to clarithromycin or benzimidazole, pregnant or lactating females, patients who received a previous eradication therapy, subjects suffering from renal or hepatic chronic diseases, as well as those suffering from neoplasm were all excluded. All criteria were assessed by means of a complete history, physical examination, endoscopy, histology and analysis of biochemical blood samples. We did not test Hp clarithromycin resistance on account of the very low level of primary resistance (,2%) recently demonstrated in our region [19]. The study was performed according to the principles of good clinical research practice, the Declaration of Helsinki and all patients included in the study had given oral informed consent on entering the study. Informed consent including general explanations concerning the rationale and importance of an accurate drug intake, detailed description of the treatment and daily table consumption schedules were all given to the patients. Compliance was evaluated by pill count and side effects were recorded by means of a structured clinical interview immediately after the end of the schedule or at any time if required. Study subjects were considered to be noncompliant if less than 90% of study medication was taken. Data were analysed by a blinded statistician (G.L). Pearson chi-square tests with standardised deviates were applied for statistical analysis. Both per protocol analysis (PP) (based on patients who completed the study) and intention to treat analysis (ITT) (a restrictive analysis which considers all drop outs as ‘failure’ of the given treatment) were performed. A p value less than 0.05 was considered statistically significant. For all calculations Bio Medical Data Processing (BMDP–Dynamic version 7, University of California, Los Angeles, CA) was used.

3. Results Major side effects leading to treatment discontinuation were observed in six patients: two patients in group A (dizziness, headache, fatigue, nausea), one patient in group

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B (nausea, hypotension and taste disturbance) and three patients in group C (fatigue, nausea and diarrhea); all but six patients completed treatment with good compliance (more than 90% of study drugs taken). Out of 150 patients [76 males, 74 females with a mean age of 50.9614.1 years (mean6SD)] enrolled in the study, 26 had peptic ulcer, 124 had gastritis, without statistically significant difference in the three groups as well as for male / female ratio, smoking habits and alcohol consumption (see Table 1). Specifically, the group A consisted of 51 cases (25 male, 26 female), with a mean age of 50.1611.1 years (mean6SD), including 42 cases of gastritis and nine cases of ulcer disease; the group B consisted of 52 cases (26 male, 26 female), with a mean age of 50.5615.3 years (mean6SD), including 43 cases of gastritis and nine cases of ulcer disease; the group C consisted of 47 cases (23 male, 24 female), with a mean age of 52.3615.8 years (mean6SD), including 39 cases of gastritis and eight cases of ulcer disease. All but 27 patients resulted negative to 13 C-UBT or HpSA at the end of the study (2 months after the end of the therapy). Summarised results in the three groups are shown in the Table 2. The eradication rates in the group A were 92.2% (47 / 51), based on ITT-analysis, and 95.9% (47 / 49), based on PP-analysis. In the group B, the eradication rates were 71.2% (37 / 52) according to ITTanalysis and 72.5% (37 / 51) according to PP-analysis. The eradication rates in the group C were 70.2% (33 / 47), based on ITT-analysis and 75% (33 / 44), based on PPanalysis. Both ITT- and PP-analyses demonstrated an eradication rate significantly higher in the group A compared with the other two regimens ( p50.01, ITT-analysis; p50.005, PPanalysis). Smoking habits and alcohol intake did not Table 1 Baseline demographic and clinical characteristics

Number of patients Median age (6S.D) (years) Female / males Smoking Yes No

Group Aa

Group B b

Group C c

51 50.1611.1

52 50.5615.3

47 52.3615.8

26 / 25

26 / 26

24 / 23

15

12

11 40

37

35

Alcohol intake: .40 g / day female .60 g / day male

3 3

3 4

4 6

Peptic ulcer Gastritis

9 42

9 43

8 39

a

Rabeprazole mg b.i.d. and b Rabeprazole 500 mg b.i.d. c Rabeprazole 500 mg b.i.d.

20 mg b.i.d., clarithromycin 500 mg b.i.d., tinidazole 500 bLf 200 mg b.i.d. for 7 days. 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole for 7 days. 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole for 10 days.

Table 2 Results

PP 95% CI ITT 95% CI Dropout

Group Aa

Group B b

Group C c

p

47 / 49 (95.9%) 90.4–1.00 47 / 51 (92.2%) 84.8–99.5 2

37 / 51 (72.5%) 60.3–84.8 37 / 52 (71.2%) 58.8–83.5 1

33 / 44 (75%) 62.2–87.8 33 / 47 (70.2%) 57.1–83.3 3

0.005 0.01 NS

NS, not significant; PP, per protocol analysis; ITT, intention-to-treat analysis. a Rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d., tinidazole 500 mg b.i.d. and bLf 200 mg b.i.d. for 7 days. b Rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole 500 mg b.i.d. for 7 days. c Rabeprazole 20 mg b.i.d., clarithromycin 500 mg b.i.d. and tinidazole 500 mg b.i.d. for 10 days.

influence eradication rate ( p50.13; p50.26, respectively, based on ITT-analysis). Adverse, mild events (fatigue, dizziness, headache, diarrhoea, tiredness, bitter taste, skin rash) were recorded in 24 patients (7, 8 and 9 in group A, B and C, respectively), however, they completed eradication schedule with spontaneously disappearance of symptoms.

4. Discussion To the best of our knowledge, our work is the first in-vivo study concerning bLf added to the ‘classical’ regimen for H. pylori eradication. Preliminary results concerning bovine lactoferrin in curing H. pylori infection had shown promising eradication rate [20]. In this open, prospective, randomised single centre study the supplementation of the standard 1-week triple therapy with bLf significantly increased the H. pylori eradication rate with respect to that of the non-supplemented regimens by both ITT- and PP-analysis, with few minor side effects and a good compliance to the schedule as the other regimens used. Recently, Lactobacillus acidophilus added to a 7-day triple therapy showed an additional positive effect on H. pylori eradication rates [21]. In the present study we used clarithromycin for its effectiveness and on account of the very low level of primary resistance (,2%) in our region [19] as well. These are the reasons why we chose bLf: (1) antimicrobial activity against Helicobacter species in vitro and in vivo and effectiveness at inhibiting its growth at pH 6 [17]; (2) antibiotic actions of bLf attributed to its ability to bind to iron with great affinity and prevent iron utilisation by bacteria; in fact, deferoxamine, another iron chelator, inhibits the growth of H. pylori [16,22,23]; (3) a non-iron dependent component of bLf activity against H. pylori not yet identified [17]; (4) bLf may also have other actions on the gastric mucosa that could reduce stomach size in an infection, such as its immune-modulatory activity

F. Di Mario, et al. / Digestive and Liver Disease 35 (2003) 706–710

[16,22,24]; (5) lactoferrin has been proposed to act as an antioxidant [25,26] and to exert a significant inhibitory effect on the attachment of the H. pylori colonizing the stomach in vivo with a reduction in bacterial numbers and associated inflammation [12]. However, a number of other potential mechanisms by which lactoferrin inhibits the growth of several microorganisms have been suggested, including structural changes in the microbial cell wall, complete loss of membrane potential and integrity, indirect effects on enzyme activation, an increased generation of metabolic by-products of aerobic metabolism, iron deprivation and combination of these factors [27–31]. Moreover, by using different points of attach against the bacterium, clarithromycin, tinidazole and bLf exert an optimal synergic anti H. pylori activity, that could lead to a complete destruction of the structure of the bacterium. Several in vivo and in vitro studies concerning bLF as monotherapy against Helicobacter pylori infection did not reveal always univocal results. A recent study, to assess the safety and efficacy of recombinant human lactoferrin in vivo, as monotherapy, to cure H. pylori infection in adult subjects, did not reveal a beneficial effect regarding the elimination of the bacterium [32]; possibly, as the authors suggested, the results could be different with more subjects, with one more long-lasting therapy or with concomitant acid-suppressing therapy; moreover, it is controversial whether human lactoferrin is inhibitory to H. pylori. Human lactoferrin acts as an iron transport protein for the bacteria and supports its growth, whereas bovine lactoferrin does not [33]. It is possible to speculate concerning a synergistic action of the bLf in association with antibiotics and proton pump inhibitors to increase the effectiveness of the current eradication schedule. Our results, therefore, raise the possibility of bLf being used as a therapeutic agent to assist antimicrobials in the eradication of H. pylori to produce a combination that maximises antibiotic activity, minimises dosage and potentially lessens side effects of the other commonly used antibiotics, even if further data are needed. This schedule, therefore, could be proposed for wider trials in Helicobacter pylori eradication strategy, focusing on the promising high cure rate of 92.2 and 95.9% according to intention-to-treat and per protocol analyses, respectively, the low rate of minor side effects, the good compliance to the schedule and the low price of the bLf (13 Euros) for full treatment.

List of Abbreviations bLf, Bovine lactoferrin; HpSA, Helicobacter pylori stool antigen-test; ITT, Intention to treat.

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