Advisable including glucosaminylmuramyldipeptide in Helicobacter pylori therapy: experience of ten‐year investigation

Helicobacter pylori infection is a common bacterial infection in humans and is associated with peptic ulcer disease and chronic gastritis. The presence of natural resistance to some antibiotics in bacteria, as well as the appearance of primary and secondary resistance to antibacterial agents, complicates treatment and determines the search for new methods of therapy. The aim of this study was to evaluate the efficacy and safety of 10-year complex treatment of patients with duodenal ulcer associated with H.pylori, 136 patients (96 men, 40 women; mean age 45.8 ± 14.8 years; 18–65 years). H.pylori was determined morphologically and by rapid urease test one day before the start of therapy, after 1, 6, 12 months, 2 years, 5 and 10 years. Patients of the first group received basic therapy: omeprazole 0.02 g 2 times a day, clarithromycin 0.5 g 2 times a day, amoxicillin 1 g 2 times a day, for 10 days (OCA group 1; n = 98). Patients of the second group, in addition to the basic therapy, took 1 mg per day drug Liсopid (group 2 OСAL; n = 38). At the 1st stage of the clinical study, 130 patients completed eradication therapy. Tracking completeness was 96 %. The frequency of H.pylori eradication after per protocol treatment: OCA – 83 % (95 % CI: 75 %–91 %), OCAL – 97 % (95 % confidence interval (CI): 92 %–100 %). The incidence of adverse reactions after treatment (per protocol): OCA – 26 % (95 % CI: 17–35 %; nausea; n = 24), discontinued treatment – 5 % (95 % CI: 0.8 %–10 %; diarrhea; n = 5); OCAL – 3 % (95 % CI: 0.01 %–8 %; nausea; n = 1), all were treated. Taking the drug Liсopid 1 mg (glucosaminyl muramyl dipeptide, JSC Peptek, Russia) as part of complex therapy contributed to the elimination of H.pylori and the absence of relapses for 2 years. Observation of patients in the next 5 and 10 years also showed the advantage of including the immunomodulator in therapy: a significant 15 % decrease in H.pylori reinfection (P <0.05), a 23 % decrease in the frequency of gastrointestinal adverse reactions (P<0.01), compared with a 10-day standard triple regimen without immunomodulatory therapy with glucosaminylmuramyl dipeptide. When using several antibiotics in H.pylori eradication therapy, not only pathogenic, but also commensal microorganisms are destroyed, the waste products of which are vital and maintain immune homeostasis, including through the NOD2 receptors of innate immunity. The effectiveness of the complex therapy of H.pylori infection can be explained by the fact that the drug Liсopid compensates for the signal for innate immunity receptors that is missing due to the absence of commensals, providing an adequate immune response and preventing chronicity and recurrence of infection.

Заявление о конфликте интересов. Авторы заявляют об отсутствии конфликта интересов. Since the identification of Helicobacter pylori in 1982, and much has been learned about this bacterium, it remains one of the most common bacterial infection in humans [1][2][3][4]. The bacterium causes a diverse pathology of the upper gastrointestinal tract from H.pylori-induced gastroduodenitis and H.pylori-associated dyspepsia to gastroduodenal ulcer, MALT lymphoma and gastric cancer. This requires appropriate anti-Helicobacter therapy [2,5]. H.pylori eradication is a first-line therapy for H.pylori-infected patients with dyspepsia (Kyoto Global Consensus on Helicobacter pylori; Regulation 9). H.pylori-infected patients should be offered eradication therapy unless otherwise stated (Kyoto Global Consensus on Helicobacter pylori; Regulation 17) [5]. Eradication of bacteria is also necessary to control complications and reduce the number of relapses of gastroduodenal ulcer associated with H.pylori infection. The clinical effect of successful H.pylori eradication is manifested by a sharp drop in the recurrence rate of this disease after elimination of the bacterium [6].
The presence of natural resistance to some antibiotics in bacteria, as well as the appearance of primary and secondary resistance to antibacterial agents, complicates treatment and determines the search for new methods of therapy. This is reflected in current international guidelines for H.pylori eradication, which presents not only the first-line treatment regimens, but also various other treatment regimens taking into account the clarithromycin-resistant H.pylori strains in the region (Table 1).

Quadrotherapy for the CIS countries
Quadrotherapy for the CIS countries (taking into account the growth of H.pylori resistance to antibiotics and the presence of fast metabolizers (60-70 %) in the population of the Russian Federation; Megraud Francis "Approaches to the treatment and diagnosis of H.pylori. European Register data" 2015) includes Omeprazole 0.02 g x 3 times a day, Amoxicillin 1.0 g x 3 times a day, Josamycin 1.0 g x 2 times a day, De-nol 0.24 g x 2 times a day. The duration of therapy is 10-14 days.
Measures to increase the effectiveness of standard triple therapy taking into account the growth of H.pylori resistance to antibiotics (Recommendations of the Russian Gastroenterological Association, 2018): Omeprazole 0.04 g x 2 times a day, Clarithromycin 0.5 g x 2 once a day, Amoxicillin 1.0 g x 2 times a day, De-nol 0.24 g x 2 times a day. The duration of therapy is 10-14 days. Addition to the standard triple therapy of probiotic strains of Bifidobacterium and Lactobacillus [7].
It is very difficult to reach H.pylori eradication. In most patients, a year after successful eradication, reinfection of H.pylori occurs within the next 10 years [8,9]. In the Russian Federation and countries of Eastern Europe, H.pylori reinfection exceeds 5 % per year, in Western Europe and the USA -less than 3 % per year [3,10]. In order to optimize standard therapy different approaches are investigated, for example the usage of probiotics [2,11,12]. This is reflected in Provisions 9 and 10 of the Consensus of Maastricht V [2], which are formulated as follows: certain probiotics are effective in reducing gastrointestinal side effects caused by H.pylori eradication therapy. Specific strains should only be selected on the basis of proven clinical efficacy (Consensus Maastricht V; Regulation 9). Certain probiotics may have a beneficial effect on H.pylori eradication (Consensus Maastricht V; Regulation 10). It is believed that probiotic strains, in particular Lactobacillus, decrease the activity of bacterial urease, the motility of H.pylori and the adhesion of H.pylori to gastric epithelial cells [7].
The concept is formulated that the immunomodulating effect plays a significant role in the me cha nism of antimicrobial action of pro-biotics [13]. The origin of the immunomodulator and its influence on the mucosa are the main issues [14,15]. It is known that glucosaminyl muramyl dipeptide (GMDP) modulate immune answer via NOD2 receptors and YB1 protein [16,17] and is effective in the therapy of infections [18][19][20], allergy [21,22], psoriasis [23], correction of cytopenia [24] and microbiocenosis [25]. The positive effect of Licopid 10mg on the elimination of H.pylori was investigated earlier [26,27] correlates with another dosage of this drug -1mg.
During the first stage of this randomized prospective comparative clinical study of the effectiveness of H. pylori elimination in standart triple therapy with addition of GMDP 1mg was carried out. During the second stage of this investigation the frequency of relapse and reinfection of Helicobacter pylori was measured.

Material and methods
This study was approved by the Ethics Committee of the Vitebsk State Medical University (Vitebsk, Belarus) and was carried out during 2000-2020 years. Prior to the start of the study, informed consent was obtained from all patients to participate in the study and the processing of personal data.
The first stage of a prospective, randomized, comparative clinical study was conducted to evaluate the efficacy and safety of H.pylori eradication during standard triple therapy with Licopid.
Inclusion criteria: the presence of H.pylori-associated duodenal ulcer (DU).
Exclusion criteria: patients using antibacterial drugs less than a month before the start of eradication therapy or FEGDS research.
Eradication therapy was performed in 136 patients (96 men, 40 women; mean age 45.8 ± 14.8 years (mean ± SD; 18 -65 years) with a duodenal ulcer associated with H. pylori (Table 2). Patients were divided by a randomized lottery drum method into 2 groups according to treatment protocols: omeprazole 0.04 g / day, clarithromycin 1 g / day, amoxicillin 2 g / day for 10 days (OCA; n = 98); omeprazole 0.04 g / day, clarithromycin 1 g / day, amoxicillin 2 g / day, Lycopid 0.001 g / day for 10 days (OCAL; n = 38). The study completed 130 patients. Six people (4.4 %) were excluded from the general group (5 people from the OCA group and 1 person from the OCAL group) due to the lack of data on the diagnosis of H.pylori or the cessation of medication. The completeness of tracking was 95.6 %. In a randomized trial 113 patients had the following treatment: 0.04 g omeprazole, 1.0 g clarithromycin, 2.0 g amoxicillin per day dyring 10 days (group OCA ; n=77). patients from the the second group received 0.04 g omeprazole, 1.0 g clarithromycin, 2.0 g amoxicillin and 0.001 g Lycopid per day (group OCAL ; n = 36).
The tissue investigation of the duodenum was carried out by standard systematization and methods [28,29]. To identify areas of gastric metaplasia (GM) of duodenum, an additional staining of histological sections of the mucous membrane of duodenal ulcer was performed with Chic -alcian blue (Serva) pH 1.0 and 2.5 [30].
Intestinal metaplasia and all cell-and tissuemorphologic characteristics were assessed using a visual analogue scale [31][32][33] according to the histological section of the Houston modification of the Sydney classification.
During the histological examination of the duodenal mucosa standard indicators were taken into account [30,34]. Diagnostics of H.pylori was carried out by Romanovsky-Giemsa stain; assessment using a standard visual-analogue scale [35] and a quick urease test (standard test systems Jatrox®-Hp-Test, Rohm Pharma, Germany; HELPIL® and AMA RUT Pro®, LLC "AMA", Russia) [36].
For statistical processing the program «STATISTICA 10.0» and t-test were used. If the distribution of the variable was not normal, the Shapiro-Wilk test was used. The Mann-Whitney U-test was used to evaluate the differences between two independent small samples by the level of the trait, measured quantitatively. Patient age was presented as mean ± standard deviation (SD). P levels <0.05 were considered significant [37].

Results and its discussion
The results of the first stage of a prospective, randomized, comparative clinical study are represented in the Table 3.
The frequency of H.pylori eradication depending on the prescribed treatment (ITT) and the actual treatment    Table 4). As follows from the results of the study, GMDP eliminates H.pylori during 12 and 24 months and was decreased after 5 and 10 years.  Table 3). Thus, in patients taking Licopid at a dose of 1 mg per day together with anti-Helicobacter pylori therapy (the OCAL group), there was no H.pylori reinfection 2 years after per protocol treatment compared to the 10-day three-component treatment protocol without Lycopid. At the second stage of the study, it was also found that patients who took Liсopid at the above dose together with three-component anti-bacterial therapy (OCAL group) had a significantly low frequency of H. pylori reinfection for 5 years (χ2 = 4.33; P = 0.0375) and 10 years (χ2 = 6.73; P = 0.0095).
The choice of patients with localization of an ulcer in the duodenal bulb (duodenal ulcer) as participants in a clinical study was based on the fact that, with duodenal ulcer of onion localization, a maximum degree of contamination of H.pylori gastric mucosa was observed (99.0 %) [38] and sections of gastric metaplasia of the mucous membrane of the duodenal bulb (87.8 %) [39].
The choice of Liсopid as an immunomodulator therapy in the 10-day H.pylori eradication scheme was consistent with the concept of an "ideal" immunomodulator and was based on three main criteria, according to current scientific research data [40]: The first criterion includes the presence of N-acetyl-glucosaminyl-N-acetylmuramyl dipeptide. One of the reasons for the ineffectiveness of eradication therapy is the transition of H.pylori to metabolically inactive forms (coccoid and U-form) that are resistant to antibiotics. It was previously shown that glucosaminyl muramil dipeptide promoted the release of Mycobacterium tuberculosis from the dormant form, which, apparently, determines the effectiveness of Licopid therapy [41]. Similarly, it was previously shown that NOD1 and NOD2 receptor activation promotes the elimination of H.pylori [42,43]. The active substence of Lycopide, N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP, glucosaminyl muramyl dipeptide), is the main complete repeating structural unchanged fragment of the cell wall of almost all known bacteria, a ligand of NOD2 receptors.
According to the second criterion for "ideal" immunomodulator it is necessary to activate immune system through T helper 1 lymphocytes. It was shown that activation immune answer through T helper 1 lymphocytes is essential for successful treatment H. pylori [44][45][46][47]. GMDP fully complies with the second criterion -its influence on the balance T helper 1/ T helper 2 shift towards T helper 1 has been proven [21,22,48].
These meta-analyzes found that specific strains of Lactobacillus or several probiotic strains increase eradication of H.pylori by 8.1 % and reduce the number of adverse reactions when using the probiotic 14 days before eradication therapy or during eradication therapy. Bifidobacterium and Saccharomyces boulardii did not affect the level of eradication during anti-Helicobacter therapy [58,62]. The use of specific strains of probiotics (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei DN-114001, Lactobacillus gasseri, and Bifidobacterium infantis 2036) during eradication therapy can be considered as an option to increase the level of H.pylori eradication, especially when the antibiotic is not effective [63,68]. The effect of probiotics on the reduction of adverse reactions during eradication therapy has been proven [62]. A significant increase in the eradication of H.pylori by 17 % was found using mainly specific strains of Lactobacillus. When multicomponent probiotics were used as adjuvant therapy, eradication increased by only 2.8 % [54]. Monotherapy with probiotics using specific strains of Lactobacillus led to significant (P <0.001), compared with placebo, eradication of H.pylori in 16 % of patients, using multicomponent probiotics (which included Lactobacillus strains) in 14 % of patients [63].
Interestingly, that GMDP was for identified the first time as a fragment of Lactobacillus bulgaricus cell wall [64] and thus its beneficial effect in H.pylori therapy is consistent with the data of the above studies.
Based on the data obtained, it can be concluded that therapy with the immunomodulator Liсopid in a 10-day H.pylori eradication scheme demonstrated an encouraging result. GMDP maintaines the long term (2, 5 and 10 years) eradication of H.pylori in 100 %, 98 % and in 95 % of cases. The reinfection of H.pylori after 5 years of the treatment is observed in 32 %-91,4 % cases [8,9,65]. Thus, the method for optimizing H. pylori therapy proposed in this research is in demand and has practical significance.

Conclusions
GMDP at a dose of 0.001 g per day during 10-day three-component anti-Helicobacter therapy significantly increased H.pylori eradication by 16 % (according to ITT; χ2 = 3, 87; P = 0.0492) and by 14.5 % (according to PP; χ2 = 4.0; P = 0.0455), with a significant decrease in the frequency of adverse reactions from the gastrointestinal tract by 2.5 % (according to ITT; χ2 = 2.38; P = 0.0115) and 2.7 % (according to PP; χ2 = 6.56; P = 0.0105) and the completion of the course of therapy by all patients. GMDP maintain the absence of H.pylori in 100 % during 2 years, in 98 % after 5 years and in 95 % after 10 years after treatment.
Triple antibiotic eradication therapy of H. pylori, eliminates both pathogenic and commensal microorganisms, the waste products of which are vital and maintain immune homeostasis, including via NOD2 receptors of innate immunity. The success of the complex H.pylori eradication treatment can be explained by the compensatory effect of the GMDP for the missing signal from commensals for innate immunity receptors, providing an adequate immune response and preventing chronicity and recurrence of the infection.