Use of L-cysteine ​​to improve gastric mucosal structure and function

Abstract

According to one exemplary aspect of the present invention, there is provided an acetaldehyde-binding preparation for use in a method for improving gastric mucosal structure and physiology in a subject suffering from Helicobacter pylori-associated or autoimmune atrophic gastritis or chronically using a PPI.

Description

[Technical field]

[0001] The present invention relates to an acetaldehyde-binding preparation for use in a method for preventing atrophic gastritis and preventing the progression to gastric cancer by improving, repairing and / or restoring the structure and function of the gastric mucosa in subjects with acid-free stomach and / or Helicobacter pylori-associated (HP+) or autoimmune (HP-) atrophic gastritis using sustained release L-cysteine, a natural amino acid (Acetium® capsules, Biohit Oyj). A GastroPanel® study (Biohit Oyj) is used for initial screening of said subjects and to demonstrate the functional improvement of the atrophic gastric corpus mucosa after an effective period of L-cysteine ​​administration. [Background technology]

[0002] Atrophic gastritis (AG) is a disease associated with a significantly increased risk of gastric cancer and is the single most important precursor of gastric cancer (GC) known to date. On the other hand, Helicobacter pylori (HP) infection is the most important causative agent in the development of gastritis and subsequently AG. HP infection and AG in the gastric antrum can lead to GC and peptic ulcer disease. It is well known that there are few cases of AG in the gastric corpus that develop by autoimmune mechanisms. In patients with severe gastric corpus mucosal atrophy (AGC), the risk of GC is 4-5 times higher than in healthy patients. In patients with severe gastric antral atrophy (AGA), this risk is 18 times higher than in healthy subjects, and when severe atrophy affects both the gastric antrum and the gastric corpus (i.e., in the presence of severe generalized atrophy, AGP), the risk increases up to 90 times.

[0003] The prevalence of AG and GC increases with age, and the risk for both diseases is highest in subjects over 45 years of age. The majority of GC in the elderly is of the intestinal subtype, progressing through an AG-to-GC sequence (Correa cascade). Due to the high risk of GC in the elderly, current consensus recommendations recommend endoscopy for all older adults with dyspepsia and those over 45 (50) years of age.

[0004] Based on extensive documentation from numerous cohort studies conducted over the past decades, a relatively clear picture of the stepwise progression of invasive GC has emerged. 1-24 This increased understanding of the pathogenesis of GC has led the scientific community to develop international recommendations for the management of precancerous gastric conditions. 25 These management guidelines are based on three major statements that have led to a relatively good consensus among leading European gastroenterology experts: 25 .

[0005] 1) Patients with chronic atrophic gastritis (AG) or intestinal metaplasia (IM) are considered to be at high risk for gastric adenocarcinoma; 2) high-grade dysplasia and invasive carcinoma should be considered preventable outcomes if patients with chronic AG or IM are appropriately managed; and 3) patients with endoscopically detectable high-grade dysplasia or carcinoma should undergo staging and appropriate management. 25 .

[0006] In most cases, the development of so-called “intestinal” GC represents the culmination of an inflammation-metaplasia-dysplasia-carcinoma sequence known as the Correa cascade of multistep gastric carcinogenesis. 16,19-21 As consistently confirmed in several studies, the progression of the disease goes from normal mucosa through chronic nonatrophic gastritis, AG, IM, to dysplasia, and finally to GC. 26,27 .

[0007] Gastric mucosal atrophy (AG) and IM constitute precursor lesions for the development of dysplasia and intestinal-type gastric adenocarcinoma, thus increasing the risk of developing GC. 28-31 Therefore, chronic AG and IM are considered to be precursors of cancer. 25 Chronic AG is diagnosed and classified based on the presence of chronic inflammatory cells, such as lymphocytes and plasma cells, infiltrating the connective tissue stroma, associated with the loss of normal glands. 8,14,32-34 The severity of glandular loss (atrophy) should be classified as mild, moderate, or severe, although inter- and intra-observer agreement is poor. 25.

[0008] Individuals may have different phenotypes of chronic gastritis due to differences in genetic profiles and environmental exposures. Diffuse antral gastritis is defined as cases in which inflammatory changes are restricted to the gastric antrum, without gland atrophy and / or IM. 8,14 On the other hand, cases with multifocal distribution of glandular atrophy and / or IM (including the lesser curvature of the gastric body and fundus) are best defined as multifocal AG. When accompanied by AG of the gastric antrum, the condition is called atrophic pangastritis (AGP). 8 .

[0009] Gastric mucosal changes should be described in terms of i) the severity and ii) the distribution of the precancerous condition / lesion. Several classification schemes have been developed for chronic gastritis and preneoplastic changes. Currently, the updated Sydney System (USS) is widely used in both clinical practice and research, combining topographical, morphological and etiological information in a reporting system designed to include both grading and staging of gastritis. 35,36 Recently, systems known as OLGA (Operative Link for Gastritis Assessment) and OLGIM (Operative Link for Gastrointestinal Metaplasia) staging have been proposed for staging gastritis. 37 Although both classification methods are good, they suffer from poor inter- and intraobserver reproducibility.

[0010] Gastric dysplasia represents the penultimate stage of gastric carcinogenesis. 16,19~21,38 , defined as histologically unquestionable neoplastic epithelium without evidence of tissue invasion, i.e., a full-blown neoplastic precancerous lesion. It is characterized by cellular atypia reflecting aberrant differentiation and disorganized glandular architecture. 39-41 Correct diagnosis and grading of dysplasia are important because they predict both the risk of malignant transformation and the risk of metachronous GC. The reported rates of progression from dysplasia to GC vary widely, ranging from 0% to 73% per year. 8,14,25,42,43Undoubtedly, these wide variations reflect multiple factors, including differences in study design and populations, and differences in the definition and evaluation of gastric dysplasia. 2 .

[0011] There are well-known differences between Japanese and European / North American pathologists in the classification of dysplasia. 25、35、36 In Japan, noninvasive intramucosal neoplastic lesions with severe cellular and architectural atypia are called "noninvasive intramucosal carcinoma," whereas in the West, most pathologists diagnose the same lesions as "severe dysplasia." 44,45 To address this issue, the World Health Organization (WHO) recently published a new classification for precancerous gastric lesions: 46 This new WHO classification of dysplasia / intraepithelial neoplasia acknowledges the widespread use of both dysplasia and intraepithelial neoplasia (IEN), and these terms are used synonymously. According to the current WHO classification, the following diagnostic categories should be recognized: 1. Negative for intraepithelial neoplasia / dysplasia 2. Unknown intraepithelial neoplasia / dysplasia 3. Low-grade intraepithelial neoplasia / dysplasia 4. High-grade intraepithelial neoplasia / dysplasia 5. Intramucosal invasive tumor / intramucosal carcinoma

[0012] In the WHO classification, category 1 (intraepithelial neoplasia / dysplasia negative) includes two important premalignant conditions (chronic AG and IM) (see 1.1.1). When there is doubt as to whether the lesion is neoplastic or non-neoplastic, especially in small biopsies showing inflammation, WHO category 2 (intraepithelial neoplasia / dysplasia unknown) should be used. In such cases, the problem can usually be resolved by removing more sections, performing additional biopsies, or controlling the possible etiology. 25 .

[0013] Intraepithelial neoplasia / dysplasia contains frank neoplastic epithelial proliferation and is characterized by a variety of cellular and architectural atypia, but without convincing evidence of invasion. In low-grade intraepithelial neoplasia, architectural disarray is minimal, with only mild to moderate cytologic atypia. In contrast, high-grade intraepithelial neoplasia contains tumor cells that display high nuclear / cytoplasmic ratios, prominent nucleoli, more prominent architectural disarray, and numerous mitoses, many of which are atypical. As reiterated, most patients with high-grade dysplastic lesions are at high risk for either synchronous invasive GC or its rapid development. 8,14,25,42,43 .

[0014] Intramucosal invasive neoplasms / intramucosal carcinomas define carcinomas that invade the lamina propria and are distinguished from intraepithelial neoplasms / dysplasias by distinct architectural abnormalities such as prominent glandular crowding, excessive branching, budding, fusion, or cribriform glands as well as minimal or absent desmoplastic changes. 25 A diagnosis of intramucosal carcinoma (also called carcinoma in situ) indicates an increased risk of lymphatic invasion and lymph node metastasis. However, endoscopic techniques allow treatment without open surgery, especially for well-differentiated lesions less than 2 cm in size and without lymphatic invasion. 25 .

[0015] It is estimated that 50% of all GC cases develop through the "Correa cascade." 16,19,20,21 , HP-associated gastritis progresses to mucosal atrophy, IM, dysplasia, and invasive adenocarcinoma. The concept of classifying AG and IM as precancerous conditions of the gastric mucosa is based on well-documented data from long-term prospective cohort studies, demonstrating that the risk of GC is significantly increased in patients with AG who have been followed up for a sufficient period of time. 1-24、28-31 Indeed, based on this compelling evidence, AG is now considered to be the strongest independent risk factor for distal (noncardia) GC. 8,16,17,18 Because this process takes decades, there is a good chance of detecting precancerous lesions early. 22 However, the lack of suitable tests for GC screening remains a problem. 23Furthermore, most patients report only a short period of symptoms before being diagnosed with GC, and up to 40% report never having dyspeptic symptoms. 24 .

[0016] On the path to GC 16、19-21 The next step following (and often accompanying) AG is a condition known as intestinal metaplasia (IM). As with gastritis, distinct phenotypes of IM have recently been recognized. Traditionally, IM has been classified as either "complete" or "incomplete." Complete intestinal metaplasia ("small intestinal" or type I) shows goblet and absorptive cells, reduced expression of gastric mucins (MUC1, MUC5AC, and MUC6), and expression of MUC2 (intestinal mucin). Incomplete intestinal metaplasia ("enterocolonic" or types IIA / II, and "colonic" or types IIB / III) shows goblet and columnar non-absorptive cells, in which gastric mucins (MUC1, MUC5AC, and MUC6) are co-expressed with MUC2.

[0017] Recently, another pattern of metaplasia called spasm-suppressing polypeptide-expressing metaplasia (SPEM) has been reported. 47 It is characterized by expression of the TFF2 spasmolytic polypeptide, which is associated with acid secretion atrophy. SPEM characteristically occurs in the gastric body and fundus, appears to share some features with pseudogastric antral metaplasia, has a strong association with chronic HP infection and gastric adenocarcinoma, and may represent an alternative pathway to gastric tumorigenesis. However, at present, identification of SPEM is not yet included in the diagnostic routine of gastric biopsies.

[0018] There is no dispute that gastric biopsy specimens are essential for the diagnosis and grading of precancerous gastric lesions. 25,49b The updated Sydney System (USS) is the most widely accepted classification and grading system for gastritis. 35,36This system was primarily designed to standardize the reporting of gastric biopsies. The updated version recommends five biopsies: two from the gastric antrum (pylorus, 3 cm from the greater and lesser curvatures), one from the incision, and two from the gastric body (one from the lesser curvature, 4 cm proximal to the incision, and one from the middle of the greater curvature). Although this biopsy protocol generally correctly determines HP status and chronic gastritis, the number of biopsies is controversial with regard to the appropriate staging of premalignant gastric lesions, mainly due to the multifocal nature of these lesions. 48,49,49b This multifocality impacts detectability and therefore decisions regarding patient treatment and future surveillance. 19,21 .

[0019] To prevent the excessive use of this invasive and expensive procedure (endoscopy), there is an urgent need to develop noninvasive diagnostic tools that can accurately detect patients at high risk for GC, i.e., the various phenotypes of gastritis and its associated HP infections. 50,51 To this end, a Finnish biotechnology company (Biohit Oyj) has launched an ELISA-based assay designed to measure the concentrations of four stomach-specific biomarkers from a single blood sample. The test, known as GastroPanel®, combines serum pepsinogen I (PGI) and II (PGII), gastrin-17 (G-17), and HP IgG antibodies (IgG-HP). The test has been proposed as a first-line diagnostic test for dyspeptic symptoms. 52-54 The GastroPanel® test is therefore the first non-invasive diagnostic tool offering the possibility to detect not only patients at risk for GC and peptic ulcer disease, but also conditions associated with AG (malabsorption of vitamin B12, iron, magnesium, calcium, and some drugs). 54 A recent meta-analysis showed that serum PGs are not suitable for screening for GC, but they have proven useful in detecting patients at risk for GC. 55Therefore, these gastric-specific biomarkers are recommended by international experts for the diagnosis and screening of AG. 56 .

[0020] Whether gastric carcinogenesis, which progresses stepwise from HP infection through AG and IM to invasive GC, can be prevented or reversed once it has progressed remains a controversial issue. 7 This issue is of great importance from a clinical point of view, since according to our current thinking, both AG and IM, once established, are irreversible conditions that, without proper monitoring, will inevitably lead to invasive GC. 16,19-21 .

[0021] However, there have been some recent suggestions that this chain may be slowed or even reversed if HP infection is eradicated early. 7,13 , suggesting that eradicating HP may have the potential to prevent gastric cancer. 57 A recent study on the effect of eradicating HP in patients with precancerous lesions showed that eradicating HP may prevent progression. 58 It is believed that there exists a so-called "point of no return" in the histological cascade from chronic gastritis to adenocarcinoma, and subsequent eradication is unlikely to prevent GC. 7 Once IM is established, eradication may slow the progression of IM but does not appear to completely prevent gastric cancer. 59,60 However, this is not necessarily the case in the case of gastric atrophy, for which there seems to be a difference in the eradication effect in the gastric corpus and the gastric antrum. Thus, a recent meta-analysis of 12 studies including 2,658 patients showed that eradication of HP infection leads to a significant improvement in AGC but not in AGA and, importantly, does not affect IM of the gastric mucosa. 61 Furthermore, it is unclear whether there are differences in this respect between autoimmune-derived AG and AG caused by HP infection, the former of which is often associated with other autoimmune diseases such as type 1 diabetes and autoimmune thyroiditis. 73 .

[0022] In light of the above considerations, two recent reports from Italian groups present a fundamentally new idea and at the same time suggest that the function of the gastric mucosa in (HP-eradicated) AG patients may be restored by a simple treatment with the natural (semi-essential) amino acid L-cysteine ​​(Acetium® capsules, Biohit Oyj, Helsinki, Finland). 62,63 In their two separate reports, the authors used the GastroPanel® test to demonstrate that gastric mucosal function improved significantly during follow-up of AG patients after treatment with Acetium® capsules.

[0023] Therefore, the authors evaluated the changes in gastric function after HP eradication for moderate to severe atrophic gastritis of the gastric corpus by measuring sPGI levels and gastrin 17 (sG-17). A series of 74 dyspeptic patients, selected from 738 consecutive HP-positive patients, had histological features of moderate to severe AG of the gastric corpus and underwent upper gastrointestinal endoscopy with gastric biopsy and measurement of sPGI and sG-17 at baseline. All HP-associated AG patients received HP eradication therapy and serum sPGI and sG-17 were measured again 6 months after eradication therapy and 1, 2, 3, 5 and 6 years later. In the first report, data were available on 5 patients (4 women, mean age 56 years, range 49-66 years) out of 74 AG patients, who received 3 months of treatment with Acetium® 100 mg (3 capsules per day) before meals. In these subjects, mean sPGI increased from 5.3 μg / L at baseline to 7.8 μg / L after 90 days of Acetium® intake, whereas sG-17 did not change (74.6 and 79.8 pmol / L). 62 The authors concluded that after HP eradication, AG of the gastric corpus in these subjects demonstrated long-term improvement in physiological gastric function, as reflected by a significant and stable increase in sPGI levels.

[0024] In their second report, they obtained similar 3-month follow-up data from 21 of 74 of these AG patients (including both autoimmune and HP-associated AG) after 3 months of treatment with Acetium® capsules, but also 6-year follow-up data for all 74 subjects who underwent HP eradication. 63 The mean sPGI level before HP eradication was 13.4 μg / L, which increased already to 16.6 μg / L at 6 months (p=0.05) and further increased to 27.3 μg / L at the end of the study after 6 years (p=0.01). Conversely, sG-17 decreased from 84.8 pmol / L at baseline to 67.6 pmol / L after 6 years of follow-up (p<0.01). Among 21 patients treated with Acetium®, the mean sPGI level increased from 7.9 μg / L at baseline to 11.4 μg / L after 90 days of Acetium® intake, with a concomitant decrease in sG-17 from 30.3 to 25.5 pmol / L. 63 These data clearly demonstrate that eradication of HP in patients with AG in the gastric corpus leads to a long-lasting improvement in gastric mucosal function, as measured by the GastroPanel® test, with a significant and stable increase in sPGI levels and a parallel decrease in sG-17 over a 6-year follow-up period. 62,63 .

[0025] Unfortunately, this study design has several inherent weaknesses that prevent us from drawing reliable conclusions about the true effect of L-cysteine ​​(Acetium® Capsules) on the recovery of gastric mucosal function. Most importantly, biopsy confirmation of gastric mucosal structures after 3 months of Acetium® Capsules intervention (or at the end of the study) was not possible, which precludes any clear normalization of serum levels of these gastric-specific biomarkers. 52,54 This makes it impossible to determine where, in fact, the effect is related to the concomitant recovery of AG. Similarly, the lack of a placebo control means there is no way to exclude the possibility that the observed effects are due to HP eradication alone. However, because of its great conceptual and clinical importance, this concept needs to be evaluated in a thorough study design.

[0026] Acetium® capsules (and lozenges) are designed to inactivate acetaldehyde in the stomach and saliva after alcohol intake and smoking, respectively. This new concept of a potential therapeutic effect of regular ingestion of Acetium® capsules on both the structure and function of the gastric mucosa in AG subjects by removing acetaldehyde in the gastric contents would provide clear direct evidence for the concept that acetaldehyde is causally related to the development of GC.

[0027] Cysteine ​​is a non-essential amino acid that was shown (approximately 40 years ago) to have the ability to covalently bind acetaldehyde to form the stable compound 2-methylthiazolidine-4-carboxylic acid (MTCA), eliminating the toxicity of acetaldehyde. 64 MTCA is an inert, non-toxic compound that is not absorbed into the blood circulation and is eliminated from the body through stool and urine. This simple principle has been exploited in Biohit Oyj's recent innovation of Acetium® capsules containing 100 mg of L-cysteine.

[0028] Proof-of-concept studies have demonstrated that oral administration of Acetium® effectively binds acetaldehyde derived from ethanol metabolism in the acidic stomach 65 In this setting, the mean acetaldehyde level in gastric juice was 2.6-fold higher with placebo than with Acetium® (13 vs. 4.7 μM, p<0.05), implying that Acetium® can be used to reduce acetaldehyde concentrations in the non-acidic stomach during alcohol exposure.

[0029] This prompted the authors to explore the concept of whether slow-release L-cysteine ​​from a specific oral tablet (Acetium®) could be used to remove alcohol-derived acetaldehyde from saliva as well. 66Indeed, volunteer studies have shown that up to two-thirds of acetaldehyde after alcohol consumption can be removed from saliva by a sustained-release oral L-cysteine ​​formulation (Acetium®). This could have important implications, such as the prevention of upper gastrointestinal cancers in people with high exposure to acetaldehyde (heavy drinkers, smokers). 66,67,67b,67c .

[0030] Tobacco smoke contains several types of carcinogens, including polycyclic aromatic hydrocarbons, aromatic amines, and nitrosamines. Tobacco smoke also contains high concentrations of toxic aldehydes. 68 The most abundant of these is acetaldehyde, whose concentration in tobacco smoke is more than 1,000 times that of polycyclic aromatic hydrocarbons and nitrosamines specific to tobacco. 69 Acetaldehyde is also the first metabolite of ethanol oxidation. It binds to DNA and forms stable DNA adducts found in alcohol drinkers. Drinkers with alcohol dehydrogenase (ADH2) deficiency or low aldehyde dehydrogenase (ALDH1B) activity provide the most compelling evidence for the carcinogenicity of acetaldehyde. 70 This deficiency leads to local accumulation of acetaldehyde in the saliva during ethanol metabolism and significantly increases the risk of many upper gastrointestinal cancers.

[0031] Similarly, acetaldehyde from cigarette smoke has recently been shown to dissolve readily in saliva during smoking. 71 Thus, toxic aldehydes may mediate the carcinogenic effects of tobacco smoke via saliva to the oral cavity and further to the larynx, esophagus and stomach. Based on solid epidemiological and toxicological documentation, IARC (in 2009) declared acetaldehyde a Group I carcinogen, on a par with asbestos, formaldehyde, etc. 72 .

[0032] Acetium® capsules are classified as medical devices and not medicines due to their inherent design. The L-cysteine ​​in the capsules is released at a controlled rate (slowly) in the stomach, and the free L-cysteine ​​reacts with acetaldehyde in the stomach contents and is not absorbed from the duodenum. This makes a crucial difference to L-cysteine ​​obtained from food as a natural (semi-essential) amino acid that is liberated only in the duodenum (by pancreatic enzymes) and is easily absorbed into the blood circulation. This excludes the possibility of Acetium® capsules exerting a systemic effect, which is a prerequisite for classification as a medicine.

[0033] At the same time, this fact also limits the framework of new hypotheses to be tested, while precluding the assumption that the final therapeutic effect of Acetium® capsules on the atrophied gastric mucosa and its functional recovery could be mediated by some systemic effect. This leaves less room to build complex pathways regarding these mechanisms. Instead, we assume that the therapeutic effect of Acetium® capsules on the atrophied gastric mucosa (if established) must be entirely local, in other words, mediated by the established efficacy of L-cysteine ​​(Acetium®) in inactivating free acetaldehyde in the stomach, regardless of its origin (alcohol, smoking, or food). In view of this, it would be necessary for a continuous exposure of the gastric mucosa to acetaldehyde to be a prerequisite for the persistence of the so-called "Correa cascade" in the stomach, since 16,19-21,38,58 Once this cascade is initiated by HP infection, it progresses through the advanced stages of AG and IM to dysplasia (IEN) and GC unless interrupted by close monitoring of precursor lesions. To assess whether there are differences between autoimmune and HP-associated AG in this respect, we plan to include both cases in our cohort.

[0034] Apart from studies confirming the usefulness of GastroPanel® in diagnosing dyspeptic symptoms and screening asymptomatic subjects for GC risk status, GastroPanel® has also recently been studied in a longitudinal setting to evaluate biomarker profiles as long-term predictors of GC. 74,75 .

[0035] The first of these studies was a matched case-control study in a Western Siberian Caucasian population. 74 Both cases (GC) and controls (CO) were drawn from a population-based cohort (n=9,360) known as the HAPIEE (Health, Alcohol, and Psychosocial Factors in Eastern Europe) study, enrolled in Novosibirsk (Siberia) in 2003–2005. A total of 156 (52 GC and 104 CO) serum samples were collected at baseline and available for GastroPanel® analysis. Biomarker levels below cutoff values ​​predicted the development of GC as follows: PGI (2.9; 95% CI: 1.3–6.4), PGII (9.0; 95% CI: 1.8–44.3), PGI / PGII (3.3; 95% CI: 1.5–7.3), G-17 (1.8; 95% CI: 0.7–4.8), and HpIgG Ab (0.4; 95% CI: 0.1–1.3). In a multivariate model adjusting for sex, age, and all four biomarkers, the PGI / PGII ratio was the strongest independent predictor of GC (OR=2.9, 95% CI:1.01-8.0). This is the first time that PGI, PGII, and the PGI / PGII ratio have been shown to be reliable longitudinal predictors of GC development in a Caucasian population. 74 .

[0036] A second longitudinal study was completed in northern China. 75Among 12,112 participants prospectively followed up from an ongoing population-based screening program, the authors used predictive modeling to perform follow-up. At follow-up, low PGI levels and PGI / II ratios were associated with a higher risk of developing GC, as were low (<0.5 pmol / l) and high (>4.7 pmol / l) G-17 levels

[95] , and a higher serological biopsy score based on the five biomarkers at enrollment was associated with a higher risk of developing GC during follow-up (p for trend <0.001). According to the authors, the Gastro-Panel® test can be used to identify high-risk individuals who should be candidates for further diagnostic gastroscopy, thereby guiding targeted screening and customized prevention. 75 .

[0037] Combined with published meta-analyses, 76,77 These two studies suggest that the high specificity for atrophic gastritis and the long-term predictive value of the individual biomarker profile make GastroPanel a true test for gastric health and disease. A negative GastroPanel test at any point in life excludes (with a probability of more than 95%) significant gastric pathology for the next few years. 74,75 In contrast, a GastroPanel® biomarker profile suggestive of AGC is an independent predictor of increased risk of developing GC within the next decade or so. Summary of the Invention [Problem to be solved by the invention]

[0038] The invention is defined by the features of the independent claims. Some particular embodiments are defined in the dependent claims. [Means for solving the problem]

[0039] According to one aspect of the present invention there is provided an acetaldehyde-binding preparation for use in a method for improving the structure and physiology of the gastric mucosa in a subject with acid-free stomach and / or Helicobacter pylori-associated (HP+) or autoimmune (HP-) atrophic gastritis.

[0040] This and other aspects, together with the advantages thereof over known solutions, are accomplished by the present invention, as hereinafter described and claimed.

[0041] The acetaldehyde-binding preparation according to the invention is characterized mainly by what is stated in the characterizing part of claim 1.

[0042] The present invention provides considerable benefits. For example, the present invention shows that L-cysteine ​​administered by daily ingestion of Acetium® capsules is an effective therapeutic agent that improves gastric mucosal function in patients suffering from atrophic gastritis and induces simultaneous recovery of the atrophic mucosa. Moreover, this improvement and recovery is achieved without intervention by conventional medical treatment or other clinical means. [Brief description of the drawings]

[0043] [Figure 1] 1 is a flow chart of patient enrollment in the cohort study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The invention will now be described in more detail with reference to specific embodiments.

[0045] (Embodiment) The novel technology behind the present invention, simply stated, is that long-term L-cysteine ​​administered via daily ingestion of Acetium® capsules (3×200 mg / day) is an effective therapeutic agent that i) improves gastric mucosal function in AG patients who have undergone eradication of HP infection, and ii) induces a concomitant recovery (reversal) of the atrophic gastric mucosa in these subjects.

[0046] Figure 1 is a flow chart of patient enrollment in the cohort study. In the flow chart, "@" grades of AGA and AGC are confirmed by gastroscopic biopsy 1) at baseline and 2) at the end of the study. "*" Randomization occurs among subjects whose AGC progresses to moderate / severe during the first 3 years of follow-up. The intervention continues until the end of the study at 60 months.

[0047] An essential aspect of the present invention is an acetaldehyde-binding preparation for use in a method for improving the structure and physiological function of the gastric mucosa in a subject suffering from an acid-free stomach (for whatever reason, e.g. long-term use of PPIs) and / or Helicobacter pylori-associated (HP+) or autoimmune (HP-) atrophic gastritis, said method comprising at least the steps of: screening or testing symptomatic or asymptomatic subjects indicative of an autoimmune disease, dyspepsia or reflux symptoms by quantitatively measuring the concentrations of a) pepsinogen I (PGI), pepsinogen II (PGII), gastrin-17 (G-17) and Helicobacter pylori antibody (HpAb) biomarkers, or b) pepsinogen I (PGI) biomarker, or c) the PGI biomarker, the PGII biomarker and the PGI / PGII biomarker ratio, or d) the G-17 biomarker, and comparing the obtained values ​​with predefined reference ranges and / or cut-off values, Based on the above measurements and comparisons, a sustained release formulation containing 50 to 500 mg of L-cysteine ​​is administered to a subject exhibiting an acid-free stomach and / or atrophic gastritis according to a predetermined administration regimen.

[0048] According to one embodiment of the present invention, if the PGI concentration in the sample is close to the lower limit or below the reference range or cutoff value, the PGI / PGII ratio is close to the lower limit or below the reference range or cutoff value, and the G-17 concentration is close to the upper limit or above the reference range, the values ​​indicate atrophic gastritis.

[0049] According to a further embodiment of the invention, the reference range for PGI values ​​is 30-160 μg / l, for PGII 3-20 μg / l, the PGI / PGII ratio is 3 or less than 3, for G-17S (stimulated) values ​​5-30 pmol / l, for G-17B (fasted) 2-10 pmol / l and the reference range for HpAb is 0-30 EIU.

[0050] According to yet another embodiment of the invention, typical cut-off values ​​of said biomarkers are selected from the group comprising PGI 30 μg / l, PGI / PGII ratio 3, G-17S value 5 pmol / l, G-17B 2 pmol / l and HpAb 30 EIU.

[0051] In one embodiment of the present invention, "long-acting formulations with local effect on the stomach" refers to any monolithic or multiparticulate tablet, capsule or granule itself, which when moistened under the influence of gastric juices forms a gel that adheres to the gastric mucosa or floats in the stomach contents, thus prolonging the residence time in the stomach and allowing a prolonged release and local effect of the drug in the stomach. A long-acting formulation with local effect on the stomach is a liquid formulation (mixture) taken orally, the physical structure of which is a gel.

[0052] A special property required for pharmaceutical compositions acting locally on the stomach is that they remain in the stomach as long as possible. Technically, this can be achieved in two ways: by creating a preparation that adheres to the gastric mucosa or by creating a preparation that floats in the stomach contents. Preparations can be made fixable to the gastric mucosa by using cationic polymers such as chitosan of various grades as additives. Preparations that float in the stomach are obtained by using a polymer that forms a gel (for example alginic acid) and adding sodium bicarbonate to the preparation, which releases carbon dioxide under the influence of gastric acid and forms bubbles in the gel. Liquid gels that float in the stomach can also be made from sodium alginate, aluminum hydroxide, sodium bicarbonate, and water to which an acetaldehyde-binding compound can be added. Corresponding liquid preparations can also be obtained by adding an acetaldehyde-binding substance to an aqueous dispersion of chitosan. Another preparation that remains in the stomach for a long time is the one called HBS™ (hydrodynamically balanced system). This preparation can remain in the stomach for a long time if it is made into a relatively large tablet (at least 7-10 mm in diameter) and coated with a film that does not degrade in the digestive tract, but which, for example, pierces the tablet and releases the active ingredient (Oros™), provided that such a preparation is taken after a meal.

[0053] A single dose of the pharmaceutical composition having a local effect on the stomach contains 50 to 500 mg of the acetaldehyde-binding substance, preferably the amount of the acetaldehyde-binding substance is 50 to 300 mg, and most preferably 100 to 200 mg.

[0054] If necessary, update the dosage every 4 to 10 hours, preferably every 6 to 8 hours.

[0055] The amount of compound released in gastric disorders is preferably 40-80 mg per hour.

[0056] According to one embodiment, the formulations according to the invention that are released in the stomach have at least one, and often two, polymers in the stomach, whose role is to keep the drug in the stomach for as long as possible, at least 2 hours, so that the formulation adheres to the gastric mucosa or forms a gel that floats in the stomach contents. Another role of the polymer is to prolong the release of the active substance.

[0057] The preparation that locally binds acetaldehyde in the stomach can be a tablet that forms a gel in the stomach, or a capsule that contains a mixture of powder or granules that form a gel.In addition to the acetaldehyde-binding substance, the preparation contains polymers that form gels in the stomach, such as chitosan, alginate, sodium carboxymethylcellulose grade, carbomer, aluminum hydroxide, etc.The preparation can also contain sodium bicarbonate to increase floating in the stomach.

[0058] The amount of the polymer in the formulation is 10 to 50%, preferably 15 to 40%, and most preferably 20 to 30%. The amount of the polymer in the formulation is 10 to 50%, preferably 15 to 40%, and most preferably 20 to 30%.

[0059] The amount of sodium bicarbonate can be 10-30% of the amount of polymer, preferably 20%.

[0060] The preparation for locally binding acetaldehyde in the stomach is a tablet or granule preparation, in which the acetaldehyde-binding substance is mixed with the necessary filler and then granulated using an enteric polymer as a binder. The binder used can be any known enteric polymer, preferably a polymer with a solution pH of 6-7. Most preferably, the polymer is one of the methacrylate derivatives known under the trade names Eudragit L and Eudragit S. The amount of enteric polymer in the preparation is preferably 2-5%, most preferably 3-4%.

[0061] The preparation that locally binds acetaldehyde in the stomach can be a liquid preparation, i.e., a mixture that contains, in addition to the acetaldehyde-binding substance, sodium alginate, aluminum hydroxide, sodium bicarbonate, and water. The amount of water in the entire preparation is 70-90%, most preferably about 75-85%. The amount of sodium alginate in the preparation is preferably 2-10%, most preferably about 5%, and the amount of aluminum hydroxide is preferably 5-15%, most preferably about 10%.

[0062] The relative composition of a formulation containing granules may, for example, be as follows: Acetaldehyde binding substances 60 parts Chitosan 10-40 parts Calcium hydrogen phosphate 0-30 parts

[0063] In one embodiment of the present invention, "long-acting formulations having a local effect in the large intestine" refers to any monolithic or multiparticulate tablet, capsule or granule itself that does not release the dosage for an extended period of time until the formulation reaches the end of the small intestine or reaches the large intestine.

[0064] Formulations according to the invention which provide extended release of acetaldehyde-binding substances in the large intestine, by either release mechanism, deliver the acetaldehyde-binding substances to the last part of the small intestine or to the large intestine before the substance of interest is released.

[0065] Pharmaceutical compositions that bind acetaldehyde in the large intestine are administered orally. Numerous technologies are available for releasing orally administered drugs in the large intestine. The most functional solutions are based on the use of enteric polymers. Both tablets and granules can be provided with a film coating that does not dissolve in the acidic environment of the stomach, but dissolves at a pH value of 7 at the latest. In the manufacture of the formulations, it is also possible to use polysaccharides that degrade under the influence of microorganisms in the large intestine, or polymers produced by azo bonds. A form of the formulation known under the trade name Oros™ can also be used, if its opening is first covered with an enteric polymer and the pH of the solution is around 7.

[0066] Useful enteric polymers include, for example, the hydroxypropyl methylcellulose acetate succinate (HPMC-AS) grades sold under the trade name Aqoat™, particularly Aqoat AS-HF™, the cellulose acetate phthalate (CAP) grades sold under the trade name Aquateric™, and the methyl methacrylate copolymer grades sold under the trade name Eudragit-S™, particularly.

[0067] The formulation according to the invention has at least one component that regulates the release of the active substance so that it does not occur until the end of the small intestine or in the large intestine. This component can be a polymer that dissolves depending on the pH (=enteric polymer) or a polymer that degrades under the influence of enzymes secreted by bacteria in the large intestine. The polymer that controls the release location can form a film around the entire formulation. It can also form a membrane around particles (granules) contained in a multi-liquid formulation. The polymer that degrades under the influence of enzymes secreted by bacteria in the large intestine can also be used as a filler in a monolithic formulation or as a filler in granules or in multi-unit formulations prepared from these granules.

[0068] The formulation according to the invention is an enteric tablet, the film coating of which does not dissolve until the end of the small intestine or the beginning of the large intestine. The dissolution pH of the film-forming polymer is 6.0-7.5, preferably 6.5-7.0. The amount of the film-forming enteric polymer is 5-20%, preferably 10-15%, of the total mass of the tablet. The tablet filler may include a non-swelling pharmaceutical excipient such as calcium hydrogen phosphate.

[0069] The preparation according to the present invention may be a granule containing an acetaldehyde-binding substance and coated with an enteric film, the dissolution pH of the film-forming polymer being 6.0-7.5, preferably 6.5-7.0. The amount of the film-forming enteric polymer relative to the total mass of the granule is 5-30%, preferably 15-25%. The granule may contain 20-40%, preferably about 30%, of a poorly water-soluble filler such as calcium hydrogen phosphate.

[0070] According to the present invention, the binder of the enteric film coated granules may be an enteric polymer with a dissolution pH of 6.0-7.5, preferably 6.5-7.0. The amount of binder in the granules is 2-5%, preferably 3-4%.

[0071] The preparation according to the present invention may also be a tablet containing the above-mentioned enteric coated granules on which an enteric film is formed. The tablet made for such a preparation contains not only the enteric granules but also a filler suitable for direct compression such as microcrystalline cellulose, the amount of which is 30-70%, preferably 40-60% of the tablet.

[0072] A dosage unit of the pharmaceutical composition preferably contains 50 to 500 mg of the acetaldehyde-binding substance, preferably the amount of the acetaldehyde-binding substance is 50 to 300 mg, most preferably 100 to 200 mg.

[0073] The amount of the compound released in the large intestine is preferably 50 to 100 mg per hour.

[0074] If necessary, administration can be repeated at intervals of 4 to 10 hours, preferably at intervals of 6 to 8 hours.

[0075] The composition of an enteric coated tablet, which contains enteric coated granules and binds acetaldehyde in a desired manner, can be, for example, as follows: Enteric coated granules: Acetaldehyde binding substances 100mg Filler, e.g. calcium hydrogen phosphate 30-50mg Enteric polymer 40-60mg Enteric-coated tablets: Enteric coated granules 170-210mg Microcrystalline cellulose 170-210mg Lubricant (e.g., magnesium stearate or talcum) 5-10 mg Enteric polymer 30-50mg

[0076] Reference to an embodiment throughout this specification means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment. For example, when referring to a numerical value using terms such as about or substantially, the exact numerical value is also disclosed.

[0077] As used herein, a plurality of items, structural elements, components, and / or materials may be presented in a common list for convenience. However, such lists should be construed as if each member of the list were individually identified as a separate and unique member. While the foregoing examples illustrate the principles of the invention in one or more specific applications, it will be apparent to those skilled in the art that numerous changes in form, use, and details of implementation may be made without the exercise of the inventive faculty and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the scope of the claims set forth below.

[0078] In this document, the verbs "comprise" and "include" are used as open limitations that do not exclude or require the presence of unmentioned features. The features recited in the dependent claims may be freely combined with each other, unless otherwise stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. the singular form, throughout this document does not exclude the plural. EXAMPLES

[0079] Example 1 A local long-acting formulation that binds acetaldehyde in the stomach can be prepared and used to reduce the risk of cancer caused by acetaldehyde as follows. The relative composition of a formulation that locally binds acetaldehyde in the stomach is, for example, as follows:

[0080] L-Cysteine ​​60 parts Chitosan 10-40 parts Calcium hydrogen phosphate 0-30 parts

[0081] The powder mixture is mixed by a conventional mixer (such as a blender) used in the pharmaceutical industry. The powder mixture is then granulated using 2.5% acetic acid as the granulation liquid. The granulation liquid can be added to the same blender. The wet powder mass is compressed through a screen plate or a perforated plate (the diameter of the opening is 2 mm). The formed granules are dried and sieved. The 1.2-1.7 mm screen fraction is collected and dispensed into hard gelatin capsules so that the dose of cysteine ​​is 100 mg.

[0082] The tablets prepared above are taken to reduce the risk of cancer caused locally by acetaldehyde in situations favorable for an increased acetaldehyde content in the stomach, associated with the intake of alcoholic beverages. Doses are given at intervals of 4-6 hours as long as there is alcohol in the blood.

[0083] Example 2 A pharmaceutical composition that releases acetaldehyde-binding substances in the large intestine for a long period of time can be prepared as follows and used to reduce the risk of cancer caused by acetaldehyde. The composition of an enteric coated tablet, which contains enteric coated granules and binds acetaldehyde in a desired manner, is, for example, as follows:

[0084] Enteric coated granules: L-Cysteine ​​100mg Calcium hydrogen phosphate 40mg Eudragit-S 5mg Aqoat AS-HF 40mg Enteric-coated tablets: Enteric coated granules 185mg Microcrystalline cellulose 185mg Magnesium stearate 4mg Talcum 4mg Aqoat AS-HF 40mg

[0085] L-cysteine ​​is mixed with calcium hydrogen phosphate, which acts as a filler. Eudragit S is dissolved in ethanol (20% solution) and the solution is used to moisten the powder mixture. The moist mass is compressed into granules. The dried granules are sieved and the granule fraction of 1.2-1.7 mm is coated with Aqoat AS-HF. The composition of the coating liquid is as follows: Aqoat AS-HF 10%, triethyl citrate 3.5%, magnesium stearate 3%, and water 83.5%. The coated granules are mixed with microcrystalline cellulose (e.g. Emcocel LP 200™) and finally, the lubricants magnesium stearate and talc are added to the mixture. The mixture is then compressed into tablets and finally, an enteric film is created on the tablets in the same way as for the granules. Mixers, granulators, sieving equipment, film coating equipment, and tablet presses commonly used in the pharmaceutical industry can be used for the entire operation.

[0086] The composition prepared above is taken orally in conjunction with the consumption of an alcoholic beverage, and administration is repeated at 4-6 hour intervals as long as alcohol is present in the blood.

[0087] Example 3 This study is designed to i) prove the concept that GastroPanel® biomarker assay is a reliable non-invasive tool in the safe follow-up of AG patients, ii) evaluate the value of GastroPanel® biomarker profile as a predictor of disease outcome in AG patients during long-term prospective follow-up, and iii) establish whether differences in clinical outcomes exist between HP+ and HP- patients with AG. Furthermore, iv) this longitudinal study incorporates a randomized controlled trial to validate the novel concept that Acetium® capsule effectively inhibits the Correa cascade, leading to the restoration of gastric function and therefore the recovery of gastric atrophy (AG), regardless of HP-related or autoimmune origin.

[0088] A longitudinal follow-up study of a cohort of patients with biopsy-confirmed AG at baseline, designed to establish the natural history (clinical outcome) of AG without medical or other interventions. Potentially eligible AG patients will be screened among consecutive gastroscopy referred patients by GastroPanel® examination. Eligible patients will have AG (corpus, antrum, or both) classified as mild, moderate, or severe by gastroscopic biopsy and serological biomarker assays. Both patients with autoimmune AG (HP-) and those who test positive for HP by GastroPanel® examination will be enrolled. Prior to enrollment, all subjects will be asked to sign a written informed consent. No medical treatment for HP infection will be performed to avoid interfering with the natural disease outcome.

[0089] The study is overseen by a Steering Committee composed of members of our Scientific Committee and representatives from our partner clinics. These two guidelines provide detailed recommendations regarding patient selection (diagnosis), study design, and outcome evaluation, which are described below.

[0090] (Research Objectives) The general goal of this study is to establish the natural history of AG, i.e., the clinical outcome of the disease without intervention by medical treatment or other clinical measures (e.g., repeated biopsies, mucosal resections, gastric surgery). This natural history of AG will be compared with the clinical outcome of patients undergoing active clinical intervention by regular intake of Acetium® capsules, which has recently been suggested as a potential treatment for AG.

[0091] In this longitudinal setting, a cohort of 500 subjects with biopsy-confirmed AG (AGA, AGC, AGP) are prospectively followed up with annual monitoring with non-invasive serological biomarker testing (GastroPanel®), which is repeated at 12-month intervals until 5 years of follow-up are completed.

[0092] During follow-up, if the GastroPanel® test reveals progression of AG, subjects will be randomly assigned to i) placebo group or ii) Acetium® intervention group. In the former, subjects will continue the same study protocol as before with 12 months of GastroPanel® monitoring without medical intervention until the end of follow-up. In the Acetium® group, subjects will start using Acetium® capsules (200 mg three times daily with meals). Otherwise, GastroPanel monitoring will continue at 12-month intervals until the end of the study at 60 months.

[0093] This study has four specific objectives: i) to validate the concept that the GastroPanel® biomarker assay is a reliable non-invasive tool in the follow-up of patients diagnosed with AG; ii) to evaluate the value of the GastroPanel® biomarker profile as a predictor of disease outcome in patients with AG during a long-term prospective follow-up; iii) to establish whether differences in clinical outcome exist between HP+ and HP- patients with AG; and finally iv) to test the novel hypothesis that L-cysteine ​​administered by daily intake of Acetium® capsules (3 x 200 mg / day) is an effective treatment to induce recovery of the atrophied gastric mucosa.

[0094] (Patient Choice) Patients will be enrolled in the study at hospitals X, Y, and Z. Only patients with biopsy-confirmed gastric corpus aggravation (AGC) and / or antral aggravation (AGA), classified as mild, moderate, or severe using the USS classification, will be enrolled in the study cohort. 35,36 To participate in the cohort, patients must give written consent prior to enrollment in the cohort.

[0095] The flow chart of patient enrollment in the cohort is shown in Figure 1. Briefly, patients referred for gastroscopy due to various indications are enrolled in the three clinics. Apart from clinical routine and laboratory tests, all patients are examined with GastroPanel® for non-invasive diagnosis of the gastric mucosa. All patients with normal GastroPanel® results are not included in the follow-up cohort and should be returned to usual management.

[0096] (Definition of atrophic gastritis) Based on abnormal GastroPanel® test results consistent with AG (AGA, AGC, or AGP), subjects are assigned to two groups: i) HP+ patients, and ii) HP- patients. Among HP patients, only the AGC group is possible, since by definition the diagnosis of AGA requires the presence of HP. Therefore, both the AGA and AGC groups follow only the HP+ diagnosis in the flow chart (Figure 1).

[0097] In both the AGA and AGC groups, the biopsy results are correlated with the GastroPanel® biomarker profile for mild, moderate, or severe AG according to the PGI and G-17 cut-off values ​​for AGC and AGA, respectively. As for AGC, the cut-offs for moderate AGC are 70 μg / l, for moderate AGC 50 μg / l, and for severe AGC 30 μg / l. The likelihood of severe AGA increases with G-17 values ​​below 1.0 pmol / l, while moderate and mild AGA usually show G-17 values ​​of 1-2 pmol / l. Importantly, these cut-off values ​​of the individual biomarkers are based on studies mostly performed in low-risk countries for GC and are not necessarily directly applicable to the current situation. More refined data on the cut-off values ​​of the individual GastroPanel® biomarkers in different grades of AG will be obtained if the GastroPanel® results are correlated with the biopsy results at the end of the study. This is another important "side result" of this study.

[0098] (Age at time of study participation) In principle, there is no specific age limit for subjects to be included in this study. Considering that AG is a rare condition under the age of 45–50 years, it is expected that the majority of subjects enrolled will be aged 50 years or older.

[0099] (sex) Both male and female participants are eligible to participate in the cohort. Every effort has been made in this study to minimize sex selection bias by encouraging the participation of both women and men, with the exception of women who are pregnant (obviously rare) who are excluded for this reason.

[0100] (Concomitant medications, comorbidities, exclusion criteria) Because Acetium® capsules have no known interactions with other medications, subjects are usually allowed to continue their usual medications for illnesses not related to AG, although other (eventual) medications targeted at AG must be discontinued before participating in the study.

[0101] The following subjects will be excluded: patients who meet criteria for substance abuse; patients who have taken antipsychotics or antidepressants in the last month; patients who abuse alcohol or other drugs; potentially fertile and sexually active women not using contraception.

[0102] The subjects will be patients who visit the hospital outpatient department due to dyspepsia symptoms, i.e. symptomatic patients. The aim is to enroll a cohort of subjects with minimal comorbidities. However, patients with autoimmune diseases (type 1 diabetes, autoimmune thyroiditis, pernicious anemia) will need to accumulate cases with autoimmune (HP-) AG.

[0103] Specific comorbidities that exclude participation in this trial include the following categories of patients: severe psychiatric disorders, infectious diseases, malignant tumors, patients with short life expectancy, cardiovascular diseases, cerebrovascular diseases, uncontrolled hypertension, degenerative diseases of the central nervous system, and pregnant and lactating women. In addition, the following patients should be considered ineligible: 1) patients whose gastric conditions require surgical treatment or immediate follow-up treatment of the main symptoms, and 2) patients who refuse to sign a written informed consent.

[0104] (Follow-up survey) As shown in the flow chart (Figure 1), subjects with AGA or AGC graded by biopsy as mild, moderate or severe, enrolled and appropriately assigned to 1) HP+ and 2) HP- groups will constitute a cohort that will be prospectively followed for 5 years (60 months). Enrolled patients will be instructed to follow their familiar dietary habits and use prescribed medications as usual. All patients will be monitored in the clinic by GastroPanel® assays at 12-month intervals for a total of 5 years, i.e., a total of 6 tests (baseline + 5 annual tests) if they are not randomized to the Acetium intervention group (see below: 2.4).

[0105] (Acetium® Intervention) If the GastroPanel® study shows progression of AG (AGC only) during the first 3 years of follow-up, patients will be randomized to 1) the Acetium® intervention group, or 2) the non-intervention group (Figure 1). The randomization criteria include i) progression from mild to moderate AGC, and ii) progression from moderate to severe AGC, and apply equally to the HP+ and HP- study groups. The study is not interested in testing subjects with AGA, due to the lack of evidence regarding the potential usefulness of Acetium® in these patients. Therefore, randomization applies only to AGC patients.

[0106] (blinded) Because no physical placebo was used, blinding is not required during the randomization phase, except for the pathologists examining the biopsies taken during gastroscopy at the end of the study.

[0107] (Placebo control) No placebo will be used in this open-label study, and patients assigned to the non-intervention group will continue to be followed up with repeat GastroPanel examinations at 12-month intervals, similar to non-progressing patients. These patients will serve as a study cohort of AG lesions who will be followed for clinical outcomes without medical or other interventions.

[0108] (Randomization) Because subjects eligible for randomization will accumulate over time (any time between 12 and 36 months of follow-up), subjects will be randomized in relatively small blocks to avoid potential bias due to changes in selection criteria over time. In this study, randomization will be performed using a random number generator (https: / / www.sealedenvelope.com / simple-randomiser / vl / lists) with a block size of 4 to create a unique randomization code for each subject.

[0109] (layering) Randomization alone may not ensure a complete comparison between participants in the two treatment groups, and stratified randomization is necessary to correct potential imbalances between the two treatment groups. Among the baseline characteristics of AG that may affect the efficacy outcomes of the study, the most obvious are: 1) severity of AG (moderate / severe), 2) localization of AG (antrum / corpus), 3) extent of AG (antrum-only gastritis, corpus-only gastritis, vs. pangastritis), and 4) association with HP infection or not. However, according to the recommended practice, these baseline stratification variables are entered as covariates into the final multivariate model to control for potential confounding of efficacy endpoints.

[0110] (Duration of Acetium® Intervention) In the only published Acetium intervention trial, patients received Acetium capsules (100 mg three times daily) for three months. 62,63 The rationale behind this treatment is unclear. L-cysteine ​​administered in this form (Acetium® capsules) is a natural amino acid with no systemic effects or known toxicity, and therefore no upper limit on daily use. However, when used for its intended indication (inactivation of acetaldehyde in the stomach after alcohol consumption), the maximum recommended daily dose is 1000 mg (10 capsules per day).

[0111] Similarly, there is no convincing argument to substantiate the 3-month period chosen for Acetium® capsule treatment. 62,63 Similarly, it can be argued that since the regeneration time of the gastric mucosa is very short (under normal circumstances), the removal of the harmful substance (acetaldehyde) with Acetium® capsules interrupts the Correa cascade and one can expect the mucosa to heal in a much shorter period than 3 months. However, it is equally valid to argue that the development of gastric carcinogenesis, initiated by HP and advanced to the stage of AG, is a long process and that even after the elimination of HP, the recovery of the mucosa caused by a second trigger (such as acetaldehyde) is much less effective than, for example, after early eradication of active HP infection, and furthermore, requires the use of Acetium® capsules for a period longer than 3 months.

[0112] The latter view was adopted in the present study. To evaluate the potential efficacy of Acetium® in full-blown AGC, potential targets for Acetium® intervention are patients with established advanced AGC. Progression here means a confirmed progression from mild AGC (baseline) to a stage of moderate AGC during the first 3 years of follow-up. This time limit is set at 36 months to leave sufficient time for Acetium® intervention, i.e. a minimum of 2 years, given that the total study period is 5 years. This period of more than 2 years seems long enough to answer the following questions: 1) whether Acetium® intervention (3 x 200 mg / day with meals) is effective in arresting the progression of AGC, and 2) whether it induces the recovery of the atrophic mucosa of the gastric corpus.

[0113] (Follow-up surveys and completion of studies) Eligible patients (n=500) will be assigned to two main study groups: HP-positive AG and HP-negative AG. The latter likely represents autoimmune AG, whereas the former represents HP-induced AG. Enrolled patients will be instructed to follow their familiar dietary habits and use prescribed medications as usual, i.e., continue to lead their normal daily lives.

[0114] All patients are monitored by GastroPanel® assays at 12-month intervals for a total of 5 years, i.e., 6 tests in total (baseline + 5 annual tests). The follow-up protocol is the same for subjects in the Acetium® intervention group and those in the non-intervention group. At the end of the study after 60 months, all patients undergo gastroscopy and biopsy. These end-of-study biopsies are read in a blinded manner by two expert pathologists using the updated Sydney System (USS) gastritis classification. Blinding is particularly important in order not to reveal whether subjects belong to the non-intervention group or the Acetium intervention group.

[0115] (compliance) Evidence of poor compliance is not uncommon in prospective cohort studies of patients with chronic HP infection and / or AG, but longitudinal cohort studies can still be completed successfully. 14,15,17,18,25,42,43 It is therefore important to monitor patient adherence to follow-up procedures, particularly the Acetium® intervention question, throughout the 5-year study period. One such approach would be to conduct drug or tablet counts at each follow-up visit and to repeatedly emphasize the importance of adherence to protocol requirements.

[0116] (Research results) At the end of the study after 60 months, all patients undergo a final GastroPanel® examination and gastroscopy with biopsy (see biopsy protocol). These end-of-study biopsies are read in a blinded manner by two expert pathologists using the latest Sydney System (USS) classification of gastritis. In the final analysis, these end-of-study biopsies are correlated with each patient's AG status in the baseline biopsy, and three potential (mutually competing) outcomes are defined: 1) no progression; 2) progression (increase in at least one AG grade or spread of AG to another localized area); and 3) regression (decrease in at least one AG grade or disappearance of AG in another localized area). This applies essentially to both the non-intervention group and the Acetium® intervention group. In the latter group, if the progression of AG is stopped by the Acetium® intervention, outcome 1) can also be defined as cessation of disease progression. If the intervention is associated with a regression outcome 3), we can speak of mucosal recovery or healing. As with the non-intervention group, all three outcomes can be assessed separately in the HP+ and HP− groups, providing insight into the eventual differences in AG outcomes between these two disease categories.

[0117] [method] The study overview and study design are detailed above. The following methods described here will be used to test patients and their samples at baseline and follow-up surveys according to the study protocol.

[0118] (Gastroscopy and biopsy procedures to determine patient eligibility) According to the flow chart (Figure 1), gastroscopy and biopsy are the first-line diagnostic procedures for patients referred for gastroscopy. Only patients whose biopsy reveals AG are potentially eligible for the study and are interviewed for possible exclusion criteria before enrolling in the study. At baseline, a GastroPanel® examination is also performed to control for gastric biomarker status; this is an important baseline measurement that will be used as a reference in the Acetium® intervention group (see below).

[0119] According to the flow chart (Figure 1), the follow-up of study subjects in both the HP+ and HP- groups, as well as in the non-intervention and Acetium intervention groups, is based exclusively on GastroPanel® examinations repeated at 12-month intervals. Only at the final follow-up, which marks the end of the study, will all subjects undergo regular gastroscopy complemented by biopsy samples from the gastric antrum and body, according to the principles of USS.

[0120] (Biopsy Protocol) During endoscopy (esophagogastroduodenoscopy), all abnormal mucosal lesions observed are recorded, photographed and, if necessary (e.g., suspicion of malignancy), additional biopsies are taken. The optimal biopsy protocol follows the USS classification. In each patient, routine biopsy specimens are taken from the gastric antrum and body, with at least two biopsies from each. These biopsies are taken from the greater curvature and lesser curvature of the central gastric antrum and the greater curvature of the gastric body. In addition, it is recommended to take two more biopsies from the incisional margin. It is important to note that, to facilitate the pathology reading, the biopsies from the gastric antrum and incision (biopsies 1, 2, 3 and 4) should be placed in one and the same formalin tube and embedded in the same paraffin block (block no. 1; labeled ANTRUM). The two biopsies from the gastric body are placed in another formalin tube and embedded in the same paraffin block (block no. 2; labeled CORPUS).

[0121] (Preparation of microscope slides) Biopsies from formalin bottles / tubes are embedded in paraffin using routine procedures in the hospital's pathology laboratory. Blocks are cut into four μ sections and stained with hematoxylin-eosin (HE) for routine diagnosis and modified Giemsa stain for identification of HPs in the specimens.

[0122] (Biopsy interpretation) All gastroscopic biopsies are examined among the samples taken daily by expert pathologists at Hospital X. Diagnosis is reported using USS to classify gastritis into different "phenotypes" of gastritis.

[0123] In this classification, the topography of the gastritis is considered fundamental. Thus, gastritis limited to the antrum, gastritis limited to the corpus, or pangastritis should be reported separately. If the etiology of the gastritis is known, it should be added as a prefix to the diagnosis (e.g., HP antral gastritis, autoimmune corpus gastritis, etc.). As a suffix, it should be graded for each of the five morphological variables shown in Appendix 6. These are; 1) chronic inflammation (chronic gastritis); 2) activity of the gastritis measured by the presence of polymorphonuclear leukocytes in the inflammatory infiltrate; 3) intestinal metaplasia (IM); 4) atrophy manifested by loss of normal mucosal glands; and 5) presence of HP microorganisms. The guidelines recommend recording these five parameters separately in both the antrum and corpus and classifying them semiquantitatively as “absent,” “mild,” “moderate,” or “severe.”

[0124] GastroPanel® Screening of Baseline Biomarker Status The GastroPanel® test is a first-line diagnostic test used in conjunction with gastroscopy for the initial screening of potential study subjects. Due to its very high negative predictive value (NPV), 52-56 , A normal GastroPanel® examination excludes AG, whereas a test result suggestive of AG (gastric antrum, gastric corpus, or both) correlates closely with a biopsy. At follow-up intervals of 12 months, the status of the gastric mucosa is monitored exclusively using the non-invasive GastroPanel®, with particular reference to the clinical findings of AG (Figure 1).

[0125] GastroPanel® is a user-friendly ELISA technology consisting of a panel of four biomarkers specific for the gastric mucosa: 1) Pepsinogen I (P-PGI), 2) Pepsinogen II (P-PGII), 3) Gastrin-17 (PG-17) and 4) HP antibody (P-HpAb). The current test version is called Unified GastroPanel, in which all biomarkers are processed using unified processing conditions for ELISA automation.

[0126] (ELISA test for pepsinogen I and pepsinogen II) P-PGI is secreted exclusively by the chief cells of the gastric corpus mucosa (chief cells / mucous neck cells). Atrophic corpus gastritis results in the loss of these cells, resulting in a decrease in circulating P-PGI levels. P-PGII is produced by the chief and mucous neck cells of the gastric mucosa, the pyloric glands of the gastric antrum, and the Brunner's glands of the proximal duodenum. The concentration ratio of PGI to PGII in plasma of normal subjects is 3.0 or higher. Although not secreted by other cells in other anatomical sites, these two biomarkers are specific to the gastric mucosa and are therefore gastric-specific biomarkers.

[0127] In the GastroPanel® test, P-PGI and P-PGII biomarkers are measured from plasma samples according to the manufacturer's instructions: Pepsinogen I ELISA kit (Biohit catalog number 601 010.01), Pepsinogen II ELISA kit (Biohit catalog number 601 020.01). Both P-PGI and P-PGII ELISAs are based on a sandwich enzyme immunoassay technique using PGI and PGII specific capture antibodies adsorbed to a microplate and a detection antibody labeled with horseradish peroxidase (HRP).

[0128] (Gastrin-17 ELISA test) G-17 is secreted exclusively by gastrin cells (G cells) in the gastric antrum and accounts for a portion of the total gastrin concentration in the circulating blood. Resting G cells secrete only small amounts of the G-17 hormone. Maximal secretion is achieved after physiological protein stimulation or when gastric acid secretion is low or absent. As a result of atrophy (loss of glands) in the gastric antrum, the amount of G cells decreases, which results in a decrease in both basal and postprandial secretion of gastrin. GastroPanel®'s G-17 ELISA method is specific for the "amidated" G-17 molecule, which is the most important member of the gastrin / cholecystokinin family and regulates the physiology of the upper gastrointestinal tract.

[0129] (Gastrin-17 stimulation) If the GastroSoft® report from the fasting sample suggests antral AG, it is recommended to repeat the gastrin-17 test with a postprandial blood sample. Secretion of G-17 can be stimulated by the ingestion of a protein drink with an average protein content of 77% [Biohit Cat. No. 601038 (50 x 20 g), Cat. No. 601037 (5 x 20 g)]. This stimulation should not be performed in lactose-sensitive patients (lactose intolerant or hypolactosemic). To prepare the protein juice, mix 20 g of protein (one foil bag of protein powder) with 150 ml of water. The stimulated (postprandial) blood sample should be taken within 20 minutes after ingestion of the protein juice.

[0130] (ELISA test for Helicobacter pylori) The GastroPanel® test for Helicobacter pylori is performed on plasma samples. The test is based on an enzyme immunoassay in which purified H. pylori bacterial antigens are adsorbed onto microplates and a detection antibody labeled with horseradish peroxidase (HRP) is used.

[0131] Patient Preparation for GastroPanel® Testing To obtain reliable results of the GastroPanel® test, several preparatory measures of the patient are necessary: ​​detailed instructions are given to each subject at the time of consenting to participate in the GastroPanel screening phase (Figure 1).

[0132] To perform the GastroPanel® test properly and obtain reliable results, some preparatory steps are required by the patient. Usually, each subject is given detailed instructions when they consent to participate, but this was not the case in this case, as all subjects were already prepared for the gastroscopy. Before blood samples were taken, compliance with the following medications was checked:

[0133] Patients should not drink, eat, or smoke alcohol for at least 4 hours prior to sample collection; for example, a 10-hour overnight fast is optimal. Patients may take regular medications as prescribed; however, they must report in the application (Appendix 2) the use of proton pump inhibitors (PPIs such as esomeprazole, lanzoprazole, omeprazole, and rabeprazole) and when they stopped using PPIs, as these medications interfere with the output of GastroPanel® biomarkers (www.gastropanel.com / GastroPanel®Sample Collection Instructions).

[0134] (Sample Collection for GastroPanel® Testing) For each patient, two plasma tubes are collected for the GastroPanel® test. Additionally, gastrin-17 can also be measured after stimulation (see 3.1.3). A minimum of 2 ml of EDTA plasma is collected from a fasting blood sample into an EDTA tube (e.g., Biohit catalogue no. 454235, Vacuette, 4 ml tube containing K2EDTA). Using Gastrin-17 stabilizer 100 μl / 2 ml plasma (Biohit catalogue no. 601 050 or 601 051) allows the transfer of samples at room temperature (20-25 °C) and allows ELISA testing within 4 days of sample collection.

[0135] (Sample processing) Blood samples should be centrifuged within 30 minutes at 1800-2000g for 10 min (e.g., Vacuette, Biohit catalogue no. 454235) or according to the tube or centrifuge manufacturer's instructions (e.g., StatsSpin Express2, 4000g for 2 min). EDTA plasma should be immediately frozen (-70°C) if not used immediately for testing. The recommended storage temperature for samples with added gastrin-17 stabilizer is 2-8°C in the refrigerator for a maximum of 4 days. If samples cannot be analysed within 4 days, they should be stored frozen at -15-20°C, but if they are to be stored for more than 2 weeks, the temperature should be -70°C.

[0136] Mix samples well after thawing. Multiple freeze-thaw cycles should be avoided. Do not use lipemic or cloudy specimens. If a postprandial blood sample is required, collect in an EDTA tube 20 minutes after ingestion of a protein drink. For more information, see the section on Gastrin-17 Stimulation (see www.biohithealthcare.com / GastroPanelSample Collection Instructions and below).

[0137] (Evaluation of GastroPanel® results with GastroSoft® application) A prerequisite for reliable results is an adequate EDTA plasma sample taken according to the manufacturer's instructions for sampling (mentioned above) and for carrying out the ELISA test. The results of the GastroPanel® test are evaluated using the GastroSoft® software. The principles and algorithms used in the GastroSoft® software are based on the updated Sydney System for Gastritis Classification (USS). The comprehensive biomarker profiles and diagnostic equivalents in the USS are summarized in Table 1. Table 1 also shows other clinical conditions (gastric functional abnormalities) that are diagnosed by specific biomarker profiles.

[0138] [Table 1]

[0139] (statistical analysis) All statistical analyses are performed using SPSS 27.0.1.0 for Windows (IBM, NY, USA) and STATA / SE 17.0 software (STATA Corp., TX, USA). Descriptive statistics are performed according to routine procedures. Frequency tables are analyzed using likelihood ratio (LR) or chi-squared Fisher's exact test for categorical variables. Differences in the means of continuous variables (e.g. biomarker levels) are analyzed using nonparametric (Mann-Whitney or Kruskal-Wallis) tests for two and multiple independent samples, respectively.

[0140] (Follow-up survey in the non-intervention group) In a basic research setting, this longitudinal cohort study is designed to examine the clinical outcomes of AG without intervention during a 5-year prospective follow-up period. These clinical outcomes will be disclosed only at the end of the study when gastroscopy and biopsies will be available. In the final analysis, these end-of-study biopsies will be compared with each patient's baseline serologic AG status and can be classified into one of three potential (mutually competing) outcomes: 1) no progression, 2) progression, and 3) regression.

[0141] (life table technique) A longitudinal study design with clearly established clinical outcomes (study end points) produces data suitable for analysis by different life table techniques and can be used to compare the two study groups (HP+ and Hp- cases). In its simplest form, this is done using univariate survival (Kaplan-Meier) analysis, where either i) an AG progression event (at any follow-up) or ii) an AG regression event detected by the respective change in the GastroPanel biomarker profile is used as end point for the KM analysis. The follow-up date represents the event of interest and stratified (HP+ vs. HP-) estimates are calculated using the log-rank (Mantel-Cox) statistic. Using the same approach, the difference in seizure-free duration (time to progression or regression) between the two study groups can be calculated. The independent effect of Acetium has already been considered here using a multivariate Cox proportional hazards regression model, where all recorded baseline characteristics of the patients (questionnaires) can be entered as covariates.

[0142] (Generalized Linear Models (GEE and Panel Poisson)) Furthermore, the effect of HP+ and HP- can also be modeled using a regression approach based on count variables, i.e. Poisson regression, using AG progression or AG regression as the event of interest. In this approach, AG regression or AG progression is expressed as an event per person-at-risk time (months) and the two groups (HP+ and HP-) can be compared using the incidence rate ratio (IRR) statistic. When applied to panel-type data recorded at each FU survey (panel Poisson), all covariates (biomarker levels) affected by random within-subject variability (per FU survey) can be appropriately controlled in this longitudinal setting. A similar approach based on panel data, i.e. generalized estimating equation (GEE) modeling, can be used to estimate the effect of HP (HP+ / HP-) on AG persistence (= no regression, no progression) using the dichotomized yes / no of persistence recorded at each FU survey as the dependent variable and adjusted for potential confounders in the multivariate GEE. Various such potential confounders should be investigated, as recorded in the baseline questionnaire.

[0143] (Modeling AG outcomes with competing risks regression) In all study settings where the same subjects are repeatedly measured, the results tend to be correlated. In other words, if the recordings are repeated within a reasonable period (years in this case), the probability of recording persistent AG increases among subjects who reported persistent AG (no change from baseline) at the previous follow-up. i) statistical methods that do not take these correlations into account are not valid, and ii) methods that do not utilize all the recorded data (in a repeated measures setting) are inefficient. Both marginal (GEE, panel Poisson) and mixed-effects models can address these issues and have shown high efficiency compared to standard logistic regression and Cox models for studying clinical outcomes of AG. Although both methods are technically suitable for the analysis of panel data in our study setting, they only accept dichotomous (yes / no) dependent variables, thus requiring separate analyses for each of the multiple study outcomes (no progression, progression, regression).

[0144] Natural history studies of AG are more complex than simply showing progression (Correa cascade) versus non-progression as a dichotomous (yes / no) outcome. When carefully modeled, three possible outcomes are expected to be observed during AG follow-up, which must be treated as competing (mutually exclusive) events. These include: i) no change in AG (= no change in AG grade compared to baseline), ii) progression of AG (= increase in AG grade from baseline status), and iii) regression (= decrease in severity of AG grade at study conclusion compared to baseline).

[0145] To i) fully utilize longitudinal data, ii) take into account the within-subject variability of repeated measures during FU studies, and iii) handle multiple outcome dependent variables (no change, progression, regression) in a single statistical model, a special technique called competing risks regression must be applied. 78,79 This elegant approach can also be used to model the impact of Acetium® intervention on the competing risks outcome of this study, adjusting for other covariates as potential confounders. The main advantage of the traditional (Cox) model is that competing events prevent the occurrence of the event of interest, whereas in Cox the event of interest is not observed due to regular censoring (exclusion) from the study. In simple terms, competing risks regression generates a hazard for the event of interest while at the same time keeping subjects who experience a competing event "at risk" so that they are not counted appropriately and potentially missed. 78,79 .

[0146] (Acetium intervention) The Acetium® intervention, carried out as part of a longitudinal cohort study, can be considered as a randomized controlled trial (RCT) testing the efficacy of Acetium® capsules (extended release L-cysteine) in restoring the structure and function of the gastric mucosa in patients with HP-associated (HP+) or autoimmune (HP-) AG, shown to progress during follow-up. The null hypothesis of this study suggests that the intake of Acetium® capsules is no different from no intervention in restoring the physiological function of the gastric mucosa and in restoring AG (inhibition of disease progression or mucosal healing).

[0147] (Study endpoints) Rejection of the null hypothesis will be determined by comparing the two strata (Acetium® vs. no intervention) against two primary study endpoints (efficacy measures): 1) change from baseline values ​​(diagnosis of AG) towards (or within) reference (normal) values ​​of serum levels of relevant gastric-specific biomarkers (GastroPanel® study), and 2) biopsy-confirmed recovery of atrophic gastric mucosa by at least one histological grade (e.g., from severe AG to moderate AG, from moderate AG to mild AG) based on blinded reading of biopsies taken at the end of the study compared with each subject's mucosal status at baseline (GastroPanel® study).

[0148] (Traditional statistical techniques) Descriptive statistics are performed according to routine procedures. Frequency tables are analyzed using chi-squared tests with likelihood ratios (LR) or Fisher's exact test for categorical variables. Standard statistics are used to compare the effectiveness of the two study arms on the observed changes in serum levels of GastroPanel biomarkers before and after Acetium® intervention. The effects of the trial preparation and non-intervention can be analyzed separately with a non-parametric paired-samples t-test (Wilcoxon signed-rank test) by comparing pairs of baseline and post-trial values. Another approach is to calculate the effect size for both arms (i.e. increase / decrease in biomarker levels due to Acetium treatment compared to baseline) and compare this effect size with the effect size observed in the non-intervention group.

[0149] (Univariate and multivariate logistic regression models) To compare categorical binary outcomes (stop / recovery, Y / N) between Acetium intervention and non-intervention groups, traditional regression models can be used, where the results are expressed as crude ORs (odds ratios) and their 95% confidence intervals (95%CI). The independent effect of Acetium® (adjusted for potential confounders) can be analyzed using multivariate logistic regression models. In this model, all recorded baseline characteristics (sex, age, etc.) can be entered as covariates, including four stratification variables: i) severity of AG, ii) localization of AG, iii) extent of AG, iv) association with HP. For categorical binary endpoints (stop / recovery; Y / N), the results of these multivariate models are expressed as adjusted ORs and their 95%CI.

[0150] (Test performance indicators and longitudinal predictive value of biomarkers) Of secondary importance in this study are the performance indicators (sensitivity, specificity, positive predictive value, PPV, negative predictive value, NPV, and their 95% CI) of the individual biomarkers (PGI, PGI / PGII, G-17) and the GastroPanel® test diagnostic report for predicting the appropriate endpoints (AGA, AGC) at the end of the study. These are calculated using STATA / SE software and the diagti algorithm introduced by Seed et al. (2001). This algorithm also calculates the area under the ROC (Receiver Operating Characteristic), called AUC, for each biomarker. As GastroPanel® is a quantitative ELISA test, these ROC curves can be used to identify the optimal sensitivity / specificity balance at which each biomarker can optimally detect the study endpoints (AG grade, localization, extent). The significance of the differences in AUC values ​​can be estimated using the ROC test in STATA with 95% CI.

[0151] Of interest is the value of individual GastroPanel® biomarkers as predictors of AG progression during long-term follow-up, as suggested by two recent studies showing that GastroPanel® biomarkers are powerful long-term predictors of GC. 74,75 The current study design offers the perfect possibility to evaluate whether values ​​below the cut-off values ​​of G-17, PGI, and PGI / PGII are predictive of progression of AGA and AGC, respectively. According to the definition of the three clinical outcomes (no change, regression, progression), the calculations are performed by traditional univariate regression (using non-progressors as reference) and the results are expressed as ORs with 95% CI. If a more advanced approach is indicated, i.e., if there are markers showing significant predictive value, the incorporated case-control setting can be designed to have a 1:1 ratio of cases (progressors) and controls selected from non-progressors, matched for age and sex, and analyzed using conditional logistic regression to control for potential confounding factors. 74 .

[0152] (Power analysis) Due to the complexity of study designs, several options exist for estimating the statistical power of a study, which are briefly described below.

[0153] (Follow-up study of the non-intervention group regarding clinical outcomes of AG) The statistical analysis of this part of the longitudinal study will use both traditional methods (e.g., Kaplan-Meier (log-rank) test, Cox proportional hazards) and more advanced methods (competing risks regression).

[0154] In this setting, the power of the study is most easily calculated based on the estimates of the rate of progression, e.g., mild AGC, during follow-up, assessed by life table (Kaplan-Meier) analysis. Using the Kaplan-Meier (log-rank, Mantel-Cox) comparison statistics for the two study groups (HP- and HP+), if there are 252 patients in the HP+ and 252 patients in the HP- group, the study is adequately powered (type II error 0.80, type I error 0.05) to detect a true difference in progression rate of 10%. With such a cohort of 504 subjects (equal numbers in the HP+ and HP- groups), the study is adequately powered for a true difference in AGC progression rate of 20% in one group (rate 0.2) and 30% in the other group (rate 0.3). While the unequal distribution of study subjects in the HP+ and HP- groups impairs power, an increase in effect size increases power as well.

[0155] (Acetium intervention) Calculating the power is not straightforward, since GastroPanel® consists of four gastric-specific biomarkers, three of which are relevant for this purpose (PGI, PGII, G-17), and their combination is key for the proper diagnosis of the endpoints (AGA, AGC). In principle, the power should be calculated separately for the biomarkers of AGC (PGI, PGII, PGI / PGII ratio) and for the biomarker related to AGA (G-17).

[0156] As no data are available on the variation of biomarker levels after the intervention, power calculations must be based on the changes in biomarker values ​​(after Acetium® treatment) reported in the Italian pilot study. 62,63 However, it is important to note that this study only included cases of AGC (low PGI and PGI / II ratio and elevated G-17) and did not include patients with AGA (low G-17 and PGI within normal range). Therefore, we calculated the power of this study based only on these body part-specific biomarkers and used this to estimate the appropriate cohort size required for both the Acetium® and non-intervention groups.

[0157] Thus, the Italian study reported an increase in PGI levels from 7.9 μg / L to 11.4 μg / L after treatment (3 months) (a difference of 3.5 μg / L). Unfortunately, the SD of these two values ​​was not reported, but based on estimates from another cohort of AG (gastric corpus) patients (n=42) with similar baseline PGI values, these SD values ​​are estimated to be 7.5 and 10 μg / L, respectively. Using a two-sample mean test for paired samples, the study would be sufficiently powered (type II error 0.80, type I error 0.05) to detect a true difference in PGI increase of this magnitude (3.5 μg / L) if there were 55 patients in the Acetium® study arm. This estimate is sensitive to the SD as well as the correlation between paired samples. An increasing SD would require a larger cohort size, but if the correlation is higher than the default 0.5, a significantly smaller cohort size would be sufficient.

[0158] To be on the safe side, a cohort of 60 patients would provide 84% power to detect differences in PGI increase after Acetium® treatment, therefore the current randomization objective is to reach a cohort of 60 subjects in both the Acetium® and non-intervention groups (Appendix 1). Using a 2-sample mean test (for independent samples) for effect differences (e.g. PGI increase during the intervention) with a cohort of 120 subjects randomized 1:1 to Acetium® and non-intervention, it is estimated that the study has adequate power (type II error 0.80, type I error 0.05) to detect true differences in effect size estimates (pre- vs. post-trial) of 3.5 μg / L (Acetium® group) and 2.15 μg / L (non-intervention group), with estimated SDs of 3.0 and 2.0, respectively. Even if the effect in the non-intervention group were slightly smaller, the power of the study would be close to 100% and the range of SD values ​​would be much wider.

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Claims

1. 1. An acetaldehyde-binding preparation for use in a method for improving the structure and physiology of the gastric mucosa in a subject suffering from an acid-free stomach and / or Helicobacter pylori-associated (HP+) or autoimmune (HP-) atrophic gastritis, comprising: The method includes at least: a) pepsinogen I (PGI), pepsinogen II (PGII), gastrin-17 (G-17) and Helicobacter pylori antibody (HpAb) biomarkers, or b) the pepsinogen I (PGI) biomarker, or c) a PGI biomarker, a PGII biomarker and a PGI / PGII biomarker ratio, or d) screening or testing symptomatic or asymptomatic subjects for autoimmune disease, dyspepsia or reflux symptoms by quantitatively measuring the concentration of the G-17 biomarker; and comparing the obtained values ​​with predetermined reference ranges and / or cut-off values, The acetaldehyde-binding preparation is characterized in that a sustained-release preparation containing 50 to 500 mg of L-cysteine ​​is administered to a subject exhibiting an acid-free stomach and / or atrophic gastritis based on the measurement and comparison in accordance with a predetermined administration regimen.

2. The acetaldehyde-binding preparation for use according to claim 1, characterized in that if the PGI concentration in the sample is close to the lower limit or below the reference range or cut-off value, the PGI / PGII ratio is close to the lower limit or below the reference range or cut-off value, and the G-17 concentration is close to the upper limit or above the reference range, the values ​​indicate atrophic gastritis.

3. 3. An acetaldehyde-binding preparation for use according to claim 1 or 2, characterized in that the reference ranges are 30-160 μg / l for PGI values, 3-20 μg / l for PGII values, 3 or less for PGI / PGII ratio, 5-30 pmol / l for G-17S (stimulated) values, 2-10 pmol / l for G-17B (fasting), and the reference ranges for HpAb are 0-30 EIU.

4. The acetaldehyde-binding preparation for use according to any one of claims 1 to 3, characterized in that the typical cut-off values ​​for the biomarkers are selected from the group comprising PGI 30 μg / l, PGI / PGII ratio 3, G-17S value 5 pmol / l, G-17B 2 pmol / l, and HpAb 30 EIU.

5. 5. The acetaldehyde-binding preparation for use according to any one of claims 1 to 4, characterized in that the preferred dosage regimen is to administer a sustained release capsule containing 50 to 500 mg, more preferably 50 to 300 mg, most suitably 100 to 200 mg of L-cysteine ​​three times a day for a period of at least one month, more preferably at least two months, most suitably at least three months.

6. 6. The acetaldehyde-binding preparation for use according to any one of claims 1 to 5, characterized in that the L-cysteine ​​is bound to a pharma- ceutically acceptable carrier that controls the release rate of the L-cysteine.

7. The acetaldehyde-binding preparation for use according to claim 6, characterized in that the pharma- ceutically acceptable carrier comprises one or more substances selected from the group consisting of various chitosans, alginates such as sodium alginate, aluminum hydroxide, sodium bicarbonate, sodium carboxymethylcellulose, and sodium bicarbonate.

8. The acetaldehyde-binding preparation for use according to any one of claims 1 to 7, characterized in that the L-cysteine ​​contained in one capsule is released in the stomach over a period of 0.5 to 8 hours.

9. 9. An acetaldehyde-binding preparation for use according to any one of claims 1 to 8, characterized in that the amount of L-cysteine ​​released in gastric disorders is between 40 and 80 mg per hour.

10. 10. The acetaldehyde-binding preparation according to any one of claims 1 to 9 for use in preventing atrophic gastritis and its progression to gastric cancer.

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