A method for eradicating Helicobacter pylori, comprising the step of orally administering to a subject a composition comprising a complex of a non-absorbable antibiotic and a clay mineral.

Abstract

The present application relates to a pharmaceutical composition or kit for oral administration for eradicating Helicobacter pylori, comprising a complex of a non-absorbable antibiotic and a clay mineral. The pharmaceutical composition and kit of the present invention may further comprise a β-lactam antibiotic and / or a gastric acid suppressant. [Selected figure] Figure 8a

Description

[Technical field]

[0001] The present invention relates to a method for eradicating Helicobacter pylori, comprising a step of orally administering to a subject a composition comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also relates to a method for eradicating Helicobacter pylori, comprising a step of orally administering to a subject a composition comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also relates to a method for preventing or treating a gastric disease caused by Helicobacter pylori, comprising a step of orally administering to a subject a composition comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also relates to a method for preventing or treating a gastric disease caused by Helicobacter pylori, comprising a step of orally administering to a subject a non-absorbable antibiotic and a clay mineral complex. The present invention also relates to a pharmaceutical composition for oral administration for eradicating Helicobacter pylori, comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also relates to a kit for oral administration for eradicating Helicobacter pylori, comprising a non-absorbable antibiotic and a clay mineral complex. [Background technology]

[0002] H. pylori is a gram-negative, microaerophilic, spiral-shaped bacillus that affects the gastric mucosa and can be found attached to epithelial cells in the human stomach. Drug-resistant H. pylori strains are the most common cause of treatment failure. Approximately 50% of the world's population is H. pylori positive, with a prevalence of 80%-90% in developing countries and 35%-40% in developed countries. Since the first draft recommendation on H. pylori eradication treatment was published in Korea in 1998, the standard triple drug regimen recommended as first-line treatment to date is a combination of clarithromycin, amoxicillin, and a gastric acid suppressant. The eradication effect of this standard triple drug regimen is currently very low, at around 70%, but no treatment better than the existing triple drug regimen has yet been developed, and triple drug therapy is recommended as the first-line treatment despite its low eradication effect (Korean J Gastroenterol 2013;62:3-26). Thus, in conventional treatment regimens, including the standard triple drug regimen, the orally administered antibiotics are dissolved in the stomach, absorbed in the intestine, transported through the bloodstream, and finally released to the gastric mucosa to eradicate H. pylori (Figure 1). However, because the antibiotics administered are absorbed in the intestine and released to the gastric mucosa to perform the eradication action, the antibiotics are exposed to the entire body, which poses the risk of antibiotic side effects and places a burden on the patient.

[0003] Smectite is a phyllosilicate mineral in which two octahedral sheets of Al, Mg, and Fe sandwich a tetrahedral sheet of Si, Al, and Fe above and below to form one unit layer (2:1 layer). The smectite unit layer has a negative charge, which occurs when a tetrahedral Si with a quadruple positive charge is isomorphically substituted with an Al or Fe with a triple positive charge, or when an octahedral Al or Fe3+ with a triple positive charge is isomorphically substituted with an Mg or Fe2+ with a double positive charge. Cations are induced between the unit layers through the negative charge generated in the unit layers, which means that smectite can be used as a drug carrier. Thus, the use of smectite as a drug delivery vehicle has recently attracted much interest, and various studies have reported drug-intercalated smectite hybrids for controlled delivery and release of drugs such as donepezil, lincomycin, chlorhexidine acetate, and tetracycline.

[0004] Aminoglycosides are representative antibiotics used to treat gram-negative bacteria, but there are no oral drugs, and only injections are available, and they have never been used in H. pylori eradication therapy. Some studies have confirmed that aminoglycosides have low minimum inhibitory concentrations in in vitro activity evaluations against H. pylori. The most active aminoglycosides tested are gentamicin, tobramycin, and netilmicin, which have MIC90 and MIC50 values ​​of 0.25-0.5 and 0.125-1.00 mg / L, respectively (Brenciaglia MI, Fornara AM, Scaltrito MM et al. Activity of amoxicillin, metronidazole, bismuth salicylate and six aminoglycosides against Helicobacter pylori. J Chemother 1996;8:52-4). However, because aminoglycosides are hardly absorbed in the gastrointestinal tract when administered orally, they have only been used parenterally up until now (Turnidge J. Pharmacodynamics and dosing of aminoglycosides. Infect Dis Clin North Am 2003; 17: 503-28). In fact, there have been no reports of aminoglycoside antibiotics showing efficacy in eradicating H. pylori in in vivo evaluations.

[0005] In the course of researching an H. pylori eradication agent that can be orally administered and effectively eradicates the bacteria in the gastrointestinal tract, the inventors discovered that the composition for eradicating H. pylori of the present invention is not absorbed in the intestine and released in the stomach via the bloodstream, but rather, after oral administration, the antibiotic reaches the stomach via the esophagus and adheres to the gastric mucus layer, effectively eradicating H. pylori, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a composition for eradicating H. pylori which is orally administered and then applied to the gastric mucosa, a method for eradicating H. pylori using the same, and a method for preventing and treating gastric diseases caused by H. pylori. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention provides a method for eradicating Helicobacter pylori, comprising a step of orally administering to a subject a composition comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also provides a method for eradicating Helicobacter pylori, comprising a step of orally administering to a subject a composition comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also provides a method for preventing or treating a gastric disease caused by Helicobacter pylori, comprising a step of orally administering to a subject a composition comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also provides a method for preventing or treating a gastric disease caused by Helicobacter pylori, comprising a step of orally administering to a subject a non-absorbable antibiotic and a clay mineral complex. The present invention also provides a pharmaceutical composition for oral administration for eradicating Helicobacter pylori, comprising a non-absorbable antibiotic and a clay mineral complex. The present invention also provides a kit for oral administration for eradicating Helicobacter pylori, comprising a non-absorbable antibiotic and a clay mineral complex. Effect of the Invention

[0008] The pharmaceutical composition and kit of the present invention contain a clay platelet carrier, which can be applied to the gastric mucosa, enabling targeted therapy of the gastric mucosa. In addition, the destruction of antibiotics in the low acidic gastric environment is reduced, minimizing the effect on intestinal bacteria. [Brief description of the drawings]

[0009] [Figure 1] A diagram showing the process of eradicating H. pylori using conventional triple therapy. [Diagram 2] 1 is a diagram showing the process of eradicating H. pylori using the complex of a non-absorbable antibiotic and a clay mineral of the present invention. [Diagram 3] A diagram showing the eradication activity of gentamicin-smectite complex (S-GEN) against Helicobacter pylori. Top left disk: smectite, top right disk: S-GEN, bottom disk: gentamicin. [Figure 4] Figure showing the eradication activity of netilmicin-smectite complex (S-NET) against Helicobacter pylori. Left disk: smectite, right disk: S-NET. [Diagram 5] Protocol for confirming anti-H. pylori efficacy in vivo. The protocol involves inoculation of C57BL / 6 mice with H. pylori and infection development, followed by treatment. [Figure 6] Gastric mucosal tissue and PCR results after a study on the efficacy of treatment of H. pylori infection using groups 1-8. [Figure 7] A diagram showing the resistance rate of H. pylori to antibiotics. [Figure 8a] Comparison of the in vitro antibacterial effects of CLR and S-GM disks against clarithromycin-resistant strains. [Figure 8b] Comparison of the in vitro antibacterial effects of CLR and S-GM disks against clarithromycin-resistant strains. BEST MODE FOR CARRYING OUT THEINVENTION

[0010] The present invention relates to a method for eradicating Helicobacter pylori, which comprises the step of orally administering to a subject a composition containing a complex of a non-absorbable antibiotic and a clay mineral.

[0011] The present invention also relates to a method for eradicating Helicobacter pylori, which comprises the step of orally administering a complex of a non-absorbable antibiotic and a clay mineral to a subject.

[0012] The present invention also relates to a method for preventing or treating a gastrointestinal disease caused by Helicobacter pylori, comprising the step of orally administering to a subject a composition comprising a complex of a non-absorbable antibiotic and a clay mineral.

[0013] The present invention also relates to a method for preventing or treating a gastrointestinal disease caused by Helicobacter pylori, comprising the step of orally administering to a subject a complex of a non-absorbable antibiotic and a clay mineral.

[0014] The present invention also relates to a pharmaceutical composition for oral administration for eradicating Helicobacter pylori, comprising a complex of a non-absorbable antibiotic and a clay mineral.

[0015] The present invention also relates to a pharmaceutical composition for oral administration for eradicating antibiotic-resistant Helicobacter pylori, comprising a complex of a non-absorbable antibiotic and a clay mineral.

[0016] The present invention also relates to use of a pharmaceutical composition for oral administration, comprising a complex of a non-absorbable antibiotic and a clay mineral, for eradicating Helicobacter pylori.

[0017] The present invention also relates to use of an oral pharmaceutical composition comprising a complex of a non-absorbable antibiotic and a clay mineral for eradicating antibiotic-resistant Helicobacter pylori.

[0018] The present invention also relates to an oral administration kit for eradicating Helicobacter pylori, which comprises a complex of a non-absorbable antibiotic and a clay mineral.

[0019] The present invention also relates to an oral administration kit for eradicating antibiotic-resistant Helicobacter pylori, comprising a complex of a non-absorbable antibiotic and a clay mineral.

[0020] The present invention also relates to a method for eradicating Helicobacter pylori, which comprises the step of administering the pharmaceutical composition of the present invention to a subject.

[0021] The present invention also relates to a method for eradicating antibiotic-resistant Helicobacter pylori, which comprises the step of administering the pharmaceutical composition of the present invention to a subject.

[0022] The present invention also relates to a method for preventing or treating a disease caused by antibiotic-resistant Helicobacter pylori, comprising the step of administering the pharmaceutical composition of the present invention to a subject.

[0023] The present invention will be described in detail below.

[0024] Non-absorbable antibiotics The non-absorbable antibiotic of the present invention is orally administered in the form of a complex with a clay mineral, and then applied to the gastric mucosa as it passes through the esophagus and reaches the stomach. The antibiotic is released from the complex applied to the gastric mucosa, eradicating H. pylori.

[0025] When the non-absorbable antibiotic of the present invention is orally administered and not used for eradicating H. pylori in the stomach, it is excreted without being absorbed, thereby reducing the side effects of the antibiotic and the burden on the patient. In this case, the non-absorbable antibiotic of the present invention is in the form of a complex bound to a clay mineral, so that it has little effect on intestinal bacteria and minimizes the destruction of the antibiotic under gastric acid conditions.

[0026] The non-absorbable antibiotic of the present invention is not particularly limited, but may be an aminoglycoside compound.

[0027] The aminoglycoside compound of the present invention may be gentamicin, tobramycin, amikacin, neomycin, netilmicin, etc., and preferably gentamicin.

[0028] Clay Minerals Generally, clay minerals have a layered structure, i.e., a plate-like structure, in which crystal units formed by bonding silica sheets and alumina sheets are stacked, but among such clay minerals, clay minerals with interlayer expansion do not have hydrogen bonds between the crystal units and the bonding strength between the crystal units is weak, so when moisture is absorbed between these crystal units, they can expand. Therefore, even ions with a relatively large size can easily flow between the crystal units of clay minerals with interlayer expansion. On the other hand, in clay minerals that have interlayer expansion properties, negative layer charges are generated when tetrahedral Si, which has a quadruple positive charge, is isomorphically substituted with Al or Fe, which has a triple positive charge, or when octahedral Al or Fe3+, which has a triple positive charge, is isomorphically substituted with Mg or Fe2+, which has a double positive charge. However, cations such as calcium ions (Ca2+), magnesium ions (Mg2+), sodium ions (Na+), and potassium ions (K+) bind between the layers or on the surface, making the mineral electrically neutral overall.

[0029] The clay mineral of the present invention has a plate-like structure, specifically, an interlayer expansion property, and can be used as a carrier by inserting an antibiotic therein. The clay mineral of the present invention may be a smectite mineral, such as montmorillonite, bentonite, beidellite, nontronite, saponite, hectorite, etc.

[0030] Complexes of non-absorbable antibiotics and clay minerals. In the complex of the non-absorbable antibiotic and clay mineral of the present invention, the clay mineral is used as a carrier, i.e., a vehicle, that delivers the non-absorbable antibiotic to the gastric mucus layer. The complex has a structure in which the non-absorbable antibiotic is inserted into the clay mineral. The complex of the non-absorbable antibiotic and clay mineral is orally administered, passes through the esophagus to the stomach, and then adheres to the gastric mucus layer to release the antibiotic (FIG. 2). That is, the complex of the present invention is not orally administered, passes through the esophagus to the stomach, and then the antibiotic is dissolved in the stomach, where it moves to the intestine, is absorbed in the intestine, travels through the blood vessels, and is released to the gastric mucosa to eradicate H. pylori.

[0031] The clay mineral of the present invention has a plate-like structure, which allows it to be applied to the gastric mucus layer (mucosa), and has excellent drug-carrying capacity, which allows targeted therapy of the gastric mucus layer. In addition, it is possible to effectively maintain the minimum inhibitory concentration (MIC) at the affected area, which reduces the burden on patients from antibiotics.

[0032] The complex of the non-absorbable antibiotic and clay mineral of the present invention can be prepared using a conventional method disclosed in the art. Methods for preparing a complex in which a clay mineral and a drug are combined have been disclosed in a number of papers, and those skilled in the art can prepare the complex of the present invention by appropriately using these methods. For example, the complex of the non-absorbable antibiotic and clay mineral of the present invention can be prepared by the method of Korean Patent No. 10-1541876, the entirety of which is incorporated herein by reference. In this case, the complex of the present invention can be prepared using a method for preparing a complex, which includes the steps of: supplying a dispersion solution of clay mineral microparticles having an expansive lattice structure and an antibiotic solution to a first space and a second space separated from each other by an ion exchange membrane, respectively, and maintaining the solution for a first time; and removing the antibiotic solution from the second space and then supplying an antibiotic ion washing solution to the second space.

[0033] β-lactam antibiotics The complex of the non-absorbable antibiotic and the clay mineral of the present invention can be administered to a subject together with a β-lactam antibiotic and / or a gastric acid suppressant. Thus, the pharmaceutical composition and kit of the present invention may further comprise a β-lactam antibiotic and a gastric acid suppressant in addition to the complex of the non-absorbable antibiotic and the clay mineral.

[0034] The β-lactam antibiotic may be penicillin, methicillin, ampicillin, amoxicillin, cephalosporin, carbapenem, etc., and preferably amoxicillin.

[0035] Acid suppressants The complex of the non-absorbable antibiotic and clay mineral of the present invention can be administered to a subject together with a β-lactam antibiotic and / or a gastric acid suppressant. The gastric acid suppressant of the present invention is a proton pump inhibitor. The gastric acid suppressant of the present invention may be a normal gastric acid suppressant and is not particularly limited. For example, the gastric acid suppressant of the present invention may be omeprazole, esomeprazole, rabeprazole, lansoprazole, pantoprazole, etc. If the subject using the pharmaceutical composition or kit of the present invention has a weak stomach or secretes too much gastric acid, the complex of the non-absorbable antibiotic and clay mineral can be administered in combination with a gastric acid suppressant.

[0036] kit The kit of the present invention comprises a complex of a non-absorbable antibiotic and a clay mineral. Preferably, the kit of the present invention further comprises a β-lactam antibiotic and / or a gastric acid suppressant. The kit of the present invention is an oral administration kit for eradicating H. pylori.

[0037] Pharmaceutical Compositions The composition of the present invention may be a pharmaceutical composition, and the terms "composition" and "pharmaceutical composition" may be used interchangeably herein. The pharmaceutical composition of the present invention comprises a complex of a non-absorbable antibiotic and a clay mineral. Preferably, the pharmaceutical composition of the present invention further comprises a β-lactam antibiotic and / or a gastric acid suppressant.

[0038] The complex of the non-absorbable antibiotic and clay mineral of the pharmaceutical composition of the present invention is orally administered, passes through the esophagus and reaches the stomach, and adheres to the gastric mucus layer to release the antibiotic. The pharmaceutical composition has urease inhibitory activity and may be a pharmaceutical composition for preventing or treating gastric diseases caused by Helicobacter pylori.

[0039] The pharmaceutical composition of the present invention may be a pharmaceutical composition for preventing, improving or treating a disease caused by Helicobacter pylori. The disease caused by Helicobacter pylori may be gastrointestinal damage, gastritis, gastric ulcer, duodenal ulcer, gastritis, gastric cancer or MALT lymphoma.

[0040] The pharmaceutical composition of the present invention may contain the complex of the non-absorbable antibiotic and clay mineral, the β-lactam antibiotic and / or the gastric acid suppressant in an amount of 0.01 to 80% by weight, preferably 0.02 to 65% by weight. However, this amount can be increased or decreased according to the needs of the person administering the medicine, and can be increased or decreased appropriately depending on the situation, such as age, diet, nutritional state, and degree of progression of the disease. The ratio of the complex of the non-absorbable antibiotic and clay mineral, the β-lactam antibiotic and / or the gastric acid suppressant in the pharmaceutical composition of the present invention, i.e., the composition, can be appropriately determined by those skilled in the art.

[0041] The pharmaceutical composition of the present invention can be orally administered and can be used in the form of a general pharmaceutical preparation.Preferred pharmaceutical preparations include oral administration preparations such as tablets, hard or soft capsules, liquids, suspensions, syrups, chewing tablets, etc., and these pharmaceutical preparations can be prepared using pharma- ceutical acceptable conventional carriers, such as excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives, or bulking agents in the case of oral administration preparations.

[0042] The dosage of the pharmaceutical composition of the present invention may be determined by a specialist depending on various factors such as the patient's condition, age, sex, and other diseases, but it can usually be administered at a dosage of 0.1 mg to 10 g, preferably 10 mg to 5 g per kg for adults. In addition, the pharmaceutical composition can be administered 1 to 6 times a day so that each unit dosage form contains a daily dose of the pharmaceutical composition or 1 / 2, 1 / 3, or 1 / 4 of that dose. However, in the case of long-term intake for the purpose of health and hygiene or health regulation, the amount may be less than the above range, and the doctor in charge can adjust it appropriately.

[0043] Diseases caused by Helicobacter pylori The pharmaceutical composition and kit of the present invention may be for preventing, improving or treating a disease caused by Helicobacter pylori. The disease caused by Helicobacter pylori may be gastrointestinal tract damage, gastritis, gastric ulcer, duodenal ulcer, gastritis, gastric cancer or MALT lymphoma. The Helicobacter pylori may be normal Helicobacter pylori or antibiotic-resistant Helicobacter pylori. In this case, the antibiotic-resistant Helicobacter pylori may be clarithromycin-resistant Helicobacter pylori. The pharmaceutical composition and kit of the present invention have eradication activity against clarithromycin-resistant Helicobacter pylori.

[0044] Treatment or sterilization method The present invention relates to a method for eradicating H. pylori, comprising a step of orally administering the pharmaceutical composition of the present invention to a subject.The present invention also relates to a method for eradicating antibiotic-resistant H. pylori, comprising a step of orally administering the pharmaceutical composition of the present invention to a subject.The present invention also relates to a method for preventing, ameliorating, or treating a disease caused by H. pylori, comprising a step of orally administering the pharmaceutical composition of the present invention to a subject.The present invention also relates to a method for preventing, ameliorating, or treating a disease caused by antibiotic-resistant H. pylori, comprising a step of orally administering the pharmaceutical composition of the present invention to a subject.

[0045] The subject may be a mammal diagnosed as infected with H. pylori or at risk of being infected with H. pylori. The subject may also be a mammal diagnosed as infected with antibiotic-resistant H. pylori or at risk of being infected with antibiotic-resistant H. pylori. The mammal may or may not include a human. Thus, the treatment method of the present invention can treat subjects infected with antibiotic-resistant H. pylori or at risk of being infected with antibiotic-resistant H. pylori, and thus has a wider range of subjects to be treated than conventional H. pylori eradication drugs. The eradication method of the present invention can also eradicate antibiotic-resistant H. pylori, and thus has a wider range of subjects to be eradicated than conventional H. pylori eradication drugs.

[0046] Antibiotic-resistant Helicobacter pylori The complex of the non-absorbable antibiotic and clay mineral of the present invention, or the pharmaceutical composition containing the complex, has an eradicating activity against antibiotic-resistant Helicobacter pylori. The antibiotic-resistant Helicobacter pylori is preferably Helicobacter pylori resistant to clarithromycin. For example, the antibiotic-resistant Helicobacter pylori may be Helicobacter pylori having single resistance to clarithromycin, or Helicobacter pylori having simultaneous resistance to clarithromycin and metronidazole, or Helicobacter pylori having resistance to one or more antibiotics selected from metronidazole, tetracycline, levofloxacin, etc., in addition to clarithromycin, that is, Helicobacter pylori having multiple resistance to multiple antibiotics. While amoxicillin resistance is less than 10%, resistance of Helicobacter pylori to clarithromycin has been increasing recently, and the main cause of failure of eradication therapy of H. pylori is clarithromycin resistance. Therefore, the complex of the present invention of a non-absorbable antibiotic and a clay mineral having eradication activity against clarithromycin-resistant Helicobacter pylori, or a pharmaceutical composition containing the complex, is particularly useful for subjects who have failed conventional eradication therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The advantages and features of the present invention, as well as the methods for achieving them, will become apparent from the following detailed description of the embodiments. However, the present invention is not limited to the embodiments disclosed below, and may be embodied in various different forms. However, the embodiments are provided to fully disclose the present invention and to fully inform those skilled in the art of the present invention of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0048] Materials and Methods <Experimental Ethics> Animal studies were performed on rats as approved by the Institutional Animal Care and Use Committee of the National Center of Efficacy Evaluation for the Development of Health Products Targeting Digestive Disorders, Incheon, Korea. Mouse experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Wonkwang University College of Pharmacy. Mice were treated in accordance with the Guidelines and Regulations for the Use and Care of Animals of Wonkwang University, Iksan, Korea, and were provided with water and standard laboratory chow ad libitum.

[0049] <Statistical analysis> Data are presented as mean ± SD, and experimental groups were compared using the nonparametric Mann-Whitney-U test. 95% CIs for the detection rates were obtained using the MINITAB statistical software program (Minitab Inc., PA, USA). If the 95% CIs of both values ​​did not overlap, they were considered to be significantly different. A P value < 0.05 was considered statistically significant. Results were analyzed using the Statistics Package for Social Science (SPSS 12.0 for Windows; SPSS Inc., Chicago, IL, USA).

[0050] <Experimental Example 1> Antibacterial activity of antibiotic-clay mineral complex <1-1> Antibiotic insertion Gentamicin solution (2 mg / mL) was prepared using United States Pharmacopeia (USP) grade gentamicin sulfate manufactured by Bio Basic Inc.

[0051] Ca-smectite was prepared by purifying bentonite from Gyeongbuk, Korea. S-GEN (gentamicin-intercalated smectite hybrid), a gentamicin-smectite complex, was prepared by mixing 250 ml of gentamicin solution per gram of Ca-smectite and stirring vigorously for 24 hours. In this specification, the term S-GEN (gentamicin-intercalated smectite hybrid), a gentamicin-smectite complex, is used interchangeably with the term S-GM. After mixing, the hybrid solution was dialyzed against 5 L of distilled water at 50°C for 8 hours, which was repeated three or four times until sulfate ions were not detected in PbCl2. Hybrid powder was obtained by freeze-drying the hybrid solution dialyzed for 2 to 3 days. The amount of gentamicin released from the hybrids was determined by batch-release testing, where 25 mL of pH 1.2 solution was repeatedly added to the same 100 mg of hybrid powder. The gentamicin concentration from the supernatant was measured using LC-MS. LC analysis was performed using a Thermo Scientific ICS system. MS analysis was performed using electrospray ionization and a Thermo Scientific MSQ Plus quadrupole mass spectrometer. The total amount of gentamicin released within 1 hour was determined to be ~5.0 mg per 100 mg of hybrid.

[0052] On the other hand, a netilmicin-smectite complex, S-NET, was prepared by the same method using netilmicin instead of gentamicin.

[0053] <1-2> In vitro evaluation of disinfection activity The eradication activity of S-GEN and S-NET was evaluated. After Helicobacter pylori was applied to the medium (red color), a disk-shaped sample was made and placed on the medium to measure the eradication efficacy. When the red color disappears around the disk, it means that the eradication efficacy has been achieved.

[0054] As a result, it was confirmed that both the gentamicin-smectite complex (S-GEN) and the netilmicin-smectite complex (S-NET) have eradication activity against Helicobacter pylori (Figures 3 and 4; upper left corner of Figure 3: smectite, upper right corner: S-GEN, lower part: gentamicin; left: smectite, right: S-NET in Figure 4).

[0055] <Experimental Example 2> Application to the gastric mucosa in rats Seven-week-old male Sprague-Dawley rats (body weight 220 g ± 20%) were purchased from Samtako Ltd., Osan, Korea, to test the application to the gastric mucosa. The rats were fasted for 24 hours before the experiment. Two groups of 10 rats were ingested 10 mL / kg (150 mg / kg) of smectite or S-GEN and euthanized after 1 hour. To analyze the application efficiency to the gastric mucosa, the stomachs of the rats were excised, cut along the greater curvature, and pinned. The stomach distribution percentage was calculated according to the following formula 1:

[0056] <Expression 1> Stomach distribution percentage (%) = {stomach distribution area (cm 2 ) / Entire stomach area (cm 2 )}×100

[0057] The percentage and area were analyzed using Leica Application Suite V4 (Leica Microsystems Ltd., Korea). The stomach distribution percentage is shown as the mean (± standard deviation).

[0058] <Experimental Example 3> Anti-H. pylori efficacy in vivo <3-1> Inoculation of experimental animals Four-week-old male C57BL / 6 mice were purchased from Japan SLC, Inc., Shizuoka, Japan, to evaluate anti-H. pylori activity. The mice were 5 weeks old and weighed 18-20 g at the time of the start of the experiment. H. pylori SS1 was used for inoculation. The bacteria were cultured on Brucella blood agar (Merck, Germany) at 37°C under microaerobic conditions (10% CO2, 85% N2 and 5% O2) for 72 h. To evaluate anti-H. pylori activity in vivo, 80 mice were adapted for 1 week before the experiment.

[0059] After adaptation, the animals were fasted for 12 hours, and 70 were infected with 0.5 mL of 2.0 × 109 cfu / mL H. pylori suspension, administered intragastrically via oral feeding every 48 hours, three times per week. The day of inoculation was considered as day 0, and the following days were considered as days 1 to 21. The uninfected group served as the normal control group, and was given an equivalent volume of PBS and distilled water.

[0060] <3-2> Treatment of experimental animals The mice were divided into 8 groups of 10 each and were allowed to rest for 1 week after the last inoculation:

[0061] Group 1: Normal group of uninfected mice. Group 2: Untreated control group with distilled water added. Group 3: treated with amoxicillin (14.25 mg / kg), clarithromycin (7.15 mg / kg) and a proton pump inhibitor (PPI, omeprazole was used at 400 μmol / kg in all groups with PPI added) served as the positive control group. Group 4: treated with amoxicillin (14.25 mg / kg), gentamicin (4 mg / kg) and PPI (400 μmol / kg). Group 5: treated with amoxicillin (14.25 mg / kg), S-GEN (78 mg / kg) and PPI (400 μmol / kg). Group 6: treated with gentamicin (4 mg / kg) and PPI (400 μmol / kg). Group 7: treated with S-GEN (78 mg / kg) and PPI (400 μmol / kg). Group 8: treated with amoxicillin (14.25 mg / kg) and PPI (400 μmol / kg).

[0062] The treatment was administered orally once a day for 7 consecutive days. To confirm the serological status of H. pylori in the infected mice, H. pylori immunoglobulin G (IgG) levels were determined by ELISA kit (Cusabio Biotech Co., USA) before treatment.

[0063] <Experimental Example 4> Bacteria confirmation Twelve hours after the last dose, the mice were euthanized and stomach tissues were extracted. Gastric mucosa from the pylorus was biopsied for Campylobacter-like organism (CLO) testing and PCR for H. pylori. In addition, 0.5 g of feces per mouse was collected from the rectum and colon, suspended in an equal volume of distilled water, and filtered for detection of H. pylori antigen (Ag) and H. pylori PCR in feces.

[0064] <4-1> CLO Testing Pyloric gastric mucosa samples were analyzed with a CLO kit (Asan Pharmaceutical Co., Seoul, Korea) and incubated at 37°C for 12 hours to test for urease activity. The reaction (color change) was considered negative when it was light yellow, or positive when it was dark red. The reaction score ranged from 0 to 3, with 0 being no color change, 1 being bright red, 2 being bright purple, and 3 being dark red.

[0065] <4-2> H. pylori PCR in gastric mucosa H. pylori DNA was prepared using the beadbeater / phenol extraction method (Kim BJ, Lee SH, Lyu M-A et al. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 1999;37:1714-20). The bacterial suspension was placed in a 2.0 mL screw-cap microcentrifuge tube filled with 200 μL of phenol, chloroform, isoamyl alcohol (50:49:1) and 200 μL (paced volume) of glass beads (0.1 mm diameter; Biospec Products, Bartlesville, OK, USA). The tube was shaken for 30 s with a mini beadbeater (Biospec Products) and centrifuged (12,000 g for 15 min) for phase separation. The aqueous phase was then transferred to another clean tube, and 10 μL of 3 M sodium acetate and 250 μL of very cold absolute ethanol were added. To precipitate the DNA, the mixture was kept at 20°C for 10 min. The harvested DNA pellet was dissolved in 60 μL of Tris-EDTA buffer (pH 8.0) and used as template DNA for PCR. PCR was performed using AccuPower PCR Premix (Bioneer, Daejeon, Korea).After an initial denaturation / activation step (95°C for 5 min), DNA (50 ng) was amplified in a 20 μL volume for 35 cycles of denaturation (94°C for 60 s), annealing (62°C for 60 s) and elongation (72°C for 90 s) using the following primers: H. pylori-specific ureA and ureC, sense 50-TGATGCTCCACTACGCTGGA-30 (SEQ ID NO: 1) and antisense 50-GGGTATGCACGGTTACGAGT-30 (SEQ ID NO: 2) (expected product 265 bp) (Kim YB, Kim ST, Lee SW et al. The influence of number of gastroscopic biopsy specimens on follow-up Campylobacter-like organism (CLO) test. Korean J Gastroenterol 2000;35:422-8), and GAPDH, sense 50-TGGGGTGATGCTGGTGCTG-AG-30 (sequence no. 3) and antisense 50-GGTTTCTCCAGGCGGCATGTC-30 (sequence no. 4) (expected product 497 bp) (Kundu P, Mukhopadhyay AK, Patra R et al. Cag pathogenicity is and independent up-regulation of matrix metalloproteinases-9 and -2 secretion andexpression in mice by Helicobacter pylori infection. J Biol Chem 2006;281:34651-62). PCR products were analyzed by electrophoresis on 1.5% agarose gel.

[0066] <4-3> Detection of H. pylori antigen in mouse feces H. pylori antigen was assessed using the commercially available SD Bioline H. pylori Ag kit (Standard Diagnostics, Inc.) according to the manufacturer's guidelines. Specimens (250 mg) were incubated with the diluent for 30 min at room temperature, and then 100 μL was placed in the H. pylori antigen test device. Results were checked after 15 min. A single red line indicated a negative result, and a red line indicated a positive H. pylori result (Moon DI, Shin EH, Oh H-G et al. Usefulness of a Helicobacter pylori stool antigen test for diagnosing H. pylori infected C57BL / 6 mice. Lab Anim Res 2013;29:27-32).

[0067] <4-4> H. pylori PCR in mouse feces Genomic DNA was extracted from stool samples using the AccuPrep Stool DNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer's instructions (Lee JU, Jung K, Kim O. Absence of vertical transmission of Helicobacter pylori in an experimental murine model. JVetSci 2006;7:225-8). Primer sets with sequence numbers 1 and 2 were used to amplify H. pylori-specific ureA and ureC (265 bp) (Kim YB, Kim ST, Lee SW et al. The influence of number of gastroscopic biopsy specimens on follow-up Campylobacter-like organism (CLO) test. Korean J Gastroenterol 2000;35:422-8). Template DNA (50 ng) and 20 pmol of each primer were added to a PCR mixture tube containing 1 U of Taq DNA polymerase, 250 μM of each deoxynucleoside triphosphate, 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 1.5 mM MgCl2, and gel loading dye. The volume was adjusted to 20 μL with distilled water. After an initial denaturation at 95°C for 5 min, the reaction mixture was subjected to 35 amplification cycles (94°C for 60 s, 62°C for 60 s, then 72°C for 90 s), followed by a 10 min extension at 72°C (GeneAmp 9700, Perkin Elmer, USA).

[0068] The PCR products were electrophoresed on a 1.5% agarose gel (Lee H-A, Park YS, Kim O. Prevalence of Helicobacter species in feces of dogs using polymerase chain reaction analysis. Lab Anim Res 2007;23:339-44).

[0069] <Experimental Example 5> Quantification of inflammatory cytokines Plasma was obtained for IL-8 and TNF-α analysis on day 21 by insertion of heparinized microhaematocrit tubes into the ophthalmic venous plexus of the mice. Plasma IL-8 and TNF-α levels were measured using mouse ELISA kits (R&D Systems, Minneapolis, MN, USA).

[0070] <Experimental Example 6> In vivo anti-H. pylori efficacy according to S-GEN treatment conditions <6-1> Inoculation of experimental animals Male C57BL / 6 mice were purchased from Japan SLC, Inc., Shizuoka, Japan, for anti-H. pylori evaluation. After 4-week-old mice were adapted, they were infected with H. pylori SS1 three times for 1 week and maintained for 2 weeks, after which the test substances were suspended in the designated doses and orally administered at the same time every day at 10 ml per kg of mice, once a day for 7 days.

[0071] <6-2> Treatment of experimental animals The mice were divided into 5 groups of 10 each and were allowed to rest for 1 week after the last inoculation:

[0072] Normal control group: vehicle (not infected), PBS, and distilled water (10 mg / kg) were added. Negative control group: vehicle (H. pylori-infected) distilled water was added at 10 mg / kg.

[0073] Test group 1: (H. pylori infection) AMX (14.25 mg / kg / day), CLR (14.3 mg / kg / day), PPI (138 mg / kg / day) Test group 2: (H. pylori infection) AMX (14.25 mg / kg / day), S-GM (202 mg / kg / day), PPI (138 mg / kg / day) (Gentamicin 8 mg / kg at this time) Study group 3: (H. pylori infection) AMX (14.25 mg / kg / day), S-GM (101 mg / kg / day)

[0074] AMX: Amoxicillin CLR: Clarithromycin S-GM: gentamicin-intercalated smectite hybrid PPI: Gastric acid suppressant

[0075] <Experimental Example 7> Eradication effect of S-GM against antibiotic-resistant H. pylori <7-1> Evaluation of antibiotic resistance rate The in vitro MICs of antibiotics currently in clinical use for Helicobacter eradication therapy (amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline) and a group of candidate non-absorbable antibiotics (gentamicin, netilmicin, tobramycin, amikacin) to be used as new therapeutic agents by compounding them with smectite were measured to confirm the MIC50 and MIC90. For this purpose, 187 culture-positive clinical strains obtained from 1,265 patient samples were used.

[0076] <7-2> In vitro evaluation of disinfection activity The eradication activity of S-GM and clarithromycin against clarithromycin-resistant strains (CLR MIC: 48mg / L) was evaluated. After spreading clarithromycin-resistant Helicobacter pylori on the medium (red color), a disk-shaped sample was made and placed on the medium to measure the eradication efficacy. When the red color disappears around the disk, it means that the eradication efficacy is achieved.

[0077] <Result> Results of Experimental Example 2 Application to gastric mucus layer in rats The experimental condition of 10mL / kg (150mg / kg) was selected, and the experimental rats were euthanized 1 hour after oral administration, taking into account the drug release time from the hybrid. S-GEN showed an application rate of 60.2% (±14.3%).

[0078] Results of Experimental Example 3 In vivo anti-H. pylori evaluation To evaluate the in vivo efficacy of S-GEN against H. pylori, an H. pylori infection model was established (Figure 5). To confirm H. pylori infection, H. pylori IgG levels were measured before and after treatment (Table 1: Plasma concentration of H. pylori IgG in each group).

[0079] [Table 1]

[0080] AMX: Amoxicillin CLR: Clarithromycin GEN: Gentamicin Data shown are the mean ± standard deviation of 70 infected mice (groups 2 to 8). *Significantly different from Group 1 (p<0.01) **Significantly different from Group 2 (p<0.01)

[0081] Results of Experimental Example 4 Gastric mucosa H. pylori PCR and CLO testing In mice, repeated intragastric inoculation with H. pylori produced a positive reaction (red color) in the CLO test of the gastric mucosa (Table 2: Results of the CLO test in treated gastric mucosa).

[0082] [Table 2]

[0083] a Each group consisted of 10 mice. bIncidence percentages (95% CI) were calculated with the MiniTab statistical program. *Significantly different from Group 2 (p<0.05)

[0084] The healing rates of gastric mucosa (100 minus positive reaction) were 70%, 60%, 80%, 50%, 60%, and 60%, respectively, in groups 3 to 8. The CLO score in group 5 was the lowest among the H. pylori-infected groups and was considerably lower than that in group 2.

[0085] The H. pylori-specific ureA and ureC (265 bp) PCR products were electrophoresed on a 1.5% agarose gel and visualized (Fig. 6).

[0086] Cure rates were identical to those confirmed by CLO testing.

[0087] H. pylori antigens and PCR in mouse stool A stool antigen kit was used to detect H. pylori in stool. Positive results in group 2 and negative results in the other groups were consistently observed.

[0088] To evaluate the therapeutic effect in H. pylori-infected mice, H. pylori PCR was performed (Table 3: PCR analysis of H. pylori in stool after treatment). The cure rates were 90% and 100% in group 3 (standard treatment) and group 5 (treated with amoxicillin / S-GEN / PPI), respectively.

[0089] [Table 3]

[0090] a Each group consisted of 10 mice. bIncidence percentages (95% CI) were calculated with the MiniTab statistical program. *Significantly different from Group 2 (p<0.05)

[0091] Results of Experimental Example 5 Quantification of inflammatory cytokines To confirm the effect of anti-H. pylori containing S-GEN on cytokine production, the plasma concentrations of inflammatory cytokines were measured in mice (Table 4). The levels of IL-8 and TNF-a in the treatment groups were significantly lower than those in group 2 (Table 4: Plasma concentrations of IL-8 and TNF-a). The plasma levels of IL-8 and TNF-a in group 5 were the lowest among the treatment groups.

[0092] [Table 4]

[0093] Data are presented as mean ± standard deviation for 10 mice per group (μg / ml). a Each group consisted of 10 mice. *Significantly different from Group 2 (p<0.05) **Significantly different from Group 2 (p<0.01)

[0094] Results of Experimental Example 6 The experimental animals treated in Experimental Example 6 are as shown in Table 5 below.

[0095] [Table 5]

[0096] [Single dose] (**Once a day) AMX: Amoxicillin CLR: Clarithromycin S-GM: gentamicin-intercalated smectite hybrid PPI: Gastric acid suppressant

[0097] Blood H. pylori IgG antibody titer 1) Measurement results for confirming H. pylori infection and for group composition H. pylori antibodies were measured by ELISA, and the mean and standard deviation were calculated. The non-infected group had a mean value of 0.04±0.01, while the infected group had a mean value of 0.10±0.10. H. pylori antibodies were statistically significantly increased by 150.0% in the infected group compared to the non-infected group (p<0.05) (Table 6).

[0098] [Table 6]

[0099] One week after infection, H. pylori antibody IgG test value Data are shown as mean ± standard deviation. Statistical analysis was performed by Sigma plot statistic. *Compared to the non-infected group. *p<0.05.

[0100] 2) Final H. pylori antibody IgG measurement results At the time of autopsy, H. pylori antibodies were measured in the blood and the mean and standard deviation were calculated as follows: non-infected group (normal control group): 0.33±0.13, infected control group (negative control group): 0.63±0.29, test group 1 (AMX+CLR+PPI administration group): 0.27±0.07, test group 2 (AMX+S-GM+PPI administration group): 0.19±0.04, test group 3 (AMX+S-GM administration group): 0.22±0.06.

[0101] The negative control group infected with H. pylori showed a 90.9% or higher increase in H. pylori antibodies compared to the normal control group, which was a non-infected group not infected with H. pylori, and it was confirmed that H. pylori infection was maintained with a statistical significance (p<0.01). In test group 1 (AMX+CLR+PPI administration group), which was the positive control substance, there was a statistically significant decrease in H. pylori antibodies of 57.1% compared to the negative control group (p<0.01). In test group 2 (AMX+S-GM+PPI administration group) and test group 3 (AMX+S-GM administration group), which were test substance groups, there was a statistically significant decrease in H. pylori antibodies of 69.8% and 65.1%, respectively, compared to the negative control group (p<0.01). Test groups 2 and 3, which were test substance groups, showed statistically significant reductions of 29.6% and 18.5%, respectively, compared to test group 1, the positive control group, and the reduction effect of test group 2 was particularly excellent (p<0.01, p<0.05).

[0102] Pathological analysis of stomach tissue Visual observation of the gastric tissues removed after autopsy revealed no specific lesions in the non-infected group (normal control group), the infected negative control group, and test groups 1 to 3. Histopathological findings in the gastric tissues were scored for damage to the surface epithelium, inflammatory cell infiltration, and submucosal edema. If no findings were observed for each item, they were classified as 0 points, Mild 0.5 points, and Moderate 1 point.

[0103] As a result, in the normal control group, epithelial cell loss was observed at 0.10±0.21 points, inflammatory cell infiltration at 0.15±0.24 points, and submucosal edema at 0.10±0.32 points, for a total score of 0.35±0.41 points. In the negative control group, epithelial cell loss was observed at 0.65±0.34 points, inflammatory cell infiltration at 0.40±0.32 points, and submucosal edema at 0.55±0.37 points, for a total score of 1.60±0.81 points. In test group 1 (AMX+CLR+PPI administration group), epithelial cell loss was observed at 0.30±0.42 points, inflammatory cell infiltration at 0.40±0.46 points, and submucosal edema at 0.60±0.46 points, for a total score of 1.30±1.14 points. In test group 2 (AMX+S-GM+PPI group), epithelial cell loss was 0.30±0.35 points, inflammatory cell infiltration was 0.15±0.24 points, and submucosal edema was 0.35±0.34 points, for a total score of 0.80±0.79 points. In test group 3 (AMX+S-GM+PPI group), epithelial cell loss was 0.60±0.39 points, inflammatory cell infiltration was 0.40±0.46 points, and submucosal edema was 0.65±0.41 points, for a total score of 1.65±1.11 points (Table 7).

[0104] [Table 7]

[0105] Data are shown as mean ± standard deviation. Statistical analysis was performed using Sigma plot statistics. *Compared to non-infected group. **p<0.01, #Compared to negative control group, ##p<0.01.

[0106] Comparing the scores for the "Damage of the surface epithelium" item by group, a statistically significant increase was observed in the negative control group compared to the normal control group (p<0.01), and only in test group 1 (AMX+CLR+PPI administration group) and test group 3 (AMX+S-GM+PPI administration group) were there statistically significant decreases of 53.8% and 53.8%, respectively, compared to the negative control group (Table 7).

[0107] Comparing the scores for the "inflammatory cell infiltration" item by group, a statistically significant increase was observed in the negative control group compared to the normal control group (p<0.01), and only in test group 2 (AMX+S-GM+PPI administration group) was there a statistically significant decrease of 62.5% compared to the negative control group (Table 7).

[0108] Comparing the scores for the "Submucosal edema" item by group, a statistically significant increase was observed in the negative control group compared to the normal control group (p<0.01), but no statistically significant differences were observed between the other groups (Table 7).

[0109] Comparing the total score calculated by adding up the scores of the three types of histopathological findings (epithelial cell loss, inflammatory cell infiltration, and submucosal edema) in each group, a statistically significant increase was observed in the negative control group compared to the normal control group (p<0.01), and only in test group 1 (AMX+S-GM+PPI administration group) was there a statistically significant decrease of 50.0% compared to the negative control group (Table 7).

[0110] CLO rapid element decomposition enzyme test results Gastric tissue was tested for CLO rapid degrading enzyme activity and graphed to calculate the percentage of positive samples relative to the total number of samples.

[0111] As a result, if animals showing an intermediate color (partially positive) are considered to have responded to treatment, the treatment rates were 50% for test group 1 (AMX+CLR+PPI administration group), which was the standard treatment group, 70% for test group 2 (AMX+S-GM+PPI administration group), and 10% for test group 3 (AMX+S-GM administration group), with test group 2 showing the best treatment rate (Table 8).

[0112] [Table 8]

[0113] Results of Experimental Example 7 Antibiotic resistance rate Clarithromycin showed a very high resistance rate of 29.8%, and the highest resistance rate was 37.2% for levofloxacin, which is not widely used in clinical practice to eradicate Helicobacter. Therefore, it was determined that clarithromycin is not suitable for producing clay mineral complexes. As for aminoglycoside antibiotics, which are candidate antibiotics used in clay mineral complexes, gentamicin and netilmicin showed relatively low MIC and resistance rates (2.78% and 1.43%, respectively) when MIC 1 μg / mL was used as the standard, and they were determined to be suitable candidate antibiotics. However, tobramycin (TOB) showed a resistance rate of 36.3%, and amikacin (AMK) also showed a resistance rate of 61.3%, and they were determined to be unsuitable as candidate antibiotics (Figure 7). AMX: amoxicillin, CLR: clarithromycin, MTZ: metronidazole, TET: tetracyclin, LEV: levofloxacin.GM: gentamicin, NET: netilmicin, TOB: tobramycin, AMK: amikacin.

[0114] Eradication effect against clarithromycin-resistant bacteria The in vitro eradication effect against clarithromycin-resistant H. pylori was evaluated. As a result of two repeated tests, a clear zone appeared around the disk of the gentamicin-smectite complex (S-GM). Therefore, it was confirmed that the gentamicin-smectite complex (S-GM) has eradication activity against clarithromycin-resistant H. pylori. On the other hand, no clear zone appeared around the clarithromycin (CLR) disk. The results of two repeated tests are shown in the same manner, and each result is shown in Figure 8A and Figure 8B (Figure 8A and Figure 8B: top: CLR: clarithromycin, bottom: S-GM: gentamicin-smectite complex).

[0115] Examination of experimental results In the present invention, the anti-H. pylori efficacy of S-GEN was evaluated in a mouse model. The experimental results of the present invention demonstrated the significantly improved antimicrobial efficacy of S-GEN in reducing H. pylori load in the mouse stomach compared with other therapeutic regimens, including triple therapy, which is currently the global standard for H. pylori treatment.

[0116] The present inventors developed S-GEN and evaluated whether it is effective in treating H. pylori infection. H. pylori mainly resides in the mucus layer attached to the epithelial surface of the gastric mucosa. Therefore, for effective treatment, S-GEN must cross the mucus layer and remain on the stomach wall. In the present invention, S-GEN was well distributed on the distal stomach wall, and 60.2% of S-GEN was observed to be present, indicating that S-GEN was effectively maintained for up to 1 hour. This result suggests that S-GEN can be used for direct eradication of H. pylori.

[0117] After treatment, the results of H. pylori PCR and CLO tests on the gastric mucosa showed that Group 5 showed the highest cure rate (80%) among the cured groups. Furthermore, the CLO value of Group 5 was the lowest among the cured groups (0.6±1.3). The efficacy of anti-H. pylori was clear in Group 5. After treatment for H. pylori infection, negative results continued to be observed only in Group 5 for H. pylori PCR tests on stool. Therefore, the excellent anti-H. pylori activity conferred by S-GEN triple therapy can be explained by its direct eradication effect and the fact that it is applied to the gastric mucosa for a longer time and the eradication effect lasts longer compared to other therapies of conventional antibiotics.

[0118] Interestingly, S-GEN-cured mice had significantly reduced H. pylori-induced proinflammatory cytokines (IL-8 and TNF-α) compared to H. pylori-infected but non-cured mice. IL-8 attracts neutrophils and promotes inflammation, and TNF-α along with IL-1β induces gastrin secretion, suggesting a role for these cytokines in H. pylori-induced hypergastrinaemia and inflammatory responses. Immune responses to H. pylori contribute to the pathogenesis of disease. Thus, a reduction in the proinflammatory response, as observed here, is expected to reduce the proinflammatory response responsible for perpetuating tissue damage. After S-GEN treatment, gastric inflammation could be reduced, with better anti-H. pylori effects and early elimination of colonization than those achieved by other therapies.

[0119] Treatment with S-GEN in the present invention did not affect the body weight of mice. The safety of oral administration of smectite has been demonstrated, and gentamicin was not absorbed through the gastric mucosa.

[0120] The inventors demonstrated the strong antimicrobial activity of S-GEN against H. pylori and the effect of long-term application of S-GEN in the gastric mucosal layer. S-GEN treatment reduced the bacterial burden in vivo compared to mice cured with double or triple therapy including PPIs or uncured mice. In addition to the direct eradication effect, S-GEN helped to reduce the inflammatory response by suppressing the production of inflammatory cytokines.

[0121] In addition, it was confirmed that when S-GEN was administered together with a β-lactam antibiotic and a gastric acid suppressant, the eradication effect of Helicobacter pylori was significantly higher than the eradication effect when S-GEN was administered together with a β-lactam antibiotic alone.

[0122] Furthermore, the resistance rate of H. pylori to clarithromycin, an antibiotic widely used in clinical practice, is high at 29.8%, but S-GEN has eradication activity even against clarithromycin-resistant H. pylori, and therefore S-GEN is considered to be useful even for patients in whom clarithromycin is not effective in eradicating the bacteria.

[0123] The present invention relates to a method for eradicating Helicobacter pylori, comprising the step of administering to a subject a complex of a non-absorbable antibiotic and a clay mineral, or a composition containing the same. The present invention also relates to a method for preventing or treating a gastrointestinal disease caused by Helicobacter pylori, comprising the step of administering to a subject a complex of a non-absorbable antibiotic and a clay mineral, or a composition containing the same. The present invention also relates to a pharmaceutical composition or kit for oral administration for eradicating Helicobacter pylori, comprising a complex of a non-absorbable antibiotic and a clay mineral. The composition and kit of the present invention may further comprise a β-lactam antibiotic and / or a gastric acid suppressant. Rank List FreeText

[0124] SEQ ID NO:1 is the base sequence of the sense primer for amplifying H. pylori-specific ureA and ureC. SEQ ID NO:2 is the base sequence of the antisense primer for amplifying H. pylori-specific ureA and ureC. SEQ ID NO:3 is the base sequence of the sense primer for amplifying GAPDH. SEQ ID NO: 4 is the base sequence of the antisense primer for amplifying GAPDH.

Claims

1. a non-absorbable antibiotic and clay mineral complex, a beta-lactam antibiotic, and a gastric acid suppressant; the non-absorbable antibiotic is gentamicin; The clay mineral has interlayer expansion properties, is used as a carrier by inserting an antibiotic, and is a smectite; A pharmaceutical composition for oral administration for eradicating clarithromycin-resistant Helicobacter pylori.

2. The pharmaceutical composition is characterized in that the complex of the non-absorbable antibiotic and the clay mineral is orally administered, passes through the esophagus to the stomach, and then adheres to the gastric mucus layer to release the antibiotic.

2. The pharmaceutical composition of claim 1.

3. The pharmaceutical composition is characterized in that it has urease inhibitory activity.

2. The pharmaceutical composition of claim 1.

4. The pharmaceutical composition is a pharmaceutical composition for preventing or treating gastrointestinal diseases caused by Helicobacter pylori.

2. The pharmaceutical composition of claim 1.

5. a non-absorbable antibiotic and clay mineral complex, a beta-lactam antibiotic, and a gastric acid suppressant; the non-absorbable antibiotic is gentamicin; The clay mineral has interlayer expansion properties, is used as a carrier by inserting an antibiotic, and is a smectite; An oral administration kit for eradicating clarithromycin-resistant Helicobacter pylori.

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