Patent Description:
In the course of the last few decades various pharmacological approaches have been developed for the pharmacological treatment of gastric hyperacidity, a condition which, if present to a marked degree and for prolonged periods, can give rise to various complications or pathologies such as peptic ulcer and gastroesophageal reflux disease.

Among the drugs most widely used are those based on active principles capable of inhibiting inhibitors of the histamine receptor H<NUM> such as, for example, cimetidine, famotidine, nizatidine, ranitidine, or based on active principles capable of inhibiting prostaglandins such as, for example, misoprostol. Another category of drugs is based on active principles which perform the function of protectors of the gastric mucosa such as, for example, bismuth salts, sucralfate or antimuscarinic or parasympatholytic drugs based on pirenzepine and pipenzolate. Finally there are also antacids such as, for example, sodium bicarbonate, aluminium hydroxide or magnesium hydroxide and proton pump inhibitors based on Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole.

Proton pump inhibitors (PPI) are a group of molecules whose principal action consists in a pronounced reduction in the acidity of the gastric juices for a fairly long period of time (<NUM> to <NUM> hours).

The group containing PPIs is the successor to H<NUM> antihistamines, and PPI inhibitors are broadly more widespread than the latter because of their greater effectiveness.

The medicines mentioned above are used in the symptomatic and aetiological treatment of various syndromes, such as: (i) dyspepsia; (ii) gastro-duodenal ulcer. PPIs are used for treating or preventing gastric and duodenal ulcers. They are also used in association with certain antibiotics in the treatment of gastritis from Helicobacter pylori, (iii) Zollinger-Ellison syndrome and (iv) gastroesophageal reflux disease.

PPIs are also used in patients treated long-term with acetylsalicylic acid or other NSAIDs. By inhibiting the function of the enzyme cyclooxigenase <NUM> (COX <NUM>), these drugs have the side effect of reducing the synthesis of prostaglandin, a process which depends on the same enzyme. Since one of the functions of prostaglandin is the protection of the gastric mucosa from acidity, PPIs are used in order to reduce acidity and protect the gastric mucosa.

This type of medicine inhibits the gastric enzyme H+/K+-ATPase (the proton pump), catalyst of the H+and K+ion exchange. This creates effective inhibition of acid secretion.

In the micro-channel where the pH is low, close to <NUM>, these inhibitors are ionised and transformed into molecules capable of establishing covalent bonds with the cysteine thiol group (SH) of the pump sub-unit. The pump is thus irreversibly inhibited. Renewal of pumping activity requires the production of new pumps, an event which requires <NUM> to <NUM> hours on average. A single dose of PPI, therefore, enables inhibition of the gastric secretion of about <NUM> hours.

The fact that the inhibitors are active only in an acid environment explains how they have a minimal effect on the extragastric H+/K+ -ATPase situated at the level of the rectum and the colon.

In any case, apart from the specific action mechanism, the final effect of almost the totality of these classes of drugs for the treatment of gastric hyperacidity, or other pathological conditions mentioned above, is the raising of the gastric pH according to kinetics and intensities dependent on the specific molecule taken and its dosage. One exception, in this sense, is the prostaglandins and protector drugs for the gastric mucosa which, instead of reducing the intraluminal hydrogen ion concentration, increase the synthesis of mucus and bicarbonate ion by the cells of the gastric wall, thus increasing the protection of the mucosa against acidity of the lumen. In any case, drugs capable of reducing gastric hyperacidity constitute the treatment of choice in cases of peptic ulcer or gastroesophageal reflux, while mucosal protectants represent a complementary therapy.

It is known, furthermore, that normal gastric acidity constitutes an effective barrier against potential harmful organisms or pathogens ingested with the normal diet. Many of them, in fact, are particularly sensitive to acidity and are not capable of surviving for more than five minutes, sometimes even less, at pH values below <NUM>. It follows that many pathogens, among them those belonging to the genus Salmonella, do not reach the intestine alive and, setting aside harmful effects on the human organism mediated by any toxins secreted and already present in food, are not capable of giving rise to an intestinal infection and, therefore, to full-blown food poisoning.

It has to be said, however, that raising the gastric pH values typically found in patients who take drugs to reduce or treat gastric hyperacidity makes these patients more exposed to dietary toxic infections caused especially by consumption of raw food, particularly fish, meat and eggs.

Patients who take drugs to reduce or treat gastric hyperacidity, such as proton pump inhibitors for example, have a stomach pH value of around <NUM>.

This pH value allows Enterobacteriaceae, and particular strains of E. Coli with pronounced decarboxylasic action, to pass through the degraded gastric barrier. Proteins ingested during eating are enzymatically degraded to amino acids which, in the presence of decarboxylasic action, are modified into a series of biogenic amines ranging from potentially dangerous to highly dangerous such as for example histamine, tyramine, putrescine and cadaverine. The most common symptoms which can cause these biogenic amines have a complete overlap with the secondary effects caused by the use of proton pump inhibitors (PPIs), and are as follows: diarrhoea, headache, nausea, abdominal pains and flatulence. When certain biogenic amines then react with nitrites, we have the formation of N-nitrosamines. These nitrosamines cause a genetic mutation through alkylation of the DNA, and their presence is associated with cancer of the stomach, the intestine, the pancreas and the bladder, and also with leukaemia.

One possible solution for these patients does not, obviously consist of suspension of the pharmacological treatment because this would expose the gastric or oesophageal mucosa once again to the harmful effects mediated by the gastric juices. On the other hand it is not even thinkable to continue the pharmacological treatment and leave the patients exposed to these risks of infection.

There remains, therefore, a need to allow patients in need, on the one hand, to take drugs for reducing or treating gastric hyperacidity and, on the other hand, to avoid being exposed to highly dangerous pathogenic infections or to risks of recurrent pathogenic infections.

In particular, it remains necessary to be able to respond to the above-mentioned needs by means of a composition of natural origin, free of side-effects, with an improved and selective antimicrobial efficacy against pathogens, such as for example coliforms which are a group of bacteria belonging to the family of Enterobacteriaceae and which includes E. coli, including serotype O157:H7. The applicant has responded to the above-mentioned needs with a composition which, on the one hand, is capable of restoring the functionality of the gastric barrier, having a protective effect against pathogenic or harmful micro-organisms and, on the other, is capable of having an improved and selective efficacy against the pathogens themselves.

The composition of the present invention is capable of restoring the functionality of the gastric barrier, normally exercised by the gastric juices, which is particularly reduced in patients who take drugs to reduce or treat gastric hyperacidity. Said composition is capable of minimising the secondary effects associated with pharmacological intake based on proton pump inhibitor drugs (PPIs for short). Said composition, furthermore, demonstrates improved efficacy against pathogenic or harmful micro-organisms.

After intense research activity, the Applicant has surprisingly found that a selected combination (or mixture) of probiotic bacteria comprising or, alternatively, consisting of at least one strain of bacteria belonging to one or more of the species stated below is capable of allowing patients in need, on the one hand, to take drugs for reducing or treating gastric hyperacidity and, on the other hand, to avoid being exposed to highly dangerous pathogenic infections or to risks of recurrent pathogenic infections.

The antibacterial efficacy shown by each individual strain of bacteria, the subject of the present invention, proves to be, in said composition, increased and more selective against pathogens as a result of the presence of N-acetylcysteine and/or lysozyme; or N-acetylcysteine and/or microencapsulated lysozyme. In a preferred embodiment, the lysozyme is microencapsulated in a lipid matrix. Advantageously, the lipid matrix is of vegetable origin and has a melting point comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM>, even more preferably between <NUM> and <NUM>.

The subject of the present invention consists of a composition having the characteristics stated in the attached independent claim.

Other preferred embodiments of the present invention will be claimed in the attached dependent claims.

Table <NUM> shows, by way of example, a group of micro-organisms which have a valid application.

Table <NUM> shows a group of micro-organisms which have a valid application.

Table <NUM> shows the results of the species-specific PCR assays carried out for identifying the bacterial species administered.

Table <NUM> shows the quantification of the total bacterial cells and of the total Lactobacillus (value ± SEM, log10 CFU/ml of the gastric juice or gram of material from brushing the duodenum) at d0 (all groups) and at d10 (Group B).

Table <NUM> shows the results of the species-specific PCR assay in Group B at d0 and at d<NUM>. The presence of correlated species is shown by a "+", while their absence is shown by a "-".

Table <NUM> shows the quantification of the specific microbial groups in faecal samples at d0 (all groups) and d10 (Group B). The results are expressed as log10 of CFU/gram of faeces (value ± SEM).

<FIG> refers to the total bacterial count present in the samples taken from the subjects of the clinical study (Figure A and Figure B).

<FIG> shows the comparison between subjects chronically treated with PPIs (PPI group totals: PPI + "PPI plus probiotics") and the control group. The data are expressed as an average of the colony-forming units (CFU). <FIG> shows the comparison between subjects chronically treated with PPIs and those treated with "PPIs plus probiotics") and the control group. The data are expressed as an average ± S. of the colony-forming units (CFU).

<FIG> shows the quantities of bacteria found in the gastric juice and after duodenal brushing in the subjects treated.

The Applicant has performed intense research and selection activity, at the end of which it found that the strains of probiotic bacteria belonging to at least one species chosen from the group comprising or, alternatively, consisting of Lactobacillus pentosus LPS01 DSM <NUM>; Lactobacillus rhamnosus LR06 DSM <NUM>; and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (MB386) DSMZ <NUM> DSM <NUM> have a valid application in the treatment of subjects who are taking proton pump inhibitors (PPIs) to reduce or treat gastric hyperacidity. Furthermore, the Applicant has found that the antibacterial efficacy demonstrated by the strains of bacteria which are the subject of the present invention is increased and more selective against pathogens as a result of the presence of N-acetylcysteine (NAC) in said composition.

Furthermore, the Applicant has found that the antibacterial efficacy demonstrated by the strains of bacteria which are the subject of the present invention is increased and more selective against pathogens as a result of the presence of microencapsulated gastroprotected lysozyme in said composition. The lysozyme is microencapsulated in a lipid matrix. Advantageously, the lipid matrix is of vegetable origin and has a melting point comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM>, even more preferably between <NUM> and <NUM>.

Furthermore, the Applicant has found that the antibacterial efficacy demonstrated by the strains of bacteria which are the subject of the present invention is increased and more selective against pathogens as a result of the presence of N-acetylcysteine and microencapsulated gastroprotected lysozyme in said composition. The lysozyme is microencapsulated in a lipid matrix. Advantageously, the lipid matrix is of vegetable origin and has a melting point comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM>, even more preferably between <NUM> and <NUM>.

The composition herein described comprises N-acetylcysteine in association with the strains of bacteria of the present invention: N-acetylcysteine which is an N-acetylate derivative of the amino acid cysteine.

The composition herein described comprises microencapsulated gastroprotected lysozyme in association with the strains of bacteria of the present invention:
The composition herein described comprises N-acetylcysteine and/or microencapsulated gastroprotected lysozyme in association with the strains of bacteria of the present invention.

The Applicant has found that the use of N-acetylcysteine in association with one or two or three or four or five or six strains of bacteria, described in Tables <NUM> and <NUM>, or in the various preferred embodiments here described, is capable of dissolving the bacterial biofilm produced by the pathogenic bacteria themselves and which is used by said pathogens as protection. In practice it has been seen that the pathogenic bacteria are capable of forming a protective coating (biofilm) around the cells. The biofilm makes the cells of the pathogens more difficult to attack and better protected. N-acetylcysteine is capable of penetrating the biofilm of the cells and dissolving it, facilitating the attack on the pathogenic cells by means of the bacteriocins and/or the metabolites and/or the oxygenated water produced by the strains of bacteria which are the subject of the present invention.

The Applicant has found, furthermore, that the use of microencapsulated gastroprotected lysozyme makes it possible to pass the gastro-duodenal barrier and arrive complete in the colon where it succeeds in exercising its action of inhibiting the Clostridiaceae, including C. difficile, thanks to the lytic action of the enzyme on the spore, in association with one or more of the strains of bacteria which are the subject of the present invention.

The quantity of N-acetylcysteine present in the composition which is the subject of the present invention is comprised between <NUM> and <NUM>,<NUM>/day, preferably between <NUM> and <NUM>/day, even more preferably between <NUM> and <NUM>/day. N-acetylcysteine, which is available on the market in non-microencapsulated form and in a pharmaceutically acceptable form, preferably in solid form, is mixed with the probiotic bacteria, preferably in solid or lyophilised form, using techniques and equipment known to experts in the field to give a homogeneous composition.

The quantity of microencapsulated gastroprotected lysozyme present in the composition which is the subject of the present invention is comprised between <NUM> and <NUM>,<NUM>/day, preferably between <NUM> and <NUM>,<NUM>/day, even more preferably between <NUM> and <NUM>/day, preferably in solid form; it is mixed with the probiotic bacteria, preferably in solid or lyophilised form, using techniques and equipment known to experts in the field, to give a homogeneous composition. Lysozyme is available on the market in a pharmaceutically acceptable form.

The strains of bacteria were selected because they are capable of colonising the stomach at a pH value comprised between <NUM> and <NUM>; preferably between <NUM> and <NUM>. At this pH value the selected strains act by means of the production of active substances such as bacteriocins and/or metabolites and/or oxygenated water.

The composition of the present invention can be a dietary composition, for example a symbiotic composition, or a supplement or a pharmaceutical composition or a medical device. The composition herein described comprises among those listed in Table <NUM> or, alternatively, in Table <NUM>, the following strains of bacteria:.

Lactobacillus delbrueckii subsp. delbrueckii LDD01 (MB386) DSMZ <NUM> DSM 22106in association with N-acetylcysteine (NAC) and/or lysozyme, preferably microencapsulated lysozyme.

The strains of Table <NUM> have been individually tested for the purpose of identifying the pathogen which they are capable of antagonising (inhibiting the growth or reducing the number of one or more harmful or pathogenic microbial species/genus), as stated in column <NUM> of Table <NUM>.

Table <NUM> shows that the bacteria are capable of producing oxygenated water or at least one bacteriocin with an inhibiting action on one or more harmful or pathogenic microbial species/genus.

All the strains described and/or claimed in the present patent application have been deposited in accordance with the Treaty of Budapest and are made available to the public on request to the competent Depositing Authority.

The compositions herein described have a valid application for use both in the treatment of subjects who are taking drugs to reduce and/or treat gastric hyperacidity and in the treatment of an ulcer caused by a deficiency in the protective mechanisms of the mucosa (e.g. reduced secretion or responsiveness to prostaglandin E, as in the case of taking aspirin or other NSAls) or by an infection by H. In other words, the composition herein described has a valid application also for those subjects who are prescribed PPls/other antacid drugs although not showing gastric hyperacidity, but with a lesion of the gastric and/or duodenal mucosa consequent on an altered ratio of gastric acidity/mechanisms protecting the mucosa.

It has been found that the compositions herein described are capable of being validly used in the treatment of peptic ulcer or gastroesophageal reflux.

The composition herein described comprises or, alternatively, consists of from two to four strains, chosen from the group comprising or, alternatively, consisting of:.

in association with N-acetylcysteine and/or lysozyme; or N-acetylcysteine and microencapsulated lysozyme.

The compositions herein described may comprise a single strain belonging to each individual species listed above or, alternatively, may comprise more than one strain belonging to the same species, as for example two strains, or three strains, or four strains, all belonging to the same species, as shown above.

In one embodiment, the composition comprises Lactobacillus pentosus LPS01 DSM <NUM> and/or Lactobacillus plantarum LP01 LMG P-<NUM> and/or Lactobacillus rhamnosus LR06 DSM <NUM> and/or Lactobacillus delbrueckii subsp. delbrueckii (MB386) LDD01 DSMZ <NUM> (DSM <NUM>) in a quantity comprised between <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM> CFU/strain/dose, preferably between <NUM> and <NUM>×<NUM><NUM> CFU/strain/dose; NAC in a quantity comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM>/dose, even more preferably between <NUM> and <NUM>/dose; potato maltodextrin in a quantity comprised between <NUM> and <NUM> grams/dose, preferably between <NUM> and <NUM> grams/dose.

The compositions described above are for use in the preventive and/or curative treatment of infections, disturbances or illnesses caused by the presence of Helicobacter pylori, in particular in the preventive and/or curative treatment of recurrences from infections caused by Helicobacter pylori; they are furthermore for use in the treatment of peptic ulcer or gastroesophageal reflux.

The selected strains are capable of producing bacteriocins and/or metabolites and/or oxygenated water, these being substances which are capable of effectively combating, inhibiting or reducing pathogenic bacteria. These strains find valid application and use in the preventive and/or curative treatment of infections and/or pathologies connected with pathogenic gram-negative bacteria.

The pathogenic bacteria are chosen from the group comprising the coliforms. The coliforms are a group of bacteria belonging to the family of Enterobacteriaceae. The group comprises E. coli including the serotype O157:H7
It has been found that a composition comprising Lactobacillus pentosus LPS01 DSM <NUM>, Lactobacillus plantarum LP01 LMG P-<NUM>, Lactobacillus rhamnosus LR06 DSM <NUM> and Lactobacillus delbrueckii LDD <NUM> (MB386) DSM <NUM> Lactobacillus delbrueckii subsp. delbrueckii LDD01 DSMZ <NUM> DSM <NUM> in a quantity in weight comprised in the ratio <NUM>:<NUM>:<NUM>:<NUM> to <NUM>:<NUM>:<NUM>:<NUM> (for example <NUM>×<NUM><NUM> CFU/strain/dose and <NUM>×<NUM><NUM> CFU/strain/dose) and a quantity of NAC comprised between <NUM> and <NUM> exerts strong antagonistic action.

In the composition of the present invention, the mixture of strains of bacteria is present in a quantity comprised between <NUM>% and <NUM>% by weight, compared with the total weight of the composition, preferably of between <NUM>% and <NUM>%.

In a preferred embodiment, the composition can furthermore comprise at least one prebiotic fibre and/or carbohydrates with bifidogenic action. The prebiotic fibre which has an application in the composition of the present invention is a fibre which must be used by the strains of bacteria present in the composition, but not by the pathogens which it is intended to antagonise. In the event that the pathogen to be antagonised belongs to the genus Candida, the fructo-oligosaccharides (FOS) and the galacto-oligosaccharides (GOS) have a valid application because said fibres are not used by Candida; whereas the gluco-oligosaccharides (GOSa) are capable of directly inhibiting E. coli by means of several metabolites. The prebiotic fibre can therefore be chosen, according to the needs of the case and the pathogen to be antagonised, between: inulin, fructo-oligosaccharides (FOS), galacto- and transgalacto-oligosaccharides (GOS and TOS), gluco-oligosaccharides (GOSa), xylo-oligosaccharides (XOS), chitosan-oligosaccharides (COS), soya-oligosaccharides (SOS), isomalto-oligosaccharides (IMOS), resistant starch, pectin, psyllium, arabino-galactanes, gluco-mannanes, galacto-mannanes, xylanes, lactosaccharose, lactulose, lactitol and various other types of rubbers, acacia fibre, carruba fibre, oat fibre, bamboo fibre, fibres from citruses and, in general, fibres containing a soluble portion and an insoluble portion, in variable ratios to each other. In a preferred embodiment of the invention, the composition comprises at least one prebiotic fibre chosen from among those mentioned above and/or suitable mixtures between them in any relative percentage. The quantity of prebiotic fibres and/or of carbohydrates with bifidogenic action, if present in the composition, is comprised between <NUM>% and <NUM>% by weight, preferably between <NUM>% and <NUM>% and even more preferably between <NUM>% and <NUM>%, compared with the total weight of the composition. In this case the composition or supplement has a symbiotic action and functional properties.

Furthermore, the composition can also comprise other active ingredients and/or components such as vitamins, minerals, bioactive peptides, substances with anti-oxidising action, hypocholesterolaemic agent, hypoglycaemic agent, antiinflammatory and anti-sweetening agents in a quantity generally comprised between <NUM>% and <NUM>% by weight, preferably between <NUM>% and <NUM>% by weight, in any event depending on the type of active component and its recommended daily dose if any, compared with the total weight of the composition.

The dietary composition which is the subject of the present invention (for example, a symbiotic composition, or a supplement or a pharmaceutical composition) is prepared according to the techniques and the equipment known to experts in the field.

In a preferred embodiment, the composition contains bacteria in a concentration comprised between <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM> CFU/g of mixture of bacteria, preferably between <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM> CFU/g of mixture of bacteria.

In a preferred embodiment, the composition contains bacteria in a concentration comprised between <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM> CFU/dose, preferably between <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM> CFU/dose. The dose can be comprised between <NUM> and <NUM>, for example it is of <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. The probiotic bacteria used in the present invention can be in solid form, in particular in the form of powder, dehydrated powder or lyophilized form. All the compositions of the present invention are prepared according to techniques known to experts in the field and by the use of known equipment.

The composition herein described comprises furthermore a drug for reducing or treating gastric hyperacidity. Said drug is chosen from the group comprising or, alternatively, consisting of: inhibitors of receptor H2, preferably cimetidine, famotidine, nizatidine or ranitidine; prostaglandins preferably misoprostol; protectors of the gastric mucosa, preferably bismuth salts or sucralfate; antimuscarinic or parasympatholytic drugs, preferably pirenzepine or pipenzolate; antacids, preferably sodium bicarbonate, aluminium hydroxide or magnesium hydroxide; proton pump inhibitors, preferably Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole. Preferably, said drug is chosen from the group comprising or, alternatively, consisting of: inhibitors of receptor H2, preferably cimetidine, famotidine, nizatidine or ranitidine; antimuscarinic or parasympatholytic drugs, preferably pirenzepine or pipenzolate; antacids, preferably sodium bicarbonate, aluminium hydroxide, magnesium hydroxide; proton pump inhibitors, preferably chosen from the group comprising Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole.

Even more preferably, said drug is chosen from the group comprising or, alternatively, consisting of: inhibitors of receptor H2, preferably cimetidine, famotidine, nizatidine or ranitidine; proton pump inhibitors, preferably chosen from the group comprising Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole. Advantageously, the drug is a proton pump inhibitor chosen from the group comprising Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole. Both the bacteria and the drug are intimately present in the said composition. For example, the bacteria and the drug are present together in a tablet, a pastille or a granulate in a pharmaceutical form suitable for oral administration. It is essential that the bacteria and the drug are administered simultaneously and act simultaneously because it is necessary to restore the barrier effect removed by the proton pump inhibitors (PPIs), thanks to the action of the probiotic bacteria of the present invention, which produce bacteriocins and are capable of colonising the stomach as a result of the fact that the proton pump inhibitors have raised the pH to a value of about <NUM> to <NUM>; preferably of <NUM> to <NUM>.

In another preferred embodiment, the composition of the present invention is comprised in a medical device. In this case the bacteria are present in a composition suitable for oral administration such as for example a tablet, a pastille or a granulate and, separately, the drug indicated for reducing or treating gastric hyperacidity, as described above, is present in another composition suitable for oral administration. Advantageously, the drug is a proton pump inhibitor chosen from the group comprising Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole.

Two tablets, for example, are therefore administered, one containing the bacteria and the other containing the drug. In any event the two tablets must be administered simultaneously, given that it is necessary for the bacteria to act simultaneously with the action of the proton pump inhibitors. In the case of the medical device, too, it is essential that the bacteria and the drug are administered at a short distance in time because it is necessary to restore the barrier effect removed by the proton pump inhibitors (PPIs), thanks to the action of the bacteria which produce bacteriocins which are capable of colonising the intestine as a result of the fact that the proton pump inhibitors have raised the pH to a value of about <NUM> to <NUM>; preferably of <NUM> to <NUM>.

The Applicant has found that the bacteria selected and listed above, are capable of colonising in the stomach at a pH value of around <NUM> so as to restore the barrier effect reduced or eliminated by the raising of the pH following the action of the drugs indicated for reducing or treating gastric hyperacidity such as, for example, a proton pump inhibiting drug chosen from the group comprising Lansoprazole, Esometazole, Rabeprazole, Pantoprazole and Omeprazole.

Herein described is a composition comprising at least one strain of bacteria as mentioned above, for use in the preventive and/or curative treatment of infections, disturbances or illnesses caused by the presence of Helicobacter pylori, in particular in the preventive and/or curative treatment of recurrences from infections caused by Helicobacter pylori.

In the broadest sense of the term, antibiotics are defined as molecular species produced by an organism and active against the growth of other organisms. In practice, however, antibiotics are generally considered as secondary metabolites active at low concentrations in blocking the growth of micro-organisms. The secondary products of the metabolism such as organic acids, ammonia and oxygenated water are not to be included in the category of antibiotics. Antibiotics are molecules, which may be peptide molecules (penicillin), produced by multi-enzymatic systems and whose biosynthesis is not blocked by protein synthesis inhibitors. Bacteriocins, on the other hand, are products of ribosomal synthesis. Bacteriocins are peptide molecules produced by ribosomal synthesis which can also be associated with lipids or carbohydrates. Although some bacteriocins produced by Gram-positive bacteria (Lactobacillus, Lactococcus) have inhibition spectra limited to certain strains belonging to the same species as the producing micro-organism, the majority of them show a broad spectrum of action against various bacterial species, both Gram-positive and Gram-negative. The current classification of the bacteriocins is based both on their chemical nature and on their spectrum of action.

The present pilot clinical study was conducted on <NUM> subjects, <NUM> of whom had been taking PPIs for more than a month. The group made up of subjects treated with PPIs was further divided into two subgroups: patients treated with PPIs plus a mixture of strains of selected lactobacilli (<NUM> billion L. rhamnosus LR06 DSM <NUM>, <NUM> billion L. plantarum LP01 LMG P-<NUM>, <NUM> billion L. pentosus LPS01 DSM <NUM> and <NUM> billion L. delbrueckii subsp. delbrueckii LDD01) for <NUM>-<NUM> days before the endoscopic examination. The biological samples, made up of gastric juice and material from duodenal brushing, were taken during the gastroscopy carried out on the patients who had been fasting for <NUM>-<NUM> hours. The biological materials, conserved in Amies liquid, were subjected to microbiological analyses suitable for evaluating the bacterial load. Non-selective culture medium (LaptG) was used to obtain the total bacterial load, while, to select the heterofermenting lactobacilli, MRS broth medium was used with the addition of the antibiotic vancomicin (<NUM>µg/ml), preparing serial dilutions of the starting sample. The last dilution which was found to be positive to bacterial growth (using optical density) made it possible to deduce the order of magnitude of the load itself.

To verify the presence of the probiotic strains administered, PCR assays were carried out with the following primer sets: Rhall/Prl for L. rhamnosus; pREV/pentF for L. pentosus; pREV/planF for L. plantarum and SS1/DB1 for L. delbruckii LDD01.

The results for the total bacterial load demonstrated that the subjects treated with PPIs (PPI group totals: PPIs + "PPIs plus probiotics") show a large number of bacteria, both in the gastric juice and in duodenal brushing, in comparison with the control group (no PPI, no probiotics) which was found to be practically sterile (<FIG>). Analysis of the bacterial load of the subjects treated with PPIs plus probiotics revealed a considerable difference between the two groups analysed (<NUM> Log; <FIG> ).

<FIG> shows the comparison between subjects chronically treated with PPIs (PPI group totals: PPI + "PPI plus probiotics") and the control group. The data are expressed as an average of the colony-forming units (CFU). <FIG> refers to the comparison between subjects chronically treated with PPIs and those treated with "PPI plus probiotics". The data are expressed as an average ± S. M of the colony-forming units (CFU).

The selection of the heterofermenting lactobacilli, by growth in MRS broth with the addition of the antibiotic vancomicin in serial dilutions, allowed us to demonstrate that the majority of the bacteria found in the subjects treated with "PPI plus probiotics", belonged to the heterofermenting group, as shown in the pie chart reproduced in <FIG> , in which the area is proportional to the total microbial population.

Analysis using species-specific PCR assay showed the presence of the species L. rhamnosus, L. plantarum and L delbr. delbrueckii in all the subjects treated with "PPI plus probiotics", while the species L. pentosus was not found (Table <NUM>). Probably this species does not possess the characteristics necessary for its survival in the gastric environment. The positive result for the species L. plantarum, shown in a subject treated with PPIs only is probably to be attributed to the subject's dietary habits.

A total of <NUM> individuals (<NUM> men and <NUM> women) aged between <NUM> and <NUM> years and treated with PPIs were spontaneously enrolled (February-March <NUM>). Another <NUM> individuals (<NUM> men and <NUM> women) aged between <NUM> and <NUM> years who did not make use of PPIs (proton pump inhibiting drugs) were enrolled as a control group representative of people with normal gastric acidity. The inclusion criteria for taking part in the study comprised: age between <NUM> and <NUM> years, chronic treatment with PPIs for at least <NUM> to <NUM> consecutive months (for the first three groups), no other health problem known at the time of enrolment, no pathology requiring treatment with antibiotics; they were informed and gave their consent to taking part in the pilot study. The individuals were also selected on the basis of certain exclusion criteria: age below <NUM> years, pregnancy in progress or breastfeeding, serious chronic degenerative illnesses, serious cognitive deficits, previous abdominal surgery, diverticulitis, immunodeficiency states, concomitant organic intestinal disease, antibiotic treatment. After informed consent was obtained, the individuals were divided into four groups (A, B, C, and D).

Groups A and B included subjects who had undergone long-term treatment with PPIs (of at least <NUM> consecutive months), while Group C included subjects who had undergone a short treatment with PPIs, from <NUM> to <NUM> consecutive months. Finally, Group D included the control individuals who had not been treated with PPIs and with physiological gastric barrier effect. Group A (<NUM> individuals) was the control group for long-term treatment with PPIs and received no treatment. Each subject in Group B (<NUM> individuals) received <NUM> sachets containing <NUM> each of L. rhamnosus LR06 (DSM <NUM>), L. pentosus LPS01 (DSM <NUM>), and L. plantarum LP01 (LMG P-<NUM>) corresponding to <NUM>×<NUM><NUM> CFU/strain/sachet, and <NUM> of micro-organism L. delbrueckii subsp. delbrueckii LDD01 (DSM <NUM>) equivalent to <NUM>×<NUM><NUM> CFU/sachet, <NUM> of N-acetylcysteine (NAC) and <NUM> grams of potato maltodextrin. The total number of vital cells per sachet was <NUM> billion (<NUM>×<NUM><NUM> CFU). Group C (<NUM> individuals) was the study group for short-term treatment with PPIs and received no probiotics. The object of this group was to compare the bacterial growth in Group C compared with Group A, because it was assumed that the bacterial concentration in the gastric lumen and in the duodenal mucosa should be greater in subjects who had undergone long-term treatment with PPIs than in patients who had undergone treatment with PPIs for not longer than <NUM> months. The individuals in Group B consumed one sachet/day during the main meal, preferably at supper, with the object of allowing the bacteria to remain longer in the stomach lumen and to be distributed homogeneously together with the N-acetylcysteine. The contents of the sachet were dissolved in half a glass of cold water before taking. Administration lasted <NUM> days. The gastric juice and the material from duodenal brushing were collected during gastroscopy on the subjects after a fast of at least <NUM> hours from the last time that the probiotics were taken. In this way, no less than half a day had passed since the last time that the probiotics were taken by the individuals. More specifically, the gastroscopy was conducted at time zero (d<NUM>) in all the Groups (A, B, C and D) and after <NUM> days (d<NUM>); i.e. after the end of taking the probiotics with reference to Group B only. The faecal samples were collected on d0 in all the groups (A, B, C and D) and on d10 for Group B only. The subjects in Groups A, B and C continued the treatment with their specific PPI drugs at the same dose for the entire duration of the pilot study.

The faeces were collected at the beginning of the study (d0 ) in all the groups (A, B, C and D) and in Group B on d<NUM>. The faecal samples for the count of the specific groups of bacteria in the intestinal flora (about <NUM> grams) were collected from the volunteers in sterile plastic containers previously filled with <NUM> of Amies liquid transport medium (BD Italy, Milan, Italy), kept at <NUM> at the volunteer's home and delivered to the laboratory within <NUM> hours of collection.

The gastric juice and duodenal brushing material were collected during a gastroscopy carried out on patients who had been fasting for <NUM>-<NUM> hours. The gastroscopies were performed at the Gastroenterology Department of the Ospedale Maggiore della Carità at Novara. The samples of brushing material (about <NUM>-<NUM> grams) were conserved in sterile plastic containers previously filled with <NUM> of Amies liquid transport medium (BD Italy, Milan, Italy). All the samples were kept at <NUM> and delivered to the laboratory within the <NUM> hours following their collection.

The samples were analysed as soon as they were received by the laboratory and in any event within <NUM> hours of collection. The samples were weighed and transferred to a sterile container (Stobag), diluted <NUM>:<NUM> weight/volume with Amies medium, and homogenised with a Stomacher apparatus for <NUM> minutes at <NUM> rpm. The samples were subjected to a serial decimal dilution using <NUM> of a saline solution in each dilution (<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM> for the counts of total vital cells and total cells of Lactobacillus). The samples were plated on specific agar culture mediums. In Group D, the dilutions from <NUM>-<NUM> to <NUM>-<NUM> were plated because the bacterial counts were expected to be significantly lower than those of other groups. The non-selective culture medium LAPTG was used for total vital cells, while the selective count of the total Lactobacillus was performed by means of the culture Rogosa Acetate Agar (Oxoid, Milan, Italy). All the plates seeded with lactobacilli were incubated for <NUM> to <NUM> hours at <NUM> in anaerobic conditions (GasPak) with an Anaerocult kit (Merck, Darmstadt, Germany), while the plates with LAPTg were incubated in aerobic conditions for <NUM> to <NUM> hours at <NUM>. The species-specific PCR assay was conducted on an extract of total genomic DNA obtained from the samples of gastric juice processed and from the duodenal brushing material, with the object of verifying and quantifying the presence of the probiotic bacteria administered to the volunteers. In particular, the primers used were as follows: L. rhamnosus (Rha/PRI), L. pentosus (PENT f/PLAN f/pREV), L. plantarum (LFPR /PLAN II), and L. delbrueckii subsp. delbrueckii (Ldel7/Lac2). The quantification of the total population of bacteria and the total of lactobacilli in the gastric juice and in the duodenal brushing material, and also the species-specific PCR assay, were conducted at the Biolab Research Srl Laboratory at Novara, Italy.

The samples were examined as soon as they reached the laboratory. The samples were weighed (about <NUM> grams) and transferred to a sterile container (Stobag), diluted with Amies liquid to obtain a <NUM>:<NUM> weight/volume dilution and were subsequently homogenised in a Stomacher apparatus for <NUM> minutes at <NUM> rpm. The samples were then subjected to a serial decimal dilution using a sterile saline solution and <NUM> of the appropriate dilution (<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM> for total coliforms, Escherichia coli and enterococci; <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM> for the yeasts and moulds). The samples were plated on agar culture mediums. The Enterococci were counted using Slanetz-Bartley (SB) agar (Oxoid, Milan, Italy); total coliforms and Escherichia coli were counted on Petrifilm CC (<NUM>, Segrate, Milan, Italy) and on Chromo IDCPS (BioMerieux, Florence, Italy), the total yeasts and the moulds on Yeast Extract Dextrose Chloramphenicol (YGC) agar (Sigma-Aldrech, Milan, Italy). The Enterococci, the total coliforms and the Escherichia coli were incubated in aerobic conditions at <NUM> for <NUM> to <NUM> hours, while the yeasts and moulds were incubated in aerobic conditions at <NUM> for <NUM> to <NUM> hours.

Quantification of the microbial groups listed above in the faecal samples was executed at the Biolab Research Srl Laboratory in Novara, Italy.

All the values obtained on the concentration of the total bacterial population and on total lactobacilli in the gastric juice and in the duodenal brushing material are expressed as the average of the number of vital cells per ml or per gram of sample ± the average standard error (m ± SEM). All the values relating to the concentration of specific faecal microbial groups are expressed as the average number of vital cells/gram of faeces ± standard error of the average (m ± SEM). The paired or independent t-tests of the statistical analyses were used to evaluate the results and compare them between d<NUM> and d<NUM> in group B (paired) and d<NUM> between the various groups (independent). In particular, the results of Group A were compared with Groups B, C, and D at d<NUM> (baseline). The differences were considered significant with p ≤ <NUM>.

All the <NUM> individuals were subjected to gastroscopy at time zero (d<NUM>), while Group B was also subjected to gastroscopy at the end of supplementation with probiotics (d<NUM>), No withdrawals were recorded, as the preparation had been very well tolerated and accepted by each participant in Group B, the only one which received probiotic supplements between d<NUM> and d<NUM>,.

The results regarding the total bacterial cells and the total Lactobacillus in the gastric juices and in the duodenal brushing material are shown in Table <NUM>.

It is interesting to note that a significant reduction in the total bacterial parameters is present at d<NUM> in Group B in comparison with the baseline (Table 1c).

The results of the species-specific PCR assay in Group B at d<NUM> compared with d<NUM> further confirmed the presence of the four species of probiotics administered. A general panorama is shown in Table <NUM>.

In the gastric juice, L. plantarum and L. delbrueckii subsp. delbrueckii were the two most representative species since <NUM> and <NUM> individuals, respectively, out of a total of <NUM> individuals were positive compared with <NUM> and <NUM> at the baseline (d<NUM>). In the duodenal brushing, L. plantarum and L. rhamnosus were present in <NUM> and <NUM> subjects, respectively, out of a total of <NUM> subjects compared with <NUM> and <NUM> at the baseline (d<NUM>).

The results on total Enterococcus, total coliforms, Escherichia coli, yeasts and moulds in the faecal samples are shown in Table <NUM>.

The study confirmed a significant bacterial growth in the upper gastro-intestinal tract in subjects who had been taking PPIs for more than <NUM> consecutive months (p=<NUM> and p=<NUM> for total bacteria in the gastric juice and in the duodenal brushing material, respectively, in Group A versus Group D which represents the general population; similar statistical results were found from the comparison off Group B and Group D in the same way). Comparison between groups A and C (subjects treated with PPIs for a period of from <NUM> to <NUM> months) demonstrated statistical significance in <NUM> out of <NUM> parameters. In this way, the duration of the PPI treatment is a factor which can determine the degree of bacterial proliferation in the upper gastrointestinal tract. The individuals treated in the short term seem to be more similar to the general population rather than to subjects who had undertaken long-term treatment with PPIs.

An interesting aspect refers to the higher percentage of total Lactobacillus in the gastric juice of subjects treated in the short term (<NUM>%, <NUM> log<NUM> CFU/ml in Group C) compared with subjects treated long-term (<NUM>%, <NUM> log<NUM> CFU/ml in Group A; <NUM>%, <NUM> log<NUM> CFU/ml in Group B). This higher concentration, however, does not reflect the results of the duodenal brushing (<NUM>%, <NUM> log<NUM>CFU/ml in Group C).

The administration of the <NUM> strains of bacteria listed above, i.e. L. rhamnosus LR06, L. pentosus LPS01, L. plantarum LP01 and L. delbrueckii subsp. delbrueckii LDD01, including <NUM> of NAC for <NUM> days was sufficient to significantly change the typical bacterial growth in the subjects treated with PPIs for more than <NUM> months, so as to restore a protective barrier against possible pathogens of dietary origin (p=<NUM> and p=<NUM> for the total of bacteria in the gastric juice and the duodenal brushing material, respectively, in Group B at d<NUM> compared with d<NUM> , Table 4c.

Another interesting result was the percentage of total bacteria represented by lactobacilli in the various groups. In control subjects who were not taking PPIs, the bacteria belonging to the genus Lactobacillus represent about <NUM>% of the total of the gastric microflora, while in patients treated with PPIs for more than <NUM> months, lactobacilli represented only about <NUM>% of the total bacteria, suggesting therefore that the great majority of the gastric micro-organisms were composed of other, potentially harmful, microbial groups. At the end of the period of supplementation by probiotics (d<NUM> ) in Group B, lactobacilli constituted <NUM>% of the total bacteria in the gastric juice, and an increase in their concentration compared with time zero was recorded, although it is not statistically significant (p = <NUM>). The lack of statistical significance could be explained in the light of the significant parallel reduction in total gastric bacteria (<NUM> log<NUM> CFU/ml compared with <NUM> log10 CFU/ml, p = <NUM>). On the other hand, the percentage of Lactobacillus in the duodenal brushing material was significantly higher at d<NUM> compared with the baseline (p = <NUM>).

The results of the species-specific PCR assay, furthermore, confirmed the capacity of the probiotics administered together with NAC to effectively colonise the gastric lumen and the duodenal mucosa in the subjects treated with PPIs for more than <NUM> consecutive months (Tables 5a and 5b). This aspect may help to inhibit and replace the possibly harmful pathogens bacteria or indeed those which are commonly present in subjects treated long-term with PPIs. This datum is more significant if it is considered that the gastroscopies were all executed at least <NUM> hours after the last time that probiotics had been taken, thus demonstrating the capacity of these beneficial bacteria to persist significantly in the stomach and on the surface of the duodenal mucosa. NAC was used for its mechanical effects against bacterial biofilms, in order to prevent a possible new formation of biofilms in subjects undergoing long-term treatment with PPIs.

The results of the faecal samples demonstrated, on the one hand, a significant increase in all the microbial parameters taken into consideration in the individuals treated with PPIs for a period of at least <NUM> months (comparison between Groups A and D): p=<NUM>, p=<NUM>, p=<NUM>, p=<NUM> and p=<NUM> for Enterococcus spp. , total coliforms, E. coli, yeasts and moulds, respectively). In any case, a short-term administration of PPIs, from <NUM> to <NUM> months, was sufficient to induce a significant faecal increase in all the five parameters, although the statistical significance was lower (see data for Group C compared with D: p=<NUM>, p=<NUM>, p=<NUM>, p=<NUM>, and p=<NUM>, respectively) (Table <NUM>). On the other hand, the statistical comparison between the subjects PPI treated long-term and short-term was significant only for the yeasts and moulds (p=<NUM> and p=<NUM>, respectively), thus suggesting that for Enterococcus spp. and for Gram-negative bacteria, taking minimal quantities of PPIs for three months is sufficient to mediate the majority of the increase observed after <NUM> months of treatment. Yeasts and moulds very probably need more time to colonise the intestinal flora after the alteration of the gastric barrier, since a significant additional increase was recorded in long-term subjects compared with short-term subjects (Group A compared with Group C).

The total coliforms usually represent about <NUM>% of the total population of human faecal bacteria in concentrations of around <NUM><NUM> bacteria per gram (<NUM>). Another interesting result is the percentage of total coliforms constituted by Escherichia coli. It is known, in fact, that this bacterium represents the majority of the total population of coliforms in the human intestine, generally amounting to <NUM>-<NUM>% (<NUM>). The total coliform bacteria present in the human intestine are made up of four genera of the family of the Enterobacteriaceae, in particular Escherichia, Klebsiella, Enterobacter and Citrobacter, with Klebsiella normally amounting to about <NUM>% and Enterobacter /Citrobacter spp. representing together about <NUM>%. The results for Group D substantially confirmed this evidence, since <NUM>% of total coliforms was made up of E. In the subjects who had undergone long-term treatment with PPIs, however, this percentage was reduced to <NUM>% (Group A) and to <NUM>% (Group B), thus suggesting an abnormal excessive growth of the genera Klebsiella and/or Enterobacter/Cítrobacterin the intestine as a consequence of the destruction of the gastric barrier. This increase could be considered harmful since some species such as Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter cloacae could exert significant pathogenic action on the host, ranging from hospital infections of the blood (BSI) through to acute appendicitis and antibiotic-associated haemorrhagic colitis (AAHC).

The Enterococcus spp. are normally present in human faeces in concentrations from <NUM><NUM> to <NUM> bacteria per gram. The data obtained on the control population confirmed this evidence, as <NUM> log<NUM>CFU/ml were counted in the faecal samples. Long-term treatment with PPIs caused a significant increase in this microbial genus in the human intestine (<NUM> log<NUM> CFU/ml in Group A and <NUM> log<NUM> CFU/ml in Group B).

The most important question represented by Enterococcus spp. , in particular by Enterococcus faecium, is their intrinsic antibiotic resistance, specially towards penicillin and vancomicin. The enterococci are the third most common cause of infective endocarditis, and the effect of tolerance to penicillin on therapeutic results has been evident since the end of the <NUM>. In any case, epidemiological studies have demonstrated that the strains of E. faecium associated with nosocomial infections, including endocarditis, are types of sequences different from the commensal strains which colonise the gastrointestinal tract of healthy human beings, even though the possibility cannot be excluded that some harmful biotypes may have colonised the human bacterial flora of the subjects treated with PPIs.

The complex analyses of the faeces at baseline time confirmed the weakening or indeed the complete interruption of the gastric barrier effect, since the composition of the intestinal flora showed that it is profoundly modified in persons who take PPIs for at least three months. Gram-negative bacteria, such as total coliforms and Escherichia coli, were significantly higher than in the controls, while yeasts and moulds increased by about <NUM> log<NUM>. Faecal Enterococci were up by more than <NUM> log<NUM>. It is also interesting to note the correlation between the duration of taking PPIs and the size of the faecal increases in the five microbial groups analysed, chosen as evidence of a potential dysmicrobism.

The four probiotics studied in association with NAC were able to reduce all the faecal parameters (p=<NUM>, p=<NUM>, p=<NUM>, p=<NUM>, and p=<NUM> for Enterococcus spp. , total coliforms, E. coli, yeasts and moulds, respectively, at d<NUM> compared with the baseline value). In particular, the reduction in total coliforms, E. coli, yeasts and moulds was more than one log10 after <NUM> days of supplementation with the probiotics. At the end of the supplementation with the probiotics in Group B, total coliforms and concentrations of E. coli were significantly lower than values found in the general population (Group D) (p=<NUM> and p=<NUM>, respectively), thus confirming the considerable antagonistic action of the probiotic bacteria against Escherichia coli.

In conclusion, the administration of an association of specific strains of L. rhamnosus LR06, L. pentosus LPS01, L. plantarum LP01, and L. delbrueckii subsp. delbrueckii LDD01, including also an efficacious quantity of N-acetylcysteine, is capable of significantly reducing bacterial proliferation at the level of the stomach and duodenum, reducing Gramnegative bacteria, Enterococcus spp. , yeasts and moulds in the intestinal flora after <NUM> days of oral supplementation, thus rapidly rebalancing its composition and restoring a protective barrier against harmful bacteria, especially at stomach level. N-acetylcysteine (NAC) was used because of its capacity to mechanically prevent the possible formation of a bacterial biofilm, and showed itself to be effective since the concentration of the various bacteria other than lactobacilli both in the gastric juice and in the samples from brushing the duodenum was significantly reduced.

All the probiotic strains used in this study have previously demonstrated a significant antagonistic action in vitro on specific strains of Escherichia coli, among them the enterohaemorrhagic serotype O157:H7, and could therefore be used to effectively prevent infections mediated by these harmful or pathogenic microbes.

In the light of an actually more widespread use of PPIs, concomitant oral supplementation with probiotics and NAC as used in this pilot study represents an innovative strategy capable of restoring, at least partially, a normal gastric barrier effect, thus reducing the threat of gastrointestinal infections of dietary origin in a large part of the population with reduced intragastric acidity.

Claim 1:
A pharmaceutical or dietary composition or a supplement or a medical device for use in the treatment of a subject who is taking a proton pump inhibitor (PPI) to reduce or treat gastric hyperacidity, which comprises at least one strain of bacteria selected from:
- Lactobacillus pentosus LPS01 DSM <NUM>;
- Lactobacillus rhamnosus LR06 DSM <NUM>; and
- Lactobacillus delbrueckii subsp. delbrueckii LDD01 (MB386) DSMZ <NUM> DSM <NUM>,
wherein said treatment is the preventive and/or curative treatment of infections and/or pathologies caused by pathogenic gram-negative bacteria E. coli serotype O157:H7,