KR20150003908A - Applications of alpha-ketoglutarate for manufacturing organic biofuel - Google Patents

Abstract

The present invention relates to the use of an undesirable medical condition associated with the presence and / or activity of urea-degrading bacteria in living organisms including humans, pets and farm animals, such as mammals, birds, amphibians, fish, mollusks or arthropods Wherein said undesirable medical condition is selected from the group consisting of the following bacteria in the gastrointestinal tract, respiratory system and / or genitourinary system: < RTI ID = 0.0 > Is a disease associated with the presence and / or activity of urea-degrading bacteria: Helicobacter pylori), brucellosis (Brucella) genus (genus) bacterial strains, Urea plasma urea duck tikum (Ureaplasma ureolyticum) and urease from the same-positive bacteria and other bacteria arc alkali, such as Bacillus Pas toeri (Bacillus pasteurii ), urease-producing enterococci ( Yersinia enterocolica ) is a urea-degrading bacterium that forms biofilms on bacilli, catheters, and other medical equipment and is responsible for the mineralization of sediments, such as urea-degrading bacteria, oral mucous membrane infections, gum disease and dental caries, , Proteus (Proteus), urea plasma (Ureaplasma), Klebsiella (Klebsiella), Pseudomonas (Pseudomonas), Stability Philo nose kusu (Staphylococcus), Providencia (Providencia), Corynebacterium (Corynebacterium) in, in particular blood. Urea-degrading bacteria that cause the formation of infectious stones in the process of urinary mechanical infection of P. mirabilis , and mycoplasma, mycoplasma, which is mycoplasma, hominis ) and oil. U. urealyticum . The invention also relates to the formation of colonies by urea-degrading bacteria which are harmful in living organisms including humans, pets and farm animals, such as mammals, birds, amphibians, fish, mollusks or arthropods, Dietary supplements, medicated food and food / feed additives containing alpha-ketoglutarate useful in preventing and inhibiting the formation of colonies of H. pylori in E. coli. In addition, according to the present invention, alpha-ketoglutarate is used not only in the methods of preventing and treating the above-mentioned diseases and diseases associated with urea-degrading bacteria, but also in converting biomass containing lignin and cellulose by bacterial enzymes The present invention relates to a process for producing organic biofuels comprising the steps of: (a) preparing an organic biofuel, (b) preparing an organic biofuel, 0.0 > rutarate. ≪ / RTI >

Description

The use of alpha ketoglutarate for the manufacture of organic biofuels {APPLICATIONS OF ALPHA-KETOGLUTARATE FOR MANUFACTURING ORGANIC BIOFUEL}

The present invention is used for the prevention and treatment of undesirable conditions in living organisms including humans, plants and animals, especially pets and/or farm animals, i.e. mammals, birds, amphibians, fish, mollusks or arthropods Novel pharmaceutical uses of alpha-ketoglutarate, including the application of alpha-ketoglutarate in the manufacture of therapeutic and prophylactic formulations, and the use of alpha-ketoglutarate in the therapeutic and prophylactic treatment of living organisms It is about.

Alpha-ketoglutarate-salt of alpha-ketoglutaric acid

Alpha-ketoglutarate occurs as an endogenous molecule in living organisms.

Salts of alpha-ketoglutaric acid have been known for more than 60 years since the discovery of the Krebs cycle. Endogenous alpha-ketoglutaric acid salts play a fundamental role in human and animal bodies, along with salts of oxaloacetic acid and pyruvic acid in the citric acid cycle. By reproducible reactions, fatty acids, sterols, cholesterol (by association of citrate), porphyrin, heme, chloroporphyll (activation of succinyl-CoA), glutamate, amino acids, nucleotide bases (alpha-ketoglu) The activity of the salt of taric acid) is synthesized.

The alpha-ketoglutaric acid anion plays an important role in metabolism, primarily in aerobic organisms. Alpha-glutarate is produced in the process of oxidative decarboxylation, which is implicated in the cellular enzyme of isocitrate dehydrogenase, and is also produced by glutamate dehydrogenase in another metabolic pathway. It is produced by oxidative deamination of catalyzed glutamate.

In the basic life cycle, aka the tricarboxylic acid cycle, i.e., the Krebs cycle, the intermediate compound alpha ketoglutarate is subject to continuous conversion within the cell, which is somewhat in the form of an anion in the peripheral blood due to its total metabolism in the organism. It is present in low concentration.

In organisms, alpha-ketoglutarate also acts as a natural scavenger-decontaminant through nitrogen transport by transamination with the effect of transporting amino groups originating from catabolic amino acids. Do it. This process occurs in the liver, which is referred to as the ornithine cycle or urea cycle.

During the aminotransmission of alpha-ketoglutarate and glutamine, a neurotransmitter called glutamate is formed. In the presence of the B6 vitamin, glutamate is an inhibitor of glutamate neurotransmitters and can be decarboxylated to produce a compound called GABA (gamma-aminobutyric acid) that blocks neurotransmitters.

It has also been found that the alpha-ketoglutarate enzyme is involved in the removal of free radicals from living organisms—incompletely metabolized products and highly toxic compounds.

In addition, one of the alpha-ketoglutarate-dependent oxygenases is a molecular oxygen sensor, i.e. an indicator of the level of oxygen in the environment.

Alpha-ketoglutarate can also be introduced into organisms via known routes of administration, such as oral, inhaled, intravenous or other routes.

The daily diet of humans and animals does not contain alpha-ketoglutarate.

On the market, especially in the U.S., salts of alpha-ketoglutaric acid, primarily arginine, pyridoxine, ornithine, creatine, as ready-made products aimed at both humans or household animals. There are various commercially available dietary supplements containing salts of, histidine and citrulline. Also available are sodium, potassium and calcium salts of alpha-ketoglutaric acid.

In the Dietary Supplement Register listed by the National Nutritional Foods Association (NNFA), alpha-ketoglutaric acid itself and alpha-ketoglutaric acid in combination with pyridoxine (vitamin B6) were listed before September 15, 1994. It was also listed as a biochemical product group. Such long-term presence in the market has led to a number of dietary supplements containing these compounds. The main reason for the existence of derivatives or salts of alpha-ketoglutaric acid salts in all commercially available products is that alpha-ketoglutarate as an intermediate compound in the Grebs cycle is one of the substances responsible for cellular respiration, thereby This is because it is believed to improve the quality of life.

The most diverse group of products discussed herein are those that contain L-arginine along with alpha-ketoglutarate. These products, according to the manufacturer, promote the retention of energy during and after physical activity, improve nitrogen oxide synthesis, and, in addition, along with nitrogen oxides, they increase the level of such oxides in living organisms and nutrients. It not only promotes the transport of blood, but also promotes metabolism in the muscles. Along with other substances, they also increase energy levels and promote amino acid metabolism.

The next largest group of commercially available products contains ornithine along with alpha-ketoglutaric acid. In this form, alpha-ketoglutarate not only increases energy production, but also protects the muscle against the breakdown of branched amino acids to produce glutamine, an energy boosting molecule. In addition, it is a compound that enhances the secretion of growth hormone and optimizes muscle metabolism. This increases the activity of insulin and polyamines in a stable manner. It also promotes neurotransmission, helping to maintain a good mental state of the organism, supporting the endurance of the muscles to maximize the exercise effect, affecting the fat burning ability of the organism, increasing libido, and , Has a beneficial effect on immunological function and reduces oxygen stress.

Another group of food formulations used as dietary supplements include pyridoxine and pyridoxyl in combination with alpha-ketoglutarate. The ingredients in these products promote metabolic activity in cells, balance the organism's effort to generate energy, and protect the liver.

There are also products on the market that contain creatine along with alpha-ketoglutarate. They act as glutamine precursors and are involved in the synthesis of proteins.

Others, i.e., non-specific alpha-ketoglutarate, stimulate fat loss in living organisms and are essential to ensure the integrity of muscle tissue.

On the other hand, alpha-ketoglutaric acid itself is a component of another type of agent, i.e., a component of agents exhibiting natural detoxifying function, recommended for use in metabolic deficiencies and chronic fatigue, often diagnosed by amino acid analysis. Administration of this type of agent increases stamina and enhances energy. Interesting products of this group are the calcium and magnesium salts of al-ketoglutaric acid. Due to the fact that alpha-ketoglutaric acid belongs to the group of strong organic acids, it irritates the esophagus and stomach when administered orally. The use of calcium and magnesium allows the production of buffered bi-component alpha-ketoglutaric acid compounds that are unpleasant and do not cause excessive acidity sensation.

A number of patents and patent applications indicate that alpha-ketoglutarate can be administered for metabolic brain dysfunction, disorders of the nervous system, blood circulation and skeletal muscle system, or to enhance cellular mitochondrial function.

Drinking water containing alpha-ketoglutaric acid salts are also available, especially for energizing organisms before, during and after physical activity. These beverages as an energy source can also be administered in humans and other mammals under fasting and long-lasting energy demands.

In addition, alpha-ketoglutarate can be used as a non-steroidal anabolic product that increases muscle mass without converting muscle to fat. The effects of these dietary supplements are similar to those obtained with synthetic anabolic steroids, but there are no undesirable side effects.

Supplements comprising thiamine, lipoic acid, creatine derivatives and L-arginine along with alpha-ketoglutarate can also be administered orally. These dietary supplements are for lowering blood glucose levels and maintaining low glucose levels in the course of treatment of diabetic neuropathy, as well as improving blood circulation and muscle efficiency.

Alpha-ketoglutaric acid salts have been found to have interesting applications as agents aimed at reducing nitrogen release in humans and animals and also maintaining protein synthesis in food microbiology.

In the food industry, these salts are used as ingredients to improve and enhance the aroma and taste of fermentation products (eg vinaigrette) and dairy products, especially cheese. The alpha-ketoglutaric acid salt affects lactic acid bacterial fermentation, thereby altering the metabolism of amino acids, the level of catabolic metabolites, and the activity of aminotransferases. In practice, the alpha-ketoglutaric acid salt shortens the cheese ripening period by accelerating the formation of compounds that ensure high commercial quality of food products. Reference: [Williams AG, Noble J, Banks JM., The effect of alpha-ketoglutaric acid on amino acid utilization by nonstarter Lactobacillus spp. isolated from Cheddar cheese. Lett. Appl. Microbiol. 2004;38:289-295].

A number of patents and patent applications relate to the use of alpha-ketoglutarate as a pharmaceutical preparation or as a component of a pharmaceutical preparation.

The use of alpha-ketoglutaric acid, glutamine, glutamic acid and salts thereof as well as amides and dipeptides and tripeptides as pharmaceutical preparations for the treatment and prevention of arthrosis, rheumatoid arthritis and cartilage damage due to inflammation and other reasons are disclosed in Publication WO 2007/058612 is known.

Publication No. WO 2005/123056 discloses alpha-ketoglutaric acid, glutamine, glutamic acid as pharmaceutical preparations, food and animal feed additives in the treatment and prevention of excess plasma levels of one or more of the parameters of cholesterol, LDL and glycerides. And their pharmaceutically acceptable salts, amides and dipeptides and the use of tripeptides. Formulations can also be used to elevate HDL levels.

EP 0 922 459 discloses alpha-ketoglutaric acid with predetermined amounts of D-galactose and ornithine in the form of tablets, powders, infusions and syrups, and the alpha-keto as calcium, potassium, magnesium, zinc and calcium salts. It has been described that salts of glutaric acid can elevate the amino acid profile in the blood, especially in patients in metabolic stressful situations. The described agents can be used for the treatment of liver diseases in alcoholism, and for the treatment and prevention of liver diseases to maintain the function and structure of the liver and to regenerate the liver.

In Publication No. WO 2006/016143, formulations consisting of alpha-ketoglutarate and a number of derivatives thereof previously used to activate HIFa hydroxylase to increase alpha-ketoglutarate levels are currently in cancer and It is mentioned that it is used to treat angiogenesis.

According to published publication WO 2006/062424, 3-hydrogen together with alpha-ketoglutarate and a number of derivatives thereof in the process of growth and mineralization of the skeleton under physiological conditions and in the process of osteochondrosis in adult humans and animals. It is possible to use oxy-3-methylbutyrate. The same product can be added to functional foods and drug-added foods.

Publication No. WO 2006/016828 discloses pharmaceutical preparations that improve the function of nerve cells and the entire nervous system, minimize and prevent apoptosis of nerve cells, and protect against diseases of the nervous system in adults and fetuses, and also food and animal feed additives. The use of alpha-ketoglutarate and a number of derivatives thereof is described.

Publication No. WO 2007/082914 discloses diseases and disorders of the gastrointestinal tract associated with low levels of alpha-ketoglutaric acid (AKG) in humans or animals, for example, in particular Helicobacter pylori ( Helicobacter pylori ) related gastritis, gastric and duodenal ulcers, peptic ulcers, gastric cancer and gastric mucosa related lymphoid tissue lymphoma. Humans or animals with levels of AKG lower than the normal average AKG levels should be treated with a pharmaceutical preparation or food or feed supplement comprising AKG, its derivatives, metabolites, derivatives or salts. In the above publication, animals with low blood levels of AKG of less than 0.1 ug/ml in the control group of test animals did not survive the experiment, whereas similar initial blood levels of AKG provided with a feed supplemented with _{Na 2} AKG*2H _{2 O.} Some results confirming that the test animals having a survived the experiment are described. However, no evidence was provided demonstrating that the test animals were infected with H. pylori. Thus, there is no evidence to show that low levels of AKG are in some way related to the H. pylori related diseases and disorders mentioned above. The data provided as an example of the correlation between AKG blood levels and various diseases of different age groups in humans are the health status of each individual patient on the one hand and the low blood levels of AKG on the other and age, sex, weight, and Failure to provide a sufficient amount of data relating to a particular disease or correlation between disorders was due to the fact that the relevant data were different for each test patient. It is important that none of the patients tested were classified as exhibiting severe signs of any one of the diseases mentioned in the above study, and that none of the patients tested were classified as displaying any more or less severe symptoms than the “mild” signs of gastritis. In addition, no evidence of H. pylori colonization was provided in the patient's gastrointestinal tract, even in patients showing "light" signs of gastritis. In addition, there is no mention of how much pathogenic strains of H. pylori have been identified in this publication. The experimental data only indicates that the range of physiological AKG blood levels is very broad and does not necessarily indicate any of the diseases listed in this publication. Based on the test results obtained in this small patient, the conclusion was drawn that statistical analysis could not provide any reliable data.

Pyridoxine alpha-ketoglutarate is known as an agent used in the prevention of acidosis in human medicine and veterinary medicine, prevention of all diseases that cause acidosis, as well as the prevention of beds in which medicines that reduce the level of lactic acid in the blood are used. have.

Alpha-ketoglutarate is also used as an antidote in medical practice in case of poisoning of organisms. The detoxification action of alpha-ketoglutarate has been used, for example, in the treatment of cyanide poisoning. Alpha-ketoglutarate as an anti-toxic agent prevents muscle catabolism after surgery, which is also used in patients recommended for parenteral feeding in hospitals, and alpha-ketoglutarate is one of the compounds of the applied bolus. Alpha-ketoglutarate is also recommended for patients suffering from cerebrovascular stroke, patients with burn wounds, patients with hypoxia, and patients with X-ray irradiation, as well as patients with cataracts resulting from selenite poisoning.

Alpha-ketoglutarate in combination with ornithine effectively protects organisms after small intestine transplantation against any translocation of bacteria as tested in mesenteric lymph nodes, liver and spleen. See: [de Oca J, Bettonica C, Cuadrado S, Vallet J, Martin E, Garcia A, Montanes T, Jaurrieta E. Effect of oral supplementation of ornithine-alpha-ketoglutarate on the intestinal barrier after orthotopic small bowel transplantation. Transplantation. 1997;63:636-639].

In rats in experimentally induced post-traumatic conditions, administration of ornithine-alpha-ketoglutarate reduces the spread of E. Coli and tissue destruction after LPS. It is assumed that administration of the product in post-traumatic humans can prevent sepsis and its consequences. Reference: [Schlegel L, Coudray-Lucas C, Barbut F, Le Boucher J, Jardel A, Zarrabian S, Cynober L. Bacterial dissemination and metabolic changes in rats induced by endotoxemia following intestinal E. coli overgrowth are reduced by ornithine alpha-ketoglutarate administration. J Nutr. 2000;130:2897-2902].

Alpha-ketoglutarate is also used in animal breeding to improve amino acid absorption. It is administered to piglets to accelerate the absorption of iron ions.

Urea-degrading bacteria

The range of nitrogen assimilation bacteria ranges from non-pathogenic convenient symbiotic bacteria such as cutaneous colony-forming bacteria and non-pathogenic symbionts living in the mucous membrane of the gastrointestinal tract to pathogenic bacteria including Helicobacter pylori and bacteria that cause urogenital infections.

The most common infection caused by urea-degrading bacteria is H. pylori infection.

A common feature of urea-degrading bacteria is the ability to use urea mainly present in the bacterium's environment as a source of nitrogen necessary for survival by urease. Bacterial urease (urea aminohydrolase EC3.5.1.5) is a nickel-dependent polymer composed of 2 or 3 subunits. The 3D crystal structure of some bacterial ureases has been revealed (H. pylori, Klebsiella aerogenes (Klebsiella aerogenes), Bacillus Paz toeri (Bacillus pasteurii )). The high degree of similarity in the amino acid sequence is that all types of ureases are derived from one parent protein, which probably have a similar three-dimensional structure, and they retain their catalytic activity while hydrolyzing urea to ammonia and carbon dioxide. Show.

Examples of nitrogen-assisted bacteria that use their own urease are Streptococcus salivarius, which are common in the oral cavity and form a biofilm. salivarius ) and Actinomyces naeslundii .

The gastrointestinal tract has the greatest concentration of urea-degrading bacteria. Microorganisms, including urea-degrading microorganisms, permanently colonize the epithelial surface, and they are recognized as natural gut microbiota. This means that access to urea is one of the important factors of the microbial ecological environment in the gastrointestinal tract, that is, access to urea affects the quantity and quality of bacteria in the area. In maintaining tissue integrity, access to urea is one of the health conditions of macroorganism. The same mode of homeostasis governs the development of body surface colony-forming microorganisms.

Urea is also important as a substrate for the pathogen's urease in the stabilizing phase of inflammation during H. pylori infection.

A common route of entry of pathogens into human and animal organisms is through the gastrointestinal tract with food, regardless of where the infection further develops. Infection of urea-degrading microorganisms through the gastrointestinal tract indicates that urease plays a role in the pathogenesis of the infection. Bacterial strains, for example bacterial strains of the urease-producing Brucella strain, have been demonstrated to be resistant to the bactericidal action of gastric juice with strong acidic conditions in a urea environment. The urease negative mutation of the bacteria, on the other hand, is sensitive to the condition, which reduces the number of bacteria after passage through the stomach. Under the above conditions, urease may include protecting Brucella against the acidic effect of gastric juice when microorganisms enter the organism through the oral cavity. Upon crossing the gastric barrier, bacteria are freed, for example, to invasion of the respiratory and genitourinary systems, which cause symptoms common for brucellosis.

Although not a major etiological factor in urinary system infections in healthy organisms, urea-degrading bacteria are often associated with infections in people with urinary system disorders. The result of the urinary system infected with urease-producing microorganisms is staghorn calculus, which accompanies pathological processes in the kidney as well as supersaturation of urine with magnesium ammonium phosphate (struvite) and calcium phosphate salts. Under physiological conditions, urine does not contain these salts.

Another interesting mechanism in the pathogenesis of urogenital infections is urease-positive bacteria, such as Ureaplasma ureoliticum (Ureaplasma). ureolyticum ) and other alkaline bacteria such as Bacillus pasteurii . In a urea environment, the pathogen uses urealysis to produce its ATP, whereby the pathogen reproduces permanently. Although U. urealiticum and other mycoplasmas are relatively rare, they can also pose risks and make the treatment of infections of the respiratory system difficult in humans and animals, including fish.

Yersinia enterocolica, which produces urease, a gastrointestinal pathogen ( Yersinia enterocolica) can cause reactive arthritis in several genetically impaired people, and it has also been found that its reactivity is related to the chemical structure of the enzyme, particularly its subunit UreB.

It should be recognized that many of the movable bacteria are urea-degrading organisms responsible for the formation of biofilms and mineralization of sediments on catheters and other mechanical medical implements.

Urea metabolism is also thought to be associated with infections within the oral mucosa, including gum disease, dental caries, and the formation of tartar.

Formation of infectious stones (infectious stone) is often associated with infection urinary system caused by the following bacteria genus of: Proteus (Proteus), Urea plasma (Ureaplasma), Klebsiella (Klebsiella), Pseudomonas (Pseudomonas), Stability Philo nose kusu (Staphylococcus), Providencia (Providencia), Corynebacterium (Corynebacterium). The most common cause of infectious stone formation is P. mirabilis bacteria. Another cause of the formation of these infectious stones is mycoplasma, which is commonly reported to be associated with infections of the reproductive tract, particularly of the lower reproductive area, mainly vaginal infections. From the male urethra, the following microorganisms are isolated: Mycoplasma hominis hominis ) and U. urealiticum. Colonization of the genitourinary system in both women and men by bacteria leads to an infection of the urogenital system accompanied by the formation of infectious stones in the bladder.

H. pylori infection

H. pylori rodents in humans are usually isolated from the stomach and duodenum. The gram negative spiral bacterium, formerly known as Campylobacter pylori , is one of the etiological factors of gastritis and ulcer formation in the stomach and duodenum. In 1983, Warren and Marschall, who won the 2005 Nobel Prize, uncovered a causal relationship between the development of H. pylori in the gastrointestinal tract and chronic gastritis. Reference: [Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;1:1311-1315]. Due to the fact that long-lasting infection clearly increases the risk of intestinal-type adenocarcinoma, H. pylori rodents have been promulgated by WHO as carcinogenic factors in recent years (IARC. Schistosomes, liver flukes and Helicobacter). pylori . IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7-14 June 1994. IARC Monogr Eval Carcinog Risks Hum. 1994;61:1-241.). In addition, a correlation has been found between H. pylori infection and pathologies in tissues and non-gastric organs, such as the heart.

For a long time, the fact that the pH is too low for the growth of most microorganisms makes permanent colonization of the mucous membrane of the stomach difficult, so the stomach was thought to be free of any concerns of bacterial infection. However, the urease produced by H. pylori makes it possible for these organisms to form permanent colonies in the stomach and intestines, and urea breakdown is a major factor in causing the pathology associated with H. pylori infection. The urease-deposited outer surface of bacterial cells can constitute up to 20% of the bacterial protein. Enzymes protect bacteria against the acidic environment and prevent fatal destruction of the bacterial cell envelope. In addition, during H. pylori infection, ammonium ions released by urease during the process of urea degradation have a cytotoxic effect on gastric epithelial cells. In the presence of leukocytes and urea, bacterial urease generates monochloramine, which can induce DNA mutagenesis, one of the factors associated with the development of cancer in the chronic H. pylori infection pathway.

A novel species of urealytic spiral bacteria is being isolated from humans and animals. However, to date on how many different Helicobacter species occur in humans (e.g. H. heilmannii ), intestines (e.g. H. cinaed , H. canadene) Cis ( H. canadensis )), liver (e.g. H. hepaticus , H. bilis ), or systemic infections (e.g. H. fulorum) ( H. pullorum ) , Helicobacter spp. flexispira taxon 8 " F. rappini ") is not clear.

In recent years, it has been found that in laboratory animals the gastrointestinal tract (intestine, stomach, liver and pancreas) can be colonized naturally by bacteria of the genus Helicobacter, such as: H. Billis, H. ganmani , H. H. party kusu (H. hepaticus), H. mu room Florida (H. muridarum), H. Mars Tommy Linus (H. mastomyrinus), H. La pinion (H. rappini), H. Rhodes tium (H . rodentium), H. Flo tee your mouse (H. typhlonius). Animals of both wild and domestic animals permanently colonized by the above microorganisms do not show any symptoms of infection, and no inflammatory processes are found in the internal organs during the necropsy process. Microorganisms sensitive to environmental conditions continue to thrive due to the permanent horizontal transmission of bacteria from one individual to another through feces and saliva.

It has been found that H. pylori infection in humans is present in 50% of the population and may be related to the economic status and age of society. This is increasing in adults, including almost all groups in less developed countries. H. pylori is found in 90-100% of gastritis patients. Often, it is found that chronic infection turns into atrophic inflammation. In 10% of infected people, serious conditions developed. H. pylori infection is common in both children and adults. The most common infection occurs in children, and colonization of H. pylori in the gastric mucosa is maintained throughout life.

It is known that factors such as malnutrition, vitamin deficiency and smoking play a role in infection.

In 10% of patients, the conventional therapy applied is not effective due to the presence and increasing resistance of bacteria to standard drugs. Some patients have also been shown to be reinfected with drug-resistant bacteria. Another about 10% of patients are not resistant to formulations from the group of proton pump inhibitors. In these patients, side effects appear.

Due to the systemic declining efficacy of the treatment and its difficulties, the regional gastrointestinal association in Europe, following the recommendations of the report "Maastricht 2-2000", has implemented an appropriate diagnostic procedure to detect H. pylori , It is recommended that treatment be initiated only when certain disease symptoms occur.Examples of such disease symptoms include gastritis, gastric and duodenal ulcer, peptic ulcer identified during interviews, surgery due to peptic ulcer, precancerous deformity (atrophic Inflammation, metabolism, dysplasia), gastric resection due to early cancer, familial gastric cancer (up to second-generation blood relatives), gastric hyperplasia adenomastic polyp (after removal), MALT lymphoma, and long-term treatment with NSAID.

H. pylori infection in humans has been found to be accompanied by certain diseases such as peptic ulcer, gastric lymphoma, chronic atrophic gastritis with intestinal metaplasia, and gastric cancer.

In the diagnosis of Helicobacter infection, a general invasive test, and a non-invasive test that does not require testing of bioptate from the patient's gastric mucosa, are used in conjunction with endoscopy.

Testing of mucosal fragments allows the separation of microorganisms (selective growth medium) or the application of appropriate techniques (specific and non-specific staining) indicating the presence of H. pylori cells. Identification of H. pylori cells in the process of mucosal biopsy can be accomplished by both traditional microbiological methods (phenotyping, measuring drug resistance in isolates), and biochemical methods ( enzymatic activity of H. pylori -urease, catalase, oxidase). Generation) and molecular biological methods (PCR test, real-time PCR using primers specific for selected portions of bacterial DNA).

The most commonly used non-invasive methods include identification of serological tests and Helicobacter antigen-stool antigen tests, and urea levels in the air expelled by the patient in excess of values presumed to be standard urea concentrations. A urea breath test to detect is included. A typical serological test is an ELISA-type test that detects G-class antibodies specific for H. pylori bacteria.

In the first stage of H. pylori infection in humans, the inflammation of the mucous membranes progressing in the stomach and duodenum must be completely eradicated by pharmacological methods. Treatment of H. pylori infection is still based on the introduction of substances that reduce the secretion of gastric juice and raise the pH level in the stomach. Such substances include proton pump inhibitors, and certain antibiotics that kill bacteria, clarithromycin, amoxicillin and compounds-metronidazole (from the group of nitroimidazole derivatives).

In general, current treatments for diseases caused by ureolytic bacteria include appropriate antisense administered orally, intravenously, intravaginally, anus and externally in the form of pills, ointments, suppositories and powders. This includes the application of bacterial drugs. The selection of a therapeutically effective drug is determined after the following factors: the biochemical activity of urea-degrading bacteria, their resistance to antibiotics and chemotherapeutic agents (antibiograms) are determined, and after the cell envelope (e.g. For example, the structure of mycoplasma) is determined.

In Europe, the list of recommendations in the "Maastricht 2-2000" report published in 2002 contains any chemotherapeutic compounds that are substances other than nitroimidazole derivatives without the use of antibacterial means such as antibiotics. H. Pylori therapy is not included. These therapies are complex, expensive, sometimes very tolerated and not always effective. Treatment according to recommendations of regional gastrointestinal associations in Europe will be based on antibiotics and chemotherapeutic compounds (claritromycin (2 × 500) and amoxicillin (2 × 1 g) or metronidazole (2 × 500 mg), and proton pump inhibitors. I can.

For example,

First-line therapy. Treatment cycle for 7 days:

1. Drugs that reduce the secretion of gastric juice; Two doses of a compound belonging to the group of proton pump inhibitors (PPI), e.g., omeprazole 2 × day, 20 mg

2. Antibiotic I-for example, amoxicillin, 2 x 1 g per day

3. Antibiotic II-for example, clarithromycin, 2 × 0.5 g per day

Second-line therapy.

1. Drugs that reduce the secretion of gastric juice; Two doses of a compound belonging to the group of proton pump inhibitors (PPI), e.g., Ranzoprazole 2 × day, 30 mg

2. Antibiotic I-for example, amoxicillin, keep 2 x 1 g per day

3. Antibiotic II-other antibiotics or compounds, eg, metronidazole, 0.5 g per 2 x

4. Bismuth compound (citrate).

With the contagious spread of H. pylori in many parts of the world, there is an enormous and permanent need for agents that reduce infection-related pathologies; The pressure on the medical community to identify these formulations is also quite high.

The main object of the present invention is to control the urea-degrading microbiota of the intestine in humans and animals, especially dogs and cats, and other livestock, to regulate the urea-degrading microbiota of the oral cavity, and to control pathogenic urea through the digested stomach. It is to provide a novel formulation for the treatment and prevention of diseases caused by urea-degrading bacteria, which are used to inhibit the passage of degrading bacteria and prevent the formation of deposits and infectious stones in the urinary system.

The subject of the present invention is to control the undesirable growth of urea-degrading bacteria in living organisms including humans, plants and animals, especially pets and farm animals, such as mammals, birds, amphibians, fish, mollusks or arthropods. It is to provide a new formulation for.

A specific object of the present invention is to provide a novel formulation for the treatment and prevention of diseases caused by H. pylori.

The subject of the present invention is also a method for treating and preventing diseases caused by urea-degrading bacteria in humans and animals, especially dogs and cats, and other livestock, in particular a method for controlling urea-degrading microbial cells in the intestine, oral To provide a method of controlling urea-degrading microbial cells, a method of inhibiting the passage of pathogenic urea-degrading bacteria through the digested stomach, and a method of inhibiting the formation of deposits and infectious stones in the urinary system.

A further object of the present invention is to inhibit the growth of urea-degrading bacteria, in particular disease-causing ureaplasma and other mycoplasma in fish, in particular caused by urea-degrading bacteria in carp and carp fry and other freshwater and seawater fish. It is to provide a novel formulation for use as an agent for preventing gill inflammation.

Moreover, it is an object of the present invention to provide a novel formulation for preventing biofilm formation and mineralization of deposits on catheters and other medical equipment.

In addition, an object of the present invention is to provide a novel formulation for reducing calculus formation and inhibiting dental caries progression.

In addition, a further object of the present invention is the colonization of living organisms including humans, pets and farm animals such as mammals, birds, amphibians, fish, mollusks or arthropods by undesirable urea-degrading bacteria ( colonization), in particular to provide novel dietary supplements, special medicinal foods and food/feed additives that prevent and/or inhibit colonization in humans and livestock by H. pylori.

Given the beneficial effect of alpha-ketoglutarate on intestinal microbial cells, the subject of the present invention is also to produce organic biofuels based on the conversion of biological resources including lignin and cellulose by bacterial enzymes. It is to provide an improved way for you.

These objects and problems are achieved by providing a solution according to the invention as set forth in the appended claims.

The achievement of the above-mentioned tasks is the production of therapeutic or prophylactic pharmaceutical preparations or dietary supplements, special medicinal foods and food/feed additives or personal hygiene products for daily use, as stipulated in the appended claims. In order to do so, it is guaranteed by using alpha-ketoglutarate as the active substance in a therapeutically or prophylactically effective dose.

The therapeutically and/or prophylactically effective dose is in the range of 0.001 g to 0.2 g/kg body weight/day when administered intragastrically or orally. As far as topical topical administration is concerned, the effective dose is 0.01 to 10 g/m 2 /day of tissue surface.

To date, it has not been reported that salts of alpha-ketoglutaric acid can be effectively used in humans or animals to treat urealytic bacterial infections, including H. pylori infection.

The bioavailability of alpha-ketoglutarate, its well-studied activity in organisms, and the fact that these substances are approved for use in other medical and prophylactic applications contribute to a number of advantages of the present invention.

The present invention will be further described with reference to the following detailed description and the accompanying drawings.
In the accompanying drawings, Figure 1 shows colonies of mouse stomach mucosa (n = 28) by H. pylori and the formation level, alpha-used to study the association between gastric treated mice as keto-gluconic acid salt of the tar H . shows the design of the experimental mouse model infected with Helicobacter bacteria.
Figure 2 is a colony forming level of mouse stomach mucosa (n = 48) by H. pylori and alpha-keto-gluconic acid into a salt of the tar with the stomach used to study the association between the treated mice H. pylori bacteria The design of an experimental mouse infection model is shown.
3 shows the mobility of PCR products related to 16S rDNA Helicobacter genus bacteria in an electric field analyzed by DGGE technology.

Terms used throughout the detailed description and appended claims have the following meanings:

As used herein, the term "alpha-ketoglutarate" refers to 2-oxo-pentanedionic acid, 2-oxoglutaric acid, alpha-oxoglutaric acid, alpha-oxopentanedionic acid, 2-ketoglutaric acid, It refers to a compound that releases the active anion of an acid known as 2-oxo-1,5-pentanedionic acid, 2-oxopentanedionic acid, or 2-oxo-glutaric acid. Examples of such compounds are salts of alpha-ketoglutaric acid, additive salts, esters, amides, imides, and prodrugs thereof. Alpha-ketoglutarate exhibits an inhibitory action on colonization and in living organisms including humans, plants and animals, especially pets and farm animals, such as mammals, birds, amphibians, fish, mollusks or arthropods. Prevents colony formation of mucous membranes by urea-degrading bacteria. As far as the salt of alpha-ketoglutaric acid is concerned, the term ketoglutarate, as used herein, is an alkali metal salt and/or alkaline earth metal salt and/or chitoate of the acid, or an alkali metal and alkaline earth metal and chitosan and alpha -Contains mixed salts of ketoglutaric acid. Particularly preferred are sodium salts and calcium salts or mixtures thereof.

The term "pharmaceutical agent" as used herein refers to a composition comprising a therapeutically effective amount of alpha-ketoglutarate for use in the novel medical or prophylactic indications encompassed by the present invention. The pharmaceutical preparations may be other active ingredient(s), and/or additional beneficial pharmaceutically acceptable substances that are compatible with the active ingredient(s), such as vehicles, diluents intended for the preparation and suitable for the route of administration selected. , Excipients, adjuvants and auxiliary additives. As other active ingredient(s), the pharmaceutical preparations comprising alpha-ketoglutarate may contain vitamins, for example.

The term “therapeutically effective” refers to living, including humans, pets and farm animals, such as mammals, birds, amphibians, birds, mollusks or arthropods, which have a therapeutic effect under the in vivo conditions provided herein. It refers to a derivative of such a specific amount, in particular a salt of alpha-ketoglutaric acid, which reduces and inhibits the colonization of mucous membranes by urea-degrading bacteria in organisms. Therapeutic or prophylactic effectiveness may be achieved by using the aforementioned pharmaceutical preparations in solid or liquid form with or without vehicles, diluents, additives or as a component of a pharmaceutical composition, such as humans, pets and farm animals, such as mammals, birds. , By introduction into living organisms including amphibians, birds, mollusks or arthropods. The formulation is administered in an amount sufficient to reduce and prevent urea-degrading bacterial infection. Alternatively, a therapeutically effective amount of the pharmaceutical agent cures infections caused by urealytic bacteria.

Depending on the desired effect, the amount of the agent may vary depending on its specific action on the urea-degrading bacteria at the target site. The dosage of the formulation may contain a defined amount of substance calculated in such a way to produce the expected therapeutic result. Dosages are given in terms of purely active substances taking into account their chemical structure and the presence of additional substances such as vehicles, diluents, adjuvants and other acceptable pharmaceutically acceptable additives. The desired therapeutic effect and thus the recommended dose are methods known by practitioners in the field of medical or veterinary services in accordance with good medical practice, mainly based on parameters such as patient age, weight, sex, and other concomitant infections and diseases. Can be determined.

The term “administration of a pharmaceutical agent” refers to a prophylactic or therapeutic agent for the aforementioned diseases, having an appropriate route of administration controlled for the site, type and extent of infection, taking into account the route of entry of harmful urea-degrading bacteria into an organism. Means reaction.

The term "inhibition of colony formation" reduces the spread and/or severity of infection in mucous membranes or other tissues caused by urealytic bacteria, or in humans, pets and farm animals such as mammals, birds, amphibians, It relates to the complete eradication of infectious agents that reduce and/or prevent further progression of infection in living organisms, including birds, mollusks or arthropods.

The term “prevention of colonization” refers to urea that is harmful when bacteria come into contact with the mucous membranes of living organisms, including humans, pets and farm animals, such as mammals, birds, amphibians, birds, mollusks or arthropods. It means preventing the development of degrading bacteria. In the case of effectively preventing colony formation, infection does not occur, or mucous membranes of living organisms including humans, pets and farm animals such as mammals, birds, amphibians, birds, mollusks or arthropods prevent colony formation. It is infected with a significantly delayed degree compared to the scenario that does not.

The term "dietary supplement" refers to a concentrated source of nutrients or other substances that have a nutritional or physiological effect, the use of which contributes to supplementing the daily diet deficient in certain beneficial ingredients. Dietary supplements are produced in easy-to-use form, for example in tablets, capsules or liquids, preferably in single-dose packages.

The term "medicinal food" means a food product having a typical form and recipe for the product, containing additional substances of particular therapeutic or prophylactic value.

The term "food/feed additive" is a vehicle, buffer, detergent, solubilizer, antioxidant, which contains the active substance, corresponds to the active substance profile, in pure or in composition, in solid or liquid form and is approved for use in food. It relates to products with or without agents, preservatives and/or other additive substances.

Detailed description of the invention

The present invention relates primarily to the novel pharmaceutical use of alpha-ketoglutarate.

In another aspect, the present invention also provides a method for producing an organic biofuel based on the conversion of biological resources including lignin and cellulose by a bacterial enzyme, comprising: a urea-degrading microorganism in the back of a higher termite that feeds on wood. It relates to a method for producing an organic biofuel in which the enzyme produced by the cells is used in the presence of alpha-ketoglutarate.

In this method, alpha-ketoglutarate is used in a proportion sufficient to increase the yield of the method by at least 5%. The enzyme can be used in purified or unpurified form that does not contain the destroyed urea-degrading bacterial cell fragments, i.e. in a mixture with the destroyed bacterial cell fragments, or can be released by the urea-degrading bacteria.

Termites are known for their ability to degrade wood. The natural colony-forming bacteria of termite intestine have been identified as a factor in converting wood into biofuel. Recently, it has been identified that there are more than 250 bacterial species identified in the termite back intestine with hundreds of genes encoding enzymes that degrade cellulose and xylan during ligno-cellulose digestion. Large amounts of urease-positive, highly motile spirochete in the termite intestine can participate in the initial hydrolysis of wood polysaccharides [Wernecker et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. 2007. Nature, 450, 7169, 560-565].

In accordance with the present invention, alpha-ketoglutarate is a biofuel in a quality way to exchange urease-positive microbial populations of bacteria symbiotic in the intestine to achieve a more effective and higher level of ligno-cellulose degradation. It is proposed to accelerate and enhance the enzymatic activity of bacteria and their products, which determines the performance of.

For the prevention and/or treatment of diseases caused by pathogenic urealytic bacteria in living organisms, including humans, plants and animals, especially pets and farm animals, such as mammals, birds, amphibians, birds, mollusks or arthropods To prepare pharmaceutical preparations for use, alpha-ketoglutarate is used in the form of a single compound or a mixture of various compounds.

The prophylactic or therapeutic effect on the administration of the formulation prepared according to the present invention is obtained when 0.001 to 0.2 g of alpha-ketoglutarate is used per 1 kg of body weight per day. When used topically and in body cavities, alpha-ketoglutarate is particularly effective at doses of 0.01 to 10 g/m 2 of tissue surface/day.

The present invention is based on the observation that H. pylori bacteria can survive in a highly acidic environment of higher organisms.

A well-known property of H. pylori is its ability to survive in low pH environments due to the activity of urease to hydrolyze urea present in the gastric mucosa and gastric juice (Saidijam M, Psakis G, Clough JL. , Meuller J, Suzuki S, Hoyle CJ, Palmer SL, Morrison SM, Pos MK, Essenberg RC, Maiden MC, Abu-bakr A, Baumberg SG, Neyfakh AA, Griffith JK, Sachs G, Scott D, Weeks D, Melchers K Gastric habitation by Helicobacter pylori : insights into acid adaptation. Trends Pharmacol Sci. 2000;21:413-416, Sachs G, Weeks DL, Melchers K, Scott DR. The gastric biology of Helicobacter pylori . Annu Rev Physiol. 2003;65:349-369, Sidebotham RL, Worku ML, Karim QN, Dhir NK, Baron JH. How Helicobacter pylori urease may affect external pH and influence growth and motility in the mucus environment: evidence from in - vitro studies. Eur J Gastroenterol Hepatol. 2003;15:395-401). The reaction scheme of the individual reactions is as follows:

(1) H ₂ NCONH ₂ + H ₂ O → CO ₂ + 2 NH ₃

(2) CO ₂ +H ₂ O → H ₂ CO ₃

_{(3) H 2 CO 3 +} 2NH 3 → NH 4 + + HCO 3 - + NH 3

^{(4) H + Cl - +} NH 3 → NH 4 + + Cl -

As a result of the urea decomposition process, ammonia is formed, which immediately reacts with hydrochloric acid, locally increasing the pH of the microenvironment in the tissues (mucosa of the stomach). As is known, in the pristine environment, the gastric mucosa is acidic due to HCl production in parietal cells.

Although H. pylori (H. pylori) bacilli are sensitive organisms difficult though growing in vitro, but the low pH in the stomach - lethal to other bacteria - may paradoxically support is formed by a colony of H. pylori (H. pylori) have. This is primarily due to the presence of endogenous urea, which is broken down into ammonia, which increases the pH of the bacterial microenvironment by bacterial urease (an enzyme produced by H. pylori). Thus, the lethal influence of the acidic environment on H. pylori is eliminated. Even in the gastric mucosa layer, the growth cycle of H. pylori is believed to be stimulated by the microbial urealytic properties (Nakazawa T. Growth cycle of Helicobacter). pylori in gastric mucous layer. Keio J Med. 2002;51,S2:15-19). It has been demonstrated at the cellular level that urease mRNA is stabilized and destabilized depending on the pH of the environment. It has been assumed that bacteria grow using nutrients from degraded cells and colonize areas where the pH has already been altered. Urease activation is accompanied by the opening of pH-dependent UreI channels in bacterial cells that promote urea hydrolysis (Weeks DL, Eskandari S, Scott DR, Sachs G. A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science. 2000;287:482-485, Weeks DL, Sachs G. Sites of pH regulation of the urea channel of Helicobacter pylori . Mol Microbiol. 2001;40:1249-1259). It has also been pointed out that H. pylori has the ability to move toward a higher concentration of urea, and that this chemotaxis accelerates the urea hydrolysis process. This explains the stabilization of the infection in the second round of the growth cycle and consequently in the stomach (Scott DR, Marcus EA, Weeks DL, Sachs G. Mechanisms of acid resistance due to the urease system of Helicobacter pylori . Gastroenterology. 2002;123:187-195, Scott DR, Marcus EA, Weeks DL, Lee A, Melchers K, Sachs G. Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease. Infect Immun. 2000;68:470-477, Voland P, Weeks DL, Marcus EA, Prinz C, Sachs G, Scott D. Interactions among the seven Helicobacter pylori proteins encoded by the urease gene cluster. Am J Physiol Gastrointest Liver Physiol. 2003;284:96-106).

On the other hand, it is known that during amino acid decomposition-as a result of oxidative deamination, the α-amino group moves to the α-keto acid, which is accompanied by desorption of ammonium ions. Ammonium ions produced as a result of amino acid decomposition are partially involved in the biosynthesis of nitrogen compounds, and the rest are removed from living organisms after transformation into urea. In urea, one of the nitrogen atoms comes directly from the ammonium ion.

Under other natural conditions, urea can diffuse freely from the site where it is produced, ie from hepatocytes to the entire digestive system, due to the low molecular weight of this compound (M _r 60). Because of the strong toxicity of ammonium ions, organisms protect themselves by using the ions in the synthesis of low-toxic compounds such as urea (urea-releasing organisms-mammals). Urea-releasing organisms cannot store the nitrogen associated with proteins, amino acids and ammonia.

In living organisms, free ammonia occurs in small amounts despite successive deamination of amino acids. Some ammonia binds immediately to glutamate and asparaginate. Some ammonia is secreted through the kidneys in the form of ammonium ions. Most of the toxic ammonia is converted to urea by the ornithine cycle in the liver.

Currently, it was unexpectedly found that after administration of alpha-ketoglutarate in the stomach, the population of H. pylori is reduced. Under the conditions of in vivo experiments, the level of ammonium ions is relatively low, and its removal by alpha-ketoglutarate (immediately binds glutamate and asparaginate) despite improved anion transport to gastric mucosa cells (DC trans Urea-degrading (via porter) causes the killing of bacteria. The production of ammonium ions from urea, which is essential for the survival of H. pylori in the stomach, is not sufficient for the needs of bacillus, because ammonium ions are administered in the synthesis of glutamate and other amino acid compounds. Because it is implicated in rutarate and is used immediately.

It is still unclear why H. pylori produces excess urease in relation to the -substrate. Bacterial urease is a nickel-dependent enzyme with a nickel divalent cation incorporated after translation. It is believed that H. pylori accumulates in order to maintain the proper amount of active enzyme and protects the urealytic activity of progeny cells growing even under nickel-deficient conditions. On alpha-intensive due to the absorption of the ketoglutaric acid salts, H. pylori (H. pylori) may also compete for access to these salts which causes an intensive production of bacterial urease in the first stage of infection .

Due to the pandemic spread of H. pylori infection, the study of agents that limit disease resulting from infection is the subject of numerous reports. See: El-Omar EM. Mechanisms of increased acid secretion after eradication of Helicobacter pylori infection. Gut. 2006;55:144-146., Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, Isaacson PG. Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, Isaacson PG. Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma. Lancet. 1991;338:1175-1176.

Alpha-ketoglutarate together with ammonium ions are involved in the synthesis of certain amino acids, such as glutamic acid, and then glutamine in the following reaction:

This is the ability of alpha-ketoglutarate to bind to ammonium ions, which has spurred research into the current new medical applications of these widely-known substances.

The above-mentioned reaction is to be a combined path of ammonium ions which compete for the synthesis of the H. pylori (H. pylori) or, for example, causes an essential nitrogen supply to other urea-decomposing bacteria in the urinary germline urea in the above environment, Was guessed.

Due to the lack of reports relating to the harmful effects of alpha-ketoglutarate on the mucous membranes of the stomach and small intestine in healthy volunteers, alpha-ketogluta on the formation of H. pylori colony in the stomach and small intestine in healthy laboratory animals. A series of experiments were conducted on healthy laboratory animals to establish the influence of rate.

Unexpectedly, the results confirmed the above-mentioned assumptions and suggested:

1.In the thickening of the gastric mucosa of mice infected with H. pylori and inoculated with alpha-ketoglutarate, or in the thickening of the small intestine mucosa and the width and depth of the villi of the tested animals. There was no morphological change.

2. Amount of lactic acid bacteria isolated from the gastric mucosa (vestibular region) scraped in tested animals, including samples from mice infected with H. pylori and inoculated with alpha-ketoglutarate. There was no change.

3. Production of gastrin -gastric hormone- localized in the parietal region of the gastric glands in mice infected with H. pylori and inoculated with alpha-ketoglutarate compared to the control (phosphate buffer-challenged with PBS). A decrease of 17% (p<0.01) was observed in the number of cells. In animals infected with H. pylori and inoculated with alpha-ketoglutarate, the level of gastrin in the blood was decreased (from 21.8 pM-24.7 pM to 12.8 pM-15.6 pM), which is probably in the gastric mucosa. This is the result of a decrease in the number of gastrin-producing cells. This fact represents an indirect (via histamine) inhibitory interaction between alpha-ketoglutarate and proton pump activity. Its overactivity is evident in reflux disease.

4. Cholecystokinin (CCK) tissue hormone-producing Some tendency to reduce the number of cells has been recognized in the small intestine of animals treated with alpha-ketoglutarate. There is a slope of 30% compared to the same intestinal segment of uninfected mice inoculated with PBS alone (no statistical significance, p=0.1). There was no significant difference in the number of CCK-producing cells in the small intestine of mice infected with H. pylori and challenged with alpha-ketoglutarate compared to mice inoculated with only alpha-ketoglutarate (p =0.25). H. pylori (H. pylori) infection is a H. pylori (H. pylori) followed by the alpha - Ke to 1.9 pM-2.5 pM from the lower CCK blood levels (3.1 pM-4.0 pM in the animals inoculated with the toggle rate doubles ) (p < 0.05) may be associated with a reduced number of CCK producing cells in the small intestine. Thus, low CCK levels can stimulate an empty stomach with increased frequency; This is referred to as a factor avoiding further colony formation and infections caused by H. pylori (H. pylori) by the medical service.

Bactericidal effect of alpha-ketoglutarate against H. pylori bacillus

At present, it was unexpectedly discovered that alpha-ketoglutarate inhibits the process of H. pylori colony formation in the gastrointestinal tract of mammals. In this study, the average number of colonies isolated from mucous membranes in the gastric flax of mice infected only with H. pylori ^{-7.8 x 10 2} ±5.0 30 days after administration of the first dose of suspension of bacterial cells. x 10 ¹ . In a group of laboratory animals that received an intragastric dose of alpha-ketoglutarate for 9 consecutive days after 14 days of infection, the average number of isolated colonies was only 3.8 x 10 ² ± 5.0 x 10 ¹ . Nine intragastric administrations of alpha-ketoglutarate for 9 consecutive days, initiated 14 days after the last infectious dose of H. pylori, reduced the gastric mucosal colonization by bacteria by 49%. This result demonstrates that alpha-ketoglutarate inhibits gastric colonization by H. pylori.

In another experiment, the average number of colonies isolated from mucous membranes in the nasolabial area of mice infected with H. pylori alone 20 days after the first administration of the bacterial cell suspension ^{was 4.3 x 10 2} ±5.0 x 10 It turned out to be ^1. Alpha for three consecutive days after eight days the infection - the cake toggle rutile rate in the group of laboratory animals treated into the above, H. pylori (H. pylori) colony was found. H. pylori disclosed in eight of the last infection dose (H. pylori), for 3 consecutive days alpha - Ke toggle three intragastric administration of rutile rate is completely stopped colony formation and H. pylori in the gastric mucosa of mice (H a. pylori) eradication was sufficient.

During observation, the gastric microbiota was altered and the DNA of H. bilis was also found in mice infected with H. pylori; Alpha in mice inoculated with additional toggle doubles as Kane rate only DNA of H. Rhodes tium (H. rodentium), H. Billy's (H. bilis), H. H. party Syracuse (H. hepaticus) was found; In the control group of mice administered with alpha-ketoglutarate, DNA of H. hepaticus and H. rodentium were identified. In mice treated with PBS, no DNA of Helicobacter bacteria was found.

These results were obtained by routine diagnostic methods demonstrated by traditional techniques that assumed that living microorganisms had to be cultured to confirm the presence of bacteria in the tissue (fulfilment of Koch's postulates). Additionally, this study was demonstrated as a method of detecting DNA against H. pylori and other non-pathogenic bacteria of the genus Helicobacter. Electrophoretic separation of PCR products by PCR followed by denaturation gradient gel electrophoresis (DGGE), and sequencing of products to investigate DNA composition, are methods for detecting the changes disclosed herein. In fact, during the sequencing alpha - H. pylori in mice treated with any cake toggle rutile rate was not also found (H. pylori) DNA (sensitive level: ethidium bromide staining).

Therefore, alpha-ketoglutarate is an ideal active substance for a large and diverse group of patients in need of prophylaxis and treatment against H. pylori, and urogenital infections caused by urea-degrading bacteria.

A mechanism of bactericidal action of alpha-ketoglutarate against urea-degrading bacteria, which is completely different from such mechanisms known to date, will safely eradicate these microorganisms without the risk of inducing an increase in drug resistance in urea-degrading bacteria. Indeed, reinfection, superinfection and difficulty in therapy with antibiotics will be reduced.

As established to date, alpha-ketoglutarate supports or is an alternative to standard antibiotic therapy. In addition, in the case of cachexia, it can function to balance the natural urea-degrading microbiota.

According to the present invention, use in the prevention and/or treatment of diseases caused by urea-degrading bacteria by using alpha-ketoglutarate and/or a suitable precursor that releases anions of alpha-ketoglutaric acid under in vivo conditions. I will prepare a medical formulation that will be used.

To treat infections caused by urealytic bacteria and diseases associated with infections, alpha-ketoglutarate with catheter and urinary system infections by administering an alpha-ketoglutarate solution to a catheter according to the present invention now. It is recommended to be administered to patients to ensure direct contact with alpha-ketoglutarate and bacteria that colonize the host mucosa or urealytic bacteria that form infectious stones.

As proposed according to the present invention, it is also recommended to introduce alpha-ketoglutarate into the body cavity in the form of suppositories and irrigation in case of urogenital infections.

Suppositories containing alpha-ketoglutarate, as suggested in the present invention, with anal gland infection (in animals), and also with time-dependent hemorrhoids, rupture or ulceration of the distal mucosa of the digestive system (including the rectum). Should be administered as an anus.

In the case of bacteremia (sepsis) arising as a result of systemic infection of urea-degrading bacteria, alpha-ketoglutarate is now administered according to the invention directly into the blood, for example in the form of an intravenous infusion solution. It is recommended to apply ketoglutarate treatment.

On the outside, to the rupture and ulceration of skin with infection caused by urea-degrading bacteria, alpha-ketoglutarate should be administered in the form of powders, dressings and ointments, as suggested in accordance with the present invention.

The solid alpha-ketoglutarate should be administered to infected fish during feeding according to the invention.

In each of the above-mentioned exemplary routes of administration of alpha-ketoglutarate, irritation can lead to increased severity of infection, so the pH of the formulation containing alpha-ketoglutarate as the active ingredient and the form of the drug are infected. The necessary measurements should always be taken to ensure that it is not irritating to the tissue or organ.

The formulations obtained according to the invention are particularly useful for inhibiting and/or inhibiting H. pylori colony formation.

The following salts are preferably used as alpha-ketoglutarate: mono- and di-substituted of alpha-ketoglutaric acid and alkali metal and/or alkaline earth metal and/or chitosan salt. Preferred salts are sodium salts and/or calcium salts.

Alpha-ketoglutarate is conditionally used as a medical preparation, dietary supplement, special medicinal food and/or food/feed additive for the inhibition of H. pylori colony formation in humans and animals according to the present invention. is used in the human or animal to prevent H. pylori (H. pylori) infection and its prognosis, or H. pylori (H. pylori) can be reduced the infection and its prognosis.

Alpha-ketoglutarate can be administered with known vehicles and additives that are approved for pharmaceutical use and are compatible with selected alpha-ketoglutarate precursors. Suitable additives are, for example, water, saline, dextrose, glycerol, ethanol or other similar substances or combinations thereof. Moreover, if desired, the formulations may contain additional substances such as, for example, wetting agents, emulsifying agents, pH adjusting agents, buffering agents and the like.

Alpha-ketoglutarate can be administered with other known active substances that are approved for pharmaceutical use and are compatible with selected alpha-ketoglutarate precursors. Among other valuable active ingredients are vitamins, especially vitamin C and many others.

According to the present invention, formulations containing alpha-ketoglutarate may be solid and/or liquid depending on the intended route of administration.

The invention also relates to the use of alpha-ketoglutarate for the manufacture of agents for treating and preventing other urealytic bacterial infections. Examples of additional medical indications for alpha-ketoglutarate include agents that inhibit the passage of pathogenic urealytic bacteria through the stomach, agents that interfere with the formation of deposits and infectious stones in the urinary system, biologic on catheters and other medical equipment. Urethral infusions, tablets, irrigation liquids, intravaginal tablets for the manufacture of agents that reduce film formation and mineralization of sediments and also inhibit the growth of other pathogenic urea-degrading bacteria in the genitourinary system. There are uses of the form of alpha-ketoglutarate.

In accordance with the present invention, alpha-ketoglutarate can also be used in the form of bladder injection in bacterial infections caused by urealytic bacteria, in dogs, cats and domesticated animals.

A further example of a novel use of alpha-ketoglutarate is a formulation that regulates the urea-degrading microbiota of the oral cavity, reduces the formation of tartar, and inhibits the development of dental caries. It is used for the manufacture of. These products can take the form of chewing gum or toothpaste.

In addition, alpha-ketoglutarate has a further novel use thereof according to the present invention for preparing a preparation that inhibits the growth of urea-degrading bacteria , especially in ureaplasma and other mycoplasma, which causes infectious diseases in fish. Is found. In this respect, the novel use of alpha-ketoglutarate is to prevent inflammation of the baby tail caused by the aforementioned urea-degrading bacteria in carp and carp fry, and in other freshwater and seawater fish. It is a use for manufacturing a product for.

The present invention also encompasses the use of alpha-ketoglutarate in the production of dietary supplements, specially prescribed foods, and food/feed additives used to prevent and/or inhibit H. pylori colony formation. do.

The following examples provide a better explanation of the invention.

Example 1.

Experimental animals: 28 6-week-old BALB/cA (female) mice weighing 25±2 g (FIG. 1 ). Fourteen mice were challenged with H. pylori cells, 0.2 ml suspension of strain 119/95 through a tube (outer diameter 1.3 mm) at ^{a concentration of 10 9} cfu/ml three times at daily intervals. Two weeks after the last challenge, 7 mice were inoculated intragastrically for 9 consecutive days with a calcium or sodium salt solution (0.2 ml, concentration 30 mM) of alpha-ketoglutaric acid (group IA). The remaining 7 mice were subjected to sham-treatment with 0.01 M phosphate buffer-PBS as in the animals of group IA (group IB).

In addition, 14 mice of groups II A and II B were treated with 0.2 ml PBS according to the scheme carried out with infection of the above animals with H. pylori. Two weeks after the treatment, 7 mice were continuously inoculated with PBS (0.2 ml) for 3 consecutive days. The remaining 7 mice were treated with a salt solution of alpha-ketoglutaric acid (0.2 ml, concentration 30 mM) (Group II A).

* On the 30th day of the experiment, all mice were excised using _{CO 2.} Blood and gastric samples from mice infected with H. pylori with or without subsequent inoculation of a salt of alpha-ketoglutaric acid were collected for further analysis. The scheme of the experimental mouse infection model with H. pylori bacteria is presented in FIG. 1. In this experiment, the relationship between the level of colony formation in the gastric mucosa (n=28) of the mouse gastric mucosa (n=28) following H. pylori and the mice treated intragastrically with a salt of alpha-ketoglutaric acid was studied according to the time regime described above.

In Figure 1, the following abbreviations were used. Mice from the experimental group (n=28) were inoculated intragastric with the following preparations: Suspension of H. pylori cells: #; Solution of alpha-ketoglutaric acid: ◆; PBS solution: ●. The name, concentration and volume of the inoculum (H. pylori) of the strain and the salt of alpha-ketoglutaric acid and the concentration and dose of PBS are consistent with the above information. The letter S accompanying the drawings in bold print indicates the date of the autopsy. Autopsy was performed according to common binding standards.

Blood samples from all tested animals were applied onto GAB-CAMP agar plates and incubated at 37° C. for 7-10 days under microaerophilic conditions. The gastric mucosa was scraped from 1/2 of the antrum and mixed with sterile 500 μl PBS. After homogenate balancing, the mucous membrane scraped from half of the cavity was found to be generally between 40 μg and 50 μg. For counting the number of H. pylori cells, 100 μl of gastric mucosa homogenate was applied to a GAB-CAMP agar plate and incubated at 37° C. for 5-10 days under microaerobic conditions. Homogenized mucosal samples from each mouse were tested in triplicate. Results are presented as mean values ± SD. The presence of H. pylori was confirmed by morphological tests of cells by gram-staining, including colony morphology and urease-, oxidase- and catalase tests.

There was no H. pylori isolated from the tissues of animals inoculated intragastric with a salt of PBS and alpha-ketoglutaric acid (group II A in FIG. 1) or PBS (group II B in FIG. 1).

H. pylori was inoculated and then bacteria were isolated from 5 to 10 days of inoculation from gastric samples of animals inoculated with a salt of alpha-ketoglutaric acid (group IA in FIG. 1) or PBS (group IB in FIG. 1). I did.

The number of colonies isolated from the cavity of the gastric mucosa from mice infected exclusively with H. pylori (mean ± SD) was 7.8 x 10 ² ± 5.0 x 10 ¹ , whereas according to the presented protocol, alpha-ketoglutaric acid After intragastric treatment of the mouse with the salt of-The number of separated colonies (mean ± SD) was 3.8 x 10 ² ± 5.0 x 10 ¹ (Fig. 3). For 9 consecutive days, 9 times, the induction of a salt of alpha-ketoglutaric acid (the introduction was initiated 14 days apart from the last infectious dose of H. pylori bacterial infection in the case of mice) in H. The degree of gastric mucosal colonization by pylori was reduced by 49%. This result is interpreted as the inhibitory action of the salt of alpha-ketoglutaric acid on the gastric colony formation step by H. pylori.

H. pylori and any other bacteria were not isolated from blood samples taken from mice.

The results obtained are shown in Table 1 below.

Table 1. H. pylori bacterium isolated from gastric mucosa homogenate obtained after subsequent inoculation or non-inoculation of a salt of alpha-ketoglutaric acid to mice and infected mice uninfected with H. pylori

Animals were infected entirely with H. pylori (H. pylori + PBS), infected with H. pylori and subsequently inoculated with a salt of alpha-ketoglutaric acid (H. pylori + AKG), and control groups of non-infected animals were inoculated. PBS alone or a salt of PBS and alpha-ketoglutaric acid (PBS + AKG) was inoculated.

Example 2

Experimental animals: 48 6-week-old BALB/cA (female) mice weighing 25±2 g (FIG. 2 ). Twenty-four mice from groups III B and III (FIG. 2) were challenged with H. pylori cells, a 0.2 ml suspension of strain 119/95 at ^{a concentration of 10 9} cfu/ml three times at daily intervals. Eight days after the last challenge, 16 mice were inoculated with 0.2 ml of a 30 mM salt solution of alpha-ketoglutaric acid intragastrically via a tube for 3 consecutive days (Group III B). The remaining 8 mice were gastric-treated with 0.2 ml of 0.01 M PBS according to the procedure of Group III (Group III).

The remaining 24 mice (FIG. 2) were inoculated intragastrically with 0.2 ml 0.01 M PBS three times at daily intervals. After 8 days of treatment, 16 mice were inoculated with a salt of alpha-glutaric acid (0.2 ml, concentration 30 mM) using a tube for 3 consecutive days (Group IV B). The remaining 8 mice were treated with 0.2 ml of 0.01 M PBS (Group IV).

On the 20th day of the experiment, all mice were dissected using _{CO 2.} Blood and gastric samples were collected for further study.

In Figure 2, the following abbreviations were used. Mice from the experimental group (n=76) were inoculated intragastric with the following preparations: Suspension of H. pylori cells: #; Solution of alpha-ketoglutaric acid: ◆; PBS solution: ●. The name, concentration and volume of the inoculum (H. pylori) of the strain and the concentration and dose of the salt of alpha-ketoglutaric acid and PBS are consistent with the above description. The bold letters identified by the letter S indicate the date of the autopsy. Autopsy was performed according to a common principle.

From the collected samples, as described in Example 1, H. pylori bacteria were cultured on GAB-CAMP agar plates. In addition, DNA was isolated and PCR was performed with primers (5′-CTATGACGGGTATCCGGC-3′ and 5′-CTCACGACACGAGCTGAC-3′) that recognize 16S rDNA fragments in bacterial DNA from Helicobacter genus. After PCR, a 470 bp-sized product was isolated and sequenced according to a denaturating gradient gel electrophoresis technique (DGGE). DGGE analysis was performed in 9% polyacrylamide gel (acrylamide/bisacrylamide solution in a ratio of 37.5:1). Electrophoresis was performed at 60° C. for 16 hours at 125V.

The results are presented in Table 2 below.

Table 2. Bacteria of the genus Helicobacter, including H. pylori, in the gastric mucosa (cavity) homogenate obtained after subsequent inoculation or non-inoculation of a mouse infected with H. pylori with a salt of alpha-ketoglutaric acid.

Animals were infected exclusively with H. pylori (H. pylori + PBS), infected with H. pylori and subsequently inoculated with a salt of alpha-ketoglutaric acid (H. pylori + AKG), and a control group of uninfected animals. Only PBS was inoculated or a salt of PBS and alpha-ketoglutaric acid (PBS + AKG) was inoculated.

H. pylori was isolated from 8 gastric samples of 8 mice infected with H. pylori + PBS (group III in FIG. 2) (Table 2). Among the DNA isolated from the same gastric sample, a 16S rDNA H. pylori-specific fragment was confirmed by performing PCR.

From gastric samples obtained from 16 mice (group III B in Figure 2) infected with H. pylori and treated with a salt of alpha-ketoglutaric acid, H. pylori culture or H. pylori specific H. pylori in PCR products No sequence was found (Table 2).

H. pylori rods were cultured from blood samples taken from these animals. Using the selected primers, DNA isolated from the blood and stomach of 8 mice challenged with PBS (group IV in Fig. 2) was not amplified.

In the accompanying drawings, Figure 3 shows the mobility of typical PCR products for 16S rDNA fragments of bacteria from the genus Helicobacter in an electric current field evaluated by the DGGE technique. A: H. bunch FIG room (H. muridorum), B: H. Billy's (H. bilis), C: H. pool rooms (H. pullorum), D: H. pylori, E: Helicobacter spp. Plexisspira) ( Helicobacter spp . flexispira ) Taxon 8” F. La pinion (F. rappini) ", F: H. H. party kusu (H. hepaticus), G: H. non - zero you to serve as markers (H. bizzozeronii). Arrows represent the DNA of H. Billis. Letters 1 to 8 are indicated by estimating the path through which the PCR product from Example 2 is moved.

H. pylori was infected and then in 16 PCR products extracted from the stomach cavity from mice treated or untreated with a salt of alpha-ketoglutaric acid or uninfected mice, 19 DNA fragments of size 470 bp were detected. . The results are shown in Figure 3 and Table 3 below. Sequencing of the DNA segments (n=19) identified in these 16S DNA fragments (separated by DGGE previously) was performed by H. Pylori (n=8), H. Rhodentium (n=4), and H. Billis. (n=3) and H. hepatitis (n=4) were consistent (Fig. 3).

Table 3. Sequence analysis of PCR products obtained after amplification of homogenate of gastric mucosa (cavity)

Animals were infected exclusively with H. pylori (H. pylori + PBS), infected with H. pylori and subsequently inoculated with a salt of alpha-ketoglutaric acid (H. pylori + AKG), and non-infected animals were alpha-infected. The salt of glutaric acid (PBS + AKG) was inoculated.

From Table 3 above, the following points are identified. DNA of two Helicobacter species: H. rhodentium and H. bilis, including H. pylori and H. bilis, were detected in three different mice. In gastric samples of 2 mice from group III (H. pylori + PBS), H. pylori and H. bilis were identified. In gastric samples of mice from group III B (H. pylori + salt of alpha-ketoglutaric acid), H. rhodentium, H. bilis were detected (Table 3). Subsequently, the DNA of H. hepatitis was confirmed in 4 animals, and 2 mice belonged to group III B (H. pylori + salt of alpha-ketoglutaric acid), PBS + alpha-ketoglutar It was identified in two mice inoculated with the salt of the acid (group IV B) (Table 3).

H. Billis' DNA did not appear separately in any of the PCR products, whereas H. Rhodentium's DNA was present in three samples without concomitant sequences, and together with H. Billis' DNA in one sample. Was present (Fig. 3, Table 3).

In summary, the number of colonies (mean ± SD) isolated from the cavity portion of the gastric mucosa of mice infected entirely with H. pylori (group III) was 4.3 x 10 ² ± 5.0 x 10 ¹ , whereas alpha-ketoglutar After additional gastric treatment of mice with acid salts (Group III B), no H. pylori colonies were formed (Table 2). Over three consecutive days, three intragastric injections of salts of alpha-ketoglutaric acid (the injections were initiated 14 days apart from the last infectious dose of H. pylori bacteria injection in the case of mice) were total for bacterial colonization. It resulted in an inhibitory effect and caused complete eradication of H. pylori from the gastric mucosa.

Moreover, during the time of the experiment, the composition of the above urea-degrading microbiota was changed, and the DNA of H. bilis was well identified in mice infected with H. pylori (Group III), whereas salts of alpha-ketoglutaric acid were found. In the subsequently inoculated mice (group III B), the DNA of H. rhodentium, H. bilis, H. hepaticus was exclusively identified and control mice treated with a salt of alpha-ketoglutaric acid (group IV In B), H. hepatitis and H. rhodentium were identified.

*Example 3.

Mixed or separate aqueous solutions of calcium and sodium salts of alpha-ketoglutaric acid were prepared and used as additives for diary products and beverages.

Salts of alpha-ketoglutaric acid 0.001 g-0.2 g

20 g of glucose

Up to 100 g of water

A therapeutically and/or prophylactically effective amount is from 0.001 g to a maximum of 0.2 g per kilogram of body weight per day dose.

With the use of model animals (mouse) and the participation of volunteers, intragastric or oral administration of sodium and/or calcium of alpha-ketoglutarate in the form of an aqueous solution shows prophylactic activity, alleviation of the course of infection, and H. pylorier It has been demonstrated that a reduction in colonization of the gastrointestinal tract is also achieved.

Example 4

Ten people (body weight) with H. pylori infection confirmed by microbiological methods (endoscopy), who experience acute symptoms of the gut intestinal tract at this time between September and November, each year at this time. 60 to 95 kg, 45 to 60 years old, 6 males and 4 females) were given a voluntary intake of alpha-ketoglutaric acid (2 g) every morning in combination with a milk drink. After a two-week treatment period, symptoms of pyrosis and other features of indigestion disappeared in selected volunteers. The disappearance of dyspeptic symptoms was still maintained for 1 month after the completion of AKG administration.

The examples presented illustrate how urea-degrading bacteria-in the present case, H. pylori-compete with host individual cells for access to the substrate, ie urea. The incorporation of salts of alpha-ketoglutaric acid into the stomach is urea degraded to ammonia by urea-degrading bacteria for the needs of macro organisms (i.e., in the synthesis of glutamate to alpha-glutarate as one of several substances). Promoted the use of. Despite the activity of bacterial urease, the acidic pH of the gastric environment consistently prevented the formation of micro-niche for H. pylori. Thus, colonization of the mucous membrane of giant organisms for urea-degrading bacteria was hindered and stopped. Also, the use of alpha-ketoglutarate prevents infections caused by urea-degrading bacteria.

SEQUENCE LISTING <110> KRUSZEWSKA, Danuta <120> NEW MEDICAL APPLICATIONS OF ALPHA-KETOGLUTARATE <130> I/148900-PCT <140> not known yet <141> 2007-12-31 <150> EP07460015.6 <151> 2007-07-03 <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 18 <212> DNA <213> Helicobacter sp. <400> 1 ctatgacggg tatccggc 18 <210> 2 <211> 18 <212> DNA <213> Helicobacter sp. <400> 2 ctcacgacac gagctgac 18

Claims (5)

Claims 1. A method for producing an organic biofuel based on the conversion of biomass comprising lignin and cellulose by bacterial enzymes, characterized in that the enzymes produced by the posterior urea digesting microorganism gun of higher termites, A method for producing an organic biofuel used in the presence of ketoglutarate. The method of claim 1, wherein the alpha-ketoglutarate is used in a ratio sufficient to increase the process yield by at least 5%. 3. The method according to claim 1 or 2, wherein the enzyme is used in a purified form containing no urea-degrading bacterial cell fragments that have been destroyed. 3. The method according to claim 1 or 2, wherein the enzyme is used in an unpurified form, that is, mixed with a broken bacterial cell fragment or in a form released by urea-degrading bacteria. A conversion enhancer for producing a biofuel according to any one of claims 1 to 4, which is based on the conversion of biomass including lignin and cellulose by bacterial enzymes, Rutarate and optionally a bulking component (s).

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