Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/04—Drugs for disorders of the urinary system for urolithiasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to new medical applications of alpha-ketoglutarate including an application of alpha-ketoglutarate for manufacturing of therapeutic and prophylactic preparations for use in prophylaxis and treatment of undesired conditions of living organisms including human being, plants and animals, especially pet and/or farm animal, namely mammal, bird, amphibian, fish, molluse or arthropod, as well as the use of alpha- ketoglutarate in therapeutic and prophylactic treatment of the living organisms.
• Alpha-ketoglutarate occurs in living organisms as an endogenous molecule.
• Salts of alpha-ketoglutaric acid have been known for at least 60 years, i.e. since the Krebs cycle was discovered. Endogenous alpha-ketoglutaric acid salts play in humans and in animals a fundamental role, together with the salts of oxaloacetic and pyruvic acid, in the citric acid cycle. As a result of reproducible reactions there are synthesised fatty acids, sterols, cholesterol (with involvement of citrate), porphiryn, heme, chlorophyll (activity of succinyl-CoA), glutamate, amino acids, nucleotide bases (activity of alpha-ketoglutaric acid salts).
• Alpha-ketoglutaric acid anion plays a key role in metabolism, mainly in aerobic organisms.
• Alpha-ketoglurate is produced in a process of oxidative decarboxylation involving cellular enzyme of isocitrate dehydrogenase, and also in another metabolic pathway, by an oxidative deamination of glutamate catalyzed by glutamate dehydrogenase.
• Alpha ketoglutarate - being an intermediate compound in the basic life cycle, namely the tricarboxylic acid cycle, i.e. the Krebs cycle, is a subject of intracellular permanent transformations and it is present in rather low concentrations in peripheral blood in an anion form due to its full metabolisation in the organism.
• alpha-ketoglutarate also plays a role of a natural scavenger - a decontaminant, through transportation of nitrogen by means of transamination in effect of a transfer of amino groups originating from catabolised amino acids. This process occurs in the liver and it is called the ornithine cycle or urea cycle.
• glutamate a neurotransmitter named glutamate is formed.
• glutamate may be decarboxylated while producing a compound referred to as GABA (gamma-aminobutyric acid) which is an inhibitor of the glutamate neurotransmitter and blocks the neurotransmitter.
• GABA gamma-aminobutyric acid
• alpha-ketoglutarate enzymes participate in the removal of free radicals - being incompletely metabolised products and very toxic compounds, from the organism.
• alpha-ketoglutarate-dependent oxygenases is a molecular oxygen sensor, i.e. an indicator of oxygen level in the environment.
• Alpha-ketoglutarate may also be introduced into an organism through known routes of administration, for instance: orally, through inhalation, intravenously or via other routes.
• the everyday diet of both humans and animals does not comprise alpha- ketoglutarate.
• alpha-ketoglutaric acid itself and alpha-ketoglutaric acid combined with pyridoxine (vitamin B6) was listed in the group of biochemical products even before September 15, 1994.
• NFA National Nutritional Foods Association
• alpha-ketoglutaric acid salts are present in all the products available on the market is that alpha-ketoglutarate - as an intermediate compound in the Krebs cycle — is one of the substances responsible for cellular respiration and therefore it is believed to improve quality of life.
• alpha-ketoglutarate not only increases energy production, but also protects muscles against decomposition of branched amino acids in order to generate glutamine, the energy boosting molecule. Besides, it is a compound which enhances secretion of growth hormone and optimises muscle metabolism. It increases the activity of insulin and polyamines in a safe way. It also enhances neurotransmission, thus helping to maintain a good mental status of the organism, supports endurance of muscles maximising athletic effects, influences the organism's fat burning capability, increases libido, has a beneficial influence on immunological functions and reduces oxygen stress.
• Another group of food preparations used as dietary supplements comprises pyridoxine and pyridoxyl that are combined with alpha-ketoglutarate. Components of these products enhance metabolic activity within a cell, balance out the organism's efforts to produce energy and protect the liver.
• alpha-ketoglutaric acid salts stimulate fat reduction in the organism and are necessary for ensuring integrity of muscle tissue.
• alpha-ketoglutaric acid itself is a component of another kind of preparations, namely those exhibiting a natural detoxifying function, recommended for use in chronic fatigue and in metabolic deficiencies often diagnosed by amino acids analysis. Administration of this type of preparations results in increase of stamina and boosts energy.
• An interesting product of this group is a calcium and magnesium salt of alpha- ketoglutaric acid. Due to the fact that alpha-ketoglutaric acid belongs to a group of strong organic acids, it irritates oesophagus and stomach when administered orally. Use of calcium and magnesium allows production of a buffered bi-component alpha-ketoglutaric acid compound that does not cause the unpleasant feeling of excessive acidity. Numerous patents and patent applications show that alpha-ketoglutarate may be administered in metabolic brain dysfunction, in disorders of the nervous system, blood circulation and the skeletal muscle system, in order to strengthen cell mitochondrial functions.
• Drinks comprising alpha-ketoglutaric acid salts are also available and are intended to supply energy to the organism, in particular before, during and after physical activity.
• Such a drink - as a source of energy - may be administered also in states of fasting and long-lasting demand for energy in humans and other mammals.
• alpha-ketoglutarate may be used as a non-steroid, anabolic product increasing muscle mass without transforming muscles into fat.
• the effect of such a dietary supplement is similar to the effect obtained with synthetic anabolic steroids, however without the unfavourable side effects.
• a supplement comprising tiamin, lipoic acid, creatine derivatives and L-arginine together with alpha-ketoglutarate may also be administered orally.
• This dietary supplement is intended to lower blood glucose levels and to maintain low glucose levels in the course of treatment of diabetic neuropathy, as well as to improve blood circulation and muscle efficiency.
• Alpha-ketoglutaric acid salts were found to have an interesting application as agents aiming to reduce nitrogen emission in humans and animals and to maintain protein synthesis, and also in food microbiology.
• the salts are used as a component improving and enhancing aroma and taste of fermentation products (e.g. vinaigrette) and dairy products, inter alia cheese.
• Alpha-ketoglutaric acid salts influence lactic acid bacteria fermentation thus altering the metabolism of amino acids, levels of catabolites and activity of aminotransferases. In practice, it leads to shortening of the cheese ripening period by accelerating formtion of those compounds which guarantee high commercial quality of food products . See: 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.
• alpha-ketoglutarate as a pharmaceutical preparation or as a component of a pharmaceutical preparation.
• alpha-ketoglutaric acid, glutamine, glutamic acid and salts thereof, as well as amides and di- and tripeptides as pharmaceutical preparations for treatment and prophylaxis of arthrosis, rheumatoid arthritis and cartilage damage due to inflammation and other reasons is known from the publication WO 2007/058612.
• Publication WO 2005/123056 discloses a use of alpha-ketoglutaric acid, glutamine, glutamic acid and. pharmaceutically acceptable salts, amides and di- and tripeptides thereof as a pharmaceutical preparation, as food and animal feed additive in the treatment and prophylaxis of excessive plasma levels of at least one of the following parameters: cholesterol, LDL 5 glycerides.
• the preparation may also be used to raise HDL level.
• EP 0 922 459 discloses that alpha-ketoglutaric acid together with D-galactose and ornithine and such alpha-ketoglutaric acid salts as sodium, potassium, magnesium, zinc and calcium salts in a defined dosage, in form of tablets, powders, infusions, syrups may serve to raise amino acid profiles in blood, in particular in patients under metabolic stress situations.
• the disclosed preparation may be used in the treatment of liver diseases, in therapy and prevention of liver diseases in alcohol addicts in order to maintain the function and structure of the liver, as well as to regenerate the liver.
• the publication WO 2006/016828 indicates the use of alpha-ketoglutarates and a number of derivatives thereof as a pharmaceutical preparation and also as a food and animal feed additive improving functioning of nerve cells and the whole nervous system, minimising and preventing apoptosis of nerve cells and protecting against diseases of the nervous system in adults and foetuses.
• the publication WO 2007/082914 discloses a method for diagnosing higher susceptibility for diseases and conditions of the gastrointestinal tract associated with low levels of alpha-ketoglutaric acid (AKG) in a human or animal, such as among others Helicobacter pylori related gastritis, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma. It is suggested that humans or animals with low levels of AKG as compared to a normal average AKG levels should be treated with a pharmaceutical preparation or food or feed supplement comprising AKG, its derivatives, metabolites, analogues or salts thereof.
• AKG alpha-ketoglutaric acid
• the publication discloses some results confirming that in the control group of test animals (2-3 years old rats) those with a low blood level of AKG - below 0.1 ug/ml, did not survive the experiment whereas the test animals with similar initial blood level of AKG receiving feed with an addition of Na 2 AKG*2H 2 O survived the experiment. No evidence however has been given to demonstrate that the test animals were H. pylori infected. There is therefore also no evidence showing that low levels of AKG correlate in any respect with the above mentioned H. pylori related diseases and conditions.
• Pyridoxine alpha-ketoglutarate is known as an agent used in prophylaxis of acidosis in human medicine and veterinary medicine, in prophylaxis of all conditions leading to acidosis, as well as in pathologies where medicines decreasing lactic acid levels in blood are used.
• Alpha-ketoglutarate is also used in medical practice as a detoxifying agent in case of intoxication of an organism.
• the detoxifying action of alpha-ketoglutarate was used, for instance, in treating cyanide poisonings.
• Alpha-ketoglutarate as an anti-toxic agent prevents post-operational muscle catabolism and it is also used in hospitals in patients with parenteral feeding recommendation, where alpha-ketoglutarate is one of the compounds of the applied bolus.
• Alpha-ketoglutarate is also recommended for patients that suffer cerebrovascular stroke, for patients with burn wounds, for patients with hypoxia and for those irradiated with X-rays, as well as in case of cataracts resulting from selenite poisoning.
• Alpha-ketoglutarate combined with ornithine effectively protects the organism after small bowel transplantation against any translocation of bacteria, as examined in mesenteric lymph nodes, the liver and spleen. See: de Oca J, Bettonica C, Cuadrado S, Vallet J, Martin E, Garcia A 5 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.
• Alfa-ketoglutarate is also used in animal breeding to improve amino acid absorption. It is administered to piglets to accelerate absorption of iron ions.
• nitrogen assimilating bacteria is wide: from non-pathogenic commensals, such as skin-colonising bacteria, and non-pathogenic symbionts inhabiting mucous membrane of the gastrointestinal tract, to pathogenic bacteria, including Helicobacter pylori and bacteria causing urogenital system infections.
• pathogenic bacteria including Helicobacter pylori and bacteria causing urogenital system infections.
• the most common infection caused by ureolytic bacteria is the H. pylori infection.
• ureolytic bacteria The common feature of ureolytic bacteria is their ability to make use of urea present in their environment, by means of urease - mainly as a source of nitrogen necessary for survival live.
• Bacterial urease (urea aminohydrolase E.C.3.5.1.5) is a nickel- dependent multimer consisting of 2 or 3 subunits.
• the 3D crystallographic structure of some bacterial ureases has been discovered (H. pylori, Klebsiella aerogenes, Bacillus pasteurii).
• a high degree of similarity in amino acid sequences indicates that all types of ureases originate from one parent protein, that they probably have a similar three- dimensional structure and that they maintain catalytic activity while hydrolysing urea to ammonia and carbon dioxide.
• Examples of nitrogen-assimilating bacteria by means of their own ureases are Streptococcus salivarius and Actinomyces naeslundii commonly present in the oral cavity and forming a biofilm.
• the gastrointestinal tract has the biggest concentration of ureolytic bacteria.
• Microorganisms including ureolytic ones, permanently colonise a surface of epithelium, and they are recognised as a natural intestinal microbiota. This means, that access to urea is one of the critical factors of microorganism ecology in the gastrointestinal tract, i.e. it influences the quantity and quality of bacteria in this area.
• access to urea is one of the conditions of the macroorganism's health.
• the same rule of homeostasis governs the occurrence of microorganisms colonising body surfaces.
• Urea is also important as a substrate for pathogens' urease in the stabilisation phase of inflammation during H. pylori infection.
• Ureolytic bacteria even though they are not the main etiological factor in urinary system infections in healthy organisms, they are often associated with infections in humans with urinary systems disorders.
• the consequence of urinary system infections with urease- producing microorganisms is staghorn calculus accompanied by supersaturation of urine with ammonium magnesium phosphate salts (struvite) and phosphate calcium salts, as well as by pathological processes within kidneys. Under physiological conditions urine does not contain these salts.
• Ureaplasma ureolyticum and other alkalophilic bacteria e.g. Bacillus pasteurii.
• Ureaplasma ureolyticum and other alkalophilic bacteria e.g. Bacillus pasteurii.
• Ureaplasma ureolyticum and other alkalophilic bacteria e.g. Bacillus pasteurii.
• pathogens use ureolysis to generate their own ATP and thus they reproduce permanently.
• U. urealyticum and other mycoplasmas are relatively rare, they may also cause dangerous and difficult to cure infections of the respiratory system in humans and animals, including fish.
• urease-producing Yersinia enterocolica - a gastrointestinal tract pathogen may in some genetically handicaped people cause reactive arthritis and its reactivity is related to the chemical structure of the enzyme, specifically its subunit UreB.
• bacteria capable of movement are ureolytic organisms responsible for formation of biofilm and for mineralisation of deposits on catheters and other mechanical medical implements.
• Urea metabolism is also believed to be related to infections in the mucous membranes of the oral cavity, including gingival diseases, formation of dental caries and tartar.
• Formation of infectious stones is associated with urinary system infections caused by bacteria of the following genii: Proteus, Ureaplasma, Klebsiella, Pseudomonas, Staphylococcus, Providencia, Corynebacterium.
• the most common cause of formation of infectious stones is the P. mirabilis bacteria.
• Another factor in formation of the stones are mycoplasmas usually reported to be associated with genital tract infections, in particular with infections of the lower genital regions - mainly with vaginal infections. From the urethra in men the following organisms are isolated: Mycoplasma hominis and U. urealyticum. Colonisation of the urogenital system in both women and men by the bacteria leads to urinary system infections accompanied by formation of infectious stones in the bladder.
• H. pylori rods in humans are usually isolated from the stomach and duodenum.
• These Gram-negative, ureolytic, spiral-shaped bacteria formerly known as Campylobacter pylori are one of the etiological factors of gastritis and the formation of ulcers in stomach and duodenum.
• Warren and Marschall, honoured in 2005 with a Nobel Prize showed the cause-effect relationship between the occurrence of H. pylori bacteria in the gastrointestinal tract and chronic gastritis. See: Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984; 1 : 1311-1315.
• H. pylori rods were declared by WHO as a carcinogenic factor (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.). Moreover, a relationship was found between H. pylori infection and pathologies in tissues and organs other than the stomach, for example in heart.
• stomach is free from bacteria because the permanent colonisation of mucous membrane in stomach is difficult due to the fact, that the pH is too low for growth of the majority of microorganisms.
• the urease produced by H. pylori allows this organism to form permanent colonies in stomach and intestine, while ureolysis is the main factor causing pathologies associated with H. pylori infections.
• Urease deposited outside of the bacteria cell may constitute even up to 20 % of bacterial protein. The enzyme protects the bacteria against the acidic environment and prevents the lethal destruction of the bacterial cell envelope. Additionally, during the H.
• ammonium ions released by urease during the course of urea decomposition have cytotoxic effects on stomach epithelium cells.
• bacterial urease generates monochloramine that may induce DNA mutagenesis, one of the factors involved in the development of cancer in the course of chronic H. pylori infection.
• New species of ureolytic spiral bacteria are being isolated in humans and animals. However, so far it is not clear how other Helicobacter species occurring in humans should be connected with diseases of stomach (for instance H. heilmanni ⁇ ), intestines (for instance H. cinaed, H. canadensis), liver (for instance H. hepaticus, H. bilis) or with systemic infections (for example H. pullorum, Helicobacter spp.flexispira taxon 8 "F. rappini").
• stomach for instance H. heilmanni ⁇
• intestines for instance H. cinaed, H. canadensis
• liver for instance H. hepaticus, H. bilis
• systemic infections for example H. pullorum, Helicobacter spp.flexispira taxon 8 "F. rappini"
• H. pylori infection is present in 50% of the population and it seems to be related to the economic status and age of communities. It increases in adults, including almost entire populations in poorly developed countries. H. pylori is found in 90 — 100% of gastritis cases. Often chronic infection is found to be transformed into atrophic inflammation. In 10 % of those infected, serious pathologies develop. Infection with H. pylori is common in both children and adults. Most often infection occurs in a childhood and the colonisation of the mucous membrane of the stomach with H. pylori rods is maintained throughout the life-time.
• pylori and starting a treatment only where certain disease symptoms occur, such as: gastritis, stomach and duodenal ulcers, peptic ulcer confirmed in course of interviews, surgery due to peptic ulcer, pre-cancer changes (atrophic inflammation, metaplasia, dysplasia), stomach resection due to an early cancer, stomach cancer in family (up to 2 nd level of consanguinity), gastric hyperplastic adenomatous polyposis (after removal thereof), MALT lymphoma, long-term treatment with NSAIDs.
• certain disease symptoms such as: gastritis, stomach and duodenal ulcers, peptic ulcer confirmed in course of interviews, surgery due to peptic ulcer, pre-cancer changes (atrophic inflammation, metaplasia, dysplasia), stomach resection due to an early cancer, stomach cancer in family (up to 2 nd level of consanguinity), gastric hyperplastic adenomatous polyposis (after removal thereof), MALT lymphoma, long-term treatment with NSAID
• H. pylori infection is accompanied by certain diseases, such as peptic ulcers, gastric lymphomas, chronic atrophic gastritis with intestinal metaplasia and stomach cancer.
• Testing of mucosa fragments enables isolation of the microorganism (selective growing media) or application of appropriate techniques (specific and non-specific staining) that indicate the presence of H. pylori cells.
• Identification of H. pylori cells in the course of mucosa biopsy is subjected to verification by means of both classical microbiological methods (phenotype determination, drug resistance determination in the isolate), as well as biochemical methods (enzymatic activity of H. pylori - production of urease, catalase, oxidase), and molecular biology methods (PCR testing, Real-time PCR with specific primers for selected segment of the bacterial DNA).
• the most commonly used non-invasive methods include serological tests and identification of Helicobacter antigens - stool antigen test, and the urea breath test which detects levels of urea above that which is assumed to be the standard urea concentration in the air exhaled by the patient.
• Routine serological tests are ELISA-type tests detecting specific G-class antibodies to H. pylori bacteria.
• H pylori infection treatment is still based on the introduction of substances which decrease secretion of gastric juice and raise pH values in stomach.
• substances include proton pump inhibitors and certain antibiotics which eliminate the bacteria - clarythromicin, amoxicillin and chemical compounds - metronidazole (from the group of nitroimidazole derivatives).
• current therapy of diseases caused by ureolytic bacteria involves the application of an appropriate anti-bacterial drug administered orally, intravenously, intravaginally, anally and externally, in the form of pills, ointments, suppositories and powders.
• Selection of a therapeutically effective drug is made after the following factors are determined: biochemical activity of ureolytic bacteria, their resistance patterns to antibiotics and chemotherapeutic agents (antibiogram), and after the structure of the cell envelope (e.g. mycoplasmas) is determined.
• chemotherapeutic compounds substances other than nitroimidazole derivatives.
• the therapy is complex, costly, sometimes badly tolerated and not always effective.
• chemotherapeutic compounds clarythromicin (2 x 500) and amoxicillin (2 x 1 g) or metronidazole (2 x 500 mg), as well as proton pump inhibitors.
• Antibiotic I for instance amoxicillin, 2 x day 1 g
• Antibiotic II for instance clarythromicin, 2 x day 0.5 g
• Antibiotic II other antibiotic or chemical compound - for instance metronidazol, 2 x day 0.5 g
• the main object of the invention is to provide a new preparation for treatment and prophylaxis of diseases caused by urolytic bacteria, to be used in particular to regulate ureolytic microbiota of intestines, to regulate ureolytic microbiota of oral cavity, to inhibit passage of pathogenic ureolytic bacteria through a digesting stomach, to prevent formation of deposits and infectious stones in the urinary system - in humans and animals, in particular in dogs and cats, and in other domestic animals.
• the object of the invention is to provide also a new agent for control of undesired growth of ureolytic bacteria in living organisms, including a human beings, plants and animals, especially pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod .
• a specific aim of the invention is to provide a new preparation for treatment and prophylaxis of diseases caused by H. pylori.
• Still further object of the invention is to provide a new preparation for inhibiting growth of ureolytic bacteria, in particular of Ureaplasma and other mycoplasmas causing fish diseases, for use in particular as a prophylaxis of gill inflammation caused by ureolytic bacteria in carp and carp fry and other fresh water and sea fish.
• it is the object of the invention is to provide a new preparation for preventing formation of biofilm and mineralisation of deposits on catheters and other medical equipment.
• It is also the aim of the invention is to provide a new preparation for reduction of tartar formation and inhibition of development of caries.
• it is a further aim of the invention is to provide new dietary supplements, special medicated food products and food/feed additives preventing and/or inhibiting colonisation of living organisms, including a human being, pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod, by undesired ureolytic bacteria, in particular colonisation of humans and of domestic animals by H. pylori.
• Therapeutic and/or prophylactically effective dosages range from 0.00 Ig to 0.2 g/kg of body mass/day when administered intragastrically or orally. As far as local topical administration is concerned, the effective dosages range from 0.01 to 10 g/m 2 of tissue surface/day.
• salts of alpha-ketoglutaric acid may be effectively used in humans or in animals to treat ureolytic bacterial infections, including H. pylori infections.
• Fig.3 shows mobility of PCR products related to 16S rDNA Helicobacter genus bacteria in electrical field assayed with DGGE technique.
• alpha-ketoglutarate refers to the compound releasing an active anion of the acid known as 2-okso-pentanedioic, 2-oksoglutaric, alpha-oksoglutaric, alpha-oksopentanedioic, 2-ketoglutaric, 2-okso-l,5-pentanedioic, 2-oksopentanedioic, or 2- okso-glutaric.
• the examples of such compounds are salts, additive salts, esters, amides, imides of alpha-ketoglutaric acid and prodrugs thereof.
• Alpha-ketoglutarate exerts an inhibitory action on colonisation and prevents mucosa colonisation by ureolytic bacteria in living organisms, including a human being, plant and animal, especially pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod.
• the term alpha-ketoglutarate - as used herein covers alkali metal salts and/or alkaline earth metal salts and/or chitosan salts of the acid, or else a mixed salts of alkali metals and alkaline earth metals and chitosan and alpha-ketoglutaric acid.
• Particularly preferred are sodium salt and calcium salt or a mixture thereof.
• the term "medical preparation” as used herein refers to a composition comprising a therapeutically effective quantity of alpha-ketoglutarate to be used in new medical or prophylactic indications covered by this invention.
• the medical preparation may comprise other active ingredient(s) and/or additional beneficial pharmaceutically acceptable and compatible with the active ingredient(s) substances such as vehicles, diluents, excipients, adjuvants and auxilliary additives suitable for the selected administration route intended for the preparation.
• the present medical preparation comprising alpha-ketoglutarate may contain for example vitamins.
• terapéuticaally effective denotes such a specific quantity of a derivative, in particular, a salt of alpha-ketoglutaric acid, that under in vivo conditions presented in this description has a therapeutic effect, i.e. reduces and inhibits colonisation of the mucous membrane by ureolytic bacteria in living organisms, including a human being, pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod.
• Therapeutic or prophylactic effectiveness is achieved by introducing the above mentioned medical preparation in a solid or liquid form, with or without a vehicle, diluent, additive, or else as a component of a pharmaceutical composition, into living organisms, including a human being, pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod.
• the preparation is administered in quantities sufficient to reduce and prevent ureolytic bacteria infections.
• therapeutically effective quantity of the medical preparation heals the infections caused by ureolytic bacteria.
• the quantity of the preparation may differ, according to its specific action on the ureolytic bacteria in the target place.
• a dose of the preparation may comprise a defined quantity of a substance calculated in such a way as to bring about the expected therapeutic results. Dosage is given in terms of a pure active substance taking into account its chemical structure and presence of additional substances, such as vehicle, diluent, adjuvants and other permitted pharmaceutically acceptable additives. Desired therapeutic effect and thus the recommended dose may be determined by means of known methods by an employee of medical or veterinary service, mainly based on such parameters as age, weight, sex of the patient, other accompanying infections and diseases, in accordance with good medical practice.
• administration of the medical preparation refers to prophylactic or therapeutic reaction to the abovementioned diseases with an appropriate route of administration adjusted to the place, type and intensity of infection, taking into the account the route of entry of harmful ureolytic bacteria into the organism.
• inhibiting of colonisation relates to the reduction of spread and/or severity of infection within mucous membranes or other tissues, caused by ureolytic bacteria, or complete eradication of the infecting factor - leading to the reduction and/or prevention of further development of infection in living organisms, including a human being, pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod.
• preventing of colonisation refers to the prevention of the development of harmful ureolytic bacteria, when the bacteria get into contact with the mucous membrane of a living organism, including a human being, pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod.
• infection does not occur or mucous membranes of the living organisms, including a human being, pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod - are infected with considerable delay in comparison to a scenario without preventing of colonisation.
• dietary supplement means a concentrated source of nutrients or other substances with nutritious or physiological effect, the use of which contributes to supplementing everyday diet deficient in certain beneficial components. Dietary supplements are produced in an easy-to-use form, e.g. tablets, capsules or liquids, preferably in one-dose packages.
• immediate food product means a food product with form and recipe typical for the product, containing an additional substance of specific therapeutic or prophylactic value.
• food/feed additive relates to a product containing an active substance, pure or in a composition, in solid or liquid form, with or without vehicles, buffers, detergents, solubilizers, antioxidants, preservatives and other additives substances corresponding with the active substance profile, and approved for usage in food.
• the invention relates mainly to new medical applications of alpha-ketoglutarate.
• the invention relates also to a process for manufacturing an organic biofuel, based on the conversion of biomass comprising of lignin and cellulose by means of bacterial enzymes, wherein the enzymes produced by hindgut ureolytic microbiota of wood-feeing higher termites are used in presence of alpha-ketoglutarate.
• alpha-ketoglutrate is used in a ratio sufficient to increase the yield of the process by at least 5 %.
• the enzymes may be used in a purified form free of disrupted ureolytical bacterial cell fragments or in a nonpurified form, i.e. in admixture with disrupted bacterial cell fragments or released by ureolytical bacteria.
• Termites are known for their wood-degradation ability. Natural colonizing bacteria of termite guts have been identified as a factor which converts wood into biofuels. It has been shown recently that there are more than 250 bacterial species found in termites' hindgut with hundreds of genes encoding enzymes that break down cellulose and xylan during lingo-cellulose degradation. A high amount of urease-positive highly motile spirochetes present in termites' intestine can take part in the initial hydrolysis of wood polysaccharides (Wernecke et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. 2007. Nature, 450, 7169, 560-565.)
• alpha-ketoglutarate is proposed for speeding up and intensifying enzymatic activities of bacteria and their products determining performance of biofuels in a quality way exchanging urease-positive microbial community of symbiotic bacteria in the gut to achieve more effective and higher level of lingo- cellulose degradation.
• alpha-ketoglutarate is used in the form of a single compound or a mixture of various compounds, for manufacturing of medical preparation for use in prophylaxis and/or treatment of diseases caused by pathogenic ureolytic bacteria in living organisms, including a human being, plant and animal, especially pet and farm animal, such as mammal, bird, amphibian, fish, molluse or arthropod.
• Prophylactic or therapeutic effect of the administration of the preparation manufactured in accordance with the invention is reached, when from 0.001 to 0.2 g of alpha-ketoglutarate is used per 1 kilogram of body mass/day.
• the alpha-ketoglutarate is particularly effective in the dosage from 0.01 to 10 g/m 2 of tissue surface/day.
• the present invention is based on the observation that H. pylori bacteria are able to survive in the highly acidic environment of the higher organisms' stomachs.
• K pylori A well known property of K pylori is its ability to survive in low pH environment due to the activity of urease which hydrolyses urea present in the mucous membrane of stomach and in the gastric juice (Saidijam M, Psakis G, Clough JL, Meuller J, Suzuki S, Hoyle CJ, Palmer SL, Morrison SM 5 Pos MK, Essenberg RC, Maiden MC, Abu-bakr A 5 Baumberg SG 5 Neyfakh AA, Griffith JK 5 Sachs G 5 Scott D 5 Weeks D 5 Melchers K. Gastric habitation by Helicobacter pylori: insights into acid adaptation. Trends Pharmacol Sci.
• H. pylori rods are sensitive organisms difficult to be grown in vitro, the low pH in the stomach - lethal to other bacteria — may paradoxically support H. pylori colonisation. This is mainly due to the presence of endogenous urea decomposed by bacterial urease (an enzyme produced by H. pylori) to ammonia which increases pH in the micro-environment of the bacteria. Thus, the lethal influence of the acidic environment on H. pylori is eliminated. It is even believed (Nakazawa T. Growth cycle of Helicobacter pylori in gastric mucous layer. Keio J Med. 2002;51,S2:15-19.) that the H.
• Urease activation is accompanied by the opening of a pH-dependent Urel channel in bacterial cell which facilitates urea hydrolysis (Weeks DL 5 Eskandari S 5 Scott DR, Sachs G. A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science.
• urea may diffuse freely from the place where it is produced, i.e. from hepatocytes, to the entire digestive system, this is due to the low molecular mass of this compound (M r 60). Due to the strong toxicity of the ammonium ion the organism protects itself by employing the ion in the synthesis of low-toxicity compounds, such as urea (ureotelic organisms - mammals). Ureotelic organisms are not able to store nitrogen: which relates to proteins, amino acids and ammonia.
• H. pylori produces excessive - in relation to the substrate, quantities of urease.
• Bacterial urease is a nickel-dependent enzyme with nickel divalent cation posttranslationally incorporated. It is assumed, that in order to maintain an appropriate quantity of active enzyme H. pylori accumulates it to safeguard ureolytic activity of descendant cells growing even under conditions of nickel deficiency. Due to the intensive up-take of alpha-ketoglutaric acid salts in the stomach, H. pylori may also compete for access to these salts which causes an intensive production of bacterial urease in the first stage of infection.
• Alpha-ketoglutarate participates, alongside ammonium ions, in the synthesis of certain amino acids, such as glutamic acid, and then glutamine, in the following reaction:
• reaction is a pathway of binding ammonium ions competitive to synthesis of urea being a necessary source of nitrogen for H. pylori in the gastric environment or for other ureolytic bacteria, for instance in the urogenital system.
• Alpha-ketoglutarate is thus an ideal active substance for a large and varied group of patients that require prophylaxis and treatment against H pylori, and urogenital system infections caused by ureolytic bacteria.
• alpha-ketoglutarate supports, or is an alternative to, standard antibiotic treatment. Also, in cases of cachexia it may serve to balance the natural ureolytic microbiota.
• alpha-ketoglutarate and/or appropriate precursors releasing under in vivo conditions anions of alpha-ketoglutaric acid will be used to manufacture a medical preparation to be used in prophylaxis and/or treatment of diseases caused by ureolytic bacteria.
• alpha-ketoglutarate As proposed in accordance with the invention it is also recommended to introduce alpha-ketoglutarate into body cavities in the case of urogenital system infections in the form of suppositories and irrigation.
• Suppositories containing alpha-ketoglutarate should be, as proposed in accordance with the present invention, administered anally in anal glands infections (in animals), and also in resistant infections that form with time alongside haemorrhoids, rupture or ulceration of mucosa of the distal part of the digestive system (including the rectum).
• Alpha-ketoglutarate in solid state should be administered, in accordance with the present invention, to infected fish during feeding.
• alpha-ketoglutarate In each above mentioned exemplary route of administration of alpha-ketoglutarate, one should always take necessary measures to ensure that the form of the drug and pH of preparation containing alpha-ketoglutarate as the active ingredient is not irritating to the infected tissue or organ, as irritations may lead to an increased severity of the infection.
• the preparation obtained in accordance with the invention is useful in particular to prevent and/or inhibit H. pylori colonisation.
• the following salts are preferably used as alpha-ketoglutarate: mono- and di- substituted salts of alpha-ketoglutaric acid and alkali metals and/or alkaline earth metals and/or chitosan.
• the preferred salts are sodium salt and/or calcium salt.
• Alpha-ketoglutarate in accordance with the invention, may be used in humans and animals as a medical preparation, dietary supplement, special medicated food product and/or food/feed additive, depending on conditions, for inhibition of H. pylori colonisation in humans and animals, in order to prevent H. pylori infection and consequences thereof, or in order to alleviate H. pylori infection and consequences thereof.
• Alpha-ketoglutarate may be administered together with known vehicles and additives that are approved for pharmaceutical use and are compatible with the selected alpha-ketoglutarate precursors.
• Appropriate additives include, for instance, water, saline, dextrose, glycerol, ethanol or other similar substances or combinations thereof.
• the preparation may contain additional substances such as, for instance, wetting agents, emulsifiers, pH modulators, buffers and other.
• Alpha-ketoglutarate may be administered together with other known active substances that are approved for pharmaceutical use and are compatible with the selected alpha-ketoglutarate precursors.
• active substances include vitamins, vitamin C in particular, and many others.
• the preparation containing alpha-ketoglutarate may be solid and/or liquid, depending on the intended route of administration.
• alpha-ketoglutarate for manufacturing preparations for treatment and prophylaxis of other ureolytic bacteria infections.
• examples of further medical applications of alpha-ketoglutarate include the use of alpha- ketoglutarate to manufacture a preparation that inhibits passage of pathogenic ureolytic bacteria through the stomach, a preparation preventing formation of deposits and infectious stones in the urinary system, a preparation reducing formation of biofilm and mineralisation of deposits on catheters and other medical equipment, and also a preparation inhibiting growth of other pathogenic ureolytic bacteria in the urogenital system, to be used in the form of urethral infusions, tablets, irrigation liquids, intravaginal tablets.
• alpha-ketoglutarate may also be used in dogs, cats and domesticated animals, in the form of bladder infusions in bacterial infections caused by ureolytic bacteria.
• a further example of a new use of alpha-ketoglutarate is a use for manufacturing of a preparation regulating ureolytic microbiota of the oral cavity, reducing formation of tartar and inhibiting development of dental caries.
• This product may have the form of a chewing gum or a tooth paste.
• Alpha-ketoglutarate finds also its further new use according to the invention, for manufacturing a preparation inhibiting growth of ureolytic bacteria, in particular Ureaplasma and other mycoplasmas causing infections in fish, hi this respect, the new use of alpha-ketoglutarate is use for manufacturing a preparation for preventing inflammation of gills caused by the above mentioned ureolytic bacteria in carp and carp fry, and in other fresh water and sea fish.
• the invention also covers the use of alpha-ketoglutarate in production of dietary supplements, special medicated food products, and food/feed additives used to prevent and/or inhibit H. pylori colonisation.
• mice twenty eight 6 week-old BALB/cA (female) mice weighing 25 ⁇ 2 g (fig. 1). Fourteen mice were challenged through a tube (outer diameter 1.3 mm) 3 times with one day intervals with 0.2 ml suspension of H. pylori cells, strain 119/95 at the concentration 10 9 cfu/ml. Two weeks after the last challenge, seven mice were inoculated intragastrically for nine successive days by the same method with a solution of calcium or sodium salt of alpha-ketoglutaric acid (0.2 ml, at concentration 30 mM) (group I A). The remaining seven mice were sham-treated with 0.01 M phosphorate buffer - PBS (group I B) as animals from group I A.
• mice from group II A and II B were treated with 0.2 ml PBS according to the scheme operating by the infection of the animals with H. pylori.
• Two weeks after the above treatment seven mice were continuously inoculated with PBS (0.2 ml) for three consecutive days. The remaining seven mice were treated with a solution of salts of alpha-ketoglutaric acid (0.2 ml, concentration 30 mM) (group II A). On the 30 th day of the experiment all mice were sacrificed using CO 2 .
• blood and stomach samples from mice infected with H. pylori with or without the following inoculation with salts of alpha-ketoglutaric acid were collected. Scheme of the experimental mice infectious model with H.
• H. pylori was identified by colony morphology and urease-, oxidase- and catalase tests, as well as by morphological examination of cells by Gram-staining.
• mice From the blood samples taken from the mice neither H. pylori nor any other bacteria were isolated.
• H. pylori + PBS Animals exclusively infected with H. pylori (H pylori + PBS), infected with H. pylori with following inoculation of salts of alpha-ketoglutaric acid (H. pylori + AKG) and control groups non infected animals, inoculated only with PBS or with PBS and salts of alpha-ketoglutaric acid (PBS + AKG).
• Example 2 Example 2.
• mice forty eight, 6 weeks old BALB/cA (female) mice weighing 25 ⁇ 2 g (fig. 2). Twenty four mice (fig. 2) from group III B and III were challenged intragastrically 3 times with one day intervals with 0.2 ml suspension of H. pylori cells, strain 119/95 at the concentration 10 9 cfu/ml. Eight days after the last challenge, sixteen mice were inoculated intragastrically through the tube for three successive days with 0.2 ml of 30 mM solution of salts of alpha-ketoglutaric acid (group III B). The remaining eight mice were sham-treated with 0.2 ml of 0.01 M PBS (group III) according to the procedure with group III.
• mice The remaining twenty four mice (fig. 2) were inoculated intragastrically 3 times with one day intervals with 0.2 ml 0.01 M PBS. Eight days after above treatment sixteen mice were inoculated with the tube for three consecutive days with salts of alpha-glutaric acid (0.2 ml, concentration 30 mM) (group IV B). The remaining eight mice were treated with 0.2 ml 0.01 M PBS (group IV).
• mice On the 20 th day of the experiment all mice were sacrificed using CO 2 . Stomach and blood were samples were collected for the further studies.
• H. pylori + PBS Animals exclusively infected with H. pylori (H. pylori + PBS), infected with H. pylori with following inoculation of salts of alpha-ketoglutaric acid (H. pylori + AKG) and control groups of non infected animals, inoculated only with PBS or with PBS and salts of alpha-ketoglutaric acid (PBS + AKG).
• H. pylori was isolated from 8 stomach samples of 8 mice infected with H. pylori + PBS (Group III in fig. 2) (Table 2). In DNA isolated from the same stomach samples 16S rDNA H. pylori specific fragment was identified employing PCR.
• H. pylori rods were cultured from blood samples taken from these animals.
• DNA isolated from blood and from stomach of 8 mice challenged with PBS did not amplify.
• Fig. 3 mobility of PCR products typical for 16S rDNA fragment of bacteria from Helicobacter genus in a field of electrical current evaluated with DGGE technigue was illustrated.
• DNA of H. bilis did not appear separately in any PCR products, whereas DNA of H. rodentium was present in 3 samples without sequences accompanying, and in one sample together with DNA of H. bilis (fig. 3. Table 3).
• Aqueous solutions of calcium and sodium salt of alpha-ketoglutaric acid in admixture or separately were prepared and used as an additive for diary products and beverages.
• Therapeutically and /or prophylactically effective amount is from O.OOlg up to 0.2 g/kg body weight in one day dose.
• ureolytic bacteria - in this case H. pylori - compete against host organism cells for access to substrate, i.e. urea.
• Intragastric introduction of salts of alpha-ketoglutaric acid facilitated the use of urea decomposed to ammonia by ureolytic bacteria for the needs of macroorganism, i.e. in synthesis of glutamate with alpha-ketoglutarate as one of the agents.
• the acidic pH of the gastric environment was maintained preventing formation of a micro-niche for H. pylori.
• the colonisation of mucous membrane of macroorganisms by ureolytic bacteria was hindered and stopped.
• Use of alpha-ketoglutarate also prevents infections caused by ureolytic bacteria.

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