WO2008058993A2 - Use of alpha ketogluaric acid for the treatment of infection with h. pylori - Google Patents

Use of alpha ketogluaric acid for the treatment of infection with h. pylori Download PDF

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Publication number
WO2008058993A2
WO2008058993A2 PCT/EP2007/062333 EP2007062333W WO2008058993A2 WO 2008058993 A2 WO2008058993 A2 WO 2008058993A2 EP 2007062333 W EP2007062333 W EP 2007062333W WO 2008058993 A2 WO2008058993 A2 WO 2008058993A2
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alpha
helicobacter pylori
ketoglutaric acid
infection
treatment
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PCT/EP2007/062333
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French (fr)
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WO2008058993A3 (en
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Stefan Pierzynowski
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Entress Ab
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    • 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
    • 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
    • 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
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • 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

Definitions

  • the present invention relates to a new use of known pharmacologically active chemical compounds. More particularly, the present invention relates to the new use of certain acids, amides, and salts and mixtures thereof, which are scavengers of ammonium ions, for the manufacture of a pharmaceutical preparation or a food or feed supplement for the in vivo therapeutic treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori.
  • Helicobacter pylori (formerly called Campylobacter pylori) is a gram- negative, S-shaped, microaerophilic bacterium that was first discovered and cultured from a human gastric biopsy specimen (Warren, J. R. and B. Marshall, (1983) Lancet 1: 1273-1275). This bacterium has been demonstrated to be associated with gastritis and peptic ulcers (Buck et al., J. Infect. Dis. 153:664-669, 1986 and Graham, Gastroenterology 96:615-625, 1989). Further, epidemiological studies have indicated that this bacterium is the causative agent for gastritis, gastric ulcers ⁇ and duodenal ulcers and is associated with disorders such as gastric cancer.
  • Helicobacter pylori bacterial cells can survive in a low pH environment because of an enzyme on their outer cell wall called urease.
  • Urease converts urea, of which there is an abundant supply in the stomach, to bicarbonate and ammonia, which are strong bases. The bicarbonate and ammonia neutralise the acid gastric juices, thereby providing a protective layer around the Helicobacter pylori.
  • Helicobacter pylori induced gastritis may not present symptoms
  • chronic infection with Helicobacter pylori can cause gastric cancer.
  • alpha-ket ⁇ glutaric acid AKG
  • pharmaceutically acceptable salts of alpha- ketoglutaric acid and amides of alpha-ketoglutaric acid may be used for the in vivo therapeutic treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori.
  • alpha-ketoglutaric acid or an alkali or alkaline earth metal salt thereof or a combination thereof is used.
  • Most preferably sodium alpha-ketoglutarate is used.
  • a method for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori particularly diseases of the gastrointestinal tract including diseases such as gastritis,, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma in birds and mammals, including man, which method comprises administering to a subject in need for such treatment or prophylaxis of an effective amount of at least one member selected from the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha-ketoglutaric acid.
  • alpha-ketoglutaric acid or an alkali or alkali or alkaline earth metal salt thereof or a combination thereof is administered.
  • Most preferably sodium alpha-ketoglutarate is administered.
  • Fig. 1 shows a scheme of the action of enteric alpha-ketoglutaric acid to eliminate ammonium ions produced by Helicobacter pylori.
  • the inhibitor of the present invention controls growth or eradicate Helicobacter pylori in order to improve the cure rate or prevent diseases related to infection of pathogenic strains of Helicobacter pylori.
  • treatment or prophylaxis in their various grammatical forms in relation to the present invention refer to preventing, curing, reversing, attenuating, alleviating, ameliorating, inhibiting, minimising, suppressing, or halting (1) the deleterious effects of a disorder associated with Helicobacter pylori infection, (2) the disorder progression, or (3) the disorder causative agent ⁇ Helicobacter pylori).
  • the new use of at least one member selected from the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha- ketoglutaric acid for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori.
  • a method for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori, particularly diseases of the gastrointestinal tract including diseases such as gastritis, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma in birds and mammals, including man which method comprises administering to a subject in need for such treatment or prophylaxis of an effective amount of at least one member selected from the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha-ketoglutaric acid.
  • Helicobacter pylori pathogenesis is related to its urease activity which converts the blood urea to ammonium ions and bicarbonate to achieve higher pH in the stomach epithelium (see reaction steps 1-4 below).
  • Helicobacter pylori is very sensitive to low pH in the stomach and becomes eliminated by low pH. Only pathogenic strains do develop mechanism switching on production of urease in low pH. This mechanism allow pathogenic Helicobacter pylori to colonise the stomach.
  • alpha-ketoglutaric acid (AKG " ) is a strong and in principle the only scavenger of ammonium ions in the metabolism. Since alpha-ketoglutaric acid is the main intermediate of the TCA cycle localised in the mitochondria, it is only present in small amounts in body tissues. If present in the stomach epithelium, alpha- ketoglutaric acid reacts with ammonium ions produced by Helicobacter pylori and this mechanism eliminates beneficial higher pH in local stomach epithelium being condition sine qua non of Helicobacter pylori colonisation in the stomach.
  • Fig. 1 shows a more detailed scheme of how enteric alpha-ketoglutar ⁇ c acid acts to eliminate ammonium ions produced by Helicobacter pylori.
  • the pharmaceutical preparation and the food or feed supplement of the present invention comprises an alpha-ketoglutaric acid, a metabolite, an analogue, an amide or a pharmaceutically acceptable salt thereof and mixtures thereof, which have ammonium ion scavenging properties.
  • the pharmaceutically acceptable salt of alpha-ketoglutaric acid of the present invention could be any monovalent metal salt of alpha-ketoglutaric acid such as sodium, potassium salt or any divalent metal salt of alpha-ketoglutaric acid such as strontium, calcium or magnesium salt.
  • alkali or alkaline earth metal salts of alpha-ketoglutaric acid are used, and most preferably sodium alpha-ketoglutarate is used.
  • the amides of alpha-ketoglutaric acid of the present invention may include any of the amino acids occurring as components in peptides in nature.
  • the amino acid or acids is/are selected from the group consisting of arginine, ornithine, leucine, isoleucine and lysine. Said amino acids are preferably used in their L- configuration.
  • amides of alpha-ketoglutaric acid with an amino acid or a di- or tripeptide include, but are not limited to, amides of alpha-ketoglutaric acid with an amino acid selected from the group consisting of glutamine, glutamic acid, arginine, ornithine, lysine, proline, isoleucine and leucine and amides of alpha-ketoglutaric acid with a dipeptide of glutamine and any of glutamic acid, arginine, ornithine, lysine, proline, isoleucine and leucine and with a dipeptide of glutamic acid and any of arginine, ornithine, lysine, proline, isoleucine and leucine.
  • the molar ratio of alpha-ketoglutaric acid or salts thereof to amino acid or amino acids of said physical mixtures will in general be within the limits of from 1:0.01 to 1 :2, preferably from 1 :0.1 to 1 :1.5 and most preferably from 1 :0.2 to 1 :1.0.
  • the food or feed supplements and the pharmaceutical preparations of the present invention may be administered to a vertebrate, including mammals and birds, such as rodent, such as a mouse, rat, guinea pig, or a rabbit; a bird, such as a turkey, hen or chicken and other broilers and free going animals; a cow., a horse, a pig or piglet and other farm animals, a dog, a cat and other pets, and humans. While many animals may be treated with the preparation of the invention, a preferred animal for treatment is a human or commercially valuable animals and livestock.
  • Administration may be performed in different ways depending on what species of vertebrate to treat, on the condition of the vertebrate in the need of said treatment, and the specific indication to treat.
  • the administration is done as a food or feed supplement, such as a dietary supplement and a component in form of solid food and beverage.
  • a food or feed supplement such as a dietary supplement and a component in form of solid food and beverage.
  • Further embodiments may be in suspensions or solutions, such as a beverage further described below.
  • the formats may be in capsules or tablets, such as chewable or soluble, e.g. effervescent tablets, as well as powder and other dry formats known to the skilled man in the art, such as pellets, micro pellets, and grains.
  • the food and feed supplement may also be emulsified.
  • the active therapeutic ingredient or ingredients may then be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, buffering agents, which enhance the effectiveness of the active ingredient.
  • Different formats of the oral food or feed supplement may be supplied, such as solid food, liquids or lyophilised or otherwise dried formulations. It may include diluents of various buffers (e.g., Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatine to Io prevent absorption to surfaces, detergents (e.g., Tween 2 0, Tween 8 0, Pluronic F68, bile acid salts), solubilising agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the composition, complexation with metal ions, or in corporation of the material into or onto particul
  • the food or feed supplement is administered in the form of a beverage, or a dry composition thereof, in any of the methods according to the invention.
  • the beverage comprises an effective amount of the active ingredient or ingredients thereof, together with a nutritionally acceptable water-soluble carrier, such as minerals, vitamins, carbohydrates, fat and proteins. All of these components are supplied in a dried form if the beverage Is provided in a dry form.
  • a beverage provided ready for consumption further comprises water.
  • the final beverage solution may also have a controlled tonicity and acidity, e.g.
  • the pH is preferably in the range of about 2-5, and in particularly about 2-4, to prevent bacterial and fungal growth.
  • a sterilised beverage may also be used, with a pH of about 6-8.
  • the beverage may be supplied alone or in combination with one or more therapeutically effective composition.
  • the pharmaceutical preparations of the invention for oral use may be in the form of tablets, lozenges, capsules, powders, aqueous or oily suspensions, syrups, elixirs, aqueous solutions and the like comprising the active Ingredient or ingredients In admixture with a pharmaceutically acceptable carrier and additives, such as diluents, preservatives, solubillsers, emulslf ⁇ ers, adjuvants and carriers useful In the methods and use disclosed in the present Invention.
  • a pharmaceutically acceptable carrier and additives such as diluents, preservatives, solubillsers, emulslf ⁇ ers, adjuvants and carriers useful In the methods and use disclosed in the present Invention.
  • Orally applied alpha-ketoglutaric acids 2" in form of salts can be absorbed to the gut and stomach eplthelia (Krlstensen, NB., et al, J. Anim. Physiol Arum. Nutr. 86, 1-7, 2002; Lambert, B., et al, J. Nutr. 132, 3383-3386, 2002) via specific co-transporters (Buddington, R.K., et al, Comparative Biochemistry and Physiology, Part A.138/2, 215-220, 2004).
  • pharmaceutically acceptable carriers are well known to those skilled in the art and may Include, but are not limited to, 0.01- 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/- aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • the dosage to be administered will vary depending on the active principle or principles to be used, the condition to be treated, the age, sex, weight etc. of the patient to be treated but will generally be within the range from 1 to 1000 mg/kg bodyweight/day 5 preferably from 10 to 100 mg/kg bodyweight/day.
  • mice A study to analyse effects of Na salts of alpha-ketoglutaric acid (AKG) on colonisation of the stomach and intestine by Helicobacter pylori and on gut morphology was performed in mice.
  • the study was conducted in two experiments on 40 and 62 mice (BALB/cA, 6 weeks old female mice (23 ⁇ 2 g) kept in standard cages with normal environment conditions). During the first 14 days, the mice were subjected to a period of acclimation to the conditions of the animal room (temperature 22°C ⁇ 2 0 C, 12hrs/12hrs day/night cycle, humidity 55% ⁇ 5%) and had permanent access to water and were fed with a standard rat pelleted diet.
  • PBS 5 0.2 ml, 0.0 IM phosphor buffer saline
  • ten mice from experimental and ten mice from control group received Na 2 AKG gavage via a stomach tube (0.2 ml, 3OmM Na 2 AKG).
  • the rest of the mice (10 + 10) in the experimental and control groups received PBS (0.2 ml) via a stomach tube.
  • all mice were sacrificed with overdose of CO 2 and cervical dislocation. Blood and stomach samples for bacteriological and PCR analyses were collected.
  • Controls (n — 32) received phosphor buffer saline (PBS, 0.2 ml, 0.01M).
  • PBS phosphor buffer saline
  • sixteen mice from experimental and sixteen mice from control group received Na 2 AKG gavage via a stomach tube (0.2 ml, 3OmM Na 2 AKG).
  • the rest of the mice (16 + 16) in the experimental and control groups received PBS (0.2 ml) via a stomach tube.
  • all mice were sacrificed with overdose of CO 2 and cervical dislocation. Blood and stomach samples for bacteriological and PCR analyses were collected. Results
  • Table 1 shows the results from an analysis of growth of Helicobacter pylori in the samples taken from the stomach or the blood from animals in experiment 1.
  • Helicobacter pylori culturing were performed on GAB-CAMP and MRSA broth.
  • Table 2 shows the results from an analysis of growth of Helicobacter pylori in the samples taken from the stomach or the blood from animals in experiment 2.
  • Helicobacter pylori culturing were performed on GAB-CAMP and MRSA broth.
  • Table 2 further shows the number of animals which were positive in a PCR analysis for Helicobacter pylori. In the PCR analysis, DNA chain reactions were performed with specific primers which detect Helicobacter spp. 16S rRNA fragments.
  • Table 3 shows the identification of PCR products of Helicobacter pylori and other bacteria strains from samples taken from the stomach of mice of experiment 2. PCR products were assayed in order to identify 16S rDNA fragments which are characteristic for the different species of bacteria.
  • Orally applied alpha-ketoglutaric acid eradicates pathogenic Helicobacter pylori previously inoculated in the stomach of experimental model mice. This event happened already after 3 days OfNa 2 AKG treatment. Ten days OfNa 2 AKG treatment was effective in 90% of the infected animals.

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Abstract

The present invention relates to Use of at least one member selected from the group consisting of alpha-ketoglutaric acid (AKG)5 pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha-ketoglutaric acid for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori in birds and mammals, including man.

Description

NEW USE OF KNOWN PHARMACOLOGICALLY ACTIVE CHEMICAL
COMPOUNDS
Field of Invention
The present invention relates to a new use of known pharmacologically active chemical compounds. More particularly, the present invention relates to the new use of certain acids, amides, and salts and mixtures thereof, which are scavengers of ammonium ions, for the manufacture of a pharmaceutical preparation or a food or feed supplement for the in vivo therapeutic treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori.
Background of the Invention
Helicobacter pylori (formerly called Campylobacter pylori) is a gram- negative, S-shaped, microaerophilic bacterium that was first discovered and cultured from a human gastric biopsy specimen (Warren, J. R. and B. Marshall, (1983) Lancet 1: 1273-1275). This bacterium has been demonstrated to be associated with gastritis and peptic ulcers (Buck et al., J. Infect. Dis. 153:664-669, 1986 and Graham, Gastroenterology 96:615-625, 1989). Further, epidemiological studies have indicated that this bacterium is the causative agent for gastritis, gastric ulcers^ and duodenal ulcers and is associated with disorders such as gastric cancer.
Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age (Taylor, D. N. and M. J. Blaser, (1991) Epidemiol. Rev 13: 42- 50). Helicobacter pylori colonises the human gastric mucosa, establishing an infection that usually persists for decades. Infection by Helicobacter pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
Helicobacter pylori bacterial cells can survive in a low pH environment because of an enzyme on their outer cell wall called urease. Urease converts urea, of which there is an abundant supply in the stomach, to bicarbonate and ammonia, which are strong bases. The bicarbonate and ammonia neutralise the acid gastric juices, thereby providing a protective layer around the Helicobacter pylori.
Current therapy is based on eradicating Helicobacter pylori through antibiotics and proton pump inhibitors. Unless Helicobacter pylori is completely eliminated from the stomach by antibiotic therapy, the infection will return to the same level as before treatment within about a month after cessation of antibiotics administration. Antibiotic treatment Is complicated by the fact that the low pH environment of the stomach tends to inactivate many antibiotics. For this reason, antibiotics are commonly administered with drugs which suppress the secretion of gastric acid (e.g., proton pump inhibitors). However, the prolonged administration of antibiotics and proton pump inhibitors results In undesirable side effects for the patient and increased antibiotic-resistant strains in the environment.
Furthermore, while Helicobacter pylori induced gastritis may not present symptoms, chronic infection with Helicobacter pylori can cause gastric cancer. A large percentage of patients remain without any treatment for Helicobacter pylori despite ulcer formation because despite ulcer formation, chronic gastritis Is not always diagnosed. In some cases, patients present mild symptoms or are asymptomatic.
At present there is a long felt but unmet need to prevent and to treat Helicobacter pylori infections on the basis of a natural supplement rather than on the basis of systemic antibiotic(s) and proton pump inhibitors.
Summary of the Invention
In accordance with the present invention, it was surprisingly found that alpha-ketαglutaric acid (AKG), pharmaceutically acceptable salts of alpha- ketoglutaric acid and amides of alpha-ketoglutaric acid may be used for the in vivo therapeutic treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori.
According to the first aspect of the present invention, there is provided the new use of at least one member selected from; the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha-ketoglutaric acid for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori, particularly diseases of the gastrointestinal tract Including diseases such as gastritis, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma, in birds and mammals, including man.
According to a preferred embodiment of the invention, alpha-ketoglutaric acid or an alkali or alkaline earth metal salt thereof or a combination thereof is used. Most preferably sodium alpha-ketoglutarate is used.
According to another aspect of the present invention, there is provided a method for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori, particularly diseases of the gastrointestinal tract including diseases such as gastritis,, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma in birds and mammals, including man, which method comprises administering to a subject in need for such treatment or prophylaxis of an effective amount of at least one member selected from the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha-ketoglutaric acid.
According to preferred embodiments of these aspects, alpha-ketoglutaric acid or an alkali or alkali or alkaline earth metal salt thereof or a combination thereof is administered. Most preferably sodium alpha-ketoglutarate is administered.
These and other purposes of the present invention will be described in more detail below.
Brief Description of Drawings
The operation of the present invention should become apparent from the following description when considered in conjunction with the accompanying drawings, in which:
Fig. 1 shows a scheme of the action of enteric alpha-ketoglutaric acid to eliminate ammonium ions produced by Helicobacter pylori.
Detailed Description of the Invention
It is an object of the present invention to provide an effective and safe inhibitor of Helicobacter pylori colonisation, which is, inter alia associated with the occurrence of peptic ulcers, without the disadvantages of side effects associated with current methods of antibiotic and proton pump inhibitor administration. Hence, the inhibitor of the present invention controls growth or eradicate Helicobacter pylori in order to improve the cure rate or prevent diseases related to infection of pathogenic strains of Helicobacter pylori.
The terms "treatment or prophylaxis" in their various grammatical forms in relation to the present invention refer to preventing, curing, reversing, attenuating, alleviating, ameliorating, inhibiting, minimising, suppressing, or halting (1) the deleterious effects of a disorder associated with Helicobacter pylori infection, (2) the disorder progression, or (3) the disorder causative agent {Helicobacter pylori).
Thus according to one aspect of the invention, there is provided the new use of at least one member selected from the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha- ketoglutaric acid for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori.
According to another aspect of the present invention there is provided a method for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori, particularly diseases of the gastrointestinal tract including diseases such as gastritis, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma in birds and mammals, including man, which method comprises administering to a subject in need for such treatment or prophylaxis of an effective amount of at least one member selected from the group consisting of alpha-ketoglutaric acid, pharmaceutically acceptable salts of alpha-ketoglutaric acid and amides of alpha-ketoglutaric acid.
Helicobacter pylori pathogenesis is related to its urease activity which converts the blood urea to ammonium ions and bicarbonate to achieve higher pH in the stomach epithelium (see reaction steps 1-4 below).
H2NCONH2 (urea) + H2O → CO2 + 2NH3 (1)
CO2 + H2O → H2CO3 (2)
H2CO3 + 2NH3 → NH4 + + HCO3 " + NH3 (3)
H + Cl" + NH3 → NH4 + + Cl" (4)
Helicobacter pylori is very sensitive to low pH in the stomach and becomes eliminated by low pH. Only pathogenic strains do develop mechanism switching on production of urease in low pH. This mechanism allow pathogenic Helicobacter pylori to colonise the stomach.
The anion of alpha-ketoglutaric acid (AKG ") is a strong and in principle the only scavenger of ammonium ions in the metabolism. Since alpha-ketoglutaric acid is the main intermediate of the TCA cycle localised in the mitochondria, it is only present in small amounts in body tissues. If present in the stomach epithelium, alpha- ketoglutaric acid reacts with ammonium ions produced by Helicobacter pylori and this mechanism eliminates beneficial higher pH in local stomach epithelium being condition sine qua non of Helicobacter pylori colonisation in the stomach.
Fig. 1 shows a more detailed scheme of how enteric alpha-ketoglutarϊc acid acts to eliminate ammonium ions produced by Helicobacter pylori.
The pharmaceutical preparation and the food or feed supplement of the present invention comprises an alpha-ketoglutaric acid, a metabolite, an analogue, an amide or a pharmaceutically acceptable salt thereof and mixtures thereof, which have ammonium ion scavenging properties.
The pharmaceutically acceptable salt of alpha-ketoglutaric acid of the present invention could be any monovalent metal salt of alpha-ketoglutaric acid such as sodium, potassium salt or any divalent metal salt of alpha-ketoglutaric acid such as strontium, calcium or magnesium salt. As a preferred embodiment of the invention, alkali or alkaline earth metal salts of alpha-ketoglutaric acid are used, and most preferably sodium alpha-ketoglutarate is used.
The amides of alpha-ketoglutaric acid of the present invention may include any of the amino acids occurring as components in peptides in nature. Preferably the amino acid or acids is/are selected from the group consisting of arginine, ornithine, leucine, isoleucine and lysine. Said amino acids are preferably used in their L- configuration.
Examples of amides of alpha-ketoglutaric acid with an amino acid or a di- or tripeptide include, but are not limited to, amides of alpha-ketoglutaric acid with an amino acid selected from the group consisting of glutamine, glutamic acid, arginine, ornithine, lysine, proline, isoleucine and leucine and amides of alpha-ketoglutaric acid with a dipeptide of glutamine and any of glutamic acid, arginine, ornithine, lysine, proline, isoleucine and leucine and with a dipeptide of glutamic acid and any of arginine, ornithine, lysine, proline, isoleucine and leucine.
The molar ratio of alpha-ketoglutaric acid or salts thereof to amino acid or amino acids of said physical mixtures will in general be within the limits of from 1:0.01 to 1 :2, preferably from 1 :0.1 to 1 :1.5 and most preferably from 1 :0.2 to 1 :1.0.
The food or feed supplements and the pharmaceutical preparations of the present invention may be administered to a vertebrate, including mammals and birds, such as rodent, such as a mouse, rat, guinea pig, or a rabbit; a bird, such as a turkey, hen or chicken and other broilers and free going animals; a cow., a horse, a pig or piglet and other farm animals, a dog, a cat and other pets, and humans. While many animals may be treated with the preparation of the invention, a preferred animal for treatment is a human or commercially valuable animals and livestock.
Administration may be performed in different ways depending on what species of vertebrate to treat, on the condition of the vertebrate in the need of said treatment, and the specific indication to treat.
In one embodiment, the administration is done as a food or feed supplement, such as a dietary supplement and a component in form of solid food and beverage. Further embodiments may be in suspensions or solutions, such as a beverage further described below. Also, the formats may be in capsules or tablets, such as chewable or soluble, e.g. effervescent tablets, as well as powder and other dry formats known to the skilled man in the art, such as pellets, micro pellets, and grains.
The food and feed supplement may also be emulsified. The active therapeutic ingredient or ingredients may then be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, buffering agents, which enhance the effectiveness of the active ingredient.
Different formats of the oral food or feed supplement may be supplied, such as solid food, liquids or lyophilised or otherwise dried formulations. It may include diluents of various buffers (e.g., Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatine to Io prevent absorption to surfaces, detergents (e.g., Tween 2 0, Tween 8 0, Pluronic F68, bile acid salts), solubilising agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the composition, complexation with metal ions, or in corporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc. or onto liposomes, micro emulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. In one embodiment, the food or feed supplement is administered in the form of a beverage, or a dry composition thereof, in any of the methods according to the invention. The beverage comprises an effective amount of the active ingredient or ingredients thereof, together with a nutritionally acceptable water-soluble carrier, such as minerals, vitamins, carbohydrates, fat and proteins. All of these components are supplied in a dried form if the beverage Is provided in a dry form. A beverage provided ready for consumption further comprises water. The final beverage solution may also have a controlled tonicity and acidity, e.g. as a buffered solution according to the general suggestions in the paragraph above. The pH is preferably in the range of about 2-5, and in particularly about 2-4, to prevent bacterial and fungal growth. A sterilised beverage may also be used, with a pH of about 6-8. The beverage may be supplied alone or in combination with one or more therapeutically effective composition.
According to a further embodiment, the pharmaceutical preparations of the invention for oral use may be in the form of tablets, lozenges, capsules, powders, aqueous or oily suspensions, syrups, elixirs, aqueous solutions and the like comprising the active Ingredient or ingredients In admixture with a pharmaceutically acceptable carrier and additives, such as diluents, preservatives, solubillsers, emulslfϊers, adjuvants and carriers useful In the methods and use disclosed in the present Invention.
Orally applied alpha-ketoglutaric acids2" in form of salts can be absorbed to the gut and stomach eplthelia (Krlstensen, NB., et al, J. Anim. Physiol Arum. Nutr. 86, 1-7, 2002; Lambert, B., et al, J. Nutr. 132, 3383-3386, 2002) via specific co-transporters (Buddington, R.K., et al, Comparative Biochemistry and Physiology, Part A.138/2, 215-220, 2004).
Further, as used herein "pharmaceutically acceptable carriers" are well known to those skilled in the art and may Include, but are not limited to, 0.01- 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
Aqueous carriers include water, alcoholic/- aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
The dosage to be administered will vary depending on the active principle or principles to be used, the condition to be treated, the age, sex, weight etc. of the patient to be treated but will generally be within the range from 1 to 1000 mg/kg bodyweight/day5 preferably from 10 to 100 mg/kg bodyweight/day.
The invention will now be further illustrated by means of a number of examples which should not be construed to limit the scope of the invention.
Examples
A study to analyse effects of Na salts of alpha-ketoglutaric acid (AKG) on colonisation of the stomach and intestine by Helicobacter pylori and on gut morphology was performed in mice. The study was conducted in two experiments on 40 and 62 mice (BALB/cA, 6 weeks old female mice (23± 2 g) kept in standard cages with normal environment conditions). During the first 14 days, the mice were subjected to a period of acclimation to the conditions of the animal room (temperature 22°C ± 20C, 12hrs/12hrs day/night cycle, humidity 55% ± 5%) and had permanent access to water and were fed with a standard rat pelleted diet.
Experiment 1
A first experimental group of mice (n = 20) were inoculated via a stomach tube at day O5 2 and 3 with pathogenic strains of Helicobacter pylori (0.2 ml suspension of Helicobacter pylori, IG9 cfu/ml). Controls (n = 20) received phosphor buffer saline (PBS5 0.2 ml, 0.0 IM). At days 19 to 27, ten mice from experimental and ten mice from control group received Na2AKG gavage via a stomach tube (0.2 ml, 3OmM Na2AKG). The rest of the mice (10 + 10) in the experimental and control groups received PBS (0.2 ml) via a stomach tube. At day 30, all mice were sacrificed with overdose of CO2 and cervical dislocation. Blood and stomach samples for bacteriological and PCR analyses were collected.
Experiment 2 A second experimental group of mice (n = 32) were inoculated via stomach tube at day 0, 2 and 3 with pathogenic strains of Helicobacter pylori (0.2 ml suspension of Helicobacter pylori, 109 cfu/ml). Controls (n — 32) received phosphor buffer saline (PBS, 0.2 ml, 0.01M). At days 13 to 15, sixteen mice from experimental and sixteen mice from control group received Na2AKG gavage via a stomach tube (0.2 ml, 3OmM Na2AKG). The rest of the mice (16 + 16) in the experimental and control groups received PBS (0.2 ml) via a stomach tube. At day 20, all mice were sacrificed with overdose of CO2 and cervical dislocation. Blood and stomach samples for bacteriological and PCR analyses were collected. Results
Table 1 shows the results from an analysis of growth of Helicobacter pylori in the samples taken from the stomach or the blood from animals in experiment 1. Helicobacter pylori culturing were performed on GAB-CAMP and MRSA broth.
Table 1
Figure imgf000010_0001
* Number of animals from which bacteria were isolated / number animals in the experiment. ** The number of colonies is presented in parentheses as cfu/ml.
Table 2 shows the results from an analysis of growth of Helicobacter pylori in the samples taken from the stomach or the blood from animals in experiment 2. Helicobacter pylori culturing were performed on GAB-CAMP and MRSA broth. Table 2 further shows the number of animals which were positive in a PCR analysis for Helicobacter pylori. In the PCR analysis, DNA chain reactions were performed with specific primers which detect Helicobacter spp. 16S rRNA fragments.
Table 2
Figure imgf000010_0002
Figure imgf000011_0001
* Number of animals from which bacteria were isolated or which were positive in the PCR analysis / number animals in the experiment.
** The number of colonies is presented in parentheses as cfu/ml.
Table 3 shows the identification of PCR products of Helicobacter pylori and other bacteria strains from samples taken from the stomach of mice of experiment 2. PCR products were assayed in order to identify 16S rDNA fragments which are characteristic for the different species of bacteria.
Table 3
Figure imgf000011_0002
Discussion
Orally applied alpha-ketoglutaric acid eradicates pathogenic Helicobacter pylori previously inoculated in the stomach of experimental model mice. This event happened already after 3 days OfNa2AKG treatment. Ten days OfNa2AKG treatment was effective in 90% of the infected animals. These findings provide evidence that oral alpha-ketoglutaric acid can be an effective drag for treatment of pathogenic strains of 'Helicobacter pylori.
While the preferred embodiment of the present invention has been described in detail by the examples, it is apparent that modifications and adaptations of the present invention will occur to those skilled in the art. It is to be expressly understood., however, that such modifications and adaptations are within the scope of the present invention, as set forth by the claims.

Claims

1. Use of sodium-alpha-ketoglutaric acid, for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori in birds and mammals, including man, wherein diseases related to infection of pathogenic strains of Helicobacter pylori include gastritis, gastric and duodenal ulcers, peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma.
2. Use of sodium-alpha-ketoglutaric acid, for the manufacture of a pharmaceutical preparation or a food or feed supplement for the treatment or prophylaxis of diseases related to infection of pathogenic strains of Helicobacter pylori in birds and mammals, including man.
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