WO2020209059A1 - Composition pharmaceutique destinée au traitement de l'hyperammoniémie - Google Patents

Composition pharmaceutique destinée au traitement de l'hyperammoniémie Download PDF

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WO2020209059A1
WO2020209059A1 PCT/JP2020/013220 JP2020013220W WO2020209059A1 WO 2020209059 A1 WO2020209059 A1 WO 2020209059A1 JP 2020013220 W JP2020013220 W JP 2020013220W WO 2020209059 A1 WO2020209059 A1 WO 2020209059A1
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hyperammonemia
glucose
acid
ketoglutaric acid
dkg
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Japanese (ja)
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信 芳野
知之 ▲高▼橋
福井　香織
直忠 石原
比呂志 長嶋
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学校法人 久留米大学
学校法人明治大学
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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
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/225—Polycarboxylic acids
    • 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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • Hyperammonemia is a disease in which blood ammonia concentration increases due to various factors including genetic disorders.
  • liver transplantation can be expected to be stable for a long period of time if engrafted, but its implementation requires a considerable preparation period.
  • hyperammonemia attacks once onset, worsen on an hourly basis and are not uncommon for death, and even if life can be saved, there is a risk of leaving neurological damage. Therefore, improvement of treatment for hyperammonemia is essential.
  • treatments for hyperammonemia include nutritional treatment (restriction of protein intake and supply of non-protein calorie), nitrogen alternative pathway activation therapy, N-carbamylglutamic acid administration, urea cycle intermediate metabolite replacement therapy, There are blood purification therapies, and in many cases, some of them are used together. However, these are often treatments that negatively affect nitrogen equilibrium, and the effect of restoring protein synthesis (anabolism) cannot be expected except for the supply of non-proteinaceous calories.
  • the object of the present disclosure is to provide a therapeutic agent for hyperammonemia.
  • the present disclosure provides a pharmaceutical composition comprising ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof for treating hyperammonemia.
  • the therapeutic agent of the present disclosure can reduce the blood ammonia concentration by a mechanism different from that of existing therapeutic agents and treatment methods, and is an effective option for the treatment of hyperammonemia.
  • FIG. 1 shows the time course of the ammonia concentration in the culture medium of wild-type mouse fetal fibroblasts (WTMEF). Each column shows a cell-free blank concentration, a gross concentration, and a net ammonia concentration.
  • FIG. 2 shows the ammonia concentration in the culture solution of WTMEF in the presence or absence of glucose at each concentration.
  • FIG. 3 is a diagram showing the effect of glucose deficiency on body protein catabolism and amino acid degradation.
  • AMPK AMP-activated protein kinase
  • CPS carbamyl phosphate synthase
  • DAc deacetylation
  • GDH glutamine dehydrogenase
  • GLS glutaminenase
  • GLUL glutamine synthase
  • OTC ornithine transcarbamylase
  • PGC-1 ⁇ peroxysome Growth factor activated receptor ⁇ (PPAR ⁇ ) coactivator-1 ⁇
  • SIRT sirtuin
  • Cit citric acid; ⁇ -KG, ⁇ -ketoglutaric acid
  • OAA oxaloacetate
  • Fum fumaric acid
  • Citr citrulin
  • AS Argininosuccinic acid
  • Orn ornithine
  • Arg arginine
  • Glu glutamine acid
  • Gln glutamine.
  • FIG. 4 shows the ammonia concentration in Atg5 -/- MEF, which is a MEF in which the genes of WT MEF and Atg5 are knocked out, under glucose sufficiency or deficiency conditions.
  • FIG. 5 shows the effect of dimethyl- ⁇ -ketoglutaric acid (DKG) on the ammonia concentration under glucose sufficiency or deficiency conditions in WTMEF.
  • FIG. 6 shows the concentration of each amino acid in the culture medium when DKG was added to WTMEF under glucose-sufficient or deficient conditions.
  • FIG. 7 shows that WTMEF was cultured under the conditions of glucose addition or non-addition and DKG was not added or added, and the cell-free blank values of ammonia and amino acid concentrations in the culture broth were shown.
  • FIG. 8 shows the gene expression levels of glutamate dehydrogenase I (Glud1), glutaminase (Gls), and glutamine synthetase (Glul) in WT MEF and Atg5 -/- MEF. Both are displayed as a relative ratio to the expression level of the Gapdh gene.
  • FIG. 9 shows the effect of rapamycin on the ammonia concentration under glucose sufficiency in WTMEF.
  • FIG. 10 shows the gene expression levels of Sirtuin (Sirt) 3, 4 and 5 in WT MEF and Atg5 -/- MEF with or without glucose.
  • FIG. 11 shows the effect of DKG infusion on blood ammonia concentration in ornithine transcarbamylase (OTC) deficiency model pigs. “Control range” indicates the range of blood ammonia concentration in healthy newborn piglets.
  • FIG. 12 shows changes in plasma ammonia concentration in ammonium chloride-loaded mice.
  • FIG. 13 shows the effect of DKG and ⁇ -ketoglutaric acid (AKG) on plasma ammonia concentration in experimental hyperammonemia model mice. S: Saline, N: Ammonium chloride, D: DKG, A: AKG.
  • FIG. 14 shows changes in amino acids in plasma in experimental hyperammonemia model mice.
  • FIG. 15 shows the activation of AMP-activated protein kinase (AMPK) under glucose-sufficient or deficient conditions in WTMEF and the effect of DKG on it. The results are shown by the ratio of protein concentration between phosphorylated (active) AMPK and non-phosphorylated (inactive) AMPK (pAMPK / AMPK).
  • FIG. 16 shows the activation of S6 kinase 1 (S6K1) under glucose sufficiency or deficiency conditions in WTMEF and the effect of DKG on it. The results are shown by the ratio of protein concentration between phosphorylated (active) S6K1 and non-phosphorylated (inactive) S6K1 (pS6K1 / S6K1).
  • S6 kinase 1 S6 kinase 1
  • FIG. 17 shows the activation of 4E-BP1 under glucose sufficiency or deficiency conditions in WTMEF and the effect of DKG on it. The results are shown by the ratio of protein concentration between phosphorylated (active) 4E-BP1 and non-phosphorylated (inactive) 4E-BP1 (p4E-BP1 / 4E-BP1).
  • FIG. 18 shows the progress of autophagy under glucose sufficiency or deficiency conditions in WTMEF and the effect of DKG on it. The results are shown by the ratio of protein concentration between non-lipidized LC3 (LC3-I) and lipidized LC3 (LC3-II) (LC3-I / LC3-II).
  • Hyperammonemia is a disease in which the concentration of ammonia in the blood increases due to various factors including genetic disorders. Ammonia in the body is produced in the process of protein metabolism and is detoxified by being synthesized into urea and excreted in the urea cycle of the liver. "Hyperammonemia" in the present disclosure is not limited to hyperammonemia due to a specific cause. Hyperammonemia is primary hyperammonemia due to genetic disorders of enzymes and transporters of the urea cycle, and secondary hyperammonemia in which the urea cycle is secondarily affected by disorders of other pathways. It is divided into. Hyperammonemia is caused by inborn errors of metabolism, liver damage, formation of portosystemic shunts, infections, and drugs.
  • Inborn errors of metabolism include urea cycle disorders, citrin deficiency, lysine protein intolerance, hyperornithineemia / hyperammonemia / homocitrullineuria syndrome (HHH syndrome), and organic acid metabolism disorders. Examples include fatty acid metabolism disorders.
  • hyperammonemia results from urea cycle abnormalities.
  • Urea cycle disorders present with hyperammonemia due to a genetic disorder in the process of producing urea in the urea cycle.
  • Enzymes involved in the urea cycle include N-acetylglutamate synthase (NAGS), carbamylphosphate synthase (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate degrading enzyme (ASL), Arginase 1 (ARG1) can be mentioned.
  • Urea cycle disorders include NAGS deficiency, CPS1 deficiency, OTC deficiency, citrullinemia type I, argininosuccinic aciduria, and arginineemia.
  • hyperammonemia results from citrine deficiency, lysine urinary protein intolerance, HHH syndrome.
  • hyperammonemia is due to an organic acid metabolism disorder.
  • Organic acid metabolic disorders are diseases in which organic acids, which are intermediate metabolites, accumulate due to enzyme abnormalities related to metabolic pathways such as amino acids and fatty acids, causing various symptoms. Examples of organic acid metabolism disorders include methylmalonic acidemia, propionic acidemia, multiple caboxylase deficiency, isovaleric acidemia, glutaric acidemia type 1 and the like.
  • hyperammonemia results from fatty acid metabolism disorders.
  • Fatty acid metabolism disorders are diseases caused by abnormalities in the carnitine circuit for the uptake of fatty acids into mitochondria or the fatty acid ⁇ -oxidation system.
  • Fatty acid metabolism disorders include very long chain acyl-CoA dehydrogenase deficiency, triad enzyme deficiency, medium chain acyl-CoA dehydrogenase deficiency, CPT1 (carnitine palmitoyl transferase 1) deficiency, and CACT (carnitine acyl).
  • Carnitine translocase deficiency
  • CPT21 carnitine palmitoyl transferase 2
  • OCTN2 deficiency systemic carnitine deficiency
  • glutaricemia type 2 and the like.
  • hyperammonemia is caused by liver damage.
  • Liver disorders as used herein include any disorder that reduces the detoxification of ammonia in the liver. Liver disorders include cirrhosis or fulminant hepatitis.
  • the subject may be a human or non-human animal such as a mouse, hamster, rat, guinea pig, rabbit, dog, monkey, etc., and is preferably a human.
  • a human or non-human animal such as a mouse, hamster, rat, guinea pig, rabbit, dog, monkey, etc.
  • the reference value of the blood ammonia concentration judged to be hyperammonemia is not limited, but for example, in the case of humans, it can be 120 ⁇ g / dL or more in newborns and 60 ⁇ g / dL or more (including adults) thereafter. ..
  • Treatment of hyperammonemia includes reduction of blood ammonia concentration and treatment of diseases or symptoms associated with hyperammonemia.
  • Treatment of a disease or symptom associated with hyperammonemia includes alleviation or elimination of the disease or symptom or suppression of its progression.
  • Symptoms associated with hyperammonemia include impaired consciousness, behavioral abnormalities, coma, cerebral edema, vomiting, and convulsions.
  • the subject may have any of the diseases or symptoms (eg, the diseases or symptoms described herein) that cause or are associated with hyperammonemia.
  • the subject suffers from liver damage (eg, cirrhosis or fulminant hepatitis).
  • the subject suffers from an inborn error of metabolism (eg, urea cycle disorder, citrine deficiency, lysine urinary protein intolerance, HHH syndrome, organic acid metabolism disorder or fatty acid metabolism disorder). There is.
  • compositions of the present disclosure are ⁇ -ketoglutaric acid (also referred to herein as AKG or ⁇ -KG) or dimethyl- ⁇ -ketoglutaric acid (also referred to herein as DKG) or pharmaceutically acceptable thereof. Contains salt to be made. ⁇ -ketoglutaric acid Dimethyl- ⁇ -ketoglutaric acid
  • salts include salts with inorganic acids (eg, hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid); organic acids (eg, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, apples).
  • inorganic acids eg, hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid
  • organic acids eg, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, apples.
  • Salts with acids citric acid, tartaric acid, lactic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid or naphthalenedisulfonic acid; alkaline earth metal salts (eg sodium salt, calcium salt, magnesium) Salt, potassium salt); salt with arginine, leucine, isoleucine, pyridoxin, chitosan, creatin or ortinin.
  • the pharmaceutically acceptable salts of ⁇ -ketoglutaric acid are sodium ⁇ -ketoglutarate, calcium ⁇ -ketoglutarate, magnesium ⁇ -ketoglutarate, potassium ⁇ -ketoglutarate.
  • the pharmaceutically acceptable salt of ⁇ -ketoglutaric acid is sodium ⁇ -ketoglutarate.
  • the pharmaceutically acceptable salt of ⁇ -ketoglutaric acid is ornithine ⁇ -ketoglutaric acid.
  • the effective amount of ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof means an amount capable of exerting a desired therapeutic effect on hyperammonemia.
  • ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof is administered in an amount appropriately selected according to the age, body weight, health condition and the like of the subject to be administered.
  • the ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof is not limited, but is, for example, 0.01 mg to 100 g, 0.1 mg to 10 g, 1 mg to 10 g, or 0.01 g to 1 kg of body weight.
  • ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof is continuously administered even if it is administered once or in multiple times (for example, 2, 3 or 4 times) per day. May be administered daily, daily or days (eg 2, 3, 4, 5 or 6 days), week or week (eg 2, 3, 4, 5 or 6 weeks), 1 month Alternatively, it may be administered at intervals of several months (for example, 2, 3, 4, 5 or 6 months).
  • the duration of administration is also not particularly limited, and is 1 day or several days (for example, 2, 3, 4, 5 or 6 days), 1 week or several weeks (for example, 2, 3, 4, 5 or 6 weeks), 1 It can be months or months (eg 2, 3, 4, 5 or 6 months) or more.
  • the pharmaceutical composition of the present disclosure may contain a pharmaceutically acceptable carrier and / or additive in addition to the active ingredient ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof.
  • Pharmaceutically acceptable carriers include sterile water, saline, propylene glycol, polyethylene glycol, vegetable oil, lactose, mannitol, crystalline cellulose, hydroxypropyl cellulose, corn starch, hydroxypropyl methylcellulose and the like.
  • Additives include disintegrants, stabilizers, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, lubricants and the like.
  • Dosage forms include, but are not limited to, tablets, capsules, powders, granules, liquids, suspensions, injections, suppositories and the like.
  • the pharmaceutical composition can be formulated by a conventional method. Examples of the administration method include oral administration and parenteral administration (for example, intravenous administration, rectal administration, oral administration, nasal administration, intramuscular administration) and the like.
  • the present disclosure provides ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof for use in the treatment of hyperammonemia.
  • the present disclosure provides the use of ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating hyperammonemia.
  • the present disclosure is a method of treating hyperammonemia that requires treatment with an effective amount of ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof. Provided are methods that include administration to.
  • ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof is (1) the production of free amino acids by suppressing the degradation of glutamine (glutaminolysis). Decrease, (2) recovery of cell proliferation and recovery of mRNA translation by supply of ⁇ -ketoglutaric acid to the TCA cycle (anaprerotic reaction), and / or (3) excess autophagy through activation of mTORC1 It is thought that the degradation (deamino acid) of amino acids, which are the source of ammonia, is reduced by the stabilization and the promotion of protein synthesis (assimilation) by stimulating translation of mRNA.
  • ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof can treat hyperammonemia by a mechanism different from that of existing therapeutic agents for hyperammonemia.
  • it is considered to be suitable for children who have a special physiological load compared to adults, which is growth, because it can be expected to have an effect of directing the synthesis of body proteins rather than promoting nitrogen excretion.
  • a pharmaceutical composition comprising ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof for treating hyperammonemia.
  • the pharmaceutical composition according to 1 above wherein hyperammonemia is caused by inborn errors of metabolism.
  • the pharmaceutical composition according to 2 above wherein the inborn error of metabolism is a urea cycle disorder.
  • the pharmaceutical composition according to 1 above wherein hyperammonemia is caused by liver damage.
  • the pharmaceutical composition according to 4 above, wherein the liver disorder is cirrhosis.
  • composition according to any one of 1 to 6 above, wherein the pharmaceutically acceptable salt of ⁇ -ketoglutaric acid is sodium ⁇ -ketoglutarate.
  • pharmaceutical composition according to any one of 1 to 6 above, wherein the pharmaceutically acceptable salt of ⁇ -ketoglutaric acid is ornithine ⁇ -ketoglutaric acid.
  • a method of treating hyperammonemia which comprises administering an effective amount of ⁇ -ketoglutaric acid or dimethyl- ⁇ -ketoglutaric acid or a pharmaceutically acceptable salt thereof to a subject in need of the treatment. ..
  • WTMEF wild-type mouse fetal fibroblasts
  • FBS fetal bovine serum
  • Gln 2 mM glutamine
  • D-MEM Dalveco modified Eagle's medium
  • the culture was carried out in (including glutamine), and the ammonia concentration in the culture solution was measured every 24 hours, and this was taken as the total ammonia concentration (gross concentration).
  • the cell-free blank concentration is defined as the ammonia concentration of the cell-free culture medium hatched in parallel, and the net concentration is the value obtained by subtracting the cell-free blank concentration from the total ammonia concentration. ) was displayed.
  • the ammonia concentration was measured by a method using glutamate dehydrogenase using ammonia and ⁇ -ketoglutaric acid as substrates. As a result, since an increase in the net ammonia concentration was observed with time in this WTMEF (Fig. 1), it was judged that the system can observe the increase and decrease in the net ammonia concentration accompanying the metabolism of cells.
  • glucose deficiency could be used as an in vitro model for inducing hyperammonemia due to starvation.
  • body protein catabolism in ammonia concentration involves two steps: the body protein is first broken down into individual amino acids, and then the amino acids are deaminated to produce free ammonia.
  • Autophagy, ubiquitin-proteasome system, intracytoplasmic protease system, etc. are known as the mechanism of body protein catabolism. The latter two of these are generally limited degradation down to the peptide level, whereas autophagy degrades individual amino acids. Therefore, when ammonia concentration is a problem, autophagy is considered to be the most directly related to the body proteolytic mechanism.
  • Atg5 -/- MEF WT MEF and MEF derived from C57BL / 6x129sv mice in which the Atg5 gene, which is one of the essential proteins for inducing autophagy, was knocked out
  • Atg5 -/- MEF provided by Professor Noboru Mizushima of the University of Tokyo. I received it
  • Atg5 -/- MEF was known to have no steady or inductive autophagy.
  • WT MEF and Atg5 -/- MEF were cultured in the same culture medium as in Experiment 1, and after reaching 50% confluency, each was replaced with a culture medium containing or without 25 mM glucose for an additional 24 hours. The culture was continued and the ammonia concentration was determined (Experiment 3).
  • Atg5 -/- MEF had a significantly lower ammonia concentration than WT MEF (Fig. 4).
  • WT MEF was cultured under the condition of glucose 25 mM or 0 mM
  • the ammonia concentration increased significantly at 0 mM.
  • the amino group of an amino acid is transferred to ⁇ -ketoglutaric acid ( ⁇ -KG) by transamination to produce glutamic acid (Glu) (Fig. 3).
  • Glutamic acid is then converted to glutamine (Gln) by 1) fixing another molecule of ammonia by the action of glutamine synthetase (GLUL), and 2) alanine and ⁇ -KG by amino group transfer reaction with pyruvate.
  • Glutamine dehydrogenase (GDH) action (oxidative deamination reaction) produces free ammonia and ⁇ -KG again. From this, the concentration of free ammonia is regulated by the equilibrium relationship between the decomposition and synthesis of glutamic acid and glutamine. Therefore, it is considered that free ammonia production can be suppressed by suppressing the decomposition of Glu or Gln or promoting the synthesis.
  • Gln the series of processes in which Gln is decomposed and its carbon skeleton is supplied to the TCA cycle is called glutaminolysis. This reaction has a physiological function (anaplerotic reaction) of supplying an oxidizing substrate to the TCA cycle.
  • DKG dimethyl- ⁇ -ketoglutaric acid
  • WTMEF was cultivated under the same conditions as in Experiment 3, and then DKG-free (0 mM) or 1.0 mM or 2.0 mM-added culture broth was added to the culture broth containing 25 mM and the culture broth not containing glucose, respectively. A total of 6 groups were set, and after culturing for 24 hours, the ammonia concentration of each was measured (Experiment 4).
  • the combination of the glucose addition concentration A mM added to the culture solution and the DKG addition concentration B mM is shown as Glc A / DKG B or A / B.
  • glucose is 25 mM
  • DKG When DKG is 0 mM, it is indicated as Glc25 / DKG0 or 25/0).
  • the ammonia concentration was significantly reduced by the addition of 2.0 mM DKG under either the condition of glucose 25 mM or 0 mM (Fig. 5).
  • the ammonia concentration was significantly reduced when comparing the DKG-free group with 1.0 mM, or the 1.0 mM-added group with 2.0 mM. This result suggests that ⁇ -KG is a regulator of ammonia production and that DKG may be a therapeutic agent for hyperammonemia.
  • WT-MEF and Atg5 -/- MEF were cultured in the same culture medium as in Experiment 1, and after reaching a confluency of about 50%, the culture medium was changed to a culture medium containing or not containing 25 mM glucose. The cells were further cultured for 24 hours. Cells were collected, cDNA was prepared according to a conventional method, and quantitative PCR was performed using this as a template. Each primer was designed by Primer3. The expression level of each gene was shown as a relative ratio of the glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh) to that of the gene (Experiment 6).
  • Gapdh glyceraldehyde-3-phosphate dehydrogenase gene
  • Glud1 was significantly increased in WT MEF when glucose was deficient, but the same tendency was observed in Atg5 -/- MEF, but no significant difference was observed (Fig. 8).
  • the expression level of Gls was almost constant regardless of the presence or absence of glucose and WT MEF or Atg5 -/- MEF.
  • the expression level of Glul was significantly lower in Atg5 -/- MEF with and without glucose than in WT MEF, but in comparison with glucose 25 mM with and without glucose, WT MEF and Atg5 -/- MEF No significant difference was observed in the expression level in any of the above.
  • Glud1 is a major regulator of glutamine and glutamate degradation and synthesis at gene expression levels.
  • Glul expression in Atg5 -/- MEF was significantly lower than in WT MEF both under glucose sufficiency and under glucose deficiency. The reason is unknown, but it may be meaningful to ensure an anaplerotic response in Atg5 -/- MEF, where autophagy is not induced even during starvation.
  • mTORC1 which is a molecular complex containing mTOR, centrally regulates the reactions of body proteins in both directions of catabolism (induction of autophagy) and assimilation (initiation of translation).
  • mTORC1 suppressively regulates Sirtuin4 (Sirt4) and Sirtuin5 (Sirt5).
  • Sirt4 and Sirt5 are known to suppress glutaminolysis (Fig. 3). Therefore, the effect of rapamycin, an mTORC1 inhibitor, on the ammonia concentration in the culture medium was investigated.
  • WT-MEF was cultured in the same culture solution as in Experiment 1, and after reaching a confluency of about 50%, a culture solution containing no rapamycin or a culture solution containing 12.5 nM or 25 nM was used for an additional 24 hours. It was cultured. After culturing, the ammonia concentration and the number of cells in the culture solution were measured (Experiment 7).
  • the blood ammonia concentration before administration was 292 ⁇ g / 100 mL.
  • DKG 250 mg / kg was intravenously injected bolus, and then continuous injection was performed at 10.4 mg / kg / hr (250 mg / kg / 24 hr) (Fig. 11).
  • the blood ammonia concentration decreased to 168 ⁇ g / 100 mL at 4 hours after the start of infusion, which was 57.5% of the value before administration.
  • the DKG was changed to a glucose-containing electrolyte solution (SOLULACT® D)
  • the blood ammonia level continued to decrease for the next 2 hours, and 147 ⁇ g / 100 mL 6 hours after the start of injection. It decreased to (50.3%) and then started to increase. From the above results, it was shown that DKG has the effect of lowering the ammonia concentration in vivo.
  • mice mice were examined using mice (Experiment 10).
  • mice we attempted to create a model system for experimental hyperammonemia by loading with ammonium chloride using male mice aged 10 to 14 and weighing 23.9 ⁇ 2.3 g.
  • the blood ammonia concentration reached 8.01 mmol / L, which was 100 times the control value, at 15 minutes after loading, but then gradually decreased, and at 120 minutes, it decreased to 0.24 mmol / L, which was 2.97 times the control value. (Fig. 12).
  • the mean value of blood ammonia concentration was 0.10 mmol / L in the S group (control group), while it was as high as 4.38 mmol / L in the N group (ammonium chloride loaded group) (Fig. 13). ).
  • the N + D group showed 2.00 mmol / L and the N + A group 0.43 mmol / L, which were significantly lower values of 45.7% and 9.8%, respectively, in the blood due to ammonium chloride loading.
  • the inhibitory effect of DKG and AKG on the increase in ammonia concentration was demonstrated. From the above results, it was confirmed that both DKG and AKG have an improving effect on acute hyperammonemia in vivo.
  • WTMEF was first cultured in a culture medium containing 25 mM glucose, and then DKG was added to 0, 1, 2, or 5 in a culture medium containing 25 mM glucose or a culture medium containing no glucose, respectively.
  • the cells were replaced with those supplemented with mM, and after further culturing for 24 hours, the cells were collected and analyzed by Western blotting.
  • AMP-activated protein kinase is activated (phosphorylated) when intracellular ADP increases or ATP / ADP ratio decreases due to glucose deficiency or other causes.
  • AMPK is known to suppress the activity of mTORC1.
  • AMPK tended to be activated under glucose-deficient conditions as compared to glucose-sufficient conditions.
  • DKG tended to suppress AMPK activity with the addition of 1 mM under both glucose-sufficient and deficient conditions, but no clear inhibitory effect was observed at higher concentrations (Fig. 15). ).
  • S6 kinase 1 is an enzyme that phosphorylates the ribosomal protein S6 and regulates protein synthesis and cell cycle. Since S6K1 is activated (phosphorylated) by mTORC1, its phosphorylation is an indicator of mTORC1 activity. Phosphorylation of S6K1 was not different between glucose-sufficient and glucose-deficient conditions, but was enhanced by the addition of DKG in both cases (Fig. 16). In addition, activation (phosphorylation) of 4E-BP1, which is another index of mTORC1 activity, was suppressed under glucose-sufficient conditions as compared with glucose-sufficient conditions, but no obvious change was observed with the addition of DKG. (Fig. 17). It was shown that DKG activates S6K1, suggesting that DKG has the effect of promoting protein synthesis.

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Abstract

L'invention fournit une composition pharmaceutique qui est destinée au traitement de l'hyperammoniémie, et qui contient soit un acide α－cétoglutarique ou un acide diméthyl-α－cétoglutarique, soit un sel pharmaceutiquement acceptable de ceux-ci.
PCT/JP2020/013220 2019-04-11 2020-03-25 Composition pharmaceutique destinée au traitement de l'hyperammoniémie WO2020209059A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3441650A (en) * 1965-07-23 1969-04-29 Lab Jacques Logeais Sa Therapy of ammonical intoxications by di-l-ornithine alpha-ketoglutarate
DE19929993A1 (de) * 1999-06-30 2001-01-11 Sueddeutsche Kalkstickstoff Kreatin-alpha-ketoglutarate, Verfahren zu ihrer Herstellung und ihre Verwendung
WO2016083399A1 (fr) * 2014-11-24 2016-06-02 Forschungsgesellschaft Für Arbeitsphysiologie Und Arbeitsschutz E. V. Alpha-cétoglutarate en combinaison avec la glutamate déshydrogénase pour le traitement de l'hyperammoniémie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3441650A (en) * 1965-07-23 1969-04-29 Lab Jacques Logeais Sa Therapy of ammonical intoxications by di-l-ornithine alpha-ketoglutarate
DE19929993A1 (de) * 1999-06-30 2001-01-11 Sueddeutsche Kalkstickstoff Kreatin-alpha-ketoglutarate, Verfahren zu ihrer Herstellung und ihre Verwendung
WO2016083399A1 (fr) * 2014-11-24 2016-06-02 Forschungsgesellschaft Für Arbeitsphysiologie Und Arbeitsschutz E. V. Alpha-cétoglutarate en combinaison avec la glutamate déshydrogénase pour le traitement de l'hyperammoniémie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DAVULURI, G. ET AL.: "Hyperammonaemia-induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress", THE JOURNAL OF PHYSIOLOGY, vol. 594, no. 24, 2016, pages 7341 - 7360, XP055748357, ISSN: 1469-7793 *

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