WO2023166071A1 - Utilisation de propionate pour diminuer la production de cétone - Google Patents

Utilisation de propionate pour diminuer la production de cétone Download PDF

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WO2023166071A1
WO2023166071A1 PCT/EP2023/055208 EP2023055208W WO2023166071A1 WO 2023166071 A1 WO2023166071 A1 WO 2023166071A1 EP 2023055208 W EP2023055208 W EP 2023055208W WO 2023166071 A1 WO2023166071 A1 WO 2023166071A1
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propionic acid
ketoacidosis
ketogenic diet
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ketone
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English (en)
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Jose Pablo Silva PATRON
Hester MEEUSEN
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N.V. Nutricia
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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
    • 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
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • This invention relates to the use of propionate for the prevention and treatment of ketoacidosis.
  • Ketosis is the state of elevated blood ketone levels resulting from ketogenic diets, caloric restriction, (therapeutic) fasting, starvation and/or supplementation with ketogenic precursors.
  • Ketone bodies represent energy substrates for both peripheral tissues and the central nervous system.
  • the two most abundant and physiologically significant ketone bodies are acetoacetate and p-hydroxybutyrate, while the third ketone body, acetone, is produced as a by-product that the lungs breathe off.
  • ketosis or "nutritional ketosis.”
  • ketoacidosis which is the runaway accumulation of ketone bodies and associated drop in blood pH.
  • Ketoacidosis typically results in blood ketone levels in excess of 3 mmol/L (Sheikh-Ali M, Diabetes Car 2008 Apr; 31 (4): 643-647) or urine ketone levels above 10 mmol/LU in combination with metabolic derangement and electrolyte imbalance.
  • Ketoacidosis can potentially be lifethreatening with initial symptoms that include extreme thirst, frequent urination, dehydration, nausea, vomiting, stomach pain, tiredness, breath that smells fruity, shortness of breath, and feelings of confusion, if not treated potentially followed by severe dehydration, coma and swelling of the brain.
  • ketoacidosis The most common cause of ketoacidosis is diabetic ketoacidosis, but ketoacidosis can also be caused by alcohol, medications, toxins, certain inborn metabolic diseases, certain micronutrient deficiencies, starvation or during the start-up phase of a ketogenic diet.
  • Diabetic ketoacidosis is associated with the absence of insulin as occurs in those suffering from type 1 diabetes or with peripheral insulin resistance as occurs in those suffering from type 2 diabetes. Insulin is necessary for glucose uptake in peripheral tissues and a potent inhibitor of fatty acid release. Consequently, a lack of insulin signalling can impair glucose uptake and utilization in different tissues and can cause an uncontrolled release of fatty acids from adipose tissue.
  • Deficient insulin action can also enhance ketone production through the increased release and availability of fatty acids, which are substrates for ketone production. This can occur during states of complete insulin deficiency such as untreated type 1 diabetes mellitus or states of peripheral insulin resistance such as untreated or undiagnosed type 2 diabetes.
  • ketoacidosis may involve intravenous fluid and electrolyte replacement and sodium bicarbonate which may result in saline overload in severe cases, and a too rapid rise in carbon dioxide level can result in an electrolyte imbalance, a dulling of the senses, coma, or death.
  • a more satisfactory treatment of ketoacidosis would be a most welcome addition to the armamentarium available in the successful management of a patient suffering from ketoacidosis.
  • ketoacidosis can, as discussed above, also occur as a result of other diseases as well as being a potential consequence of a too rapid initiation or excessive response during the initiation of a ketogenic diet. Treatment suffers from the disadvantages and possible side effects which may be severe. There remains thus a need in the art for treatment and prevention of ketoacidosis.
  • the present invention is directed at propionic acid for use in decreasing ketone body production and the treatment and/or prevention of ketoacidosis.
  • the invention provides a strategy to control and/or lower blood ketone levels and reduce the level of ketoacidosis through the administration of a composition comprising therapeutically effective amount of propionate. It was found by the inventors that a small yet therapeutically effective amount of propionic acid reduces ketone production, reduces plasma ketone levels, and thereby prevents and/or treats the occurrence of ketoacidosis, while not negatively affecting the viability of the cells.
  • the invention pertains to the use of propionic acid and compositions comprising propionic acid in the manufacture of a product for (i) decreasing ketone production and/or (ii) preventing and/or treating ketoacidosis in a subject in need thereof.
  • the invention further pertains to the use of propionic acid and compositions comprising propionic acid in the manufacture of a product for reducing the level of ketoacidosis in a subject in need thereof, wherein reducing the level of ketoacidosis is a reduction in blood ketone levels in comparison to the level of ketoacidosis without the use of propionic acid.
  • the invention pertains to a method for (i) decreasing ketone production and/or (ii) preventing and/or treating ketoacidosis in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of propionic acid.
  • the invention further pertains to a method for reducing the level of ketoacidosis in a subject in need thereof, wherein reducing the level of ketoacidosis is a reduction in blood ketone levels in comparison to the level of ketoacidosis without administering propionic acid.
  • the present invention provides for propionic acid, compositions comprising propionic acid, methods of use and use to (i) decrease ketone production and/or (ii) prevent and/or treat ketoacidosis in a subject in need thereof.
  • the subject is a human subject adhering to a ketogenic diet.
  • propionic acid for use in the prevention of diabetic ketoacidosis in a subject with diabetes mellitus adhering to a ketogenic diet.
  • the present invention further provides for a kit of parts comprising (a) propionic acid or derivatives thereof, (b) means to assess the level of blood or urine ketone bodies and (c) means to determine a propionic acid administration regimen.
  • Figure 1 The effect of C3 fatty acids in the claimed therapeutic effective amounts on ketone production by liver cells.
  • Figure 2 The cytotoxic impact of too high amounts of C3 fatty acids on ketone production by liver cells.
  • ketoacidosis in a human subject in need thereof, and wherein said subject is administered therapeutically effective amounts of propionic acid, preferably in a range of 1 mg to 1200 mg propionic acid per day.
  • Propionic acid for use according to embodiment 1 wherein (i) decreasing ketone production and/or (ii) preventing and/or treating ketoacidosis involves lowering blood ketone levels to 3 mM and lower, preferably to ketone levels between 1 .0 mM and 3.0 mM.
  • ketoacidosis is suffering from a condition selected from the list of medical conditions consisting of i) diabetes mellitus type 1 and type 2; ii) starvation ketoacidosis; iii) alcoholic ketoacidosis resulting from acute or chronic alcohol abuse and alcohol-induced liver disease; iv) inborn errors of amino acid metabolism including propionic acidemia, methylmalonic acidemia, and isovaleric acidemia; v) inborn errors of iron metabolism including hereditary hemochromatosis and aceruloplasminemia; vi) inborn errors of biotin (vitamin B7) metabolism including holocarboxylase synthetase deficiency vii) inborn errors of metabolism impairing the utilization of ketones as an metabolic substrate or nutrient including p-ketothiolase deficiency; viii) in
  • ketogenic diet has a ketogenic weight ratio between 1 :1 and 4:1.
  • the present invention provides for propionic acid, compositions comprising propionic acid, methods of use and use in therapeutically effective amounts to (i) decrease ketosis and/or (ii) prevent and/or treat ketoacidosis in a subject in need thereof.
  • the invention provides for reducing the level of ketoacidosis in a subject in need thereof.
  • the present invention further provides for lowering of plasma ketone levels below a concentration of 3mM and treat and/or prevent a blood pH below 7.4.
  • the invention further provides for propionic acid for use in therapeutically effective amounts in a subject initiating a ketogenic diet to enter a state of therapeutic ketosis, wherein initiating a ketogenic diet indicates the stage until a stage of therapeutic ketosis is reached.
  • Initiating a ketogenic diet preferably indicates the initial 1 - 8 weeks wherein the subject is adhering to the diet.
  • Propionic acid is found to be beneficial in decreasing ketone production and/or preventing and/or treating ketoacidosis. It was found that the use of propionic acid allows, when used in therapeutically effective amounts that give rise to (sub-)physiological plasma levels, decreases or diminishes the production of ketones.
  • propionic acid is turned into methylmalonyl-CoA by the enzyme propionyl-CoA carboxylase.
  • the present inventors have now observed that propionic acid is able to block ketone synthesis at (low) physiological concentrations of 1 pM to 10 pM.
  • propionic acid reduces fatty acid oxidation either by a mechanism comparable to malonyl-CoA (which is an inhibitor of carnithine palmitoyl transferase-1 , a protein that mediates the uptake of long chain fatty acids into the mitochondria for subsequent ketone production) or by a conversion through an unknown mechanism of methylmalonyl-CoA, derived from propionic acid, into malonyl-CoA.
  • malonyl-CoA which is an inhibitor of carnithine palmitoyl transferase-1 , a protein that mediates the uptake of long chain fatty acids into the mitochondria for subsequent ketone production
  • a classical ketogenic diet comprises an amount of lipids (by weight), which is typically 4-fold the weight of the sum of proteins and digestible carbohydrates.
  • the so-called ketogenic (weight) ratio is the weight ratio of the amount of lipid to the combined weight amounts of protein and digestible carbohydrates in the composition.
  • the ketogenic diet referred to in the context of the invention may be any type of ketogenic diet known, preferably characterized by a ketogenic weight ratio between 1 :1 and 4:1 which is the ratio of the amount of fat to the combined amounts of protein and digestible carbohydrates.
  • the ketogenic diet used within the context of the invention preferably comprises a lipid content that is at least twice the carbohydrate content by weight.
  • Ketosis refers to a subject having blood ketone levels above 0.5 mmol/L. Ketone levels sustained above 0.5 mmol/L and ideally in the range of 1 to 3 mmol/L appear to offer therapeutic effects in humans [Anderson JC et al. Obes Sci Pract. 2021 ; 7(5):646-656)]. Levels of ketones in the blood above 10 mmol/L are generally associated with signs of ketoacidosis. While ketosis refers to a state of elevated ketones, ketoacidosis is a pathological and potentially life-threatening condition wherein there is an abnormal increase in the ketone concentration amongst others resulting in a decrease in blood pH below 7.4. Ketoacidosis may potentially be life-threatening and may induce a coma. In the context of the invention the subject is a mammal, preferably a human.
  • short chain fatty acyl chain refers to a short chain fatty acid linked by an ester bond.
  • supplement refers to a nutritional product that provides nutrients to an individual that may otherwise not conveniently be consumed in sufficient quantities by said individual. Supplements typically provide the selected nutrients while not representing a significant portion of the overall nutritional needs of the subject. Typically, they do not represent more than 0.1 %, 1 %, 5%, 10% of the daily energy need of the subject.
  • Propionic acid as used herein means any compound that is in the body of an animal convertible to either propionic acid or its deprotonated counter-ion propionate. Examples are provided below.
  • a glyceride is an ester from glycerol and a carboxylic acid.
  • a triglyceride also known as a triacylglycerol
  • triglycerides may be a source of carboxylic acids or fatty acids.
  • tripropionin or glycerol tripropanoate is potentially a source of three moles of propionic acid per mole of tripropionin.
  • Partial glycerides are esters of glycerol with fatty acids, where not all the hydroxyl groups are esterified; mono- and di-propionin are also sources of propionic acid according to the invention, providing for one and two moles of propionic acid per mole respectively.
  • amounts are calculated in terms of the corresponding (mole) amount of propionic acid.
  • an aspect of the present disclosure is propionic acid in an amount therapeutically effective for decreasing ketosis and/or (ii) preventing and/or treating ketoacidosis in a subject in need thereof.
  • decreasing ketone production and/or (ii) preventing and/or treating ketoacidosis involves lowering blood ketone levels to 3 mM and lower, preferably to ketone levels between 1 mM and 3 mM.
  • Decreasing ketone production and/or preventing and/or treating ketoacidosis thus involves administration of propionic acid in an amount to lower blood ketone levels to 3 mM and lower, preferably to ketone levels between 1 mM and 3 mM.
  • the skilled person may monitor blood or urine ketone levels for example using testing strips, enzymatic assays and the like.
  • the subject in need is a human subject on a ketogenic diet and/or suffering from a condition selected from the list of medical conditions consisting of i) diabetes mellitus type 1 and type 2; ii) starvation ketoacidosis; iii) alcoholic ketoacidosis resulting from acute or chronic alcohol abuse and alcohol-induced liver disease iv) inborn errors of amino acid metabolism including propionic academia, methylmalonic academia, and isovaleric academia; v) inborn errors of iron metabolism including hereditary hemochromatosis and aceruloplasminemia; vi) inborn errors of biotin (vitamin B7) metabolism including holocarboxylase synthetase deficiency vii) inborn errors of metabolism impairing the utilization of ketones as an metabolic substrate or nutrient including p-ketothiolase deficiency; viii) inborn errors of thiamine (vitamin B1) metabolism including Rogers syndrome or otherwise called thiamine-responsive megaloblastic anemia;
  • the subject is a human subject on a ketogenic diet and taking drugs which are intended to lower blood glucose levels, said drugs preferably including sodium-glucose cotransporter-2 inhibitors.
  • ketoacidosis are all conditions wherein subjects may suffer from increased ketone body production and/or ketoacidosis that may benefit from propionic acid in an amount effective for decreasing ketosis and/or preventing and/or treating ketoacidosis.
  • propionic acid may also therapeutically prolong the time to reach plasma ketone levels that are associated with a state of ketosis and wherein the occurrence of ketoacidosis is treated and/or prevented.
  • propionic acid is of particular use in uncontrolled diabetes mellitus and/or in a subject adhering to a ketogenic diet.
  • the subject is starting or building up the use of a ketogenic diet to enter a state of therapeutic ketosis.
  • Propionate Propionic acid according to the invention may be present as such in free fatty acid form, as lipids comprising the fatty acids and in the form of a physiologically acceptable salt such as its sodium, potassium or calcium salt.
  • Propionic acid is preferably present in a composition wherein propionic acid/propionate is present as active ingredient.
  • such a composition is consisting essentially of propionic acid.
  • the propionic acid are lipids comprising propionic acid.
  • Lipids comprising propionic acid are preferably selected from the group consisting of triglycerides, diglycerides, monoglycerides, glycolipids, phospholipids and lysophospholipids.
  • the present composition contains triglycerides comprising fatty acyl chains and/or phospholipids comprising fatty acyl chains, more preferably triglycerides comprising fatty acyl chains wherein at least one short chain fatty acyl chain is a propionic acyl chain.
  • the present triglyceride preferably has at least one, more preferably at least two fatty acyl chains, which is preferably at the sn-3 position.
  • propionate may be present in the form of glycerol derivatives monopropionin, dipropionin or tripopionin, preferably as tripropionin.
  • Lipids comprising short chain fatty acyl chains are degraded by lingual, gastric, duodenal (i.e. pancreatic) and small intestinal lipases. Hence, administration of lipids comprising short chain fatty acyl chains results in the release of short chain fatty acids in the stomach, the duodenum, in the jejunum and ileum.
  • propionic acid is in the form of physiologically acceptable salts thereof such as calcium, potassium, sodium, magnesium, ammonium salts of propionic acid.
  • propionic acid is in the form of organic esters of propionic acid, such as for example methyl, ethyl, propyl, or iso-propyl esters such as ethyl propionate.
  • esters may be naturally occurring or may be obtained via chemical reactions for example by condensation of a SCFA and an appropriate alcohol (for example ethylic alcohol).
  • Propionic acid is provided in an amount to provide a daily dosage in the range of 1 mg to 1200 mg per day, preferably 5 mg to 600 mg per day, more preferably 10 mg to 400 mg per day of propionic acid.
  • propionic acid is provided in triglyceride form as tripropionin or glycerol tripropanoate in an amount to provide a daily dosage in the range of 1 mg to 1400 mg per day, preferably 6 to 700 mg per day, more preferably 12 to 500 mg per day.
  • the propionic acid is provided in diglyceride form as dipropionin or glycerol dipropanoate in an amount to provide a daily dosage in the range of 1 mg to 1700 mg per day, preferably 5 to 1000 mg per day, more preferably 15 to 600 mg per day.
  • the propionic acid is provided in monoglyceride form as monopropionin or glycerol monopropanoate in an amount to provide a daily dosage in the range of 2 mg to 2400 mg per day, preferably 10 to 1200 mg per day, more preferably 20 to 800 mg per day.
  • mixture of mono-, di- and/or tripropionin provide the propionic acid according to the invention.
  • propionic acid is for use in a subject adhering to a ketogenic diet.
  • Propionic acid is used and for use together with a ketogenic diet.
  • Propionic acid is preferably provided in a range of 0.002 mg to 0.3 mg propionic acid per kcal of ketogenic diet, more preferably 0.01 mg to 0.2 mg propionic acid per kcal of ketogenic diet, even more preferably 0.02 mg to 0.1 mg propionic acid per kcal of ketogenic diet. The amount calculated is based on the weight of propionate.
  • propionic acid is provided in triglyceride form as tripropionin or glycerol tripropanoate in a range of 0.002 mg to 0.4 mg tripropionin per kcal of ketogenic diet, more preferably 0.01 mg to 0.2 mg tripropionin per kcal of ketogenic diet, even more preferably 0.02 mg to 0.1 mg tripropionin per kcal of ketogenic diet.
  • propionic acid is provided in diglyceride form as dipropionin or glycerol dipropanoate in a range of 0.003 mg to 0.4 mg dipropionin per kcal of ketogenic diet, more preferably 0.01 mg to 0.2 mg dipropionin per kcal of ketogenic diet, even more preferably 0.02 mg to 0.1 mg dipropionin per kcal of ketogenic diet.
  • propionic acid is provided in monoglyceride form as monopropionin or glycerol monopropanoate in a range of 0.004 mg to 0.6 mg monopropionin per kcal of ketogenic diet, more preferably 0.02 mg to 0.3 mg monopropionin per kcal of ketogenic diet, even more preferably 0.06 mg to 0.1 mg monopropionin per kcal of ketogenic diet.
  • the propionic acid of the invention may be provided in any form suitable for enteral, oral or parenteral administration.
  • parenteral administration include intravenously, intramuscularly, intraperitoneally, subcutaneously, intraarticularly, intrasynovially, intraocularly, intrathecally, topically, and inhalation.
  • composition according to the invention may be used as a pharmaceutical product consisting essentially of propionic acid and one or more pharmaceutically acceptable carrier materials.
  • the composition according to the invention may in a preferred aspect be used as a nutritional product, for example as a nutritional supplement, e.g., as an additive to a ketogenic diet.
  • the supplement preferably for enteral application, may be a solid or liquid galenical formulation.
  • solid galenical formulations are tablets, capsules (e.g., hard- or soft-shell gelatine capsules), pills, sachets, powders, granules and the like which contain the active ingredient together with conventional galenical carriers.
  • Any conventional carrier material can be utilized.
  • the carrier material can be organic or inorganic inert carrier material suitable for oral administration and form up to 25 wt% of the supplement, preferably up to 20 wt%, more preferably up to 10 wt%.
  • additives such as flavouring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of nutritional and pharmaceutical compounding.
  • composition according to the invention may also be used as a nutritional product for use together with tube feeding, e.g., as an additive to a tube feed for intermittent or continuous administration. It can also be co-administered with a ketogenic diet.
  • the propionic acid may be provided in one or more dosage units.
  • the dosage unit may be in a liquid form or in a solid form, wherein in the latter case the daily dosage may be provided by one or more solid dosage units, e.g., in one or more capsules or tablets.
  • propionic acid is provided one to six times per day.
  • the administration occurs until a detectable improvement in the plasma level of ketone bodies occurs and, in further cases, continues to remain ameliorated.
  • the ideal duration of the administration of the composition can be determined by those of skill in the art but is typically only if and as long as needed in a subject at risk of or suffering from ketoacidosis.
  • Plasma levels of ketone bodies are determined by means known in the art such as testing strips or enzymatic assays.
  • kit of parts comprising a) propionic acid (b) means to assess the level of blood or urine ketone bodies and (c) means to determine a propionic acid administration regimen.
  • Means to assess the level of blood or urine ketone bodies are known in the art and include but are not limited to blood and urine test strips and spectrophotometric assays.
  • the kit of parts may further include (d) one or more nutritional products having a ketogenic ratio between 1 :1 and 4:1. It is particularly beneficial during the start-up phase of a ketogenic diet when and until a subject transits into ketosis to provide for said kit of parts.
  • said kit of parts for use in (i) decreasing ketosis and/or (ii) preventing and/or treating ketoacidosis in a subject in need thereof.
  • Hepa1-6 cell line An assay to measure ketone production in mouse liver cells (Hepa1-6 cell line) was used wherein cells were first grown to a monolayer (growth phase), then exposed to starvation to deplete cellular lipid stores and subsequently brought into a ketogenic phase wherein cells were cultured in presence of oleic acid or a ketogenic diet fat blend with or without the addition of propionic acid (C3).
  • the ketone body production was subsequently determined by measurement of beta-hydroxybutyrate in the cell culture medium according to the details set out below:
  • Dulbecco Modified Eagle Medium
  • FBS Fetal Bovine serum
  • FBS Fetal Bovine serum
  • antibiotics (1 % penicillin & streptomycin
  • Starvation phase At the end of the growth phase the cells formed a monolayer. The medium was exchanged for DMEM containing 1 mM glucose and antibiotics (1 % penicillin & streptomycin) but no FBS. The medium was further supplemented with L-carnitine (a required co-factor forthe uptake of fatty acids into the mitochondria, where fatty acids are turned into ketones). The cells were incubated in this medium for 24 hours. During this time, the cells were vastly deprived of glucose, lipids and hormones contained in FBS, forcing them to consume and empty their lipid stores. Thereby, any interference with the nutrient testing was avoided. Moreover, the starvation phase is thought to induce specific metabolic changes that maximize the production of ketones.
  • Ketogenic phase At the end of the starvation phase, the medium was exchanged for Krebs-Henseleit buffer (KHB) supplemented with the test nutrients (table 1 , single fatty acids or fatty acid blends) and L- carnitine.
  • KHB Krebs-Henseleit buffer
  • the fatty acid blend is contained in a similar fatty acid composition to the one used in a ketogenic diet (here denoted as ”K-one”).
  • the content of every fatty acid in the blend is given as the molar ratio to Oleic acid (OA), whose molar content is set to 1.
  • Concentrations of individual fatty acids in 100 pM of the blend are shown in Table 1 . Dilutions of this blend were used to determine the dose response curve in figure 1 D. C3 was not included in the blend.
  • Table 1 Composition of fatty acid blend.
  • KHB is composed of salts and bicarbonate as a chemical buffer to maintain the osmotic pressure and acidity (pH) at physiological levels.
  • Medium and long chain fatty acids were coupled to bovine serum albumin prior to the assay to make them soluble in KHB and allow their efficient uptake by the cells.
  • medium and long chain fatty acids are normally coupled to albumin for transport in the blood circulation.
  • Short chain fatty acids such as C3 are water-soluble and were not coupled to albumin in the assay or in humans.
  • the cells were incubated for 6 hours at 37 °C. During this time, they produced ketones from the test nutrients and secreted beta-hydroxybutyrate (p-Hb) into the medium.
  • p-Hb measurements At the end of the ketogenic phase, the whole medium was collected and dried by vacuum centrifugation for 20 hours at 21 °C. The dry pellet was resuspended in a small volume of assay buffer to yield a 10-20 fold higher p-Hb concentration than in the medium. The samples were deproteinized using spin columns to measure the free unbound fraction of p-Hb. The p-Hb concentration was then measured by an enzymatic reaction that generates a fluorescent product (Cayman Chemicals 700740). The Fluorescence was measured on a fluorescence microplate reader and is directly proportional to the p-Hb concentration in the sample within a range of 0.1 pM to 50 pM p-Hb.
  • a blend of medium, long, and very long chain fatty acids contained in a ketogenic diet resulted in top level production of pHb at a total fatty acid concentration of 100 pM (figure 1 D).
  • the total fatty acid concentration in the blood plasma of humans on a ketogenic diet vastly exceeds 100 pM.
  • C3 within a low physiological concentration of 1 pM to 10pM inhibits the top-level production of ketones (pHb) from a single fatty acid (oleic acid, OA) ( Figure 1A) and from a blend of fatty acids contained in a ketogenic diet (denoted as K-one) ( Figure 1 B).
  • the fat blend did not contain C3 (Table 1), meaning that the inhibition is indeed due to the administration of a low concentration of C3.
  • the single fatty acid (oleic acid) and the fat blend (K-one) were tested with C3 at a concentration of 100 pM, when they both maximize ketone production on their own ( Figures 1 C, D) and would maximize the ketone production of humans on a ketogenic diet.
  • C3 inhibits the top-level production of ketones from a single fatty acid or a blend of fatty acids under ketogenic diet-like treatment conditions.
  • Hepa1-6 cell line An assay to measure ketone production in mouse liver cells (Hepa1-6 cell line) was used as described above wherein cells were first grown to a monolayer (growth phase), then exposed to starvation to deplete cellular lipid stores and subsequently brought into a ketogenic phase wherein cells were cultured in presence of oleic acid or a ketogenic diet fat blend with or without the addition of propionic acid (C3). Cells were exposed to C3 during the 6-hour ketogenic phase for assessment of short-term effects of C3 or during the 24-hour starvation plus 6-hour ketogenic phase for assessment of longer-term effects of C3. The impact on cell viability was assessed by measuring the ATP content of the cells as a proxy for the number of alive cells using a bioluminescence assay.
  • DMEM Modified Eagle Medium
  • FBS Fetal Bovine serum
  • FBS Fetal Bovine serum
  • FBS Fetal Bovine serum
  • Starvation phase At the end of the growth phase the cells formed a monolayer. The medium was exchanged for DMEM containing 1 mM glucose and antibiotics (1 % penicillin & streptomycin) but no FBS.
  • the medium was further supplemented with L-carnitine (a required co-factor for the uptake of fatty acids into the mitochondria, where fatty acids are turned into ketones).
  • L-carnitine a required co-factor for the uptake of fatty acids into the mitochondria, where fatty acids are turned into ketones.
  • the cells were further supplemented with a concentration range of C3 from 300uM to 10mM. The cells were incubated in this medium for 24 hours. The cells in the control conditions were incubated in the same starvation medium without addition of C3.
  • Ketogenic phase At the end of the starvation phase, the medium was exchanged for Krebs-Henseleit buffer (KHB) supplemented with the L-carnitine and C3 in a concentration range from 300uM to 10mM.
  • KHB Krebs-Henseleit buffer
  • the cells in the control conditions were incubated in the same starvation medium without addition of C3.
  • the number of alive cells is measured by measuring the ATP content with ATP Determination Kit with recombinant firefly luciferase and its substrate luciferin (provided by ThermoFischer, ATP Determination Kit 200-1 ,000 assays catalogue number: A22066).
  • ATP is the energy source of all living cells and is involved in many vital biochemical reactions. When cells are unhealthy, they stop synthesizing ATP and the existing ATP pool is quickly degraded. This is eventually followed by the death of the cells, which is an endpoint of such unhealthy state. Therefore, ATP is widely accepted as a marker of viable (and healthy) cells. Higher ATP concentration indicates higher number of living cells. ATP assays are procedures that can measure cell viability based on detection of ATP. All living cells, including bacteria, can be detected with ATP assays. Several detection methods can be used, such as colorimetric, fluorescent and bioluminescent. In the present invention bioluminescent ATP assays were chosen to measure cell viability due to higher sensitivity, simple and homogeneous protocol, fast and accurate results.

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Abstract

La présente invention concerne de l'acide propionique pour diminuer la production de cétone et/ou prévenir et/ou traiter la cétoacidose chez un sujet humain en ayant besoin et ledit sujet étant administré en quantités thérapeutiquement efficaces d'acide propionique, de préférence dans une plage de 1 mg à 1200 mg d'acide propionique par jour.
PCT/EP2023/055208 2022-03-02 2023-03-01 Utilisation de propionate pour diminuer la production de cétone WO2023166071A1 (fr)

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Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
ANDERSON JC ET AL., OBES SCI PRACT, vol. 7, no. 5, 2021, pages 646 - 656
BRASS BIOCHEMICAL JOURNAL, 1988
BRASS E P ET AL: "Effects of propionate and carnitine on the hepatic oxidation of short- and medium-chain-length fatty acids", vol. 250, no. 3, 15 March 1988 (1988-03-15), GB, pages 819 - 825, XP055933809, ISSN: 0264-6021, Retrieved from the Internet <URL:https://portlandpress.com/biochemj/article-pdf/250/3/819/633776/bj2500819.pdf> DOI: 10.1042/bj2500819 *
BUSH CANADIAN JOURNAL OF ANIMAL SCIENCE, 1971
BUSH R. S. ET AL: "STUDY OF THE MECHANISM OF INHIBITION OF KETOGENESIS BY PROPIONATE IN BOVINE LIVER", vol. 51, no. 1, 1 April 1971 (1971-04-01), CA, pages 121 - 127, XP055933519, ISSN: 0008-3984, Retrieved from the Internet <URL:http://dx.doi.org/10.4141/cjas71-016> DOI: 10.4141/cjas71-016 *
EMMANUEL BIJAN ET AL: "Effect of Propionic Acid on Ketogenesis in Lactating Sheep Fed Restricted Rations or Deprived of Food", vol. 67, no. 2, 1 February 1984 (1984-02-01), US, pages 344 - 350, XP055933523, ISSN: 0022-0302, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0022030284813077/pdf?md5=7663573fa0e6d55536bec5a804d0cb53&pid=1-s2.0-S0022030284813077-main.pdf> DOI: 10.3168/jds.S0022-0302(84)81307-7 *
EMMANUEL BIJAN, JOURNAL OF DAIRY SCIENCE, 1984
GART EVELINE ET AL: "Propionic acid intervention in obese Ldlr-/-.Leiden mice attenuates NASH development, but negatively affects cognition", PROCEEDINGS OF THE NUTRITION SOCIETY, vol. 79, no. OCE2, 1 January 2020 (2020-01-01), GB, XP055933649, ISSN: 0029-6651, DOI: 10.1017/S0029665120004498 *
PETITET SÉBASTIEN ET AL: "Effects of propionate on rat hepatocyte metabolism", THE JOURNAL OF NUTRITIONAL BIOCHEMISTRY, vol. 9, no. 11, 1 November 1998 (1998-11-01), AMSTERDAM, NL, pages 652 - 658, XP055933702, ISSN: 0955-2863, DOI: 10.1016/S0955-2863(98)00073-4 *
PETITET, THE J OF NUTRITIONAL BIOCHEMISTRY, 1998
SHEIKH-ALI M, DIABETES CAR, vol. 31, no. 4, April 2008 (2008-04-01), pages 643 - 647
WANG JOURNAL OF DAIRY SCIENCE, 2022
WANG XINGHUI ET AL: "Propionate alleviates fatty acid-induced mitochondrial dysfunction, oxidative stress, and apoptosis by upregulating PPARG coactivator 1 alpha in hepatocytes", JOURNAL OF DAIRY SCIENCE, AMERICAN DAIRY SCIENCE ASSOCIATION, US, vol. 105, no. 5, 16 February 2022 (2022-02-16), pages 4581 - 4592, XP087031344, ISSN: 0022-0302, [retrieved on 20220216], DOI: 10.3168/JDS.2021-21198 *

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