EP4669130A1 - KETOGENIC DIET - Google Patents

KETOGENIC DIET

Info

Publication number
EP4669130A1
EP4669130A1 EP24705192.3A EP24705192A EP4669130A1 EP 4669130 A1 EP4669130 A1 EP 4669130A1 EP 24705192 A EP24705192 A EP 24705192A EP 4669130 A1 EP4669130 A1 EP 4669130A1
Authority
EP
European Patent Office
Prior art keywords
ketogenic
ketogenic composition
enteral
fatty acids
epilepsy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24705192.3A
Other languages
German (de)
French (fr)
Inventor
Jose Pablo Silva
Hester MEEUSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nutricia NV
Original Assignee
Nutricia NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nutricia NV filed Critical Nutricia NV
Publication of EP4669130A1 publication Critical patent/EP4669130A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • 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
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
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    • 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
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    • 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
    • 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
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • 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
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/717—Celluloses
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/718—Starch or degraded starch, e.g. amylose, amylopectin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/733—Fructosans, e.g. inulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • adenosine triphosphate ATP
  • the KD is thought to result in adaptive changes to energy metabolism, amongst others in the brain, that increase the energy production. In neurological disorders this is believed to help the neurons to remain functional and viable in the face of increased energy demand and may confer a neuroprotective effect.
  • Studies have shown a clear benefit of the KD and ketosis in treating intractable epilepsy in children and adults. Short-term trials have demonstrated that approximately half of those studies had at least a 50% reduction in seizures after 6 months, and approximately one third reached at least a 90% reduction.
  • the ketogenic diet is sufficiently effective to enable a reduction in, or cessation of prescribed anti-epileptic medication and to improve quality of life.
  • Ketogenic diets must be strictly controlled to ensure effective seizure prevention whilst maintaining adequate nutritional value. Although clearly effective, ketogenic diets are difficult to manage in both children and adults resulting in poor patient compliance. Current approaches to the ketogenic diet can be very restrictive and this can lead to poor compliance, particularly in adults. Furthermore, such diets can result in a number of side effects (including problems of the digestive system owing amongst others to the excess amount of fats, increased serum lipid levels, drowsiness, poor growth and increased risk of bone fractures). Accordingly, there is a continued need for compositions that do not suffer from the disadvantages while still providing the desired therapeutic effects.
  • even-chain long-chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction
  • the even-chain LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC- PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids, and wherein the ketogenic composition has a ketogenic weight ratio between 1 .5:1 and 3.0:1.
  • the inventors observed that the therapeutic benefits comprise preventing and/or treating behavioural consequences of epilepsy.
  • Attention Deficit hyperactivity Disorder [ADHD] is frequently associated with epilepsy.
  • the inventors observed that the behavioural patterns associated with ADHD are diminished and/or prevented using the enteral ketogenic composition according to the invention.
  • the behavioural patterns associated with ADHD comprise hyperactive behaviour, impulsive behaviour and/or fearless behaviour.
  • the invention also relates to a method for treatment and/or prevention of epilepsy and/or progression of epilepsy in a subject suffering from epilepsy or at risk of seizures comprising administering to the subject the enteral ketogenic composition.
  • the method for treatment and/or prevention involves preventing abnormal elevations of blood triglyceride levels, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures.
  • the method for treatment and/or prevention of epilepsy and/or progression of epilepsy further comprises diminishing and/or preventing behavioural patterns associated with ADHD.
  • the present invention provides enteral ketogenic compositions, methods of use and use of said enteral ketogenic compositions to treat and/or prevent epilepsy, the progression of epilepsy and to prevent abnormal elevations of blood triglyceride levels, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
  • Figure 2 shows the distribution of seizure scores after a number of stimulations and the latency to seizure stage 4, 5, or fully kindled status across the experimental groups.
  • FIG 4 A shows the afterdischarge pattern following initial 10s electrical stimulation
  • B shows the duration of the first part of afterdischarge (AD1) across the experimental groups
  • C shows the latency to the second part of afterdischarge (AD2) across the experimental groups.
  • Figure 5 shows the centre zone entries and time spent in the centre zone across the experimental groups.
  • Figure 6 shows activity and exploration of the animals in the different experimental groups.
  • Figure 7 shows the locomotion of the animals in the different experimental groups.
  • Figure 8 shows the blood ketone levels of the animals in the different experimental groups after 5 days on the diet.
  • Figure 9 shows blood ketone levels of the animals in the different experimental groups after 21 days on the diet in the different experimental groups.
  • Figure 10 shows triglyceride serum levels of non-fasted rats after 5 and 21 days on the diet in the different experimental groups.
  • FIG 11 shows glucose serum levels of non-fasted rats after 5 and 21 days on the diet in the different experimental groups.
  • Figure 12 shows omega-3 and omega-6 PUFA serum levels of non-fasted rats after 5 days of dietary exposure in the different experimental groups.
  • Figure 14 shows specific LCFA serum levels of non-fasted rats after 5 days of diet in the different experimental groups.
  • Figure 15 shows specific LCFA serum levels of non-fasted rats after 21 days of diet in the different experimental groups.
  • Figure 16 shows liver triglyceride content in mg per g liver in the different experimental groups.
  • Figure 17 shows body weight development over time in the different experimental groups.
  • Figure 18 shows serum essential amino acid levels after 5 days of diet in the different experimental groups.
  • the alternative ketogenic diet provided higher levels of essential amino acids than a rodent classic ketogenic diet at day 5 of diet exposure.
  • Figure 19 shows serum essential amino acid levels after 21 days of diet in the different experimental groups.
  • the alternative ketogenic diet provided higher levels of essential amino acids than a rodent classic ketogenic diet at day 21 of diet exposure.
  • Figure 21 shows serum amino acid levels of amino acids associated with blockage of seizures after 21 days of diet in the different experimental groups.
  • the alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 21 of diet exposure.
  • Figure 22 shows serum amino acid levels of amino acids associated with blockage of seizures after 5 days of diet in the different experimental groups.
  • the alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 5 of diet exposure.
  • Figure 23 shows serum amino acid levels of amino acids associated with blockage of seizures after 21 days of diet in the different experimental groups.
  • the alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 21 of diet exposure.
  • Figure 24 shows the effect of stimulation of neuroglial cell cultures with 0.1 mM Mg 2+ on oscillation frequency, amplitude and area under the curve as well as the effect on glycolysis and mitochondrial respiration as compared to unstimulated neuroglial cell cultures maintained at 0.8 mM Mg 2+ .
  • Figure 25 shows the effect of a fat blend according to the invention (the INV-KD fat blend) and a comparative fat blend with moderate amounts of MCTs (COMP-KD fat blend) on mitochondrial respiration in hyperexcited (seizure-like) neuroglial cell cultures.
  • the effect on glycolysis, ATP production, and the values for proton leak measured after exposure to each fat blend is also reported.
  • Enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, wherein the lipid fraction comprises
  • MCFAs medium-chain triglycerides fatty acids
  • MCTs medium chain triglycerides
  • LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC-PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids based on total weight of the protein fraction, and wherein the ketogenic composition has a ketogenic weight ratio between 1 .5:1 and 3.0:1
  • composition further comprises 7 - 15 wt% dietary fibers and wherein the fibers are selected from oligofructose, inulin, resistant starch, cellulose, wheat bran, gum arabic, soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin and hydrolysed pectin.
  • fibers are selected from oligofructose, inulin, resistant starch, cellulose, wheat bran, gum arabic, soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin and hydrolysed pectin.
  • the enteral ketogenic composition according to embodiment 2 wherein the fibers are a fiber mixture comprising cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch.
  • enteral ketogenic composition according to the preceding embodiments wherein the low glycemic index carbohydrates are selected from trehalose, lactose, galactose and isomaltulose.
  • enteral ketogenic composition according to the preceding embodiments wherein the protein fraction comprises intact and/or (partly) hydrolysed protein selected from pea, soy, casein and/or whey, preferably casein.
  • composition comprises mono-unsaturated fatty acids [MUFAs], and wherein at least 85 wt% of the MUFAs is oleic acid, based on total weight of the MUFAs in the lipid fraction.
  • MUFAs mono-unsaturated fatty acids
  • Enteral ketogenic composition according to the preceding embodiments for use in the treatment and/or prevention of epilepsy in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
  • the enteral ketogenic composition according to embodiment 13 for use in the treatment and/or prevention of epilepsy comprises treatment and/or prevention and/or reduction of the severity of epilepsy and prevention and/or delay of the formation of epileptiform circuits in the brain.
  • the ketogenic composition according to the invention comprises a lipid fraction, preferably a lipid fraction suitable for nutrition as known in the art.
  • the ketogenic composition preferably comprises 45 to 65 g lipid per 100 g, more preferably 50 - 60 g lipid per 100 g, even more preferably 54 - 56 g per 100 g weight of the ketogenic composition.
  • the ketogenic composition preferably comprises 45 to 65 wt% lipids, more preferably 50 -60 wt%, even more preferably 54 - 56 wt% of lipids.
  • the ketogenic composition may comprise 7 to 10 g of lipid per 100 kcal, preferably 7.5 to 9.5 g per 100 kcal, even more preferably 8 to 9 g of lipid per 100 kcal based on the total energy content of the ketogenic composition.
  • the amount of lipid fraction can be determined by applying methods known in the art for measuring fat content in a food matrix as applicable. For example, fat content for general foods is determined by applying AOACI official method 983.23, while the Roese-Gott Kunststoff method (AOACI 932.06) is better applicable for products based on dried milk (Lehner, R., Estoppey, A., (1954) Mitt. Strukturuntersuchung Hyg. 54:183-185).
  • the amount of individual lipid components can be determined by applying methods specifically designed for measuring that specific component or by fractionating the fat fraction isolated from the extraction of the chloroform-methanol fraction as given in the 983.23 method.
  • the structural composition of the lipids such as the binding at the sn-1 , sn-2 orsn-3 position to form mono-, di- ortri-acylglycerols may be determined and quantified using liquid chromatography, mass spectrometry (also called lipidomics) and other methods known in the art (Beermann C et al., Lipids, 2005;40(2): 211-8).
  • the ketogenic composition according to the invention comprises saturated medium chain fatty acids [MCFAs], Preferably at least 70wt%, more preferably at least 80 wt%, even more preferably at least 90 wt% of the MCFAs are provided in the form of medium chain triglycerides [MCTs], In some embodiment it is preferred essentially all the MCFAs are provided in the form of MCTs.
  • MCTs are a type of fat found in a small number of foods, such as coconut oil and palm kernel oil. MCTs may also be supplied by commercially available MCT oils.
  • MCFAs are defined to be linear or branched, preferably linear, saturated carboxylic acids having six to twelve carbon atoms.
  • MCTs When bound to a glycerol backbone at the sn-1 to sn-3 positions of glycerol MCFAs are present in the form of MCTs.
  • MCTs also include glycerides wherein at least one of the FAs is an MCFA, preferably at least 2, most preferably 3 of the FAs are MCFAs.
  • MCTs in the ketogenic composition is very advantageous, since they are effective in promoting a rapid induction of ketosis, that is, a rapid production and secretion of ketones by the liver into the blood circulation to create a condition of ketosis.
  • MCTs are particularly beneficial for neurons for suppressing seizures and also induce the local production of ketones by glial cells (astrocytes) and supply to neurons in the brain.
  • these oils provide concentrated sources of MCFAs with chain lengths of 6 (hexanoic acid), 8 (caprylic or octanoic acid), 10 (capric or decanoic acid) and 12 (lauric or dodecanoic acid) carbon atoms.
  • the MCFAs according to the invention are preferably provided or selected from MCTs originating from coconut oils and/or palm kernel oils.
  • the chain length of the MCFAs according to the invention is 6, 7, 8, 9, 10, 11 or 12, preferably 6, 8, 10 and/or 12 carbon atoms.
  • the amounts of MCFAs provided herein are based on the molar weight of the MCFAs present in the composition and corrected for the weight of glycerol.
  • the ketogenic composition preferably comprises 10 - 20g MCFAs per 100g composition, more preferably 12 - 18g, even more preferably 13 - 16g per 100g composition based on the total weight of the ketogenic composition.
  • the ketogenic composition preferably comprises 10 - 20 wt% MCFAs, more preferably 12 - 18 wt%, even more preferably 13 - 16 wt% based on total weight of the ketogenic composition.
  • the MCFAs preferably provide 10 - 30%, preferably 15 - 25%, more preferably 17 - 20% of the total energy content of the ketogenic composition.
  • the amount of MCFAs in the lipid fraction is about 5 - 50 g per 100 g fatty acids, preferably 10 - 45 g per 100 g fatty acids, preferably 15 - 40 g per 100 g fatty acids, more preferably 20 to 35 g per 100 g fatty acids, and even more preferably 25 - 30 g per 100 g fatty acids.
  • the lipid fraction preferably comprises 5 - 50 wt% MCFAs, preferably 10 - 45 wt%, preferably 15 - 40 wt%, more preferably 20 - 35 wt%, and even more preferably 25 - 30 wt%, based on total weight of the fatty acids in the ketogenic composition.
  • the ketogenic composition comprises 4 - 12 mol% MCFAs, preferably 5 - 10 mol% MCFAs, more preferably 6 - 9 mol % based on the total amount of fatty acids in the ketogenic composition.
  • the MCTs When calculated in terms of MCTs, in an embodiment the MCTs preferably provide 10 -35%, preferably 15 - 30%, more preferably 20 -25% of the total energy content of the ketogenic composition.
  • the lipid fraction of the ketogenic composition preferably comprises 15 -45 wt% MCTs, more preferably 20 - 40 wt%, even more preferably 25 - 35 wt% MCTs based on total weight of the lipid fraction.
  • the ketogenic composition preferably comprises 10 - 20 wt% MCTs, more preferably 12 - 18 wt% MCTs, even more preferably 13 - 17 wt% MCTs based on total weight of the ketogenic composition.
  • the MCT fraction in the ketogenic composition is beneficially rich in C8, C10 and C12 fatty acids. It is believed that the provision of an MCT blend rich in C8:0, C10:0 and C12:0 fatty acids aids in the ketogenicity of the diet.
  • the MCT fraction is rich in C8:0, C10:0 and C12:0 fatty acids, wherein "rich” is defined as together forming more than 60 wt% of the FAs in the MCT fraction, preferably more than 70 wt% of the MCT fraction, even more preferably more than 80 wt% of the MCT fraction.
  • up to 100 wt% of the FAs in the MCT fraction are C8:0, C10:0 and C12:0 fatty acids.
  • the sum of the weight of medium chain fatty acids C6:0 + C8:0 over the sum of the weight of C10:0 and C12:0 in the ketogenic composition is less than 1 :1 , preferably less than 0.8:1 , more preferably less than 0.6:1 , even more preferably less than 0.3:1.
  • the weight of C12:0 over the sum of the weight of medium chain fatty acids C6:0 + C8:0 + C10:0 is more than 1 .5:1 , preferably more than 2:1 , more preferably more than 2.3:1 , even more preferably more than 2.5:1
  • the ketogenic composition preferably comprises 8 - 12 g lauric or dodecanoic acid (C12:0), more preferably 8.5 -11 g lauric acid, even more preferably 9 - 10 g of lauric acid per 100g of the ketogenic composition.
  • the ketogenic composition preferably comprises 0.5 - 2.5 g capric acid (C10:0), more preferably 1 - 2 g capric acid, even more preferably 1.2 - 1 ,8g capric acid per 100g of the ketogenic composition.
  • the ketogenic composition further preferably comprises 0.5 - 3 g caprylic acid (C8:0), more preferably 1 - 2.5 g caprylic acid, even more preferably 1 .5 - 2g caprylic acid per 100 g weight of the ketogenic composition.
  • the ketogenic composition comprises long chain fatty acids [LCFA] and Very Long Chain fatty acids [VLCFA], wherein said long chain fatty acids are even-chain fatty acids that have a carbon length of 14 to 18, and wherein said very long chain fatty acids are even-chain fatty acids that have a carbon length of 20 or more.
  • the LCFAs having a carbon chain length of 14 to 18 and VLCFAs having a carbon length of 20 or more comprise both saturated and unsaturated fatty acids.
  • the LCFAs and VLCFAs may be provided in any form such as, but not limited to, triglycerides, diglycerides, monoglycerides, free fatty acids or their salts or esters, phospholipids, lysophospholipids, glycerol ethers, lipoproteins, ceramides, glycolipids, or combinations thereof.
  • the ketogenic composition comprises the fatty acids in triglyceride form.
  • Suitable sources of LCFAs and VLCFAs are, but are not limited to, palm oil and rapeseed oil.
  • the amounts of LCFAs and VLCFAs provided are based on the molar weight of the LCFAs present in the ketogenic composition and corrected for the weight of glycerol.
  • the amount of even-chain LCFAs having a carbon length of 14 to 18 and VLCFAs having a carbon length of 20 or more is preferably less than 95 g per 100 g fatty acids, more preferably less than 85 g per 100 g fatty acids, preferably 50 - 95 g per 100 g fatty acids, more preferably 50 - 85 g per 100 g fatty acids, more preferably 55 to 80 g per 100 g fatty acids, and most preferably 60 - 75 g per 100 g fatty acids.
  • the lipid fraction preferably comprises 50 -95 wt% LCFAs and VLCFAs, 50 -85 wt% LCFAs and VLCFAs, more preferably 55 -80 wt%, even more preferably 60 - 75 wt%, based on total weight of the fatty acids in the ketogenic composition.
  • the ketogenic composition preferably comprises 25 - 55 g LCFAs and VLCFAs per 100g composition, preferably 25 - 50 g LCFAs and VLCFAs per 100g composition, more preferably 30 - 45 g, even more preferably 35 - 40 g per 100 g composition based on the total weight of the ketogenic composition.
  • the ketogenic composition preferably comprises 25 - 50 wt% LCFAs and VLCFAs, more preferably 30 -45 wt%, even more preferably 35 - 40 wt% based on total weight of the ketogenic composition.
  • the LCFAs and VLCFAs preferably provide 40 - 70%, preferably 45 - 65%, more preferably 50 - 60% of the total energy content of the ketogenic composition. In yet another embodiment the LCFAs preferably provide 50 - 80%, preferably 55 - 75%, more preferably 60 - 70% of the total energy content of the lipids in the ketogenic composition.
  • the amount of palmitic acid (C16:0) is less than 40 g per 100 g fatty acids, preferably 10 - 40 g per 100 g fatty acids, more preferably 15 - 35 g per 100 g fatty acids, and most preferably 20 - 30 g per 100 g fatty acids.
  • the lipid fraction preferably comprises 10 - 40 wt%, more preferably 15 - 35 wt%, even more preferably 20 - 30 wt% palmitic acid.
  • the ketogenic composition preferably comprises less than 20g palmitic acid, more preferably less than 16g, even more preferably less than 14g per 100g of the ketogenic composition.
  • the ketogenic composition preferably comprises less than 20 wt%, more preferably less than 16 wt%, even more preferably less than 14 wt% of palmitic acid based on total weight of the ketogenic composition.
  • the LCFA and VLCFA fraction preferably comprises less than 45 wt% palmitic acid (C16:0), more preferably less than 40 wt%, even more preferably less than 35 wt% based on total weight of the LCFAs and VLCFAs.
  • myristic acid C14:0 and palmitic acid (C16:0) may decrease hepatic LDL receptor expression and thereby promote the formation of pro-atherogenic oxLDL, whereas stearic acid (C18:0) is associated with lower plasma LDL levels.
  • the ketogenic composition of the invention has a beneficially high stearic acid to the sum of [palmitic acid and myristic acid] ratio, such that despite having a high fat content the ketogenic composition provides a more beneficial cardiovascular risk profile.
  • the ketogenic composition preferably has a stearic acid to (palmitic acid and myristic acid) weight ratio of more than 1 : 12, more preferably more than 1 : 10, even more preferably more than 1 : 9.
  • the ketogenic composition preferably comprises MCTs, LCTs and VLCTs at a weight ratio of MCTs to the sum of LCTs and VLCTs of between 1 :2 and 1 :5, preferably 1 : 2.5 and 1 : 3, more preferably 1 : 2.6 and 1 : 2.9, even more preferably 1 : 2.7 and 1 : 2.8.
  • the ketogenic composition comprises monounsaturated fatty acids [MUFA].
  • the MUFA is at least one of palmitoleic acid (C16:1 co7), vaccenic acid (C18:1 co7), oleic acid (C18:1 co9), eicosoneic acid (C20:1 co9), erucic acid (C22:1 OJ9) and nervonic acid (C24:1 co9), preferably at least oleic acid.
  • Suitable sources of MUFA include but are not limited to tea seed oil, olive oil, canola oil, palm oil and rapeseed oil. It is believed that MUFAs as used in the ketogenic composition are not only a substrate for ketone body production but may also provide a beneficial effect on glycemic control.
  • the total amount of mono-unsaturated fatty acids is between 15 and 40 g per 100 g, more preferably between 20 and 35 g per 100 g, even more preferably between 25 and 30 g per 100 g of fatty acids.
  • the lipid fraction preferably comprises 15 - 40 wt%, more preferably 20 - 35 wt%, even more preferably 25 - 30 wt% MUFAs based on total weight of the fatty acids in the lipid fraction.
  • the total amount of MUFAs is between 5 and 25 g per 100 g, more preferably between 10 and 20 g per 100 g, even more preferably between 12 and 16 g per 100g of the ketogenic composition.
  • the ketogenic composition preferably comprises 5 - 25 wt%, more preferably 10 -20 wt%, even more preferably 12 - 16 wt% MUFAs based on total weight of the ketogenic composition.
  • the MUFAs preferably provide 10 - 30%, preferably 15 - 25%, more preferably 18 - 22 % of the total energy content of the ketogenic composition.
  • the ketogenic composition comprises at least 8 g oleic acid per 100 g of the composition, more preferably at least 10 g, even more preferably at least 13 g per 100g of the composition.
  • the ketogenic composition preferably comprises at least 8 wt%, more preferably at least 10 wt%, even more preferably at least 13 wt% oleic acid.
  • the ketogenic composition further comprises omega-3 and/or omega-6 polyunsaturated fatty acids (PUFA), preferably n-3 LC-PUFA, having a chain length of 18 carbon atoms, and n-3 VLC-PUFA, having a chain length of 20 and more carbon atoms).
  • PUFA omega-3 and/or omega-6 polyunsaturated fatty acids
  • n-3 LC-PUFA having a chain length of 18 carbon atoms
  • n-3 VLC-PUFA having a chain length of 20 and more carbon atoms
  • the ketogenic composition may comprise gamma linolenic acid (C18:3 co6).
  • GLA is an n-6 fatty acid the fatty acid provides anti-inflammatory properties.
  • omega-3 fatty acids and the reduction of omega-6 fatty acids is believed to aid in limiting neuroinflammation after seizures and therewith aid in the observed reduction of the progression of epilepsy in the in vivo experiment.
  • the ketogenic composition comprises at least one, preferably two omega-3 VLC-PUFAs selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), preferably DHA and EPA.
  • the weight ratio of EPA to DHA is preferably lower than 1 , more preferably 1 :1.1 to 1 :4, more preferably 1 :1.3 to 1 :4. In some embodiments the weight ratio of DHA to EPA is more than 2:1 .
  • ketogenic composition may comprise alpha-linolenic acid [C18:3 co3, ALA],
  • the DHA, EPA and/or DPA may be provided in any form such as, but not limited to, triglycerides, diglycerides, monoglycerides, free fatty acids or their salts or esters, phospholipids, lysophospholipids, glycerol ethers, lipoproteins, ceramides, glycolipids or combinations thereof.
  • the ketogenic composition comprises at least DHA in triglyceride form.
  • Suitable OJ-3 VLCPUFA and/or sources of DHA and EPA include tuna oil, (other) fish oils, DHA-rich alkyl esters, algae oil, egg yolk, krill oil or phospholipids enriched with OJ-3 VLCPUFA, e.g. phosphatidylserine-DHA.
  • the ketogenic composition comprises fish oil providing the omega- 3 VLCPUFA(s).
  • Another particularly suitable source for the omega-3 VLCPUFA(s) is algae oil.
  • the ketogenic composition preferably comprises 0.5 - 3 g VLC-PUFAs selected from DHA, EPA and DPA, most preferably DHA+EPA per 100g of the ketogenic composition, more preferably 1- 2.5 g, even more preferably 1 .2 - 2 g per 100g composition.
  • VLC-PUFAs selected from DHA, EPA and DPA most preferably DHA and EPA, preferably provide 1 - 5%, more preferably 1 .5 - 3.5%, even more preferably 2 - 3% of the total energy content of the ketogenic composition.
  • the total amount DHA, EPA and DPA, most preferably DHA and EPA are preferably 1.5 to 7 g per 100 g fatty acids, more preferably 2 to 6 g per 100 g fatty acids, and most preferably 3 to 5 g per 100 g fatty acids.
  • the total amount of omega-3 LCPUFAs having a carbon length of 18, and omega-3 VLCPUFAs having a carbon length of 20 to 24 in the ketogenic composition is less than 15 g per 100 g fatty acids, preferably 2-15 g per 100 g fatty acids, more preferably 3 to 10 g per 100 g fatty acids, and even more preferably 4 to 7 g per 100 g fatty acids.
  • the LA:ALA weight ratio of the ketogenic composition is preferably in the range of 2:1 to 12:1 , more preferably 4:1 to 10:1 , even more preferably 6:1 to 8:1.
  • the present ketogenic composition preferably optionally further comprises choline.
  • Choline may be present as such, or as choline equivalent in the form of a choline salt and/or choline ester.
  • the choline salt is preferably selected from choline chloride, choline bitartrate, or choline stearate, preferably choline chloride.
  • a choline ester is preferably selected from a phosphatidylcholine and lyso-phosphatidyl choline, preferably phosphatidylcholine.
  • the ketogenic composition preferably comprises less than 2000 mg, more preferably less than 1750 mg choline equivalents selected from choline, a choline salt and/or choline ester, calculated as choline, per 100 g of the ketogenic composition.
  • the ketogenic composition comprises 500 to 5000 mg choline, preferably 1000 to 4500 mg, more preferably 1500 to 4000 mg choline per 100 g of the ketogenic composition and calculated as choline.
  • the ketogenic composition comprises a carbohydrate fraction.
  • the present ketogenic composition comprises digestible carbohydrates, preferably digestible carbohydrates with a low glycemic index.
  • digestible carbohydrates preferably digestible carbohydrates with a low glycemic index.
  • the ketogenic composition comprises digestible carbohydrates rich in one or more of non-glucose and non-fructose monosaccharides such as mannose, galactose, xylulose, xylose, glucosamine, and sialic acid.
  • the composition, combination, or product according to the invention comprises one or more low glycemic index carbohydrates selected from trehalose, lactose, galactose and isomaltulose.
  • the composition, combination, or product according to the invention comprises galactose and isomaltulose.
  • such low Gl carbohydrates are carbohydrates having a Gl of 55 or less.
  • Low glycemic index carbohydrates beneficially allow the inclusion of carbohydrates in the ketogenic composition while not significantly impacting ketogenesis.
  • the ketogenic composition also comprises non-digestible carbohydrates or dietary fibers.
  • the carbohydrate fraction of the ketogenic composition comprises between about 30 and 80 wt% low glycemic index carbohydrates, more preferably between 40 and 70 wt%, more preferably between 45 and 60 wt%.
  • the amount of galactose is preferably between about 8 wt% and 30 wt% of the carbohydrate fraction, more preferably between about 10 wt% and 25 wt% of the carbohydrate fraction and in an even more preferred embodiment between about 15 wt% and 20 wt% of the carbohydrate fraction.
  • the amount of isomaltulose is in one embodiment preferably between about 20 wt% and 50 wt% of the carbohydrate fraction, more preferably between about 25wt% and 45 wt%, and in an even more preferred embodiment between about 30 wt% and 40 wt% of the carbohydrate fraction.
  • the amount of high glycaemic index carbohydrates such as glucose or rapidly digestible glucose polymers such as but not limited to sucrose is preferably below 70 wt% of the carbohydrate fraction, more preferably below 60 wt% of the carbohydrate fraction, even more preferably below 50 wt% of the carbohydrate fraction.
  • the ketogenic composition is essentially free of glucose and/or rapidly digestible glycose polymers.
  • the ketogenic composition preferably comprises 5 to 12 g digestible carbohydrates, more preferably 6 to 10 g digestible carbohydrates, even more preferably 7 to 9 g digestible carbohydrates per 100 g of the ketogenic composition, wherein said digestible carbohydrates preferably comprise low Gl carbohydrates, preferably galactose and isomaltulose.
  • the ketogenic composition thus preferably comprises 5 to 12 wt% digestible carbohydrates, more preferably 6 to 10 wt%, even more preferably 7 to 9 wt% digestible carbohydrates based on total weight of the ketogenic composition.
  • the glycaemic index (Gl) of the ketogenic composition is below 70, preferably below 65, preferably below 60, more preferably below 55. In a preferred aspect the glycaemic index is between 65 and 25, wherein the glycaemic index is based on glucose as reference value set at 100.
  • the ketogenic composition comprises fibres, preferably food-grade dietary fibres.
  • Dietary fibers are to large extent fermented in the colon by the bacterial flora.
  • SOFA short chain fatty acids
  • a preferred embodiment according to the present invention thus includes dietary fibres in the ketogenic composition.
  • the dietary fiber or fibers are selected from the group consisting of oligofructose, inulin, resistant starch, cellulose, methylcellulose preferably hydroxypropyl methylcellulose, wheat bran, gum arabic, soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin, hydrolysed pectin and mixtures thereof.
  • soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin, hydrolysed pectin and mixtures thereof.
  • the terms ‘gum arabic’ and ‘arabic gum’ are used interchangeably.
  • the ketogenic composition comprises a fiber mixture comprising cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch.
  • the fiber mixture comprises cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch in a weight ratio of 4 - 6: 1 - 3 : 1 - 3 : 0.5 - 2 : 0.1 - 0.4, more preferably 4.5 - 5.5 : 1 .5 - 2.5 : 1 .5 - 2.5 : 1 - 1 .5 : 0.2 - 0.3.

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Abstract

Provided herein is an enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, having a ketogenic weight ratio between 1.5:1 and 3.0:1 and therapeutic uses thereof.

Description

Ketogenic diet
FIELD OF THE INVENTION
The present invention relates to the field of ketogenic compositions and uses thereof.
BACKGROUND OF THE INVENTION
Epilepsy is one of the most common neurological disorders after stroke and affects at least 50 million people worldwide. It is diagnosed in a person having recurrent unprovoked seizures. These occur when cortical neurons fire excessively, hypersynchronous, or both, leading to temporary disruption of normal brain function. This might affect, for example, the muscles, the senses, consciousness, or a combination thereof. A seizure can be focal (confined to one part of the brain) or generalized (spread widely throughout the brain and leading to a loss of consciousness). Epilepsy may occur for a variety of reasons; some forms have been classified into epileptic syndromes, most of which begin in childhood. Epilepsy is considered refractory to treatment when two or three anticonvulsant drugs have failed to control it. About 60 percent of patients will achieve control of their epilepsy with the first drug they use, whereas about 30 percent do not achieve control with drugs. When drugs fail, other options include epilepsy surgery, vagus nerve stimulation and the ketogenic diet (KD).
The KD mimics aspects of starvation by forcing the body to use fats as a fuel for cellular energy production rather than carbohydrates. Normally, the carbohydrates contained in food are converted into glucose, which is then transported around the body and is particularly important in fuelling brain function. However, if there is very little carbohydrate in the diet, the liver converts fat into fatty acids and ketone bodies. The term ketone bodies typically refer to three acid compounds present in the blood in small quantities, that is, acetone (propanone), acetoacetic acid (3-oxobutanoic acid) and beta-hydroxybutyric acid (3-hydroxybutanoic). These compounds derive from the metabolism of fats introduced with food and are of fundamental importance for the correct function of brain cells, since ketone bodies are the only compounds, in addition to glucose, that can be used by these cells as an energy source.
The use of KD was initially suggested to mimic the metabolic state and biochemical changes associated with fasting, since fasting was proven to possess anticonvulsant properties. Classical ketogenic diets were first introduced in the 1920s for use in human childhood epilepsy. The classic KD typically has a ketogenic ratio between 4:1 and 3:1 , meaning such diets comprise an amount of lipids (weight), which is about 3 to 4-fold the weight of the sum of proteins and digestible carbohydrates. Various variations to said classic ketogenic diet exist wherein the fat content has been lowered such as the Medium Chain Triglyceride Ketogenic Diet (MCTKD), wherein a large percentage (30-70%) of total daily calories comes from medium chain triglycerides. Other known variants to the classic KD are the Modified Atkins Diet with a ketogenic ratio of about 1 :1 and the Low Glycaemic Index diet, both in practice diets wherein it is difficult to induce and maintain a state of ketosis. In general, reducing the fat content of the cKD may come with the trade-off of reduced ketogenesis and some studies report sub-optimal seizure suppression in epilepsy patients compared to the classic KD (Sondhi et al. JAMA Pediatrics, 2020). A further known variant to the classic KD is the mild ketogenic diet according to W02019013616 A1 comprising less than 10 kcal% of MCTs.
In subjects on a KD, fats are broken down into fatty acids and ketone bodies in the liver, and these compounds are utilised in further metabolic pathways for generating adenosine triphosphate (ATP) as a chemical energy source. The KD is thought to result in adaptive changes to energy metabolism, amongst others in the brain, that increase the energy production. In neurological disorders this is believed to help the neurons to remain functional and viable in the face of increased energy demand and may confer a neuroprotective effect. Studies have shown a clear benefit of the KD and ketosis in treating intractable epilepsy in children and adults. Short-term trials have demonstrated that approximately half of those studies had at least a 50% reduction in seizures after 6 months, and approximately one third reached at least a 90% reduction. Usually, the ketogenic diet is sufficiently effective to enable a reduction in, or cessation of prescribed anti-epileptic medication and to improve quality of life.
Ketogenic diets must be strictly controlled to ensure effective seizure prevention whilst maintaining adequate nutritional value. Although clearly effective, ketogenic diets are difficult to manage in both children and adults resulting in poor patient compliance. Current approaches to the ketogenic diet can be very restrictive and this can lead to poor compliance, particularly in adults. Furthermore, such diets can result in a number of side effects (including problems of the digestive system owing amongst others to the excess amount of fats, increased serum lipid levels, drowsiness, poor growth and increased risk of bone fractures). Accordingly, there is a continued need for compositions that do not suffer from the disadvantages while still providing the desired therapeutic effects.
Summary of the invention
A ketogenic composition is provided herein comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein (including addition of free amino acids) per 100g of the ketogenic composition, based on weight, wherein the lipid fraction of the ketogenic composition comprises
5 - 50 wt% medium-chain fatty acids [MCFAs] based on total weight of the fatty acids in the lipid fraction, wherein more than 60 wt% of said MCFAs are C8:0, C10:0 and C12:0 fatty acids and wherein at least 70 wt% the MCFAs are provided in the form of medium chain triglycerides [MCTs];
50-95 wt% even-chain long-chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction wherein the even-chain LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC- PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids, and wherein the ketogenic composition has a ketogenic weight ratio between 1 .5:1 and 3.0:1.
The ketogenic composition according to the invention has a specific combination of lipids, carbohydrates, proteins, and micronutrients allowing to provide both beneficial health effects of the ketogenic diet and does not pose the compliance problems associated with the ingestion of excessive amounts of fats.
The inventors have observed that the enteral ketogenic composition according to the invention is beneficial for therapeutic use in the treatment of epilepsy and/or prevention of progression of epilepsy. The inventors found that the ketogenic composition according to the invention is beneficial for therapeutic use in the treatment and/or prevention and/or reduction of the severity of epilepsy and in prevention and/or delay of the formation of epileptiform circuits in the brain. In addition to the beneficial therapeutic use in epilepsy the inventors observed that the enteral ketogenic composition is beneficial for use in preventing abnormal elevations of blood triglyceride levels, fatty liver development and/or metabolic syndrome development in a subject adhering to the enteral ketogenic composition. In addition to the beneficial therapeutic use in epilepsy the inventors observed that the therapeutic benefits comprise preventing and/or treating behavioural consequences of epilepsy. Attention Deficit hyperactivity Disorder [ADHD] is frequently associated with epilepsy. The inventors observed that the behavioural patterns associated with ADHD are diminished and/or prevented using the enteral ketogenic composition according to the invention. The behavioural patterns associated with ADHD comprise hyperactive behaviour, impulsive behaviour and/or fearless behaviour.
Worded differently, the invention also pertains to the use of the enteral ketogenic composition according to the invention for the treatment of epilepsy and/or prevention of progression of epilepsy in a subject suffering from epilepsy or at risk of seizures. The invention additionally pertains to the use of the enteral ketogenic composition for preventing abnormal elevations of blood triglyceride levels, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures. The invention further pertains to the use of the enteral ketogenic composition in a subject suffering from epilepsy or at risk of seizures wherein behavioural patterns associated with ADHD are diminished and/or prevented.
The invention also relates to a method for treatment and/or prevention of epilepsy and/or progression of epilepsy in a subject suffering from epilepsy or at risk of seizures comprising administering to the subject the enteral ketogenic composition. In addition, the method for treatment and/or prevention involves preventing abnormal elevations of blood triglyceride levels, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures. The method for treatment and/or prevention of epilepsy and/or progression of epilepsy further comprises diminishing and/or preventing behavioural patterns associated with ADHD.
In particular, the present invention provides enteral ketogenic compositions, methods of use and use of said enteral ketogenic compositions to treat and/or prevent epilepsy, the progression of epilepsy and to prevent abnormal elevations of blood triglyceride levels, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
List of Figures
The present invention will be discussed in more detail below, with reference to the attached drawings.
Figure 1 shows the distribution of seizure scores across stimulation sessions for animals exposed to the control diet, classic ketogenic diet, and alternative ketogenic diet.
Figure 2 shows the distribution of seizure scores after a number of stimulations and the latency to seizure stage 4, 5, or fully kindled status across the experimental groups.
Figure 3 shows the mean seizure scores of the experimental groups with increasing stimulation number. Y-axis: seizure score; x-axis: stimulation session.
Figure 4 A shows the afterdischarge pattern following initial 10s electrical stimulation, B shows the duration of the first part of afterdischarge (AD1) across the experimental groups and C shows the latency to the second part of afterdischarge (AD2) across the experimental groups.
Figure 5 shows the centre zone entries and time spent in the centre zone across the experimental groups.
Figure 6 shows activity and exploration of the animals in the different experimental groups.
Figure 7 shows the locomotion of the animals in the different experimental groups.
Figure 8 shows the blood ketone levels of the animals in the different experimental groups after 5 days on the diet.
Figure 9 shows blood ketone levels of the animals in the different experimental groups after 21 days on the diet in the different experimental groups.
Figure 10 shows triglyceride serum levels of non-fasted rats after 5 and 21 days on the diet in the different experimental groups.
Figure 11 shows glucose serum levels of non-fasted rats after 5 and 21 days on the diet in the different experimental groups.
Figure 12 shows omega-3 and omega-6 PUFA serum levels of non-fasted rats after 5 days of dietary exposure in the different experimental groups.
Figure 13 shows omega-3 and omega-6 PUFA serum levels of non-fasted rats after 21 days of dietary exposure in the different experimental groups.
Figure 14 shows specific LCFA serum levels of non-fasted rats after 5 days of diet in the different experimental groups.
Figure 15 shows specific LCFA serum levels of non-fasted rats after 21 days of diet in the different experimental groups.
Figure 16 shows liver triglyceride content in mg per g liver in the different experimental groups.
Figure 17 shows body weight development over time in the different experimental groups.
Figure 18 shows serum essential amino acid levels after 5 days of diet in the different experimental groups. The alternative ketogenic diet provided higher levels of essential amino acids than a rodent classic ketogenic diet at day 5 of diet exposure. Figure 19 shows serum essential amino acid levels after 21 days of diet in the different experimental groups. The alternative ketogenic diet provided higher levels of essential amino acids than a rodent classic ketogenic diet at day 21 of diet exposure.
Figure 20 shows serum amino acid levels of amino acids associated with blockage of seizures after 5 days of diet in the different experimental groups. The alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 5 of diet exposure.
Figure 21 shows serum amino acid levels of amino acids associated with blockage of seizures after 21 days of diet in the different experimental groups. The alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 21 of diet exposure.
Figure 22 shows serum amino acid levels of amino acids associated with blockage of seizures after 5 days of diet in the different experimental groups. The alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 5 of diet exposure.
Figure 23 shows serum amino acid levels of amino acids associated with blockage of seizures after 21 days of diet in the different experimental groups. The alternative ketogenic diet provided greater levels of seizure blocking amino acids than a rodent standard (control) diet at day 21 of diet exposure.
Figure 24 shows the effect of stimulation of neuroglial cell cultures with 0.1 mM Mg2+ on oscillation frequency, amplitude and area under the curve as well as the effect on glycolysis and mitochondrial respiration as compared to unstimulated neuroglial cell cultures maintained at 0.8 mM Mg2+.
Figure 25 shows the effect of a fat blend according to the invention (the INV-KD fat blend) and a comparative fat blend with moderate amounts of MCTs (COMP-KD fat blend) on mitochondrial respiration in hyperexcited (seizure-like) neuroglial cell cultures. The effect on glycolysis, ATP production, and the values for proton leak measured after exposure to each fat blend is also reported.
List of Embodiments
1 . Enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, wherein the lipid fraction comprises
5 - 50 wt% medium-chain triglycerides fatty acids [MCFAs] based on total weight of the fatty acids in the lipid fraction, wherein more than 60 wt% of said MCFAs are C8:0, C10:0 and C12:0 fatty acids and wherein at least 70wt% of said MCFAs are provided in the form of medium chain triglycerides [MCTs];
50 - 95 wt% even-chain long chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction, and wherein the LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC-PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids based on total weight of the protein fraction, and wherein the ketogenic composition has a ketogenic weight ratio between 1 .5:1 and 3.0:1 .
2. The enteral ketogenic composition according to embodiment 1 wherein the composition further comprises 7 - 15 wt% dietary fibers and wherein the fibers are selected from oligofructose, inulin, resistant starch, cellulose, wheat bran, gum arabic, soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin and hydrolysed pectin.
3. The enteral ketogenic composition according to embodiment 2 wherein the fibers are a fiber mixture comprising cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch.
4. The enteral ketogenic composition according to the preceding embodiments wherein the low glycemic index carbohydrates are selected from trehalose, lactose, galactose and isomaltulose.
5. The enteral ketogenic composition according to the preceding embodiments wherein the protein fraction comprises intact and/or (partly) hydrolysed protein selected from pea, soy, casein and/or whey, preferably casein.
6. The enteral ketogenic composition according to the preceding embodiments wherein the protein fraction comprises 75 - 95 wt% casein based on total weight of the protein fraction.
7. The enteral ketogenic composition according to the preceding embodiments wherein the protein fraction comprises free amino acids selected from the group consisting of serine, lysine, glycine, valine, cysteine, leucine and isoleucine, preferably leucine, isoleucine and/or cysteine.
8. The enteral ketogenic composition according to the preceding embodiments wherein the protein fraction comprises 5 - 15 wt% free leucine and 0.5 - 2.5 wt% free cysteine based on total weight of the protein fraction.
9. The enteral ketogenic composition according to the preceding embodiments wherein the OJ-3/OJ- 6 weight ratio of the lipids is between 0.2:1 to 5:1.
10. The enteral ketogenic composition according to the preceding embodiments wherein the composition comprises mono-unsaturated fatty acids [MUFAs], and wherein at least 85 wt% of the MUFAs is oleic acid, based on total weight of the MUFAs in the lipid fraction.
11 . The enteral ketogenic composition according to the preceding embodiments further comprising citrate and/or nicotinamide riboside and/or nicotinamide and/or pyruvate.
12. The enteral ketogenic composition according to the preceding embodiments wherein the composition comprises less than 20 wt% of palmitic acid based on total weight of the ketogenic composition.
13. Enteral ketogenic composition according to the preceding embodiments for use in the treatment and/or prevention of epilepsy in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
14. The enteral ketogenic composition according to embodiment 13 for use in the treatment and/or prevention of epilepsy, wherein the treatment and/or prevention of epilepsy comprises treatment and/or prevention and/or reduction of the severity of epilepsy and prevention and/or delay of the formation of epileptiform circuits in the brain.
15. The enteral ketogenic composition according to any one of embodiments 13 and 14 for use in the treatment and/or prevention of epilepsy further preventing the development of dyslipidemia, fatty liver and/or metabolic syndrome in the subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures
16. The enteral ketogenic composition according to any one of embodiments 13 to 15 wherein the treatment and/or prevention of epilepsy and/or progression of epilepsy in a subject suffering from epilepsy or at risk of seizures further comprises diminishing and/or preventing behavioural patterns associated with ADHD.
Detailed description of the invention
In a first aspect, the present invention relates to an enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, based on weight, wherein the lipid fraction of the ketogenic composition comprises
5 - 50 wt% medium-chain fatty acids [MCFAs] based on total weight of the fatty acids in the lipid fraction, wherein more than 60 wt% of said MCFAs are C8:0, C10:0 and C12:0 fatty acids and wherein at least 70 wt% the MCFAs are provided in the form of medium chain triglycerides [MCTs];
50-95 wt% even-chain long-chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction wherein the even-chain LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC- PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids based on total weight of the protein fraction, and wherein the ketogenic composition has a ketogenic weight ratio between 1.5:1 and 3.0:1 , preferably between 1.8:1 and 2.7:1.
The inventors surprisingly found that enteral, preferably oral, administration of the ketogenic composition according to the invention is effective in the treatment of epilepsy. The inventors surprisingly found a stronger therapeutic efficacy of the new ketogenic diet despite being less ketogenic than a classic ketogenic diet as compared to results obtained with a classic ketogenic diet having a 6:1 ketogenic weight ratio. The ketogenic diet according to the invention was in particular found to be useful for the treatment of epilepsy, showing in an in vivo rapid rat kindling model, which is an acute epilepsy model, a delay in seizure severity progression, a reduction in the severity of convulsions and an increased latency period (increased number of electrical stimulations) to evoke the first behavioural response. In addition to the beneficial effect on epilepsy it was found that the metabolic plasma profile is more similar to the profiles observed upon consumption of a control diet rather than a classic ketogenic diet. In particular plasma triglycerides and glucose levels and liver lipids were decreased in animals fed the ketogenic composition according to the invention compared to the animals fed the classic ketogenic diet.
The inventors have observed that the enteral ketogenic composition according to the invention is beneficial for therapeutic use in the treatment of epilepsy and/or prevention of progression of epilepsy. The inventors found that the ketogenic composition according to the invention is beneficial for therapeutic use in the treatment and/or prevention and/or reduction of the severity of epilepsy and in prevention and/or delay of the formation of epileptiform circuits in the brain. In addition to the beneficial therapeutic use in epilepsy the inventors observed that the enteral ketogenic composition is beneficial for use in preventing abnormal elevations of blood triglyceride levels and/or dyslipidaemia, fatty liver development and/or metabolic syndrome development in a subject adhering to the enteral ketogenic composition.
Worded differently, the invention also pertains to the use of the enteral ketogenic composition according to the invention for the treatment of epilepsy and/or prevention of progression of epilepsy in a subject suffering from epilepsy or at risk of seizures. The invention additionally pertains to the use of the enteral ketogenic composition for preventing dyslipidaemia, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures.
Worded differently, the invention pertains to an enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, based on weight, wherein the lipid fraction of the ketogenic composition comprises 5 - 50 wt% medium-chain fatty acids [MCFAs] based on total weight of the fatty acids in the lipid fraction, wherein more than 60 wt% of said MCFAs are C8:0, C10:0 and C12:0 fatty acids and wherein at least 70 wt% the MCFAs are provided in the form of medium chain triglycerides [MCTs];
50-95 wt% even-chain long-chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction wherein the even-chain LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC- PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids, and wherein the ketogenic composition has a ketogenic weight ratio between 1.5:1 and 3.0:1 , preferably between 1.8:1 and 2.7:1 , for use in treating and/or preventing epilepsy, treating and/or preventing the progression of epilepsy and/or preventing the development of dyslipidaemia, fatty liver and/or metabolic syndrome in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
The invention also relates to a method for treatment and/or prevention of epilepsy and/or progression of epilepsy in a subject suffering from epilepsy or at risk of seizures comprising administering to the subject the enteral ketogenic composition. In addition, the method for treatment and/or prevention involves preventing dyslipidaemia, fatty liver and/or metabolic syndrome development in a subject suffering from epilepsy or at risk of seizures
In particular, the present invention provides enteral ketogenic compositions, methods of use and use of said enteral ketogenic compositions to treat and/or prevent epilepsy, treat and/or prevent the progression of epilepsy and/or prevent development of dyslipidaemia, fatty liver and/or metabolic syndrome in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
According to one embodiment the invention also pertains to a method for treating epilepsy in a subject, comprising administering to the subject the enteral ketogenic composition according to the invention.
The invention may also be worded as the use of an enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, based on weight, wherein the lipid fraction of the ketogenic composition comprises
5 -50 wt% medium-chain fatty acids [MCFAs] based on total weight of the fatty acids in the lipid fraction, wherein more than 60 wt% of said MCFAs are C8:0, C10:0 and C12:0 fatty acids and wherein at least 70 wt% the MCFAs are provided in the form of medium chain triglycerides [MCTs];
50-95 wt% even-chain long-chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction wherein the even-chain LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [LC- PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids, and wherein the ketogenic composition has a ketogenic weight ratio between 1.5:1 and 3.0:1 , preferably between 1.8:1 and 2.7:1 , for the manufacture of a product for treating and/or preventing epilepsy, treating and/or preventing the progression of epilepsy and/or preventing the development of dyslipidaemia, fatty liver and/or metabolic syndrome in a subject suffering from epilepsy or at risk of seizures. In a particular aspect the seizures are epileptic seizures.
In a further embodiment of the invention the present method, product, use or composition for use provides for the generation of ketone bodies, in particular acetoacetate, beta-hydroxybutyrate and acetone, wherein the plasma levels of ketone bodies are sufficient to provide for the therapeutic effect on subjects suffering from epilepsy, or at risk of seizures, preferably epileptic seizures.
Definitions
Throughout this application, the following terminology and abbreviations are used:
Fibres are non-digestible carbohydrates. Non-digestible carbohydrates are carbohydrates that are resistant to digestion and absorption in the human stomach and small intestine and enter the colon intact. So, compounds like lactose, maltose, glucose, (regular, unmodified) maltodextrin, and (regular, unmodified) starch are regarded as digestible. Fibres can be fermentable in the colon, or non- fermentable. The term “fermentable” refers to the capability to undergo (anaerobic) breakdown by microorganism in the lower part of the gastro-intestinal tract, e.g. colon, to smaller molecules, in particular short chain fatty acids and lactate. The fermentability may be determined by the method described in Am. J. Clin. Nutr. 53, 1418-1424 (1991).
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. In the context of the invention, the so-called ketogenic (weight) ratio is the weight ratio of the weight amount of lipid to the combined (sum) weight amounts of protein and digestible carbohydrates in the composition.
In the context of the invention, the so-called ketogenic (weight) ratio is the weight ratio of the total amount of lipids to the combined weight amounts of protein (including the addition of free amino acids) and digestible carbohydrates in the composition. The ketogenic diet referred to in the context of the invention is characterized by a ketogenic weight ratio between 1 .5:1 and 3:1 , preferably between 1 .8:1 and 2.4:1 which is the ratio of the amount of fat to the combined amounts of protein (including the addition of free amino acids) and digestible carbohydrates. Within the aforementioned (sub)ranges of ketogenic ratios of the invention, the ketogenic diet used within the context of the invention preferably comprises a lipid content that is at least 1 .5 times, preferably at least twice the carbohydrate content by weight. The term "Ketosis" as used herein 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 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 associated with signs of ketoacidosis. While ketosis refers to a state of elevated ketones, ketoacidosis is a pathological and potentially life-threatening condition amongst others resulting in a decrease in blood pH and may induce a coma.
The term ‘seizure’ as used herein refers to a period of symptoms due to abnormally excessive or synchronous neuronal activity in the brain. A seizure often occurs when the electrical activity of the brain becomes more "synchronized" as would be the case when the person is in a drowsy state. The most common and stereotypical type of seizure is convulsive (60%). A ‘convulsion’ is a medical condition wherein body muscles contract and relax rapidly and repeatedly, resulting in uncontrolled shaking. Epileptic seizures may include convulsions. Epilepsy is the clinical diagnosis characterized by recurrent seizures, which are the outward manifestation of excessive and/or hyper-synchronous abnormal electrical activity of neurons in the brain.
The term dyslipidaemia as used herein refers to an imbalance in lipids including cholesterol, low-density lipoproteins, triglycerides, and high-density lipoproteins in the blood forming a risk factor for atherosclerotic cardiovascular disease.
Abnormal elevated triglyceride levels as used herein may occur in dyslipidaemia and is clinically defined as fasting blood triglyceride levels > 1 .7 mmol/L in humans.
The glycaemic index (Gl) of a food is a ranking from 0-100 of the postprandial effect of a specific food on blood glucose levels. High Gl foods give higher postprandial blood glucose levels than those with a low Gl. The Gl of a carbohydrate also predicts the insulin response to that food. The standard for a score of 100 is pure glucose. The glycaemic index may be determined by analysing the blood glucose levels in regular intervals for a 2-3-hour period after intake of the test carbohydrate or food and a reference food which, generally, is a similar amount of digestible carbohydrates in glucose. The areas under the blood glucose curves of the test and reference are determined. The Gl is expressed as the ratio of the area under the curve of the test and the reference multiplied by a factor of 100, or, in other words, in percent of the Gl of the reference food (FAO (1998), Carbohydrates in Nutrition, FAO Food and Nutrition Paper 66, p. 25-30). On the scale of 1 -100, scores of a food composition below 55 are generally considered low by those skilled in the art and scores between 59 and 56 moderate.
In the context of the invention a subject adhering to or taking a ketogenic diet is a mammal, preferably a human.
A glyceride is an ester from glycerol and a carboxylic acid. As an example, a triglyceride (also known as a triacylglycerol) is a triester that is derived from glycerol and three fatty acids. Linder hydrolysis conditions such as those during digestion, triglycerides may be a source of fatty acids. For instance, tributyrin is potentially a source of three moles of butyric acid per mole of tributyrin. Partial glycerides are esters of glycerol with fatty acids, where not all the hydroxyl groups are esterified; mono- and dibutyrin are also sources of butyric acid according to the invention providing for one and two moles of butyric acid per mole respectively.
Fatty acids may be either unsaturated or saturated. Fatty acids which are not attached to other molecules are referred to as free fatty acids (FFA).
Medium chain triglycerides (MCTs) are triglycerides in which all three fatty acid moieties are mediumchain fatty acid moieties. MCTs are found in a small number of foods, such as coconut oil and palm kernel oil.
As defined herein, medium-chain fatty acids are defined to be linear or branched, preferably linear, saturated carboxylic acids having six (C6:0) to twelve (C12:0) carbon atoms. Medium-chain fatty acids with 10 carbon atoms may be referred to herein as “C10 fatty acids” or“C10.”
The term "long chain polyunsaturated fatty acids" or "LCPUFA" refers to monocarboxylic acids having at least 20 carbon atoms and at least two double bonds. Examples of LCPUFA include (n-6) fatty acids, such as arachidonic acid (AA), and (n-3) fatty acids, such as eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).
The term “fish oil” means a fatty or oily extract, relatively rich in LCPUFA, whether crude or purified, obtained from a sea animal, e.g. a cold-water fish such as, but not limited to, salmon, tuna, mackerel, herring, sea bass, striped bass, halibut, catfish, and sardines, as well as shark, shrimp, and clams, or any combination thereof. Fish oil is generally a term of art used by ingredient suppliers and encompasses a range of products of varying PUFA content and purity.
In the context of the in invention, where reference is made to an amount of fatty acid, where different sources or derivatives are used the weight amounts are calculated in terms of the corresponding (mole weight) amount of the fatty acid.
In the context of the invention, where reference is made to the "total weight of the ketogenic composition” this refers to the sum of the weights of the ingredients forming the ketogenic composition without the addition of water, i.e. the water-free weight.
"Nutritional composition” means a substance or formulation that satisfies at least a portion of a subject’s nutrient requirements. The terms “nutritional(s)”, “nutritional formula(s)”, “enteral nutritional(s)”, and “nutritional supplements)” are used as non-limiting examples of nutritional composition (s) throughout the present disclosure. Moreover, “nutritional composition (s)” may referto liquids, powders, gels, pastes, solids, concentrates, suspensions, or ready-to-use forms of enteral formulas, oral formulas, formulas for infants, formulas for paediatric subjects, formulas for children, growing-up milks and/or formulas for adults.
The energy provided by nutrients is calculated using the Atwater calculation factors of 9 kcal per g lipid, 4 kcal per gram protein or gram digestible carbohydrate, 2 kcal per gram dietary fiber and zero kcal for the other components in the product.
All percentage intervals reported here are provided with the provision that the sum with respect to the overall composition is 100%, unless otherwise indicated.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. "Consisting essentially of” means that the composition contains the active ingredients and possibly additional compounds, provided these do not materially affect the essential characteristics of the composition. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. Where reference is made to % herein it means wt% unless indicated differently.
Ingredients of the enteral ketogenic composition
The present invention relates to an enteral ketogenic composition comprising (i) a lipid fraction, (ii) a carbohydrate fraction and (iii) a protein fraction.
The enteral ketogenic composition comprises (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, based on weight, wherein said ketogenic composition has a ketogenic weight ratio between 1.5:1 and 3.0:1 , preferably between 1.8:1 and 2.7:1. In an aspect of the invention the ketogenic composition has a ketogenic weight ratio below 2:1. The ketogenic composition preferably is an enteral nutritional composition. Preferably the composition is administered orally or as tube feed. The ketogenic composition may be formulated as complete nutrition, and potentially serve as the sole source of nutrition. The ketogenic composition is applied in liquid form and can be provided in solid form for dissolving in water before intake. Preferably the ketogenic composition is in liquid form. In one embodiment the present enteral ketogenic composition is a liquid. In one embodiment, preferably the present ketogenic composition is a liquid ready-to-feed composition. In a preferred embodiment, the composition is used as sole nutrition. In a further embodiment the ketogenic composition provides between 20% and 60% of the daily energy intake, more preferably between 30% and 50%. As used herein the weights are based on the total water- free of alternatively named dry weight of the enteral ketogenic composition.
Preferred daily dosages of the ketogenic composition are in the range of 500 to 4500 kcal, preferably 500 to 4000 kcal per day. The daily dosage may be provided in a single dose, but preferably in multiple doses per day. In one embodiment, the ketogenic composition is a liquid composition containing between 0.8 and 4 kcal per ml.
In an embodiment, dosing of the ketogenic composition is at least daily; for example, a subject may receive one or more doses daily, preferably 1 to 6 times per day, more preferably 3 to 4 times per day. In some embodiments, the administration continues for the remaining life of the individual. The ideal duration of the administration of the ketogenic composition can be determined by those of skill in the art.
Lipid fraction
The ketogenic composition according to the invention comprises (i) a lipid fraction.
The total amount of lipids that is present in the ketogenic composition, i.e. the combination of all lipids and phospholipids present, is referred to as the “lipid fraction” of the ketogenic composition. Suitable sources of lipids to provide for the lipid fraction of the ketogenic composition according to the invention are food-grade ingredients, for example those derived from eggs, milks, vegetables or plant material, marine organisms like fish or algae, natural- or mutated organisms or the lipids prepared by modification of the natural lipids, processed lipids from these sources such as extracts, isolates, inter-esterified lipids and the like, synthetic compounds or combinations thereof. Throughout this application the amounts of lipids are, unless otherwise indicated such as for example when reference is made to MCTs, described in terms of the molar weight of the fatty acids (FAs) present in the composition and corrected for the weight of glycerol in an oil blend.
The lipid fraction of the ketogenic composition contains a mixture of medium chain, long chain, and very long chain saturated fatty acids, as well as long chain and very long chain mono- and poly-unsaturated fatty acids. The fatty acids may be present in the ketogenic composition in either free form or bound either combined or non-combined to glycerol as mono-, di- or triacylglycerols and bound to any of the sn-1 , sn-2 or sn-3 position of glycerol.
Suitable sources of lipids for use in the ketogenic composition include, but are not limited to, fish oil, krill oil, algae oil, soybean oil, rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, coconut oil, palm oil, MCT oil, high oleic safflower oil, olive oil, poultry derived oil and egg-derived oil. An alternative option are lipids obtained from milk from non-human mammals, preferably cow's milk, goat milk, sheep milk, horse milk, buffalo milk, yak milk, reindeer milk, donkey milk and camel milk, particularly cow's milk and/or goat milk. Milk lipid is sometimes also referred to as milk fat or butterfat. In a preferred embodiment the lipid fraction of the ketogenic composition comprises coconut oil, fish oil, palm oil, rapeseed oil and/or MCT oil.
In one embodiment the lipid fraction preferably provides up to 70 % of the total amount of calories of the ketogenic composition, more preferably up to 72.5 % of the total amount of calories, more preferably up to 75% of the calories. In one embodiment the lipid fraction preferably provides between 65 and 85% of the calories, more preferably between 70 and 82.5% of the calories, even more preferably between 75 and 80% of the calories. The remainder of the calories of the ketogenic composition is provided by carbohydrates and proteins.
The ketogenic composition according to the invention comprises a lipid fraction, preferably a lipid fraction suitable for nutrition as known in the art. The ketogenic composition preferably comprises 45 to 65 g lipid per 100 g, more preferably 50 - 60 g lipid per 100 g, even more preferably 54 - 56 g per 100 g weight of the ketogenic composition. Worded alternatively the ketogenic composition preferably comprises 45 to 65 wt% lipids, more preferably 50 -60 wt%, even more preferably 54 - 56 wt% of lipids.
Based on calories, preferably the ketogenic composition may comprise 7 to 10 g of lipid per 100 kcal, preferably 7.5 to 9.5 g per 100 kcal, even more preferably 8 to 9 g of lipid per 100 kcal based on the total energy content of the ketogenic composition.
The amount of lipid fraction can be determined by applying methods known in the art for measuring fat content in a food matrix as applicable. For example, fat content for general foods is determined by applying AOACI official method 983.23, while the Roese-Gottlieb method (AOACI 932.06) is better applicable for products based on dried milk (Lehner, R., Estoppey, A., (1954) Mitt. Lebensmitteluntersuchung Hyg. 54:183-185). The amount of individual lipid components can be determined by applying methods specifically designed for measuring that specific component or by fractionating the fat fraction isolated from the extraction of the chloroform-methanol fraction as given in the 983.23 method. Additionally, the structural composition of the lipids such as the binding at the sn-1 , sn-2 orsn-3 position to form mono-, di- ortri-acylglycerols may be determined and quantified using liquid chromatography, mass spectrometry (also called lipidomics) and other methods known in the art (Beermann C et al., Lipids, 2005;40(2): 211-8).
MCT
The ketogenic composition according to the invention comprises saturated medium chain fatty acids [MCFAs], Preferably at least 70wt%, more preferably at least 80 wt%, even more preferably at least 90 wt% of the MCFAs are provided in the form of medium chain triglycerides [MCTs], In some embodiment it is preferred essentially all the MCFAs are provided in the form of MCTs. MCTs are a type of fat found in a small number of foods, such as coconut oil and palm kernel oil. MCTs may also be supplied by commercially available MCT oils. MCFAs are defined to be linear or branched, preferably linear, saturated carboxylic acids having six to twelve carbon atoms. When bound to a glycerol backbone at the sn-1 to sn-3 positions of glycerol MCFAs are present in the form of MCTs. Within the scope of this application MCTs also include glycerides wherein at least one of the FAs is an MCFA, preferably at least 2, most preferably 3 of the FAs are MCFAs. The presence of MCTs in the ketogenic composition is very advantageous, since they are effective in promoting a rapid induction of ketosis, that is, a rapid production and secretion of ketones by the liver into the blood circulation to create a condition of ketosis. In addition, MCTs are particularly beneficial for neurons for suppressing seizures and also induce the local production of ketones by glial cells (astrocytes) and supply to neurons in the brain. When hydrolysed, these oils provide concentrated sources of MCFAs with chain lengths of 6 (hexanoic acid), 8 (caprylic or octanoic acid), 10 (capric or decanoic acid) and 12 (lauric or dodecanoic acid) carbon atoms. Hence, the MCFAs according to the invention are preferably provided or selected from MCTs originating from coconut oils and/or palm kernel oils. The chain length of the MCFAs according to the invention is 6, 7, 8, 9, 10, 11 or 12, preferably 6, 8, 10 and/or 12 carbon atoms. The amounts of MCFAs provided herein are based on the molar weight of the MCFAs present in the composition and corrected for the weight of glycerol.
In an embodiment the ketogenic composition preferably comprises 10 - 20g MCFAs per 100g composition, more preferably 12 - 18g, even more preferably 13 - 16g per 100g composition based on the total weight of the ketogenic composition. Worded alternatively the ketogenic composition preferably comprises 10 - 20 wt% MCFAs, more preferably 12 - 18 wt%, even more preferably 13 - 16 wt% based on total weight of the ketogenic composition.
In an embodiment the MCFAs preferably provide 10 - 30%, preferably 15 - 25%, more preferably 17 - 20% of the total energy content of the ketogenic composition.
In an embodiment the amount of MCFAs in the lipid fraction is about 5 - 50 g per 100 g fatty acids, preferably 10 - 45 g per 100 g fatty acids, preferably 15 - 40 g per 100 g fatty acids, more preferably 20 to 35 g per 100 g fatty acids, and even more preferably 25 - 30 g per 100 g fatty acids. Alternatively, in an embodiment the lipid fraction preferably comprises 5 - 50 wt% MCFAs, preferably 10 - 45 wt%, preferably 15 - 40 wt%, more preferably 20 - 35 wt%, and even more preferably 25 - 30 wt%, based on total weight of the fatty acids in the ketogenic composition. In a preferred embodiment the ketogenic composition comprises 4 - 12 mol% MCFAs, preferably 5 - 10 mol% MCFAs, more preferably 6 - 9 mol % based on the total amount of fatty acids in the ketogenic composition.
When calculated in terms of MCTs, in an embodiment the MCTs preferably provide 10 -35%, preferably 15 - 30%, more preferably 20 -25% of the total energy content of the ketogenic composition. In a further embodiment the lipid fraction of the ketogenic composition preferably comprises 15 -45 wt% MCTs, more preferably 20 - 40 wt%, even more preferably 25 - 35 wt% MCTs based on total weight of the lipid fraction. The ketogenic composition preferably comprises 10 - 20 wt% MCTs, more preferably 12 - 18 wt% MCTs, even more preferably 13 - 17 wt% MCTs based on total weight of the ketogenic composition.
The MCT fraction in the ketogenic composition is beneficially rich in C8, C10 and C12 fatty acids. It is believed that the provision of an MCT blend rich in C8:0, C10:0 and C12:0 fatty acids aids in the ketogenicity of the diet. In an embodiment the MCT fraction is rich in C8:0, C10:0 and C12:0 fatty acids, wherein "rich” is defined as together forming more than 60 wt% of the FAs in the MCT fraction, preferably more than 70 wt% of the MCT fraction, even more preferably more than 80 wt% of the MCT fraction. In some aspects up to 100 wt% of the FAs in the MCT fraction are C8:0, C10:0 and C12:0 fatty acids. In one embodiment, the sum of the weight of medium chain fatty acids C6:0 + C8:0 over the sum of the weight of C10:0 and C12:0 in the ketogenic composition is less than 1 :1 , preferably less than 0.8:1 , more preferably less than 0.6:1 , even more preferably less than 0.3:1. In a further embodiment, the weight of C12:0 over the sum of the weight of medium chain fatty acids C6:0 + C8:0 + C10:0 is more than 1 .5:1 , preferably more than 2:1 , more preferably more than 2.3:1 , even more preferably more than 2.5:1
In an embodiment the ketogenic composition preferably comprises 8 - 12 g lauric or dodecanoic acid (C12:0), more preferably 8.5 -11 g lauric acid, even more preferably 9 - 10 g of lauric acid per 100g of the ketogenic composition. In a further embodiment the ketogenic composition preferably comprises 0.5 - 2.5 g capric acid (C10:0), more preferably 1 - 2 g capric acid, even more preferably 1.2 - 1 ,8g capric acid per 100g of the ketogenic composition. The ketogenic composition further preferably comprises 0.5 - 3 g caprylic acid (C8:0), more preferably 1 - 2.5 g caprylic acid, even more preferably 1 .5 - 2g caprylic acid per 100 g weight of the ketogenic composition.
Long Chain and Very Long Chain fatty acids
The ketogenic composition comprises long chain fatty acids [LCFA] and Very Long Chain fatty acids [VLCFA], wherein said long chain fatty acids are even-chain fatty acids that have a carbon length of 14 to 18, and wherein said very long chain fatty acids are even-chain fatty acids that have a carbon length of 20 or more. The LCFAs having a carbon chain length of 14 to 18 and VLCFAs having a carbon length of 20 or more, comprise both saturated and unsaturated fatty acids. The LCFAs and VLCFAs may be provided in any form such as, but not limited to, triglycerides, diglycerides, monoglycerides, free fatty acids or their salts or esters, phospholipids, lysophospholipids, glycerol ethers, lipoproteins, ceramides, glycolipids, or combinations thereof. Preferably, the ketogenic composition comprises the fatty acids in triglyceride form. Suitable sources of LCFAs and VLCFAs are, but are not limited to, palm oil and rapeseed oil. The amounts of LCFAs and VLCFAs provided are based on the molar weight of the LCFAs present in the ketogenic composition and corrected for the weight of glycerol.
In an embodiment of the invention the amount of even-chain LCFAs having a carbon length of 14 to 18 and VLCFAs having a carbon length of 20 or more is preferably less than 95 g per 100 g fatty acids, more preferably less than 85 g per 100 g fatty acids, preferably 50 - 95 g per 100 g fatty acids, more preferably 50 - 85 g per 100 g fatty acids, more preferably 55 to 80 g per 100 g fatty acids, and most preferably 60 - 75 g per 100 g fatty acids. Alternatively, in an embodiment the lipid fraction preferably comprises 50 -95 wt% LCFAs and VLCFAs, 50 -85 wt% LCFAs and VLCFAs, more preferably 55 -80 wt%, even more preferably 60 - 75 wt%, based on total weight of the fatty acids in the ketogenic composition.
In an embodiment the ketogenic composition preferably comprises 25 - 55 g LCFAs and VLCFAs per 100g composition, preferably 25 - 50 g LCFAs and VLCFAs per 100g composition, more preferably 30 - 45 g, even more preferably 35 - 40 g per 100 g composition based on the total weight of the ketogenic composition. Alternatively worded, in an embodiment the ketogenic composition preferably comprises 25 - 50 wt% LCFAs and VLCFAs, more preferably 30 -45 wt%, even more preferably 35 - 40 wt% based on total weight of the ketogenic composition. In an embodiment the LCFAs and VLCFAs preferably provide 40 - 70%, preferably 45 - 65%, more preferably 50 - 60% of the total energy content of the ketogenic composition. In yet another embodiment the LCFAs preferably provide 50 - 80%, preferably 55 - 75%, more preferably 60 - 70% of the total energy content of the lipids in the ketogenic composition.
In an embodiment of the invention the amount of palmitic acid (C16:0) is less than 40 g per 100 g fatty acids, preferably 10 - 40 g per 100 g fatty acids, more preferably 15 - 35 g per 100 g fatty acids, and most preferably 20 - 30 g per 100 g fatty acids. Alternatively worded, in an embodiment, the lipid fraction preferably comprises 10 - 40 wt%, more preferably 15 - 35 wt%, even more preferably 20 - 30 wt% palmitic acid.
In an embodiment the ketogenic composition preferably comprises less than 20g palmitic acid, more preferably less than 16g, even more preferably less than 14g per 100g of the ketogenic composition. Alternatively worded, in an embodiment the ketogenic composition preferably comprises less than 20 wt%, more preferably less than 16 wt%, even more preferably less than 14 wt% of palmitic acid based on total weight of the ketogenic composition.
In a preferred embodiment the LCFA and VLCFA fraction preferably comprises less than 45 wt% palmitic acid (C16:0), more preferably less than 40 wt%, even more preferably less than 35 wt% based on total weight of the LCFAs and VLCFAs.
Without being bound by theory, it is believed that myristic acid (C14:0 and palmitic acid (C16:0) may decrease hepatic LDL receptor expression and thereby promote the formation of pro-atherogenic oxLDL, whereas stearic acid (C18:0) is associated with lower plasma LDL levels. The ketogenic composition of the invention has a beneficially high stearic acid to the sum of [palmitic acid and myristic acid] ratio, such that despite having a high fat content the ketogenic composition provides a more beneficial cardiovascular risk profile.
In one embodiment the ketogenic composition preferably has a stearic acid to (palmitic acid and myristic acid) weight ratio of more than 1 : 12, more preferably more than 1 : 10, even more preferably more than 1 : 9. In an embodiment the ketogenic composition preferably comprises MCTs, LCTs and VLCTs at a weight ratio of MCTs to the sum of LCTs and VLCTs of between 1 :2 and 1 :5, preferably 1 : 2.5 and 1 : 3, more preferably 1 : 2.6 and 1 : 2.9, even more preferably 1 : 2.7 and 1 : 2.8.
Unsaturated fatty acids: MUFA In an embodiment of the invention the ketogenic composition comprises monounsaturated fatty acids [MUFA]. In a preferred embodiment the MUFA is at least one of palmitoleic acid (C16:1 co7), vaccenic acid (C18:1 co7), oleic acid (C18:1 co9), eicosoneic acid (C20:1 co9), erucic acid (C22:1 OJ9) and nervonic acid (C24:1 co9), preferably at least oleic acid. Suitable sources of MUFA include but are not limited to tea seed oil, olive oil, canola oil, palm oil and rapeseed oil. It is believed that MUFAs as used in the ketogenic composition are not only a substrate for ketone body production but may also provide a beneficial effect on glycemic control.
In an embodiment the total amount of mono-unsaturated fatty acids (MUFA) is between 15 and 40 g per 100 g, more preferably between 20 and 35 g per 100 g, even more preferably between 25 and 30 g per 100 g of fatty acids. Alternatively worded, in an embodiment, the lipid fraction preferably comprises 15 - 40 wt%, more preferably 20 - 35 wt%, even more preferably 25 - 30 wt% MUFAs based on total weight of the fatty acids in the lipid fraction.
In yet another preferred embodiment the total amount of MUFAs is between 5 and 25 g per 100 g, more preferably between 10 and 20 g per 100 g, even more preferably between 12 and 16 g per 100g of the ketogenic composition. Alternatively worded, in an embodiment, the ketogenic composition preferably comprises 5 - 25 wt%, more preferably 10 -20 wt%, even more preferably 12 - 16 wt% MUFAs based on total weight of the ketogenic composition.
In an embodiment the MUFAs preferably provide 10 - 30%, preferably 15 - 25%, more preferably 18 - 22 % of the total energy content of the ketogenic composition.
In a preferred embodiment the ketogenic composition comprises at least 8 g oleic acid per 100 g of the composition, more preferably at least 10 g, even more preferably at least 13 g per 100g of the composition. Alternatively worded, in an embodiment the ketogenic composition preferably comprises at least 8 wt%, more preferably at least 10 wt%, even more preferably at least 13 wt% oleic acid.
In an embodiment at least 85 wt% of the MUFAs is provided by oleic acid, more preferably at least 90 wt%, even more preferably at least 95 wt%, based on total weight of the MUFAs in the lipid fraction.
Unsaturated fatty acids: n-3 and n-6 PUFA
The ketogenic composition further comprises omega-3 and/or omega-6 polyunsaturated fatty acids (PUFA), preferably n-3 LC-PUFA, having a chain length of 18 carbon atoms, and n-3 VLC-PUFA, having a chain length of 20 and more carbon atoms). The provision of n-3 VLC-PUFA beneficially supports recovery of neurons after seizures and aids in reducing neuroinflammation and seizure progression.
In one embodiment, the OJ-3/OJ-6 weight ratio in the ketogenic composition of the invention is preferably in the range of 0.2:1 to 5:1 , preferably in the range of 0.3:1 to 4:1 , even more preferably in the range of 0.5:1 to 3:1. The amount of OJ-6 LCPUFAs in the ketogenic composition is preferably less than 20 weight%, preferably less than 15 weight%, more preferably less than 10 weight% of the fatty acids in the ketogenic composition. Alternatively worded the amount of OJ-6 LCPUFAs in the ketogenic composition is preferably between 0 - 20 weight %, preferably between 0 - 15 weight%, more preferably between 0 - 10 weight% of the total amount of fatty acids in the ketogenic composition
In an embodiment the ketogenic composition may comprise gamma linolenic acid (C18:3 co6). Although GLA is an n-6 fatty acid the fatty acid provides anti-inflammatory properties.
The provision of omega-3 fatty acids and the reduction of omega-6 fatty acids is believed to aid in limiting neuroinflammation after seizures and therewith aid in the observed reduction of the progression of epilepsy in the in vivo experiment.
The ketogenic composition comprises at least one, preferably two omega-3 VLC-PUFAs selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), preferably DHA and EPA. The weight ratio of EPA to DHA is preferably lower than 1 , more preferably 1 :1.1 to 1 :4, more preferably 1 :1.3 to 1 :4. In some embodiments the weight ratio of DHA to EPA is more than 2:1 .
In an embodiment the ketogenic composition may comprise alpha-linolenic acid [C18:3 co3, ALA],
The DHA, EPA and/or DPA may be provided in any form such as, but not limited to, triglycerides, diglycerides, monoglycerides, free fatty acids or their salts or esters, phospholipids, lysophospholipids, glycerol ethers, lipoproteins, ceramides, glycolipids or combinations thereof. Preferably, the ketogenic composition comprises at least DHA in triglyceride form.
Suitable OJ-3 VLCPUFA and/or sources of DHA and EPA include tuna oil, (other) fish oils, DHA-rich alkyl esters, algae oil, egg yolk, krill oil or phospholipids enriched with OJ-3 VLCPUFA, e.g. phosphatidylserine-DHA. Preferably, the ketogenic composition comprises fish oil providing the omega- 3 VLCPUFA(s). Another particularly suitable source for the omega-3 VLCPUFA(s) is algae oil.
In an embodiment the ketogenic composition preferably comprises 0.5 - 3 g VLC-PUFAs selected from DHA, EPA and DPA, most preferably DHA+EPA per 100g of the ketogenic composition, more preferably 1- 2.5 g, even more preferably 1 .2 - 2 g per 100g composition.
In an embodiment VLC-PUFAs selected from DHA, EPA and DPA, most preferably DHA and EPA, preferably provide 1 - 5%, more preferably 1 .5 - 3.5%, even more preferably 2 - 3% of the total energy content of the ketogenic composition. In a further aspect the total amount DHA, EPA and DPA, most preferably DHA and EPA are preferably 1.5 to 7 g per 100 g fatty acids, more preferably 2 to 6 g per 100 g fatty acids, and most preferably 3 to 5 g per 100 g fatty acids. In an embodiment of the invention the total amount of omega-3 LCPUFAs having a carbon length of 18, and omega-3 VLCPUFAs having a carbon length of 20 to 24 in the ketogenic composition is less than 15 g per 100 g fatty acids, preferably 2-15 g per 100 g fatty acids, more preferably 3 to 10 g per 100 g fatty acids, and even more preferably 4 to 7 g per 100 g fatty acids.
The LA:ALA weight ratio of the ketogenic composition is preferably in the range of 2:1 to 12:1 , more preferably 4:1 to 10:1 , even more preferably 6:1 to 8:1.
Choline
The present ketogenic composition preferably optionally further comprises choline. Choline may be present as such, or as choline equivalent in the form of a choline salt and/or choline ester. The choline salt is preferably selected from choline chloride, choline bitartrate, or choline stearate, preferably choline chloride. A choline ester is preferably selected from a phosphatidylcholine and lyso-phosphatidyl choline, preferably phosphatidylcholine.
The ketogenic composition preferably comprises less than 2000 mg, more preferably less than 1750 mg choline equivalents selected from choline, a choline salt and/or choline ester, calculated as choline, per 100 g of the ketogenic composition. In an embodiment the ketogenic composition comprises 500 to 5000 mg choline, preferably 1000 to 4500 mg, more preferably 1500 to 4000 mg choline per 100 g of the ketogenic composition and calculated as choline.
Carbohydrate fraction - digestible carbohydrates
The ketogenic composition comprises a carbohydrate fraction. The present ketogenic composition comprises digestible carbohydrates, preferably digestible carbohydrates with a low glycemic index. Typically, any digestible carbohydrates that are known in the art to be suitable for use in nutritional compositions may be used. Preferably, the ketogenic composition comprises digestible carbohydrates rich in one or more of non-glucose and non-fructose monosaccharides such as mannose, galactose, xylulose, xylose, glucosamine, and sialic acid. In a preferred embodiment the composition, combination, or product according to the invention comprises one or more low glycemic index carbohydrates selected from trehalose, lactose, galactose and isomaltulose. In a preferred embodiment the composition, combination, or product according to the invention comprises galactose and isomaltulose. Typically, such low Gl carbohydrates are carbohydrates having a Gl of 55 or less. Low glycemic index carbohydrates beneficially allow the inclusion of carbohydrates in the ketogenic composition while not significantly impacting ketogenesis. The ketogenic composition also comprises non-digestible carbohydrates or dietary fibers.
In a preferred embodiment the carbohydrate fraction of the ketogenic composition comprises between about 30 and 80 wt% low glycemic index carbohydrates, more preferably between 40 and 70 wt%, more preferably between 45 and 60 wt%.
The amount of galactose is preferably between about 8 wt% and 30 wt% of the carbohydrate fraction, more preferably between about 10 wt% and 25 wt% of the carbohydrate fraction and in an even more preferred embodiment between about 15 wt% and 20 wt% of the carbohydrate fraction. The amount of isomaltulose is in one embodiment preferably between about 20 wt% and 50 wt% of the carbohydrate fraction, more preferably between about 25wt% and 45 wt%, and in an even more preferred embodiment between about 30 wt% and 40 wt% of the carbohydrate fraction.
In an embodiment the amount of high glycaemic index carbohydrates such as glucose or rapidly digestible glucose polymers such as but not limited to sucrose is preferably below 70 wt% of the carbohydrate fraction, more preferably below 60 wt% of the carbohydrate fraction, even more preferably below 50 wt% of the carbohydrate fraction. In a preferred aspect the ketogenic composition is essentially free of glucose and/or rapidly digestible glycose polymers.
In one embodiment the digestible carbohydrate fraction preferably provides less than 8% of the total amount of calories of the ketogenic composition, more preferably less than 6% of the total amount of calories, more preferably less than 4% of the calories. The digestible carbohydrate fraction of the composition provides at least 1 % of the total amount of calories of the ketogenic composition, more preferably at least 2%.
In an embodiment the ketogenic composition preferably comprises 5 to 12 g digestible carbohydrates, more preferably 6 to 10 g digestible carbohydrates, even more preferably 7 to 9 g digestible carbohydrates per 100 g of the ketogenic composition, wherein said digestible carbohydrates preferably comprise low Gl carbohydrates, preferably galactose and isomaltulose. Worded alternatively, the ketogenic composition thus preferably comprises 5 to 12 wt% digestible carbohydrates, more preferably 6 to 10 wt%, even more preferably 7 to 9 wt% digestible carbohydrates based on total weight of the ketogenic composition.
In a preferred embodiment the glycaemic index (Gl) of the ketogenic composition is below 70, preferably below 65, preferably below 60, more preferably below 55. In a preferred aspect the glycaemic index is between 65 and 25, wherein the glycaemic index is based on glucose as reference value set at 100.
Fibers
Preferably the ketogenic composition comprises fibres, preferably food-grade dietary fibres. Dietary fibers are to large extent fermented in the colon by the bacterial flora. The fermentation process of the dietary fibers leads to the formation of short chain fatty acids (SOFA) that may be used as fuel by intestinal cells, provide beneficial effects on the gut microbiome and may provide beneficial effects to the ketogenicity of the ketogenic composition. A preferred embodiment according to the present invention thus includes dietary fibres in the ketogenic composition.
The ketogenic composition preferably comprises 7 - 15 g dietary fibers, preferably 8 - 14 g, more preferably 10 - 12 g fibers per 100 g of the ketogenic composition. Worded differently, the ketogenic composition preferably comprises 7 - 15 wt% fibers, preferably 8 - 14 wt%, more preferably 10 - 12 wt% of dietary fibers based on total weight of the ketogenic composition
In a preferred embodiment, the dietary fiber or fibers are selected from the group consisting of oligofructose, inulin, resistant starch, cellulose, methylcellulose preferably hydroxypropyl methylcellulose, wheat bran, gum arabic, soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin, hydrolysed pectin and mixtures thereof. The terms ‘gum arabic’ and ‘arabic gum’ are used interchangeably. In a preferred embodiment, mixtures of distinct dietary fibers are used, such as mixtures of at least 2, at least 3, at least 4, at least 5, at least 6 or even at least 7 distinct dietary fibers are used, preferably selected from the above list of preferred dietary fibers.
In an embodiment the fiber mixture comprises at least galactooligosaccharides, inulin and/or fuji soy. In a preferred embodiment the fiber mixture further comprises insoluble fibres like resistant starch and cellulose. It is preferred that the non-soluble fibres represent less than 50 wt % of the total amount of fibers in the ketogenic composition. The fiber mixture in the ketogenic composition according to the invention provides about 2 kcal per g fiber and preferably provides 2.5 - 5.5 % of the total amount of calories of the ketogenic composition, more preferably 3 - 5% of the total amount of calories, even more preferably 3.5 - 4.5 % of the calories
In an aspect the ketogenic composition comprises a fiber mixture comprising cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch. Preferably the fiber mixture comprises cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch in a weight ratio of 4 - 6: 1 - 3 : 1 - 3 : 0.5 - 2 : 0.1 - 0.4, more preferably 4.5 - 5.5 : 1 .5 - 2.5 : 1 .5 - 2.5 : 1 - 1 .5 : 0.2 - 0.3.
Protein fraction
The ketogenic composition according to the invention comprises a protein source. Protein includes all proteinaceous material, including intact and (partly) hydrolysed protein, peptides and free (added) amino acids. The total protein that is present in the ketogenic composition, i.e. the combination of all proteins and amino acids present, may be referred to as the “protein fraction” of the ketogenic composition. Any source of protein suitable to be used in a nutritional composition may be comprised in the ketogenic composition according to the invention. Preferred proteins sources include dairy proteins such as whey, casein, vegetables (beans, pea, soy, lupin, amaranth, potato, seed, grains tubers, etc.), fish, krill, algae, animals’ proteinaceous material, eggs and mushrooms.
In an embodiment the ketogenic composition preferably comprises 15 - 25 g of protein per 100 g of the composition, preferably 16 - 24 g of protein per 100 g of composition, more preferably 18 - 21 g of protein, based on total proteinaceous material of the protein fraction, per 100g composition. In an embodiment the ketogenic composition thus preferably comprises 15 - 25 wt% proteins, more preferably 16 - 24 wt%, even more preferably 18 - 21 wt% of proteins based on total weight of the ketogenic composition. Protein thus includes all proteinaceous material, including intact and (partly) hydrolysed protein, peptides and free (added) amino acids.
In an embodiment the protein provides 10 - 16%, preferably 11 - 15%, more preferably 12 - 14% of the total energy content of the ketogenic composition.
In one embodiment the protein fraction comprises at least 75 wt% intact and/or (partly) hydrolysed protein, preferably at least 80 wt%, more preferably at least 85 wt% based on total weight of the protein fraction.
In a preferred embodiment the protein fraction comprises intact and/or (partly) hydrolysed protein selected from casein and/or whey, preferably casein. In one embodiment the protein fraction comprises at least 75 wt% casein, preferably at least 80 wt%, more preferably at least 85 wt%. The protein fraction preferably comprises 75 - 95g casein per 100g, more preferably 80 - 92 g per 100g, even more preferably 85 - 90 g per 100g of the protein fraction. Worded differently the protein fraction of the ketogenic composition preferably comprises 75 - 95 wt% casein, more preferably 80 - 92 wt%, even more preferably 85 - 90 wt% based on total weight of the protein fraction. In a preferred aspect the casein is substantially intact casein derived from mammalian milk, preferably milk from a species of the genus Bos, Bison, Bubalus or Capra, more preferably from genus Bos, most preferably from cow’s milk (Bos ta urns').
In one embodiment the protein fraction comprises free amino acids selected from serine, lysine, glycine, valine, cysteine, leucine, and isoleucine, preferably leucine, isoleucine and/or cysteine. In a more preferred embodiment of the invention the protein fraction comprises the amino acids cysteine and/or leucine in free form. The protein fraction comprises at least 5 wt% of said amino acids, more preferably between 5 and 15 wt%, even more preferably between 7 and 13 wt%, particularly 8 - 12 wt%, based on the total protein content.
Without being bound by theory it is believed that the provision of leucine beneficially stimulates the reestablishment of functional connections between neurons in the brain following epileptic seizures through the activation of the cell growth-stimulating mTOR pathway. Furthermore, leucine provides antiseizure effects through modulation of neuroexcitation.
Preferably, the protein fraction of the ketogenic composition comprises at least 5 g amino acids per 100 g protein fraction, preferably at least 7.5 g amino acids per 100g protein fraction, preferably 5 - 15 g amino acids per 100g protein fraction, more preferably 7 - 13 g amino acids per 100g protein fraction, even more preferably 8 - 12 g amino acids per 100g protein fraction. Worded differently, the protein fraction thus preferably comprises at least 7.5 wt% amino acids, preferably 5 - 15 wt%, more preferably 7 - 13 wt%, even more preferably 8 - 12 wt% based on total weight of the protein fraction. In an embodiment the protein fraction comprises up to 15 g of amino acids per 100g protein fraction or up to 15 wt% of amino acids, based on total weight of the protein fraction. The amino acids are provided in free form, i.e., not part of a protein or peptide sequence. Preferably the amino acids comprise at least leucine in its free form. Per 100 g protein fraction the amount of free leucine is preferably 5 - 15 g, more preferably 7.5 - 12.5 g, even more preferably 8 - 10 g in free form. Preferably the protein fraction comprises 5 - 15 wt% free leucine, more preferably 7.5 - 12.5 wt%, even more preferably 8 - 10 wt% based on total weight of the protein fraction. In one embodiment the total amount of leucine in the protein fraction, provided by both the free leucine and protein-bound leucine preferably amounts to at least 7 wt%, preferably at least 8 wt%, more preferably at least 9 wt% based on total protein fraction. In the context of the invention, the terms ‘leucine’ and ‘L-leucine’, which is the L-enantiomer of Leucine, are used interchangeably.
Preferably the amino acids further comprise cysteine in free form. Per 100g protein fraction the amount of free cysteine is preferably 0.5 - 2.5 g, more preferably 1 - 2 g, even more preferably 1.2 - 1.6 g of cysteine. Preferably the protein fraction comprises 0.5 - 2.5 wt% free cysteine, more preferably 1 - 2 wt%, even more preferably 1 .2 - 1 .6 wt% based on total weight of the protein fraction.
Vitamins, minerals and antioxidants
In one embodiment, the ketogenic composition additionally comprises minerals, vitamins, antioxidants, and trace elements. The micronutrients are provided in amounts recommended for nutritionally complete foods and in line with the EFSA’s recommendations on required daily minerals and vitamins adjusted for age and gender.
In a preferred embodiment the ketogenic composition comprises therapeutically effective doses of vitamin B3, preferably nicotinamide riboside [NR] or nicotinamide [NAM], The ketogenic composition preferably comprises 75 - 225 mg NR per 100 g or 36 - 108 mg NAM per 100 g, more preferably 100 - 200 mg NR per 100 g or 50 - 100 mg NAM per 100 g, even more preferably 125 - 175 mg NR per 100 g or 60 - 85 mg NAM per 100 g ketogenic composition. The above captioned ranges are applicable with the recommendation made by the EFSA that the total daily dose of nicotinamide riboside for an adult subject should be limited to 300 mg per day, corresponding to 126mg NAM per day. Meanwhile, in 2002, the Scientific Committee on Food (SCF) has set a tolerable upper intake level for NAM of 900 mg/day (12.5 mg/kg bw) for the adult population, excluding pregnant and lactating women. As used herein, "nicotinamide riboside" includes derivatives thereof such as L-valine and L-phenylalanine esters of nicotinamide riboside. NR and NAM as preferable comprised in the ketogenic composition according to the invention is a NAD+ precursor, provides beneficial effects on both oxidative stress and DNA repair and therewith aids in delaying the progression of epilepsy.
In a preferred embodiment the ketogenic composition comprises therapeutically effective doses of pyruvate. Pyruvate can be incorporated e.g. as free acid or as its Ca, Na or K salt or as precursor of pyruvate. Pyruvate is preferably provided in a range of 1 .5 mg to 25 mg pyruvate per kcal of ketogenic composition, more preferably 2.5 mg to 15 mg pyruvate per kcal of ketogenic composition, even more preferably 5.8 mg to 12 mg pyruvate per kcal of ketogenic composition. Pyruvate is preferably present in the ketogenic composition at a dose corresponding to 1 wt% to 13 wt% of the total weight of the ketogenic composition, more preferably 1.5 wt% to 9 wt% of the total weight of the ketogenic composition. Any amounts of pyruvate equivalents or derivatives are recalculated to the equivalent weight amount of pyruvate, counter-ions excluded.
The weight ranges and percentages of pyruvate in the neuroprotective composition in association with a ketogenic diet are based on a daily intake of between 500 and 4000 kcal per day and a body weight between 5 kilogram and 100 kilogram.
A further antioxidant preferably comprised in the ketogenic composition according to the invention is tert-butylhydroquinone. Tert-butylhydroquinone beneficially acts as an antioxidant, aiding in preventing neuronal damage in epilepsy, prolong seizure latency and reduce the degree of epileptic seizures. In a preferred embodiment the ketogenic composition comprises 0.8 - 2 mg tert-butylhydroquinone per 100 g, more preferably 1 - 1.5 mg tert-butylhydroquinone, even more preferably 1.2 - 1.4 mg tert- butylhydroquinone per 100g of the ketogenic composition. Worded differently, the ketogenic composition preferably comprises 0.0008 - 0.002 wt% tert-butylhydroquinone, more preferably 0.001 - 0.0015 wt%, even more preferably 0.0012 - 0.0014 wt% based on total weight of the ketogenic composition.
In a preferred embodiment the ketogenic composition further comprises citrate. Citrate as used in the neuroprotective composition may be in the form of citric acid or a salt of potassium (K+), sodium (Na+), Magnesium (Mg2+) or calcium (Ca2+).
Citrate or citric acid is converted to isocitrate in the first step in the Krebs cycle (also known as the citric acid orTCA cycle). Citrate was found to increase glycolytic rates in in vitro neuroglial models and provide prevention against neuronal damage by increasing energy production, preferably through a coupled increase in glycolysis in glial cells and mitochondrial oxygen consumption in neurons in the brain. Citrate is believed to reduce and/or prevent excitotoxity associated with or following seizures, and consequently prevent and/or treat neuroinflammation associated with epileptic seizures, and consequently prevent and/or delay the formation of epileptiform circuits in the brain.
In a preferred embodiment the ketogenic composition comprises 100 - 1000 mg citrate per 100 g, more preferably 250 - 750 mg citrate, even more preferably 500 - 600 mg citrate per 100g of the ketogenic composition. Worded differently, the ketogenic composition preferably comprises 0.1 - 1 wt% citrate, more preferably 0.25 - 0.75 wt%, even more preferably 0.5 - 0.6 wt% based on total weight of the ketogenic composition. Any amounts of citrate equivalents or derivatives are calculated to the equivalent weight amount of citrate, counterions excluded.
In some embodiments it is preferred that the ketogenic composition comprises both citrate and pyruvate. EXAMPLES
For a more complete understanding of the present disclosure, reference is now made to the following examples taken in conjunction with the accompanying drawings. Example 1
An exemplary enteral ketogenic composition was formulated comprising per 100g of the composition 644 kcal and comprising 20 wt% proteins including free amino acids, 8 wt% digestible carbohydrates, 56 wt% lipids, 11 wt% dietary fibers and 5 wt% further micronutrients. The ketogenic composition comprised ingredients according to table 1 .
Table 1
Example 2
The ketogenic diet (hereinafter: alternative ketogenic diet) according to table 1 was assessed in the so- called rapid rat kindling model of temporal lobe epilepsy. Adult male rats were switched from a chow diet to a rodent standard diet one week prior to the surgical implantation of stimulation electrodes into the angular bundle. Animals were left to recover for 1 week from surgery. They were then switched to either a rodent standard diet, a rodent adapted classic ketogenic diet with a fat-to-carbohydrate plus protein ratio of 6:1 or the alternative ketogenic diet. The animals were acclimated to their diets for ~1 week during which body weights and food intake were measured. On day 5 of the diet, the glucose and ketone levels in the blood were measured to confirm ketosis induction. Thereafter the animals were subjected for one week to 36 sessions of electrical stimulations via the implanted electrodes. The seizures were behaviourally scored on a scale according to Racine from 1 (lowest severity) to 5 (highest severity). During seizures the electrical brain activity was recorded during stimulation. After the last stimulation, animals were left to recover for one week. They were then subjected for one week to behaviour analyses (open field test, Barnes Maze test, novel object recognition test). One week after the end of the behaviour studies, the animals were fasted overnight, blood samples taken for glucose and ketone level measurements and then sacrificed by decapitation, followed by the collection of brain and liver samples for molecular, biochemical, and histological analyses. The number of animals analysed in each treatment group was n=14 for the alternative ketogenic diet, n=16 for classic ketogenic diet, and n=15 for the control diet.
The alternative ketogenic diet significantly delayed the progression from mild (Racine score 1) to severe seizure scores (Racine score 5) and full kindling (Racine score 5 with subthreshold electrical stimulation), compared to the classic ketogenic diet and the rodent standard diet (Figure 1).The onset of a progressive worsening is seen from stimulation session 12 in the alternative KD group compared to session 3-5 and 3 in the classic ketogenic diet and control diet groups, respectively (Figure 1). Furthermore, rats in the alternative KD group remained in stage 1 significantly longer, and significantly more stimulations were needed to reach stages 4 and 5 (Figure 2). Also, when averaging the seizure score for each stimulation session, a reduction in mean seizure severity was seen with the alternative ketogenic diet (Figure 3, alternative KD: dark grey color; classic KD: dashed line; control diet: light grey color).
Furthermore, electroencephalography determined the duration and latency of the afterdischarge (AD), an additional measure for neuroexcitability, following the initial 10 second electrical stimulation (Figure 4A). The AD consisted of a first part (AD1) and a second part (AD2) separated by a latency period. The AD1 was significantly shorter for both KD groups compared to controls (Figure 4B). Moreover, the latency between the AD1 and AD2 was significantly longer for both KD groups compared to controls (Figure 4C).
Behavioural analyses were conducted by the open field test. With the open-field test the following was determined: (i) entries into and time spent in the centre zone as a measure of anxiety; (ii) rearing frequency and rearing duration as a measure of activity and exploration; (iii) distance and velocity moved as a measure of locomotion. Healthy, non-kindled control animals preferentially stayed close to the confinement/border of the open field test with few entries into and little time spent in the centre zone. Meanwhile kindled animals on the control diet entered and spent significantly more time in the centre zone of the open field (Figure 5). They also displayed significantly more rearing behaviour (Figure 6). They also showed increased velocity, distance moved and zone alternations (Figure 7). Kindling (or seizures) led to a hyperactive, hyper-exploratory, possibly impulsive, and fearless behaviour in the control animals. This behavioural pattern is reminiscent of an attention deficit hyperactivity disorder (ADHD). ADHD is indeed frequently associated with epilepsy and vice versa in humans. Both the alternative and classic ketogenic diets showed a significant reduction in centre zone entries and time spent in the centre zone than kindled animals on the control diet (Figure 5). Furthermore, they show a significant reduction in rearing (Figure 6) and no hyperlocomotion (Figure 7). Hence, both the alternative and classic ketogenic diets reversed the abnormal ADHD -like behaviour induced by repeated seizures.
Measurements of ketone levels in the blood of the animals were done after 5 days that is priorto kindling, and after 21 days of exposure to the ketogenic diet, i.e. about 1 weeks after the last kindling session. Ketone measurements were conducted in serum of non-fasted animals obtained during the same morning hours. These measurements showed a significant increase in blood ketone levels in both ketogenic diet groups after 5 and 21 days of ketogenic diet exposure, however, the alternative ketogenic diet increased ketone levels only by about 50% of the levels achieved by the classic ketogenic diet (Figures 8 and 9). Hence, the novel (alternative) KD was less ketogenic. This implies additional Mode of Actions of the alternative KD that are not mediated by ketones and render the alternative ketogenic diet more efficacious. Furthermore, the induction of blood ketosis was the same in kindled and nonkindled animals exposed to the alternative ketogenic diet (Figures 8 and 9).
Fatty acid and glucose concentrations were measured in the serum of non-fasted rats obtained in the mornings of day 5 and day 21 of diet exposure. Triglyceride levels were significantly increased by the classic ketogenic diet while they were not increased by the alternative ketogenic diet compared to the control diet (Figure 10) both in kindled and non-kindled animals, as well as after 5 and 21 days of diet exposure (Figure 10). Glucose levels were not significantly different across all groups, indicating that neither ketogenic diet led to visible changes in glycemia compared to the control diet (Figure 11).
Specific assessment of the levels of omega-3 and omega-6 polyunsaturated fatty acids in the serum of kindled and non-kindled rats showed that the alternative ketogenic diet increased the total omega-3 PUFA content, and decreased the total omega-6 PUFA content, leading to a significant increase in the omega-3 to omega-6 PUFA ratio compared to the classic ketogenic diet and rodent standard diet (control diet) (Figures 12 and 13). Furthermore, the serum levels of the omega-3 PUFAs DHA, EPA were dramatically increased by the alternative ketogenic diet whereas the levels of the omega-3 PUFA alpha linolenic acid (ALA) and the omega-6 PUFA linoleic acid (LA) were significantly decreased compared to the classic ketogenic diet and the rodent standard diet (control diet) in kindled and nonkindled animals at day 5 and 21 of the diet exposure (Figures 12 and 13).
Specific assessment of the serum levels of other fatty acids showed that - contrary to the classic ketogenic diet - the alternative ketogenic diet did not increase the serum concentrations of the long chain fatty acids palmitic acid (C16), stearic acid (C18), oleic acid (C18:1) compared to the rodent standard (control) diet in kindled and non-kindled animals after 5 and 21 days of diet exposure (Figure 14 and 15). Moreover, the alternative ketogenic diet decreased the serum concentrations of the long chain fatty acids myristic acid (C14), palmitic acid (C16), stearic acid (C18), and oleic acid (C18:1) compared to the rodent standard (control) diet in non-kindled animals after 5 and 21 days of diet exposure (Figure 14 and 15). These LCFAs are the most abundant LCFAs present in the alternative ketogenic diet, the classic ketogenic diet, and the rodent standard (control) diet. Notably, albeit they are more abundant in the alternative ketogenic diet than in rodent standard (control) diet, their serum levels were lower in the alternative ketogenic diet group than in the control diet group. These LCFAs are known to mediate the development of insulin resistance, a metabolic syndrome, and fatty liver. Hence, the alternative ketogenic diet protected kindled and especially non-kindled rats, against an increase of circulating long chain atty acids known to lead to fatty liver development, insulin resistance, metabolic syndrome, and consequently cardiovascular complications thereof. The protection from fatty liver development was confirmed by visual inspection of the liver at the time of harvesting the livers and by measuring their fatty acid content (Figure 16). Livers collected from animals on the classic ketogenic diet showed a pale yellowish colour, consistent with liver steatosis. Meanwhile livers collected from animals on the rodent standard (control) diet, or the alternative ketogenic diet showed a normal dark red color, consistent with normal fat content of the liver. Measurements of the triglyceride contents of these livers in non-kindled animals showed equal triglyceride content in both the alternative ketogenic diet and control diet groups. Furthermore, they showed that kindled rats on the alternative and classic ketogenic diets had accumulated more triglycerides than rats on the control diet, but that triglyceride accumulation was significantly lower in kindled rats on the alternative ketogenic diet than in kindled rats on a classic ketogenic diet (Figure 16).
In kindled and non-kindled animals, body weight measurements showed a trend for a reduction in body weight gain with the alternative ketogenic diet compared to the rodent standard (control) diet (Figure 17), consistent with a reduction in adiposity or fat mass conveyed by the alternative ketogenic diet compared to the control diet.
Serum measurements of amino acid concentrations after 5 days and 21 days of diet exposure showed that the alternative ketogenic diet provided greater levels of essential amino acids Leucine (Leu), Threonine (Thr), Tryptophan (Trp), Valine (Vai), Phenylalanine (Phe) - that is amino acids that cannot be produced endogenously and must be obtained through food intake - than the classic ketogenic diet (Figure 18 and 19). The alternative ketogenic diet has a greater protein content and provides free Leu, totalling ~20%wt compared to ~9%wt in the rodent-adapted classic ketogenic diet (with a 6:1 fat-to- protein plus carbohydrate ratio) and ~14%wt in a classic human ketogenic diet (with a 4:1 fat-to-protein plus carbohydrate ratio). Meanwhile the protein source (casein) is the same in these diets and thus unlikely to have caused differences in essential amino acid supply.
Furthermore, both the alternative and classic ketogenic diets increased the serum concentrations of several amino acids previously known to reduce neuroexcitability, and thereby helping to reduce seizure activities in neurons, namely leucine, serine, glycine, and lysine, compared to the rodent standard (control) diet, both after 5 days and 21 days of diet exposure (Figures 20 and 21).
Lastly, it was found that, the alternative ketogenic diet provided less gluconeogenic amino acids - that is, amino acids that can be used by the liver to produce glucose - compared to the rodent standard (control) diet both at day 5 and day 21 of diet exposure (Figures 22 and 23). This beneficially increased the efficacy of the diet as less glucose would be produced in the liver, forcing a higher production and utilization of ketones.
Example 3
Fatty acid blends representing the fatty acid blends in a ketogenic diet containing a moderate level of medium chain triglycerides (comparative Diet) and fatty acid blends representative for a diet according to Example 1 (inventive diet) were prepared. The fatty acid blend corresponding to the comparative diet is referred to as the COMP-KD fat blend, and that of the experimental diet of the present invention is referred to as the INV-KD fat blend. The fatty acid profiles of the COMP-KD and INV-KD are comparable to the fatty acid blends present in the corresponding nutritionally completely ketogenic diets. The COMP- KD comprises moderate amounts of MCFAs with less than 10% of the total kcal being provided by MCFA, while for the INV-KD about 20 % of the kcal is provided by MCFA. The INV-KD fat blend has a weight ratio of MCTs to the sum of [LCTs and VLCTs] of about 1 : 2.8, while the COMP-KD fat blend has a weight ratio of MCTs to the sum of [LCTs and VLCTs] of about 1 : 19.
The fatty acid profile of both fatty acid blends is shown in Table 2 (in molar percentage based on a total of 100%). These fatty acid blend are similar to the ones that can be used in the comparative diet and the diet according to the invention for preparation of a complete nutritional composition further comprising proteins, carbohydrates and micronutrients. The ketogenic responses and the direct effect of the fat blends on neuronal cell cultures can be extrapolated to effects of the corresponding ketogenic diets.
Table 2. Molar % of medium, long and very long chain fatty acids contained in the blend based on a total of 100%.
Faty acid-albumin conjugation
Conjugation of medium, long and very long chain fatty acids to bovine serum albumin (BSA) for the COMP-KD and INV-KD was performed using an adapted protocol for BSA-palmitate conjugate. Therein, each fatty acid was dissolved in a 150mM sodium chloride solution by warming up the solution to 5-8 degrees over the fatty acid’s melting point. Additionally, the pH of this solution was increased to 7.4 to deprotonate the carboxyl groups of the fatty acids thereby rendering them more hydrophilic. The fatty acid solution was then added in short bouts to a stirring Ultra Fatty acid-free BSA solution (Merck Life Science N.V., Zwijndrecht, the Netherlands), that never exceeded 40°C to avoid denaturation of the protein. After stirring for one hour, the BSA conjugated stocks were filtered through a 0.22 pm polyvinylidene difluoride (PVDF) membrane. Due to their inherent difficulty to dissolve in aqueous environments, the final concentrations of all long-chain fatty acids were measured afterwards using gas chromatography. C19:0 was used as an internal standard. Fatty acids were converted to methyl esters using methanol and sulphuric acid. After extraction using hexane, the Fatty Acid Methyl Esters were separated and quantified using a gas chromatograph GC-2025 (Shimadzu Benelux B.V., ‘s Hertogenbosch, the Netherlands). The ratio between the internal standard and fatty acid methyl esters peaks was used to calculate the concentration of the respective fatty acid stocks used.
Under these conditions and by titrating the amounts of added fatty acids, the binding ratio of fatty acid to BSA could be determined, well-controlled and reproduced with minimal inter-experimental variation and could be well compared between different fatty acid-BSA conjugates. The molar binding ratio of fatty acid to BSA was aimed at 4:1 for all conjugates. The very short chain fatty acids acetic acid, propionic acid and butyric acid were not conjugated to BSA due to their inherent high water-solubility. Aliquots of the saturated fatty acid-BSA conjugate stocks were stored at -20°C and unsaturated stocks at -80 °C. They were diluted to required concentrations in Krebs-Henseleit Buffer (KHB) prior to the ketogenic phase. The following fatty acids were used in the assay: sodium acetate, sodium propionate, sodium butyrate, Hexanoic acid, Octanoic acid, Decanoic acid, Dodecanoic acid, myristic acid, palmitic acid, oleic acid, oleic acid-Albumin from bovine serum, Linoleic acid, a-linolenic acid, Docosahexaenoic acid, Eicosapentaenoic acid (Merck Life Science N.V., Zwijndrecht, the Netherlands).
Metabolic effects
A Seahorse assay was performed on primary rat neuronal-glial cell cultures to determine the impact the INV-KD and COMP-KD fat blends on glycolytic rates, mitochondrial respiration and total (glycolytic and mitochondrial) ATP production. In the below described Seahorse assay it was found that the INV-KD fat blend increases glycolysis and respiration and ATP production in neuroglial cells under seizure-like conditions. Therefore, providing the increased proportion of MCTs in the INV-KD blend helps neurons and glial cells to meet the energy demand imposed on them by epileptic seizures and provides neuroprotective effects. These effects were not attributed to the proton leak, as explained below.
Primary neuroglial cell culture
Rat cortical cells were isolated from embryonic day 18 embryos using a combination of enzymatic and mechanical dissociation of brain cortical rat tissue. Immediately after dissociation, the cells were seeded at a density of 500’000 cells per mL in 96-well plates and grown at 37 °C for two weeks from day in vitro (DIV) 0 until DIV17 in Neurobasal medium supplemented with 2% (v/v) B27, 0.8mM Magnesium (Mg2+), 25mM glucose and 1 % (v/v) Penicillin/Streptomycin with partial medium refreshments on DIV3, DIV7 and DIV10. During this time neuronal axons grew out and formed synaptic contacts. In addition, glial cells, foremost astrocytes, grew and formed contacts with neurons. The incubation in 25mM glucose accelerated the differentiation and network growth of neurons. On DIV16, the medium was exchanged for unbuffered Hank’s Balanced Salt Solution (HBSS) (a nutrient deprived medium) supplemented with 0.1 mM Mg2+, 5mM glucose and various concentrations of the test nutrient The switch from high (0.8mM) to low (0.1 mM) Mg2+ increases the electrical firing frequency of neurons, because Mg2+ is a known inhibitor of excitatory NMDA-type ionotropic glutamate receptors, which account for most of the neuronal firing in this cell culture model. The increase in neuronal firing caused by incubation in 0.1 mM Mg2+ is reflected in an increase of the amplitude and/or frequency of calcium (Ca2+) oscillations (which arise from the influx of Ca2+ into the neurons triggering neurotransmitter release, and the subsequent efflux of Ca2+ out of the neurons) and consequently an increase of the area under the curve of these Ca2+ oscillations. Furthermore, the neuronal firing increases the energy consumption and production of neurons and glial cells. The energy production is reflected in an increase in basal respiration as oxygen is used for energy production in neurons and an increase in glycolysis, which produces energy from the conversion of glucose to lactate (also called glycolysis) in astrocytes. In turn, lactate is transferred from astrocytes to neurons where it serves as a fuel to sustain neuronal energy production through respiration. The transfer of lactate from astrocytes to neurons is known as the astrocyte-neuronal lactate shuttle as described in Magistretti et al. Nature Reviews Neuroscience, 2018.)
Seahorse assay
The oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) of cells seeded in 96-well plates were monitored in real time at 37°C in unbuffered Hanks Balanced Salt Solution (HBSS). The OCRs represent foremost mitochondrial respiration, and to a minor extent non-mitochondrial respiration. The ECARs relate foremost to the acidification of the medium with lactate produced through glycolysis and secreted by the cells into the medium (Table 3).
During the Seahorse run, both the OCRs and ECARs were measured simultaneously at baseline and following the injection of compounds by the Seahorse instrument (Table 3). HBSS was supplemented with glucose from the start and no glucose was injected through the Seahorse instruments. Measurements were repeated three times at baseline and three times after each compound injection.
Table 3. Compounds used during Seahorse assay and their effect on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
Glycolytic and mitochondrial energy production was assessed on DIV16 by exposing the primary neuroglial cultures described above to the fat blends at a concentration of 50 JJ.M. Neuroglial cell cultures were pre-incubated for 2 hours in the Seahorse medium containing the fat blends. Immediately after the end of this pre-incubation, the oxygen consumption rate [OCRs] and extracellular acidification rate [ECARs] of the neuroglial culture were measured in the Seahorse assay. The number of repeats for each fat blend was 9.
Ca2+ oscillation assay
The Ca2+ oscillation assay uses neuroglial cell cultures grown in the same manner as those used for the Seahorse assay. The neurons in these cultures form dense networks that display spontaneous synchronized activity, or oscillations. These oscillations are measured by incubating the cells with a calcium sensitive dye that produces a fluorescent signal when neurons become active. The frequency, amplitude and the area under the curve of the oscillatory pattern are measures of excitability of the neuronal network, which can be stimulated with pro-epileptic conditions such as low magnesium (0.1 mM Mg2+). The 0.1 mM Mg2+ stimulation of the neuronal network mainly increases the frequency and area under the curve of oscillatory pattern as compared to the effect under normal magnesium concentrations, i.e. at 0.8 mM Mg2+ and this can be partially reversed with nutrient interventions such as ketogenic diet fat blends.
Neuroexcitability was measured by assessing Ca2+ oscillations on DIV16, To this end, the neuroglial cell cultures described above were pre-incubated for 2 hours to the fat blends at a concentration of 25 |j.M. Immediately thereafter, the amplitude and frequency of the Ca2+ oscillations in the cell culture were recorded in real-time by a fluorescence plate reader (FlexStation, Molecular Devices) and the area under the curve quantified from these measurements with a statistics package (Prism-GraphPad) The number of repeats for each fat blend was 6.
Results
Stimulation of the neuroglial cells with 0.1 mM Mg2+ increased the frequency and area under the curve (AUC) of oscillatory patterns as compared to the control situation wherein cells are exposed to 0.8 mM Mg2+ (Figure 24 A-C). Stimulation of the neuroglial cells with 0.1 mM Mg2+ also increased energy production, as reflected in an increase in baseline glycolytic rate (ECAR), baseline mitochondrial oxygen consumption rate (OCR), baseline glycolytic ATP production and baseline mitochondrial ATP production compared to the baseline situation wherein neuroglial cells are maintained at 0.8 mM Mg2+ (Fig 24 D- G).
A moderate reduction in the frequency, amplitude and area under the curve (AUC) of Ca2+ oscillations was observed for 0.1 mM Mg2+ stimulated neuroglial cells with the INV-KD fat blend compared to the COMP-KD fat blend. This shows a moderately stronger potential of the INV-KD fat blend on dampening neuroexcitability than the COMP-KD fat blend (Fig. 25 E - G). Furthermore, the INV-KD fat blend increased baseline respiration, and oxidative capacity (FCCP response) more than the COMP-KD fat blend (Fig 25 A - B). The INV-KD fat blend also increased glycolytic rates moderately more than the COMP-KD fat blend (Fig 25 C-D). Finally, the INV-KD fat blend increased mitochondrial and glycolytic ATP production moderately more than the COMP-KD fat blend (Fig 25 H-l). The proton leak (or uncoupling effect) was the same between both blends and as a result the mitochondrial proton leak could not explain the observed differences in mitochondrial or glycolytic energy production between the KD fat blends (Fig 25J). Taken together, the INV-KD fat blend dampened neuroexcitability and increased mitochondrial and glycolytic energy production in neurons and glial cells more than the COMP-KD fat blend.
Overall these data thus show that the INV-KD fat blend composition unexpectedly has a direct and better effect on neuroexcitability and neuroglial energy production than the COMP-KD fat blend. These effects are in turn linked to better seizure control while not being linked to ketones or ketosis induction.

Claims

Claims
1 . Enteral ketogenic composition comprising (i) a lipid fraction comprising 45 to 65 g lipids, (ii) a carbohydrate fraction comprising 5 to 12 g digestible carbohydrates, and (iii) a protein fraction comprising 15 - 25 g of protein per 100g of the ketogenic composition, wherein the lipid fraction comprises
5 - 50 wt% medium-chain fatty acids [MCFAs] based on total weight of the fatty acids in the lipid fraction, wherein more than 60 wt% of said MCFAs are C8:0, C10:0 and C12:0 fatty acids and wherein at least 70wt% of said MCFAs are provided in the form of medium chain triglycerides [MCTs];
50-95 wt% even-chain long chain fatty acids [LCFAs] and very-long-chain fatty acids [VLCFAs] having a carbon length of 14 to 24 based on total weight of the fatty acids in the lipid fraction, and wherein the LCFAs and VLCFAs comprise at least one omega-3 poly-unsaturated fatty acid [PUFA] selected from the group consisting of docosahexaenoic acid (22:6, co-3; DHA), eicosapentaenoic acid (20:5, co-3; EPA) and docosapentaenoic acid (22:5 co-3; DPA), wherein the ketogenic composition comprises MCTs, long chain triglycerides (LCTs) and very long chain triglycerides (VLCTs) at a weight ratio of MCTs to the sum of [LCTs and VLCTs] between 1 : 2 and 1 : 5, wherein the carbohydrate fraction comprises between 30 and 80 wt% low glycemic index carbohydrates based on total weight of digestible carbohydrates, and wherein the protein fraction comprises 5 - 15 wt% free amino acids based on total weight of the protein fraction, and wherein the ketogenic composition has a ketogenic weight ratio between 1 .5:1 and 3.0:1 .
2. The enteral ketogenic composition according to claim 1 wherein the composition further comprises 7 - 15 wt% dietary fibers and wherein the fibers are selected from oligofructose, inulin, resistant starch, cellulose, wheat bran, gum arabic, soy polysaccharides such as fuji soy, oat fiber, galactooligosaccharides, locus bean gum, guar gum, pectin and hydrolysed pectin.
3. The enteral ketogenic composition according to claim 2 wherein the fibers are a fiber mixture comprising cellulose, inulin, galactooligosaccharides, fuji soy, and resistant starch.
4. The enteral ketogenic composition according to the preceding claims wherein the low glycemic index carbohydrates are selected from trehalose, lactose, galactose and isomaltulose.
5. The enteral ketogenic composition according to the preceding claims wherein the protein fraction comprises intact and/or (partly) hydrolysed protein selected from pea, soy, casein and/or whey, preferably casein.
6. The enteral ketogenic composition according to the preceding claims wherein the protein fraction comprises 75 - 95 wt% casein based on total weight of the protein fraction.
7. The enteral ketogenic composition according to the preceding claims wherein free amino acids in the protein fraction are selected from the group consisting of serine, lysine, glycine, valine, cysteine, leucine and isoleucine, preferably leucine, isoleucine and/or cysteine.
8. The enteral ketogenic composition according to the preceding claims wherein the protein fraction comprises 5 - 15 wt% free leucine and 0.5 - 2.5 wt% free cysteine based on total weight of the protein fraction.
9. The enteral ketogenic composition according to the preceding claims wherein the OJ-3/OJ-6 weight ratio of the lipids is between 0.2:1 and 5:1.
10. The enteral ketogenic composition according to the preceding claims wherein the composition comprises mono-unsaturated fatty acids [MUFAs] and wherein at least 85 wt% of the MUFAs is oleic acid, based on total weight of the MUFAs in the lipid fraction.
11 . The enteral ketogenic composition according to the preceding claims further comprising citrate and/or nicotinamide riboside and/or nicotinamide and/or pyruvate.
12. The enteral ketogenic composition according to the preceding claims wherein the composition comprises less than 20 wt% of palmitic acid based on total weight of the ketogenic composition.
13. Enteral ketogenic composition according to the preceding claims for use in the treatment and/or prevention of epilepsy in a subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures.
14. The enteral ketogenic composition according to claim 13 for use in the treatment and/or prevention of epilepsy, wherein the treatment and/or prevention of epilepsy comprises treatment and/or prevention and/or reduction of the severity of epilepsy and prevention and/or delay of the formation of epileptiform circuits in the brain.
15. The enteral ketogenic composition according to any one of claims 13 and 14 for use in the treatment and/or prevention of epilepsy further preventing the development of dyslipidemia, fatty liver and/or metabolic syndrome in the subject suffering from epilepsy or at risk of seizures, preferably epileptic seizures
16. The enteral ketogenic composition according to any one of claims 13 to 15 wherein the treatment and/or prevention of epilepsy and/or progression of epilepsy in a subject suffering from epilepsy or at risk of seizures further comprises diminishing and/or preventing behavioural patterns associated with ADHD.
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