Patent ID: 12257222

DETAILED DESCRIPTION

By elucidating the mechanism associated with the benefits of ketogenic diets we overcome some of the problems associated with prior compositions by providing a highly effective product which can be formulated to increase its palatability and deliver a specific level of C10. Moreover it allows diseases to be treated by the use of C10 outside of the strict confines of the classic ketogenic diet.

Briefly, the classical version of the ketogenic diet uses ratios to determine and describe fat content. Thus, the ketogenic ratio represents the relationship between the grams of fat and the combined grams of protein and carbohydrate. In a 4:1 ratio there are four times as many grams of fat for every 1 g of protein and carbohydrate combined. The ratio is traditionally intended to regulate the degree of ketosis, with higher ratios theoretically stimulating greater ketosis. The MCT version of the ketogenic diet uses percentage energy from fat to determine and describe fat content. The other two versions of the ketogenic diet are the so-called modified Atkins diet and the low glycemic (GI) index diet, which encourage people to ingest a lot of fat. In these two latter diets neither the ratio nor the percentage of fat is formally calculated although typically the ketogenic ratio is about 1:1. In all 4 versions of the ketogenic diet the percentage of total energy from fat ranges from 50-92% but is typically 70-90%. In any event whichever form of the diet one follows it is necessary to ingest a lot of fat to achieve efficacy and this can impact severely on patient compliance. However, following the teaching of the present invention it may be possible to arrive at a clinical benefit with a diet which is outside these traditional ratios provided that the fat content contains suitable levels of decanoic acid according to the present invention.

If the invention is delivered as part of a ketogenic diet, the ratio or total fat content can be altered during therapy to achieve nutritional goals and to optimize clinical benefit. The ratio can be in the range of 1.0:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1, 3.5:1, 4.0:1, 4.5:1 or 5.0:1.

In one embodiment the ratio is 2.25:1 to 3.9:1. In another embodiment the ratio is 2.26 to 3.8:1 or 2.7-3.4:1. In further embodiments the ratio is 3.21:1, 3.23:1, 3.24:1, 3.25:1, 3.26:1, 3.27:1, 3.28:1 or 3.29:1.

It should be borne in mind that two different individuals of the same age and weight may experience a different level of clinical benefit on the same ratio or quantity of fat. Thus a clinician may wish to alter the ratio to achieve the optimum clinical benefit. Thus fine tuning the ratio or total fat content and altering it at the start and end of therapy, and during the therapy, e.g. to increase compliance, is within the scope of the invention.

Decanoic acid occurs naturally in e.g. coconut oil and palm kernel oil and it can be envisaged that these products could form the key basis of the diet. In general terms decanoic acid forms around 5 to 8% of the fatty acid composition of coconut oil. Thus one can envisage a food composition in which the fatty acid composition comprises from about 5 to 8% of decanoic acid. Conversely octanoic acid comprises around 4.6 to 10% of the fatty acid composition of coconut oil. Since the present invention has identified that C8 is less beneficial than C10, one can envisage a food composition which the fatty acid composition comprises less than 10%, ideally less than 4.6% of octanoic acid.

It will be appreciated that the lipid fraction useful in the present invention can be in the form of triglycerides, diacyl-glycerides, monoacyl-glycerides, phospholipids, lyso-phospholipids, cholesterol and glycolipids, with triglycerides being generally preferred.

One can envisage the situation where the C10 is delivered to a patient as a blended product. In this case it will be appreciated that according to the present invention the amount of saturated fatty acids utilised is relatively high. In particular it is preferably between 23 and 50, more preferably 25-45, and even more preferably 33-44 g per 100 g lipids, on fatty acid basis. The saturated fatty acids have 8 to 24 carbon atoms. It is preferred that a major part of the saturated fatty acids is decanoic acid (C10:0). Decanoic acid thus provides e.g. 15-50, preferably 18-45, more preferably 23-44 g per 100 g lipids. A particular embodiment comprises 30-37 g decanoic acid per 100 g lipids. Coconut oil or palm oil is a preferred source for at least 50%, preferably between 70 and 90 of the lipid fraction. The remainder of the lipid fraction can be selected from e.g. medium-chain triglyceride sources such as fractionated coconut oil, macadamia oil, palm oil or palm kernel oil, or long-chain triglyceride sources such as safflower oil, sesame seed oil, soy oil (which may be obtained from soybean), sunflower oil, high oleic sunflower oil, corn oil, canola oil, walnut oil, evening primrose oil, peanut oil, cottonseed oil, rapeseed oil, olive oil, fish oil, palm olein or algal oil, or mixtures thereof, preferably soybean oil (preferably between 2 and 30), medium-chain triglycerides (with fatty acids having 8-12 carbon atoms; between 0 and 14), marine oils (preferably between 0 and 14 wt. %, more preferably between 2 and 12 wt. %), and phospholipids, mono- and di-glycerides.

The present invention preferably does not include mono-unsaturated and/or polyunsaturated fatty acids. However, if present the amount of mono-unsaturated fatty acids is suitably between 25 and 48, preferably 28-43, more preferably 30-40 g per 100 g lipids (fatty acid basis). If present, the amount of polyunsaturated fatty acids (i.e. having two or more unsaturated bonds), to which the trans fatty acids have been excluded, is 16-40, preferably 20-30 g per 100 g lipids. It is preferred that the lipid fraction also comprises omega.-3 polyunsaturated fatty. In particular, the polyunsaturated fatty acids comprise more than 0.5, preferably 1.0-10 wt. %. The amount of trans fatty acids is below 20, preferably 0-10, more preferably 0.2-4 g per 100 g lipids.

MCT oil is a food grade oil generally comprising more than 90 wt % fatty acids. Traditionally these fatty acids have been made up of saturated fatty acids having 8, 10 or 12 carbon atoms. Whilst MCT oil may have application in the present invention, preferred is the use of MCT based oil in which the majority of the saturated fatty acids are decanoic acid.

Preferably decanoic acid represents at least 51%, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 wt % of the fatty acid content of the composition useful in the present invention.

In one embodiment of the composition useful in the present invention the ratio of the saturated fatty acids of C10 to C8 is 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5 or 100:0.

LCT oil is defined as a food grade oil that comprises 15% of fatty acids having 20 or more carbon atoms. The present invention may utilise an LCT oil, but preferably at a level of 1%, 0.5% or 0.1% or less per 100 g.

In general terms, administration of the composition of C10 of the present invention may be by an oral route or another route into the gastro-intestinal tract or by parenteral routes. The forms for these modes of administration may include conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.

Thus, the composition of C10 of the present invention can be formulated in a suitable form, which may be a dosage form. The form is generally suitable for oral administration, but the invention is also applicable to gastric tube feeding. Suitable forms may include tablets, dragees, capsules, gel caps, powders, granules, solutions, emulsion, suspension, coated particles, spray-dried particles and pills, which contain the composition of C10 of the present invention, and optionally one or more suitable pharmaceutically acceptable carriers. In some embodiments the composition of C10 can be inserted or mixed into a food substance. In some embodiments the composition of C10 of the present invention is in the form of a nutritional product. By nutritional product we generally refer to the situation where the substance intended to supplement a diet; although in the present situation the composition may be in a form such that it is intended to be the sole item or a meal or diet, i.e. a so-called “complete” nutritional product. Thus, the present invention may be administered to subjects in the form of nutritional supplements, foods, beverages. A preferred type of food is a medical food, e.g. a food which is in a formulation to be consumed under medical supervision and which is intended for the specific dietary management of a disease or condition, such as in the ketogenic diet referred to above.

As mentioned above, the present invention may be realised in the form of a product which is suitable for complete nourishment of human beings, or as an oil emulsion or supplement or any other convenient product form. The product may be suitable for infants, children and adults. The product as well as comprising a lipid fraction in accordance with the teachings of the present invention may optionally comprise a protein fraction, a fraction of digestible carbohydrates, available and/or nonavailable carbohydrates, a nitrogen fraction and optionally, a vitamin fraction, a mineral/trace element fraction or other components as may be appropriate to provide a nutritional supplement or complete nutritional product.

The protein fraction comprises preferably peptides larger than 8 amino acids, which can make the product unsuitable for parenteral administration, due to potential allergic reactions. It is preferred to select proteins, which have strong emulating properties, such as certain caseins. However, products usable for reconstitution in water or liquid formula should preferably contain lysolecithin, tartaric esters or combinations thereof as stabilization system in order to obtain a product that is suitable for drinking.

The product may preferably be solid or semi-solid, such as a powder, bar, pudding, etcetera. A semi-solid product is understood to be a product having a solid content of more than 40 g per 100 g ready to use product. More preferably the semi-solids are supplied as powder, which can be reconstituted in water to be used as a single, complete food. The powder may comprise of primary particles, agglomerated primary particles or mixtures of particles of various size. Such powders can be manufactured using methods known in the art, such as spray drying. Spray drying is preferred when aids are used to improve flowing characteristics. The product may also be in the form of an oil which could be used for frying etc.

The dry product may be at least partly soluble in water, so as to allow ready make-up of a liquid food, if desired. Preferably at least 50 wt %, more preferably at least 75 wt % of the dry mass is soluble when dissolved as 10% (w/v) in water at 20° C.

The amount of digestible carbohydrates is 0-9, preferably 3.2-9, more preferably 4-8.6, even more preferably 5-8.2 g per 100 g dry mass. The amount of protein is 5-20, preferably 13-20, more preferably 13-18, more preferably 13.8-17, even more preferably 14.2-16.2 g per 100 g dry mass. The amount of lipids is 0, 0.1-100 g per 100 g dry mass, but may be 60-80, 63-75, or 65-72 g per 100 g dry mass.

The inclusion of alpha-lactalbumin or ingredients which comprise high amounts of protein, are particularly suitable. The presence of more than 20 wt % alpha-lactalbumin in the protein fraction of the product results in easy compliance with the requirements for leucine, lysine, methionine and cysteine, excellent palatability and digestion properties. Preferably more than 20%, more preferably 40-80 wt % of the protein fraction consists of alpha-lactalbumin.

The digestible carbohydrate fraction can comprise food grade ingredients such as glucose syrup, maltodextrins, lactose, sucrose, galactose, ribose, etc. Though excellent products can be obtained in terms of efficacy when several of the other technical features as disclosed in this description are applied, best results in terms of avoidance of side effects and efficacy are obtained if the digestible carbohydrate fraction takes a specific form. It appears beneficial if at least 20%, preferably 30-90% of the digestible carbohydrate fraction is formed by a source of galactose or ribose. Lactose is considered as suitable ingredient for this purpose. In particular oxidative stress will decrease, when such ketogenic formula is consumed, which comprises such non-glucose digestible carbohydrates. Digestibility is determined by applying the Englyst 1999 method.

The proteins, lipids, and carbohydrates preferably originate from at least two different sources, for example, the proteins at least partly from animal, especially milk, source, but optionally also partly from plant source, the lipids at least partly from vegetal source, and the carbohydrates at least partly from milk source, or from a combination of milk (lactose) and plant (glucose, maltodextrins etc.).

The amount of micro ingredients follow recommendations. However, increasing the amounts of several specific ingredients above recommendations improves efficacy and prevents side effects in paediatric epileptics.

In order to maintain normal development and growth, the energy requirement may be calculated according to an individual's energy requirements. Useful products for feeding patients, particularly paediatric epileptic patients, have an energy density of 3.8-12.6 kJ/ml preferably 4.6-8.4 kJ/ml and more preferably 5.0-7.2 kJ/ml. An energy density of 5.4-6.7 kJ/ml appears particularly useful when nourishing completely with the product. When the energy density of the liquid formula is 8.4-12.6 kJ/ml, the product may also be useful for intermediate fortification of the patient.

The product usefully is relatively energy dense. In some embodiments, it provides 2520-3780, or say 2520-3080, and preferably 2800-3040 kJ per 100 grams dry matter. The diet may provide 2500-3100 kJ per 100 grams dry matter, such as 2505, 2510, 2515, 2520, 2525, 2530, 2535 or 2540 to 3100, 3095, 3090, 3085 or 3080 kJ per 100 g. In a particularly preferred embodiment the diet comprises 2984, 2985, 2986, 2987, 2988, 2989 or 2990 kJ per 100 g.

The product, suitably after reconstitution to a liquid product, can be administered in an amount of between 50 and 200, preferably between 75 and 150 g per day, calculated as dry mass, for infants younger than 12 months, following general energy consumption recommendations, like has been described in the guidelines of the health authorities. For older children, the preferred daily amount calculated on dry mass is between 100 and 360, especially between 150 and 300 g. For adults these amounts are 100-500, most preferably 150-340 g for providing most ketogenic potential, and at the same time a sufficient amount of essential amino acids, carbohydrate skeletons, and other nutrients and being well tolerated and safe.

Examples of suitable products for use in the present invention are given below:

Spray Dried Powder Formulationper 100 gNutrition InformationEnergykj2987kcal714Proteing16Total carbohydrateg7.0sugarsg0.7Total fatg75saturatesg75monounsaturatesg0polyunsaturatesg0C10g74.9Other lipidsg0.1Linoleic acidmg0α-linolenic acidmg0Fibreg0MineralsSodiummg245mmol10.5Potassiummg1.7mmol<0.1Chloridemg1.5mmol<0.1Calciummg8.8mmol0.2Phosphorusmg140mmol4.5Magnesiummg0.7mmol0.3

Powder FormulationNUTRITIONAL INFORMATIONper 100 gper 100 ml (25%)Energykj2508627kcal600150Proteing22.05.5Carbohydrateg14.83.7Fatg53.813.5of which C10g36.09.0of which other lipidsg17.04.3Fibreg6.01.5VitaminsVitamin Aμg29358.6Vitamin Dμg5.61.12Vitamin Emg5.61.12Vitamin Cmg33.36.7Vitamin Kμg37.77.5Thiaminmg0.440.09Riboflavinmg0.660.13Niacinmg6.61.32Vitamin B6mg0.60.12Folic Acidμg14929.8Vitamin B12μg0.850.17Biotinμg11.32.26Pantothenic acidmg2.20.44Cholinemg20340.6MineralsSodiummg44488.8mmol193.8Potassiummg600120mmol153Chloridemg555111mmol15.53.1Calciummg48096Phosphorusmg44488.8Phosphatemmol14.22.84Magnesiummg12424.8mmol5.11.02Trace ElementsIronmg5.551.11Coppermg0.440.088Zincmg4.40.88Manganesemg0.740.148Iodineμg55.511.1Molybdenumμg26.45.32Seleniumμg27.45.48Chromiumμg13.22.64

Ketogenic Feedper 100 gper 100 ml (25%)Energykj2508627kcal600150Proteing307.5Carbohydrateg153.8Fatg48.612.2of which MCTg25.36.3of which LCTg23.35.8Fibreg00VitaminsVitamin Aμg9358.6Vitamin Dμg5.61.12Vitamin Emg5.61.12Vitamin Cmg33.36.7Vitamin Kμg37.77.5Thiaminmg0.440.09Riboflavinmg0.660.13Niacinmg6.61.32Vitamin B6mg0.60.12Folic Acidμg14929.8Vitamin B12μg0.850.17Biotinμg11.32.26Pantothenic acidmg2.20.44Cholinemg20340.6MineralsSodiummg44488.8mmol193.8Potassiummg600120mmol153.0Chloridemg555111mmol15.53.1Calciummg48096mmol122.4Phosphorusmg44488.8mmol14.22.84Magnesiummg12424.8mmol5.11.02Trace ElementsIronmg5.551.11Coppermg0.440.088Zincmg4.40.88Manganesemg0.740.148Iodineμg55.511.1Molybdenumμg26.65.32Seleniumμg27.45.48Chromiumμg13.22.64

NUTRITIONAL INFORMATIONper 100 mlper carton (250 ml)EmulsionEnergykJ8482121kcal180449Total proteing00Carbohydrateg00Fatg21.553.8of which saturatesg2152.5of which monosaturatesg0.30.8of which polyunsaturatesg0.20.5of which other lipidsg1.53.8of which C10g2050VitaminsVitamin Aμg<21<53MineralsSodiummg3997mmol1.74.3Fatty AcidsLinoleic Acidmg140350Alpha Linolenic Acid (18:3)mg6015050% EmulsionPer 100 mlper 250 ml cartonEnergykJ17434358kcal4151038Proteing00Total Carbohydrateg00sugarsg00Total Fatg50125of which medium chaing50125triglyceridesof which long chaing00triglycerides

Experimental Examples

Brief Experimental Approach

Mitochondrial enrichment was estimated by evaluation of citrate synthase activity (corrected for total cellular protein content). This enzyme is localised at the mitochondria, comprises part of the TCA cycle and is commonly used as a marker of mitochondrial enrichment.

In order to gain further independent insight into mitochondrial function, the activity of the respiratory chain enzyme, Complex I, was also evaluated.

A human neuroblastoma cell line (SH-SY5Y) was utilised throughout this study, except where stated. In brief, cells were exposed to a range (50-300 μM, dissolved in 0.5% DMSO) of either octanoic or decanoic acid concentrations. After 6 days, the cells were harvested and citrate synthase activity was determined. Activity was expressed as nmol/min/mg of cellular protein.

In a second experiment, cells were exposed to decanoic acid (250 μM, dissolved in 0.5% DMSO) for 6 days. The cells were subsequently harvested, and the activity of Complex I was determined. Each experiment was repeated 5 times, and the activity was expressed as nmol/min/mg of cellular protein.

Additionally, SH-SY5Y cells were prepared for electron microscopy (EM) analysis to assess mitochondrial density and morphology following treatment with decanoic acid.

Finally, the effect of decanoic acid was validated in an independent cell line. Primary cultures of human fibroblasts were exposed to decanoic acid at a concentration of 250 μM for 6 days. Following the incubation, citrate synthase activity was again assessed.

Results

Octanoic acid had no effect on the parameters studied. However, exposure of the SH-SY5Y cells to decanoic but not octanoic acid resulted in an increase in citrate synthase activity when compared to control cells (incubated with vehicle only). This effect was dose dependent (FIG.1), with a maximum, highly significant (p<0.001), 30% increase occurring at a concentration of 250 μM (expressed as nmol/min/mg cell protein):

Control (n = 8)Decanoic Acid (n = 8)105 ± 5137 ± 5

Determination of Complex I activity in SH-SY5Y cells also revealed a significant (p<0.002) increase with respect to the control experiment, following treatment with decanoic acid (FIG.2). A significant (p<0.05) increase in Complex I activity was still apparent when the data were normalised against citrate synthase activity (FIG.3). Normalisation takes into account the mitochondrial enrichment following administration of decanoic acid. The Complex I:citrate synthase activity ratio therefore provides a more accurate demonstration of mitochondrial function independently of mitochondrial content.

In all cases, the addition of 0.5% DMSO was shown to have no effect on the parameters studied, i.e. when compared to untreated cells.

The electron microscopy study revealed increased numbers of mitochondria in cells treated with 250 μM decanoic acid (FIG.4). The mitochondria are clearly visible in both images as dark circular and elongated organelles present in the cytoplasm. Treatment with decanoic acid also appears to alter the morphology of the mitochondria, with the more intense staining potentially resulting from more dense cristae within the organelles. These observations are supported by a quantitative analysis (FIG.5) of the EM data, which reveals a significant (p<0.002) increase in the number of mitochondria per cell.

Finally, the data were validated by the observation that primary human fibroblasts exposed to decanoic acid (250 μM) also revealed a 45% increase in citrate synthase activity.

CONCLUSIONS

At a concentration relevant to that achieved in the plasma by patients on the ketogenic diet, decanoic acid exposure resulted in a marked increase in citrate synthase activity in treated SH-SY5Y neuroblastoma cells. Activity of this enzyme is known to correlate with cellular mitochondrial levels, therefore these findings raise the possibility that decanoic acid exposure leads to an alteration in cellular mitochondrial function, potentially by increasing mitochondrial content. This conclusion is further supported by independent data derived from EM-based direct observation of cellular mitochondrial content. Additionally, Complex I activity was observed to increase following administration of decanoic acid. This mitochondrial respiratory enzyme is a direct marker of mitochondrial function, therefore this data independently suggests an increase in mitochondrial function results from incubation with decanoic acid. Notably, these findings do not appear to be restricted to one cell type since a similar phenomenon, with regards to increased citrate synthase activity, was apparent in primary human fibroblast cells exposed to decanoic acid.

This action of decanoic acid may therefore prove to be of therapeutic benefit for those patients with epilepsies that are responsive to the ketogenic diet. Similarly, for patients with inherited or acquired mitochondrial disorders, decanoic acid may also be beneficial. With regards to acquired disorders, this could include diabetes and neurodegenerative conditions such as Parkinson's disease and dementias including Alzheimer's disease.

It will be appreciated that any of the ranges disclosed herein may be used in suitable combinations.