THERAPEUTIC COMPOSITIONS

Abstract

Compositions comprising ketone bodies and/or their metabolic precursors are provided that are suitable for administration to humans and animals and which have the properties of, inter alia, (i) increasing cardiac efficiency, particularly efficiency in use of glucose, (ii) for providing energy source, particularly in diabetes and insulin resistant states and (iii) treating disorders caused by damage to brain cells, particularly by retarding or preventing brain damage in memory associated brain areas such, as found in Alzheimer's and similar conditions.

These compositions may be taken as nutritional aids, for example for athletes, or for the treatment of medical conditions, particularly those associated with poor cardiac efficiency, insulin resistance and neuronal damage. The invention further provides methods of treatment and novel esters and polymers for inclusion in the compositions of the invention.

Claims

1-31. (canceled)

32. A method of treating a patient for insulin insensitive diabetes comprising orally administering to that patient a therapeutically effective amount of a metabolic precursor of D--hydroxybutyric acid or acetoacetate, such as to elevate the patient's blood levels of ketone bodies, defined as the sum total of D--hydroxybutyric acid and acetoacetate, to from 0.3 mM to 20 mM, wherein the metabolic precursor is selected from the group consisting of esters of D--hydroxybutyric acid or its oligomers with alcohols selected from the group consisting of C.sub.1-C.sub.4 alkyl alcohols, (R)-1,3-butandiol and glycerol.

33. A method as claimed in claim 32 wherein the metabolic precursor is an (R)-1,3-butandiol ester.

34. A method as claimed in claim 32 wherein the patient is treated for Type II diabetes

Description

FIGURES

[0118] FIG. 1 is a graph showing blood (R)-3-hydroxybutyrate level produced after time after gavage of (R)-3-hydroxybutyrate, an oligomer of this as produced in Example 1 and an acetoacetyl monomer thereof as produced in Example 2.

[0119] FIG. 2 is a graph showing blood (R)-3-hydroxybutyrate level produced after time after feeding rats with the triolide of (R)-3-hydroxybutyrate, a cyclic oligomer produced in Example 1 in yoghurt and controls fed yoghurt alone.

EXAMPLES

Example 1

[0120] Preparation of Oligomers of (R)-3-hydroxybutyric Acid (D--hydroxybutyrate).

[0121] (R )-3-hydroxybutyric acid (Fluka-5.0 g: 0.048 mole), p-toluene sulphonic acid (0.025 g) and benzene (100 ml) were stirred under reflux within a Dean-Stark trap arrangement for 24 hours. The reaction mixture was cooled and the benzene evaporated in vacuo (0.5 mm Hg). 4.4 g of colourless oil was obtained of which a 20 mg sample was converted to the methyl ester for analysis of number of monomer repeats using NMR. These studies show that the product is a mixture of oligomers of D--hydroxybutyrate of average number of repeats 3.75, being mainly a mixture of trimers, tetramers and pentamers with the single most abundant material being the tetramer. The product mixture was soluble in 1 equivalent of sodium hydroxide.

Example 2

[0122] Preparation of Acetoacetyl Ester of Oligomeric (R)-3-hydroxybutyric Acid.

[0123] A further batch of the colourless oil product from Example 1 (4.5 g) was heated for 1 hour at 60 C. with diketene (3.8 g) and sodium acetate (0.045 g) under nitrogen. Further diketene (3.8 g) was added and the reaction heated for a further hour, cooled and diluted with ether, washed with water and then extracted with saturated sodium bicarbonate (5100 ml). Combined extract was washed with ether then acidified with concentrated HCl (added dropwise). Ethyl acetate extraction (350 ml) was followed by drying over magnesium sulphate and evaporation in vacuo. A yellow solid/oil mixture was obtained (7.6 g) which was chromatographed on a silica column using dichloromethane/methanol (98:2) to give a light amber oil product. Faster moving impurities were isolated (1.6 g) and after recolumning carbontetrachloride methanol (99:1) 0.8 g of oil was recovered which was shown by NMR and Mass spectrometry to be the desired mixture of acetoacetylated oligomers of D--hydroxybutyrate. The product mixture had an Rf of 0.44 in dichloromethane/methanol (90:1) and was soluble in 1 equivalent of sodium hydroxide. Both products of Example 1 and Example 2 are susceptible to separation of individual components by preparative HPLC.

Example 3

[0124] Oral Administration of D--hydroxybutyrate, Oligomers and Acetoacetyl D--hydroxybutyrate Oligomers to Rats.

[0125] The ability of orally administered D--hydroxybutyrate and the oligomers of Examples 1 and 2 to raise blood ketone body levels was investigated as follows. Rats were starved overnight and then savaged with 100 l/100 g bodyweight of 4M D--hydroxybutyrate brought to pH 7.74 using methyl glucamine. Blood levels of D--hydroxybutyrate measured using and NAD+/EDTA assay of Anal. Biochem. 131, p478-482 (1983). 1.0 ml of a solution made up from 2-amino-2-methyl-1-propanol (100 mM pH 9.9, 0.094 g/10 ml). NAD (30 mM, 0.199 g/10 ml) and EDTA (4 mM, 0.015 g/10 ml) was added to each of a number of cuvettes and 4 l sample or D--hydroxybutyrate control.

[0126] As the rats had been fasted the initial levels of D--hydroxybutyrate were elevated from the 0.1 mM fed state. However, consistent serum increases of D--hydroxybutyrate, between 1 and 3.2 mM increase in each case, were provided.

[0127] This procedure was repeated with 2 M solutions of the mixtures of D--hydroxybutyrate oligomers and their acetoacetyl esters described in Examples 1 and 3. The D--hydroxybutyrate oligomer (19/1 in FIG. 1) and the acetoacetyl ester (20/4 in FIG. 1) were both brought to pH 7.6 with methyl glucamine and the blood D--hydroxybutyrate level monitored using the aforesaid assay procedure. Increases in serum D--hydroxybutyrate were shown to be of 0.5 to 1.2 mM at 60 and 120 minutes after gavaging. These results demonstrate the efficacy of orally administered D--hydroxybutyrate and its metabolic precursors of the invention in raising blood levels significantly for a period of hours after intake.

[0128] It was noted that the oligomeric esters 19/1 and 20/4, while not elevating the blood ketone body level as high as the monomer itself, did result in elevation for a much longer period of time and thus are suited to administration less frequently than the monomer.

Example 4

[0129]

TABLE-US-00003 TABLE 2 Sample 1500 calorie ketogenic diet using ketone bodies, their esters or polymers. The ketones were assumed to contain 6 kcal/g, fats 9 kcal/g, carbohydrate and protein 4 kcal/g. Ketones have been substituted to give equivalent calories. Amount Ketones (g) Fat (g) Protein (g) CHO (g) (g) Breakfast Egg 32 4 4 apple juice 70 7 ketones 66 66 skim milk 92 0 2 3 Total Breakfast 4 6 10 66 Lunch Lean beef 12 1.75 3.5 cooked carrots 45 0.6 3 canned pears 40 4 ketones 69.75 69.75 skim milk 92 2 3 Total Lunch 1.75 6.1 10 69.75 Supper Frankfurter 22.5 6 3 cooked broccoli 50 1 2 watermelon 75 5 ketones 62.25 62.25 skim milk 92 2 3 Total Supper 6 6 10 62.25 Daily Total 11.75 18.1 30 198

Example 5

[0130] Effect of Increased Blood D--hydroxybutyrate Levels On Whole Brain GABA Levels.

[0131] To assess the effect of D--hydroxybutyrate on whole brain GABA levels, and thus provide an indication of antiepileptic effect of ketone body or precursor treatment aimed at increasing blood ketone body levels, whole rat brain was frozen at set times after administration of D--hydroxybutyrate as described in Example 3. GABA was assayed using standard HPLC technique and related to protein content using standard protein assay. At t=0 GABA levels were 191 pmoles/g protein while at 120 minutes this was elevated at 466 pmoles, g protein, demonstrating antepileptic potential.

Example 6

[0132] Effect of D--hydroxybutyrate on -amyloid Toxicity to Hippocampal Cells In Vitro Culture Medium and Chemicals

[0133] The serum free medium used from 0 to day 4 contained Neurobasal medium with B27 supplement diluted 50 fold (Life Technology, Gaithersburg, Md.) to which was added: 0.5 mM L-glutamine, 25 M Na L-glutamate, 100 U/ml penicillin and 100 g/ml streptomycin. After day 4, DMEM/F12 medium containing 5 M insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Na selenite 100 U/ml penicillin and 100 g/ml streptomycin were used.

Hippocampal Microisland Cultures

[0134] The primary hippocampal cultures were removed from Wistar embryos on day 18 and dispersed by gentle agitation in a pipette. The suspension was centrifuge at 1,500 g for 10 min and the supernatant discarded. New media was make 0.4-0.510.sup.6 cells/ml. Ten l of this suspension was pipetted into the center of poly D-lysine coated culture wells and the plates incubated at 38 C. for 4 hrs and then 400 l of fresh Neurobasal media was added. After 2 days of incubation, half of the media was exchanged for fresh media and the incubation continued for 2 more days. After day 4, the medium was changed with DMEM/F12 medium containing 5 M insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Na selenite 100 U/ml penicillin and 100 g/ml streptomycin. The wells were divided into 4 groups: half the wells received Na D--hydroxybutyrate to a final concentration of 8 mM while and half of the wells received 5 nM amyloid .sub.1-42 (Sigma). These media were exchanged 2 days later (day 8) and the cells were fixed on day 10 and stained with anti MAP2 (Boehringer Manheim, Indianapolis, Ind.) to visual neurons and vimentin and GFAP (Boehringer) to visualize glial cells.

Results

Cell Counts

[0135] Addition of D--hydroxybutyrate to the incubation resulted in an increase in the neuronal cell number per microisland from a mean of 30 to mean of 70 cells per microisland. Addition of 5 nM amyloid .sub.1-42 to the cultures reduced the cell numbers from 70 to 30 cells per microisland, confirming the previous observations of Hoshi et al. that amyloid .sub.1-42 is toxic to hippocampal neurons. Addition D--hydroxybutyrate to cultures containing amyloid .sub.1-42 increased the cell number from a mean of 30 to 70 cells per microisland. From these data we conclude that addition of substrate level quantities of D--hydroxybutyrate, to media whose major nutrients are glucose, pyruvate and L-glutamine, slows the rate of cell death in culture. We further conclude that D--hydroxybutyrate can decrease the increased rate of hippocampal cell death caused by the addition of amyloid .sub.1-42 in culture.

[0136] The number of dendritic outgrowths and the length of axons were both observed to have increased with presence of D--hydroxybutyrate, whether .sub.1-42 was present or not. This is indicative of nerve growth factor like behaviour.

Example 7

[0137] Preparation of (R,R,R)-4, 8, 12-trimethyl-1, 5, 9-trioxadodeca-2, 6, 10-trione: Triolide of (R)-3-hydroxybutyric Acid.

##STR00006##

[0138] Synthesis was as described in Angew. Chem. Int. Ed. Engl. (1992), 31, 434. A mixture of poly[(R)-3-hydroxybutyric acid] (50 g) and toluene-4-sulphonic acid monohydrate (21.5 g, 0.113 mole) in toluene (840 ml) and 1,2-dichloroethane (210 ml) was stirred and heated to reflux for 20 hours. The water was removed by Dean-stark trap for 15 hours whereafter the brown solution was cooled to room temperature and washed first with a half saturated solution of sodium carbonate then with saturated sodium chloride, dried over magnesium sulphate and evacuated in vacuo. The brown semi-solid residue was distilled using a Kugelrohr apparatus to yield a white solid (18.1 g) at 120-130 C./0.15 mmHg. Above 130 C. a waxy solid began to distilldistillation being stepped at this point. The distilled material had mp 100-102 C. (literature mp 110-110.5 C.). Recrystallisation from hexane gave colourless crystals in yield 15.3 g. Mp=107-108 C.; [].sub.D-35.1 (c=1.005, CHCl.sub.3), (lit.=33.9). .sup.1H NMR (300 MHz, CDCl.sub.3): =1.30 (d, 9H, CH.sub.3): 2.4-2.6 (m, 6H; CH.sub.2); 5.31-5.39 (M, 3H; HCO). .sup.13C NMR (CDCl.sub.5): =20.86 (CH.sub.3), 42.21 (CH.sub.2), 68.92 (CH), 170.12 (CO). Elemental analysis: calculated for C.sub.12H.sub.15O.sub.5: C, 55.81; H 7.02: Found: C, 55.67; H. 7.15.

Example 8

[0139] Oral Administration of Triolide of D--hydroxybutyrate of Example 1 to Rats.

[0140] The ability of orally administered triolide to raise blood ketone levels was investigated as follows. The day before the experiment commenced, 12 Wistar rats weighing 31610 g were placed in separate cages. They had no access to food for 15 hours prior to presentation with triolide containing compositions, but water was provided ad libitum.

[0141] On the morning of the experiment 0.64 g of triolide was mixed with 5 g Co-op brand Black Cherry yoghurt in separate feeding bowls for 9 of the rats. The remaining 3 rats were given 5 g of the yoghurt without the triolide as controls. The yoghurt containing bowls were placed in the cages and the rats timed while they ate. Two of the three control rats ate all the yoghurt and four of the six triolide yoghurt rats ate approximately half the provided amount. The remaining six rats slept.

[0142] Control rats (n=2) were killed at 60 and 180 minutes after ingestion of yoghurt while triolide fed rats were killed at 80, 140, 150 and 155 minutes. Blood samples were taken for assay of D--hydroxybutyrate. Brains were funnel frozen and later extracted in perchloric acid and extracts neutralised and assayed. Blood levels of (R)-3-hydroxybutyrate were measured using a NAD.sup./EDTA assay of Anal. Biochem (1983) 131, p478-482. 1.0 ml of a solution made up from 2-amino-2-methyl-1-propanol (100 mM pH 9.9, 0.094 g/10 ml), NAD.sup. (30 mM, 0.199 g/10 ml) and EDTA (4 mM, 0.015 g/10 ml) was added to each of a number of cuvettes and 4 l sample or D--hydroxybutyrate control.

[0143] The two control rats ate 5.20.1 g yoghurt and their plasma (R)-3-hydroxybutyrate concentrations were about 0.45 mM at 60 minutes and 180 minutes. The four triolide fed rats ate 0.390.03 g of the triolide and 2.60.2 g of yoghurt. Their plasma D--hydroxybutyrate concentrations were 0.8 mM after 80 minutes and 1.1 mM for the group sacrificed at about 150 minutes. All rats displayed no ill effects from ingestion of triolide.

[0144] The test rats thus showed increase in plasma D--hydroxybutyrate over at least 3 hours with no ill effects. It should be noted that two other rats fed approximately 1.5 g triolide each in Hob-Nob biscuit showed no ill effects after two weeks.

[0145] For all examples given above, it should be noted that the increased levels of (R)-3-hydroxybutyrate will also be mirrored in acetoacetate levels, not measured here, as there is a rapid establishment of equilibrium between the two in vivo such that acetoacetate levels will be between 40 and 100% of the (R)-3-hydroxybutyrate levels.

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