KETOGENlC SACCHARIDES. The present invention relates to novel compounds which have utility as nutraceuticals and medicaments for producing ketosis in humans and animals for nutraceutical or therapeutic purposes It is known that ketone bodies, particularly (R)-3-hydroxybutyrate (r>β- hydroxybutyrate) and acetoacetate have both nutritional and therapeutic application m man and many animals. US 6,136,862 and US 6,232,345 (incorporated herein by reference) relate to the use of D-β-hydroxybutyrate, oligomers, esters and salts thereof, inter alia, m the treatment of cerebral edema and cerebral infarction. US 6,207,856 and PCT/US99/21015 also refer to β-hydroxybutyrate and its oligomers, esters and salts thereof in protecting against other forms of neurodegeneration inter aha, through their proposed ability to activate the TCA cycle and, through favourable redox reactions in cells and antioxidant activity, scavenge free radicals β- hydroxybutyrate has also been demonstrated to have cardωprotectant effect and can increase cardiac efficiency (Sato et al FASEB J 9: 651-658, 1995) US6,207,S56, US6,136,862, US6,207,856 and PCT/US99/21015, incorporated herein by reference, teach that preferred ketogenic precursors for producing such ketosis are monohydnc-, dihydric and trihydnc alcoholic esters of (R)-3-hydroxybutyrate, but particularly a (R)-3-hydroxybutyry) ester of (R)- 1,3- butandiol, more preferably the dicster formed from two molecules of (R)-3- hydroxybutyrate and one molecule of (R)-1 ,3-butandiol. However, it is also known that production of oligomers of (R)-3- hydroxybutyrate in pure form is problematic PCTλJS99/21015 exemplifies a ketogenic oligomer with a mixture of (R)-3-hydroxybutyrate tnmer, tetramer and pentamer and exemplifies esters thereof with acetoacetyl (rimer, tetrarner and pentamer of (R)-3-hydroxybutyrate. Similarly, Hiraidc et al al (1999) J. Parenteral and Enteral Nutrition VoI 23 No 6 discloses ube of a mixture of dimer and tnmer of (R)-3-hydroxybutyrate for studies in ability of plasma to degrade these to the monomer Copendmg provisional patent applications of Richard Gross (US provisional filings 60/5883156 and US60/588990) claim compounds comprising fixed length oligomers of (R)-3-hydrυxybutyrate esteπfed to monohydnc and dihydric alcohols, methods for syπthesising these m pure form and methods of treatment using these. These compounds are either water soluble syrups or water insoluble waxy solids. Henderson et al WO2004077938, postulate use of saccharides which have been substituted on all of their free hydroxy! groups by fatty acyl, acetoacetyl or hydroxybutryl groups as compounds for producing ketosis in subjects m need of ketogeruc therapy The formulae taught in that document are fully substituted, no synthetic routes are discussed and no biological date provided The present inventors have now determined that in order to produce useful ketosis in a subject it 13 in fact necessary to use saccharides that are not fully substituted by ketogenic precursor moieties. It is believed that it is important that some significant level of hydroxylatioπ remain on the saccharide in order for efficient metabolism of the compounds to proceed and useful ketosis occur. The present invention provides a ketogenic saccharide material which is suitable for use in animals and man for therapeutic purposes Preferred compounds of the present invention are soluble in water and other aqueous liquids and therefor have application in beverages and liquid, semi-solid or gelled orally administerable medicaments. Preferred compounds are of single component constituent In a first aspect of the present invention there is provided a compound of general formula (R(OCH(CH3)CH2C<O))nO)m-A wherein n is a integer between 1 and 10, m is an integer of 1 to 200,000, A is a monsacchaπde, oligosaccharide or polysaccharide residue and R is selected from the group consisting of H, Ci-Ce alkyl and acetoacetyl-, wherein m is such that the number of free hydroxyl groups on the compound is at least an average of 0.3 free hydroxyls per saccharide moiety in residue A. Thus the group R(OCH(CHj)CH7C(O))nO- is esterified to the moiety A by substituting for the H on a number 'm' of saccharide hydroxy groups. N is preferably 1 to 3.. Preferably m is an integer of 1 to 20,000 more preferably 1 to 200, still more preferably 1 to 100, eg. 3 to 100. Clearly the precise number 'm1 will depend upon whether the compound is a monosaccharide, where m cannot be more than 4 or 5 for hexoses and heptoses; an oligosaccharide, where m cannot be more than 3 or 4 for hexoses and heptoses. Where the saccharide is a polysaccharide m is proportionately able to be a multiple of the number of monomers in the polymer Preferably there is at least one free hydroxy! group in the compound for each saccharide nng in the compound. It will be realised that this may be an average number of hydroxyl groups, wherein some rings will have no free hydroxyls on each saccharide nng of the compound whilst others have more than one. In a preferred group of compounds at least one hydroxyl group on each nng remains unsubstituted. In one preferred aspect of the present invention A is a monosaccharide, oligosaccharide or polysaccharide and m is equal the number of repeat sugar monomer moieties in the saccharide multiplied by a substitution factor (aka degree of substitution) of between 0 5 and 4 the substitution factor being an indication of the average number of the free hydroxyl groups situated on each saccharide moiety of monosaccharide, oligosaccharide or polysaccharide, ie. that have been substituted; more preferably being a number of between 0.6 and 4 for every saccharide moiety in the molecule, more typically between 1 and 3, eg. 1 and 2 for every such moiety. Preferred monosaccharides are tetroses, pentoses, hexoses, heptoses; preferred oligosaccharides are disaccharides and higher oligomers of these the monosaccharides. Prefeiτed polysaccharides are those used in foodstuffs, particularly preferred being glucose based saccharides, eg pullulans Pullulan is a linear homopolysacchaπde of glucose that is an α-(l-6)-l inked polymer of maltotnose subunits. It has adhesive properties and is suitable for forming a variety of forms and derivatiscs easily such that its solubility can be controlled. In a second aspect of the present invention there is provided a nutraceutnal or pharmaceutical composition comprising a compound of the first aspect together with a foodstuff component or a pharmaceutically acceptable earner, diluent or excipient. Suitable foodstuff components may, but are not limited to, edible oils, emulsions, gels or solids and drinkable liquids, including suspensions and solutions In a third aspect of the present invention there is provided the use of a compound of the first aspect of the present invention for the manufacture of a medicament for producing a physiologically acceptable ketosis Such medicament will be suitable for treating a number of debilitating conditions, including trauma, haemorrhagic shock, neurodegeneration, diabetes, and epilepsy, stroke, head trauma, myocardial infarction, congestive heart failure, pulmonary failure, kidney failure and obesity In a fourth aspect of the present invention there is provided a method for the manufacture of a compound of formula (R(OCH(CH,)CH2C(O))nO),n-A wherein n is a integer between 1 and 10, m is an integer of ) to 200,000, A is a monsacchande, polysaccharide or oligosaccharide residue and R is selected from the group consisting of H, CI-CJ alkyl and acetoacetyl- wherein m is such that the number of free hydroxyl groups on the compound is at least an average of 0 3 free hydroxyls per saccharide moiety in residue A compπsing reacting (R)-3-hydroxybutyrate or an oligomer thereof containing between 2 and 10 (R)-3-hydroxybutyrate moieties with a monosaccharide, oligosaccharide or polysaccharide m the presence of an acid and in an organic solvent Preferably the solvent provides the acid, more preferably then solvent is an organic acid, more particularly being toluene sulphonic acid, eg Para-toluene sulphonic acid The reaction mixture may advantageously also include dimethylsulphoxide. In a fifth aspect of the present invention there is provided a method for (he manufacture of a compound of formula (R(OCH(CH3)CH2C(O))nO)m-A wherein n JS a integer between 1 and 10, m is an integer of 1 to 200,000, A is a monsacchaπde, polysaccharide or oligosaccharide residue and R >s selected from the group consisting of H, CpCn alkyl and acetoacetyl- wherein in is such that the number of free hydroxyl groups on the compound is at least an average of 0 3 free hydroxyls per saccharide moiety in residue A. compπsing reacting (R)-3-hydroxybutyrate or an oligomer thereof containing between 2 and 10 (R)-3-hydroxybutyrate moieties with a monosaccharide, oligosaccharide or polysaccharide in the presence of dimethylsulphoxide (DMSO) in an organic solvent. Preferably the solvent is DMSO. The compounds where n is more than 1 may be made alternately by reacting a monosaccharide, oligosaccharide or polysaccharide having been substituted with H(OCH(CH3)CH2C(O))nO-, wherein π is 1 , with a cyclic oligomer of (R)- hydroxybutyrate in the presence of a lipase, a reaction disclosed in my copending provisional applications related to esteπfication of mono-ols and diols Such reaction is conveniently carried out in THF with Novozym 435 (a CAL B enzyme). Where a tnmer of (R)-3-hydroxybutyrate is to be added to the HOCH(CH3)CH2C(O)O- substituted saccharide, the tnohde of (R)-3-hydroxybutyrate is employed. Compounds where R is Ci-C^ alkyl and acatoacctyl can be made from the corresponding compound where R is H by simple esterification with the acetoacetate or use of an alkylating agent. Regarding starting materials for producing the compounds of the present invention, various cyclic esters of (R)-3-hydroxybutyrate are known in the art and are readily produced by known methods: see for example sec for example Seebach et al. Helvetia Chimica Acta VoI 71 (1988) pages 155-167, and Seebach et al. Helvetia Chimica Acta, VoI 77 (1994) pages 2007 to 2033. The present invention will now be described further by reference to the following non-Iirmtmg Examples, Schemes and Figures. Further embodiments falling within the scope of the claim will occur to those skilled in the art in the hght of these.

FIGURES FIGURE 1 : General scheme showing the synthesis of KTX 0310 by the esterification of glucose with (R)-3-hydrθxybutyric acid in the presence of CAL-B

FIGURE 2. General scheme showing the synthesis of KTX 0311 by the esterification of fructose with (R)-3-hydroxybutyric acid in the presence of CAL-B.

FIGURE 3: General scheme showing the synthesis of KTX 0312 by the esterification of ardbinose with (R)-3-hydroxybutyric acid in the presence of CAL-B.

FIGURE 4: General scheme showing the synthesis of KTX 0313 by the εstenncation of sorbitol with (R)-3-hydroxybutyric acid in the presence of CAL-B.

FIGURE 5: General scheme showing the synthesis of KTX 0301 and poly(3-hydroxybutyrate) oligomers by the estenfication of pullulan with (R)-3-hydroxybutyπc acid in the presence of para-toluene sulphonic acid.

FIGURE 6: General scheme showing the synthesis of KTX 0321 by the esterification of pullulan with (R)-3-hydroxybutyπc acid in the presence of para-toluene sulphonic acid and dimethylsulphoxide FIGURE 7: General scheme showing the synthesis of KTX 0322 by the esterification of soluble starch with (R)-3-hydroxybutyric acid in the presence of para-toluene sulphonic acid.

FIGURE 8: Effect of oral administration KTX 0310 (glucose (R)-3-hydroxybutyrate ester) as determined by increases of β-hydroxybutyrate concentrations in rat plasma.

FIGURE 9: Effect of oral administration KTX 03 H (fructose (R)-3-hydroxybutyrate ester) as determined by increases of β-hydroxybutyrate concentrations in rat plasma.

FIGURE 10: Effect of oral administration KTX 0312 (axabinose (R)-3-hydroxybutyrate ester) as determined by increases of β-hydroxybutyrate concentrations in rat plasma.

FIGURE 1 1 Effect of oral administration KTX 0313 (the sorbitol tn-ester) as determined by increases of β-hydroxybutyrate concentrations in rat plasma.

FIGURE 12: Effect of oral administration KTX 0301 (a pullulan (R)-3-hyduroxybutyrate ester •*• PHB oligomers) as determined by increases of β-hydroxybutyrate concentrations in rat plasma.

FIGURE 13: Effect of oral administration KTX 0321 (a purified pullulan (R)-3-hydrσxybutyrate ester) as determined by increases of β-hydroxybutyrate concentrations in rat plasma.

FIGURE 14: Effect of oral administration KTX 0322 (a puπfied soluble starch (R)-3-hydroxybutyrate ester) as determined by increases of β-hydroxybutyτate concentrations in rat plasma. EXAMPLES Procedure for the Synthesis of Methyl |Rl-3-Hydroxybutyratc To a 1 L, one-neck round bottom flask equipped with condenser and magnetic stirring was charged 62.5 g poly((R]-3-hydroxybutyrate) (PHB) Julich, Germany, and 350 ml 1,2-dichloroethane A solution of acidic methanol was prepared by the careful addition of 12 5 mL con. H2SO4 to 250 mL methanol and this was added to the reaction slurry Stirring was maintained with heating at 80 0C (reflux) for 120 hrs. The slurry was cooled to room temperature, extracted with 200 ml 'δ saturated NaCI and 50 mL saturated NaCl. The organic material was re-extracted with 100 ml NaHCθ3 to a pH 6.0, followed by 2X50 ml saturated NaCl The organic material was dried over MgSO4, filtered, and the solvent was removed by rotary evaporation. The organic material was fractionally distilled at 0.3 mmHg, 45 0C to give 46 g (73% yield based on the initial polymer charge) of a clear colorless liquid. NMR was used to characterize the product.

Procedure for the Synthesis of [RI-3-Hydroxybutyric Acid Into a 50 ml flask was added 5N KOH (10 ml) which was cooled to 0 0C. Methyl [R]- 3-Hydroxybutyrate (5 g) was then added with stirring over 1 5-h time period, and the temperature was maintained at 0 0C for 24 h. The reaction was terminated by the slow addition of 6N HCl (8.3 ml) with stirring at 5 0C The resultant aqueous solution was then saturated with solid NaCl and extracted 20 times with 20 mL portions of diethyl ether. The organic extract was dried over anhydrous MgSO4 and the ether removed by rotary evaporation. The product was a white crystalline solid (3 8 g, yield 87%). NMR and IR were used to characterize the product.

EXAMPLE 1. The synthesis of KTX 0310 by the esterification of glucose with (R)-3-hydroxybutyric acid in the presence of CAL-B. To a round-bottomed flask, Ig glucose and 4 6g 3-hydroxybutyπc acid were added. The mixture was heated at 8O0C to obtain a homogenous solution The temperature was lowered to 7O0C and 1 1 g (20% w/v of the total mixture) CAL B was added. The mixture was stirred at 7O0C for 48 hrs to yield glucose 3-hydroxybutyrate tri- and tetra-esters as shown in Figure 1.

The material was separated by column chromatography based on its polarity The column was packed in pure chloroform and the polarity was increased using methanol The desired product was elutod using chloroform methanol: water (9 2: 0.3) The product was a water-soluble syrup and was obtained at a yield of 0.3g (30%). A mixture of tπ- and tetra-substituted products was formed (substitution factor between 3 and 4 with 1 to 2 free hydroxyls left per monosaccharide ring). The structure of the compound was verified by LC/MS.

EXAMPLE Z. The synthesis of KTX 0311 by the esterification of fructose with (RV-3-hydroxybutyric acid in the presence of CAL-B. To a round-bottomed flask, 5g fructose and 23g 3-hydroxybutyric acid were added. The mixture was heated at 8O0C to obtain a homogenous solution The temperature was lowered to 7O0C and 5.6g (20% w/v of the total mixture) CAL B was added. The mixture was stirred at 7O0C for 48 hrs to yield the fructose 3-hydroxybutyτate tri- and tetra-esters as shown in Figure 2

The material was separated by column chromatography based on its polarity. The column was packed in pure chloroform and the polarity was increased using methanol The desired product was eluted using chlorofomrmethanol : water (9 : 2 " 0 3) The product was a water-soluble syrup and was obtained at a yield of 1.1 g (22%). A mixture of tri- and tetra-substituted products was formed (substitution factor between 3 and 4- 1 to 2 free hydroxyls left on the monosaccharide ring) The structure of the compound was verified by LC/MS.

EXAMPLE 3. The synthesis of KTX 0312 by the esterification of arabinose with (RV-3-hγdroxybutyric acid in the presence of CAL-B.

To a round -bottomed flask, Ig arabinose and 5.5g 3-hydroxybutync acid were added The mixture was heated at 8O0C to obtain a homogenous solution The temperature was lowered to 7O0C and 1.3g (20% w/v of the total mixture) CAL B was added. The mixture was stirred at 7O0C for 48 hrs to yield the arabinose 3-hydroxybutyrate di- and tn-esters as shown in Figure 3

The material was separated by column chromatography based on its polarity The column was packed in pure chloroform and the polarity was increased using methanol. The desired product was elutcd using chloroform. methanol ■ water (9 • 2 0.3). The product was a water-soluble syrup and was obtained at a yield of 0.2g (20%). A mixture of di- and tn-substituted products was formed (substitution factor 2 to 3 leaving 1 to 2 free hydroxyls per monosaccharide moiety The structure of the compound was verified by LC/MS and by 1H NMR (300MHz, CDCl3) and 13C NMR (75 5 MHz, CDCl5) spectroscopy EXAMPLE 4. The synthesis of KTX 0313 by the esterification of sorbitol with (R>-3-hvdroxybutyric acid in the presence of CAL-B. To a round-bottomed flask, 5g sorbitol and 8.Og 3-hydroxybutync acid were added. The mixture was heated at 8O0C to obtain a homogenous solution. The temperature was lowered to 7O0C and 2.7g (20% w/v of the total mixture) CAL B was added. The mixture was stirred at 7O0C for 48 hrs to yield the sorbitol 3-hydroxybutyrate tn-ester as shown in Figure 4.

The material was separated by column chromatography based on its polarity The column was packed m pure chloroform and the polarity was increased using methanol. The desired product was eluted using chloroform methanol water (9 : 2 : 0.3).

The product was a water-soluble syrup and was obtained at a yield of Ig (20%) The product had a degree of substitution of 3, (leaving 3 free hydroxyls per monosaccharide moiety). The structure of the compound was verified by MALDI mass spectrometry and 1H NMR (300 MHz, CDCl3).

EXAMPLE S. The synthesis of KTX 0301 and oϋgo(3-hγdroxyburyrate') oligomers by the esterificatioπ of pullulan with (RV3-hvdroxybutyric acid in the presence of para-toluene sulphonϊc acid. To a 100ml round- bottomed flask were added with constant stirring, 5.3g pullulan, 21.2g (R)-3-hydroxybutyπc acid and 79.5mg p-toluene sulphonic acid. The flask was capped with a rubber septum and vacuum and dry nitrogen were applied alternately to the flask via a 3-way connector to remove any moisture and to fill the flask with dry nitrogen. The flask contents were heated to a constant 80 C in an oil bath with continuous stirring. PuHulan was dispersed m lhe melt of (R)-3-hydroxybucyπc acid. After 2 hrs, the reaction mixture was stirred under vacuum for 19 hxs. The reaction mixture was then ground to a fine powder and stiπed in acetone overnight. The mixture was filtered, washed with acetone and then dried under reduced pressure at room temperature. The scheme for the synthesis of KTX 0301 and its chemical structure are shown m Figure 5. Yield- 8.5g of water-insoluble grey solid The structure of the acylated puUulan was determined by 1H NMR (300 MHz, CDCU) and 13C NMR (75.5 MHz, CDCI3) spectroscopy. The degree of esterification of pullulan by (R)-3-hydroxybutyrate was 0.33, leaving relatively high amounts of free hydroxyl groups on the KTX0301 compound. The oligomers of (R)-3-hydroxybutyrate contained in the mixture had an average degree polymerisation of 13. The mixture contained by weight: 33% 3-hydroxybutync acid, 12% esterified pullulan and 21% PHB oligomers.

EXAMPLE 6. The synthesis of KTX 0321 _ by the esterification of puUulan with (RV-3-hγdroxybutyric acid in the presence of para-toluene sulphonic acid and dimethylsulphoxide. To a 100ml round-bottomed flask were added with constant stirring, 7.75g pullulan and 19.4ml anhydrous dimethylsulphoxide (DMSO). The flask was capped with a rubber septum and vacuum and dry nitrogen were applied alternately to the flask via a 3-way connector to remove any moisture and to fill the flask with dry nitrogen. The flask contents were heated to a constant 80 C in an oil bath with continuous stirring.

The flask was cooled to room temperature and 24.Og (R)-3-hydroχybutyric acid and 1.16g p-toluene sulphonic acid were added to the mixture. The flask was capped with a rubber septum and vacuum and dry nitrogen were applied alternately to the flask via a 3-way connector to remove any moisture and to fill the flask with dry nitrogen. The nasK contents were neateα to a constant sυ L in an oil bath with continuous shmng. After the solution had had become clear, the reaction mixture was kept under vacuum for 38 hrs. The reaction mixture was added to a large amount of acetone with stirring and the precipitate was separated by centrifugation More acetone was added to the precipitate and the centrifugation step was repeated several times. The product was then dried under reduced pressure at room temperature for 3 days. The scheme for the synthesis of KTX 0321 and its chemical structure are shown in Figure 6 Yield 5.1g of water- insoluble white solid The structure of the acylated pullulan was determined by 1H NMR (300 MHz, CDCIj) spectroscopy. The degree of substitution of pullulan by (R)-3-hydroxybutyτate was 0.64 Elemental analysis. C = 43.33%, H = 6 4% by weight.

EXAMPLE 7. The synthesis of KTX 0322 by the esterifϊcation of soluble starch with (R)-3-hvdroxybutyric acid in the presence of para-toluene sulphonic acid. To a 100ml round-bottomed flask were added with constant stimng 7 75g soluble starch and 35ml anhydrous dimethylsulphoxide (DMSO). The flask was capped with a rubber septum and vacuum and dry nitrogen were applied alternately to the flask via a 3-way connector to remove any moisture and to fill the flask with dry nitrogen. The flask contents were heated to a constant 80 C in an oil bath with continuous stirring. The flask was cooled to room temperature and 24.Og (R)-3-hydroxybutyπc acid and 1.16g p-toluene sulphonic acid were added to the mixture The flask was capped with a rubber septum and vacuum and dry nitrogen were applied alternately to the flask via a 3-way connector to remove any moisture and to fill the flask with dry nitrogen. The flask contents were heated to a constant 80 C in an oil bath under vacuum for 46 hrs. The reaction mixture was added to a large amount of acetone with stimng and the precipitate was separated by centnfugauon. More acetone was added to the precipitate and the centrifugation step was repeated several times. The product was then dried under reduced pressure at room temperature for 3 days The scheme for the synthesis of KTX 0322 and its chemical structure are shown in Figure 7. Yield 3.9g of water-insoluble white solid The structure of the acylated soluble starch was determined by 1H NMR (300 MHz, CDCl3) spectroscopy The degree of substitution of pullulan by (R)-3-hydroxybutyrate was 0.60. Elemental analysis C = 43 94%, H = 6.49% by weight

EXAMPLE 8. Modification of Starch with IRl-3-Hydroχybutγric Acid Procedure for Modification of Starch Nanosphercs witb fRI-3-Hydroxybutyric Acid 250 mg of starch nanosphere (from Ecosynthetix), I 0 g of [Rj-3-hydroxybutyric acid and 37 5 mg p-toluene-sulfonic acid were added to a 50 mL flask with stimng bar The flask was capped by a rubber septum. Vacuum and dry nitrogen were applied to the flask alternately via three way connector to remove any moisture and fill the flask with dry nitrogen The flask was placed into a constant temperature (80 0C) oil bath with stirring After 2 h, the reaction mixture was subjected to reduced pressure for 19 h. The reaction product(s) were ground to a fine powder and stirred in acetone for 2 h. The mixture was then filtered. The white solid was washed with acetone and then dπed under reduced pressure at room temperature to give a product yield of 408 mg. NMR, IR was used to characterize the product. The degree of substitution was measured as 1 2.

EXAMPLE 9. Modification of Soluble Starch with lR|-3-Hvdroxy butyric Acid Soluble Starch, an was sourced as A. C. S. reagent, from Sigma-Aldπch The method of esteπficatioπ used was that of Example 1. NMR was used to characterize the product. The degree of substitution attained was 0 7 EXAMPLE 10. Modification of PuHiUan with IRl-3-Hydroxybutyric Acid Pullulan was sourced from Pfanstiehl Laboratories, Inc. The method of estcπfication used was that of Example 1. NMR was used to characterize the product. The degree of substitution was measured as 1 1

EXAMPLE 11. Modification of Pectin and with IRl-3-Hydroxybutyric Acid Pectin, from citrus fruits, was sourced ordered from Sigma The method of Example 8 was used to modify these polysaccharides. The product was water soluble indicative of a low degree of substitution

EXAMPLE 12 Modification of Locugt Bean Gum. Locust bean gum was treated as described in Example 1 The product was water soluble indicating a low degree of substitution

EXAMPLE 13. Ketogenesis in rats in vivo Male Sprague-Dawley rats (weight range 200-250g Charles River, Margate, Kent) were group housed in polypropylene cages at a temperature of 21±4°C and 55±2O% humidity and on a standard light/dark cycle Animals had free access to a standard pelleted rat diet and tap water at all times. Animals were accustomed to these conditions for at least one week before experimentation.

Compounds were administered by oral gavage (po) Control animals received the appropπatc vehicle via the same route The experiment was performed over 2 days (ie 2 compounds were tested per day) Blood samples were taken by cardiac puncture after the animals were killed by a British Home Office Schedule 1 method. The terminal blood sample was collected into suitable plasma preparation tubes (EDTA- coated rubes). Plasma samples were initially frozen on dry ice and transferred to a -750C freezer until required for subsequent analysis (spectrophotometry anaylsis of (R)-3-bydroxybutyrate) Protocol for KTX 0301, KTX 0313 only

Protocol for KTX 03 IJ only

Protocol for KTX 0321, KTX 0310, KTX 0312 and KTX 0322

Sodium DL-β-hydroxybutyrate (H-6501 Lot 1 1 1K2618) was obtained from Sigma. A stock solution of β-hydroxybutyrate (4OmM DL racemate equivalent to 2OmM D-isomer) was prepared in 0.9% saline solution, kept at 4CC and used to generate appropriate dilutions for an assay standard curve. Such solutions have been shown to be stable for at least 2 months Commercial clinical assay lots for the determination of D-β-hydroxybutyrate were obtained from Randox Laboratories (Antrim, UK.). Kirs were obtained in two pack sizes (Ranbut RB 1007- I Ox 10ml and RB 1008 10x50mI) but were otherwise identical Each kit contained a standard solution of ImM D-β-hydroxybutyrate that was assayed every time to confirm the assay was performing correctly The kit rehes on measuring the appearance of NADH via the activity of β-hydroxybutyrate dehydrogenase measured as an increase of OD340nrn. An alkaline pH is necessary to dnve the reaction equilibrium towards the production of NADH and acetoacetate;

pH 8.5 Hydroxybutyrate + NAD+ * NADH + Acetoacetate pH 7.0

The protocol supplied with the Ranbut kits was for a discrete (cuvette-based) spectrophotometπc assay, so the protocol was modified for suitability with a 96- well microplate format using blank, flat-bottomed microplates (Greiner PS 655101 Lot 98 35 01) Assays were performed in triplicate using a sample volume of lOμl to each well for the standards and usually 20μl for plasma samples (though this was varied for some experiments). Standard dilutions and samples were pipetted a single plate at a time and premcubated at 37°C for 15 minutes in the sample compartment of a Molecular Devices VERSAmsx tunable microplate reader The appropriate volume of assay reagent was reconstituted, according to instructions, using distilled water and premcubated at 370C for 15 minutes using a static water bath. The assay plate was ejected and the reaction started by adding rapidly 25Oμ! of reagent to each well (avoiding air bubbles). The plate was reloaded, mixed and then the change in OD340nm followed in kinetic mode with a reading at every 15 seconds for a total of 2 minutes The reaction rate was then determined from the OD increase over a suitable 1 minute peπod, after allowing a necessary period for the reaction rate to settle. The rate between 45 seconds and 105 seconds was used as the default measuring penod, though occasionally a different period was used as necessary (eg if an aberrant reading was obtained at one of these time-points) Statistical tests were employed in-house using Graph Pad Prism for this preliminary study (i e. not using an independent qualified statistician). ANOVA followed by Dunnerf s test was used to compare the vaπous time-points with baseline. P<0.05 was considered to be statistically significant. Baseline values were combined for each day (ie n=8) to increase the power of the analysis.

After oral administration, the (R)-3-hydroxybutyrate ester derivatives of the monosaccharides, ie tn- and tetra-(R)-3-hydroxybutyrate ester derivatives of fructose (KTX 031 1) and the tπ-(R)-3-hydroxybutyrate ester derivative of sorbitol (KTX 0313), were found to produce significant increases in plasma 3-hydroxybutyrate concentrations In contrast, the tn- and tetra- (R)-3-hydroxybutyrate ester derivatives of glucose (KTX 0310) and the di- and tn-(R)-3-hydroxybutyrate ester derivatives of arabinose (KTX 03! 2) did not evoke significant kctogenesis in rats after oral administration at the doses and times tested. KTX 0301 (a mixture of a (R)-3-hydroxybutyrate ester derivatives of pullulan and a poly-3-hydroxybutyrate oligomer) also produced significant increases in plasma 3-hydroxybutyrate concentrations after oral administration, whereas KTX 0321 (a different (R)-3-hydroxybutyrate ester derivative of pullulan) and KTX 0322 (a (R)-3-hydroxybutyrate ester derivative of soluble starch) did not evoke significant ketogenesis in rats after oral administration at the doses and times used