Use of neotrehalose for the manufacture of a medicament for supplementing energy to living body

Abstract: A composition for supplementing energy to a living body, which contains neotrehalose as effective ingredient, is disclosed. Neotrehalose is a disaccharide, therefore, when used in the composition, enables 2-fold higher energy supplement than in the case of using monosaccharide. Neotrehalose has non-reducing properties, and this renders the composition stable for a relatively-long time period.

Description: The present invention relates to use of a composition comprising neotrehalose for the manufacture of a medicament for supplementing energy to a living body and to the preparation of said composition comprising neotrehalose (designated as "COMPOSITION" hereinafter) Glucose and fructose, which exhibit reducing properties, have been used from old times. These saccharides are, however, unstable because of their inherent reducing properties, and usually, the unstableness is more augmented in the presence of other nutritional substances such as amino acids and vitamins. Therefore, there has been a great demand for the establishment of non-reducing saccharides with a satisfiable stability, for example, xylitol, sorbitol, maltitol, lactitol, sucrose and trehalose. The monosaccharide alcohols (hydrogenated monosaccharides) such as xylitol and sorbitol have, however, a drawback: They give an acute diarrhea when the dose and administration method are mistaken. As described in Japanese Patent Publication Nos.13,699/72 and 42,506/72, the disaccharide alcohols (hydrogenated disaccharides) such as maltitol and lactitol are not readily metabolized and utilized in vivo, and actually, they have been used as a low-caloric sweetener and are not suitable for use in the present invention. Sucrose has a drawback: It is readily hydrolyzed under acidic conditions into glucose and fructose which exhibit reducing properties, and this hinders the storage stability. As Japanese Patent Laid-Open No. 240,758/88 describes "Trehalose is a low-caloric sweetener which is not readily metabolized and absorbed by the human body". And "Trehalose is not readily hydrolyzed by enzymes such as amylases.", it has been recognized that trehalose is a saccharide which does not release energy in a living body, and that a large amount of the preparation has not been studied in detail. JP-A-6324075B discloses a food material comprising neotrehalose and lactose. It is disclosed that this food material prevents tooth decay and promotes the growth of bifidobacterium. There has been a great demand for the realization of a composition which is free of reducing properties, satisfiable in storage stability, and usable in a variety of fields, as well as for the realization of a composition which contains neotrehalose as effective ingredient. The present inventors have studied saccharides. In particular, the present inventors studied non-reducing disaccharides such as trehalose and its relating substances. As a result, the present inventors found that unexpectedly unlike trehalose (α,α-trehalose) and isotrehalose (β,β-trehalose), neotrehalose (O-α-D-glucopyranosyl β-D-glucopyranoside or α,β-trehalose) was readily metabolized and utilized by a living body into energy, and that neotrehalose can be used in a variety of fields because of its stability and non-reducing properties, and the present inventors accomplished the present COMPOSITION which contains neotrehalose as effective ingredient. Thus, the present inventors accomplished the present invention. Since neo-trehalose has a relatively-high storage stability and does not have reducing properties, the present COMPOSITION can be advantageously prepared into a synthetic nutritional composition and a pharmaceutical composition with a higher therapeutic-effect by combining the present COMPOSITION with other nutritional- and/or pharmaceutical-substances. The invention will now be described further by way of example only. In the first instance, the present invention establishes use of a composition comprising neotrehalose for the manufacture of a medicament for supplementing energy in a living body. In the second instance, the present invention provides a process for preparing a composition for supplementing energy in a living body, wherein the composition contains neotrehalose as an effective ingredient, the process comprising: (a) providing an aqueous neotrehalose solution; (b) purifying the aqueous neotrehalose solution to remove pyrogens; and, (c) distributing the resultant solution into a container. Any preparation of the present composition can be used in the invention, as long as it produces the present composition. Neotrehalose which is prepared, for example, either of by the method disclosed in Japanese Patent Laid-Open No.216,492/88, wherein cyclodextrin-synthesizing enzyme is allowed to act on starch or partial starch hydrolysate, and the method, disclosed by the present inventors in Japanese Patent Application No.307,054/90, wherein β-galactosidase is allowed to act on lactoneotrehalose, can be used in the invention after the treatment of purification. Neotrehalose preparations, which have been purified to the possible highest level, are preferably used. in the present invention. Usually, neotrehalose preparations in the form of syrup or powder with a neotrehalose content of 50 w/w % or higher, preferably, those in the form of syrup or crystalline powder with a neotrehalose content of 80 w/w % or higher, more preferably, those in the form of crystalline powder or crystal with a neotrehalose content of 90 w/w % or higher are suitably used in the invention. Any COMPOSITION can be used in the invention, as long as it contains neotrehalose as effective ingredient and can supply energy to a living body, and usually, in order to higher the effect, COMPOSITIONs with a neotrehalose concentration of 10 w/w % or higher, preferably, 20 w/w % or higher on the dry solid basis (abbreviated as "d.s.b." hereinafter) can be favorably used in the invention. A simple composition consisting of neotrehalose can be used in the invention, and usually, the present COMPOSITION can be prepared by combining neotrehalose with one or more of other substances, for example, nutritional substances such as proteins, amino acids, lipids, other saccharides, vitamins and minerals; and effective substances such as antibacterial substances, enzymes, hormones and cytokines. If necessary, one or more of other appropriate substances such as taste-improving agents and coloring agents, flavor-imparting agents, stabilizers, vehicles and fillers, can be used in combination, and the COMPOSITION thus obtained can be formed to meet to its final use. The COMPOSITION is parenterally administered without exhibiting toxicity and fear of causing side effect, and satisfiably metabolized and utilized in vivo . Thus, the COMPOSITION can be advantageously used to supplement energy to a living body. The dose of the present composition is chosen from the range of about 1-1,000 g/day/adult, preferably about 1-500g/day/adult even more preferably of about 5-5000 g/day/adult based on the weight of neotrehalose, d.s.b., as effective ingredient. The present COMPOSITION is advantageously administrable to human, as well as to domestic-and pet-animals such as cow, horse, dog and cat. The following Experiments will explain the present invention in detail. Example A1 Neotrehalose Example Al-1 Preparation of lactoneotrehalose Fifty parts by weight of commercially-available lactose and 50 parts by weight of "PINE-DEX #1", a dextrin product (DE 8) commercialized by Matsutani Chemical Ind., Co., Ltd., Hyogo, Japan, were dissolved in 150 parts by weight of water while heating, and the solution was heated to 60°C, adjusted to pH 6.0, added with 300 units/g dextrin of a cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus commercialized by Hayashibara Biochemical Laboratories Inc., Okayama, Japan, allowed to react for 20 hours, and heated at 100°C for .30 minutes to inactivate the remaining enzyme. Thereafter, the mixture was cooled to 55°C, adjusted to pH 5.0, added with 15 units/g dextrin of "Glucozyme", a glucoamylase specimen commercialized by Nagase Biochemicals Ltd., Kyoto, Japan, allowed to react for 16 hours, and heated at 100°C for 15 minutes to inactivate the remaining enzyme. The resultant solution containing about 24 w/w % lactoneotrehalose, d.s.b., was decolored with activated charcoal, treated with ion exchange (H + - and OH - -form) for deionization and purification, concentrated to give a concentration of about 45 w/w % and subjected to column chromatography, followed by the recovery of lactoneotrehalose-rich fractions. As the fractionating resin was used "Amberlite XT-1016 (Na + -form)", a strongly-acidic cation exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan, which was suspended in water and packed in jacketted-stainless steel columns, inner diameter of 5.4 cm each. In this case, 4 columns, gel-bed depth of 5 m each, were cascaded to give a total gel-bed depth of about 20 m. While keeping the inner temperature of the columns at 55°C, 5 v/v % of a material saccharide solution was added thereto, and 55°C.water was then passed through the columns at a flow rate of SV 0.3 to effect fractionation, thus obtaining lactoneotrehalose-rich fractions. A portion of lactoneotrehalose-rich fractions, lactoneotrehalose content of about 67 w/w %, d.s.b., which had been pooled through the repetition of the above method, was concentrated to give a concentration of 75 w/w %, and allowed to stand overnight at 20°C to effect crystallization. A high-lactoneotrehalose content solution, which had been prepared by concentrating the lactoneotrehalose-rich fractions to give a concentration of 70 w/w %, was added with the resultant crystal as seed to effect crystallization under gentle-stirring conditions. The resultant massecuite was separated, and the formed crystal was washed by spraying thereto a small amount of water to obtain a high-purity crystalline lactoneotrehalose which was then dissolved in water and treated similarly as above to effect recrystallization. Thus, about 3 parts by weight of a high-purity crystalline lactoneotrehalose, purity of 99.8 w/w % or higher, was obtained. Example A1-2 Preparation of neotrehalose One part by weight of a crystalline lactoneotrehalose specimen obtained by the method in Example Al-1 was dissolved in 30 parts by weight of water while heating, and the solution was adjusted to 40°C and pH 4.5, added 10 units/g lactoneotrehalose of "LACTASE-LP", a β-galactosidase specimen commercialized by K·I Chemical Industry, Co., Ltd., Shizuoka, Japan, allowed to react for 20 hours, and heated at 100°C for 10 minutes to inactivate the remaining enzyme. Similarly as in Experiment 1, the resultant solution containing about 66 w/w % neotrehalose and about 33 w/w % galactose, d.s.b., was decolored, deionized, purified, concentrated, and subjected to column chromatography using a strongly-acidic cation exchange resin, followed by the recovery of neotrehalose-rich fractions. A part of the neotrehalose-rich fractions containing about 88 w/w % neotrehalose, d.s.b., was concentrated to give a concentration of 75 w/w %, and allowed to stand overnight at 20°C to effect crystallization. A solution having a concentration of 70 w/w %, which had been prepared by concentrating the neotrehalose-rich fractions, was added with the resultant crystal as seed to effect crystallization under gentle stirring conditions. The resultant massecuite was separated, and the formed crystal was washed by spraying thereto a small amount of water to obtain a high-purity crystal which was then dissolved in water and recrystallized similarly as above to obtain about 0.15 parts by weight of a high-purity crystalline neotrehalose, purity of 99.8 w/w % or higher. Parenterally administrable nutritional - and pharmaceutical-compositions can be advantageously prepared by incorporating the above produced neotrehalose therein. Example A2 Neotrehalose A solution containing about 66 w/w % neotrehalose, d.s.b., prepared by using the reaction- and purification-methods in Example A1-2, as a material saccharide solution, was concentrated to give a concentration of about 45 w/w %. In order to higher the neotrehalose content in the resultant solution, which was subjected to column chromatography similarly as the method in Example Al-1 except that "DOWEX 50W×4 (Ca ++ -form)", a strongly-acidic cation exchange resin commercialized by Dow Chemical Company, Midland, Michigan, USA, was used as a resin for fractionation to obtain a neotrehalose-rich fraction containing about 85 w/w % neotrehalose, d.s.b. The fraction was concentrated to give a concentration of about 83 w/w %, and the resultant solution was transferred to a crystallizer and admixed with an about one w/w % seed. The mixture was transferred to a tray and allowed to stand at 20°C for 4 days to effect crystalization and solidification. Thereafter, the resultant solid was pulverized with a cutting-type pulverizer and dried to obtain a crystalline neotrehalose powder containing molasses in the yield of about 70 w/w %, d.s.b. Example B1 Solid intubation-nutrient A composition was prepared by mixing 500 parts by weight of a crystalline neotrehalose specimen obtained by the method in Example A1-2, 270 parts by weight of dried yolk, 209 parts by weight of defatted milk, 4.4 parts by weight of sodium chloride, 1.85 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate, 0.6 parts by weight of vitamin E acetate, and 0.04 part by weight of nicotinamide. Twenty-five g aliquots of the composition were distributed into laminated-aluminum small bags, and heat sealed to obtain the captioned product. In use, one bag of the product is first dissolved in an about 150-300 ml water to prepare an intubation nutrient solution, which is then administered through intubation feeding, for example, into nasal cavity. The product can be advantageously used as COMPOSITION. Example B2 Infusion solution A crystalline neotrehalose specimen prepared by the method in Example A1-2 was dissolved in water to give a concentration of about 10 w/v %, and the solution was subjected, in an usual manner, to membrane filtration, aseptically distributed into a plastic bottle, and sealed to obtain the captioned product. The product is a stable agent free of change on standing and can be favorably used in intravenous- and intraperitoneal-injections which supplement energy to a living body. The product gives isotonicity at a concentration of 10 w/v % to the blood and supplies energy to a living body at 2-fold higher concentration than in the case of glucose. Example B 3 Infusion solution A crystalline neotrehalose specimen was dissolved in water to give a concentration of 20 w/v %, and the solution was subjected, in an usual manner, to membrane filtration, aseptically distributed into a plastic bag and sealed to obtain the captioned product. The product is stable and free of change on standing and can be favorably admixed with other medicaments and/or COMPOSITIONs and adjusted to an appropriate concentration, prior to administration. Example B4 Infusion solution A crystalline neotrehalose and a crystalline sorbitol were mixed and dissolved in water to give concentrations of 5 w/v % and 2.5 w/v % respectively, and the mixture was distributed, similarly as in Example B2, into a bottle and sealed to obtain the captioned product. The product is stable and free of change on standing and can be favorably used in intravenous- and intraperitoneal-injections which supplement amino acids and energy to a living body. Example B5 Infusion solution A crystalline neotrehalose specimen prepared by the method in Example A1-2 and an amino acid composition having the following components were dissolved in water to give concentrations of 5 w/v % and 3.0 w/v % respectively, and the mixture was purified, similarly as in Example B2, distributed into a bag and sealed to obtain the captioned product. Components of amino acid composition mg/100ml L-Isoleucine 180 L-Leucine 410 L-Lysine hydrochloride 620 L-Methionine 240 L-Phenylalanine 290 L-Threonine 180 L-Tryptophan 60 L-Valine 200 L-Arginine hydrochloride 270 L-Histidine hydrochloride 130 L-Glycine 340 Although the product is a complex agent which contains saccharide and amino acids, it is stable and free of change on standing because of the non-reducibility of neotrehalose, and can be favorably used in intravenous- and intraperitoneal-injections. The product is suitably used as COMPOSITION to supplement both energy and amino acids to a living body. Example B6 Infusion solution One hundred parts by weight of a 10 w/v % aqueous neotrehalose solution was added with 5 parts by weight of soybean and 1.5 parts by weight of lecithin from yolk, and the mixture was subjected to a mixer to obtain a partial homogenate which was then mixed to homogeneity with a device commercialized by Gaulin Co., U.S.A., under nitrogen atmosphere and at a pressure of 600kg/cm 2 into minute droplets of O/W emulsion, average particle size of 0.2µm or lower. Similarly as in Example B3, the emulsion was distributed into a bag and sealed to obtain the captioned product. Although the product is a complex COMPOSITION which contains a saccharide and lipids, it is stable and free of change on standing and can be favorably used in intravenous- and intraperitoneal-injections for COMPOSITION. Example B 7 Infusion solution An aqueous solution containing as minerals 0.136 w/v % sodium dihydrogenphosphate, 0.098 w/v % potassium acetate, 0.031 w/v % magnesium chloride hexahydrate, 0.022 w/v % calcium chloride dihydrate, 1.59 w/v % sodium lactate was prepared by adding and dissolving the minerals in a 10 w/v % aqueous neotrehalose solution. The aqueous solution was adjusted to pH 5.5, and distributed, similarly as in Example B3, and sealed to obtain the captioned product. The product is stable and free of change on standing and can be favorably used in intravenous- and intraperitoneal-injections which supplement energy and minerals to a living body. Example B8 Infusion solution An aqueous solution containing 0.1 w/v % human serum albumin and 100,000 international units per ml of human interferon-α was prepared by dissolving human serum albumin and human interferon-α in a 20 w/v % aqueous neotrehalose solution. The aqueous solution was subjected to membrane filtration, aseptically distributed into a 10ml-bottle and sealed to obtain the captioned product. The neotrehalose in the product stabilizes the human interferon-α as biologically active substance, and this renders the activity of the human interferon-α stable for a relatively-long time period when stored under light-shielded conditions. The product can be advantageously used to attain a therapeutic effect exerted by the biologically active substance, as well as to supplement energy to a living body by dissolving the product in distiled water for injection or by using the product with other COMPOSITION in combination, prior to use. Example B9 . Traumatic in the form of ointment Five hundred parts by weight of a crystalline neotrehalose specimen, prepared by the method in Example A2, was admixed with 50 parts by weight of a methanol solution which had been prepared by dissolving 3 parts by weight of iodine in methanol, and the resultant mixture was admixed with 200 parts by weight of a 10 w/w % aqueous pullulan solution to obtain the captioned product having an appropriate spreadability and adhesiveness. The product exerts an antibacterial action inherent to iodine and acts as COMPOSITION via the action of neotrehalose, and these shorten the healing time period and cure the affected part as beautiful as before. Example B10 Sugar-coated tablet A plain tablet (150 mg in weight) as a core tablet was coated with the first sugar-coating composition consisting of 40 parts by weight of a crystalline neotrehalose specimen obtained by the method in Example A1-2, 2 parts by weight of pullulan (average molecular weight of 200,000), 30 parts by weight of water, 25 parts by weight of talc, and 3 parts by weight of titanium oxide until the tablet weighed about 230 mg. Thereafter, the resultant tablet was first coated with the second sugar-coating composition consisting of 65 parts by weight of the same crystalline neotrehalose specimen as used in the above, one part by weight of pullulan (average molecular weight of 200,000) and 34 parts by weight of water, then coated with a wax solution to impart a gloss. Thus, the captioned product having a gloss and satisfiable appearance was obtained. The handleability in the sugar-coating step for the product is satisfactory, and the product has a satisfiable shock-resistance and retains its quality at a relatively high-level over a relatively-long time period. The product can be advantageously used as COMPOSITION. Experiment 1 A neotrehalose specimen prepared by the method in Example A1-2 was allowed to remove pyrogen in an usual manner, and 50 g of the resultant neotrehalose specimen was prepared, in accordance with the method reported in Matsuzaki, Yakubutsu Ryoho , Vol.6, No.2, pp.65-72 (1973), into a 10 w/v % aqueous solution which was then intravenously administered to 2 healthy volunteers (37- and 49-year old men) through instillation and their bloods were sampled at prescribed time intervals and subjected to the measurements of the blood sugar and insulin levels and the amount of neotrehalose secreted in their urine. As control, glucose and maltose were used. As a result, glucose extremely increased the blood sugar and insulin levels, while neotrehalose exhibited the same dynamics as maltose, i.e. neotrehalose slightly increased the blood sugar level but almost not increased the insulin level. The amount of glucose in the urines of the volunteers, who had been administered with glucose, was less than 10%, while those with neotrehalose and maltose were less than 20%. These results confirm that neotrehalose is a saccharide which is well metabolized and utilized by a living body, and this. renders neotrehalose very useful in SACCHARIDEs and COMPOSITIONs. Experiment 2 Acute toxicity A crystalline neotrehalose specimen, prepared by the method in Example A1-2, was orally administered to 7 week-old dd mice for acute toxicity test. As a result, no animal death was noted with the administration of up to 5 g of the specimen, and higher dose was difficult to do. Therefore, the toxicity of the tested substance appears to be extremely low. The production of neotrehalose suitable for incorporation in the composition of the present invention and the composition itself are illustrated by the preceding Examples. [Effect of the invention] As evident from above, the present composition which comprises neotrehalose is a stable and non-reducing saccharide, and, entirely different from trehalose (α,α-trehalose) and isotrehalose (β,β-trehalose) which are similar to neotrehalose, the saccharide used in the present invention is readily metabolized and utilized in vivo . The present COMPOSITION containing neotrehalose as effective ingredient dose not show reducing properties and has a relatively-high storage stability. Thus, the present COMPOSITION has a feature of that it is readily preparable into a more favorable synthetic-nutritional-composition and a pharmaceutical composition with a higher therapeutic-effect by combining the present COMPOSITION with other nutritional- and/or effective-substances.

Claims: Use of a composition comprising neotrehalose for the manufacture of a medicament for supplementing energy in a living body. Use of a composition according to claim 1, wherein the medicament is to be administered parenterally. Use of a composition according to claim 1, wherein the medicament is in the form of an intubation nutrient, infusion solution, ointment or tablet. Use of a composition according to any one of claims 1 to 3, wherein the composition contains at least 10 w/w % neotrehalose, on a dry solid basis. Use of a composition according to any one of claims 1 to 4, wherein the neotrehalose is obtainable from neotrehalose in a syrup or powder form. Use of a composition according to any one of claims 1 to 4, wherein the neotrehalose is obtainable from neotrehalose in a crystalline form. Use of a composition according to any one of the preceding claims, wherein the composition additionally comprises one or more members selected from the group consisting of proteins, amino acids, saccharides, vitamins, minerals, antibacterial substances, enzymes, hormones, cytokines, and mixtures thereof. Use of a composition according to any one of the preceding claims, wherein the body is that of a human or animal. Use of a composition according to any one of the preceding claims, wherein the composition is formulated for administration at a dose of about 1-1,000 g/day/adult based on the weight of neotrehalose, on a dry solid basis. Use of a composition according to any one of the preceding claims, wherein the composition is pyrogen-free. A process for preparing a composition for supplementing energy in a living body, wherein the composition contains neotrehalose as an effective ingredient, the process comprising: (a) providing an aqueous neotrehalose solution; (b) purifying the aqueous neotrehalose solution to remove pyrogens; and (c) distributing the resultant solution into a container. A process according to claim 11, wherein the aqueous neotrehalose solution in step (a) is prepared by using crystalline neotrehalose. A process according to claim 11 or 12, wherein the composition contains at least 10 w/w % of neotrehalose, on a dry solid basis. A process according to claim 11, 12 or 13, wherein step (a) additionally comprises the step of adding to the aqueous solution one or more members selected from the group consisting of proteins, amino acids, saccharides, vitamins, minerals, antibacterial substances, enzymes, hormones, cytokines, and mixtures thereof A process according to any one of claims 11 to 14, wherein the composition is formulated for administration at a dose of about 1-500 g/day/adult based on the weight of neotrehalose, on a dry solid basis.

Inventors: CHAEN HIROTO, MIYAKE TOSHIO, SAKAI SHUZO, SHIBUYA TAKASHI, CHAEN, HIROTO, MIYAKE, TOSHIO, SAKAI, SHUZO, SHIBUYA, TAKASHI
Applicants: HAYASHIBARA BIOCHEM LAB, KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO
Application year: 1991
Main classification: A61K31
IPC codes: A61K31, A23L1