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WO2019034112A1 - 一种包含左旋肉碱和β-羟基丁酸化合物的组合物 - Google Patents

一种包含左旋肉碱和β-羟基丁酸化合物的组合物 Download PDF

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WO2019034112A1
WO2019034112A1 PCT/CN2018/100836 CN2018100836W WO2019034112A1 WO 2019034112 A1 WO2019034112 A1 WO 2019034112A1 CN 2018100836 W CN2018100836 W CN 2018100836W WO 2019034112 A1 WO2019034112 A1 WO 2019034112A1
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Prior art keywords
hydroxybutyrate
carnitine
hydroxybutyric acid
compound
composition
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French (fr)
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袁建栋
孙占莉
丁海峰
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Brightgene Bio Medical Technology Co Ltd
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Brightgene Bio Medical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a composition comprising L-carnitine and ⁇ -hydroxybutyric acid compound, and the invention also relates to preparation of L-carnitine- ⁇ -hydroxybutyrate in medicine and food additive Application in technology.
  • Obesity has become a serious public health problem. In recent years, obesity patients are increasing all over the world. The number of deaths due to obesity is second only to smoking every year. The International Obesity Conference released a report on the number of deaths caused by obesity worldwide. The number of people who have starved to death in the world has been exceeded. Obesity can also cause severe diabetes, cardiovascular disease and other diseases, and is the enemy of human health and longevity.
  • Therapeutic drugs for obesity include: appetite suppressing drugs, drugs for increasing energy consumption, drugs for inhibiting intestinal digestion and absorption, insulin sensitizers, biological peptides, and agonists or inhibitors thereof. These drugs have many adverse reactions, such as heart valve damage, pulmonary hypertension and finger necrosis, elevated blood pressure, gastrointestinal dysfunction, respiratory infections, headache, menstrual disorders, anxiety, fatigue, flatulence, abdominal pain, diarrhea, etc. Individual patients may also have hypoglycemia and hepatotoxicity.
  • L-carnitine The main physiological function of L-carnitine is to promote the conversion of fat into energy. L-carnitine can reduce body fat and reduce body weight without reducing water and muscle. It was recognized as the safest and no side effect by the International Obesity Health Organization in 2003. Weight loss nutritional supplements. L-carnitine is a key substance in the process of fat metabolism, which can promote the oxidative decomposition of fatty acids into mitochondria. L-carnitine is a carrier for transporting fatty acids. In long-term high-intensity exercise, L-carnitine increases the rate of fat oxidation, reduces the consumption of glycogen, and also delays fatigue. Many weight loss products use L-carnitine as one of the main components of weight loss.
  • L-carnitine has been used in the fields of medicine, health care and food, and has been regulated as a legal multi-purpose nutrient by Switzerland, France, the United States and the World Health Organization.
  • China's food additive health standard GB2760-1996 stipulates that L-carnitine tartrate is a food nutrition fortifier, which can be applied to chewable tablets, drinking liquids, capsules, milk powder and milk drinks.
  • medical effects including cardiovascular disease, liver disease, kidney disease, hyperlipidemia, diabetes, neuromuscular diseases, etc. can be improved by taking L-carnitine and its products.
  • the pharmaceutically acceptable salt of L-carnitine has the same therapeutic or nutritional effect as its internal salt. And these pharmaceutically acceptable salts can improve the stability and moisture absorption of the inner salt, such as L-carnitine tartrate (US4602039) and L-fumarate (US2009281183A1), L-carnitine mucate commonly used in the market. (US5952379). It has also been reported that L-carnitine is combined with metal ions such as calcium and magnesium ions to form a salt which has good water solubility and is easily absorbed by the human body. For example, US Pat. No.
  • 6,051,608 discloses magnesium levoth fumarate and alkanoyl L-carnitine magnesium fumarate
  • WO 98/45250 discloses L-carnitine magnesium tartrate and alkanoyl L-carnitine magnesium citrate
  • L-carnitine tartrate still has a large moisture absorption, relative humidity above 60% will deliquesce; and L-carnitine and metal ions such as Salts formed by the combination of calcium and magnesium ions also have some defects.
  • L-carnitine and metal ions such as Salts formed by the combination of calcium and magnesium ions also have some defects.
  • a large amount of calcium lactate may cause fatigue; excessive calcium gluconate and calcium chloride are unfavorable for diabetes; in addition, L-carnitine calcium galactate is in the preparation process.
  • Nutritional or therapeutic ketosis is a physiological condition caused by a ketogenic diet, calorie restriction, therapeutic fasting, and/or supplementation with ketogenic precursors resulting in elevated blood ketone levels (typically above 0.5 mmol/L).
  • Ketone bodies represent alternative energy substrates for peripheral tissues and the central nervous system. Beta-hydroxybutyrate and acetoacetate are the two most abundant and physiologically significant ketone bodies. Glucose storage in the body is quickly used and becomes rapidly depleted during fasting, extreme sports, and/or low carbohydrate consumption. Failure to replenish glucose storage as they become depleted causes the body to resort to alternative methods to produce energy by making ketone bodies.
  • Ketone bodies can be used as an alternative fuel by every cell in the body to meet the body's energy needs, including the needs of the brain.
  • blood ketone levels will increase up to 2 mmol/L or 3 mmol/L.
  • ketone bodies ⁇ -hydroxybutyric acid and acetoacetic acid
  • This state is called Ketoacidosis or "nutritional ketosis”.
  • ketosis the body burns fat essentially, so once in ketosis, one can induce body fat loss by reducing dietary fat intake and adjusting carbohydrate intake to a low enough to maintain ketosis.
  • ketogenic fats such as medium chain triglycerides (MCT oils) are usually not required by the gastrointestinal system to help induce ketosis.
  • MCT oils medium chain triglycerides
  • ⁇ HB beta-hydroxybutyrate
  • acetoacetate in its free acid form is expensive and ineffective in the manufacture of persistent ketosis.
  • the sodium salt is used to buffer the free acid form of [beta]HB, but this causes potentially harmful sodium overload and mineral balance imbalance at the therapeutic level of ketosis.
  • the ketogenic diet raises concerns about increasing total cholesterol and triglycerides while lowering high-density lipoprotein (HDL) levels, leading to cardiovascular disease.
  • HDL high-density lipoprotein
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, to provide a novel composition comprising L-carnitine; and at least one ⁇ -hydroxybutyric acid compound comprising ⁇ -hydroxyl groups Butyrate, beta-hydroxybutyrate precursor or a combination thereof.
  • Another aspect of the present invention also provides a composition comprising L-carnitine and a ⁇ -hydroxybutyric acid compound in the form of an intramolecular salt.
  • a third aspect of the invention provides a process for the preparation of the composition and the use of the composition.
  • the present invention provides a composition comprising L-carnitine; and at least one ⁇ -hydroxybutyric acid compound comprising ⁇ -hydroxybutyrate, ⁇ -hydroxybutyric acid Precursor or a combination thereof.
  • the at least one ⁇ -hydroxybutyric acid compound comprises one or more of the following: sodium ⁇ -hydroxybutyrate, potassium ⁇ -hydroxybutyrate, calcium ⁇ -hydroxybutyrate, magnesium ⁇ -hydroxybutyrate , lithium beta-hydroxybutyrate, or a mixture thereof;
  • the at least one ⁇ -hydroxybutyric acid compound comprises
  • the at least one ⁇ -hydroxybutyric acid compound is racemic DL- ⁇ -hydroxybutyric acid or the single isomer R- ⁇ -hydroxybutyric acid.
  • the molar ratio of the ⁇ -hydroxybutyric acid compound to the L-carnitine is from 20:1 to 1:1.
  • the present invention provides a composition
  • A is L-carnitine
  • B is ⁇ -hydroxybutyrate arginine, ⁇ -hydroxybutyrate lysine, ⁇ -hydroxyl Butyric acid histidine, ⁇ -hydroxybutyrate ornithine, ⁇ -hydroxybutyrate sarcosine, ⁇ -hydroxybutyrate decyl butylamine or ⁇ -hydroxybutyrate citrulline
  • m, n independently selected from An integer of from 1 to 4; preferably B is ⁇ -hydroxybutyrate arginine, ⁇ -hydroxybutyrate lysine, ⁇ -hydroxybutyrate histidine, or ⁇ -hydroxybutyrate ornithine, and preferably m is 1 or 2, and n is 2 or 1.
  • composition comprising L-carnitine- ⁇ -hydroxybutyrate, and wherein the L-carnitine and the ⁇ -hydroxybutyric acid compound are intramolecular salts.
  • the form exists and has the following structure:
  • M is Na, K or Li
  • the ratio of m to n is from 0.8:10 to 10:0.8, preferably from 1:4 to 4:1, more preferably 1:1, most preferably m and n are both 1, or ,
  • M is Ca or Mg
  • the ratio of m to n is from 0.8:10 to 10:0.8, preferably from 1:4 to 4:1, more preferably 1:1, and most preferably m and n are both 1.
  • the composition comprises L-carnitine- ⁇ -hydroxybutyrate, and the L-carnitine- ⁇ -hydroxybutyrate is a combination of one or more of the following:
  • the ratio of values of m to n is from 0.8:10 to 10:0.8, preferably from 1:4 to 4:1, more preferably 1:1, and most preferably m and n are both 1.
  • composition comprising L-carnitine- ⁇ -hydroxybutyrate, the L-carnitine- ⁇ -hydroxybutyrate being the following compound ( a mixture of Ia) and compound (Ib),
  • ratio of m to n is 1:4 to 4:1, preferably 1:1; more preferably, m and n are 1 in the compounds Ia and Ib.
  • a composition comprising one or more compounds A m B n and/or compound I and/or compound II.
  • the composition comprises one or more compounds A m B n and one or more compounds I; the composition comprising one or more compounds A m B n and one or more Compound II; the composition comprising one or more compounds A m B n and one or more compounds I and one or more compounds II;
  • M is Na, K or Li
  • the ratio of m to n is from 0.8:10 to 10:0.8, preferably from 1:4 to 4:1, more preferably both m and n are 1;
  • M is Ca or Mg
  • the ratio of m to n is from 0.8:10 to 10:0.8, preferably from 1:4 to 4:1, more preferably both m and n are 1.
  • compositions for the preparation of a medicament for the promotion of ketosis in a mammal, preferably a L-carnitine, and at least one of the compositions
  • the ⁇ -hydroxybutyric acid compound is used as an active ingredient, or the compound A m B n and/or the compound I and/or the compound II are used as an active ingredient.
  • composition provided by the present invention may also be used for the preparation of a slimming or health food, a diet/nutrition supplement, a veterinary product or a feed, preferably, the composition has L-carnitine, and at least one ⁇ -hydroxyl group.
  • a butyric acid compound is used as an active ingredient, or a compound A m B n and/or a compound I and/or a compound II as an active ingredient.
  • compositions provided by the present invention are also advantageous for treating hyperglycemia or type II diabetes and improving the general health of the user in a short period of time. It can also be used as a brain-enhancing medicine to improve athlete performance, to prevent diseases associated with metabolic disorders, mitochondrial defects and insulin resistance, as an adjunct to the ketogenic diet, as an anti-aging supplement, and for Other uses associated with increased metabolic health.
  • composition of the present invention When the above composition of the present invention is applied, it can be formulated into an oral dosage form including: a tablet, a capsule, a granule, a pill, and an oral solution.
  • the composition further comprises one or more pharmaceutically acceptable or edible excipients, which are formed into a powder mixture, as a ready-to-drink liquid, into hard or soft capsules, tablets, concentrated gels or in the field. Any other suitable dosage form known to the skilled person.
  • compositions described herein and optional pharmacologically acceptable excipients can be combined.
  • the composition is equivalent to 20-2000 mg/day, or 50 mg to 1000 mg, or 40-800 mg/day, per unit weight of L-carnitine.
  • the composition comprises, by weight, L-carnitine in a unit dose equivalent to 50-3000 mg/kg, or 100-1000 mg/kg; as a drug for promoting ketosis, the composition is by weight
  • the ⁇ -hydroxybutyric acid in a unit dose corresponds to 1 g to 50 g, or 2 g to 30 g, or 2 g to 10 g, or 5 g to 8 g.
  • the frequency of use and the specific amount of the preferred preparation may vary depending on various factors such as the degree of susceptibility of the individual, the age, sex and weight of the individual, the specific reaction of the individual, etc., and optimization of these factors can be determined by those skilled in the art as needed.
  • Another aspect of the present invention provides a method for preparing the compound A m B n , the compound I, and the compound II in the composition;
  • a m B n can be prepared by the following method, wherein m and n have a value of 1:4 to 4:1, preferably 1:1.
  • L-carnitine preferably in a molar ratio of 1 to 1.2:1
  • an equimolar amount of a base such as sodium hydroxide or potassium hydroxide is slowly added dropwise, and then the prepared L-carnitine solution is added to the above-mentioned organic compound of the ⁇ -hydroxybutyric acid amino acid salt.
  • the reaction is carried out in a solvent.
  • the organic solvent is preferably acetone; the base used is selected according to the corresponding metal in the compound I or the compound II. For example, when M in the compound I is Na, the base used is preferably sodium hydroxide.
  • the present invention provides L-carnitine; and at least one ⁇ -hydroxybutyric acid compound comprising ⁇ -hydroxybutyrate, ⁇ -hydroxybutyrate precursor or a combination thereof
  • the composition is capable of significantly promoting fat metabolism, and synergistically promotes a fat loss effect by significantly increasing blood ketone and lowering blood sugar level, and the use of the composition does not adversely affect the lipid mass spectrum, by using the composition,
  • the rapid initiation of ketosis and the rate at which ketone adaptation is accelerated is beneficial to avoid the symptoms of glucose withdrawal that are routinely experienced by individuals who initiate a ketogenic diet. It is more easily accepted by the human body than the existing L-carnitine salt, and is more likely to participate in physiological metabolism of the human body, and has stronger nutrition and therapeutic effects.
  • the human body takes these compositions containing L-carnitine and ⁇ -hydroxybutyric acid compounds, the compound A m B n , the ⁇ -hydroxybutyric acid precursor or the ⁇ -hydroxybutyrate in the compound I and the compound II structure. Part of it will not accumulate in the human body, but can be used as a ketone supplement to enable patients to quickly establish ketosis and maintain ketosis with little or no perception of the patient's physical and psychological comfort; The ketone body also did not find a decrease in high-density lipoprotein (HDL) levels.
  • HDL high-density lipoprotein
  • the L-carnitine part promotes the decomposition of the fat while promoting the ketone body supplement (such as ⁇ -hydroxybutyric acid compound, ⁇ -hydroxybutyrate or Oxidation of ⁇ -hydroxybutyric acid precursors, thereby promoting the utilization and elimination of these ketone bodies, reducing metabolic toxicity caused by acyl accumulation during ketone body utilization; in particular, A m B n , Compound I
  • the values of m and n are 1:4 to 4:1, especially when m and n are simultaneously 1, the ketogenic and weight loss effects are the best;
  • L-carnitine and ⁇ -hydroxybutyric acid compound are present in the form of intramolecular salt compounds A m B n , Compound I and Compound II, which are more stable in nature, are not hygroscopic, and are bioavailable in vivo. Higher degrees.
  • composition of the present invention comprises the compound A m B n and when B is a ⁇ -hydroxybutyric acid amino acid (such as ⁇ -hydroxybutyrate arginine, ⁇ -hydroxybutyrate lysine, ⁇ - when using hydroxybutyrate histidine, beta-hydroxybutyrate ornithine, beta-hydroxybutyrate sarcosine, beta-hydroxybutyrate decylamine or beta-hydroxybutyrate citrulline, etc.
  • ⁇ -hydroxybutyric acid amino acid such as ⁇ -hydroxybutyrate arginine, ⁇ -hydroxybutyrate lysine, ⁇ - when using hydroxybutyrate histidine, beta-hydroxybutyrate ornithine, beta-hydroxybutyrate sarcosine, beta-hydroxybutyrate decylamine or beta-hydroxybutyrate citrulline, etc.
  • ⁇ -hydroxybutyric acid amino acid such as ⁇ -hydroxybutyrate arginine, ⁇ -hydroxybut
  • composition comprising the compound A m B n and when B is the above-mentioned ⁇ -hydroxybutyric acid amino acid, on the other hand, also solves the possibility that the presence of ⁇ -hydroxybutyric acid in the form of a mineral salt may cause potential Mineral salt overload, such as sodium salt overload, the risk of cardiovascular effects of calcium salts.
  • composition provided by the invention has simple and reasonable production process, and reacts under normal pressure, mild reaction conditions, and is suitable for industrial large-scale production.
  • the " ⁇ -hydroxybutyric acid” and the “3-hydroxybutyrate” described in the present invention are the same compound and are mutually replaceable.
  • the "L-carnitine- ⁇ -hydroxybutyrate” refers to an internal salt formed by L-carnitine and a ⁇ -hydroxybutyric acid compound; and the “ ⁇ -hydroxybutyrate” refers to ⁇ -hydroxybutyric acid.
  • a metal salt such as a sodium salt, a potassium salt, a calcium salt, a magnesium salt, and a salt of ⁇ -hydroxybutyric acid and an amino acid;
  • the “ ⁇ -hydroxybutyric acid amino acid salt” refers to a ⁇ -hydroxybutyric acid and an amino acid. salt.
  • Example 3 Preparation of L-carnitine- ⁇ -hydroxybutyrate arginine (in which the molar ratio of L-carnitine to ⁇ -hydroxybutyrate arginine is 1:1):
  • Example 4 Preparation of L-carnitine- ⁇ -hydroxybutyrate lysine (in which the molar ratio of L-carnitine to ⁇ -hydroxybutyrate lysine is 1:1):
  • Example 5 Preparation of L-carnitine- ⁇ -hydroxybutyrate ornithine (in which the molar ratio of L-carnitine to ⁇ -hydroxybutyrate ornithine is 1:1):
  • Example 6 Preparation of L-carnitine- ⁇ -hydroxybutyrate amdecylamine (in which the molar ratio of L-carnitine to ⁇ -hydroxybutyrate is 2:1)
  • Example 8 Preparation of L-carnitine- ⁇ -hydroxybutyrate histidine (in which the molar ratio of L-carnitine to ⁇ -hydroxybutyrate histidine is 1:2)
  • Example 9 Study on weight loss effect, ketogenic effect and lipid mass spectrometry:
  • SPF male SD rats were purchased from Shanghai Slack Laboratory Animals Co., Ltd.
  • the experimental animals were kept in the SPF animal room.
  • the animal room was well ventilated and equipped with air conditioning.
  • the temperature was kept at 20-25 °C
  • the humidity was kept at 40%-70%
  • the air exchange times were 10-15 times/h
  • the light and dark lighting was 12 hours each.
  • Experimental animals were given free access to food and water, and each rat was labeled with an ear tag.
  • Eight blank rats were fed normal diet, and 48 model rats were fed a high-fat and high-cholesterol diet (Nantong Trophy Feed Technology Co., Ltd.) for 60 days to establish an obesity model.
  • mice were randomly divided into six groups: model group, model + exercise group, composition (compound prepared in Example 1 (Iaa)) + exercise group, physical mixing (L-carnitine and ⁇ - Sodium hydroxybutyrate molar ratio 1:1 mixed) + exercise group, L-carnitine + exercise group and ⁇ -hydroxybutyrate + exercise group, 8 / group.
  • the blank control group and the model group were only routinely reared, and there was no other intervention.
  • the rats in the exercise group were forced to exercise for half an hour every day. Rats in the blank control group, model group and model + exercise group were given normal saline, 10 mL/kg once a day for 4 weeks.
  • Composition (Compound (Iaa) prepared in Example 1) + exercise group, physical mixing (mixed L-carnitine with ⁇ -hydroxybutyrate molar ratio 1:1) + exercise group, L-carnitine + exercise group and ⁇ - The hydroxybutyrate + exercise group was intragastrically administered with 15 mmol/kg of the composition, physical mixture, L-carnitine and ⁇ -hydroxybutyric acid once a day for 4 weeks.
  • the body weight of the animals was measured four weeks after the administration, and the ketone body level was measured using a Yicheng T-1 blood ketone body tester 4 hours after the last administration, and serum triglyceride (TG) and cholesterol (TCHO) were determined by blood separation serum. , low density lipoprotein (LDL) and high density lipoprotein (HDL) levels.
  • Table 1 The experimental results are shown in Table 1 below.
  • L-carnitine- ⁇ -hydroxybutyrate arginine prepared in Example 3 L-carnitine- ⁇ -hydroxybutyrate ornithine prepared in Example 5 was substituted for the above compound (Iaa), and the corresponding L-carnitine was used.
  • L-carnitine- ⁇ -hydroxybutyrate arginine or L-carnitine- ⁇ -hydroxybutyrate ornithine + exercise group had the best weight loss effect, and the ketogenic effect was the most significant and increased to some extent.
  • HDL; L-carnitine and ⁇ -hydroxybutyrate (such as ⁇ -hydroxybutyrate ornithine, ⁇ -hydroxybutyrate arginine) + exercise group weight loss and ketone effect is also significantly higher than the use of left-handed meat alone In the base + exercise group or the ⁇ -hydroxybutyrate + exercise group, it was found that the ⁇ -hydroxybutyrate + exercise group reduced HDL to a certain extent.
  • SPF male SD rats were purchased from Shanghai Slack Laboratory Animals Co., Ltd.
  • the experimental animals were kept in the SPF animal room.
  • the animal room was well ventilated and equipped with air conditioning.
  • the temperature was kept at 20-25 °C, the humidity was kept at 40%-70%, the air exchange times were 10-15 times/h, and the light and dark lighting was 12 hours each.
  • Experimental animals were given free access to food and water, and each rat was labeled with an ear tag. Animals before the administration were fasted overnight, and were randomly divided into 4 groups and 3 groups/group according to body weight.
  • Three rats were given 1 mg/kg L-carnitine intravenously, 3 rats were given 5 mg/kg L-carnitine- ⁇ -hydroxybutyrate, and 3 rats were given 5 mg/kg L-carnitine and ⁇ -.
  • Physical mixture of hydroxybutyrate, 3 rats were intragastrically administered with 5 mg/kg L-carnitine, before the administration and 5, 15, 30, 1, 2, 4, 8, 12 and 24 h after the completion of the bolus, by the neck 0.2 mL of venous blood was collected and placed in a test tube containing K2-EDTA anticoagulant. The whole blood sample collected was placed on ice before centrifugation. The whole blood sample collected at the same time point was centrifuged within half an hour after the collection was completed.
  • L-carnitine- ⁇ -hydroxybutyrate prepared in Examples 3 to 8 of the present invention was physically mixed with L-carnitine and its corresponding ⁇ -hydroxybutyrate to carry out the above bioavailability study, and the experimental results showed similar to Table 2
  • the trend is that L-carnitine- ⁇ -hydroxybutyrate has the highest bioavailability, followed by a physical mixture, which is more bioavailable than L-carnitine alone.
  • the compound (Iab), the compound (IIaa), and the L-carnitine- ⁇ -hydroxybutyric acid prepared in Examples 3 to 8 were tested according to the Guiding Principles of Drug Hygroscopicity (Chinese Pharmacopoeia 2015 Edition, Part 4, 9103). Salt, and the corresponding physical mixture of L-carnitine and ⁇ -hydroxybutyrate, and L-carnitine-calcium fumarate, the hygroscopicity results are shown in Table 3.

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Abstract

一种包含左旋肉碱和至少一种β-羟基丁酸化合物的组合物，所述的β-羟基丁酸化合物包含β-羟基丁酸盐、β-羟基丁酸前体或其组合，优选所述组合物中左旋肉碱与β-羟基丁酸氨基酸以分子内盐的形式存在。一种左旋肉碱-β-羟基丁酸盐的制备方法。所述组合物具有减肥和生酮效果。

Description

一种包含左旋肉碱和β-羟基丁酸化合物的组合物 技术领域
本发明属于医药技术领域，具体的本发明涉及一种包含左旋肉碱和β-羟基丁酸化合物的组合物，本发明还涉及左旋肉碱-β-羟基丁酸盐在医药及食品添加剂的制备技术中的应用。
背景技术
肥胖已经成为一个严重的公众健康问题，近年来全世界肥胖症患者日益增多，每年因肥胖死亡的人数仅次于吸烟，国际肥胖症大会发布报告，全世界因肥胖症引起的有关疾病的死亡人数已超过同期全球饿死的人数。肥胖症还可诱发严重的糖尿病、心血管病等疾患，是人类健康长寿的大敌。
肥胖症的治疗药有：食欲抑制药、增加能量消耗的药物、抑制肠道消化吸收的药物、胰岛素增敏剂、生物肽类及其激动剂或抑制剂等。这些药物有较多的不良反应，例如心脏瓣膜损害、肺动脉高压和手指坏死、升高血压、胃肠道功能紊乱、呼吸道感染、头痛、月经失调、焦虑、易疲劳、肠胀气、腹痛、腹泻等，个别患者还可出现低血糖、肝毒性反应等。
左旋肉碱的主要生理功能是促进脂肪转化成能量，服用左旋肉碱能够在减少身体脂肪、降低体重的同时，不减少水分和肌肉，在2003年被国际肥胖健康组织认定为最安全无副作用的减肥营养补充品。左旋肉碱是脂肪代谢过程中的一种关键的物质，能够促进脂肪酸进入线粒体氧化分解。左旋肉碱是转运脂肪酸的载体。在长时间大强度运动中，左旋肉碱提高了脂肪的氧化速率，减少了糖原的消耗，同时也延缓了疲劳。很多减肥品都把左旋肉碱作为减肥的主要成分之一。
目前左旋肉碱已应用于医药、保健和食品等领域，并已被瑞士、法国、美国和世界卫生组织规定为法定的多用途营养剂。我国食品添加剂卫生标准GB2760－1996规定了左旋肉碱酒石酸盐为食品营养强化剂，可应用于咀嚼片、饮液、胶囊、乳粉及乳饮料等。此外，在医药方面的疗效包括心血管疾病，肝脏疾病，肾脏疾病，高血脂症，糖尿病，神经肌肉疾病等均可通过服用左旋肉碱及其系列产品而使病症得到改善。
还已知左旋肉碱的药学可接受盐和其内盐一样具有同样的治疗或营养作用。且这些药学可接受盐能够改善内盐的稳定性及易吸潮性，如现在市场上普遍使用的左旋肉碱酒石酸盐(US4602039)和左旋富马酸盐(US2009281183A1)，左旋肉碱粘酸盐(US5952379)。也有报道将左旋肉碱与金属离子如钙、镁离子结合，形成具有良好水溶性，易于被人体吸收的盐，例如US6051608公开了左旋富马酸镁和烷酰基左旋肉碱富马酸镁，WO 98/45250公开了左旋肉碱酒石酸镁和烷酰基L-肉碱柠檬酸镁；也有 左旋肉碱半乳糖二酸钙和左旋肉碱富马酸钙(WO2008080287)的专利报道。
但是现有的这些左旋肉碱药学可接受盐仍存在一些问题和缺陷，如左旋肉碱酒石酸盐仍有较大的吸潮性，相对湿度超过60％会潮解；而左旋肉碱与金属离子如钙、镁离子结合形成的盐，也存在一些缺陷，如，大量摄取乳酸钙会引起疲劳；葡萄糖酸钙、氯化钙过多对糖尿病不利；另外，左旋肉碱半乳糖二酸钙在制备过程中还存在原料供应不足的风险，并且成本高，因为乳糖二酸在国内没有大批量生产，价格较昂贵；左旋肉碱富马酸钙(CN101209975)的制备过程中需要冷冻，这在大生产过程中操作不便，增加了生产成本。
也有研究表明，通过生酮饮食追求减肥，在可能导致损失脂肪存储同时维持和保护肌肉质量；一些研究也已经表明，生酮饮食的肌肉保护性质导致身体性能的提高。
营养性或治疗性酮症是由生酮饮食、卡路里限制、治疗性禁食和/或补充生酮前体导致血酮水平升高(通常高于0.5mmol/L)的生理状态。酮体代表外周组织和中枢神经系统的替代能量底物，β-羟基丁酸和乙酰乙酸是两种最丰富和生理显著的酮体。在禁食、极限运动和/或低碳水化合物消耗期间，身体中的葡萄糖储存被快速使用并变得快速耗尽。没能在它们变得耗尽时补充葡萄糖储存造成身体求助于替代方法以通过制造酮体来生产能量。酮体可被身体的每一个细胞用作代替燃料以满足身体的能量需求，包括大脑的需求。在延迟的禁食期间，例如血酮水平将增加至高达2mmol/L或3mmol/L。常规上理解并同意的是，当血酮升高超过0.5mmol/L时，心脏、大脑和外周组织使用酮体(β-羟基丁酸和乙酰乙酸)作为主要燃料源，这一状态被称作酮症或“营养性酮症”。当在酮症中时，身体基本上燃烧脂肪，因此一旦在酮症中，人们可以通过降低饮食性脂肪摄入和将碳水化物摄取调节至足够低以维持酮症来诱发体脂损失。
虽然通过生酮饮食追求减重，具有诸多优势，然后，在追求和保持生酮的生活方式时，面临显著障碍，如，难以过渡到生酮状态，大部分人受到难以进入酮症的能力限制；损耗身体中葡萄糖储存的最快方式是通过禁食与运动结合，而对从吃蛋糕、糖果、面包等获取快感的人来讲，这种饮食调整，这在身体和情感上是苛刻的，并且具有极大的挑战；在进入酮症的过渡时会有昏睡和头晕目眩等不舒服的生理和精神状态。
虽然有报道，生酮医疗食品或外源补充酮可以有助于过渡到酮症，但生酮脂肪如中链甘油三酯(MCT油)通常不被肠胃系统以有助于诱发酮症的必要量很好的耐受；而β-羟基丁酸(βHB)和乙酰乙酸以其游离酸形式口服在制造持久的酮症时是昂贵和无效的。使用钠盐缓冲βHB的游离酸形式，但这在酮症的治疗水平造成潜在有害的钠过载和矿物质平衡失调。也已经提出生酮饮食会增加总胆固醇和甘油三酸酯同时降低高密度脂蛋白(HDL)水平的担忧，从而引发心血管病变。
发明内容
本发明的目的就在于克服现有技术存在的上述不足，提供新的组合物，包含左旋肉碱；和至少一种β-羟基丁酸化合物，所述的β-羟基丁酸化合物包含β-羟基丁酸盐、β-羟基丁酸前体或其组合。
本发明的另一方面还提供了一种组合物，包含左旋肉碱与β-羟基丁酸化合物以分子内盐的形式存在。
本发明第三方面提供了所述组合物的制备方法和所述组合物的用途。
首选，本发明提供了一种组合物，其包含左旋肉碱；和至少一种β-羟基丁酸化合物，所述的β-羟基丁酸化合物包含β-羟基丁酸盐、β-羟基丁酸前体或其组合。
其中，所述至少一种β-羟基丁酸化合物包括如下中的一种或多种：β-羟基丁酸钠、β-羟基丁酸钾、β-羟基丁酸钙、β-羟基丁酸镁、β-羟基丁酸锂、或其混合物；
β-羟基丁酸精氨酸、β-羟基丁酸赖氨酸、β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸；
1,3-丁二醇、乙酰乙酸乙酯或β-羟基丁酸乙酯；或，
β-羟基丁酸盐与1,3-丁二醇、乙酰乙酸乙酯或β-羟基丁酸乙酯的组合；或，
β-羟基丁酸盐混合物与1,3-丁二醇、乙酰乙酸乙酯或β-羟基丁酸乙酯的组合。
进一步优选的，所述至少一种β-羟基丁酸化合物包括
(1)β-羟基丁酸钠、β-羟基丁酸精氨酸或其组合；或
(2)β-羟基丁酸钠盐与β-羟基丁酸钾盐的组合；或
(3)β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸。
其中，所述的至少一种β-羟基丁酸化合物是外消旋的DL-β-羟基丁酸或单个的同分异构体R-β-羟基丁酸。
优选，上述所述组合物中，β-羟基丁酸化合物与左旋肉碱的摩尔比为20:1～1:1。
另外，本发明还提供了一种组合物，其包含化合物A mB n，其中A为左旋肉碱，B为β-羟基丁酸精氨酸、β-羟基丁酸赖氨酸、β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸；m，n独立的选自1～4的整数；优选的B为β-羟基丁酸精氨酸、β-羟基丁酸赖氨酸、β-羟基丁酸组氨酸，或β-羟基丁酸鸟氨酸，并且，优选的m为1或2，n为2或1。
在本发明的另一优选技术方案中，提供了一种组合物，其包含左旋肉碱-β-羟基丁酸盐，并且，所述左旋肉碱与β-羟基丁酸化合物以分子内盐的形式存在，具有如下结构：
Figure PCTCN2018100836-appb-000001
其中M为Na,K或Li，m与n的取值比为0.8:10～10:0.8，优选1:4～4:1，更优选1:1，最优选m与n均为1，或者，
Figure PCTCN2018100836-appb-000002
其中M为Ca或Mg，m与n的取值比为0.8:10～10:0.8，优选1:4～4:1，更优选1:1，最优选m与n均为1。
进一步的，优选所述的组合物，包含左旋肉碱-β-羟基丁酸盐，所述左旋肉碱-β-羟基丁酸盐是下列一种或多种的组合：
Figure PCTCN2018100836-appb-000003
Figure PCTCN2018100836-appb-000004
其中，m与n的取值比为0.8:10～10:0.8，优选1:4～4:1，更优选1:1，最优选m与n均为1。
在本发明的另一个优选实施方式中，提供了一种组合物，所述组合物包含左旋肉碱-β-羟基丁酸盐，所述左旋肉碱-β-羟基丁酸盐是下列化合物(Ia)和化合物(Ib)的混合物，
Figure PCTCN2018100836-appb-000005
Figure PCTCN2018100836-appb-000006
其中m与n的取值比1:4～4:1，优选1:1；更优选所述化合物Ia和Ib中m，n均为1。
在本发明的另一个优选技术方案中，提供了一种组合物，包含一种或多种化合物A mB n和/或化合物I和/或化合物II。优选地，例如，所述组合物包含一种或多种化合物A mB n和一种或多种化合物I；所述组合物包含一种或多种化合物A mB n和一种或多种化合物II；所述组合物包含一种或多种化合物A mB n和一种或多种化合物I和一种或多种化合物II；
其中化合物I为：
Figure PCTCN2018100836-appb-000007
其中M为Na,K或Li，m与n的取值比为0.8:10～10:0.8，优选1:4～4:1，更优选m与n均为1；
化合物II为：
Figure PCTCN2018100836-appb-000008
其中M为Ca或Mg，m与n的取值比为0.8:10～10:0.8，优选1:4～4:1，更优选m与n均为1。
在本发明的第二方面还提供了上述所述组合物用于制备在哺乳动物中促进酮症的药物//营养增补剂的用途，优选，所述组合物中左旋肉碱，和至少一种β-羟基丁酸化合物作为活性成分，或化合物A mB n和/或化合物I和/或化合物II作为活性成分。
本发明提供的上述组合物，还可以用于制备减肥药物或保健食品、饮食/营养增补剂、兽医产品或饲料的用途，优选，所述组合物中左旋肉碱，和至少一种β-羟基丁酸化合物作为活性成分，或化合物A mB n和/或化合物I和/或化合物II作为活性成分。
初步研究发现，本发明提供的组合物，还有利于治疗高血糖或II型糖尿病，并且在短时间内改进使用者的一般健康。也可以用作补脑药，用于提高运动员性能，帮助预防与代谢功能紊乱、线粒体缺陷和抗胰岛素性相关的疾病，用作生酮饮食的辅助剂，用作抗老化补充物，和用于与提高的代谢健康相关的其他用途。
在应用本发明所述的上述组合物时，可将其制成口服剂型，包括：片剂、胶囊剂、颗粒剂、丸剂、口服液。所述组合物，还包含一种或多种可药用或可食用的辅料，将其做成粉末混合物，作为即饮液体，做成硬或软的胶囊、片剂、浓缩凝胶或本领域技术人员已知的任何其他合适的剂型。
在应用时，可将本发明所述的组合物和任选的药理学上可以接受的赋形剂进行组合。根据初步测算，作为减肥药物或保健食品，所述组合物以重量计，单位剂量中左旋肉碱相当于20-2000mg/天，或者50mg～1000mg，或者40-800mg/天。作为饮食/营养增补剂，所述组合物以重量计，单位剂量中左旋肉碱相当于50-3000mg/kg，或者100-1000mg/kg；作为促进酮症的药物，所述组合物以重量计， 单位剂量中β-羟基丁酸相当于1g～50g，或者2g～30g，或者2g～10g，或者5g～8g。优选制剂的使用频率和具体用量可根据种种因素而变化，如个体的易感程度，个体年龄、性别和体重，个体的特异反应等，这些因素的优化是本领域技术人员根据需要可以确定的。本发明另一方面还提供了所述组合物中化合物A mB n、化合物I、化合物II的制备方法；
A mB n可采用如下方法制备，其中m与n取值为1:4～4:1，优选1:1。
以有机溶剂如丙酮，异丙醇等为溶剂加入少量水，将β-羟基丁酸氨基酸盐与左旋肉碱(优选按照1～1.2:1的摩尔比)投料进行反应，更优选，先将左旋肉碱溶解在少量纯水中，然后再慢慢滴加等摩尔的碱如氢氧化钠或氢氧化钾，随后将该配好的左旋肉碱溶液加入到上述β-羟基丁酸氨基酸盐的有机溶剂中进行反应。反应结束后，反应液在40℃的水浴中减压除去溶剂，往烧瓶中加入有机溶剂如丙酮，异丙醇等，搅拌，析出固体，过滤，真空干燥得到产品。
化合物I和化合物II可采用如下方法制备：
将3-羟基丁酸溶于有机溶剂，如丙酮、异丙醇或二氯甲烷中，得到3-羟基丁酸的溶液；另将左旋肉碱溶解在纯水中，往该溶液里加入碱(如氢氧化钠、氢氧化钾、氢氧化锂、氢氧化钙或氢氧化镁)的水溶液。将配好的左旋肉碱溶液缓慢的滴入3-羟基丁酸的溶液中，然后将混合的反应液在35℃～45℃，优选40℃的水浴中减压除去溶剂，再往残留物中加入对应的有机溶剂(如丙酮、异丙醇或二氯甲烷)，搅拌析晶，过滤，滤饼用有机溶剂(如丙酮、异丙醇或二氯甲烷)洗涤后真空干燥得对应的化合物I、化合物II。其中所述有机溶剂优选为丙酮；所用碱的依据化合物I、化合物II中对应的金属选择，例如当化合物I中的M为Na时，优选所用碱为氢氧化钠。本发明所述提供的包含左旋肉碱；和至少一种β-羟基丁酸化合物，所述的β-羟基丁酸化合物包含β-羟基丁酸盐、β-羟基丁酸前体或其组合的组合物，能够显著促进脂肪代谢，并通过显著的升高血酮和降低血糖水平达到协同促进减脂效果，并且所述组合物的使用没有不利的影响脂质谱，通过使用所述的组合物，快速发起酮症和加快酮适应的速率，有利于避免常规由发起生酮饮食的个体所经历的葡萄糖撤回症状。比现有的左旋肉碱盐更容易为人体接受，更易参与人体生理代谢，并具有更强的营养及治疗作用。
本发明提供的包含一种或多种化合物A mB n和/或化合物I和/或化合物II的组合物中这些以分子内盐形式的化合物具有更加显著的优势：
首选，当人体服用这些包含左旋肉碱和β-羟基丁酸化合物的组合物时，化合物A mB n，化合物I和化合物II结构中的β-羟基丁酸前体或β-羟基丁酸盐部分不会在人体蓄积，而是可作为酮补充物，使患者快速建立酮症，并对患者的生理和心理舒适有较小或没有感知的情况下维持酮症；而以这种形 式补充的酮体也并没有发现高密度脂蛋白(HDL)水平降低的现象。
其次，A mB n、化合物I，化合物II进入体内后，其中的左旋肉碱部分在促进脂肪分解的同时，促进酮体补充物(如β-羟基丁酸化合物，β-羟基丁酸化盐或β-羟基丁酸前体)的氧化，从而在这些酮体的利用和消除中起到促进作用，减少酮体利用过程中因酰基积累而造成的代谢毒性；特别是A mB n、化合物I，化合物II中m与n的取值为1:4～4:1，尤其是m，n同时为1时，生酮和减肥效果最佳；
第三，所述药物组合物中，左旋肉碱与β-羟基丁酸化合物以分子内盐化合物A mB n，化合物I和化合物II形式存在，性质更加稳定，不易吸湿，在体内的生物利用度更高。
第四，当本发明所述的组合物，包含化合物A mB n，并且当B为β-羟基丁酸氨基酸(如β-羟基丁酸精氨酸、β-羟基丁酸赖氨酸、β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸等)时，在使用时，同时增加了人体内氨基酸的补充，这对需要减肥或生酮的使用者来说是非常有益的。
第五，所述的组合物，包含化合物A mB n，并且当B为上述β-羟基丁酸氨基酸时，在另一方面也解决了以矿物盐形式存在的β-羟基丁酸可能造成潜在的矿物盐过载，如钠盐超载，钙盐对心血管影响的风险。
第六，本发明提供的组合物，生产工艺简单、合理，常压下反应，反应条件温和，适合工业化大生产。
具体实施方式
以下结合具体实施例对本发明的技术方案的优点做具体阐释，应当理解，这些实施例仅为示例性的，并非意在限制本发明。
本发明所述的“β-羟基丁酸”与“3-羟基丁酸盐”为同一化合物，可相互替换。所述的“左旋肉碱-β-羟基丁酸盐”是指左旋肉碱与β-羟基丁酸化合物形成的内盐；所述“β-羟基丁酸盐”是指β-羟基丁酸的金属盐如钠盐，钾盐，钙盐，镁盐，以及β-羟基丁酸与氨基酸形成的盐；所述的“β-羟基丁酸氨基酸盐”是指β-羟基丁酸与氨基酸形成的盐。
实施例1、化合物(Iaa)的制备方法：
Figure PCTCN2018100836-appb-000009
向100mL的圆底烧瓶中加入1.04g(10mmol)3-羟基丁酸、8mL丙酮。取1.61g(10mmol)左旋肉碱溶解在3mL纯水中，往该溶液里加入1mL 10N(10mmol)的氢氧化钠水溶液。将配好的左旋肉碱溶液缓慢的滴入反应瓶中，滴毕后反应5分钟，将反应液在40℃的水浴中减压除去溶剂，得到无色油状物，往烧瓶中加入80mL丙酮，搅拌析晶，过滤，滤饼用丙酮洗涤后真空干燥得2.58g白色固体，收率90％。
Chemical Formula:C 11H 22NO 6Na；1HNMR(D 2O):δ1.12(3H,d),2.12～2.41(4H,m),3.14(9H,s),3.34(2H,dd),4.04～4.09(1H,m),4.47～4.50(1H,m)。
实施例2：下式化合物(IIaa)的制备
Figure PCTCN2018100836-appb-000010
向100mL的圆底烧瓶中加入1.04g(10mmol)3-羟基丁酸、16mL丙酮。取1.61g(10mmol)左旋肉碱溶解在16mL纯水中，往该溶液里加入0.37g(5mmol)氢氧化钙粉末，搅拌加热至80℃2小时后，将此悬浊的左旋肉碱溶液滴入反应瓶中，加热至60℃搅拌2小时后溶清，将反应液在40℃的水浴中减压除去溶剂，得乳白色油状物，往烧瓶中加入80mL丙酮，搅拌析晶，过滤，滤饼用丙酮洗后真空干燥得1.76g白色产品，收率86％。
Chemical Formula:C 22H 44N 2O 12Ca； 1HNMR(D 2O):δ1.13(3H,d),2.11～2.40(8H,m),3.15(18H,s),3.36(4H,dd),4.05～4.13(2H,m),4.47～4.51(2H,m)。
实施例3：左旋肉碱-β-羟基丁酸精氨酸(其中左旋肉碱与β-羟基丁酸精氨酸的摩尔比为1:1)的制备：
往100mL的圆底烧瓶中加入2.78g(11mmol)β-羟基丁酸精氨酸，加入10mL丙酮和3mL纯化水。取1.61g(10mmol)左旋肉碱溶解在3mL纯水中，往该溶液里加入1mL 10N(10mmol)的氢氧化钠溶液。将配好的左旋肉碱溶液缓慢的滴入反应瓶中，滴完后，反应5分钟，反应液在40℃的水浴 中减压除去溶剂，得到无色油状物，往烧瓶中加入80mL丙酮，搅拌20分钟，析出白色固体，过滤，滤饼用丙酮洗后真空干燥得3.92g白色产品，收率89％。
实施例4：左旋肉碱-β-羟基丁酸赖氨酸(其中左旋肉碱与β-羟基丁酸赖氨酸的摩尔比为1:1)的制备：
往100mL的圆底烧瓶中加入11mmolβ-羟基丁酸赖氨酸，加入10mL丙酮和3mL纯化水。取10mmol左旋肉碱溶解在3mL纯水中，往该溶液里加入1mL 10N(10mmol)的氢氧化钠溶液。将配好的左旋肉碱溶液缓慢的滴入反应瓶中，滴完后，反应5分钟，反应液在40℃的水浴中减压除去溶剂，得到无色油状物，往烧瓶中加入80mL丙酮，搅拌20分钟，析出白色固体，过滤，滤饼用丙酮洗后真空干燥得3.92g白色产品，收率87％。
实施例5：左旋肉碱-β-羟基丁酸鸟氨酸(其中左旋肉碱与β-羟基丁酸鸟氨酸的摩尔比为1:1)的制备：
往100mL的圆底烧瓶中加入11mmolβ-羟基丁酸鸟氨酸，加入10mL丙酮和3mL纯化水。取10mmol左旋肉碱溶解在3mL纯水中，往该溶液里加入1mL 10N(10mmol)的氢氧化钠溶液。将配好的左旋肉碱溶液缓慢的滴入反应瓶中，滴完后，反应5分钟，反应液在40℃的水浴中减压除去溶剂，得到无色油状物，往烧瓶中加入80mL丙酮，搅拌20分钟，析出白色固体，过滤，滤饼用丙酮洗后真空干燥得3.92g白色产品，收率83％。
实施例6：左旋肉碱-β-羟基丁酸胍基丁胺(其中左旋肉碱与β-羟基丁酸胍基丁胺的摩尔比为2:1)的制备
往100mL的圆底烧瓶中加入10.5mmolβ-羟基丁酸胍基丁胺，加入10mL丙酮和3mL纯化水。取10mmol左旋肉碱溶解在3mL纯水中，往该溶液里加入1mL 10N(10mmol)的氢氧化钠溶液。将配好的左旋肉碱溶液缓慢的滴入反应瓶中，滴完后，反应5分钟，反应液在40℃的水浴中减压除去溶剂，往烧瓶中加入80mL丙酮，搅拌20分钟，析出白色固体，过滤，滤饼用丙酮洗后真空干燥得3.92g白色产品，收率80％。
实施例8：左旋肉碱-β-羟基丁酸组氨酸(其中左旋肉碱与β-羟基丁酸组氨酸的摩尔比为1:2)的制备
往100mL的圆底烧瓶中加入11mmolβ-羟基丁酸组氨酸，加入10mL丙酮和3mL纯化水。取10mmol左旋肉碱溶解在3mL纯水中，往该溶液里加入1mL 10N(10mmol)的氢氧化钠溶液。将配好的左旋肉碱溶液缓慢的滴入反应瓶中，滴完后，反应5分钟，反应液在40℃的水浴中减压除去溶剂，往烧瓶中加入80mL丙酮，搅拌20分钟，析出白色固体，过滤，滤饼用丙酮洗后真空干燥得3.92 g白色产品，收率85％。
实施例9：减肥效果、生酮效果及脂质谱影响研究：
56只SPF级雄性SD大鼠，购自上海斯莱克实验动物有限责任公司。实验动物饲养在SPF动物房内，动物房通风良好，装备空调，温度保持在20～25℃，湿度保持在40％～70％，换气次数10～15次/h，明暗照明各12小时，实验动物自由进食和饮水，每只大鼠均用耳标标记。8只空白组大鼠饲喂正常饲料，48只造模组大鼠饲喂高脂高胆固醇饲料(南通特洛菲饲料科技有限公司)60天建立肥胖模型。按照体重将造模成功48只大鼠随机分为六组：模型组、模型+运动组、组合物(实施例1制备的化合物(Iaa))+运动组、物理混合(左旋肉碱与β-羟基丁酸钠摩尔比1:1混合)+运动组，左旋肉碱+运动组和β-羟基丁酸+运动组，8只/组。空白对照组和模型组仅为常规饲养，无其它干预，运动组大鼠每天强迫运动半小时。空白对照组、模型组和模型+运动组大鼠灌胃给予生理盐水，10mL/kg，每天一次，连续4周。组合物(实施例1制备的化合物(Iaa))+运动组、物理混合(左旋肉碱与β-羟基丁酸钠摩尔比1:1混合)+运动组、左旋肉碱+运动组和β-羟基丁酸+运动组分别灌胃给予15mmol/kg组合物、物理混合物、左旋肉碱和β-羟基丁酸，每天一次，连续4周。给药四周后测量动物体重，于最后一次给药后4小时使用怡成T-1型血酮体测试仪测量酮体水平，取血分离血清测定血清甘油三酯(TG)、胆固醇(TCHO)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)水平。实验结果如下表1所示。
表1：减肥效果、生酮效果及脂质谱影响研究结果：
Figure PCTCN2018100836-appb-000011
分别将实施例3制备的左旋肉碱-β-羟基丁酸精氨酸，实施例5制备的左旋肉碱-β-羟基丁酸鸟氨酸替代上述化合物(Iaa)，对应的将左旋肉碱与β-羟基丁酸精氨酸摩尔比1:1物理混合物，左旋肉碱 与β-羟基丁酸鸟氨酸摩尔比1:1物理混合物替代上述物理混合物，进行相同的操作，实验结果得到如上类似的趋势；其中左旋肉碱-β-羟基丁酸精氨酸或左旋肉碱-β-羟基丁酸鸟氨酸+运动组减肥效果最佳，并且生酮效果最显著，并且一定程度升高HDL；左旋肉碱与β-羟基丁酸盐(如β-羟基丁酸鸟氨酸，β-羟基丁酸精氨酸)+运动组的减肥效果和升酮效果也明显高于单独使用左旋肉碱+运动组或β-羟基丁酸+运动组，同时发现β-羟基丁酸+运动组反而在一定程度降低HDL。
实施例10：体内生物利用度研究
12只SPF级雄性SD大鼠，购自上海斯莱克实验动物有限责任公司。实验动物饲养在SPF动物房内，动物房通风良好，装备空调，温度保持在20～25℃，湿度保持在40％～70％，换气次数10～15次/h，明暗照明各12小时，实验动物自由进食和饮水，每只大鼠均用耳标标记。给药前动物过夜禁食后，按照体重随机分为4组，3只/组。3只大鼠静脉注射给予1mg/kg左旋肉碱，3只大鼠灌胃给予5mg/kg左旋肉碱-β-羟基丁盐，3只大鼠灌胃给予5mg/kg左旋肉碱和β-羟基丁酸盐物理混合物，3只大鼠灌胃给予5mg/kg左旋肉碱，于给药前及推注完成后5、15、30min、1、2、4、8、12和24h，由颈静脉采血0.2mL，置于含K2-EDTA抗凝剂试管中，采集的全血样品在离心前都置于冰上，同一时间点采集的全血样品需在采集完成半小时内离心完，低温离心(5500rpm)10min后分离血浆，保存在-70℃冰箱。使用LC-MS/MS分析测定大鼠血浆中左旋肉碱浓度，采用WinNonlin非房室模型计算药代动力学参数，并计算绝对生物利用度，结果见表2。
表2：生物利用度
Figure PCTCN2018100836-appb-000012
将本发明实施例3～8制备的左旋肉碱-β-羟基丁酸盐以左旋肉碱与其对应的β-羟基丁酸盐进行物理混合进行上述生物利用度研究，实验结果显示类似表2的趋势，其中左旋肉碱-β-羟基丁酸盐生物利用度最高，其次是物理混合物，均比单独使用左旋肉碱生物利用度高。
实施例11：稳定性研究
按《药物引湿性指导原则》(中国药典2015年版四部9103)进行试验，检测本发明提供的化合物(Iab)、化合物(IIaa)、实施例3～8制备的左旋肉碱-β-羟基丁酸盐、以及对应的左旋肉碱与β-羟基 丁酸盐物理混合物，及左旋肉碱-富马酸钙，引湿性结果见表3。
表3：引湿性结果
产品 引湿性
化合物(Iaa) 0.1％
化合物(IIaa) 0.2％
左旋肉碱-β-羟基丁酸盐 ＜0.3％
物理混合物 ＜0.5％
左旋肉碱-富马酸钙 0.8％

Claims (11)

  1. 一种组合物，其包含左旋肉碱，和至少一种β-羟基丁酸化合物，所述的β-羟基丁酸化合物包含β-羟基丁酸盐、β-羟基丁酸前体或其组合。
  2. 根据权利要求1所述组合物，其特征在于，所述至少一种β-羟基丁酸化合物包括如下中的一种或多种：β-羟基丁酸钠、β-羟基丁酸钾、β-羟基丁酸钙、β-羟基丁酸镁、β-羟基丁酸锂、或其混合物；β-羟基丁酸精氨酸、β-羟基丁酸赖氨酸、β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸；1,3-丁二醇、乙酰乙酸乙酯或β-羟基丁酸乙酯；或β-羟基丁酸盐与1,3-丁二醇、乙酰乙酸乙酯或β-羟基丁酸乙酯的组合；或β-羟基丁酸盐混合物与1,3-丁二醇、乙酰乙酸乙酯或β-羟基丁酸乙酯的组合。
  3. 根据权利要求1所述的组合物，其特征在于，所述至少一种β-羟基丁酸化合物包括
    (1)β-羟基丁酸钠、β-羟基丁酸精氨酸或其组合；或
    (2)β-羟基丁酸钠盐与β-羟基丁酸钾盐的组合；或
    (3)β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸。
  4. 根据权利要求1所述的组合物，其特征在于，所述的至少一种β-羟基丁酸化合物是外消旋的DL-β-羟基丁酸或单个的同分异构体R-β-羟基丁酸。
  5. 根据权利要求1～4任一项所述的组合物，其特征在于，所述组合物中β-羟基丁酸化合物与左旋肉碱的摩尔比为20:1～1:1，优选1:4～4:1。
  6. 一种组合物，其包含化合物A mB n，其中A为左旋肉碱，B为β-羟基丁酸精氨酸、β-羟基丁酸赖氨酸、β-羟基丁酸组氨酸、β-羟基丁酸鸟氨酸、β-羟基丁酸肌氨酸、β-羟基丁酸胍基丁胺或β-羟基丁酸瓜氨酸；m，与n的取值为1:4～4:1。
  7. 根据权利要求6所述的组合物，其特征在于，所述m，n均为1。
  8. 根据权利要求7所述的组合物，其特征在于，还包含下式化合物I或II：
    Figure PCTCN2018100836-appb-100001
    其中M为Na,K或Li，m与n的取值比为1:4～4:1，优选为1:1；或者，
    Figure PCTCN2018100836-appb-100002
    其中M为Ca或Mg，m与n的取值比为1:4～4:1，优选为1:1。
  9. 权利要求1～8任一项所述组合物用于制备在哺乳动物中促进酮症的药物//营养增补剂的用途。
  10. 权利要求1～8任一项所述组合物用于制备减肥药物或保健食品、饮食/营养增补剂、兽医产品或饲料的用途。
  11. 根据权利要求9～10任一项所述组合物的用途，其特征在于，所述组合物制成口服剂型，包括：片剂、胶囊剂、颗粒剂、丸剂、口服液。
PCT/CN2018/100836 2017-08-17 2018-08-16 一种包含左旋肉碱和β-羟基丁酸化合物的组合物 Ceased WO2019034112A1 (zh)

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