WO2016150378A1 - 一种含有水飞蓟宾、左旋肉碱的药物组合物 - Google Patents

一种含有水飞蓟宾、左旋肉碱的药物组合物 Download PDF

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WO2016150378A1
WO2016150378A1 PCT/CN2016/077037 CN2016077037W WO2016150378A1 WO 2016150378 A1 WO2016150378 A1 WO 2016150378A1 CN 2016077037 W CN2016077037 W CN 2016077037W WO 2016150378 A1 WO2016150378 A1 WO 2016150378A1
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Prior art keywords
preparation
silybin
pharmaceutical composition
carnitine
phospholipid
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PCT/CN2016/077037
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English (en)
French (fr)
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孙鹤
闫希军
吴迺峰
闫凯境
朱永宏
章顺楠
白晓林
马晓慧
何毅
李婷
李磊
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天士力制药集团股份有限公司
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Priority to CA2980225A priority Critical patent/CA2980225A1/en
Priority to AU2016236607A priority patent/AU2016236607B2/en
Priority to DK16767755.8T priority patent/DK3275455T3/da
Priority to KR1020177030490A priority patent/KR102583409B1/ko
Priority to EP16767755.8A priority patent/EP3275455B1/en
Priority to JP2017548886A priority patent/JP6794366B2/ja
Priority to US15/560,560 priority patent/US10307395B2/en
Priority to RU2017136979A priority patent/RU2699011C2/ru
Publication of WO2016150378A1 publication Critical patent/WO2016150378A1/zh

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/221—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having an amino group, e.g. acetylcholine, acetylcarnitine
    • A—HUMAN NECESSITIES
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    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
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    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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    • A61K9/08—Solutions
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    • 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
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    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
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    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions

  • the invention relates to the field of medicine, in particular to a pharmaceutical composition of an aqueous silybin for treating liver diseases.
  • liver damage caused by various poisons such as carbon tetrachloride, thioacetamide, muscarinic, phalloidin and porcine. It can be used for acute and chronic hepatitis, early cirrhosis, fatty liver, toxic or drug-induced liver disease.
  • the phospholipid complex is a relatively special solid dispersion, which has a fixed melting point and is a molecular compound (complex) which is chemically stable and different from drugs and phospholipids.
  • the class of compounds differs depending on the type of phospholipid and the ratio of the phospholipid of the drug, and one phospholipid molecule can bind to a different number of drug molecules.
  • the spectral characteristics of the complex are inferred that the drug interacts strongly with the polar group of the phospholipid, inhibiting the free rotation of the single chain in the molecule, while the two long fatty acid chains of the phospholipid do not participate in the complex reaction and can move freely.
  • the polar portion encapsulating the phospholipid forms a lipophilic surface, rendering the complex exhibits strong fat solubility. It changes the physicochemical properties of the drug, increases the fat solubility of the drug, and reduces the water solubility. Promote the binding of drug molecules to the cell membrane to promote absorption and improve the bioavailability of the drug.
  • Pu'er tea is a famous local tea in Yunnan. The climate of the producing area is mild, the rainfall is abundant, and the clouds are haunted. Pu'er tea. It is a series of large-leaf sun-dried tea from Yunnan origin and its re-processing: it is directly reprocessed into the finished product of Shengpu and matured after being artificially fermented and fermented. And pressing the tea two types; after the finished product, it continues to carry out the natural aging process, with the unique quality of the more Chen Yuexiang.
  • Pu'er tea is the only post-fermentation type of tea. Its harmful substances such as theophylline and tea polyphenols are differentiated in the long-term fermentation process. Therefore, the quality is mild, it is not irritating to the human body, and it can also promote metabolism. Accelerate the digestion and transformation of fat and toxins in the body, and now it is a problem that alleviates urban obesity and high-fat, Pu'er tea can play a good role in relieving, such as detoxification, stomach, anti-inflammatory, lowering cholesterol, and eliminating fat. Greasy, beauty to lose weight. Modern technology shows that Pu'er tea set The effects of improving insulin resistance, regulating blood lipids and leptin levels can block the body's parenchymal cell fat accumulation caused by insulin resistance.
  • Non-alcoholic fatty liver disease is a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility. Its pathological changes are similar to alcoholic fatty liver disease. It is a clinicopathic syndrome in which the main body of the lesion is characterized by hepatic steatosis and fat accumulation in the hepatic lobules but has no history of excessive drinking. NAFLD shows different degrees of liver lesions, from simple fatty liver without any inflammation to significant fibers. 3. The severe inflammatory response of liver cirrhosis mainly includes simple fatty liver, steatohepatitis and fatty liver cirrhosis.
  • Weight Loss All patients with overweight, visceral obesity, and short-term weight gain in NAFLD need to change their lifestyle to control weight and reduce waist circumference.
  • Basic treatment 6 months weight loss monthly ⁇ 0.45kg, or body mass index (BMI)>27kg/m2 with blood lipids, blood sugar, blood pressure and other two abnormal indicators, consider adding sibutramine or orlistat
  • BMI body mass index
  • the weekly weight loss should not exceed 1.2Kg (children do not exceed 0.5Kg per week); BMI>40kg/m2 or BMI>35kg/m2 combined with sleep apnea syndrome and other obesity-related diseases, consider the proximal stomach Bypass surgery to lose weight (II-1, II-2, II-3, III).
  • Insulin sensitizer combined with type 2 diabetes, impaired glucose tolerance, fasting blood glucose, and visceral obesity, metformin and thiazolidinediones may be considered to improve insulin resistance and control blood glucose (II-1, II) -2, II-3).
  • hypolipidemic drugs dyslipidemia after basic treatment and / or application of weight loss hypoglycemic drugs for more than 3-6 months, still mixed hyperlipidemia or hyperlipidemia combined with more than two risk factors, need to consider Add lipid-lowering drugs (II-1, II-2, II-3) such as fibrates, statins or probucol.
  • liver disease NAFLD with abnormal liver function, metabolic syndrome, 3-6 months after basic treatment is still ineffective, and liver biopsy confirmed to be NASH and the course of chronic progressive progression, liver disease can be used
  • Drug-assisted treatment, anti-oxidation, anti-inflammatory, anti-fibrosis can choose polyene phospholipid choline according to drug performance, disease activity and disease period, Vitamin E, silymarin and ursodeoxycholic acid (II-1, II-2, II-3, III), but it is not advisable to use multiple drugs at the same time.
  • Liver transplantation It is mainly used for the treatment of NASH-related end-stage liver disease and partial hidden source J cirrhosis with liver function decompensation. The metabolic status should be screened before liver transplantation (III). BMI>40kg/m2 is a contraindication for liver transplantation (III).
  • the present invention provides a pharmaceutical composition and a preparation thereof, which are effective for nonalcoholic fatty liver.
  • the present invention provides a process for the preparation of the pharmaceutical composition and its preparation.
  • a pharmaceutical composition made of the following bulk drug substance
  • the phospholipid of the present invention is a soybean phospholipid or lecithin mainly composed of phosphatidylcholine, preferably soybean phospholipid.
  • phospholipids in the present invention are to promote drug dissolution and absorption, silybin low-insoluble hypotonic drugs, phospholipids and When combined with the formation of a phospholipid complex, the solubility of the silybin can be improved, thereby improving the bioavailability of the drug.
  • the silybin, phospholipids are all prior art or commercially available.
  • the silybin of the present invention is preferably prepared by the following method, taking silymarin, respectively, dissolving with 80% ethanol, filtering, and precipitating and dissolving three times with 95% ethanol to collect a precipitate.
  • the precipitate is dissolved in absolute ethanol, filtered, and a certain amount of water is added to the filtrate to precipitate a precipitate, which is filtered, and the precipitate is collected, dried under reduced pressure, pulverized, and mixed.
  • the Pu'er tea extract is commercially available, preferably in the form of Dibao Pu'er tea. It can also be prepared according to the prior art. In order to better exert the therapeutic effect of the present invention, it is preferred that the Pu'er tea or Pu'er tea extract is prepared according to Patent Publication Nos. CN101961061A, CN101961061B, CN101961425A, CN101961425B, CN101961060A, CN101961059A, CN101961059B.
  • Step 2 the concentrated liquid is centrifuged by a centrifuge, and the concentrated liquid is concentrated under reduced pressure to a specific gravity of 1.1-1.25 at 45-65 ° C.
  • the concentrated paste is spray-dried or microwave-dried.
  • the steps are as follows:
  • Step 2 the concentrated liquid is centrifuged by a three-leg centrifuge, and the three-legged centrifugation liquid is centrifuged by a tube centrifuge, and the centrifugal liquid is concentrated under reduced pressure to a specific gravity of 1.1-1.25 at 45-65 ° C, and the concentrated paste is spray-dried or microwave-dried;
  • tubular centrifugal conditions centrifuge speed: 15000-19000 rev / min; spray drying conditions: inlet air temperature: 140-190 ° C, outlet air temperature: 75-95 ° C.
  • the steps are as follows: Pu'er tea is added with water vigorously boiling and boiling for 3 times, the first boiling for 1.5 hours, adding water 10 times volume; the second boiling for 1.5 hours, adding water 8 times volume; the third boiling for 1 hour 8 times the volume of water, the extract is filtered, and the filtrate is concentrated under reduced pressure at ⁇ 70 °C to the weight of the tea: the volume of the concentrate is 1:2-1:3, the concentrate is centrifuged with a three-leg centrifuge, and the three-legged centrifuge is used. Centrifuge in a tube centrifuge, concentrate the concentrated solution to a specific gravity of 1.1-1.25 at 45-65 ° C under reduced pressure, and spray or dry the concentrated paste to obtain a solution;
  • tubular centrifugal conditions centrifuge speed: 15000-19000 rev / min; spray drying conditions: inlet air temperature: 140-190 ° C, outlet air temperature: 75-95 ° C.
  • the L-carnitine is any one of L-carnitine and L-carnitine tartrate which are commercially available for dietary supplementation or medicinal use.
  • the above composition is based on the weight, and can be increased or decreased according to the corresponding proportion during production.
  • mass production can be in kg or in t (ton); small-scale preparation can also be in g. .
  • the weight can be increased or decreased, but the ratio of the weight ratio of the raw herbs to the composition is constant.
  • the ratio of the above weight ratio is scientifically selected.
  • the proportion of the composition can be adjusted accordingly, increasing or decreasing by no more than 10%. constant.
  • Any pharmaceutically acceptable dosage form can be prepared in the preparation of a pharmaceutical preparation, and these dosage forms are selected from the group consisting of: tablets, sugar-coated tablets, film-coated tablets, enteric coated tablets, capsules, hard capsules, soft capsules, Oral liquid, buccal, granules, pills, powders, ointments, dans, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, patches.
  • composition of the present invention may be added with some pharmaceutically acceptable carriers as needed, and the pharmaceutical preparation may be prepared by conventional techniques such as mixing the pharmaceutically active substance with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is selected from the group consisting of: mannitol, sorbitol, sorbic acid or potassium salt, sodium metabisulfite, sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin A, Vitamin C, vitamin E, vitamin D, azone, EDTA disodium, EDTA calcium sodium, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, chlorine Sodium, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mann
  • a unit dose of the medicament may contain 0.1 to 1000 mg of the pharmaceutically active substance of the present invention, and the balance is a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may range from 0 to 99.9% by weight of the total formulation.
  • the pharmaceutically acceptable carrier may be from 40 to 70% by weight based on the total weight of the formulation.
  • the traditional Chinese medicine composition or preparation of the present invention determines the usage amount according to the condition of the patient at the time of use.
  • the pharmaceutical composition preparation of the present invention such as a tablet, a capsule, a granule, etc.
  • a dissolution condition a slurry method, a rotation speed of 100 rpm, a temperature of 37 ° C, and a release medium of 1000 ml of a hydrochloric acid solution having a pH of 1.2.
  • Amount 1 capsule / 1 tablet / 1 bag of granules.
  • the cumulative dissolution in vitro for 2h is not less than 80%, and the dissolution at 30min is greater than or equal to 15%.
  • a method for preparing a pharmaceutical composition of the present invention comprises the following steps:
  • silybin complex liquid Preparation of silybin complex liquid: Weigh the prescription amount of silybin and phospholipid into absolute ethanol, heat and reflux to make the solution clarify, then continue to heat for a certain period of time, and then concentrate the clear solution to a certain volume under reduced pressure. Obtaining the silybin complex solution and standby;
  • the present invention also encompasses Formulation Step 5, which takes the step 4 of the pharmaceutical composition together with a pharmaceutically acceptable carrier in a pharmaceutically acceptable dosage form according to conventional formulation procedures.
  • the preparation method of the present invention comprises the following steps:
  • silybin complex liquid Preparation of silybin complex liquid: Weigh the prescription amount of silybin and phospholipid into absolute ethanol, heat and reflux to make the solution clarify, then continue to heat for a certain period of time, and then concentrate the clear solution to a certain volume under reduced pressure. Obtaining the silybin complex solution and standby;
  • L-carnitine and step 3 particles are mixed in an equal amount to obtain a pharmaceutical composition
  • a pharmaceutical preparation and a pharmaceutically acceptable carrier are prepared into a conventional preparation.
  • the heating time described in the step 2 is 0.5 to 1.5 hours; the concentrated volume is 5% to 20% of the original volume, and the concentration under reduced pressure is 60 to 80 °C.
  • the parameters of the fluidized bed in step 3 are: the material temperature is 40-65 ° C, the granulation process adjusts the fan frequency, the inlet air temperature and the infusion frequency and other parameters to keep the material in a good fluidized state.
  • the granulation After the granulation is completed, it is dried for 10 to 60 minutes, and the drying temperature is 55 to 65 °C.
  • Pu'er tea can improve insulin resistance, regulate blood lipids and leptin levels, etc. It can block the accumulation of fat in the liver parenchyma caused by insulin resistance in the body, and combines the strong free radical scavenging and anti-oxidative stress of silybin. Ability, both have better anti-NAFLD (non-alcoholic fatty liver disease) effect.
  • the dissolution of the silybin-phospholipid-Purin tea-L-carnitine composition prepared in Examples 17-21 was determined under the following conditions: the selection of the dissolution method was based on the nature of the main component of the silybin in the composition. Ding, the fourth category of silybin biopharmaceutics classification system (BCS), low solubility and low permeability drugs, dissolution and absorption are all speed-limiting steps, improve drug bioavailability It is solved simultaneously from the two aspects of improving drug dissolution and absorption.
  • the dissolution phase of silybin is mainly carried out in the stomach, and the absorption phase is mainly carried out in the small intestine.
  • the in vitro dissolution of the drug is helpful to improve the bioavailability of the drug.
  • composition dissolution evaluation slurry method, rotation speed of 100 rpm, temperature of 37 ° C, release medium: 1000 ml of hydrochloric acid solution of pH 1.2, dosage: 1 capsule / 1 tablet / 1 bag of granules.
  • the sampling points are: 15, 30, 45, 60, 90, 120 min.
  • the cumulative dissolution was measured. The results are shown in Table 1 below.
  • the silybin-phospholipid-Purin tea-L-carnitine composition prepared by the preparation method of the invention has significantly better in vitro release than the reference preparation silybin-phospholipid complex, surprisingly,
  • the cumulative dissolution rate of the composition in hydrochloric acid solution at pH 1.2 reached 80%, nearly completely dissolved, and the dissolution rate of the reference preparation was doubled, which solved the small solubility and bioavailability of silybin.
  • the low difficulty will provide a basis for the study of the dose setting and safety and effectiveness of the subsequent silybin composition.
  • the present invention improves the drug absorption by continuing the compatibility of the silybin with the phospholipid to improve the compatibility of the drug with the biofilm, and further by using the silybin-phospholipid complex with After the combination of Pu'er tea and L-carnitine, the dissolution of the drug is improved, and the bioavailability of the main component, silybin, is improved from the aspects of improving dissolution and absorption.
  • mice 80-year-old male C57 BL/6J leptin-deficient mice (ob/ob) mice, SPF grade, 6-week old male C57 BL/6J (ob/m) mice, SPF grade, by Beijing Hua Provided by Fukang Biotechnology Co., Ltd., it is kept in the barrier animal room of the Institute of Pharmacology and Toxicology of Tasly Research Institute.
  • the temperature is 20 °C ⁇ 25 °C
  • the relative humidity 60%
  • 5 cages per cage 5 cages per cage
  • the illumination time is 12 hours.
  • Ob/ob mice were fed with high-fat diet (HFD, D12492), and C57 BL/6J mice were fed with normal feed, all provided by Beijing Huakang Biotechnology Co., Ltd., free drinking water, and daily replacement of litter.
  • HFD high-fat diet
  • Silibinin phospholipid complex supplied by Tasly Pharmaceutical Group Co., Ltd., batch number 500902031; Pu'er tea, tan powder, supplied by Tasly Pharmaceutical Group Co., Ltd., batch number Z001 PE(2014)C06(H); Alkali tartrate (containing 68.4% L-carnitine), supplied by Northeast Pharmaceutical Group Co., Ltd., batch number 0661504001; the above test materials are stored in the sample cabinet of the sample room of the Pharmacology Institute, and stored at room temperature in the dark.
  • the silybin phospholipid complex was calculated according to the daily dose of 3 g (water silibin 420 mg, soybean phospholipid 504 mg), and the experimental animal was administered.
  • the Pu'er tea extract was administered to the experimental animal according to the daily dose of 1.2 g.
  • Dosage; the compatibility ratio of the five different compositions and the experimental dose design are shown in Table 2.
  • the doses of the experimental animals are set to the clinical equivalent dose of the corresponding test substance, and the calculation formula is as follows:
  • mice After 1 week of adaptive feeding, 80 6-week-old ob/ob mice were randomly divided into 8 groups: model group, silybin phospholipid complex group, Pu'er tea extract group, combination group 1, Two groups were combined, three groups were combined, four groups were combined, and five groups were combined, with 10 in each group. Another 10 6-week-old C57BL/6J mice were in the normal group. Normal mice were fed with normal diet, and the model group and the drug-administered group were fed with high-fat diet (HFD, D12492). In addition, mice in different drug intervention groups were given the corresponding doses of the drugs by intragastric administration. The doses of the five compositions were shown in Table 1. The normal group and the model group were given the same amount of distilled water for 6 weeks.
  • HFD high-fat diet
  • mice were free to eat and drink during the experiment, weighed once a week, and adjusted the dose according to body weight. After the last dose Fasting for 12 hours, weighing the body, taking the eyeball to take the blood and then smashing the neck, quickly dissecting the liver, rinsing with normal saline, blotting the filter paper and weighing it in a refrigerator at -20 °C.
  • mice The body weight of each group of mice was weighed once a week during the experiment.
  • liver index (%) liver wet weight / body weight * 100%.
  • mice All mice were bled by eyeballs, centrifuged at 3000 r/min for 15 minutes, serum was separated, collected in EP tubes, and stored in a refrigerator at -20 °C until use. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were measured by 7020 automatic biochemical analyzer.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • TC total cholesterol
  • LDL-C low-density lipoprotein cholesterol
  • Serum FINS was measured using the Elisa kit and the insulin resistance index was calculated by the formula.
  • Frozen sections were prepared from frozen liver tissue, and the degree of hepatic steatosis was observed by oil red o staining.
  • Oil red o dyeing operation steps frozen section ⁇ distilled water fully washed ⁇ oil red O dilution solution to avoid light staining for 10-15 minutes ⁇ remove oil red O saturated liquid 6ml, add distilled water 4ml, let stand for 5-10 minutes, filter and use ⁇ 60% ethanol microscopically differentiated to interstitial clear ⁇ washed ⁇ hematoxylin counterstained nuclei ⁇ washed ⁇ neutral gum seals ⁇ microscopic observation.
  • mice The body weight of each group of mice was weighed once a week during the experiment, and the effects of body weight on the non-alcoholic fatty liver mice in each group were examined. As shown in Table 3, the weight gain of the mice in the normal group was slow, and the weight of the mice in the model group increased rapidly. After 6 weeks of administration, except for the silybin complex, the other groups could inhibit the weight gain of mice in different degrees (P ⁇ 0.01), and there was no significant difference between the groups.
  • the serum TC, LDL, ALT, AST and insulin resistance indexes of the nonalcoholic fatty liver model mice were significantly increased (P ⁇ 0.05); the silybin phospholipid complexes were abnormal. There was no significant improvement in the elevated index (P>0.05); different proportions of silybin phospholipid complex, Pu'er tea extract and L-carnitine compatibility group, except composition 5 showed no significant improvement in serum AST ( P>0.05), other compositions can significantly reduce TC, LDL-C, ALT, AST and insulin resistance index (P ⁇ 0.05), and the effect is better than the silybin phospholipid complex, Pu'er tea extract alone .
  • Composition 1 6.56 ⁇ 0.89** 1.05 ⁇ 0.15** 217.11 ⁇ 109.25* 190.02 ⁇ 22.29** 0.89 ⁇ 0.13**
  • Composition 2 8.90 ⁇ 1.02* 1.73 ⁇ 0.32* 260.00 ⁇ 41.34* 172.23 ⁇ 36.88** 0.97 ⁇ 0.29**
  • Composition 3 7.45 ⁇ 1.32** 1.33 ⁇ 0.14** 250.93 ⁇ 60.02* 201.24 ⁇ 29.07** 0.93 ⁇ 0.11**
  • Composition 4 6.74 ⁇ 1.05** 1.10 ⁇ 0.11** 195.64 ⁇ 62.02** 158.29 ⁇ 37.78** 0.77 ⁇ 0.20**
  • Composition 5 7.72 ⁇ 1.07** 1.28 ⁇ 0.14** 237.54 ⁇ 91.63* 258.89 ⁇ 35.96 0.92 ⁇ 0.18**
  • Oil red O staining According to the size and number of red particles in hepatocytes under the light microscope of liver frozen tissue oil red staining, it is divided into mild, moderate and severe three types. Mild, that is, 1/3-2/3 red particles per unit area under light microscope, rated as 1 point; moderate, ie 2/3 or more, containing red particles in liver cells, rated as 2 points; severe, That is, almost all of the liver cells had red particles, which were rated as 3 points; no fat degeneration was evaluated as 0 points.
  • Fig. 1 is an in vitro release curve, wherein each sample is: a reference preparation Shuilinjia, Shuilinjia without Pu'er tea, and a silybin-phospholipid-Puer tea composition prepared in Examples 16-20.
  • silybin complex liquid Weigh the prescribed amount of silybin and soybean phospholipid dissolved in absolute ethanol, heat and reflux to clarify the solution, continue heating for 1 hour, and concentrate and recover ethanol to 15% of original volume under reduced pressure. ,spare;
  • Step 3 Mixing the prescribed amount of L-carnitine with the granules obtained in Step 2 in an equal amount of increments, bagging, and making 1000 bags of granules.
  • silybin complex liquid Preparation of silybin complex liquid: Weigh the prescribed amount of silybin and soybean phospholipid into absolute ethanol, heat and reflux to clarify the solution, continue heating for 1.5 hours, and concentrate and recover ethanol to 20% of the original volume under reduced pressure. ,spare;
  • Step 3 Mixing the prescribed amount of L-carnitine with the granules obtained in Step 2 in an equal amount of increments, bagging, and making 1000 bags of granules.
  • silybin complex liquid Preparation of silybin complex liquid: Weigh the prescribed amount of silybin and soybean phospholipid into absolute ethanol, heat and reflux to clarify the solution, continue heating for 0.5 hours, and concentrate and recover ethanol to 5% of original volume under reduced pressure. ,spare;
  • Step 3 Mixing the prescribed amount of L-carnitine with the granules obtained in Step 2 in an equal amount of increments, bagging, and making 1000 bags of granules.
  • 26 bags of granules were prepared according to the method of Example 1 by taking 26.25 g of silibinin, 195 g of phospholipid, 75 g of Pu'er tea extract, and 180 g of L-carnitine.
  • 180 g of granules were prepared by taking 180 g of silybin, 45 g of phospholipid, 75 g of Pu'er tea extract, and 15.6 g of L-carnitine, according to the method of Example 1.
  • 180 g of granules were prepared by taking the method of the method of Example 1 by taking 180 g of silibinin, 45 g of phospholipid, 450 g of Pu'er tea extract, and 180 g of L-carnitine.
  • 180 g of granules were prepared by taking 180 g of silibinin, 195 g of phospholipid, 75 g of Pu'er tea extract, and 180 g of L-carnitine according to the method of Example 1.
  • 26 bags of granules were prepared according to the method of Example 1 by taking 26.25 g of silybin, 48.75 g of phospholipid, 75 g of Pu'er tea extract, and 62.5 g of L-carnitine.
  • 26 bags of granules were prepared according to the method of Example 1 by taking 26.25 g of silybin, 48.75 g of phospholipid, 300 g of Pu'er tea extract, and 62.5 g of L-carnitine.
  • 5 bags of granules were prepared by taking 52.5 g of silibinin, 97.5 g of phospholipids, 300 g of Pu'er tea extract, and 15.625 g of L-carnitine, according to the method of Example 1.
  • 75 g of granules were prepared by taking 75 g of silibinin, 90 g of phospholipid, 240 g of Pu'er tea extract, and 105 g of L-carnitine according to the method of Example 1.
  • 90 g of granules were prepared according to the method of Example 1 by taking 90 g of silibinin, 108 g of phospholipid, 270 g of Pu'er tea extract, and 120 g of L-carnitine.
  • 105 g of granules were prepared by taking 105 g of silybin, 126 g of phospholipid, 300 g of Pu'er tea extract, and 125 g of L-carnitine according to the method of Example 1.
  • 120 g of granules were prepared by taking 120 g of silybin, 150 g of phospholipid, 360 g of Pu'er tea extract, and 150 g of L-carnitine according to the method of Example 1.
  • Example 8 The granules of Example 8 were added, and 523 g of microcrystalline cellulose and 64 g of sodium carboxymethyl starch were added thereto, uniformly mixed, and filled into capsules to prepare 1,000 tablets.
  • Example 9 The granules of Example 9 were added, and 298 g of microcrystalline cellulose and 64 g of sodium carboxymethyl starch were added thereto, uniformly mixed, and filled into capsules to prepare 1,000 tablets.
  • Example 10 The granules of Example 10 were added, and 270 g of microcrystalline cellulose and 64 g of sodium carboxymethyl starch were added thereto, uniformly mixed, and filled into capsules to prepare 1,000 tablets.
  • Example 13 The pellet of Example 13 was taken, and 144 g of microcrystalline cellulose was added, mixed uniformly, and filled into capsules to prepare 1,000 pellets.
  • Example 14 The granules of Example 14 were taken, and 11 g of microcrystalline cellulose and 64 g of sodium carboxymethyl starch were added, uniformly mixed, and filled into capsules. 1000 capsules.
  • Example 8 The composition of Example 8 was added, and 400 g of lactose, 123 g of starch, and 64 g of sodium carboxymethyl starch were mixed, uniformly, and filled into capsules to prepare 1,000 tablets.
  • Example 13 The composition of Example 13 was added, and 70 g of lactose, 10 g of talc, and 64 g of low-substituted hydroxypropylcellulose were added, uniformly mixed, and filled into capsules to prepare 1,000 tablets.
  • Example 13 The composition of Example 13 was taken, and 144 g of microcrystalline cellulose was added, mixed uniformly, and tableted to prepare 1,000 pieces.

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Abstract

一种治疗非酒精性脂肪肝的药物组合物，所述药物组合物由水飞蓟宾8.75-60重量份，磷脂15-65重量份，普洱茶提取物25-150重量份，左旋肉碱10.425-60重量份制成。

Description

一种含有水飞蓟宾、左旋肉碱的药物组合物 技术领域
本发明涉及医药领域，尤其是治疗肝病的含水水飞蓟宾的药物组合物。
背景技术
20世纪60-80年代末，以H.wagner为代表的西德药学家从菊科水飞蓟属植物水飞蓟(Silybummarianum)果实中提取分离其有效成分，称之为水飞蓟素，是一类新型的具有C－9取代基的黄酮类化合物，即以二氢黄酮醇和苯丙素类衍生物缩合而成的黄酮木脂素类。水飞蓟宾(Silybin，silibinin)系水飞蓟的主要成分之一。经药理和毒理研究，表明其具有明显的保护及稳定肝细胞膜，促进肝细胞恢复，改善肝功能的作用。对各种毒物，如四氯化碳、硫代乙酰胺、毒蕈碱、鬼笔碱、猪屎豆碱等引起的肝损伤均具有不同程度的保护治疗作用。可用于急慢性肝炎、早期肝硬化、脂肪肝、中毒性或药物性肝病。
由于水飞蓟宾难溶于水及一般有机溶剂，口服吸收差，其生物利用度较低,从而影响了其临床疗效。为改善其生物利用度，国内外药学工作者作了大量的工作。改善难溶性药物吸收的措施一般有超细粉碎、成盐，添加助溶剂等。近年来采用制成环糊精包合物、固体分散体，合成磷脂复合物，制成不同剂型等方法，研究表明，溶出度及生物利用度大大提高。
从固体制剂角度来看，磷脂复合物为一种较为特殊的固体分散体，他有固定的熔点，是一种其化学本质较稳定，不同于药物和磷脂的分子化合物(络合物)，该类化合物随磷脂种类及药物磷脂比例的不同而不同，一个磷脂分子可与不同数目的药物分子结合。复合物光谱学特征推断，药物与磷脂的极性基团部分发生了较强的相互作用，抑制了分子中单链的自由转动，而磷脂的两个长脂肪酸链不参与复合反应，可自由移动，包裹了磷脂的极性部分形成一个亲脂性的表面，使复合物表现出较强的脂溶性。它改变了药物的理化性质，使药物的脂溶性增加，水溶性减少。促进药物分子与细胞膜结合而促进吸收，提高药物的生物利用度。普洱茶是云南特有的地方名茶。产地气候温和，雨量充沛，云雾缭绕。普洱茶。是以云南原产地的大叶种晒青茶及其再加工而成两个系列：直接再加工为成品的生普和经过人工速成发酵后再加工而成的熟普，型制上又分散茶和紧压茶两类；成品后都还持续进行着自然陈化过程，具有越陈越香的独特品质。
普洱茶是惟一的后发酵型的茶，它的茶碱、茶多酚等对人体有害的物质在长期的发酵过程中被分化掉了，因此品性温和，对人体不刺激，还能够促进新陈代谢，加速身体内脂肪、毒素的消解和转化，现在困扰都市人的肥胖、三脂高等问题，普洱茶都能够起到很好的缓解作用，如排毒、养胃、消炎、降低胆固醇、消脂去腻、美容减肥。现代技术表明，普洱茶具 有改善胰岛素抵抗、调节血脂和瘦素水平等作用可一定程度阻断机体胰岛素抵抗所带来的肝脏实质细胞脂肪堆积等作用。
非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)一种与胰岛素抵抗和遗传易感密切相关的代谢应激性肝脏损伤，其病理学改变与酒精性脂肪肝病相似。是一种病变主体在肝小叶以肝细胞脂肪变性和脂肪贮积为病理特征但无过量饮酒史的临床病理综合征.NAFLD表现不同程度的肝脏病变,从没有任何炎症的单纯脂肪肝至显著纤维化甚至肝硬化的严重炎症反应,主要包括单纯性脂肪肝、脂肪性肝炎、脂肪性肝硬化3种类型.
非酒精性脂肪肝病治疗方法
1.防治原发病或相关危险因素
2.基础治疗：制定合理的能量摄人以及饮食结构调整、中等量有氧运动、纠正不良生活方式和行为。
3.避免加重肝脏损害：防止体重急剧下降、滥用药物及其他可能诱发肝病恶化的因素。
4.减肥：所有体重超重、内脏性肥胖以及短期内体重增长迅速的NAFLD患者，都需通过改变生活方式控制体重、减少腰围。基础治疗6个月体重下降每月<0.45kg，或体重指数(BMI)>27kg/m2合并血脂、血糖、血压等两项以上指标异常者，可考虑加用西布曲明或奥利司他等减肥药物，每周体重下降不宜超过1.2Kg(儿童每周不超过0.5Kg)；BMI>40kg/m2或BMI>35kg/m2合并睡眠呼吸暂停综合征等肥胖相关疾病者，可考虑近端胃旁路手术减肥(Ⅱ-1，Ⅱ-2，Ⅱ-3，Ⅲ)。
5.胰岛素增敏剂：合并2型糖尿病、糖耐量损害、空腹血糖增高以及内脏性肥胖者，可考虑应用二甲双胍和噻唑烷二酮类药物，以期改善胰岛素抵抗和控制血糖(Ⅱ-1，Ⅱ-2，Ⅱ-3)。
6.降血脂药：血脂紊乱经基础治疗和(或)应用减肥降糖药物3-6个月以上，仍呈混合性高脂血症或高脂血症合并2个以上危险因素者，需考虑加用贝特类、他汀类或普罗布考等降血脂药物(Ⅱ-1，Ⅱ-2，Ⅱ-3)。
7.针对肝病的药物：NAFLD伴肝功能异常、代谢综合征、经基础治疗3～6个月仍无效，以及肝活体组织检查证实为NASH和病程呈慢性进展性经过者，可采用针对肝病的药物辅助治疗，以抗氧化、抗炎、抗纤维化，可依药物性能以及疾病活动度和病期合理选用多烯磷脂酞胆碱、 维生素E、水飞蓟素以及熊去氧胆酸等相关药物(Ⅱ-1，Ⅱ-2，Ⅱ-3，Ⅲ)，但不宜同时应用多种药物。
8.肝移植：主要用于NASH相关终末期肝病和部分隐源J睦肝硬化肝功能失代偿患者的治疗，肝移植前应筛查代谢情况(III)。BMI>40kg/m2为肝移植的禁忌证(Ⅲ)。
在上述治疗方法还没有将几种治疗方法混合使用的方法，例如降糖和肝病药物同用，或者降血脂和肝病药物同用的方法。因此，寻找一个能够具有多种保健功能的药物是迫不及待的。
发明内容
为了解决上述技术问题，本发明提供一种药物组合物及其制剂，对非酒精性脂肪肝的有疗效。
本发明提供了该药物组合物及其制剂的制备方法。
本发明是通过以下技术方案实现的：
一种药物组合物，其是由下述重量比的原料药制成
Figure PCTCN2016077037-appb-000001
优选其是由下述重量比的原料药制成：
Figure PCTCN2016077037-appb-000002
最优选其是由下述重量份的原料制成
Figure PCTCN2016077037-appb-000003
本发明所述的磷脂是以磷脂酰胆碱为主的大豆磷脂或卵磷脂，优选大豆磷脂。
磷脂在本发明中的作用在于：促进药物溶解与吸收，水飞蓟宾属低溶低渗药物，磷脂与 其结合形成磷脂复合物后，可提高水飞蓟宾溶解性，从而提高药物的生物利用度。
所述的水飞蓟宾，磷脂均为现有技术，或者市购。为了更好的发挥本发明的疗效，本发明的水飞蓟宾优选如下方法制备，取水飞蓟素，分别用80％乙醇溶解，过滤，沉淀用95％乙醇溶解洗脱三次，收集沉淀。沉淀用无水乙醇溶解，过滤，滤液加入一定量水使沉淀析出，过滤，收集沉淀，减压干燥，粉碎，混合即可。
所述的普洱茶提取物市购，优选帝泊洱普洱茶珍。还可以按照现有技术制备，为了更好的发挥本发明的疗效，优选普洱茶珍或普洱茶提取物是按照专利公开号CN101961061A、CN101961061B、CN101961425A、CN101961425B、CN101961060A、CN101961059A、CN101961059B制备。
例如，普洱茶珍生产工艺
步骤1，普洱茶叶加水煎煮提取2-4次，每次0.5～2小时，6-12倍体积的水，提取液过滤，滤液在≤70℃条件下减压浓缩至茶叶重量：浓缩液体积＝1:2-1:3；
步骤2，浓缩液用离心机离心，离心液减压浓缩至45-65℃比重1.1-1.25，浓缩膏喷雾干燥或微波干燥，即得。
优选的，步骤如下：
步骤1，普洱茶叶加水剧烈沸腾煎煮提取3次，每次0.5～2小时，加水量6-12倍体积的水；提取液过滤，滤液在≤70℃条件下减压浓缩至茶叶重量：浓缩液体积＝1:2-1:3；
步骤2，浓缩液用三足离心机离心，三足离心液用管式离心机离心，离心液减压浓缩至45-65℃比重1.1-1.25，浓缩膏喷雾干燥或微波干燥，即得；
其中管式离心条件：离心机转速：15000-19000转/min；喷雾干燥条件：进风温度：140-190℃，出风温度：75-95℃。
最优选的，步骤如下：普洱茶叶加水剧烈沸腾煎煮提取3次，第1次煎煮1.5h，加水10倍体积；第2次煎煮1.5h，加水8倍体积；第3次煎煮1h，加水8倍体积，提取液过滤，滤液在≤70℃条件下减压浓缩至茶叶重量：浓缩液体积＝1：2-1：3，浓缩液用三足离心机离心，三足离心液用管式离心机离心，离心液减压浓缩至45-65℃比重1.1—1.25，浓缩膏喷雾干燥或微波干燥，即得；
其中管式离心条件：离心机转速：15000-19000转/min；喷雾干燥条件：进风温度：140-190℃，出风温度：75-95℃。
所述的左旋肉碱，为市售可供膳食补充或药用的左旋肉碱，左旋肉碱酒石酸盐中的任意一种。
以上组成是按重量作为配比的，在生产时可按照相应比例增大或减少，如大规模生产可以以kg为单位，或以t(吨)为单位；小规模制剂也可以以g为单位。重量可以增大或者减小，但各组成之间的生药材重量配比的比例不变。
以上重量配比的比例是经过科学筛选得到的，对于特殊病人，如重症或轻症，肥胖或瘦小的病人，可以相应调整组成的量的配比，增加或减少不超过10％，药效基本不变。
在制成药物制剂时可以制成任何可药用的剂型，这些剂型选自：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、丸剂、散剂、膏剂、丹剂、混悬剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、贴剂。优选的是口服制剂形式，最佳优选的是片剂，胶囊剂，颗粒剂。
本发明的组合物，根据需要可以加入一些药物可接受的载体，可以采用制剂学常规技术制备该药物制剂，如将药物活性物质与药物可接受的载体混合。所述药物可接受的的载体选自：甘露醇、山梨醇、山梨酸或钾盐、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素A、维生素C、维生素E、维生素D、氮酮、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、丙二醇、乙醇、土温60-80、司班-80、蜂蜡、羊毛脂、液体石蜡、十六醇、没食子酸酯类、琼脂、三乙醇胺、碱性氨基酸、尿素、尿囊素、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β－环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。优选所述的载体为微晶纤维素、乳糖、淀粉、羧甲基纤维素钠、低取代羟丙基纤维素、滑石粉中不含或含有一种或以上。
本发明的组合物，在制成药剂时，单位剂量的药剂可含有本发明的药物活性物质0.1-1000mg，其余为药学上可接受的载体。药学上可接受的载体以重量计可以是制剂总重量的0-99.9％。优选所述的药学上可接受的载体以重量计可以是制剂总重量的40-70％。
本发明的中药组合物或制剂在使用时根据病人的情况确定用法用量。
本发明所述的药物组合物制剂，例如片剂、胶囊剂、颗粒剂等，在溶出条件：浆法，转速为100rpm，温度为37℃，释放介质为：pH1.2的盐酸溶液1000ml，投药量：1粒胶囊/1片/1袋颗粒。其体外溶出2h累积溶出度不低于80％，30min时溶出度大于等于15％。
本发明的一种药物组合物的制备方法，包括如下步骤：
①取处方量的原料备用；
②水飞蓟宾复合液的制备：称取处方量水飞蓟宾、磷脂溶解到无水乙醇中，加热回流使溶液澄清后再继续加热一定时间，再将澄清溶液减压浓缩至一定体积，得到水飞蓟宾复合液，备用；
③制粒：称取处方量普洱茶提取物作为底料，以②中制备得水飞蓟宾复合液为料液，以流化床流化喷液方式制颗粒，待液态复合物全部喷入后，干燥，备用；
④总混：取左旋肉碱与步骤③颗粒按等量递增方式混合均匀即得药物组合物。
本发明还包括制剂步骤⑤，取步骤④药物组合物与药学上可接受的载体，按照常规制剂工艺制成药学上可接受的剂型。
进一步优选本发明的制备方法，包括如下步骤：
①取处方量的原料备用；
②水飞蓟宾复合液的制备：称取处方量水飞蓟宾、磷脂溶解到无水乙醇中，加热回流使溶液澄清后再继续加热一定时间，再将澄清溶液减压浓缩至一定体积，得到水飞蓟宾复合液，备用；
③制粒：称取处方量普洱茶提取物作为底料，以②中制备得水飞蓟宾复合液为料液，以流化床流化喷液方式制颗粒，待液态复合物全部喷入后，干燥，备用；
④总混：左旋肉碱与步骤③颗粒按等量递增方式混合均匀即得药物组合物；
⑤制剂：取药物组合物与药学上可接受的载体制成常规制剂。
其中步骤②中所述的加热时间为0.5～1.5小时；所述的浓缩的体积为原体积的5％～20％，减压浓缩温度为60～80℃。
其中步骤③中流化床的参数为：物料温度40～65℃，制粒过程调节风机频率，进风温度和输液频率等参数，保持物料良好流化状态。
制粒完成后，干燥10-60分钟，干燥温度为55～65℃。
普洱茶可改善胰岛素抵抗、调节血脂和瘦素水平等作用可一定程度阻断机体胰岛素抵抗所带来的肝脏实质细胞脂肪堆积，同时联合水飞蓟宾较强的自由基清除和抗氧化应激能力，二者具有较好的抗NAFLD(非酒精性脂肪性肝病)效果。
增加Ve，进一步提升产品美容养颜的功效，增加左旋肉碱，强化产品的降脂减肥功效。
试验例一体外溶出试验
在以下条件下测定实施例17-21制得的水飞蓟宾-磷脂-普洱茶-左旋肉碱组合物的溶出：溶出方法的选择是基于组合物中主要组份水飞蓟宾的性质而定，水飞蓟宾属生物药剂学分类系统(BCS)中第四类，低溶低渗药物，溶出、吸收均为限速步骤，提高药物生物利用度应 当从提高药物溶出与吸收两方面同时解决。水飞蓟宾的溶出阶段主要在胃中进行，吸收阶段主要在小肠中进行，检测药物体外溶出度有助于提高药物生物利用度。因此选择以下溶出方法进行组合物溶出评价：浆法，转速为100rpm，温度为37℃，释放介质为：pH1.2的盐酸溶液1000ml，投药量：1粒胶囊/1片/1袋颗粒。取样点分别为：15、30、45、60、90、120min。测定其累积溶出度。结果如下述表1所示。
表1
Figure PCTCN2016077037-appb-000004
对参比制剂(水飞蓟宾-磷脂复合物制剂，实验室自制)进行溶出测定，与实施例17～21制备的水飞蓟宾-磷脂-普洱茶-左旋肉碱组合物进行比较，结果见图1。从表1的数据及图1的曲线可以看出：
采用本发明的制备方法制得的水飞蓟宾-磷脂-普洱茶-左旋肉碱组合物，其体外释放均显著优于参比制剂水飞蓟宾-磷脂复合物，令人惊奇的是，组合物在pH1.2的盐酸溶液中2h的累积溶出度达到80％，近乎完全溶出，较参比制剂溶出度提高了一倍，解决了水飞蓟宾一直以来存在的溶解度小，生物利用度低的难题，将为后续水飞蓟宾组合物的剂量设定及体内安全性和有效性的研究提供依据。
综合体外溶出试验和体内药效研究的数据，本发明通过延续水飞蓟宾与磷脂复合后提高药物与生物膜的相容性改善药物的吸收，进一步通过将水飞蓟宾-磷脂复合物与普洱茶、左旋肉碱组合后提高药物的溶出，从提高溶出与吸收两方面共同提高主要组份水飞蓟宾的生物利用度。
试验例二体内药效试验
1实验动物
6周龄雄性C57 BL/6J瘦素缺失小鼠(ob/ob)小鼠80只，SPF级，6周龄雄性C57 BL/6J(ob/m)小鼠10只，SPF级，由北京华阜康生物科技股份有限公司提供，饲养于天士力研究院药理毒理研究中心屏障动物房，温度20℃～25℃，相对湿度60％，每笼5只，照明时间12小时，定时定量添加饲料，ob/ob小鼠采用高脂饲料喂养(HFD,D12492)，C57 BL/6J小鼠采用普通饲料喂养，均由北京华阜康生物科技股份有限公司提供，自由饮水，每日更换垫料。
2受试物
水飞蓟宾磷脂复合物，由天士力制药集团股有限公司提供，批号500902031；普洱茶珍，棕褐色粉末，由天士力制药集团股有限公司提供，批号Z001 PE(2014)C06(H)；左旋肉碱酒石酸盐(含左旋肉碱68.4％)，由东北制药集团股份有限公司提供，批号0661504001；以上受试物均存放于药理所供试品室样品柜内，避光室温下保存。
3实验方法
3.1实验剂量设计及分组
水飞蓟宾磷脂复合物按人日用量为3g(含水飞蓟宾420mg、大豆磷脂504mg、)计算该实验动物给药剂量；普洱茶提取物按人日用量为1.2g计算该实验动物给药剂量；5种不同组合物的配伍比例以及实验剂量设计见表2，实验动物的剂量均设为相应受试物的临床等效剂量，计算公式如下：
动物实验剂量＝推荐人用剂量/60kg*12.3
表2不同组合物的给药剂量
Figure PCTCN2016077037-appb-000005
3.2受试物给予
经适应性喂养1周后，将80只6周龄的ob/ob小鼠随机分为8组，分别为模型组、水飞蓟宾磷脂复合物组、普洱茶提取物组、组合1组、组合2组、组合3组、组合4组、组合5组，每组10只。另外10只6周龄的C57BL/6J小鼠为正常组。正常组小鼠给予普通饲料喂养，模型组和给药组均以高脂饲料喂养(HFD,D12492)。另外，不同药物干预组小鼠灌胃给予相应剂量的药物，五种组合物剂量见表1，正常组和模型组均给予等量蒸馏水，连续灌胃6周。
小鼠在实验期间自由进食和饮水，每周称体重一次，根据体重调整用药量。末次给药后 禁食不禁水12h，称量体重，摘眼球取血后断颈处死小鼠，迅速解剖摘取肝脏，生理盐水漂洗，滤纸吸干称重后置于-20℃冰箱中保存。
3.3检测指标及方法
3.3.1一般情况的观察
实验期间每周称一次各组小鼠的体重。
3.3.2计算肝指数并观察肝脏大体形态
实验结束后取肝脏称重，计算肝脏指数，肝指数(％)＝肝湿重/体重*100％。
3.3.3血清生化指标检测
所有小鼠经摘眼球取血，3000r/min离心15分钟，分离血清，收集于EP管中，置于-20℃冰箱保存备用。采用7020全自动生化仪检测血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)、总胆固醇(TC、)、低密度脂蛋白胆固醇(LDL-C)含量。
3.3.4胰岛素抵抗指数
采用Elisa试剂盒检测血清FINS，并通过公式计算胰岛素抵抗指数。
Figure PCTCN2016077037-appb-000006
3.3.5肝脏组织病理学检测
取冰冻肝组织制备冰冻切片，釆用油红o染色观察肝脏脂肪变性程度。油红o染色操作步骤：冰冻切片→蒸馏水充分洗涤→油红O稀释液避光染10-15分钟→取出油红O饱和液6ml，加蒸馏水4ml，静置5-10分钟后过滤后使用→60％乙醇镜下分化至间质清晰→水洗→苏木素复染核→水洗→中性树胶封片→显微镜观察。
3.4数据处理
统计学方法采用SPSS 15.0统计学软件进行分析，数据以均数±标准差表示，采用t检验，分析比较各组治疗前后以及组间各项观察指标有无差异，以P<0.05为差异有统计学意义。
4实验结果
4.1各受试物对体重的影响
实验期间每周称一次各组小鼠的体重，考察各组药物非酒精性脂肪肝小鼠体重的影响。如表3，正常照组小鼠体重增长缓慢，模型组小鼠体重增长较快。给药6周后，除水飞蓟宾磷脂复合物组外，其他各给药组均能不同程度地抑制小鼠体重增长(P＜0.01)，各组间没有明显差异。
表3各受试物对小鼠体重的影响(g，n＝10，
Figure PCTCN2016077037-appb-000007
)
组别 给药前 给药2周 给药4周 给药6周
正常 21.34±0.69 24.92±1.95 26.57±2.06 27.38±2.33
模型 44.35±1.66 51.43±4.02 58.09±3.37 61.23±3.67
水飞蓟宾磷脂复合物 44.52±2.04 49.45±2.13 56.68±5.07 61.31±564
组合物1 44.31±1.03 47.68±2.18* 54.05±2.27** 56.29±2.49**
组合物2 44.24±2.46 48.81±1.48* 54.72±3.10** 57.39±1.71**
组合物3 44.03±1.58 48.39±1.87* 54.12±3.53** 56.40±2.61**
组合物4 46.81±2.07 45.29±2.33* 51.26±2.57** 53.47±2.88**
组合物5 45.22±1.99 46.87±1.57* 55.67±3.08** 57.04±2.47**
*与模型组比较P＜0.05；**与模型组比较P＜0.01；
4.2各受试物对肝脏指数的影响
如表4所示，与正常组比较，模型组小鼠体重，肝脏湿重及肝脏指数均明显增大(P＜0.01)，各受试物均能明显降低小鼠肝脏湿重及肝脏指数(P＜0.01)。
表4各受试物对小鼠肝脏指数的影响
组别 体重(g) 肝湿重(g) 肝指数％
正常 27.93±1.53 1.12±0.11 4.17±0.35
模型 63.97±2.97 4.51±0.49 7.06±0.74
水飞蓟宾磷脂复合物 63.38±2.54 3.74±0.30** 5.91±0.48**
组合物1 58.55±2.21*** 2.710±0.37** 4.63±0.57**
组合物2 59.60±2.66** 2.90±0.63** 4.87±1.07**
组合物3 59.02±2.59*** 2.74±0.24** 4.64±0.38**
组合物4 54.66±2.38*** 2.235±0.47** 4.09±0.97**
组合物5 59.01±2.75*** 2.667±0.28** 4.52±0.88**
*与模型组比较P＜0.05；**与模型组比较P＜0.01；
4.3各受试物对血脂、肝功能及胰岛素抵抗指数的影响
如表5所示，与正常组比较，非酒精性脂肪肝模型小鼠血清TC、LDL、ALT、AST及胰岛素抵抗指数均明显升高(P＜0.05)；水飞蓟宾磷脂复合物各异常升高指标均未见明显的改善(P＞0.05)；不同比例的水飞蓟宾磷脂复合物、普洱茶提取物、左旋肉碱配伍组，除组合物5对血清AST未见明显改善外(P＞0.05)，其他各组合物均可显著降低TC、LDL-C、ALT、AST及胰岛素抵抗指数(P＜0.05)，且效果优于水飞蓟宾磷脂复合物、普洱茶提取物单独使用。
表5各受试物对小鼠血脂、肝功能及胰岛素抵抗指数的影响
组别 TC LDL-C ALT AST 胰岛素抵抗指数
正常 2.93±0.38 0.26±0.05 40.00±9.70 96.00±14.01 0.62±0.08
模型 9.97±1.04 2.19±0.53 336.48±94.15 312.00±76.26 1.25±0.24
水飞蓟宾磷脂复合物 10.17±1.18 2.25±0.32 310.93±65.87 327.85±90.80 1.15±0.26
组合物1 6.56±0.89** 1.05±0.15** 217.11±109.25* 190.02±22.29** 0.89±0.13**
组合物2 8.90±1.02* 1.73±0.32* 260.00±41.34* 172.23±36.88** 0.97±0.29**
组合物3 7.45±1.32** 1.33±0.14** 250.93±60.02* 201.24±29.07** 0.93±0.11**
组合物4 6.74±1.05** 1.10±0.11** 195.64±62.02** 158.29±37.78** 0.77±0.20**
组合物5 7.72±1.07** 1.28±0.14** 237.54±91.63* 258.89±35.96 0.92±0.18**
*与模型组比较P＜0.05；**与模型组比较P＜0.01；
4.4各受试物对小鼠肝脏病理的影响
油红O染色：根据肝冰冻组织油红O染色光镜下肝细胞内红色颗粒的大小与多少，分为轻度、中度、重度三型。轻度，即光镜下每单位面积见1/3-2/3的红色颗粒，评为1分；中度，即2/3以上的肝细胞内含红色颗粒，评为2分；重度，即几乎所有的肝细胞内均有红色颗粒，评为3分；未见脂肪变性评为0分。
如表6所示，模型组肝脏组织几乎所有的肝细胞均发生脂肪变性，病理评分较正常组明显升高(P＜0.01)；单独使用水飞蓟宾磷脂复合物或普洱茶提取物对肝脏病理评分未见明显改善(P＞0.05)；不同比例的普洱茶提取物、水飞蓟宾磷脂复合物、左旋肉碱搭配使用可明显改善肝脏脂肪变性，降低病理评分(P＜0.05)，效果优于水飞蓟宾磷脂复合物、普洱茶提取物单独使用。
表6各受试物对小鼠肝脏病理的影响
组别 油红O染色病理评分
正常 0.000±0.000
模型 2.889±0.333
水飞蓟宾磷脂复合物 2.500±0.535
普洱茶提取物 2.429±0.787
组合物1 2.222±0.667*
组合物2 2.250±0.707*
组合物3 2.300±0.675*
组合物4 1.750±0.707**
组合物5 1.889±0.782**
*与模型组比较P＜0.05；**与模型组比较P＜0.01；
5实验结论
以上实验结果表明：与空白组相比，非酒精性脂肪肝模型组小鼠体重、肝脏指数、血脂、ALT、AST、胰岛素抵抗指数均明显升高，肝脏组织重度脂肪变性。普洱茶可改善胰岛素抵抗、调节血脂，左旋肉碱也具有较好的降血脂功效，同时联合水飞蓟宾较强的自由基清除和抗氧化应激能力，三者配合使用对肝脏脂肪变性具有明显的改善，效果优于二者单独使用。
附图说明
图1为体外释放曲线，其中，各样品为：参比制剂水林佳，无普洱茶的水林佳，实施例16～20制备的水飞蓟宾‐磷脂‐普洱茶组合物。
具体实施例
通过以下具体实施例对本发明作进一步的说明，但不作为本发明的限制。
实施例1
取水飞蓟宾26.25g、大豆磷脂45g、普洱茶提取物75g、左旋肉碱15.6g。
①水飞蓟宾复合液的制备：称取处方量水飞蓟宾、大豆磷脂溶解到无水乙醇中，加热回流使溶液澄清后继续加热1小时，减压浓缩回收乙醇至原体积的15％，备用；
②制粒：称取处方量普洱茶提取物作为底料，以①中制备得水飞蓟宾复合液为料液，以流化床流化喷液方式制粒，控制物料温度为40摄氏度，待液态复合物全部喷入后，干燥20min，干燥温度为60摄氏度，备用；
③将处方量的左旋肉碱与步骤②制得的颗粒按等量递增方式混合均匀，装袋，制成1000袋颗粒。
实施例2
取水飞蓟宾180g、大豆磷脂195g、普洱茶提取物450g、左旋肉碱180g。
①水飞蓟宾复合液的制备：称取处方量水飞蓟宾、大豆磷脂溶解到无水乙醇中，加热回流使溶液澄清后继续加热1.5小时，减压浓缩回收乙醇至原体积的20％，备用；
②制粒：称取处方量普洱茶提取物作为底料，以①中制备得水飞蓟宾复合液为料液，以流化床流化喷液方式制粒，控制物料温度为65摄氏度，待液态复合物全部喷入后，干燥60min，干燥温度为65摄氏度，备用；
③将处方量的左旋肉碱与步骤②制得的颗粒按等量递增方式混合均匀，装袋，制成1000袋颗粒。
实施例3
取水飞蓟宾26.25g、磷脂195g、普洱茶提取物450g、左旋肉碱15.6g。
①水飞蓟宾复合液的制备：称取处方量水飞蓟宾、大豆磷脂溶解到无水乙醇中，加热回流使溶液澄清后继续加热0.5小时，减压浓缩回收乙醇至原体积的5％，备用；
②制粒：称取处方量普洱茶提取物作为底料，以①中制备得水飞蓟宾复合液为料液，以流化床流化喷液方式制粒，控制物料温度为50摄氏度，待液态复合物全部喷入后，干燥10min，干燥温度为55摄氏度，备用；
③将处方量的左旋肉碱与步骤②制得的颗粒按等量递增方式混合均匀，装袋，制成1000袋颗粒。
实施例4
取水飞蓟宾26.25g、磷脂195g，普洱茶提取物75g,左旋肉碱180g,按照实施例1的方法制备，制成1000袋颗粒。
实施例5
取水飞蓟宾180g、磷脂45g、普洱茶提取物75g,左旋肉碱15.6g,按照实施例1的方法制备，制成1000袋颗粒。
实施例6
取水飞蓟宾180g、磷脂45g、普洱茶提取物450g,左旋肉碱180g,按照实施例1的方法制备，制成1000袋颗粒。
实施例7组合物的制备
取水飞蓟宾180g、磷脂195g、普洱茶提取物75g,左旋肉碱180g,按照实施例1的方法制备，制成1000袋颗粒。
实施例8组合物的制备
取水飞蓟宾26.25g、磷脂48.75g、普洱茶提取物75g,左旋肉碱62.5g,按照实施例1的方法制备，制成1000袋颗粒。
实施例9组合物的制备
取水飞蓟宾26.25g、磷脂48.75g、普洱茶提取物300g,左旋肉碱62.5g,按照实施例1的方法制备，制成1000袋颗粒。
实施例10组合物的制备
取水飞蓟宾52.5g、磷脂97.5g、普洱茶提取物300g,左旋肉碱15.625g,按照实施例1的方法制备，制成1000袋颗粒。
实施例11组合物的制备
取水飞蓟宾75g、磷脂90g、普洱茶提取物240g,左旋肉碱105g,按照实施例1的方法制备，制成1000袋颗粒。
实施例12组合物的制备
取水飞蓟宾90g、磷脂108g、普洱茶提取物270g,左旋肉碱120g,按照实施例1的方法制备，制成1000袋颗粒。
实施例13组合物的制备
取水飞蓟宾105g、磷脂126g、普洱茶提取物300g,左旋肉碱125g,按照实施例1的方法制备，制成1000袋颗粒。
实施例14组合物的制备
取水飞蓟宾105g、磷脂195g、普洱茶提取物300g,左旋肉碱125g,按照实施例1的方法制备，制成1000袋颗粒。
实施例15组合物的制备
取水飞蓟宾120g、磷脂150g、普洱茶提取物360g,左旋肉碱150g,按照实施例1的方法制备，制成1000袋颗粒。
实施例16组合物的制备
取水飞蓟宾105g、磷脂126g、普洱茶提取物300g,左旋肉碱酒石酸盐226.6g,按照实施例1的方法制备，制成1000袋颗粒。
实施例17
取实施例8的颗粒，加入微晶纤维素523g，羧甲基淀粉钠64g，混合均匀，装入胶囊，制成1000粒。
实施例18
取实施例9的颗粒，加入微晶纤维素298g，羧甲基淀粉钠64g，混合均匀，装入胶囊，制成1000粒。
实施例19
取实施例10的颗粒，加入微晶纤维素270g，羧甲基淀粉钠64g，混合均匀，装入胶囊，制成1000粒。
实施例20
取实施例13的颗粒，加入微晶纤维素144g，混合均匀，装入胶囊，制成1000粒。
实施例21
取实施例14的颗粒，加入微晶纤维素11g，羧甲基淀粉钠64g，混合均匀，装入胶囊，制成 1000粒。
实施例22
取实施例8的组合物，加入乳糖400g，淀粉123g，羧甲基淀粉钠64g，混合均匀，装入胶囊，制成1000粒。
实施例23
取实施例13的组合物，加入乳糖70g，滑石粉10g，低取代羟丙基纤维素64g，混合均匀，装入胶囊，制成1000粒。
实施例24
取实施例13的组合物，加入微晶纤维素144g，混合均匀，压片，制成1000片。

Claims (10)

  1. 一种药物组合物，其是由下述重量比的原料药制成
    Figure PCTCN2016077037-appb-100001
  2. 如权利要求1所述的一种药物组合物，其是由下述重量比的原料药制成
    Figure PCTCN2016077037-appb-100002
  3. 如权利要求2所述的一种药物组合物，其是由下述重量比的原料药制成
    Figure PCTCN2016077037-appb-100003
  4. 如权利要求1-3任意一项所述的药物组合物，其特征在于，所述的左旋肉碱还可以是左旋肉碱酒石酸盐。
  5. 一种药物制剂，其特征在于，包括如权利要求1-3任意一项所述的药物组合物，和药学上可接受的载体；
    优选药学上可接受的载体以重量计可以是制剂总重量的0.1-99.9％；
    优选药学上可接受的载体以重量计可以是制剂总重量的40-70％。
  6. 如权利要求5所述的制剂，其特征在于，所述的载体包括甘露醇、山梨醇、山梨酸或钾盐、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素A、维生素C、维生素E、维生素D、氮酮、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、丙二醇、乙醇、土温60-80、司班-80、蜂蜡、羊毛脂、液体石蜡、十六醇、没食子酸酯类、琼脂、三乙醇胺、碱性氨基酸、尿素、尿囊素、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β－环糊精、磷脂类 材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁、微晶中一种或以上；优选所述的载体为微晶纤维素、乳糖、淀粉、羧甲基纤维素钠、低取代羟丙基纤维素、滑石粉中一种或以上。
  7. 如权利要求5所述的制剂，其特征在于，所述的制剂为片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、丸剂、散剂、膏剂、丹剂、混悬剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、贴剂任意一种；优选所述的制剂为胶囊剂、颗粒剂或片剂。
  8. 如权利要求5所述制剂的制备方法，其特征在于：包括如下步骤：
    ①取处方量的原料备用；
    ②水飞蓟宾复合液的制备：称取处方量水飞蓟宾、磷脂溶解到无水乙醇中，加热回流使溶液澄清后再继续加热一定时间，再将澄清溶液减压浓缩至一定体积，得到水飞蓟宾复合液，备用；
    ③制粒：称取处方量普洱茶提取物作为底料，以②中制备得水飞蓟宾复合液为料液，以流化床流化喷液方式制颗粒，待液态复合物全部喷入后，干燥，备用；
    ④总混：左旋肉碱与步骤③颗粒按等量递增方式混合均匀即得药物组合物；
    ⑤制剂：取药物组合物与药学上可接受的载体制成常规制剂。
  9. 权利要求8的方法，其特征在于，步骤②中所述的加热时间为0.5～1.5小时；所述的浓缩的体积为原体积的5％～20％，减压浓缩温度为60～80℃；
    步骤③中流化床的参数为：物料温度40～65℃，制粒过程调节风机频率，进风温度和输液频率等参数，保持物料良好流化状态；制粒完成后，干燥10-60分钟，干燥温度为55～65℃。
  10. 权利要求1-3任意一项所述的药物组合物或权利要求5所述制剂在制备治疗非酒精性脂肪肝和/或减肥降脂、美容养颜药物中的应用。
PCT/CN2016/077037 2015-03-23 2016-03-22 一种含有水飞蓟宾、左旋肉碱的药物组合物 WO2016150378A1 (zh)

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