WO2012113117A1 - Oral preparation comprising protein or polypeptide, preparation method thereof, and use thereof - Google Patents

Oral preparation comprising protein or polypeptide, preparation method thereof, and use thereof Download PDF

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Publication number
WO2012113117A1
WO2012113117A1 PCT/CN2011/000815 CN2011000815W WO2012113117A1 WO 2012113117 A1 WO2012113117 A1 WO 2012113117A1 CN 2011000815 W CN2011000815 W CN 2011000815W WO 2012113117 A1 WO2012113117 A1 WO 2012113117A1
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weight
parts
emulsion
fatty acid
polyethylene glycol
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PCT/CN2011/000815
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French (fr)
Chinese (zh)
Inventor
马二利
郑昌学
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美迪思生物科技(北京)有限公司
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Publication of WO2012113117A1 publication Critical patent/WO2012113117A1/en

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    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/23—Calcitonins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH] (Somatotropin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention is in the field of pharmacy and formulation, and relates to an oral preparation containing a protein or a polypeptide, a preparation method thereof and use thereof. Background technique
  • Insulin in, parathyroid hormone, ca lci tonin, growth hormone, incretin-like peptide-1 (GLP-1) and other protein or peptide drugs clinical An important drug in treatment.
  • GLP-1 incretin-like peptide-1
  • other protein or peptide drugs clinical An important drug in treatment.
  • a common feature of these drugs is that oral administration is almost ineffective and requires frequent injections.
  • Protein shield polypeptides limit their use due to the lack of oral administration routes in the clinic.
  • the degradation of proteases in the gastrointestinal tract and the intestinal epithelial barrier function limit the oral bioavailability of these drugs.
  • researchers have used strategies such as covalent modification, enzyme inhibitors, absorption enhancers, microsphere microspheres, and W/0 (water-in-oil) emulsions to address the low oral bioavailability of protein peptides. .
  • Car ino GP et al (2000) J. Control. Release. 65: 261-269; Kathryn Wehi tehead, et al (2004) J. Control. Release. 98: 37-45; Yan Pan, et al (2002) Int J.
  • covalent modification may reduce the activity of the drug, and may change the pharmacology and toxicology of the drug molecule, resulting in high preparation cost and serious safety hazard; the enzyme inhibitor can effectively prevent the hydrolysis of the protease,
  • enzyme inhibitors also affect the digestion of food proteins. Long-term use will cause digestion and absorption disorders and even pancreatic enlargement or hyperplasia; absorption enhancers may cause irreversible damage to the cell membrane while increasing intestinal permeability. Long-term use can lead to membrane poisoning; micro-sphere microsphere preparation engineering often requires the participation of organic solvents, and requires high-speed centrifugation and other steps, increasing the complexity of the process.
  • the drug used has a molecular weight of less than 700 Daltons, and the drug can be bioavailable up to 2% after oral administration, with protein
  • P. Constantinides obtained a combination of high HLB (hydrophilic-lipophilic balance) surfactants and low HLB surfactants to obtain different hydrophilic molecules including RGD, descending hormone, and insulin.
  • the W/0 formulation was emulsified, but its oral bioavailability effect was not reported publicly.
  • Mao-Bo Cheng et al. prepared an oral W/0 microemulsion preparation containing guanidine kinase (Mao-Bo Cheng, et al (2008) J. Control. Release. 129: 41-48).
  • microemulsion as a carrier to achieve the encapsulation of proteins or peptide drugs.
  • these preparations have poor emulsifying effect, large difference in particle size, and stratification of the emulsion after emulsification (after dilution); or the clear area is narrow and cannot be uniformly dispersed after dilution, thereby limiting its oral biology. Utilization effect. It may even cause emulsion demulsification, protein peptide molecules encapsulated in the inner aqueous phase will be released quickly, degraded by proteases in the gastrointestinal tract, or incompatible with substances such as medium chain fatty acid esters.
  • the protein peptide molecules are flocculated and precipitated, resulting in loss of drug activity and difficulty in being absorbed by the body. Moreover, after oral administration of these products, the drug has a slow onset of action, and it usually takes 4 hours or even 8 hours to reach the peak plasma concentration, which is greatly different from the existing drugs, which brings inconvenience to clinical application (G. Sharma , et al (2010) EUR. J. PHARM. BIOPHARM. 76: 159-169; Amani El sayed, et al (2009) EUR. J. PHARM. BIOPHARM. 73: 269 - 279 ) 0 SUMMARY
  • the inventors have surprisingly found through a large amount of trials and creative labor that the compatibility of the obtained protein or polypeptide-containing emulsion is obtained by selecting a high HLB surfactant and a medium chain fatty acid ester (especially when a phospholipid is added). It is very stable and the emulsion maintains excellent bioavailability while maintaining excellent emulsifying properties. Moreover, when the emulsion is diluted by water, it can be uniformly dispersed, and the generated droplet size distribution is relatively uniform, which does not cause the emulsion to be unclear. Moreover, the emulsion of the invention can be quickly absorbed after oral administration, and has a blood concentration or a peak effect time similar to that of the similar injection drug, which will greatly facilitate clinical application.
  • An aspect of the invention relates to an emulsion comprising a protein or polypeptide, an aqueous phase, an oil phase, an emulsifier, characterized in that the oil phase contains a medium chain fatty acid ester (for example, a medium chain) a fatty acid glyceride), and the emulsifier comprises an emulsifier having an HLB value greater than 12; Specifically, the oil phase is a medium chain fatty acid ester (for example, a medium chain fatty acid glyceride), and the emulsifier is an emulsifier having an HLB value of more than 12.
  • the protein or polypeptide is an active ingredient (medicinal ingredient) or an active ingredient of the emulsion.
  • the protein or polypeptide may be isolated from a living organism, or may be obtained by genetic engineering means such as genetic recombination or chemical synthesis.
  • the protein or polypeptide is calcitonin, insulin, GLP-1, parathyroid hormone, parathyroid hormone 1-84, parathyroid hormone 1- 34, or growth hormone.
  • the calcitonin comprises salmon, salmon, and human calcitonin; the insulin, including pig, cow, human, and recombinant insulin; the GLP-1, including recombinant and synthetic GLP-1; the parathyroid hormone, including human parathyroid hormone 1-84, recombinant and/or synthetic parathyroid hormone 1-34; the long hormones, including pig, cow, human body and recombination Growth hormone.
  • the medium chain fatty acid ester is a medium chain fatty acid glyceride, a pegylated medium chain fatty acid glyceride, an acetylated medium chain fatty acid glyceride, a medium chain fatty acid Propylene glycol ester, medium chain fatty acid butylene glycol ester, or medium chain fatty acid polyethylene glycol ester.
  • the medium chain fatty acid glyceride may be one or more of a medium chain fatty acid monoglyceride, a medium chain fatty acid diglyceride, and a medium chain fatty acid triglyceride.
  • the medium chain fatty acid in the medium chain fatty acid glyceride may be bonded to any one or more hydroxyl positions in the glycerol molecule, and when forming a medium chain fatty acid diglyceride or a medium chain fatty acid triglyceride, the hydroxyl group in the glycerol molecule
  • the bound medium chain fatty acid molecules can be the same or different.
  • the medium chain fatty acid means a fatty acid having 6 to 12 carbon atoms, which may be saturated or unsaturated, and may be linear or branched.
  • the medium chain fatty acid is octanoic acid and/or citric acid.
  • the medium chain fatty acid ester is a carbon number a monoglyceride, a diglyceride, and/or a triglyceride of a fatty acid of 6 to 12; more specifically, a glyceryl caprylate, a polyglyceryl octanoate/capric glyceride, a caprylic acid glyceride, a hydrazine One or more of the acid glycerides.
  • the emulsion according to any one of the present invention, wherein the emulsifier having an HLB value of more than 12 is selected from the group consisting of poloxamer 188, poloxamer 407, Tween 20, Tween 80, and polyethylene glycol.
  • the emulsifier having an HLB value of more than 12 is selected from the group consisting of poloxamer 188, poloxamer 407, Tween 20, Tween 80, and polyethylene glycol.
  • the medium-chain fatty acid glycerol for example, medium-chain fatty acid glycerol
  • the emulsifier having an HLB value of more than 12 is from 20 to 2000 parts by weight; specifically, the protein or polypeptide is from 0.01 to 0.5 parts by weight. 01 ⁇
  • the weight of the protein is 0. 01 - 0. 3 parts by weight, the protein or the polypeptide is 0. 01 - 0.
  • the emulsifier having an HLB value of more than 12 is 50 - 1 000 parts by weight;
  • the medium chain fatty acid ester (for example, a medium chain fatty acid glyceride) is from 100 to 300 parts by weight, and the emulsifier having an HLB value of more than 12 is from 50 to 500 parts by weight.
  • the emulsion further contains a phospholipid, and the molar ratio of the phospholipid to the protein or polypeptide is from 10:1 to 1 000:1.
  • the molar ratio of the phospholipid to the protein or polypeptide is preferably from 20:1 to 500:1, more preferably from 60:1 to 200:1.
  • the phospholipid is preferably soy lecithin and/or egg yolk lecithin.
  • the emulsion according to any one of the present invention which further comprises a pharmaceutically acceptable excipient such as a dispersion medium, an isotonic agent, an antioxidant, a lyoprotectant, or a pH adjuster.
  • a pharmaceutically acceptable excipient such as a dispersion medium, an isotonic agent, an antioxidant, a lyoprotectant, or a pH adjuster.
  • dispersion medium is selected from the group consisting of Chitosan, silica, magnesium aluminum silicate, sodium carboxymethyl cellulose, crosslinked cellulose sodium, croscarmellose sodium, magnesium sulfate, magnesium stearate, polyethylene glycol 400, and One or more of glycerin.
  • the antioxidant is selected from the group consisting of L-cysteine hydrochloride, sodium sulfite, sodium hydrogen sulfite, propyl gallate, glutathione, sodium metabisulfite, One or more of potassium metabisulfite, and vitamin E.
  • lyoprotectant is selected from one or more of sucrose, lactose, trehalose, glucose, glycine, arginine, and xylitol.
  • pH adjusting agent is selected from the group consisting of hydrochloric acid, sulfuric acid, lactic acid, malic acid, acetic acid, citric acid, phosphoric acid, sodium hydroxide, sodium carbonate, sodium hydrogencarbonate One or more of disodium hydrogen phosphate, and sodium dihydrogen phosphate.
  • Microemulsion is an isotropic, transparent, thermodynamically stable dispersion consisting of water, oil, or emulsifier, usually having a particle size of less than 500 nm.
  • An emulsion according to any one of the invention which is administered orally.
  • the emulsion according to any one of the present invention the components and contents thereof are as follows: Recombinant human insulin 0. 008 - 0. 012 parts by weight Hydrochloric acid
  • physiological saline 0. 4 - 0. 6 parts by weight of medium chain fatty acid triglycerides (eg Crodamol ⁇ & ⁇
  • the present invention also relates to the use of the emulsion for the preparation of a medicament for treating or preventing diabetes, or lowering blood sugar.
  • the invention further relates to a method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion.
  • the components and levels of the emulsion are as follows:
  • Polyethylene glycol dodecyl stearate for example
  • Vitamin E 0.1 The emulsion according to any one of the present invention, the components and contents thereof are as follows:
  • Poloxamer 188 eg Lutrol F68, BASF
  • soy lecithin eg Epikuron 170
  • the present invention also relates to the use of the emulsion in the preparation of a medicament for treating or preventing diabetes, or lowering blood sugar.
  • the invention further relates to a method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion.
  • the components and levels of the emulsion are as follows:
  • Polyethylene glycol octanoate / glyceryl citrate for example
  • Poloxamer 188 eg Lutrol F68, BASF 0.2 parts by weight
  • Soy lecithin eg Epikuron 170
  • PTH 1-34 0.0008-0.0012 parts by weight PBS buffer 0.4-0.6 parts by weight glyceryl caprylate (eg Miglyol ® 812,
  • Polyethylene glycol dodecyl stearate for example
  • Tocopheryl succinate polyethylene glycol ester eg TPGS
  • the present invention also relates to the use of the emulsion in the preparation of a medicament for treating or preventing osteoporosis.
  • the invention further relates to a method of treating or preventing osteoporosis comprising the step of administering an effective amount of the emulsion.
  • the components and levels of the emulsion are as follows:
  • Caprylic glyceryl citrate (eg Miglyol ® 812,
  • Polyethylene glycol dodecyl stearate for example
  • Tocopheryl succinate polyethylene glycol ester eg TPGS
  • Vitamin E 0.1 parts by weight.
  • GLP-1 0.08-0.12 parts by weight Deionized water 55 - 65 parts by weight Caprylic glyceryl citrate (eg Miglyol ® 812,
  • Poloxamer 188 eg Lutrol F68, BASF
  • Soybean egg fat eg Epikuron 170
  • Glycerol 8- 12 parts by weight Polyethylene glycol 400 6-8 parts by weight
  • the present invention also relates to the use of the emulsion in the preparation of a medicament for treating or preventing diabetes, or lowering blood sugar.
  • the invention further relates to a method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion.
  • the components and levels of the emulsion are as follows:
  • Poloxamer 188 eg Lutrol F68, BASF 1.5 parts by weight
  • Soybean egg fat (eg Epikuron 170,
  • An emulsion according to any one of the invention which is a lyophilized emulsion (lyophilized formulation).
  • a lyophilized preparation of the above emulsion can be prepared, for example, by adding an appropriate amount of a lyoprotectant to the above emulsion, and then removing the moisture by freeze-drying (for example, temperature - 40 ° C, working vacuum of 30 Pa). Thereby obtaining a dried protein or polypeptide lyophilized emulsion. After the freezing, the emulsion does not contain a water phase, and can be formed into a final dosage form such as a tablet or a capsule.
  • the method of preparing the emulsion of the present invention for example 1 using the following steps:
  • step 1) Add the product of step 1) to the product of step 2) at 4 - 37 ° C, and continue stirring at 200 - 800 rpm until a clear and clear homogeneous preparation is formed, and finally a protein or peptide containing drug is obtained.
  • the component dissolved in the aqueous phase further comprises a pH adjusting agent and/or an isotonic agent.
  • step B it is possible to prepare the phospholipid complex for those skilled in the art.
  • the following method is used: the phospholipid and the protein are respectively dissolved in the organic phase and the aqueous phase, and after mixing, freeze-dried (-40 to -150 ° C, or even lower; pressure is lower than -100 Pa, lower than -200 Pa , or less than -300 Pa; lyophilization time of 12 hours or more, such as 12 - 24 hours, or more than 24 hours, such as 24-48 hours), to obtain a protein phospholipid complex.
  • the phospholipids and eggs The white matter or polypeptide molar ratio is from 10:1 to 1000:1.
  • the molar ratio of the phospholipid to the protein or polypeptide is more preferably from 20:1 to 500:1, further preferably from 60:1 to 200:1.
  • the phospholipid is preferably soy egg yolk and/or egg yolk lecithin.
  • the organic phase is dichloromethane, acetone, or dimethyl sulfoxide, preferably dimethyl sulfoxide.
  • the mixed mixture further includes deionized water; optionally, a dispersing shield and/or an antioxidant.
  • the preparation method further comprises the step 4): lyophilizing the emulsion prepared in the step 3) to obtain a lyophilized emulsion.
  • the lyophilization step described in step B) and step 4) may employ the lyoprotectant described herein.
  • the term "mice l les" refers to a molecularly ordered aggregate of a hydrophobic group as a core and a hydrophilic group as a shell formed by association of several solute molecules or ions in a solution. Micellar is a colloidal dispersion system and belongs to a class of associative colloids. When the amphiphilic polymer substance is dispersed in a solution to a critical micelle concentration (CMC), the molecular association self-assembles to form a micelle.
  • CMC critical micelle concentration
  • a nonionic surfactant having an HLB value (hydrophilic-lipophilic balance) of 12 or more is used as an emulsifier
  • a medium-chain fatty acid ester is an oil phase
  • an aqueous solution containing a protein-peptide drug is an aqueous phase, and is prepared.
  • the remarkable feature of the invention is that the problem of emulsifying performance of the protein polypeptide emulsion is solved, the oral bioavailability of the protein or polypeptide drug can be significantly increased, the application prospect is very promising, and the preparation process is simple, and is suitable for industrial production, the dosage form of the product. It may be an emulsion, a micelle solution, a self-emulsifying administration system, a paste substance or a solid powder.
  • Fig. 1 Phase diagram of blood glucose in rats after ileal administration.
  • Fig. 2 PTH1-34 blood concentration.
  • Fig. 3 PTH1-34 efficacy test.
  • the four columnar figures from left to right represent the oral group, the injection group, the control group, and the sham operation group.
  • Example 1 Preparation of Oral Insulin Formulation (Sample 1)
  • Poloxamer 188 (Lutrol F68, BASF) 0.2 g
  • Soybean Eggs ( Epikuron 170, Degussa ) 0 ⁇ 5 grams
  • the preparation method of the preparation is as follows:
  • porcine insulin soy lecithin complex is added to the product in step 3) at room temperature, and the mixture is stirred at a speed of 200 r/min for 15 min;
  • step 5 After mixing the above weight of silica, crosslinked cellulose sodium and stearic acid at room temperature, the product of step 4) is added dropwise at a speed of 200 r/min, and then the rotation speed is increased to 300 r/min, and After 30 minutes of lyophilization (temperature - 40 ° C, working vacuum of 30 Pa), the oral preparation of insulin was finally obtained as a sample.
  • Sample 1 can be formulated into a final dosage form such as a tablet or a capsule.
  • Example 2 Preparation of an oral insulin preparation (Sample 2)
  • step 3 A solution was added dropwise to the product of step 2) at room temperature, and the mixture was stirred until clear and clear at 200 r/min, and finally an insulin oral preparation was obtained as sample 2.
  • Example 3 Preparation of recombinant human parathyroid hormone 1-34 oral preparation (Sample 3)
  • caprylic caproic acid glyceride polyethylene glycol dodecyl stearate, tocopherol succinate polyethylene glycol ester, Tween 20, vitamin E at 60 ° C To clear and clear;
  • step 3 At room temperature, add the A solution dropwise to the product of step 2), stir the mixture for 15 min at lOOr/min, and finally obtain the PTH 1-34 oral preparation as sample 3.
  • Example 4 Preparation of GLP-1 Oral Formulation (Sample 4)
  • Soybean egg fat (Epikuron 170, Degussa) 7 g
  • Vitamin E 0.3 g
  • the preparation method of the preparation is as follows:
  • GLP-1 soybean phospholipid complex 1) The above weight of GLP-1 is dissolved in 5 g of deionized water at room temperature, and the above-mentioned weight of soybean lecithin is dissolved in 30 ml of dichlorosilane. After mixing the two phases, it is placed in liquid nitrogen for 30 min, and the freezing is performed. Dry, the obtained product is GLP-1 soybean phospholipid complex;
  • step 3 At room temperature, at 500r / min, the GLP-1 soy lecithin complex is added to the product of step 2), stirring is continued for 5min, to obtain colostrum B;
  • Example 4 Stability study of samples 2, 3, and 4.
  • the stability of the oral preparation samples 2, 3, and 4 was determined by visually observing whether the preparation exhibited flocculent substances or produced an "oil-water" interface.
  • the stability of insulin in samples 1, 2 was studied by high performance liquid chromatography.
  • the color meter used was the Agilent 1200.
  • the mobile phase is a linear gradient elution, in which mobile phase A is acetonitrile and mobile phase B is 0.05 mol/L sodium dihydrogen phosphate (pH 3.0): 0 - lOmin mobile phase A is increased from 20% to 50%, mobile phase B Change from 80% to 50%; 11 - 15min mobile phase A changes from 50% to 80%, mobile phase B changes from 50% to 20%; 21 - 25min mobile phase A changes from 80% to 20%, mobile phase B Change from 20% to 80%.
  • the flow rate is 1.0 mL/min
  • the detection wavelength is 214 nm
  • the column temperature is 40° (:.
  • sample 2 preparation, sample 3 preparation, and sample 4 preparation of C did not produce macroscopic flocculent substances, nor did they produce an "oil-water" interface.
  • the resulting preparation was placed in a refrigerator at 4 ° C, and the drug content was determined at 0 days, 3 months, and 6 months after the placement.
  • HPLC HPLC method was used to determine the content of active pharmaceutical ingredients. The results are shown in Table 1.
  • Example 2 Samples prepared in Example 3 2, 3, placed in a refrigerator at 4 ° C, and measured the particle size and appearance of the emulsion at 0 days, the third month, and the sixth month.
  • the particle size was measured with a Malvern Zetasizer 3000HSA particle size analyzer. Before the measurement, samples 2 and 3 were diluted 5, 30, and 100 times with Millipore ultrapure water, and the appearance of each dilution was visually determined. The results are shown in Table 2 below.
  • Test group 1 Male SD rats weighing 180-220 g, 6 rats, were administered the sample 1 prepared in Example 1.
  • Test group 2 Male SD rats having a body weight of 180-220 g, 6 rats, were administered the sample 2 prepared in Example 2.
  • Control group Male Sprague-Dawley rats weighing 180-220 g, 6 doses For insulin aqueous solution, subcutaneous injection.
  • mice Male Sprague-Dawley rats weighing 180-220 g were fasted for 12 h and the test was started; the animals were anesthetized with 50 mg of sodium pentobarbital before the experiment. After the animals were anesthetized, they were tied in a supine position, and a small opening was made in the abdomen, and the ileum was administered by a dispenser. After administration, the incision was sutured, and at the time of 0.5, 1, 2, 3, and 4 h, blood was taken from the tail vein, blood glucose was measured using a Roche blood glucose meter, and the average blood glucose level of 6 rats in each group was calculated.
  • test group 1 is administered as sample 1, according to insulin, the dose is 0.3 mg/kg; the test group 2 is administered as sample 2, and the insulin is administered at a dose of 1 mg/kg; 02mg/ ⁇ The drug was administered in a dose of 0. 02mg / kg.
  • Figure 1 shows that both Sample 1 and Sample 2 are effective in reducing blood glucose in rats.
  • the blood glucose concentration was reduced to the lowest value 1 hour after the administration.
  • Example 9 Gavage absorption test of PTH1-34 mouth Yueliang preparation
  • Test group Male Sprague-Dawley rats weighing from 180 to 220 g, 12, and the sample prepared in Example 3, were administered to the stomach.
  • Control group Male SD rats weighing 180-220 g, 10 rats, were administered PTH1-34 PBS solution and injected subcutaneously.
  • the test group was administered with stomach, and the control group was treated with PTH1-34.
  • Serum PTH 1-34 concentration with PTH 1-34 Sensitivity EIA kit (Alpco Di agnos t ic, Cat. No. 31-IPTHUU-E01) detected that the kit reacts exclusively with exogenous PTH1-34, but does not react with the rat itself, so it can be detected.
  • PTH1-34 which is ⁇ 1-34 into the blood of rats after administration. There were 12 animals in the gavage group and 10 animals in the injection group. After administration, the PTH1-34 plasma concentration changes as shown in Figure 2.
  • FIG. 2 shows that after intragastric administration, PTH1-34 can rapidly enter the rat body, and the plasma concentration peak time is very similar (the peak time of the blood concentration in the experimental group is 18.3 ⁇ 1. 2 minutes, control The bioavailability of the intragastric administration group relative to the injection group was 6.73%.
  • Example 10 Efficacy test of PTH1-34 oral preparation
  • Oral group Six female rats of 2 months old were selected, and the bilateral ovaries of the female rats were excised, and the sample prepared in Example 3 was administered and administered by oral gavage.
  • Injection group Six female rats of 2 months old were selected, and the bilateral ovaries of the female rats were excised, and the sample prepared in Example 3 was administered, PTH1-34 PBS injection, subcutaneously.
  • Control group Six female rats of 2 months old were selected, and the bilateral ovaries of the female rats were excised, and the sample 3 prepared in Example 3 was administered without administration.
  • Sham-operated group Five female rats of 2 months old were selected, the ovaries were preserved, and the same size of fat near the egg nest was removed, and no drug was administered.
  • Two-month-old female rats were randomly divided into the castration group (castrated animals, bilateral ovaries by surgery, including the oral group, the injection group, the control group) and the sham operation group for 6 weeks, followed by oral administration.
  • the group and the injection group were continuously administered for 8 weeks, administered 6 days a week, once a day, and the rats were fasted for 10 hours before administration.
  • BMD Bone mineral density
  • BMC bone mineral content

Abstract

An emulsion for oral administration comprising protein or polypeptide, a preparation method thereof, and the use thereof are disclosed. The emulsion comprises protein or polypeptide, water phase, oil phase and emulsifier, wherein the oil phase comprises medium-chain aliphatic ester, the emulsifier includes emulsifier with HLB value greater than 12. The emulsion can improve bioavailability. The emulsion can be uniformly dispersed and generates emulsion drops with uniform grain size when it is diluted with water.

Description

一种含有蛋白质或多肽的口服制剂、其制备方法及用途 技术领域 Oral preparation containing protein or polypeptide, preparation method and use thereof
本发明属于药物学和制剂学领域， 涉及一种含有蛋白质或多 肽的口服制剂、 其制备方法及用途。 背景技术  The present invention is in the field of pharmacy and formulation, and relates to an oral preparation containing a protein or a polypeptide, a preparation method thereof and use thereof. Background technique
胰岛素 ( insul in ) ， 甲状旁腺激素（parathyroid hormone) , 降 素（ca lci tonin) , 生长激素（growth hormone)， 肠促胰岛素样 肽 -1 ( GLP-1 ) 等蛋白质或多肽药物， 是临床治疗中的重要药物。 但这类药物的共同特点是口服给药几乎无效， 需要频繁的注射给 药。  Insulin in, parathyroid hormone, ca lci tonin, growth hormone, incretin-like peptide-1 (GLP-1) and other protein or peptide drugs, clinical An important drug in treatment. However, a common feature of these drugs is that oral administration is almost ineffective and requires frequent injections.
蛋白盾多肽类药物由于在临床中缺乏口服给药途径而限制了 它们的应用， 胃肠道中蛋白酶的降解以及肠上皮细胞屏障作用等多 种因素限制了这类药物的口服生物利用度。 几十年来， 研究人员采 用共价修饰、 酶抑制剂、 吸收促进剂、 微嚢微球包裹、 W/0 (油包 水）乳剂等策略， 以期解决蛋白质多肽类药物口服生物利用度低的 问题。 Car ino G. P. et al (2000) J. Control. Release. 65: 261-269; Kathryn Wehi tehead, et al (2004) J. Control. Release. 98: 37-45; Yan Pan, et al (2002) Int. J. Pharm 249: 139-147; Nerurkar M. M. et a l (1996) Pharm. Res. 13: 528-534; Marschutz M. K. et a l (2000) Biomateria l s 21: 1499-1507; Chri s topher J. H. et al (1997) Adv. Drug. Del iver. Rev. 25: 71-89; Jeff Wang, et a l (2003) J. Control. Release. 88: 369-380; Roger R. C. New, et al (1997) Adv. Drug. Del iver. Rev. 25: 59-69; Anja Graf, et al (2008) Int. J. Pharm. 350: 351-360; R. Nes l ihan Gursoy et al (2004) Biomed. Pharmacother. 58: 173-182; CN01115327. X, US4849405, US5824638。 Protein shield polypeptides limit their use due to the lack of oral administration routes in the clinic. The degradation of proteases in the gastrointestinal tract and the intestinal epithelial barrier function limit the oral bioavailability of these drugs. For decades, researchers have used strategies such as covalent modification, enzyme inhibitors, absorption enhancers, microsphere microspheres, and W/0 (water-in-oil) emulsions to address the low oral bioavailability of protein peptides. . Car ino GP et al (2000) J. Control. Release. 65: 261-269; Kathryn Wehi tehead, et al (2004) J. Control. Release. 98: 37-45; Yan Pan, et al (2002) Int J. Pharm 249: 139-147; Nerurkar MM et al (1996) Pharm. Res. 13: 528-534; Marschutz MK et al (2000) Biomateria ls 21: 1499-1507; Chri s topher JH et al (1997) Adv. Drug. Del iver. Rev. 25: 71-89; Jeff Wang, et al (2003) J. Control. Release. 88: 369-380; Roger RC New, et al (1997) Adv. Drug. Del Iver. Rev. 25: 59-69; Anja Graf, et al (2008) Int. J. Pharm. 350: 351-360; R. Nes l ihan Gursoy Et al (2004) Biomed. Pharmacother. 58: 173-182; CN01115327. X, US 4849405, US 5824638.
上述策略存在明显的不足， 如共价修饰会降低药物活性， 并可 能改变药物分子的药理、 毒理， 带来高昂的制备成本和严重的安全 隐患； 酶抑止剂能有效避免蛋白酶的水解作用， 但酶抑制剂同时也 影响了食物蛋白的消化， 长期服用将造成消化吸收紊乱甚至胰脏的 肿大或增生； 吸收促进剂在增加肠道通透性的同时， 有可能对细胞 膜造成不可逆的破坏， 长期服用会导致膜中毒； 微嚢微球制备工程 中常需要有机溶剂的参与， 并需要高速离心等步骤， 增加了工艺的 复杂性。  There are obvious deficiencies in the above strategies. For example, covalent modification may reduce the activity of the drug, and may change the pharmacology and toxicology of the drug molecule, resulting in high preparation cost and serious safety hazard; the enzyme inhibitor can effectively prevent the hydrolysis of the protease, However, enzyme inhibitors also affect the digestion of food proteins. Long-term use will cause digestion and absorption disorders and even pancreatic enlargement or hyperplasia; absorption enhancers may cause irreversible damage to the cell membrane while increasing intestinal permeability. Long-term use can lead to membrane poisoning; micro-sphere microsphere preparation engineering often requires the participation of organic solvents, and requires high-speed centrifugation and other steps, increasing the complexity of the process.
另外有研究表明， 中链脂肪酸可以作为底物引发级联反应并最 终打开细胞间紧密连接，从而增加肠道的渗透性（ Masahiro Hayashi: et al (1997) ADV. DRUG. DELIVER. REV. 28: 191-204. ) 。 Panayiostis等人以中链脂肪酸甘油酯作为吸收促进剂，研究了 W/0 型微乳制剂对亲水性小分子药物的吸收效果 （Panayiostis P. Constantinides, et al (1994) PHARM RES 11: 1385-90; Panayiostis P. Constantinides, et al (1996) PHARM RES 13: 210-215。 ) , 但其所用药物分子量不足 700道尔顿， 并且该类药 物直接口服后生物利用度可达 2%，与蛋白质多肽类药物存在本质差 别。 经过进一步的研究， P. Constantinides通过联合应用高 HLB 值（亲水亲油平衡值 )表面活性剂和低 HLB值表面活性剂， 获得了 能够包含 RGD、降 素、胰岛素等不同亲水分子的自乳化 W/0制剂， 但其口服生物利用度效果却没有公开的报道。 Mao-Bo Cheng等人制 备了含蚓激酶的口服 W/0型微乳制剂（Mao-Bo Cheng, et al( 2008 ) J. Control. Release. 129: 41 - 48 ) 。 In addition, studies have shown that medium-chain fatty acids can act as substrates to initiate cascades and eventually open tight junctions between cells, thereby increasing intestinal permeability (Mashiro Hayashi : et al (1997) ADV. DRUG. DELIVER. REV. 28: 191-204. ). Panayiostis et al. used medium-chain fatty acid glycerides as absorption enhancers to study the absorption of hydrophilic small molecule drugs by W/0 microemulsions (Panayiostis P. Constantinides, et al (1994) PHARM RES 11: 1385- 90; Panayiostis P. Constantinides, et al (1996) PHARM RES 13: 210-215. ) , but the drug used has a molecular weight of less than 700 Daltons, and the drug can be bioavailable up to 2% after oral administration, with protein There are essential differences in peptide drugs. After further research, P. Constantinides obtained a combination of high HLB (hydrophilic-lipophilic balance) surfactants and low HLB surfactants to obtain different hydrophilic molecules including RGD, descending hormone, and insulin. The W/0 formulation was emulsified, but its oral bioavailability effect was not reported publicly. Mao-Bo Cheng et al. prepared an oral W/0 microemulsion preparation containing guanidine kinase (Mao-Bo Cheng, et al (2008) J. Control. Release. 129: 41-48).
上述文献以微乳为载体， 实现了对蛋白质或多肽药物的包裹， 但存在一个最大的问题就是这些制剂乳化效果差，粒径大小悬殊很 大， 并且乳化后 （稀释后） 乳液出现了分层； 或者清亮区域狭小， 稀释后不能均匀分散， 从而会限制其口服生物利用度效果。 甚至可 能导致乳滴破乳， 包裹于内层水相中的蛋白质多肽类分子会被快速 释放出来， 被胃肠道中的蛋白酶降解， 或与中链脂肪酸酯等物质由 于不相容性的问题， 蛋白质多肽类分子发生絮集、 沉淀， 造成药物 活性丟失且难以被机体吸收。 并且这些产品口服给药后药物起效 慢，通常需要 4个小时，甚至 8个小时以后才能达到血药浓度峰值， 与现有药物作用方式差别巨大， 为临床应用带来诸多不便 （G. Sharma , et a l (2010) EUR. J. PHARM. BIOPHARM. 76: 159-169; Amani El sayed, et a l (2009) EUR. J. PHARM. BIOPHARM. 73: 269 - 279 ) 0 发明内容 The above literature uses microemulsion as a carrier to achieve the encapsulation of proteins or peptide drugs. However, one of the biggest problems is that these preparations have poor emulsifying effect, large difference in particle size, and stratification of the emulsion after emulsification (after dilution); or the clear area is narrow and cannot be uniformly dispersed after dilution, thereby limiting its oral biology. Utilization effect. It may even cause emulsion demulsification, protein peptide molecules encapsulated in the inner aqueous phase will be released quickly, degraded by proteases in the gastrointestinal tract, or incompatible with substances such as medium chain fatty acid esters. The protein peptide molecules are flocculated and precipitated, resulting in loss of drug activity and difficulty in being absorbed by the body. Moreover, after oral administration of these products, the drug has a slow onset of action, and it usually takes 4 hours or even 8 hours to reach the peak plasma concentration, which is greatly different from the existing drugs, which brings inconvenience to clinical application (G. Sharma , et al (2010) EUR. J. PHARM. BIOPHARM. 76: 159-169; Amani El sayed, et al (2009) EUR. J. PHARM. BIOPHARM. 73: 269 - 279 ) 0 SUMMARY
本发明人通过大量的试验和创造性的劳动， 惊奇地发现， 通 过选用高 HLB 值表面活性剂和中链脂肪酸酯 （特别再添加磷脂 时） ， 得到的含有蛋白质或多肽的乳剂的相容性和稳定性很好， 乳剂在保持较高的生物利用度的同时， 保持优异的乳化性能。 并 且乳剂在被水稀释时， 能均勾分散， 生成的乳滴粒径分布比较均 匀， 不会导致乳液不清。 并且本发明的乳剂经口服给药后， 能被 快速吸收，有着与同类注射药物相近的血药浓度或药效达峰时间， 这将为临床应用带来极大便利。 由此提供了下述发明： 本发明的一个方面涉及一种乳剂， 包含蛋白质或多肽、 水相、 油相、 乳化剂， 其特征在于， 所述油相含有中链脂肪酸酯（例如中 链脂肪酸甘油酯），并且所述乳化剂包括 HLB值大于 12的乳化剂； 具体地， 所述油相为中链脂肪酸酯（例如中链脂肪酸甘油酯）， 所 述乳化剂为 HLB值大于 12的乳化剂。 The inventors have surprisingly found through a large amount of trials and creative labor that the compatibility of the obtained protein or polypeptide-containing emulsion is obtained by selecting a high HLB surfactant and a medium chain fatty acid ester (especially when a phospholipid is added). It is very stable and the emulsion maintains excellent bioavailability while maintaining excellent emulsifying properties. Moreover, when the emulsion is diluted by water, it can be uniformly dispersed, and the generated droplet size distribution is relatively uniform, which does not cause the emulsion to be unclear. Moreover, the emulsion of the invention can be quickly absorbed after oral administration, and has a blood concentration or a peak effect time similar to that of the similar injection drug, which will greatly facilitate clinical application. The invention thus provided provides: An aspect of the invention relates to an emulsion comprising a protein or polypeptide, an aqueous phase, an oil phase, an emulsifier, characterized in that the oil phase contains a medium chain fatty acid ester (for example, a medium chain) a fatty acid glyceride), and the emulsifier comprises an emulsifier having an HLB value greater than 12; Specifically, the oil phase is a medium chain fatty acid ester (for example, a medium chain fatty acid glyceride), and the emulsifier is an emulsifier having an HLB value of more than 12.
所述蛋白质或多肽为乳剂的有效成分（药效成分）或者活性 成分。 所述蛋白质或多肽可以从生物机体中分离获得， 也可以通 过基因工程手段（例如基因重组） 或化学合成的方式获得。  The protein or polypeptide is an active ingredient (medicinal ingredient) or an active ingredient of the emulsion. The protein or polypeptide may be isolated from a living organism, or may be obtained by genetic engineering means such as genetic recombination or chemical synthesis.
根据本发明的任一项所述的乳剂， 其中， 所述蛋白质或多肽 为降钙素、 胰岛素、 GLP-1、 曱状旁腺激素、 甲状旁腺激素 1-84、 甲状旁腺激素 1-34、 或生长激素。 在一些实施方案中， 所述降钙 素， 包括鲑鱼、 鰻鱼和人的降钙素； 所述胰岛素， 包括猪、 牛、 人和重组的胰岛素； 所述 GLP-1 , 包括重组和合成的 GLP-1 ; 所述 曱状旁腺激素， 包括人体甲状旁腺激素 1-84、 重组和 /或合成的 曱状旁腺激素 1-34 ; 所述长激素， 包括猪、 牛、 人体和重组的生 长激素。  The emulsion according to any one of the present invention, wherein the protein or polypeptide is calcitonin, insulin, GLP-1, parathyroid hormone, parathyroid hormone 1-84, parathyroid hormone 1- 34, or growth hormone. In some embodiments, the calcitonin comprises salmon, salmon, and human calcitonin; the insulin, including pig, cow, human, and recombinant insulin; the GLP-1, including recombinant and synthetic GLP-1; the parathyroid hormone, including human parathyroid hormone 1-84, recombinant and/or synthetic parathyroid hormone 1-34; the long hormones, including pig, cow, human body and recombination Growth hormone.
根据本发明的任一项所述的乳剂， 其中， 所述中链脂肪酸酯 为中链脂肪酸甘油酯、 聚乙二醇化的中链脂肪酸甘油酯、 乙酰化 中链脂肪酸甘油酯、 中链脂肪酸丙二醇酯、 中链脂肪酸丁二醇酯、 或中链脂肪酸聚乙二醇酯。 所述中链脂肪酸甘油酯可以为中链脂 肪酸甘油一酯、 中链脂肪酸甘油二酯、 以及中链脂肪酸甘油三酯 中的一种或多种。 所述中链脂肪酸甘油酯中的中链脂肪酸可以结 合在甘油分子中的任何一个或多个羟基位置， 当形成中链脂肪酸 甘油二酯或中链脂肪酸甘油三酯时， 甘油分子中的羟基上结合的 中链脂肪酸分子可以相同或者不同。 在本发明中， 中链脂肪酸是 指碳原子数 6 - 12的脂肪酸， 其可以是饱和的， 也可以是不饱和 的， 可以是直链的， 也可以是支链的。 具体地， 所述中链脂肪酸 为辛酸和 /或癸酸。  The emulsion according to any one of the present invention, wherein the medium chain fatty acid ester is a medium chain fatty acid glyceride, a pegylated medium chain fatty acid glyceride, an acetylated medium chain fatty acid glyceride, a medium chain fatty acid Propylene glycol ester, medium chain fatty acid butylene glycol ester, or medium chain fatty acid polyethylene glycol ester. The medium chain fatty acid glyceride may be one or more of a medium chain fatty acid monoglyceride, a medium chain fatty acid diglyceride, and a medium chain fatty acid triglyceride. The medium chain fatty acid in the medium chain fatty acid glyceride may be bonded to any one or more hydroxyl positions in the glycerol molecule, and when forming a medium chain fatty acid diglyceride or a medium chain fatty acid triglyceride, the hydroxyl group in the glycerol molecule The bound medium chain fatty acid molecules can be the same or different. In the present invention, the medium chain fatty acid means a fatty acid having 6 to 12 carbon atoms, which may be saturated or unsaturated, and may be linear or branched. Specifically, the medium chain fatty acid is octanoic acid and/or citric acid.
在本发明的一个实施方案中， 所述中链脂肪酸酯为碳原子数 6 - 12的脂肪酸的甘油一酯、 甘油二酯、 和 /或甘油三酯； 更具体 地， 为选自辛酸癸酸甘油酯、 聚乙二醇辛酸 /癸酸甘油酯、 辛酸甘 油酯、 癸酸甘油酯中的一种或多种。 In one embodiment of the invention, the medium chain fatty acid ester is a carbon number a monoglyceride, a diglyceride, and/or a triglyceride of a fatty acid of 6 to 12; more specifically, a glyceryl caprylate, a polyglyceryl octanoate/capric glyceride, a caprylic acid glyceride, a hydrazine One or more of the acid glycerides.
根据本发明的任一项所述的乳剂， 其中， 所述 HLB值大于 12 的乳化剂为选自泊洛沙姆 188、 泊洛沙姆 407、 吐温 20、 吐温 80、 聚乙二醇十二羟基硬脂酸酯、 聚氧乙烯醚（35)蓖麻油、 聚氧乙烯 醚（40)氢化蓖麻油、 生育酚琥珀酸聚乙二醇酯、 聚乙二醇 -32-月 桂酸甘油酯、 聚乙二醇- 32-硬脂酸甘油酯中的一种或多种。  The emulsion according to any one of the present invention, wherein the emulsifier having an HLB value of more than 12 is selected from the group consisting of poloxamer 188, poloxamer 407, Tween 20, Tween 80, and polyethylene glycol. Dodecyl stearate, polyoxyethylene ether (35) castor oil, polyoxyethylene ether (40) hydrogenated castor oil, tocopherol succinate polyethylene glycol ester, polyethylene glycol-32-lauric acid glyceride And one or more of polyethylene glycol-32-glyceryl stearate.
根据本发明的任一项所述的乳剂， 其中， 所述蛋白质或多肽 为 0. 01 - 1重量份， 所述中链脂肪酸酯（例如中链脂肪酸甘油酯） 为 5 0 - 1 000重量份， 所述 HLB值大于 12 的乳化剂为 20 - 2000 重量份； 具体地， 所述蛋白质或多肽为 0. 01 - 0. 5重量份， 所述 中链脂肪酸酯（例如中链脂肪酸甘油酯）为 1 00一 5 00重量份，所述 HLB值大于 12的乳化剂为 50 - 1 000重量份； 更具体地， 所述蛋 白质或多肽为 0. 01 - 0. 3重量份，所述中链脂肪酸酯（例如中链脂 肪酸甘油酯）为 1 00 - 300重量份，所述 HLB值大于 12的乳化剂为 5 0 - 5 00重量份。  The emulsion according to any one of the present invention, wherein the protein or polypeptide is 0.01 to 1 part by weight, and the medium chain fatty acid ester (for example, medium chain fatty acid glyceride) is 50 to 1 000 by weight. The medium-chain fatty acid glycerol (for example, medium-chain fatty acid glycerol), wherein the emulsifier having an HLB value of more than 12 is from 20 to 2000 parts by weight; specifically, the protein or polypeptide is from 0.01 to 0.5 parts by weight. 01重量。 The weight of the protein is 0. 01 - 0. 3 parts by weight, the protein or the polypeptide is 0. 01 - 0. 3 parts by weight, the emulsifier having an HLB value of more than 12 is 50 - 1 000 parts by weight; The medium chain fatty acid ester (for example, a medium chain fatty acid glyceride) is from 100 to 300 parts by weight, and the emulsifier having an HLB value of more than 12 is from 50 to 500 parts by weight.
根据本发明的任一项所述的乳剂， 优选地， 所述乳剂还含有 磷脂，并且磷脂与蛋白质或者多肽的摩尔比为 1 0 : 1至 1 000: 1。 所述磷脂与蛋白质或者多肽的摩尔比优选为 20: 1至 5 00: 1， 更 优选为 60: 1至 200 : 1。 所述磷脂优选为大豆卵磷脂和 /或蛋黄 卵磷脂。  The emulsion according to any one of the present invention, preferably, the emulsion further contains a phospholipid, and the molar ratio of the phospholipid to the protein or polypeptide is from 10:1 to 1 000:1. The molar ratio of the phospholipid to the protein or polypeptide is preferably from 20:1 to 500:1, more preferably from 60:1 to 200:1. The phospholipid is preferably soy lecithin and/or egg yolk lecithin.
根据本发明的任一项所述的乳剂， 其还包含药学上可接受的 辅料， 例如分散介质、 等渗剂、 抗氧化剂、 冻干保护剂、 或 pH 调节剂。  The emulsion according to any one of the present invention, which further comprises a pharmaceutically acceptable excipient such as a dispersion medium, an isotonic agent, an antioxidant, a lyoprotectant, or a pH adjuster.
根据本发明的任一项所述的乳剂， 其中， 所述分散介质选自 壳聚糖、 二氧化硅、 硅酸铝镁、 羧曱基纤维素钠、 交联纤维素钠、 交联羧曱基纤维素钠、 硫酸镁、 硬脂酸镁、 聚乙二醇 400、 和甘 油中的一种或多种。 The emulsion according to any one of the present invention, wherein the dispersion medium is selected from the group consisting of Chitosan, silica, magnesium aluminum silicate, sodium carboxymethyl cellulose, crosslinked cellulose sodium, croscarmellose sodium, magnesium sulfate, magnesium stearate, polyethylene glycol 400, and One or more of glycerin.
根据本发明的任一项所述的乳剂， 其中， 所述等渗剂选自木 糖醇、' 氯化钠、 果糖、 山梨醇、 和葡萄糖中的一种或多种。  The emulsion according to any one of the present invention, wherein the isotonic agent is one or more selected from the group consisting of xylitol, 'sodium chloride, fructose, sorbitol, and glucose.
根据本发明的任一项所述的乳剂， 其中， 所述抗氧化剂选自 L-半胱氨酸盐酸盐、 亚硫酸钠、 亚硫酸氢钠、 没食子酸丙酯、 谷 胱甘肽、 焦亚硫酸钠、 焦亚硫酸钾、 和维生素 E中的一种或多种。  The emulsion according to any one of the present invention, wherein the antioxidant is selected from the group consisting of L-cysteine hydrochloride, sodium sulfite, sodium hydrogen sulfite, propyl gallate, glutathione, sodium metabisulfite, One or more of potassium metabisulfite, and vitamin E.
根据本发明的任一项所述的乳剂， 其中， 所述的冻干保护剂 选自蔗糖、 乳糖、 海藻糖、 葡萄糖、 甘氨酸、 精氨酸、 和木糖醇 中的一种或多种。  The emulsion according to any one of the invention, wherein the lyoprotectant is selected from one or more of sucrose, lactose, trehalose, glucose, glycine, arginine, and xylitol.
根据本发明的任一项所述的乳剂，其中， 所述的 pH值调节剂 选自盐酸、 硫酸、 乳酸、 苹果酸、 醋酸、 枸橼酸、 磷酸、 氢氧化 钠、 碳酸钠、 碳酸氢钠、 磷酸氢二钠、 和磷酸二氢钠中的一种或 多种。  The emulsion according to any one of the invention, wherein the pH adjusting agent is selected from the group consisting of hydrochloric acid, sulfuric acid, lactic acid, malic acid, acetic acid, citric acid, phosphoric acid, sodium hydroxide, sodium carbonate, sodium hydrogencarbonate One or more of disodium hydrogen phosphate, and sodium dihydrogen phosphate.
根据本发明的任一项所述的乳剂， 其为微乳剂。 微乳是指由 水、 油、 乳化剂组成的， 各向同性、 透明、 热力学稳定的分散体 系， 通常粒径小于 500nm。  An emulsion according to any one of the invention, which is a microemulsion. Microemulsion is an isotropic, transparent, thermodynamically stable dispersion consisting of water, oil, or emulsifier, usually having a particle size of less than 500 nm.
根据本发明的任一项所述的乳剂， 其通过口服给药。  An emulsion according to any one of the invention, which is administered orally.
根据本发明的任一项所述的乳剂， 其各组分及含量如下： 重组人胰岛素 0. 008 - 0. 012重量份 盐酸 适量  The emulsion according to any one of the present invention, the components and contents thereof are as follows: Recombinant human insulin 0. 008 - 0. 012 parts by weight Hydrochloric acid
0. 04一 0. 06重量份 生理盐水 0. 4 - 0. 6重量份 中链脂肪酸甘油三酯 （例如 Crodamol ^ & ι 0. 04-0. 06 parts by weight of physiological saline 0. 4 - 0. 6 parts by weight of medium chain fatty acid triglycerides (eg Crodamol ^ & ι
0. 8 - 1. 2重量份  0. 8 - 1. 2 parts by weight
GTCC ) 聚乙二醇十二羟基硬脂酸酯 （例如 GTCC) Polyethylene glycol dodecyl stearate (for example
0.8 - 1.2重量份  0.8 - 1.2 parts by weight
Solutol HS 15, BASF)  Solutol HS 15, BASF)
吐温 80 1.2 - 1.8重量份 维生素 E 0.08 - 0.12重量份; 本发明还涉及该乳剂在制备治疗或预防糖尿病、 或者降低血 糖的药物中的用途。 本发明还涉及一种治疗或预防糖尿病、 或者 降低血糖的方法， 包括给予有效量的该乳剂的步骤。 在本发明的 一个实施方案中， 所述乳剂的组分及含量如下：  Tween 80 1.2 - 1.8 parts by weight Vitamin E 0.08 - 0.12 parts by weight; The present invention also relates to the use of the emulsion for the preparation of a medicament for treating or preventing diabetes, or lowering blood sugar. The invention further relates to a method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion. In one embodiment of the invention, the components and levels of the emulsion are as follows:
重组人胰岛素 0.01重
Figure imgf000008_0001
Recombinant human insulin 0.01 weight
Figure imgf000008_0001
盐酸 适量  Hydrochloric acid
0.05重量份  0.05 parts by weight
生理盐水 0.5重量份  Saline 0.5 parts by weight
中链脂肪酸甘油三酯 （例如 Crodamol  Medium chain fatty acid triglycerides (eg Crodamol
1重量份  1 part by weight
GTCC) GTCC)
聚乙二醇十二羟基硬脂酸酯 （例如  Polyethylene glycol dodecyl stearate (for example
1重量份  1 part by weight
Solutol HS 15, BASF) Solutol HS 15, BASF)
吐温 80 1.5  Tween 80 1.5
维生素 E 0.1
Figure imgf000008_0002
根据本发明的任一项所述的乳剂， 其各組分及含量如下： Vitamin E 0.1
Figure imgf000008_0002
The emulsion according to any one of the present invention, the components and contents thereof are as follows:
0.004 - 0.006 重量 猪胰岛素  0.004 - 0.006 Weight Pig Insulin
份  Share
氢氧化钠 适量  Sodium hydroxide
PBS緩冲液 0.8 - 1.2重量份 聚乙二醇辛酸 /癸酸甘油酯（ 例如  PBS buffer 0.8 - 1.2 parts by weight Polyethylene glycol octanoate / glyceryl citrate (for example
1.8 - 2.2重量份  1.8 - 2.2 parts by weight
Sof itgen® 767, SASOL) 泊洛沙姆 188 (例如 Lutrol F68, BASF) Q.15 - 0.25重量份 大豆卵磷脂 （例如 Epikuron 170, Sof itgen® 767, SASOL) Poloxamer 188 (eg Lutrol F68, BASF) Q.15 - 0.25 parts by weight of soy lecithin (eg Epikuron 170,
0.4 - 0.6重量份  0.4 - 0.6 parts by weight
Degussa ) Degussa )
二氧化硅 （ 例如 SYLOID®, GRACE  Silica (eg SYLOID®, GRACE
0.8- 1.2重量份  0.8-1.2 parts by weight
DAVISON) DAVISON)
交联纤维素钠 0.1-Q.14重量份  Crosslinked cellulose sodium 0.1-Q.14 parts by weight
0.013 - 0.017 重量 硬脂酸镁  0.013 - 0.017 Weight Magnesium Stearate
份  Share
甘氨酸 0.25 - 0.35重量份； 本发明还涉及该乳剂在制备治疗或预防糖尿病、 或者降低血 糖的药物中的用途。 本发明还涉及一种治疗或预防糖尿病、 或者 降低血糖的方法， 包括给予有效量的该乳剂的步骤。 在本发明的 一个实施方案中， 所述乳剂的组分及含量如下：  Glycine 0.25 - 0.35 parts by weight; the present invention also relates to the use of the emulsion in the preparation of a medicament for treating or preventing diabetes, or lowering blood sugar. The invention further relates to a method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion. In one embodiment of the invention, the components and levels of the emulsion are as follows:
猪胰岛素 0.005重量份 氢氧化钠 适量  Pig insulin 0.005 parts by weight sodium hydroxide
PBS緩冲液 1重量份  PBS buffer 1 part by weight
聚乙二醇辛酸 /癸酸甘油酯（ 例如  Polyethylene glycol octanoate / glyceryl citrate (for example
2重量份  2 parts by weight
Sofitgen® 767, SAS0L)  Sofitgen® 767, SAS0L)
泊洛沙姆 188 (例如 Lutrol F68, BASF) 0.2重量份  Poloxamer 188 (eg Lutrol F68, BASF) 0.2 parts by weight
大豆卵磷脂 （例如 Epikuron 170，  Soy lecithin (eg Epikuron 170,
0.5重量份  0.5 parts by weight
Degussa )  Degussa )
二氧化硅 （ 例如 SYLOID®, GRACE  Silica (eg SYLOID®, GRACE
1重量份  1 part by weight
DAVISON) DAVISON)
交联纤维素钠 0.12重量份  Crosslinked cellulose sodium 0.12 parts by weight
硬脂酸镁 0.015重量份 甘氨酸 0.3重量份。 根据本发明的任一项所述的乳剂， 其各组分及含量如下Magnesium stearate 0.015 parts by weight of glycine 0.3 parts by weight. The emulsion according to any one of the present invention, the components and contents thereof are as follows
PTH 1-34 0.0008-0.0012重量份 PBS緩冲液 0.4-0.6重量份 辛酸癸酸甘油酯（例如 Miglyol ®812， PTH 1-34 0.0008-0.0012 parts by weight PBS buffer 0.4-0.6 parts by weight glyceryl caprylate (eg Miglyol ® 812,
0.6 - 0.8重量份 SASOL)  0.6 - 0.8 parts by weight SASOL)
聚乙二醇十二羟基硬脂酸酯 （ 例如  Polyethylene glycol dodecyl stearate (for example
1.8-2.2重量份 Solutol HS 15, BASF)  1.8-2.2 parts by weight Solutol HS 15, BASF)
生育酚琥珀酸聚乙二醇酯 （例如 TPGS,  Tocopheryl succinate polyethylene glycol ester (eg TPGS,
0.4 - 0.6重量份 Eastman )  0.4 - 0.6 parts by weight Eastman )
吐温 20 0.4-0.6重量份 维生素 E 0.08- 0.12重量份; 本发明还涉及该乳剂在制备治疗或预防骨质疏松的药物中的 用途。 本发明还涉及一种治疗或预防骨质疏松的方法， 包括给予 有效量的该乳剂的步驟。 在本发明的一个实施方案中， 所述乳剂 的组分及含量如下：  Tween 20 0.4-0.6 parts by weight Vitamin E 0.08-0.12 parts by weight; The present invention also relates to the use of the emulsion in the preparation of a medicament for treating or preventing osteoporosis. The invention further relates to a method of treating or preventing osteoporosis comprising the step of administering an effective amount of the emulsion. In one embodiment of the invention, the components and levels of the emulsion are as follows:
PTH 1-34 0.001重量份  PTH 1-34 0.001 parts by weight
PBS緩冲液 0.5重量份  PBS buffer 0.5 parts by weight
辛酸癸酸甘油酯（例如 Miglyol ®812,  Caprylic glyceryl citrate (eg Miglyol ® 812,
0.7重量份  0.7 parts by weight
SASOL) SASOL)
聚乙二醇十二羟基硬脂酸酯 （例如  Polyethylene glycol dodecyl stearate (for example
2重量份  2 parts by weight
Solutol HS 15， BASF) Solutol HS 15, BASF)
生育酚琥珀酸聚乙二醇酯（例如 TPGS,  Tocopheryl succinate polyethylene glycol ester (eg TPGS,
0.5重量份  0.5 parts by weight
Eastman ) Eastman )
吐温 20 0.5重量份  Tween 20 0.5 parts by weight
维生素 E 0.1重量份。 根据本发明的任一项所述的乳剂， 其各组分及含量如下 Vitamin E 0.1 parts by weight. The emulsion according to any one of the present invention, the components and contents thereof are as follows
GLP-1 0.08-0.12重量份 去离子水 55 - 65重量份 辛酸癸酸甘油酯（例如 Miglyol ®812,  GLP-1 0.08-0.12 parts by weight Deionized water 55 - 65 parts by weight Caprylic glyceryl citrate (eg Miglyol ® 812,
12 - 18重量份  12 - 18 parts by weight
SASOL) SASOL)
泊洛沙姆 188(例如 Lutrol F68, BASF ) 1.3- 1.7重量份 大豆卵碑脂 （例如 Epikuron 170，  Poloxamer 188 (eg Lutrol F68, BASF) 1.3-1.7 parts by weight Soybean egg fat (eg Epikuron 170,
6-8重量份  6-8 parts by weight
Degussa ) Degussa )
甘油 8- 12重量份 聚乙二醇 400 6-8重量份  Glycerol 8- 12 parts by weight Polyethylene glycol 400 6-8 parts by weight
维生素 E 00..2255-- 00..3355重重量份； 本发明还涉及该乳剂在制备治疗或预防糖尿病、 或者降低血 糖的药物中的用途。 本发明还涉及一种治疗或预防糖尿病、 或者 降低血糖的方法， 包括给予有效量的该乳剂的步骤。 在本发明的 一个实施方案中， 所述乳剂的组分及含量如下：  Vitamin E 00..2255-- 00..3355 parts by weight; The present invention also relates to the use of the emulsion in the preparation of a medicament for treating or preventing diabetes, or lowering blood sugar. The invention further relates to a method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion. In one embodiment of the invention, the components and levels of the emulsion are as follows:
GLP-1 0.1重量份  GLP-1 0.1 parts by weight
去离子水 60重量份  Deionized water 60 parts by weight
辛酸癸酸甘油酯（例如 Miglyol ®812  Caprylic glyceryl citrate (eg Miglyol ® 812
15重量份  15 parts by weight
SASOL)  SASOL)
泊洛沙姆 188(例如 Lutrol F68, BASF ) 1.5重量份  Poloxamer 188 (eg Lutrol F68, BASF) 1.5 parts by weight
大豆卵麟脂 （例如 Epikuron 170，  Soybean egg fat (eg Epikuron 170,
7重量份  7 parts by weight
Degussa ) Degussa )
甘油 10重量份  Glycerin 10 parts by weight
聚乙二醇 400 7重量份  Polyethylene glycol 400 7 parts by weight
维生素 E 0.3重量份 < 根据本发明的任一项所述的乳剂，其为冻干乳剂（冻干制剂）。 对本领域技术人员而言， 能够制得上述乳剂的冻干制剂， 例如在 上述乳剂中加入适量的冻干保护剂， 然后通过冷冻干燥（例如温 度- 40'C， 工作真空度 30pa)后去除水分， 从而得到干燥的蛋白 质或多肽冻干乳剂。 冻千后乳剂不含水相， 可以做成片剂、 胶嚢 剂等终剂型。 本发明所的乳剂的制备方法， 例如可以1采用下面的步骤：Vitamin E 0.3 parts by weight < An emulsion according to any one of the invention, which is a lyophilized emulsion (lyophilized formulation). For those skilled in the art, a lyophilized preparation of the above emulsion can be prepared, for example, by adding an appropriate amount of a lyoprotectant to the above emulsion, and then removing the moisture by freeze-drying (for example, temperature - 40 ° C, working vacuum of 30 Pa). Thereby obtaining a dried protein or polypeptide lyophilized emulsion. After the freezing, the emulsion does not contain a water phase, and can be formed into a final dosage form such as a tablet or a capsule. The method of preparing the emulsion of the present invention, for example 1 using the following steps:
1 )如下的步骤 A或 B: 1) The following steps A or B:
A. 在 4 - 25°C奈件下， 将蛋白质或多肽分子溶于水相；  A. Dissolving the protein or polypeptide molecule in the aqueous phase at 4-25 °C;
B. 制备蛋白质或多肽的磷脂复合物；  B. preparing a phospholipid complex of a protein or polypeptide;
2 ) 在 20 - 70°C条件下， 将中链脂肪酸酯、 HLB值大于 12的 乳化剂相混合， 并在 200 - 800πηρ搅袢奈件下持续搅拌， 直至整 个体系呈透明清亮状；  2) Mix the medium-chain fatty acid ester and the emulsifier with HLB value greater than 12 at 20 - 70 °C, and continue stirring under 200 - 800πηρ stirring until the whole system is transparent and clear;
3) 在 4 - 37°C条件下， 将步骤 1 ) 产物加入到步骤 2 ) 产物 中， 并在 200 - 800rmp条件下继续搅拌， 直至形成透明清亮的均 匀制剂， 最终获得含蛋白质或多肽药物的乳剂。  3) Add the product of step 1) to the product of step 2) at 4 - 37 ° C, and continue stirring at 200 - 800 rpm until a clear and clear homogeneous preparation is formed, and finally a protein or peptide containing drug is obtained. Emulsion.
关于步骤 A， 可选地， 溶于水相的成分还包括 pH调节剂和 / 或等渗剂。  With regard to step A, optionally, the component dissolved in the aqueous phase further comprises a pH adjusting agent and/or an isotonic agent.
关于步骤 B， 为对本领域技术人员而言， 能够制备所述的磷 脂复合物。 例如采用下面的方法： 分别将磷脂和蛋白溶于有机相 和水相， 混合后， 冷冻干燥（-40 至- 150°C, 甚至更低； 压力为 低于 -100帕， 低于 -200帕， 或者低于 -300帕； 冻干时间为 12小 时以上， 例如 12 - 24 小时， 或者为 24 小时以上， 例如 24 - 48 小时） ， 获得蛋白磷脂复合物。 在优选的实施方案中， 磷脂与蛋 白质或者多肽的摩尔比为 10: 1至 1000: 1。 所述磷脂与蛋白质 或者多肽的摩尔比更优选为 20: 1至 500: 1， 进一步优选为 60: 1至 200: 1。 所述磷脂优选为大豆卵嶙脂和 /或蛋黄卵磷脂。 有机 相为二氯甲烷、 丙酮、 或二甲基亚砜， 优选二甲基亚砜。 With regard to step B, it is possible to prepare the phospholipid complex for those skilled in the art. For example, the following method is used: the phospholipid and the protein are respectively dissolved in the organic phase and the aqueous phase, and after mixing, freeze-dried (-40 to -150 ° C, or even lower; pressure is lower than -100 Pa, lower than -200 Pa , or less than -300 Pa; lyophilization time of 12 hours or more, such as 12 - 24 hours, or more than 24 hours, such as 24-48 hours), to obtain a protein phospholipid complex. In a preferred embodiment, the phospholipids and eggs The white matter or polypeptide molar ratio is from 10:1 to 1000:1. The molar ratio of the phospholipid to the protein or polypeptide is more preferably from 20:1 to 500:1, further preferably from 60:1 to 200:1. The phospholipid is preferably soy egg yolk and/or egg yolk lecithin. The organic phase is dichloromethane, acetone, or dimethyl sulfoxide, preferably dimethyl sulfoxide.
关于步骤 2 ) ， 可选地， 相混和的还包括去离子水； 可选地， 还包括分散介盾和 /或抗氧化剂。  With respect to step 2), optionally, the mixed mixture further includes deionized water; optionally, a dispersing shield and/or an antioxidant.
在本发明的一个实施方案中， 所述制备方法还包括步骤 4 ) ： 将步骤 3 ) 中制得的乳剂进行冻干， 得到冻干乳剂。 步骤 B )和步 骤 4 ) 中涉及的冻干步骤可以使用本发明描述的冻干保护剂。 在本发明中， 术语 "胶束（mice l les ) " 是指在溶液中由若 干溶质分子或离子締合形成的以疏水基团为内核、 亲水基团为外 壳的分子有序聚集体。 胶束是一种胶体分散体系， 属于一类締合 胶体。 两亲性高分子物质在溶液中分散达到临界胶束浓度（CMC ) 时， 分子締合自组装形成胶束。 发明的有益效果  In one embodiment of the present invention, the preparation method further comprises the step 4): lyophilizing the emulsion prepared in the step 3) to obtain a lyophilized emulsion. The lyophilization step described in step B) and step 4) may employ the lyoprotectant described herein. In the present invention, the term "mice l les" refers to a molecularly ordered aggregate of a hydrophobic group as a core and a hydrophilic group as a shell formed by association of several solute molecules or ions in a solution. Micellar is a colloidal dispersion system and belongs to a class of associative colloids. When the amphiphilic polymer substance is dispersed in a solution to a critical micelle concentration (CMC), the molecular association self-assembles to form a micelle. Advantageous effects of the invention
在本发明中通过选用 HLB值（亲水亲油平衡值）为 12以上的 非离子表面活性剂为乳化剂， 中链脂肪酸酯为油相， 含蛋白质多 肽药物的水溶液为水相， 制备出分散性好， 粒径均勾， 乳化性能 良好的蛋白质多肽的乳剂。 本发明的显著特点， 是解决了蛋白质 多肽类乳剂乳化性能的问题， 能显著增加蛋白质或多肽药物的口 服生物利用度， 极具应用前景， 并且制备工艺简单， 适于工业化 生产， 该产品的剂型， 可以是乳剂、 胶束溶液、 自乳化给药体系、 膏状物质或固体粉末。 附图说明 In the present invention, a nonionic surfactant having an HLB value (hydrophilic-lipophilic balance) of 12 or more is used as an emulsifier, a medium-chain fatty acid ester is an oil phase, and an aqueous solution containing a protein-peptide drug is an aqueous phase, and is prepared. An emulsion of protein peptides with good dispersibility, particle size, and good emulsifying properties. The remarkable feature of the invention is that the problem of emulsifying performance of the protein polypeptide emulsion is solved, the oral bioavailability of the protein or polypeptide drug can be significantly increased, the application prospect is very promising, and the preparation process is simple, and is suitable for industrial production, the dosage form of the product. It may be an emulsion, a micelle solution, a self-emulsifying administration system, a paste substance or a solid powder. DRAWINGS
Fig.1: 回肠给药后大鼠血糖化时相图。  Fig. 1: Phase diagram of blood glucose in rats after ileal administration.
Fig.2: PTH1- 34血药浓度。  Fig. 2: PTH1-34 blood concentration.
Fig.3: PTH1-34药效试验。对于胫骨近端 BMD、胫骨近端 BMC、 胫骨 BMD、 胫骨 BMD四组中的每一组， 从左至右的四个柱状图形 分别表示口服组、 注射组、 对照组、 假手术组。 具体实施方式  Fig. 3: PTH1-34 efficacy test. For each of the four groups of BMD, proximal humeral BMC, tibia BMD, and tibia BMD, the four columnar figures from left to right represent the oral group, the injection group, the control group, and the sham operation group. detailed description
下面将结合实施例对本发明的实施方案进行详细描述， 但是 本领域技术人员将会理解， 下列实施例仅用于说明本发明， 而不 应视为限定本发明的范围。 实施例中未注明具体条件者， 按照常 规条件或制造商建议的条件进行。 所用试剂或仪器未注明生产厂 商者， 均为可以通过市购获得的常规产品。 实施例 1: 口服胰岛素制剂 （样品 1) 的制备  The embodiments of the present invention are described in detail below with reference to the accompanying drawings. Those who do not specify the conditions in the examples are subject to the usual conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that are commercially available. Example 1: Preparation of Oral Insulin Formulation (Sample 1)
其组成如下：  Its composition is as follows:
猪胰岛素 0.005克  Pig insulin 0.005 g
5摩尔氢氧化钠 适量  5 moles of sodium hydroxide
PBS緩冲液（北京索莱宝科技有限公司） 1克  PBS buffer (Beijing Suo Laibao Technology Co., Ltd.) 1 g
聚 乙 二 醇 辛 酸 / 癸 酸甘 油 酯 2克 Polyethylene glycol caprylic acid / glyceryl citrate 2 g
( Sofitgen® 767， SAS0L)  ( Sofitgen® 767, SAS0L)
泊洛沙姆 188 (Lutrol F68, BASF) 0.2克  Poloxamer 188 (Lutrol F68, BASF) 0.2 g
大豆卵御旨 （ Epikuron 170， Degussa ) 0· 5克  Soybean Eggs ( Epikuron 170, Degussa ) 0 · 5 grams
二氧化硅（ SYLOID®, GRACE DAVISON) 1克  Silica (SYLOID®, GRACE DAVISON) 1 g
交联纤维素钠 Q.12克  Crosslinked cellulose sodium Q.12g
硬脂酸镁 0.015克 甘氨酸 0. 3克 Magnesium stearate 0.015g Glycine 0. 3 g
该制剂的制备方法如下：  The preparation method of the preparation is as follows:
1 ) 室温下， 将上述重量的猪胰岛素溶解于 0. 2克 PBS緩冲 液， 加入适量氢氧化钠， 所得溶液搅拌至澄清， 得 A溶液；  1) The above weight of porcine insulin is dissolved in 0.2 gram of PBS buffer, an appropriate amount of sodium hydroxide is added, and the resulting solution is stirred until clarified to obtain a solution A;
2 ) 室温下， 将上述重量的大豆卵磷脂溶解于 60ml的二甲基 亚砜， 搅拌至澄清后， 加入步骤 1 ) 中的 A溶液， 在 300r/min条 件下搅拌 5fflin， 随后放置到液氮中 30min, 所得产物经冷冻干燥 后， 得到猪胰岛素大豆卵磷脂复合物；  2) Dissolve the above-mentioned weight of soybean lecithin in 60 ml of dimethyl sulfoxide at room temperature, stir until clarified, add the solution A in step 1), stir 5 fflin at 300 r/min, and then place it in liquid nitrogen. After 30 min, the obtained product was freeze-dried to obtain a porcine insulin soy lecithin complex;
3 ) 70 °C下， 将上述重量的甘氨酸、 泊洛萨姆 188 溶于剩余 PBS緩冲液， 与上述重量的聚乙二醇辛酸 /癸酸甘油酯， 混合并搅 拌至透明清亮；  3) at 70 ° C, the above weight of glycine, poloxamer 188 is dissolved in the remaining PBS buffer, mixed with the above weight of polyethylene glycol caprylic / glyceryl citrate, and stirred until clear and clear;
4 ) 室温下， 猪胰岛素大豆卵磷脂复合物， 加入到步驟 3 ) 中 的产物中， 在 200r/min转速下将混合物搅拌 15min;  4) porcine insulin soy lecithin complex is added to the product in step 3) at room temperature, and the mixture is stirred at a speed of 200 r/min for 15 min;
5 )室温下， 将上述重量的二氧化硅、 交联纤维素钠、 硬脂酸 混合后， 在 200r/min转速下， 逐滴加入步骤 4 ) 的产物， 随后提 高转速至 300r/min， 并保持 30min; 所得产物经冷冻干燥（温度 - 40'C， 工作真空度 30pa )后， 最终获得胰岛素口服制剂， 为样 丄。  5) After mixing the above weight of silica, crosslinked cellulose sodium and stearic acid at room temperature, the product of step 4) is added dropwise at a speed of 200 r/min, and then the rotation speed is increased to 300 r/min, and After 30 minutes of lyophilization (temperature - 40 ° C, working vacuum of 30 Pa), the oral preparation of insulin was finally obtained as a sample.
样品 1可以做成片剂、 胶嚢剂等终剂型。  Sample 1 can be formulated into a final dosage form such as a tablet or a capsule.
本领域技术人员可以理解， 尽管实施例 1中的各组分的重量 单位是克， 但是也可以理解为重量份， 即各组分之间满足上面的 实施例 1中的重量比例就可以。 实施例 2: 胰岛素口服制剂 （样品 2 ) 的制备  It will be understood by those skilled in the art that although the weight unit of each component in Example 1 is gram, it can be understood as a part by weight, i.e., the weight ratio in the above Example 1 is satisfied between the components. Example 2: Preparation of an oral insulin preparation (Sample 2)
其组成如下：  Its composition is as follows:
重组人胰岛素 0. 01克 1摩尔盐酸 Recombinant human insulin 0. 01 g 1 mole of hydrochloric acid
壳聚糖 0.05克 生理盐水 0.5克 中链脂肪酸甘油三酯 （Crodamol GTCC) 1克  Chitosan 0.05 g saline 0.5 g medium chain fatty acid triglyceride (Crodamol GTCC) 1 g
聚乙二醇十二羟基硬脂酸酯（Solutol HS  Polyethylene glycol dodecyl stearate (Solutol HS
15， BASF)  15, BASF)
吐温 80 1.5克 维生素 E 0。 1克 该制剂的制备方法如下：  Tween 80 1.5 g Vitamin E 0. 1 g The preparation method of the preparation is as follows:
1 )室温下， 将重组胰岛素、 壳聚糖溶于生理盐水， 加入适量 盐酸、 搅拌至澄清， 得 A溶液；  1) Recombinant insulin and chitosan are dissolved in physiological saline at room temperature, and an appropriate amount of hydrochloric acid is added thereto, and stirred until clarification to obtain a solution A;
2) 40°C下， 将上述重量的中链脂肪酸甘油三酯、 聚乙二醇十 二羟基硬脂酸酯、 吐温 80和维生素 E， 混合并搅袢至透明清亮； 2) mixing and weaving the above-mentioned medium-chain fatty acid triglyceride, polyethylene glycol decahydroxystearate, Tween 80 and vitamin E at 40 ° C until transparent and clear;
3) 室温下， 将 A 溶液， 逐滴加入到步骤 2 ) 的产物中， 在 200r/min转速下将混合物搅袢至透明清亮， 最终获得胰岛素口服 制剂， 为样品 2。 3) A solution was added dropwise to the product of step 2) at room temperature, and the mixture was stirred until clear and clear at 200 r/min, and finally an insulin oral preparation was obtained as sample 2.
本领域技术人员可以理解， 尽管实施例 2中的各组分的重量 单位是克， 但是也可以理解为重量份, 即各組分之间满足上面的 实施例 1中的重量比例就可以。 实施例 3: 重组人曱状旁腺激素 1-34口服制剂 （样品 3) 的 制备 It will be understood by those skilled in the art that although the weight unit of each component in Example 2 is gram, it can be understood as a part by weight, that is, the weight ratio in the above Example 1 is satisfied between the components. Example 3: Preparation of recombinant human parathyroid hormone 1-34 oral preparation (Sample 3)
其组成如下：  Its composition is as follows:
PTH 1-34 0.001克 PTH 1-34 0.001 g
PBS緩冲液（北京索莱宝科技有限公司 ) 0.5克 辛酸癸酸甘油酯 （ Miglyol ®812， 0.7克 SASOL) PBS buffer (Beijing Solabao Technology Co., Ltd.) 0.5 g octanoic acid glyceride (Miglyol ® 812, 0.7 g SASOL)
聚乙二醇十二羟基硬脂酸酯（Solutol HS  Polyethylene glycol dodecyl stearate (Solutol HS
2克  2 grams
15， BASF)  15, BASF)
生育酚琥珀酸聚乙二醇酯 （ TPGS， ^  Tocopheryl succinate polyethylene glycol ester (TPGS, ^
、 0· 5克 , 0·5 grams
Eastman ) Eastman )
吐温 20 0.5克 维生素 E 0.1克 该制剂的制备方法如下：  Tween 20 0.5 g Vitamin E 0.1 g The preparation method of the preparation is as follows:
1 )室温下， 将上述重量的 PTH1-34溶于 PBS緩冲液， 搅拌至 澄清， 得 A溶液；  1) The above weight of PTH1-34 is dissolved in PBS buffer at room temperature, stirred until clear, to obtain A solution;
2) 60°C下， 将上述重量的辛酸癸酸甘油酯、 聚乙二醇十二羟 基硬脂酸酯、 生育酚琥珀酸聚乙二醇酯、 吐温 20、 维生素 E, 混 合并搅袢至透明清亮；  2) Mix and agitate the above weight of caprylic caproic acid glyceride, polyethylene glycol dodecyl stearate, tocopherol succinate polyethylene glycol ester, Tween 20, vitamin E at 60 ° C To clear and clear;
3) 室温下， 将 A 溶液， 逐滴加入到步骤 2) 的产物中， 在 lOOr/min转速下将混合物搅拌 15min, 最终获得 PTH 1-34口服制 剂， 为样品 3。  3) At room temperature, add the A solution dropwise to the product of step 2), stir the mixture for 15 min at lOOr/min, and finally obtain the PTH 1-34 oral preparation as sample 3.
本领域技术人员可以理解， 尽管实施例 3中的各组分的重量 单位是克， 但是也可以理解为重量份， 即各组分之间满足上面的 实施例 3中的重量比例就可以。 实施例 4: GLP-1口服制剂 （样品 4) 的制备  It will be understood by those skilled in the art that although the weight unit of each component in Example 3 is gram, it can be understood as a part by weight, i.e., the weight ratio in the above Example 3 is satisfied between the components. Example 4: Preparation of GLP-1 Oral Formulation (Sample 4)
其组成如下  Its composition is as follows
GLP-1 0.1克  GLP-1 0.1 g
去离子水 60克  Deionized water 60g
辛酸癸酸甘油酯 （ Miglyol ®812, ^  Caprylic glyceryl citrate ( Miglyol ® 812, ^
、 15克  15 grams
SAS0L) 泊洛沙姆 188 (Lutrol F68, BASF) 1.5克 SAS0L) Poloxamer 188 (Lutrol F68, BASF) 1.5 g
大豆卵麟脂（Epikuron 170， Degussa ) 7克  Soybean egg fat (Epikuron 170, Degussa) 7 g
甘油 10克  Glycerin 10 g
聚乙二醇 400 7克  Polyethylene glycol 400 7 g
维生素 E 0.3克  Vitamin E 0.3 g
该制剂的制备方法如下：  The preparation method of the preparation is as follows:
1 )室温下， 将上述重量的 GLP-1溶于 5克去离子水， 将上述 重量的大豆卵磷脂溶于 30ml的二氯曱烷中， 两相混合后， 置于液 氮 30min， 冷冻千燥， 所得产物为 GLP- 1大豆磷脂复合物；  1) The above weight of GLP-1 is dissolved in 5 g of deionized water at room temperature, and the above-mentioned weight of soybean lecithin is dissolved in 30 ml of dichlorosilane. After mixing the two phases, it is placed in liquid nitrogen for 30 min, and the freezing is performed. Dry, the obtained product is GLP-1 soybean phospholipid complex;
2) 70°C下， 将上述重量的辛酸癸酸甘油酯、 泊洛萨姆 188、 5克去离子水， 混合并搅拌至透明清亮；  2) at 70 ° C, the above weight of octanoic acid glyceride, poloxamer 188, 5 grams of deionized water, mixed and stirred until clear and clear;
3) 室温下， 在 500r/min转速下， 将 GLP- 1大豆卵磷脂复合 物加入步骤 2) 的产物中， 继续搅拌 5min， 得初乳 B;  3) At room temperature, at 500r / min, the GLP-1 soy lecithin complex is added to the product of step 2), stirring is continued for 5min, to obtain colostrum B;
4) 室温下， 将上述重量的甘油、 聚乙二醇、 维生素 E， 和剩 余的 50克水混合， 在 500r/mifi转速下， 逐滴加入初乳 B, 继续 搅拌 5min后， 经高压均质机（意大利 Niro Soavi )处理（ Obar, 循环 5次） ， 最终获得的透明清亮的胶束溶液， 即为 GLP-1 口服 制剂， 为样品 4。  4) Mix the above weight of glycerin, polyethylene glycol, vitamin E, and the remaining 50 grams of water at room temperature, add colostrum B dropwise at 500r/mifi, continue stirring for 5min, and homogenize by high pressure. The machine (Niro Soavi, Italy) was treated (Obar, cycled 5 times), and the finally obtained clear and clear micelle solution, namely GLP-1 oral preparation, was sample 4.
本领域技术人员可以理解， 尽管实施例 4中的各组分的重量 单位是克， 但是也可以理解为重量份， 即各组分之间满足上面的 实施例 4中的重量比例就可以。 实施例 5: 样品 2、 3、 4的稳定性研究  It will be understood by those skilled in the art that although the weight unit of each component in Example 4 is gram, it can be understood as a part by weight, i.e., the weight ratio in the above Example 4 is satisfied between the components. Example 5: Stability study of samples 2, 3, and 4.
口服制剂样品 2、 3、 4的稳定性， 通过肉眼观察制剂是否出 现絮状物质或产生 "油一水" 界面而判定。  The stability of the oral preparation samples 2, 3, and 4 was determined by visually observing whether the preparation exhibited flocculent substances or produced an "oil-water" interface.
样品 1、 2中胰岛素的稳定性， 采用高效液相色谱进行研究， 所用色镨仪为 Agilent 1200。 The stability of insulin in samples 1, 2 was studied by high performance liquid chromatography. The color meter used was the Agilent 1200.
色谱洗脱条件：  Chromatographic elution conditions:
流动相为线性梯度洗脱， 其中流动相 A为乙腈， 流动相 B为 0.05 mol/L磷酸二氢钠 （pH3.0) ： 0 - lOmin流动相 A从 20 %升 至 50%， 流动相 B从 80%变至 50%; 11 - 15min流动相 A从 50% 变至 80%， 流动相 B从 50%变至 20%; 21 - 25min流动相 A从 80%变至 20%, 流动相 B从 20%变至 80%。 流速 1.0mL/min， 检测 波长为 214nm, 柱温 40° (：。  The mobile phase is a linear gradient elution, in which mobile phase A is acetonitrile and mobile phase B is 0.05 mol/L sodium dihydrogen phosphate (pH 3.0): 0 - lOmin mobile phase A is increased from 20% to 50%, mobile phase B Change from 80% to 50%; 11 - 15min mobile phase A changes from 50% to 80%, mobile phase B changes from 50% to 20%; 21 - 25min mobile phase A changes from 80% to 20%, mobile phase B Change from 20% to 80%. The flow rate is 1.0 mL/min, the detection wavelength is 214 nm, and the column temperature is 40° (:.
在 6个月的考察期内， 放置于 4。C的样品 2制剂、 样品 3制 剂、 样品 4制剂没有产生肉眼可见的絮状物质， 也没有产生 "油 —水" 界面。  Placed at 4 during the 6-month study period. The sample 2 preparation, sample 3 preparation, and sample 4 preparation of C did not produce macroscopic flocculent substances, nor did they produce an "oil-water" interface.
另外对照高效液相色谱图，我们发现样品 1、 2在不同时间（ 0、 3m、 6m) 其药效成分胰岛素的保留时间、 峰面积， 均没有发生显 著变化， 这就表明， 在 4'C条件下， 所得样品至少能保持 6个月 的稳定性。 实施例 6: 样品 1 - 4的药物成分的含量变化试验  In addition, according to the high performance liquid chromatogram, we found that the retention time and peak area of the medicinal components of the samples 1 and 2 at different times (0, 3m, 6m) did not change significantly, which indicated that at 4'C Under the conditions, the obtained sample can maintain stability for at least 6 months. Example 6: Variation test of the content of the pharmaceutical ingredients of samples 1 - 4
所得制剂至于 4°C冰箱， 并在放置后的 0天、 第三个月、 第 六个月测定药物含量。 釆用 HPLC 法进行药物活性成分含量的测 定， 结果如表 1所示。  The resulting preparation was placed in a refrigerator at 4 ° C, and the drug content was determined at 0 days, 3 months, and 6 months after the placement. HPLC HPLC method was used to determine the content of active pharmaceutical ingredients. The results are shown in Table 1.
表 1: 样品 1-4药物成分含量的变化  Table 1: Samples 1-4 Changes in the content of pharmaceutical ingredients
Figure imgf000019_0001
样品 4 95.0 93.8 94.6 实施例 7: 乳剂稀释液粒径及外观研究
Figure imgf000019_0001
Sample 4 95.0 93.8 94.6 Example 7: Study on particle size and appearance of emulsion diluent
实施例 2、 实施例 3制备的样品 2、 3， 放置 4°C冰箱， 并于 0 天、 第三个月、 第六个月测定乳剂粒径及外观清亮情况。 用 Malvern Zetasizer 3000HSA 粒度分析仪检测的粒径， 测定前， 样品 2、 3用 Millipore超纯水分别稀释 5、 30、 100倍， 并肉眼 测定各稀释液外观是否清亮。 结果如下面的表 2所示。  Example 2, Samples prepared in Example 3 2, 3, placed in a refrigerator at 4 ° C, and measured the particle size and appearance of the emulsion at 0 days, the third month, and the sixth month. The particle size was measured with a Malvern Zetasizer 3000HSA particle size analyzer. Before the measurement, samples 2 and 3 were diluted 5, 30, and 100 times with Millipore ultrapure water, and the appearance of each dilution was visually determined. The results are shown in Table 2 below.
表 2: 乳剂稀释液粒径及外观的结果  Table 2: Results of particle size and appearance of emulsion diluent
Figure imgf000020_0001
实施例 8: 胰岛素口服制剂药效研究
Figure imgf000020_0001
Example 8: Study on the efficacy of oral insulin preparations
试验组 1: 体重为 180-220克范围的雄性 SD大鼠， 6只， 给 药为实施例 1制备的样品 1。  Test group 1: Male SD rats weighing 180-220 g, 6 rats, were administered the sample 1 prepared in Example 1.
试验组 2: 体重为 180-220克范围的雄性 SD大鼠， 6只， 给 药为实施例 2制备的样品 2。  Test group 2: Male SD rats having a body weight of 180-220 g, 6 rats, were administered the sample 2 prepared in Example 2.
对照组： 体重为 180- 220克范围的雄性 SD大鼠， 6只， 给药 为胰岛素水溶液， 皮下注射。 Control group: Male Sprague-Dawley rats weighing 180-220 g, 6 doses For insulin aqueous solution, subcutaneous injection.
试验方法：  experiment method:
将体重为 180-220克范围的雄性 SD大鼠禁食 12h后开始试 验； 实验前用剂量为 50mg的戊巴比妥钠对动物进行麻醉。待动物 麻醉后， 按仰位方式将其捆绑， 并在腹部切一小口， 用给药器对 回肠给药。 给药后， 缝合切口， 并在 0. 5、 1、 2、 3、 4h时刻点， 由尾静脉取血， 使用罗氏血糖测定仪测定血糖并计算每组内 6只 大鼠的平均血糖值。 其中试验组 1中给药为样品 1， 按胰岛素计， 给药剂量为 0. 3mg/kg; 试验组 2中给药为样品 2， 按胰岛素计， 给药剂量为 lmg/kg; 对照组为皮下注射对照药物， 给药剂量按胰 岛素计为 0. 02mg/kg。 给药后， 大鼠血糖变化如图 1 所示。 图 1 显示样品 1和样品 2均能够有效地降低大鼠的血糖。 其中样品 1 与对照组在给药后 1小时， 血糖浓度均降至最低值。 实施例 9 : PTH1-34口月良制剂的灌胃吸收试验  Male Sprague-Dawley rats weighing 180-220 g were fasted for 12 h and the test was started; the animals were anesthetized with 50 mg of sodium pentobarbital before the experiment. After the animals were anesthetized, they were tied in a supine position, and a small opening was made in the abdomen, and the ileum was administered by a dispenser. After administration, the incision was sutured, and at the time of 0.5, 1, 2, 3, and 4 h, blood was taken from the tail vein, blood glucose was measured using a Roche blood glucose meter, and the average blood glucose level of 6 rats in each group was calculated. The test group 1 is administered as sample 1, according to insulin, the dose is 0.3 mg/kg; the test group 2 is administered as sample 2, and the insulin is administered at a dose of 1 mg/kg; 02mg/公斤。 The drug was administered in a dose of 0. 02mg / kg. After administration, the changes in blood glucose in rats are shown in Figure 1. Figure 1 shows that both Sample 1 and Sample 2 are effective in reducing blood glucose in rats. In the sample 1 and the control group, the blood glucose concentration was reduced to the lowest value 1 hour after the administration. Example 9: Gavage absorption test of PTH1-34 mouth Yueliang preparation
试验组： 体重为 180-220克范围的雄性 SD大鼠， 12只， 给 药为实施例 3制备的样品 3， 胃部给药。  Test group: Male Sprague-Dawley rats weighing from 180 to 220 g, 12, and the sample prepared in Example 3, were administered to the stomach.
对照组： 体重为 180-220克范围的雄性 SD大鼠， 10只， 给 药为 PTH1- 34 PBS溶液， 皮下注射。  Control group: Male SD rats weighing 180-220 g, 10 rats, were administered PTH1-34 PBS solution and injected subcutaneously.
试验方法：  experiment method:
所有大鼠在禁食 12h后开始试验。试验组采用胃部给药方式， 对照组选用 PTH1- 34注射液。 胃部给药： 采用给药器， 以 PTH1-34 计， 按 5(Vg/kg剂量对大鼠胃部灌注 PTH1-34口服制剂； 注射给 药： 以 PTH1- 34计， 按 l(Vg/kg剂量， 皮下注射 PTH1-34注射液。  All rats started the test after 12 h of fasting. The test group was administered with stomach, and the control group was treated with PTH1-34. Gastric administration: PTH1-34 oral preparation was administered to the rat stomach by a dose of 5 (Vg/kg) using an applicator; injection administration: PTH1-34, press 1 (Vg/ Kg dose, subcutaneous injection of PTH1-34 injection.
给药后 15、 30、 45、 60、 75、 90、 105min时刻点， 由尾静脉 取血， 分离出血清备测。 血清中 PTH 1-34浓度用 PTH 1-34 高灵 敏度 EIA试剂盒（美国 Alpco Di agnos t ic , 货号 31-IPTHUU-E01 ) 检测， 该试剂盒仅与外源 PTH1-34特异性反应， 而不与大鼠本身 PTH反应， 因而所能检测到的 PTH1-34 , 即为给药后进入大鼠血液 中的 ΡΤΗ1-34。 灌胃组动物为 12只， 注射组动物为 10只。 给药 后， PTH1-34血药浓度变化如图 2所示。 图 2显示， 灌胃给药后， PTH1-34 能够迅速进入大鼠体内， 两者血药浓度达峰时间极其相 近（试验组血药浓度达峰时间为 18. 3 ± 1. 2分钟， 对照组达峰时 间为 17. 4 ± 0. 9分钟）按血药浓度计， 灌胃给药组相对于注射组 的生物利用度为 6. 73%。 实施例 10: PTH1-34口服制剂的药效试验 At the time of 15, 30, 45, 60, 75, 90, and 105 minutes after administration, blood was taken from the tail vein, and serum was separated for testing. Serum PTH 1-34 concentration with PTH 1-34 Sensitivity EIA kit (Alpco Di agnos t ic, Cat. No. 31-IPTHUU-E01) detected that the kit reacts exclusively with exogenous PTH1-34, but does not react with the rat itself, so it can be detected. PTH1-34, which is ΡΤΗ 1-34 into the blood of rats after administration. There were 12 animals in the gavage group and 10 animals in the injection group. After administration, the PTH1-34 plasma concentration changes as shown in Figure 2. Figure 2 shows that after intragastric administration, PTH1-34 can rapidly enter the rat body, and the plasma concentration peak time is very similar (the peak time of the blood concentration in the experimental group is 18.3 ± 1. 2 minutes, control The bioavailability of the intragastric administration group relative to the injection group was 6.73%. Example 10: Efficacy test of PTH1-34 oral preparation
通过建立去势雌鼠模型， 研究口服 PTH1-34制剂对动物骨密 度、 骨矿含量的影响。  The effects of oral PTH1-34 preparation on animal bone density and bone mineral content were studied by establishing a castrated female model.
口服组： 选取 2月龄的雌性大鼠 6只， 切除雌鼠双侧卵巢， 给药为实施例 3制备的样品 3 , 口服灌胃方式给药。  Oral group: Six female rats of 2 months old were selected, and the bilateral ovaries of the female rats were excised, and the sample prepared in Example 3 was administered and administered by oral gavage.
注射组： 选取 2月龄的雌性大鼠 6只， 切除雌鼠双侧卵巢， 给药为实施例 3制备的样品 3， PTH1-34 PBS注射液， 皮下注射。  Injection group: Six female rats of 2 months old were selected, and the bilateral ovaries of the female rats were excised, and the sample prepared in Example 3 was administered, PTH1-34 PBS injection, subcutaneously.
对照组： 选取 2月龄的雌性大鼠 6只， 切除雌鼠双侧卵巢， 给药为实施例 3制备的样品 3， 不给药。  Control group: Six female rats of 2 months old were selected, and the bilateral ovaries of the female rats were excised, and the sample 3 prepared in Example 3 was administered without administration.
假手术组： 选取 2月龄的雌性大鼠 5只， 保留卵巢， 切除卵 巢附近同等大小的脂肪， 不给药。  Sham-operated group: Five female rats of 2 months old were selected, the ovaries were preserved, and the same size of fat near the egg nest was removed, and no drug was administered.
试验方法：  experiment method:
选取 2月龄的雌性大鼠， 随机分为去势组 （去势动物， 通过 手术切除大鼠双侧卵巢， 包括口服组、 注射组、 对照组） 与假手 术组， 饲养 6周， 随后口服组和注射组连续给药 8周， 每周给药 6 天， 每天一次， 给药前对大鼠禁食 10 小时。 其中口服组剂量 10(Vg/kg (n=6 ),注射组给药剂量 l(^g/kg( n=6 ),对照组（n-6)、 假手术组（n-5 )不给药。 然后后测定左侧胫骨的骨密度（BMD) 、 骨矿含量 （ BMC ) (双能 X 射线吸收法检测， dual X-ray absorptimetry ) ， *P<0.01， **Ρ<0· 05。 结果如图 3所示。 Two-month-old female rats were randomly divided into the castration group (castrated animals, bilateral ovaries by surgery, including the oral group, the injection group, the control group) and the sham operation group for 6 weeks, followed by oral administration. The group and the injection group were continuously administered for 8 weeks, administered 6 days a week, once a day, and the rats were fasted for 10 hours before administration. Oral dose 10 (Vg/kg (n=6), the injection group dose was 1 (^g/kg (n=6), the control group (n-6), the sham operation group (n-5) was not administered. Then after Bone mineral density (BMD) and bone mineral content (BMC) of the left tibia were measured (dual energy X-ray absorptiometry, dual X-ray absorptimetry), *P<0.01, **Ρ<0· 05. The results are shown in Fig. 3. Shown.
由图 3可知， 与对照组相比， 经过治疗， 骨质疏松模型大鼠 的胫骨及胫骨近端 BMD (单位 g/cra2) 、 BMC (单位 g) 均有显著 增加， 这就表明， PTH1-34注射液及 PTH1-34 口服制剂， 均能促 进骨骼生成， 对骨质疏松有着显著的疗效。 As can be seen from Fig. 3, compared with the control group, the BMD (unit g/cra 2 ) and BMC (unit g) of the proximal tibia and tibia of the osteoporosis model rats were significantly increased, indicating that PTH1 Both -34 injection and PTH1-34 oral preparations promote bone formation and have a significant effect on osteoporosis.
尽管本发明的具体实施方式已经得到详细的描述， 本领域技 术人员将会理解。 根据已经公开的所有教导， 可以对那些细节进 行各种修改和替换， 这些改变均在本发明的保护范围之内。 本发 明的全部范围由所附权利要求及其任何等同物给出。 Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

权利要求 Rights request
1. 一种乳剂， 包含蛋白质或多肽、 水相、 油相、 乳化剂， 其 特征在于，所述油相含有中链脂肪酸酯，并且所述乳化剂包括 HLB 值大于 12的乳化剂。 An emulsion comprising a protein or polypeptide, an aqueous phase, an oil phase, an emulsifier, characterized in that the oil phase contains a medium chain fatty acid ester, and the emulsifier comprises an emulsifier having an HLB value of more than 12.
2. 根据权利要求 1所述的乳剂， 其中， 所述蛋白质或多肽为 降钙素、 胰岛素、 GLP- 1、 曱状旁腺激素、 曱状旁腺激素 1-84、 甲状旁腺激素 1-34、 或生长激素。  The emulsion according to claim 1, wherein the protein or polypeptide is calcitonin, insulin, GLP-1, parathyroid hormone, parathyroid hormone 1-84, parathyroid hormone 1- 34, or growth hormone.
3. 根据权利要求 1所述的乳剂， 其中， 所述中链脂肪酸酯为中 链脂肪酸甘油酯、 聚乙二醇化的中链脂肪酸甘油酯、 乙酰化中链脂 肪酸甘油酯、 中链脂肪酸丙二醇酯、 中链脂肪酸丁二醇酯、 中链脂 肪酸聚乙二醇酯；具体地，为碳原子数 6 - 12的脂肪酸的甘油一酯、 甘油二酯、 和 /或甘油三酯； 更具体地， 为选自辛酸癸酸甘油酯、 聚乙二醇辛酸 /癸酸甘油酯、 辛酸甘油酯、 癸酸甘油酯中的一种或 多种。  The emulsion according to claim 1, wherein the medium chain fatty acid ester is a medium chain fatty acid glyceride, a pegylated medium chain fatty acid glyceride, an acetylated medium chain fatty acid glyceride, or a medium chain fatty acid propylene glycol. An ester, a medium chain fatty acid butylene glycol ester, a medium chain fatty acid polyethylene glycol ester; specifically, a monoglyceride, a diglyceride, and/or a triglyceride of a fatty acid having 6 to 12 carbon atoms; more specifically It is one or more selected from the group consisting of glyceryl caprylate, glyceryl octanoate/capric acid glyceride, caprylic glyceride, and glyceryl citrate.
4. 根据权利要求 1所述的乳剂， 其中， 所述 HLB值大于 12的 乳化剂为选自泊洛沙姆 188、 泊洛沙姆 407、 吐温 20、 吐温 80、 聚 乙二醇十二羟基硬脂酸酯、聚氧乙烯醚（35)蓖麻油、聚氧乙浠醚（40) 氢化蓖麻油、 生育酚琥珀酸聚乙二醇酯、 聚乙二醇 -32-月桂酸甘油 酯、 聚乙二醇 -32-硬脂酸甘油酯中的一种或多种。  The emulsion according to claim 1, wherein the emulsifier having an HLB value of more than 12 is selected from the group consisting of poloxamer 188, poloxamer 407, Tween 20, Tween 80, and polyethylene glycol ten. Dihydroxystearate, polyoxyethylene ether (35) castor oil, polyoxyethylene ether (40) hydrogenated castor oil, tocopherol succinic acid polyethylene glycol ester, polyethylene glycol-32-lauric acid glyceride One or more of polyethylene glycol-32-glyceryl stearate.
5. 根据权利要求 1所述的乳剂， 其中， 所述蛋白质或多肽为 0. 01 - 1重量份，所述中链脂肪酸酯为 50 - 1000重量份，所述 HLB 值大于 12的乳化剂为 20 - 2000重量份； 具体地， 所述蛋白质或 多肽为 0. 01 - 0. 5重量份，所述中链脂肪酸酯为 100 - 500重量份， 所述 HLB值大于 12的乳化剂为 50 - 1000重量份； 更具体地， 所 述蛋白质或多肽为 0. 01 - 0. 3重量份， 所述中链脂肪酸酯为 100 一 300重量份， 所述 HLB值大于 12的乳化剂为 50 - 500重量份。 The emulsion according to claim 1, wherein the protein or polypeptide is 0.01 to 1 part by weight, the medium chain fatty acid ester is 50 to 1000 parts by weight, and the emulsifier having an HLB value of more than 12 Specifically, the protein or polypeptide is 0.01 to 0.5 parts by weight, the medium chain fatty acid ester is 100 to 500 parts by weight, and the emulsifier having an HLB value of more than 12 is重量重量的优选为中中的脂脂酯为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为为 One 300 parts by weight of the emulsifier having an HLB value of more than 12 is 50 to 500 parts by weight.
6. 根据权利要求 1所述的乳剂， 其中， 所述乳化剂还含有磷 脂。  The emulsion according to claim 1, wherein the emulsifier further contains a phospholipid.
7. 根据权利要求 6所述的乳剂， 其中， 磷脂与蛋白质或者多 肽的摩尔比为 10: 1至 1000: 1， 优选为 20: 1至 500: 1， 更优 选为 60: 1至 200: 1。  The emulsion according to claim 6, wherein the molar ratio of the phospholipid to the protein or polypeptide is from 10:1 to 1000:1, preferably from 20:1 to 500:1, more preferably from 60:1 to 200:1 .
8. 根据权利要求 6所述的乳剂， 其中， 所述磷脂为大豆卵磷 脂和 /或蛋黄卵磷脂。  The emulsion according to claim 6, wherein the phospholipid is soybean lecithin and/or egg yolk lecithin.
9. 根据权利要求 1至 8中任一项所述的乳剂， 其还包含药学 上可接受的辅料， 例如分散介质、 等渗剂、 抗氧化剂、 冻干保护 剂、 或 pH调节剂。  The emulsion according to any one of claims 1 to 8, which further comprises a pharmaceutically acceptable adjuvant such as a dispersion medium, an isotonic agent, an antioxidant, a lyoprotectant, or a pH adjuster.
10. 根据权利要求 2 所述的乳剂， 其各组分及含量如下面的  10. The emulsion according to claim 2, the components and contents of which are as follows
(1) - (6) 组中的任一组所示：  (1) - (6) As shown in any of the groups:
( 1)  ( 1)
重組人胰岛素 0.008 - 0.012重量份 盐酸 适量 Recombinant human insulin 0.008 - 0.012 parts by weight hydrochloric acid
0.04 - 0.06重量份 生理盐水 0.4- 0.6重量份 中链脂肪酸甘油三酯 0.8 -1.2重量份 聚乙二醇十二羟基硬脂酸酯 0.8 -1.2重量份 吐温 80 1.2 -1.8重量份 维生素 E 0.08- 0.12重量份； (2)  0.04 - 0.06 parts by weight of physiological saline 0.4 - 0.6 parts by weight of medium chain fatty acid triglyceride 0.8 - 1.2 parts by weight of polyethylene glycol dodecyl stearate 0.8 - 1.2 parts by weight of Tween 80 1.2 - 1.8 parts by weight of vitamin E 0.08 - 0.12 parts by weight; (2)
重组人胰岛素 0.01重
Figure imgf000025_0001
Recombinant human insulin 0.01 weight
Figure imgf000025_0001
盐酸 适量 Hydrochloric acid
壳聚糖 0.05重量份 生理盐水 0.5重量份 中链脂肪酸甘油三酯 1重量份 Chitosan 0.05 parts by weight 0.5 parts by weight of physiological saline, 1 part by weight of medium chain fatty acid triglyceride
聚乙二醇十二羟基硬脂酸酯 1重量份 Polyethylene glycol dodecyl stearate 1 part by weight
吐温 8G 1.5重量份 维生素 E 0.1重量份； Tween 8G 1.5 parts by weight Vitamin E 0.1 parts by weight;
(3)  (3)
0.004 - 0.006 重量 猪胰島素  0.004 - 0.006 Weight Pig Insulin
份  Share
氢氧化钠 适量 Sodium hydroxide
PBS緩沖液 0.8 -1.2重量份 聚乙二醇辛酸 /癸酸甘油酯 1.8 - 2.2重量份 泊洛沙姆 188 0.15 - 0.25重量份 大豆卵磷脂 0.4 - 0.6重量份 二氧化硅 0.8 -1.2重量份 交联纤维素钠 0.1 - 0.14重量份 PBS buffer 0.8 - 1.2 parts by weight of polyethylene glycol caprylic acid / glyceryl citrate 1.8 - 2.2 parts by weight of poloxamer 188 0.15 - 0.25 parts by weight of soy lecithin 0.4 - 0.6 parts by weight of silica 0.8 - 1.2 parts by weight Sodium cellulose sodium 0.1 - 0.14 parts by weight
0.013 - 0.017 重量 硬脂酸镁  0.013 - 0.017 Weight Magnesium Stearate
份  Share
甘氨酸 0.25 - 0.35重量份； (4) Glycine 0.25 - 0.35 parts by weight; (4)
猪胰岛素 0.005重量份 氢氧化钠 适量 Pig insulin 0.005 parts by weight sodium hydroxide
PBS緩沖液 1重量份 PBS buffer 1 part by weight
聚乙二醇辛酸 /癸酸甘油酯 2重量份 Polyethylene glycol caprylic acid / glyceryl citrate 2 parts by weight
泊洛沙姆 188 0.2重量份 Poloxamer 188 0.2 parts by weight
大豆卵磷脂 0.5重量份 Soy lecithin 0.5 parts by weight
二氧化硅 1重量份 交联纤维素钠 0.12重量份 硬脂酸镁 0.015重量份 甘氨酸 0.3重量份; 1 part by weight of silica Crosslinked cellulose sodium 0.12 parts by weight magnesium stearate 0.015 parts by weight glycine 0.3 parts by weight;
(5)  (5)
GLP-1 0.08-0.12重量份 去离子水 55 - 65重量份 辛酸癸酸甘油酯 12-18重量份 泊洛沙姆 188 1.3- 1.7重量份 大豆卵磷脂 6-8重量份 甘油 8-12重量份 聚乙二醇 400 6-8重量份 维生素 E 0.25 - 0.35重量份 (6) GLP-1 0.08-0.12 parts by weight of deionized water 55 - 65 parts by weight of caprylic caproic acid glyceride 12-18 parts by weight of poloxamer 188 1.3-1.7 parts by weight of soy lecithin 6-8 parts by weight of glycerin 8-12 parts by weight Polyethylene glycol 400 6-8 parts by weight of vitamin E 0.25 - 0.35 parts by weight (6)
GLP-1 0.1重量份 去离子水 60重量份 GLP-1 0.1 parts by weight Deionized water 60 parts by weight
辛酸癸酸甘油酯 15重量份 Caprylic citrate 15 parts by weight
泊洛沙姆 188 1.5重量份 大豆卵磷脂 7重量份 Poloxamer 188 1.5 parts by weight Soy lecithin 7 parts by weight
甘油 10重量份 Glycerin 10 parts by weight
聚乙二醇 400 7重量份 Polyethylene glycol 400 7 parts by weight
维生素 E 0.3重量份。 Vitamin E 0.3 parts by weight.
11. 根据权利要求 2所述的乳剂 其各组分及含量如下：  11. The emulsion according to claim 2, the components and contents of which are as follows:
0.0008-0.0012 重 0.0008-0.0012 heavy
PTH 1-34 PTH 1-34
份  Share
PBS緩冲液 0.4- 0.6重量份 辛酸癸酸甘油酯 0.6- 0.8重量份 聚乙二醇十二羟基硬脂酸酯 1.8-2.2重量份 生育酚琥珀酸聚乙二醇酯 0.4- 0.6重量份 吐温 20 0.4 - 0.6重量份 维生素 E 0.08一 0.12重量份; 具体地， 所述乳剂的组分及含量如下 PBS buffer 0.4-0.6 parts by weight glyceryl caprylate 0.6-0.8 parts by weight Polyethylene glycol dodecyl stearate 1.8-2.2 parts by weight tocopherol succinic acid polyethylene glycol ester 0.4-0.6 parts by weight Tween 20 0.4 - 0.6 parts by weight of vitamin E 0.08 to 0.12 parts by weight; The composition and content of the emulsion are as follows
PTH 1-34 0.001重量份  PTH 1-34 0.001 parts by weight
PBS緩冲液 0.5重量份  PBS buffer 0.5 parts by weight
辛酸癸酸甘油酯 0.7重量份 Caprylic glyceryl citrate 0.7 parts by weight
聚乙二醇十二羟基硬脂酸酯 2重量份 Polyethylene glycol dodecyl stearate 2 parts by weight
生育酚琥珀酸聚乙二醇酯 0.5重量份 Tocopherol succinic acid polyethylene glycol ester 0.5 parts by weight
吐温 20 0.5重量份 Tween 20 0.5 parts by weight
维生素 E 0.1重量份。 Vitamin E 0.1 parts by weight.
12. 根据权利要求 1至 11中任一项所述的乳剂， 其为微乳剂 或冻干乳剂。  The emulsion according to any one of claims 1 to 11, which is a microemulsion or a lyophilized emulsion.
13. 权利要求 1-12 项任一项所述的乳剂的制备方法， 包括 下述步驟：  The method of preparing an emulsion according to any one of claims 1 to 12, comprising the steps of:
1 ) 如下的步驟 A或 B:  1) The following steps A or B:
A. 在 4-25°C条件下， 将蛋白质或多肽分子溶于氷相；  A. Dissolving the protein or polypeptide molecule in the ice phase at 4-25 ° C;
B. 制备蛋白质或多肽的磷脂复合物；  B. preparing a phospholipid complex of a protein or polypeptide;
2) 在 20 - 70。C条件下， 将中链脂肪酸酯、 HLB值大于 12的乳化 剂相混合， 并在 200 - 800rmp搅拌条件下持续搅拌， 直至整个体 系呈透明清亮状；  2) At 20 - 70. Under the condition of C, the medium chain fatty acid ester and the emulsifier having an HLB value of more than 12 are mixed, and stirring is continued under stirring at 200 - 800 rpm until the whole system is transparent and clear;
3) 在 4- 37。C条件下， 将步驟 1) 产物加入到步骤 2 )产物中， 并在 200 - 800rmp条件下继续搅拌， 直至形成透明清亮的均匀制 剂， 最终获得含蛋白质或多肽药物的乳剂。 3) At 4-37. Under the conditions of C, the product of step 1) is added to the product of step 2 ), and stirring is continued at 200 - 800 rpm until a clear and clear homogeneous preparation is formed, and finally an emulsion containing the protein or polypeptide drug is obtained.
14. 权利要求 10所述的乳剂在制备治疗或预防糖尿病、 或者 降低血糖的药物中的用途。 14. The emulsion of claim 10 in the manufacture of a treatment or prevention of diabetes, or Use in drugs that lower blood sugar.
15. 权利要求 11所述的乳剂在制备治疗或预防骨质疏松的药 物中的用途。  15. Use of an emulsion according to claim 11 for the manufacture of a medicament for the treatment or prevention of osteoporosis.
16. 一种治疗或预防糖尿病、 或者降低血糖的方法， 包括给 予有效量的权利要求 10所述的乳剂的步骤。  16. A method of treating or preventing diabetes, or lowering blood glucose, comprising the step of administering an effective amount of the emulsion of claim 10.
17. 一种治疗或预防骨质疏松的方法， 包括给予有效量的权 利要求 11所述的乳剂的步骤。  17. A method of treating or preventing osteoporosis comprising the step of administering an effective amount of the emulsion of claim 11.
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CN102125520A (en) * 2011-02-24 2011-07-20 美迪思生物科技(北京)有限公司 Emulsion containing hydrophilic biological macromolecule, preparation method and application thereof
CN102552174B (en) * 2011-11-18 2015-07-22 中山大学 Preparation method of polypeptide/proteinic drug nanoparticle with high drug loading and high encapsulation efficiency
CN103861090B (en) * 2012-12-18 2017-06-13 美迪思生物科技(北京)有限公司 Hydrophobic sol, Preparation Method And The Use containing albumen or polypeptide
CN103655487B (en) * 2013-11-15 2015-05-06 西安力邦制药有限公司 Injection alprostadil freeze-dried emulsion
CN110639425B (en) * 2019-10-22 2021-09-17 湖北文理学院 Preparation method of composite emulsifier
CN113952301A (en) * 2021-11-17 2022-01-21 胡振华 Application of medium-chain fatty acid as absorption enhancer to preparation of pharmaceutical composition emulsion

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CN1456351A (en) * 2003-05-26 2003-11-19 上海医药工业研究院 Self-emulsifying agglomerant of oral polypeptide medicine
CN101422431A (en) * 2007-12-28 2009-05-06 上海医药工业研究院 Insulation administration preparation through nose

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CN101422431A (en) * 2007-12-28 2009-05-06 上海医药工业研究院 Insulation administration preparation through nose

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