WO2012113116A1

WO2012113116A1 - Emulsion containing hydrophilic biological macromolecule, preparation method and application thereof

Info

Publication number: WO2012113116A1
Application number: PCT/CN2011/000814
Authority: WO
Inventors: 马二利; 郑昌学
Original assignee: 美迪思生物科技(北京)有限公司
Priority date: 2011-02-24
Filing date: 2011-05-10
Publication date: 2012-08-30
Also published as: CN102125520A

Abstract

An emulsion containing hydrophilic biological macromolecule, preparation method and application thereof are provided. The emulsion contains hydrophilic biological macromolecule, water phase, oil phase, emulsifying agent, vitamin E and/or ester derivatives of vitamin E.

Description

Emulsion containing hydrophilic biomacromolecules, preparation method and use thereof

The invention belongs to the field of pharmacy and preparation, and relates to an emulsion containing a hydrophilic biomolecule, a preparation method thereof and use thereof. Background technique

Hydrophilic biomacromolecules, including proteins or peptides, polysaccharides, and nucleic acids, are an increasingly important class of drugs in the pharmaceutical field. However, a common feature of these substances is that oral administration is ineffective and clinically needed. Frequent injection administration. Therefore, it is a common ideal in the medical field to develop a drug delivery system that is highly patient-compliant and suitable for oral administration.

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 oral bioavailability of these hydrophilic biomacromolecules. Low problem. Carino GP et al (2000) J. Control. Release. 65: 261-269; Kathryn Wehitehead, 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) Biomaterials 21: 1499-1507; Christopher JH et al (1997) Adv. Drug. Deliver. Rev. 25: 71-89; Jeff Wang, et al (2003) J. Control. Release. 88: 369-380; Roger RC New, et al (1997) Adv. Drug. Deliver. Rev. 25: 59 -69; Anja Graf, et al (2008) Int. J. Pharm. 350: 351-360; R. Neslihan Gursoy et al (2004) Biomed. Pharmacother. 58: 173-182; CN01115327. X, US4849405, US5824638 ₀ However, the above-mentioned conventional safety problems for the oral administration of hydrophilic substances, for example, covalent modification may reduce the activity of the drug, and may change the pharmacology and toxicology of the drug molecule, causing serious safety hazards; enzyme inhibitors, long-term use It will cause digestive and absorption disorders and even enlargement or hyperplasia of the pancreas; absorption enhancers may cause irreversible damage to the cell membrane, and long-term use may cause membrane poisoning.

In addition, data on oral bioavailability of biomacromolecules that have been reported so far are not ideal. Oral insulin microemulsion bioavailability is only 4.44% (Amani Elsayed, et al, European Journal of Pharmaceutics and Biopharmaceutics 73 (2009) 269 - 279 ); oral PTH formulation bioavailability is only 2.1% ( Andrea Leone-Bay, et Al, Pharmaceutical Research, Vol. 18, No. 7, 2001).

Vitamin E, also known as tocopherol, is a common fat-soluble vitamin E. When used as an antioxidant, the concentration is generally less than 1%. Vitamin E is also often used as a nutritional supplement for a wide range of applications in health care, food, medical, and beauty. Due to its special structure, vitamin E has good solubility for some hydrophobic molecules such as paclitaxel, cyclosporine and body substances. According to this, Constantinides et al., in addition to "water-soluble" and "oil-soluble", The concept of "vitamin E dissolution" (Constantinides, PP, et al. (2004) Adv Drug Deliv Rev. 56: 175-82).

A new drug delivery vector for hydrophobic molecules using vitamin E as a solvent was first seen in the study by Y. Kato et al., and subsequent research has emerged in an endless stream (Y. Kato, et al. (1993) Chem. Pharm. Bull. ( Tokyo) . 41: 599 - 604; Constantinides, PP, et al. (2000) Pharm Res. 17: 175-82; PB Nielsen, et al. (2001) Int. J. Pharm. 222: 217-24; Constantinides , PP, et al. (2006) Pharm Res. 23: 243-55; WO 95/11039; WO97/03651; WO 99/04787; US 6,479,540; US 6, 458, 373; US7030155B2). Vitamin E is highly safe as a food-borne substance and a nutritional supplement. However, the current study only uses vitamin E as a carrier for hydrophobic drugs. Vitamin E has not yet been used to achieve oral administration of hydrophilic biomacromolecules. Report of the drug. Summary of the invention

The inventors have conducted extensive experiments and creative labor to obtain an emulsion containing a hydrophilic biomacromolecule in which an ester derivative of vitamin E or vitamin E is used as a carrier of a hydrophilic biomacromolecules. Vitamin E or its ester derivatives are not toxic by themselves, and can effectively solve the safety hazards of currently administered hydrophilic biomacromolecules. The present inventors have surprisingly found that the use of an ester derivative of vitamin E or vitamin E as a carrier for the administration of a hydrophilic biomacromolecule can significantly improve the oral bioavailability of these hydrophilic biomacromolecules.

The following invention is thus provided:

An emulsion containing a hydrophilic biomacromolecule comprising a hydrophilic biomacromolecule, an aqueous phase, an oil phase, an emulsifier, characterized in that the emulsion further comprises an ester derivative of vitamin E and/or vitamin E .

The emulsion according to any one of the invention, wherein the hydrophilic biomacromolecule is selected from the group consisting of a protein or a polypeptide, a polysaccharide, and a nucleic acid. The hydrophilic biomolecule can be isolated from a living organism, or can be obtained by genetic engineering or organic synthesis.

The emulsion according to any one of the present invention, wherein the vitamin E is one or more selected from the group consisting of d-α-vitamin E and d-α-vitamin E, and the vitamin oxime ester derivative The substance is selected from the group consisting of d-a-vitamin E acetate, dl-a-tocopheryl acetate, da-vitamin E succinate, d-a-vitamin E succinate, da-vitamin E succinic acid polyethylene glycol One or more of an ester, and a polyethylene glycol dl-a-tocopheryl succinate. The ester derivative of vitamin E or vitamin E in the present invention may be natural or artificially synthesized.

The emulsion according to any one of the present invention, wherein the hydrophilic biomacromolecule is 0.01 to 1 part by weight, and the ester derivative of the vitamin E and/or vitamin E is 50 - 1 Q0G parts by weight, 50 - 5 QQ parts by weight, or 100 - 300 parts by weight.

The emulsion according to any one of the invention, wherein the aqueous phase, the oil phase, and the emulsifier are 20 - 2000 parts by weight, 20 - 1000 parts by weight, or 50 - 500 parts by weight in total. The amount of each of the aqueous phase, the oil phase, and the emulsifier can be readily determined by those skilled in the art.

The emulsion according to any one of the present invention, wherein the protein or polypeptide is selected from the group consisting of calcitonin, insulin, GLP-1 (glucagon-like peptide-1), parathyroid hormone, and growth hormone The polysaccharide is selected from the group consisting of heparin, low molecular weight heparin (average molecular weight of 4000-6000), chondroitin sulfate, keratan sulfate, and hyaluronic acid; the nucleic acid is selected from the group consisting of an antisense nucleic acid, a ribozyme, an interfering RNA, and a DECOY nucleic acid. And a triple-stranded nucleic acid; specifically; the calcitonin is salmon calcitonin, salmon calcitonin, or human calcitonin; the insulin is porcine insulin, bovine insulin, or human insulin; The gland hormone is human parathyroid hormone 1-84, or human parathyroid hormone 1-34, and the growth hormone is porcine growth hormone, bovine growth hormone, or human growth hormone.

The emulsion according to any one of the present invention, wherein the aqueous phase is one or more selected from the group consisting of water, physiological saline, and an aqueous phase buffer.

The emulsion according to any one of the invention, wherein the emulsifier is selected from the group consisting of soy lecithin, egg yolk lecithin, poloxamer 188, poloxamer 407, carbomer, tween 20, spit Warm 80, polyethylene glycol dodecyl stearate, polyoxyethylene ether (35) castor oil, polyoxyethylene ether (40) hydrogenated castor oil, polyglycerol-6-dioleate, polyglycerol-3 - oleate, polyethylene glycol-32-lauric acid glyceride, polyethylene glycol-32-stearic acid One or more of a glyceride, and a polyethylene glycol caprylic/capric glyceride.

The emulsion according to any one of the present invention, wherein the oil phase is selected from the group consisting of medium chain fatty acid glycerides, soybean oil, corn oil, peanut oil, oleic acid, polyethylene glycol 400, glycerin, ethanol, and propylene glycol. One or more of them. 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.

According to any of the emulsions of the present invention, further comprising a pH adjusting agent, a _P H adjusting agent selected from hydrochloric acid, lactic acid, acetic acid, citric acid, phosphoric acid, sodium hydroxide, sodium carbonate, sodium bicarbonate One or more of disodium hydrogen phosphate and sodium dihydrogen phosphate.

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.

An emulsion according to any one of the invention, which is administered orally. In a specific embodiment of the invention, the components and contents of the hydrophilic biopolymer-containing emulsion are as follows:

Salmon calcitonin 0. 001 parts by weight 1 mole of hydrochloric acid

Physiological saline 0. 35 parts by weight D-α-vitamin E 0.7 parts by weight

Oleic acid 0.3 parts by weight

Polyethylene glycol dodecyl stearate

1.5 parts by weight

Tween 80 1.5 parts by weight.

The invention further relates to the use of the emulsion in the manufacture of a medicament for the treatment or prevention of 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 hydrophilic biomacromer-containing emulsion are as follows:

Recombinant human insulin 0.03 - 0.05 parts by weight sodium hydroxide

Deionized water 0.8-1.2 parts by weight dl-α-vitamin E vinegar 0.8 - 1.2 parts by weight Medium chain fatty acid glycerides (eg Labrafac 0.6-0.7 parts by weight Lipophi le 1349, Gattefosse )

Polyethylene glycol octanoate / glyceryl citrate (eg 2.5-3.5 parts by weight Sofitgen® 767, SAS0L)

Bo, Losham 188 0.1 - 0.3 parts by weight Soy lecithin 0.4 - 0.6 parts by weight. The invention further relates to the use of the emulsion in the manufacture of a medicament for the treatment or prevention of diabetes, or for the reduction of 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 a specific embodiment of the invention, the components and contents of the hydrophilic biomacromer-containing emulsion are as follows:

Recombinant human insulin 0.04 parts by weight 1 part by weight of sodium hydroxide in deionized water

Dl-α-vitamin E acetate 1 weight * part medium chain fatty acid glyceride (eg Labrafac 0.5 parts by weight

Lipophile 1349, Gattef osse )

Polyethylene glycol caprylic acid / glyceryl citrate (for example, 3 parts by weight)

Sofitgen® 767, SAS0L)

Poloxamer 188 0.2 heavy

Soy lecithin 0.5 weight

The invention further relates to the use of the emulsion in the manufacture of a medicament for the treatment or prevention of diabetes, or for lowering blood glucose. In a specific embodiment of the present invention, the components and contents of the hydrophilic biomacromer-containing emulsion are as follows:

GLP-1 0.0005 parts by weight

PBS buffer 0.3 parts by weight d-α-tocopherol polyethylene glycol succinate 1.5 parts by weight polyethylene glycol dodecyl stearate 2 parts by weight Tween 20 0.5 parts by weight. The invention further relates to the use of the emulsion in the manufacture of a medicament for the treatment or prevention of diabetes, or for the reduction of 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 hydrophilic biomacromer-containing emulsion are as follows:

PTH 1-34 0.005 - 0.015 parts by weight Water (preferably double distilled water) 0.4-0.6 parts by weight

Dl-α-vitamin E 0.6 - 0.8 parts by weight of d-α-tocopherol polyethylene glycolate 0.4.0.6 parts by weight of soybean oil 1.0-1.4 parts by weight of soy lecithin 0.6-0.8 parts by weight of polyoxyethylene ether (40 Hydrogenated castor oil 2.5-3.5 parts by weight of polyethylene glycol 400 1.5-2.5 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 a specific embodiment of the invention, the components and contents of the hydrophilic biopolymer-containing emulsion are as follows:

PTH 1-34 0.01 parts by weight Water (preferably double distilled water) 0.5 parts by weight Acetic acid

Dl-α-vitamin Ε 0.7 parts by weight of d-α-vitamin succinic acid polyethylene glycol ester 0.5 parts by weight of soybean oil 1.2 parts by weight of soybean lecithin 0.7 parts by weight of polyoxyethylene ether (40) hydrogenated castor oil 3 parts by weight Ethylene glycol 400 2 parts by weight.

The invention further relates to the use of the emulsion in the manufacture of a medicament for the treatment or prevention of osteoporosis, in a particular embodiment of the invention, the hydrophilic biological fraction 0重量份

5重量份的聚含含-3-oleic acid ester 1. 2 parts by weight of Tween 8Q 1 part by weight of polyethylene glycol caprylic acid / glycerol citrate; 2重量份。 7 parts by weight of polyethylene glycol 400 0. 5 parts by weight. The invention also relates to the use of the emulsion in the manufacture of a medicament for anticoagulation and or thrombolysis. The invention further relates to a method of treating or preventing a thrombus, or anticoagulation, comprising the step of administering an effective amount of the emulsion. In a specific embodiment of the invention, the components and contents of the hydrophilic biopolymer-containing emulsion are as follows:

Small amount of interfering RNA (for example, 500 bp) 0. 0003 parts by weight water for injection 0. 5 parts by weight

5重量份。 According to the present invention, the present invention is a glycerol phthalic acid glyceride. In another aspect, the method for preparing a hydrophilic biomacromer-containing emulsion according to any one of the present invention comprises the steps of:

1) dissolving the hydrophilic biomacromolecules in the aqueous phase at 4 - 25 °C;

2) Mixing the ester derivatives of vitamin E and/or vitamin E with the oil phase and emulsifier at 20 - 70 ° C and stirring at 200 - 800 rpm Stir until the entire system is clear and clear;

3) At 4 - 37. Under the condition of C, the product of step 1) is added dropwise 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 a hydrophilic biomacromolecular emulsion is obtained. Still another aspect of the present invention relates to the use of the hydrophilic biomacromer-containing emulsion of the present invention for the preparation of a medicament for treating or preventing diabetes, or lowering blood glucose, wherein the hydrophilic biomacromolecule is insulin or GLP- 1. Still another aspect of the present invention relates to the use of the hydrophilic biomacromolecular-containing emulsion of the present invention for the preparation of a medicament for treating or preventing osteoporosis, wherein the hydrophilic biomacromolecule is calcitonin or scorpion Parathyroid hormone, parathyroid hormone 1-84, or human parathyroid hormone 1-34. A further aspect of the invention relates to the use of an ester derivative of vitamin E and/or vitamin E for the preparation of a hydrophilic biomolecular-containing emulsion. Specifically, the hydrophilic biomacromer-containing emulsion is the hydrophilic biomacromolecular emulsion according to any one of the inventions. Specifically, the vitamin E is one or more selected from the group consisting of d-α-vitamin E and cU-a-vitamin E, and the ester derivative of the vitamin E is selected from the group consisting of da-vitamin E acetate. d a-vitamin E acetate, d-a-tocopheryl succinate, d-a-vitamin E succinate, d-a-vitamin E succinate, and d la-vitamin E amber One or more of acid polyethylene glycol esters. Specifically, the hydrophilic biomacromolecule and the aqueous phase, the oil phase, the emulsifier, the pH adjuster, and the like, and the amounts thereof are as described above. Advantageous effects of the invention Compared with the prior art, the invention has the following advantages and outstanding effects:

1) In the present invention, oral administration of a hydrophilic biomacromolecular drug is achieved by means of an ester derivative of vitamin E and/or vitamin E, and is safe, effective, and has high oral bioavailability, and bioavailability. Degree can reach 5%, 10%, 12%, 15% or even 30%;

2) The prepared vitamin E microemulsion containing a hydrophilic bio-low molecular drug can spontaneously emulsify into a droplet of 300 nm or less after being in contact with water, and is better absorbed and utilized by the gastrointestinal tract to exert better biological activity.

3) The preparation process of the invention is simple, low in cost and suitable for large-scale industrial production. DRAWINGS

Fig. 1: Diagram of blood glucose changes in rats under different modes of administration.

Fig. 2: Phase diagram of plasma concentration of rat PTH1-34 under different modes of administration. detailed description

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 an emulsion containing salmon calcitonin (microemulsion)

Its composition is as follows:

Salmon calcitonin 0. 001 g

1 mole of hydrochloric acid Saline G.35g

D-α-vitamin E 0.7 g oleic acid Q.3 g polyethylene glycol dodecyl stearate (Solutol 1.5 g

HS 15, BASF)

Tween 80 1.5 grams.

The preparation method of the preparation is briefly described as follows:

1) 4. C, the salmon calcitonin is dissolved in physiological saline, adding an appropriate amount of hydrochloric acid, stirring to clarify, to obtain a solution;

2) at 70 ° C, the above weight of d-α-tocopherol, oleic acid, polyethylene glycol dodecyl stearate, Tween 80, mixed and stirred at 200 rpm, until transparent and clear;

3) 30. Under C, add the A solution dropwise to the product of step 2), stir the mixture to a clear and clear at 800 r/min, and finally obtain an emulsion (microemulsion) containing salmon calcitonin (called sample 1). ).

It will be understood by those skilled in the art that although the weight unit of each component in Example 1 is g, 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 emulsion containing recombinant human insulin (microemulsion)

Its composition is as follows:

Recombinant human insulin 0.04 g

5 moles of sodium hydroxide

Deionized water 1 g

(il-a-vitamin E acetate 1 g

Medium chain fatty acid glyceride (Labrafac Lipophile 0.5g 1349, Gattefosse)

Polyethylene glycol octanoate / glyceryl citrate ( Sofitgen® 3 g

767, SAS0L)

Poloxamer 188 (Lutrol F68, BASF) 0.2 g

Soy lecithin (Epikuron 170, Degussa) 0.5 g.

The preparation method of the preparation is briefly described as follows:

1) at 25 ° C, the above weight of recombinant human insulin is dissolved in 0.5 grams of deionized water, adding an appropriate amount of sodium hydroxide, stirred until clear, to obtain a solution;

2) 20. C, the above weight (Π-α-vitamin E acetate, medium chain fatty acid glyceride, polyethylene glycol caprylic/capric glyceride, poloxamer 188, soy lecithin, and the remaining aqueous phase are mixed, And stirred at 800 rpm to achieve clear and clear;

3) Add the A solution dropwise to the product of step 2) at 4 ° C, stir the mixture to clear and clear at 200 r / min, and finally obtain an emulsion (microemulsion) containing recombinant human insulin (called Sample 2).

It will be understood by those skilled in the art that although the weight unit of each component in Example 2 is g, it can be understood as a part by weight, i.e., the weight ratio in the above Example 2 is satisfied between the components. Example 3: Preparation of an emulsion containing GLP-1 (microemulsion)

Its composition is as follows:

GLP-1 0.0005 g

PBS buffer 0.3 g

d-α-Vitamin E succinic acid polyethylene glycol ester (TPGS, 1.5 g

Eastman )

Polyethylene glycol dodecyl stearate (Solutol HS 2 g

15, BASF) Tween 20 0.5 g.

The preparation method of the preparation is briefly described as follows:

1) Dissolve the above weight of GLP-1 in PBS buffer at 20 ° C, stir until clarified to obtain A solution;

2) Mix the above weight of d-α-tocopheryl succinate, polyethylene glycol dodecyl stearate, and Tween 20 at 60 ° C, and mix at 600 rpm until transparent Clear

3) Add the A solution dropwise to the product of step 2) at 37 ° C, stir the mixture to clear and clear at 500 r / min, and finally obtain a GLP-1 -containing emulsion (microemulsion) (called Sample 3).

It will be understood by those skilled in the art that although the weight unit of each component in Example 3 is g, 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 an emulsion containing PTH 1-34 (microemulsion)

Its composition is as follows:

PTH 1-34 0.01 g double distilled water 0.5 g

Acetic acid

d Bu α-Vitamin E 0.7 g

D-α-vitamin succinic acid polyethylene glycol ester (TPGS, 0.5 g

Eastman )

Soybean oil 1.2 g

Soy lecithin (Epikuron 170, Degussa) 0·7 g

Polyoxyethylene ether (40) hydrogenated castor oil (Cremophor 3 g

®RH 40, BASF) 2 g of polyethylene glycol 400.

The preparation method of the preparation is briefly described as follows:

1) 20. In C, the above weight of PTH 1-34 is dissolved in double distilled water, an appropriate amount of acetic acid is added, and the solution is stirred until clarified to obtain a solution A;

2) at 50'C, the above weight (Π-α-vitamin E, d-α-tocopheryl succinate, ethylene glycol ester, soybean oil, soy lecithin, polyoxyethylene ether (40) hydrogenated castor oil , polyethylene glycol 400, mixed and stirred at 400 rpm until clear and clear;

3) Add the A solution dropwise to the product of step 2) at 30 ° C, and continue stirring at 500 r / min to clear and clear, and finally obtain a PTH 1-34 containing emulsion (microemulsion) ( Called sample 4).

It will be understood by those skilled in the art that although the weight unit of each component in Example 4 is g, 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: Preparation of emulsion containing low molecular weight heparin (microemulsion)

Its composition is as follows:

Low molecular weight heparin 0.3g

PBS buffer 0.5 g

Dl-a-vitamin E 0.2 g

Polyglycerol-3-oleate 1.2 g

Tween 80 1 gram

Polyethylene glycol octanoate / glyceryl citrate 2.7 g

Polyethylene glycol 400 0.5 g.

The preparation method of the preparation is briefly described as follows:

1) at 10 ° C, the above weight of low molecular weight heparin is dissolved in PBS solution, stirred until clear, to obtain A solution; 2) mixing the above-mentioned weights of d-α-vitamin, polyglycerol-3-oleate, Tween 80, polyethylene glycol caprylic/capric glyceride, and polyethylene glycol 400 at 50 ° C Stir at 400 rpm until clear and clear;

3) Add the A solution dropwise to the product of step 2) at 30 ° C, and continue stirring at 500 r / min to clear and clear, and finally obtain an emulsion (microemulsion) containing low molecular weight heparin. For sample 5).

It will be understood by those skilled in the art that although the weight unit of each component in Example 5 is g, it can be understood as a part by weight, i.e., the weight ratio in the above Example 5 is satisfied between the components. Example 6: Preparation of an emulsion containing small interfering RNA (microemulsion) The composition is as follows:

Small interfering RNA (self-made, from hepatitis B disease 0. 0003 g poison P area, 500bp)

Water for injection 0. 5 grams

Dg-α-vitamin E 0. 2 g Polyglycerol-3-oleate 0. 3 g Polyethylene glycol octanoic acid / glyceryl citrate 2. 7 g Glycerol 0. 5 g.

The preparation method of the preparation is briefly described as follows:

1) Dissolving the above-mentioned small interfering RNA in water for injection at 4 ° C, stirring until clarification, to obtain A solution;

2) Mix the above weight (U-a-vitamin E, polyglycerol-3-oleate, polyethylene glycol caprylic/capric glyceride, glycerin) at 50 ° C and stir until transparent at 300 rpm Clear

3) 10. C, the A solution is added dropwise to the product of step 2), and At 200 r/min, stirring was continued until clear and clear, and finally an emulsion (microemulsion) containing small molecule interference RNA (referred to as sample 6) was obtained.

It will be understood by those skilled in the art that although the weight unit of each component in Example 6 is g, it can be understood as a part by weight, i.e., the weight ratio in the above Example 6 is satisfied between the components. Example 7: Particle size measurement test

The particle size of Samples 1-6 prepared in Examples 1-6 was measured with a Malvern Zetasizer 3000HSA Particle Size Analyzer, and the sample was diluted 30 times with Millipore Ultrapure Water before the measurement. The results are shown in Table 1.

Table 1: Samples 1-6 Average particle size after dilution

Example 8: Efficacy test of emulsion containing recombinant human insulin (microemulsion) Oral test group: 6 male SD rats in the range of 180 - 220 g, drug (test drug) was sample 2 prepared in Example 2.

Oral control group: 6 male Sprague-Dawley rats in the range of 180-220 g, the drug (control drug) was prepared according to the composition and procedure of Example 2, except that no d-α-vitamin oxime acetate was added. Intravenous control group: 6 male Sprague-Dawley rats in the range of 180-220 g, the drug was an aqueous insulin solution.

experiment method:

Male Sprague-Dawley rats weighing 180-220 g were fasted for 12 h before starting the test. The test group was administered orally, and the dose was 0.11⁄2 g/kg in terms of insulin. Rats in the experimental group were anesthetized with sodium pentobarbital at a dose of 50 rag before the test, then bundled in the supine position, and cut into a small mouth in the abdomen, and administered to the ileum by the applicator. After that, suture the incision. The control group was administered subcutaneously as a control drug, and the dose was 0.04 mg/kg based on the insulin.

The three groups were all taken from the tail vein at the time of 0.5, 1, 2, 3, and 4 h after administration. The blood glucose was measured using a Roche blood glucose meter and the average blood glucose level of 6 rats in each group was calculated. As shown in Figure 1. As can be seen from Fig. 1, the bioavailability of the test group administered orally was 12.5% relative to the control group administered by injection. Example 9: Emulsion containing PTH1-34 (microemulsion) Bioavailability test Test group: 10 male SD rats in the range of 180 - 220 g, and the drug (test drug) was the sample prepared in Example 4.

Control group: 10 male SD rats in the range of 180-220 g, the drug was PTH1-34 aqueous solution, and administered by injection.

experiment method:

Male SD rats weighing 180-220 g were fasted for 12 h before starting the test. The test group was orally administered, and the dose was 10 (^g/kg) according to PTH1-34. The animals in the oral group were anesthetized with sodium pentobarbital at a dose of 50 mg before the experiment, and then tied in an upright manner. A small opening was made in the abdomen, and the ileum was administered by the applicator. After the administration, the incision was sutured. The control group was subcutaneously injected with PTH1-34, and the dose was 10 g/kg as PTH1-34. The blood was taken from the tail vein at the time points of 15, 30, 45, 60, 75, 90, and 105 minutes after administration, and the serum was separated for testing. Serum PTH 1-34 concentrations were measured using the PTH 1-34 high sensitivity EIA kit. The results are shown in Figure 2, where the area under the PTH1-3 blood concentration curve is calculated. 2%。 The bioavailability of the test group administered orally was 23.13%.

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

Rights request

An emulsion containing a hydrophilic biomacromolecule comprising a hydrophilic biomacromolecule, an aqueous phase, an oil phase, an emulsifier, characterized in that the emulsion further comprises an ester of vitamin E and/or vitamin E derivative.

The emulsion according to claim 1, wherein the hydrophilic biomacyon is selected from the group consisting of a protein or a polypeptide, a polysaccharide, and a nucleic acid.

The emulsion according to claim 1, wherein the vitamin E is one or more selected from the group consisting of d-α-vitamin E and dl-ct-vitamin E, and the ester derivative of the vitamin E It is selected from d-α-vitamin E acetate, (U-α-vitamin oxime acetate, d-α-tocopheryl succinate, dl-α-vitamin succinate, d-α-vitamin E succinic acid One or more of polyethylene glycol ester, and dl-a-tocopheryl succinate polyethylene glycol ester.

The emulsion according to claim 1 , wherein the hydrophilic biomacromolecule is 0.01 to 1 part by weight, and the ester derivative of the vitamin E and/or vitamin E is 50 to 1000 weight. Parts, 5 Q - 5 GG parts by weight, or 100 - 3 GG parts by weight.

The emulsion according to claim 4, wherein the aqueous phase, the oil phase, and the emulsifier are 20 - 2000 parts by weight, 20 - 1000 parts by weight, or 50 - 500 parts by weight in total.

The emulsion according to claim 1, wherein the protein or polypeptide is selected from the group consisting of calcitonin, insulin, GLP-1, parathyroid hormone, and growth hormone; the polysaccharide is selected from the group consisting of heparin, low molecular weight heparin, Chondroitin sulfate, keratan sulfate, and hyaluronic acid; the nucleic acid is selected from the group consisting of an antisense nucleic acid, a ribozyme, an interfering RNA, a DECOY nucleic acid, and a triple-stranded nucleic acid; specifically; the calcitonin is salmon calcitonin, Salmon calcitonin, or human calcitonin; the insulin is porcine insulin, bovine insulin, or human insulin; the parathyroid hormone is human parathyroid hormone 1-84, or human parathyroid hormone 1-34, the growth hormone is porcine growth hormone, bovine growth hormone, or human growth hormone b

7. The emulsion according to claim 1, which satisfies any one or more of the following (1) - (3):

(1) the aqueous phase is one or more selected from the group consisting of water, physiological saline, and an aqueous phase buffer;

(2) the oil phase is one or more selected from the group consisting of medium chain fatty acid glycerides, soybean oil, corn oil, peanut oil, oleic acid, polyethylene glycol 400, glycerin, ethanol, and propylene glycol;

(3) The emulsifier is selected from the group consisting of soybean lecithin, egg yolk lecithin, poloxamer 188, poloxamer 407, carbomer, Tween 20, Tween 80, polyethylene glycol dodecyl hard Fatty acid ester, polyoxyethylene ether (35) castor oil, polyoxyethylene ether (40) hydrogenated castor oil, polyglycerol-6-dioleate, polyglycerol-3-oleate, polyethylene glycol-32 One or more of glyceryl laurate, polyethylene glycol-32-stearate, and polyethylene glycol caprylic/capric glyceride.

8. The emulsion according to claim 1, further comprising a pH adjusting agent selected from the group consisting of hydrochloric acid, lactic acid, acetic acid, citric acid, phosphoric acid, sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, phosphoric acid One or more of disodium hydrogenate and sodium dihydrogen phosphate.

9. An emulsion according to any one of claims 1 to 8 which is a microemulsion.

The emulsion according to claim 1, wherein the composition and content are as follows: recombinant human insulin 0. 03 - 0. 05 parts by weight sodium hydroxide

Desicfied water 0. 8 - 1. 2 parts by weight of dl-α-vitamin E acetate 0. 8 - 1. 2 parts by weight of medium chain fatty acid glyceride 0. 6 - 0. 7 parts by weight of polyethylene glycol octanoic acid / hydrazine Acid glyceride 2. 5 - 3. 5 parts by weight Poloxamer 188 0.1 - 0.3 parts by weight of soybean lecithin 0.4 - 0.6 parts by weight; specifically, the components and contents of the emulsion are as follows

Recombinant human insulin 0.04 parts by weight sodium hydroxide

Deionized water 1 part by weight

Dl-α-vitamin E acetate 1 part by weight

Medium chain fatty acid glyceride 0.5 parts by weight Polyethylene glycol octanoate / glyceryl citrate 3 parts by weight

Polosham 188

Soy lecithin

11. The emulsion of claim 1

PTH 1-34

Water

Acetic acid

Dl-α -Vitamin E 0.6 - 0. 8 parts by weight of d-α-vitamin E succinic acid polyethylene glycol ester 0.4- 0. 6 parts by weight of soybean oil 1.0 - 1. 4 parts by weight of soy lecithin 0.6 - 0. 8 Parts by weight of polyoxyethylene ether (40) hydrogenated castor oil 2.5 - 3. 5 parts by weight of polyethylene glycol 400 1.5 - 2. 5 parts by weight; specifically, the components and contents of the emulsion are as follows

PTH 1-34 0.01 parts by weight water 0.5 parts by weight

Acetic acid

Dl-a - vitamin E 0.7 parts by weight 5重量份。 D-α-Vitamin E succinate polyethylene glycol ester 0. 5 parts by weight

Soybean oil 1. 2 parts by weight

Soy lecithin 0. 7 parts by weight

Polyoxyethylene ether (40) hydrogenated castor oil 3 parts by weight

Polyethylene glycol 400 2 parts by weight.

The method for preparing a hydrophilic biomacromer-containing emulsion according to any one of claims 1 to 11, comprising the steps of:

1) At 4-25. Under the condition of C, the hydrophilic biological macromolecule is dissolved in the aqueous phase;

2) Mix the ester derivatives of vitamin E and/or vitamin E with the oil phase and emulsifier at 20 - 70 °C, and continue stirring under the stirring condition of 200 - 800rnip until the whole system is transparent and clear. Shape

3) At 4 - 37. Under the condition of C, the product of step 1) is added dropwise 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 a hydrophilic biomacromolecular emulsion is obtained.

13. Use of an emulsion according to claim 10 for the manufacture of a medicament for the treatment or prevention of diabetes, or for lowering blood glucose.

14. Use of an emulsion according to claim 11 for the manufacture of a medicament for the treatment or prevention of osteoporosis.

15. Use of an ester derivative of vitamin E and/or vitamin E in the preparation of a emulsion containing a hydrophilic biomolecule.

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. A method of treating or preventing osteoporosis comprising the step of administering an effective amount of the emulsion of claim 11.