CN103861090B

CN103861090B - Hydrophobic sol, Preparation Method And The Use containing albumen or polypeptide

Info

Publication number: CN103861090B
Application number: CN201210550294.6A
Authority: CN
Inventors: 马二利; 郑昌学
Original assignee: MEDIS BIO-TECHNOLOGY (BEIJING) Co Ltd
Current assignee: MEDIS BIO-TECHNOLOGY (BEIJING) Co Ltd
Priority date: 2012-12-18
Filing date: 2012-12-18
Publication date: 2017-06-13
Anticipated expiration: 2032-12-18
Also published as: CN103861090A

Abstract

The invention belongs to materia medica and pharmaceutical art, it is related to hydrophobic sol, Preparation Method And The Use containing albumen or polypeptide.In the present invention, using water-soluble co-solvents as albumen and/or the decentralized medium of polypeptide, preparation process does not need water or the aqueous solution to participate in, the decentralized medium containing albumen and/or polypeptide of gained, directly can be mixed with hydrophobic, single-phase, transparent and stable liquid solution is derived from, the fat-soluble low problem of this quasi-molecule is efficiently solved.The fat-soluble and cross-film ability of the albumen and/or the hydrophobic sol of polypeptide being made of the method, albumen and/or polypeptide is significantly improved, and bioactivity can keep muchly stability at room temperature, without the phenomenon that degraded or bioactivity loss occur.Product of the invention, preparation process is simple is with low cost, is suitable for industrialized production.

Description

Hydrophobic sol, Preparation Method And The Use containing albumen or polypeptide

Technical field

The invention belongs to materia medica and pharmaceutical art, be related to the hydrophobic sol containing albumen or polypeptide, its preparation method and Purposes.

Background technology

Some have protein and peptide such as trypsase, chymotrypsin, insulin, the parathyroid hormone of important application Deng, all it is hydrophilic, its hydrophobization has many important applications, for example：The stability of hydrophilic substance can be improved, can The fat-soluble of protein and peptide drugs is improved, increases the ability that medicine passes through membranes barriers；Albumen or peptide drug can be extended The action time of thing molecule, realize slow controlled release drug administration；Hydrophobization is carried out to protease to fix, can be greatly enhanced yield, and Be conducive to the separation of product and the recovery of protease.

Zhang Haizhou et al. passes through oily bag alcohol system, has prepared ascorbic non-aqueous solution, can greatly improve vitamin The stability of C.But oily bag alcohol system is unstable, therefore, Zhang Haizhou et al. is with cetyl polyethylene/polypropylene glycol -1,0/1 two Methylsiloxane（ABIL EM90, viscosity 400-2000cS）Make emulsifying agent, prepare cream preparation, oleyl alcohol can be avoided to separate （Zhang Haizhou, etc., for the research of ascorbic oily polyol system in stable cosmetic, 2009《Daily chemical industry》39 （4）：249-252；Patent CN200810100726.7）.United States Patent (USP) US2011/0147259A1 is dystectic auxiliary by adding Material trihydroxy stearic acid（110~140C of fusing point）With 12- hydroxy stearic acids（Fusing point 80C）To increase system viscosity（K value is situated between In 2,000cp-3,000,000cp）, and then increase the stability of oily bag alcohol.Above-mentioned technical proposal, is by increasing viscosity It is layered with the auxiliary material for overcoming different densities, the stability of the oily bag alcohol formulations of raising, cream preparation or 2,000cp-3,000, The oily bag alcohol system of 000cp, those skilled in the art know, and these systems are not the molten of clear homogeneous truly Liquid, emulsifying agent ABIL EM90 used are only limitted to be applied in cosmetics by the description of product, can not be medicinal, and 12- hydroxyls Base stearic acid is classified as indirect food additive by U.S. FDA（For food containers and packaging material）, can not be directly as medicine Thing auxiliary material is used.In addition, containing substantial amounts of polyalcohol in CN200810100726.7（40-80wt%,）, it is difficult to it is significant to improve It is ascorbic fat-soluble.

Patent CN98126744.0 and CN200910027809.2 are by way of gene mutation, protease to be carried out Transformation, produces the protease variant that can be stable in the presence of in non-aqueous media.M.Hashimoto is by palmitic acid covalently tying The mode of conjunction is connected on insulin molecule, is found by the insulin after modification, and fat-soluble and cross-film ability is dramatically increased（M. Hashimoto,etal.(1992)J.Pharm.Pharmacol.44:555-559）.But, the general character of these technological means is Industrialization high cost, and with the change of medicines structure, this is possible to change toxicity, the pharmacology of drug molecule.Comparatively, By the means of preparation, increase the fat-soluble of hydrophilic molecules, with it is safe and efficient the characteristics of.

By liposome technology, or the compound of the materials such as medicine and phosphatide is formed, hydrophilic substance can be wrapped in thin In aqueous medium, so as to improve the fat-soluble and stability of drug molecule.Correlative study is see JP Colletier, et al. (2002)BMC Biotechnol.2:9-16;CN1421246A, Zhang Lei, etc., 2009,《Pharmacy and clinical research》17：81-84； F.D.Cui,et a l.(2006)J CONTROL RELEASE.114:242-250;D.Y.Lee,et al.(2007)J CONTROL RELEASE.123:39-45.But in these researchs, the hydroaropic substance such as protein, low molecular weight heparin, and It is not dissolved in hydrophobic phase really, and whole system is also presented with solid-state, rather than be a kind of single-phase, stable, clarification liquid Liquid solution.In addition, for technological process, above-mentioned technical proposal is required to be removed by means such as rotary evaporation, freeze-dryings Solvent, thus increased equipment cost and production cost.

Patent US5719039 obtains a kind of non-aqueous solution containing protease, and it is under the conditions of suitable pH, will to contain The aqueous solution and isooctane solution containing Aerosol OT for having protease mix, protease to be formed and surfactant from After sub- compound, water phase is removed by the method for extracting, the protease being dissolved in organic phase is combined with surface active agent ion Thing forms single phase soln of stabilization in after drying, being soluble in acetone and other organic solvent.But it is organic in field of medicaments Solvent has strict residual quantity to limit.

Technical scheme disclosed in patent CN01115327.X, is that the aqueous solution containing insulin is mixed with parents' material Close, it is final to obtain the oil-phase solution containing insulin.Similar AmaniElsayed will contain insulin-chitosan complexes The aqueous solution, with parents' material mixing, final oil-phase solution of the acquisition containing insulin.Amani Elsayed,et al.(2009) EUR.J.PHARM.BIOPHARM.73:269–279.Due to using water as hydroaropic substance（Such as insulin）Decentralized medium, And not the step of above-mentioned preparation does not remove moisture removal, it is seen that these preparations are simultaneously not belonging to the hydrophobic preparations of hydrophilic molecules, but Water-In-Oil（W/O）Type emulsion.

RRC knobs are in the technical scheme disclosed in patent CN97193180.1, CN97196069.0, and C.L.Li Research（C.L.Li,et al.(2004)J.Pharma.Pharmaco l.56:1101-1107）, its hydroholic solution is able to Realization has two essential steps：With water as decentralized medium, hydroaropic substance is dissolved in water phase；Water with contain Parents' medium it is hydrophobic mix after, and by the drying means such as freeze-drying method remove moisture.From these preparation processes From the point of view of, the preparation method of above-mentioned hydrophobic sol is, by the moisture in w/o type preparation, water removal to be gone by means such as freeze-dryings Point, it is different that this has no two with the preparation method of the lyophilized emulsion formulation that those skilled in the art are understood.On the other hand, moisture removal is being removed During, freeze-drying high energy consumption and low production efficiency, and the drying means such as other spray drying processes, again can be to stabilization Property difference medicine（Such as protein）Impact.

A kind of preferable hydrophilic molecule hydrophobic preparations are preparation process is simples, without special installation, with low cost, and The auxiliary material for being used is the material that will not bring potential safety hazard.Importantly, hydrophilic molecule not with water or the aqueous solution as point Dispersion media, in hydrophobic preparations hydrophilic molecule activity will not only lose, and can in the period of more long in keep.

The content of the invention

The present inventor has obtained a kind of dredging containing albumen and/or polypeptide by in-depth study and performing creative labour The aqueous solution, surprisingly, it was found that the hydrophobic sol can efficiently solve albumen and/or polypeptide is fat-soluble low asks Topic.Preparation process of the invention does not need water or the aqueous solution to participate in, the decentralized medium containing albumen and/or polypeptide of gained, can be with Directly mixed with hydrophobic, be derived from single-phase, transparent and stable liquid solution.The present inventor be also surprising that albumen and/ Or polypeptide is in thus obtained hydrophobic sol, fat-soluble and cross-film ability significantly improves, and bioactivity is in room temperature Under can keep muchly stability, without occur degraded or bioactivity lose phenomenon.Thus provide following inventions：

One aspect of the present invention is related to a kind of hydrophobic sol containing albumen or polypeptide, including albumen as main ingredient and/ Or polypeptide, water-soluble co-solvents, emulsifying agent and oil phase.

Preferably, the albumen and/or polypeptide are hydrophilic albumen and/or polypeptide（It is able to maintain that in aqueous Stable conformation, and keep biological function）.

The albumen and/or polypeptide can be separated from living organism and obtained, it is also possible to by genetic engineering means or be had Machine synthesis mode is obtained.

Hydrophobic sol according to any one of the present invention, wherein, the molecular weight of the albumen and/or polypeptide is 1- 200kDa；Preferably 2-100kDa；More preferably 3-60kDa, particularly preferably 4-55kDa.

In order to be better described, in the embodiment of the present invention with insulin, PTH1-34, GLP-1 be polypeptide represent, its molecule Amount is less than 6kDa；Using trypsase, papain, urokinase etc. as macro-molecular protein represent, wherein urokinase point Son amount is maximum, about 54kDa.Their specific molecular weight is as shown in Table 1 below.

Table 1：The molecular weight and isoelectric point of Partial Protein or polypeptide

Hydrophobic sol according to any one of the present invention, wherein, the albumen and/or polypeptide are selected from calcitonin, pancreas islet Element, GLP-1（Glucagon-like-peptide-1）, thyroxine, parathyroid hormone, growth hormone, interferon, urokinase, earthworm swash It is enzyme, pepsin, trypsase, chymotrypsin, bromelain, papain, serrapeptass, staphylococcus lysozyme, molten Any one or more in bacterium enzyme and peroxidase；Specifically, the calcitonin be selected from salmon calcitonin, eel calcitonin and Any one or more in HCT；Specifically, the insulin is appointing in pork insulin, bovine insulin and actrapid monotard One or more；Specifically, the parathyroid hormone is HPTH 1-84 and Human Parathyroid Hormone 1-34 In any one or more；Specifically, the growth hormone is appointing in pig growth hormone, BGH and human growth hormone (HGH) One or more.

Hydrophobic sol according to any one of the present invention, wherein, the content of the albumen and/or polypeptide is 0.001- 5%（w/w）；Preferably 0.01-2%（w/w）Or 0.05-2%（w/w）；More preferably 0.05-1%（w/w）, 0.1-1% （w/w）Or 0.15-1%（w/w）；More preferably 0.1-0.8%（w/w）Or 0.15-0.8%（w/w）, for example 0.15-0.5%（w/w）, 0.3-0.8%（w/w）, 0.5-0.8%（w/w）Or 0.3-0.6%（w/w）；Again for example 0.15%th, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75% or 0.8% （w/w）.

Hydrophobic sol according to any one of the present invention, wherein, the water-soluble co-solvents are selected from ethanol, 1,2- the third two Any one or more in alcohol, glycerine, dimethyl sulfoxide (DMSO), PEG200, PEG300 and PEG400；Preferably, selected from 1,2- third Any one or more in glycol, glycerine, dimethyl sulfoxide (DMSO) and PEG400；It is highly preferred that selected from 1,2-PD, glycerine and Any one or more in dimethyl sulfoxide (DMSO).The present inventor is found through experiments that, when water-soluble co-solvents are selected from 1,2- the third two During any one or more in alcohol, glycerine and dimethyl sulfoxide (DMSO), be conducive to improving the dissolubility of albumen and/or polypeptide.

Hydrophobic sol according to any one of the present invention, wherein, the content of the water-soluble co-solvents is 0.1-15% （w/w）；Preferably 1-15%（w/w）Or 3-15%（w/w）；More preferably 3-12%（w/w）Or 5-12%（w/w）, example Such as 5-10%（w/w）, 8-12%（w/w）Or 8-10%（w/w）；Again for example, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12% （w/w）.

Hydrophobic sol according to any one of the present invention, wherein, the emulsifying agent be one or more HLB be more than or Emulsifying agent equal to 4 and less than or equal to 9, or for one or more HLB more than or equal to 12 emulsifying agent.It is preferred that Ground, the emulsifying agent is that one or more of HLB is more than or equal to 4 and the emulsifying agent less than or equal to 9；It is highly preferred that institute State the HLB that emulsifying agent is one or more and be more than or equal to 6 and the emulsifying agent less than or equal to 9；It is particularly preferred that the breast Agent is that two or more HLB is more than or equal to 6 and the emulsifying agent less than or equal to 9.

Specifically, the emulsifying agent is selected from any one or more in table 2 below：

Table 2：Emulsifying agent and its HLB（Hydrophilic lipophilic balance）

Emulsifying agent	HLB
		Soybean lecithin	6-9
Egg yolk lecithin	6-9
		Span 20	8.6
Sorbester p18	4.7
		Sorbester p17	4.3
Polyglycereol -6- dioleates	6
		Unigly GO 102S	6
Polyethylene glycol -6- glycerin mono-fatty acid esters	4
		Polyethylene glycol -6- Dimodans	4
Polysorbas20	16.7
		Tween 80	15.0
Solutol HS15	14-16
		APEO (35) castor oil	14
APEO (40) rilanit special	12-14
		Tocopherol polyethyleneglycol succinate	13.2
Polyethylene glycol caprylic/capric glyceride	14
		Gelucire 44/14	14

Preferably, the emulsifying agent is selected from soybean lecithin, polyglycereol -6- dioleates, Unigly GO 102S, poly- second two It is any one in alcohol -6- glycerin mono-fatty acid esters, Solutol HS15 and polyethylene glycol caprylic/capric glyceride Plant or various；It is highly preferred that the emulsifying agent is selected from soybean lecithin, Unigly GO 102S, the dihydroxy of polyethylene glycol ten tristearin Any one or more in acid esters and polyethylene glycol caprylic/capric glyceride.It is particularly preferred that the emulsifying agent is selected from above Emulsifying agent in two kinds or more than two kinds.

In one embodiment of the invention, the emulsifying agent includes emulsifying agent 1 and emulsifying agent 2, wherein, emulsifying agent 1 It is soybean lecithin and/or egg yolk lecithin, emulsifying agent 2 is selected from polyglycereol -6- dioleates, Unigly GO 102S, poly- second two It is any one in alcohol -6- glycerin mono-fatty acid esters, Solutol HS15 and polyethylene glycol caprylic/capric glyceride Plant or various.

In one embodiment of the invention, the emulsifying agent includes emulsifying agent 1 and emulsifying agent 2, wherein, the emulsification Agent 1 is soybean lecithin, and it is pungent that emulsifying agent 2 is selected from Unigly GO 102S, Solutol HS15 and polyethylene glycol Any one or more in acid/glycerol decanoate.

In one embodiment of the invention, the emulsifying agent is made up of above-mentioned emulsifying agent 1 and emulsifying agent 2.

In one embodiment of the invention, according to the gross weight of hydrophobic sol, the content of above-mentioned emulsifying agent 1 is 7.5-10%（w/w）, such as 7.5-8.5%（w/w）, 8.5-9.5%（w/w）Or 8-9%（w/w）；The content of emulsifying agent 2 is 2.5-5%（w/w）, such as 2.5-3.5%（w/w）, 3.5-4.5%（w/w）Or 3-4%（w/w）.

It is not limited to the water-soluble co-solvents such as the limitation of theory, glycerine, propane diols, dimethyl sulfoxide (DMSO) and oil phase is existed significantly Density contrast（Glycerol density 1.26g/ml, medium-chain fatty glyceride density 0.94g/ml）, directly can cause phase after mixing Separate.The inventors discovered that, as addition HLB（Hydrophilic lipophilic balance）When surfactant between 4-9 makees emulsifying agent, Especially after above-mentioned emulsifying agent 1 and emulsifying agent 2 is added, can effectively overcome the density contrast between different auxiliary material and bring Preparation lamination, is derived from single-phase, transparent and stable liquid solution.And work as and use glycerine, propane diols, dimethyl During the pharmaceutically acceptable water-soluble co-solvents such as sulfoxide, preparation technology removes water-soluble hydrotropy also without by drying means Agent.

Hydrophobic sol according to any one of the present invention, wherein, the content of the emulsifying agent is 0-40%（w/w）；It is excellent Elect 1-25% as（w/w）；More preferably 5-25%（w/w）；More preferably 5-20%（w/w）Or 5-18%（w/w）, Such as 5-15%（w/w）, 5-10%（w/w）, 10-18%（w/w）, 10-15%（w/w）Or 15-18%（w/w）；Example again Such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20（w/w）.

Hydrophobic sol according to any one of the present invention, wherein, the oil phase is selected from the oil phase and is selected from Medium chain fatty It is any in acid esters, soybean oil, corn oil, peanut oil, safflower oil, olive oil, oleic acid, vitamin E, vitamin A and oleogel One or more；Any one or more preferably in medium chain fatty acid ester, soybean oil, olive oil and oleogel；Specifically Ground, the medium chain fatty acid ester is medium-chain fatty glyceride and/or Medium chain fatty acid propylene glycol ester；More specifically, in described Chain fatty acid triglyceride is Miglyol 812.

Hydrophobic sol according to any one of the present invention, wherein, the content of the oil phase is 45-95%（w/w）；It is excellent Elect 65-90% as（w/w）；More preferably 70-90%（w/w）Or 80-90%（w/w）, such as 75-85%（w/w）, 70- 85%（w/w）, 70-80%（w/w）, 85-90%（w/w）Or 80-85%（w/w）；Again such as 70%, 71%, 72%, 73%th, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%th, 89% or 90%（w/w）.

Hydrophobic sol according to any one of the present invention, wherein, do not use water in the preparation process of the hydrophobic sol Or aqueous solution.

Hydrophobic sol according to any one of the present invention, it also includes pH adjusting agent, and the pH value regulator is selected from salt Acid, sulfuric acid, lactic acid, acetic acid, citric acid, phosphoric acid, triethylamine, NaOH, sodium carbonate, sodium acid carbonate, disodium hydrogen phosphate and phosphorus One or more in acid dihydride sodium；One or more preferably in hydrochloric acid, acetic acid, citric acid and triethylamine.

The limitation of theory is not limited to, accelerates large hydrophilic molecular molten in water-soluble co-solvents to avoid isoelectric point Solution, needs to add pH adjusting agent in some specific embodiments；Preferably, to adding pH adjusting agent in water-soluble co-solvents. Specific pH can be according to albumen or the PI of polypeptide（Specific isoelectric point may be referred to the document of table 1 above or this area）It is suitable Work as adjustment, pH is away from their PI for regulation.

Hydrophobic sol according to any one of the present invention, it is not aqueous, or the content of water is less than 1.0%（w/w）, example Such as it is less than 0.9%, 0.8%, 0.7% or 0.6%（w/w）；Preferably shorter than 0.5%（w/w）, such as less than 0.4%, 0.3%, 0.2% or 0.1%（w/w）；Preferably, the step of preparation method of the hydrophobic sol is not comprising removing or reducing moisture（For example rotate Evaporation, spray drying or freeze-drying）.

Hydrophobic sol according to any one of the present invention, it is single phase soln.

Hydrophobic sol according to any one of the present invention, its component and content for example following 1）- 7）Any one group in group It is shown：

1）

2）

3）

4）

5）

6）

7）

Hydrophobic sol according to any one of the present invention, its component and content are as following（1）-（7）It is any in group Shown in one group：

（1）

（2）

（3）

（4）

（5）

（6）

（7）

It should be noted that above-mentioned 1）- 7）Group or（1）-（7）Unit of gram and milligram in group represent each component Between ratio, if being revised as other unit of weights, including but not limited to, for example kilogram and gram etc., also in the present invention Protection domain within.

Another aspect of the present invention is related to the preparation method of the hydrophobic sol any one of the present invention, including following steps Suddenly：

1）At 4-25 DEG C, albumen and/or polypeptide are added into water-soluble co-solvents, under the conditions of 400-1500rpm, stirring To formation transparent clear solution；

2）Under the conditions of 20-80 DEG C, oil phase is mixed with emulsifying agent, and held under 200-8000rpm stirring conditions Continuous stirring, until the transparent limpid shape of whole system；

3）Under the conditions of 4-37 DEG C, by step 1）Product, adds（It is preferred that being added dropwise over）Step 2）In product, and Continue to stir under the conditions of 200-800rpm, until form single-phase, transparent clear homogeneous preparations, it is final obtain containing albumen and/or The hydrophobic sol of polypeptide；

Preferably, the step of preparation method is not comprising using water or aqueous solution；Preferably, the preparation method is not The step of comprising removing or reducing moisture（Such as rotary evaporation, spray drying or freeze-drying）.

Albumen and/or polypeptide, oil phase, emulsifying agent, the specific species of water-soluble co-solvents in the preparation method and contain Amount can be with as described above.

On step 1), the pH environment of albumen and/or stabilizing polypeptides is suitable to create, or avoid isoelectric point to accelerate egg Dissolving of the white and/or polypeptide in water-soluble co-solvents, alternatively, can also be to adding pH adjusting agent in water-soluble co-solvents.It is special Not, when pH adjusting agent is added in water-soluble co-solvents in form of an aqueous solutions, the content of the aqueous solution should be no more than water solubility The 3% of cosolvent（By weight percentage）；Preferably more than the 2% of water-soluble co-solvents；More preferably no more than water-soluble hydrotropy The 1% of agent.

On step 1), cosolvent is also an option that the water-soluble solvents such as acetonitrile, acetone, dimethylformamide, ethamine, is The security and preparation technology for improving product are succinct, cheap, and when product of the present invention is used for drug field, the water solubility helps Solvent is selected from one or more in pharmaceutical grade ethanol, propane diols, glycerine, dimethyl sulfoxide (DMSO), PEG200, PEG300, PEG400.

On step 1）Or step 2）Used in water-soluble co-solvents, oil phase and emulsifying agent, wherein moisture will Less than 1%, or the presence without free water.

Step 3）In product, in the case of without dried process, water content be less than 1%（By weight percentage Meter）.

When hydrophilic molecule hydrophobic sol is prepared, the auxiliary material that water content should be selected low is needed to used auxiliary in the case of necessity Material is dried dewater treatment, and to ensure not having free water in auxiliary material, and the hydrophobic sol water content for being produced is less than 1% （By weight percentage）；Preferably, the content of water is less than 0.5%；It is highly preferred that the content of water is less than 0.1%.

Another aspect of the invention is related to a kind of pharmaceutical preparation, and it includes the hydrophobic sol any one of the present invention, Or the hydrophobic sol as any one of the present invention is obtained；Specifically, the pharmaceutical preparation is oral liquid, parenteral solution, soft Capsule, emulsion, nano particle or Nano capsule.

Hydrophobic sol of the invention serves many purposes, and they can be used alone, it is also possible to will by the means of preparation Hydroholic solution further optimizes, and is prepared into the new formulations such as emulsion, nano particle, Nano capsule, wherein hydroholic solution In being disperseed or being wrapped in above-mentioned new formulation.

Another aspect of the invention is related to the hydrophobic sol any one of the present invention to be with albumen or polypeptide preparing Purposes in the medicine of active ingredient, cosmetics or health food.

Product prepared by the present invention, can be widely used for the industries such as field of medicaments, cosmetics, health food.

The explanation of part term of the present invention：

In the present invention, the implication of " hydrophily " is the dissolving generally in water, and in oil or other hydrophobic solvents not It is solvable." non-aqueous solution " or " hydrophobic formulation " or " hydrophobic sol ", refer to that hydroaropic substance contained therein is not with water Decentralized medium, and form is single-phase liquid solution；Preferably, the content of the content water of preparation reclaimed water is less than 1.0%；More preferably Ground, the content of water is less than 0.5%, it is further preferred that the content of water is even lower less than 0.1% or be zero.When of the invention After preparation mixes with water, it is impossible to form the uniform mixed system of stabilization, and water-oil separating phenomenon can occur, and hydrophily medicine Thing more than 70% is distributed in oil phase layer.Preferably, it is the solution of transparent and stable.

In the present invention, for the percentage of the content of each component, if not otherwise specified, refer both to account for hydrophobic sol total The percentage by weight of weight（w/w）.

The beneficial effect of the invention

The present invention can be mixed directly using water-soluble co-solvents as albumen and/or the decentralized medium of polypeptide with hydrophobic, It is derived from single-phase, transparent and stable hydrophobic sol.Distinguishing feature of the invention is, albumen and/or polypeptide in hydrophobic sol, Fat-soluble and cross-film ability significantly improves, and bioactivity can keep muchly stability at room temperature, without hair The phenomenon that raw degraded or bioactivity are lost.Additionally, research also found, the mobility of hydrophobic sol of the invention very well, is not hung Wall.

Preparation process of the present invention does not need water or the aqueous solution to participate in, it is not required that drying means removes solvent, with preparation Process is simple, the advantage of low production cost, great application prospect.

In pharmaceutical preparation application aspect, product of the invention can directly with oral liquid, parenteral solution, filling agent, soft capsule Used etc. formulation, it is also possible to further optimize hydroholic solution, be prepared into emulsion, nano particle, Nano capsule etc. new Preparation.

Brief description of the drawings

Fig. 1：The blood glucose curve figure of SD rats.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are Can by city available from conventional products.

Embodiment 1：Insulin hydrophobic sol（Sample 1）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）Under the conditions of 4 DEG C, during the rh-insulin of above-mentioned weight added into 1 gram of dimethyl sulfoxide (DMSO), and aequum is added Hydrochloric acid, under the conditions of 700rpm stir, to the transparent limpid shape of system；

2）Under the conditions of 65 DEG C, by the MCT Oil of above-mentioned weight, polyethylene glycol caprylic/capric glyceride, Soybean lecithin, Unigly GO 102S mixing, and persistently stirred under 3000rpm stirring conditions, to the transparent limpid shape of system；

3）Under the conditions of 4 DEG C, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 200rpm Mix, until single-phase, transparent clear homogeneous preparations are formed, the final hydrophobic sol for obtaining insulin-containing.

Embodiment 2：PTH1-34 hydrophobic sol（Sample 2）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）Under the conditions of 25 DEG C, by PTH1-34 1 gram of propane diols of addition of above-mentioned weight, stirred under the conditions of 400rpm, to body It is transparent limpid shape；

2）Under the conditions of 20 DEG C, the soybean oil of above-mentioned weight, soybean lecithin, Unigly GO 102S are mixed, and Persistently stirred under 8000rpm stirring conditions, to the transparent limpid shape of system；

3）Under the conditions of 25 DEG C, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 800rpm Mix, until single-phase, transparent clear homogeneous preparations are formed, it is final to obtain the hydrophobic sol containing PTH1-34.

Embodiment 3：GLP-1 hydrophobic sols（Sample 3）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）At room temperature, by GLP-1 0.2 gram of propane diols of addition of above-mentioned weight, under the conditions of 800rpm, stirring is saturating to being formed Bright clear solution；

2）60 DEG C, the Miglyol 812 of above-mentioned weight, soybean lecithin, polyglycereol -6- dioleates are mixed, And persistently stirred under 2000rpm stirring conditions, until the transparent limpid shape of whole system；

3）Under room temperature condition, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 600rpm Mix, until single-phase, transparent clear homogeneous preparations are formed, it is final to obtain the hydrophobic sol containing GLP-1.

Embodiment 4：Trypsase hydrophobic sol（Sample 4）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）At 20 DEG C, by trypsase 1 gram of glycerine of addition, under the conditions of 1500rpm, stirring is molten to transparent clear is formed Liquid；

2）Under the conditions of 80 DEG C, the Miglyol 812 of above-mentioned weight, Unigly GO 102S, soybean lecithin are mutually mixed Close, and persistently stirred under 200rpm stirring conditions, until the transparent limpid shape of whole system；

3）Under the conditions of 37 DEG C, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 600rpm Mix, until single-phase, transparent clear homogeneous preparations are formed, it is final to obtain the hydrophobic sol containing trypsase.

Embodiment 5：Papain, bromelain hydrophobic sol（Sample 5）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）At 25 DEG C, the papain of above-mentioned weight, bromelain, hydrochloric acid are added glycerine, third of above-mentioned weight In glycol, under the conditions of 800rpm, stirring to formation transparent clear solution；

2）Under the conditions of 50 DEG C, by the olive oil of above-mentioned weight, polyethylene glycol caprylic/capric glyceride, polyethylene glycol 12 Hydroxy stearic acid ester is mixed, and is persistently stirred under 600rpm stirring conditions, until the transparent limpid shape of whole system；

3）Under room temperature condition, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 600rpm Mix, until forming single-phase, transparent clear homogeneous preparations, final acquisition contains papain, the hydrophobic sol of bromelain.

Embodiment 6：Lysozyme, staphylococcus lysozyme hydrophobic sol（Sample 6）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）At room temperature, by the lysozyme of above-mentioned weight, staphylococcus lysozyme 1 gram of glycerine of addition, under the conditions of 800rpm, stir Mix to formation transparent clear solution；

2）At room temperature, by the safflower oil of above-mentioned weight, Miglyol 812, polyglycereol -6- dioleates, poly- second two Alcohol -6- glycerin mono-fatty acid esters are mixed, and are persistently stirred under 200rpm stirring conditions, until whole system is transparent limpid Shape；

3）Under room temperature condition, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 600rpm Mix, until forming single-phase, transparent clear homogeneous preparations, finally obtain lysozyme, the hydrophobic sol of staphylococcus lysozyme.

Embodiment 7：Urokinase hydrophobic sol（Sample 7）Preparation

Its constituent is as follows：

The preparation method of the hydrophobic sol is as follows：

1）At room temperature, during the urokinase of above-mentioned weight to add the propane diols and glycerine of above-mentioned weight, 1200rpm conditions Under, stirring to formation transparent clear solution；

2）60 DEG C, the safflower oil of above-mentioned weight, polyglycereol -6- dioleates, Unigly GO 102S are mixed, and Persistently stirred under 1500rpm stirring conditions, until the transparent limpid shape of whole system；

3）Under room temperature condition, by step 1）Product, is added dropwise over step 2）In product, and continue to stir under the conditions of 600rpm Mix, until single-phase, transparent clear homogeneous preparations are formed, most

The hydrophobic sol containing urokinase is obtained eventually.

Embodiment 8：Spectrodensitometry is tested

With Hitachi's U-2001 ultraviolet-uisible spectrophotometers, optical density is determined at 600nm.Wherein hydrophobic sol be according to Sample 1-7 obtained by embodiment 1-7 preparation processes；Physical mixed sample is consistent with embodiment product form；Blank sample group Into as corresponding hydrophobic sol, but without hydrophilic substance, as a result as shown in table 3.

Table 3：The influence of different sample solution states and physical mixed to optical density

Optical density represents the permeability of light, and numerical value is lower, shows that sample gets over clarification homogeneous.

From result, the fat-soluble of hydroaropic substance in product of the present invention is greatly improved, hydroaropic substance energy " dissolve " in corresponding hydrophobic sol well.

Embodiment 9：Fat-soluble experiment

Hydrophobic sol can increase the fat-soluble of hydroaropic substance, after hydrophobic sol is directly mixed with water, hydrophily thing Matter is still wrapped in hydrophobic sol, only can enter water phase on a small quantity；The organic solvents such as ethyl acetate, can destroy hydrophobic sol Solubilization to hydroaropic substance so that hydroaropic substance is discharged into water phase from hydrophobic environment.

The fat-soluble change of hydroaropic substance in product of the present invention, can be by determining different pretreatments under the conditions of, water phase In the changes of contents of hydroaropic substance that can be detected verified, select embodiment 1-3 to be produced as polypeptide in this experiment Product are represented；Embodiment 4-7 is represented as protein：

Preprocess method one：Sample prepared by embodiment 1,2,3,4,5,6,7 is respectively taken 1 gram, 10ml0.001N salt is added Acid, magnetic agitation 30min is centrifuged 5min under the conditions of 6000rpm, phase of fetching water, and determines the content of medicine in water phase.

Preprocess method two：Sample prepared by embodiment 1,2,3,4,5,6,7 is respectively taken 1 gram, 5ml0.001N is separately added into Hydrochloric acid and 5ml ethyl acetate, magnetic agitation 30mi n, are placed in extraction flask, after the phase, 6000rpm of being fetched water after organic-aqueous separation Under the conditions of 5min is centrifuged, for sample determination.

Wherein, sample 1, sample 2, sample 3 carry out the measure of active constituents of medicine content using HPLC methods；Sample 4, sample 5th, sample 6, sample 7 carry out the measure of active constituents of medicine content using enzymatic reaction method, as a result such as table 4.

Table 4：The each sample drug content that be can be detected in water phase

As can be seen from Table 4, after hydrophobic sol is mixed with water, the hydroaropic substance in hydrophobic sol is dissolved in, only Having small part can enter water phase, fully show that hydrophobic sol can be fat-soluble by increasing, and dissolve hydroaropic substance well. By pretreatment, ethyl acetate can destroy solubilization of the hydrophobic sol to hydroaropic substance so that hydroaropic substance is from dredging Water environment is discharged into water phase.

Embodiment 10：Stability test（1）

Products obtained therefrom is placed in 40 DEG C ± 2 DEG C, and humidity is 75% ± 5%, accelerated test is carried out under the conditions of lucifuge, and placing 1st month afterwards, March and 6th month measure medicament contg or activity.Wherein, sample 1, sample 2, sample 3 use HPLC Method carries out the measure of active constituents of medicine content；Sample 4, sample 5, sample 6, sample 7 carry out medicine work using enzymatic reaction method The measure of property component content；Result is as shown in table 5.

Following demulsification treatment is carried out to sample before determining：1 gram of testing sample is taken, 5ml0.001N hydrochloric acid and 5ml is separately added into Ethyl acetate, magnetic agitation 30min, is placed in extraction flask, after phase of being fetched water after organic-aqueous separation, for sample determination.

Protein and peptide drugs are easily inactivated to thermo-responsive（An Furong etc., insulin solutions chemical stability, 1993《Shenyang Pharmacy College's journal》10：165-170）, dropped especially as this kind of PD enzyme of trypsase may occur from molten Low activity（The circular Dichroism Studies On of Ni Yisheng etc., title of article, Porcine trypsin and its autolytic activity product, 1986,《Beijing College journal（Natural science edition）》3：114-120）, in the product prepared by accelerated test, the present invention, due to hydrophobic The effect of environment, can greatly strengthen the long-time stability of hydrophilic medicament.

Table 5：The change of example pharmaceuticals component content

Embodiment 11：Stability test（2）

First, laboratory sample

Sample 1-7 prepared by embodiment 1-7.

2nd, experimental technique

Multigelation 6 times under -20 DEG C of -20 DEG C of environmental conditions by sample 1-7, observe sample appearance；Or respectively 4 DEG C, 25 DEG C, 60 DEG C place 6 months, observe sample appearance.

3rd, experimental result

It is shown in Table 6.

Table 6：Difference is placed with treatment conditions, and sample appearance changes

From table 6, sample 1-7 is respectively provided with fabulous stability, and by multigelation, or even 60 DEG C of high temperature are placed, outward Sight remains to keep transparent homogeneous phase, not wall built-up, not stratified, with good mobility.

Embodiment 12：Insulin hydrophobic sol orally-taken blood sugar reducing effect disquisition

Oral test group：The 180-220 grams of male SD rat of scope 6, medicine（Trial drug）For embodiment 1 is made Standby sample 1.

Injection control group：The 180-220 grams of male SD rat of scope 6, medicine is insulin solution（0.04mg/ kg）.

Test method：

Experiment will be started after male SD rat fasting 12h that body weight is 180-220 grams of scope.Test group is oral administration, Dosage is calculated as 0.4mg/kg by insulin.Rats in test groups, is carried out with the yellow Jackets that dosage is 50mg before experiment to rat After anesthesia, bundled by a mode is faced upward, and an osculum is cut in belly, ileum is administered with delivery device, after administration, suture is cut Mouthful.Control group is hypodermic injection control drug, and dosage is calculated as 0.04mg/kg by insulin.

Two groups upon administration 0.5,1,2,3,4,5h moment points, blood is taken by tail vein, surveyed using Roche Instrument for Measuring Blood Sugar Determine blood sugar and calculate 6 average blood sugar values of rat in every group, as a result as illustrated by fig. 1.As known to the researcher of this area , it is substantially invalid after insulin solution is directly oral, in this product, with the insulin that hydrophobic sol is present, can by Fig. 1 See, the test group of oral administration is 8.17% relative to the bioavilability of the control group of drug administration by injection.

Embodiment 13：Heel cracking cream containing papain, bromelain

The sample 5 prepared by 2 grams of embodiments 5 is taken, heel cracking cream is prepared according to the following steps：

1）100 milligrams of urea are taken, 5 milligrams of chlorophyllin copper complex sodiums, 30 milligrams of 4- formylphenyl boronic acids add 48ml water In, mix, and be preheated to 70 DEG C；

2）Take 7 grams of Solsperse 2000s, 10 grams of polyethylene glycol stearates, 1 gram of sorbester p18,12 grams of propane diols, 12 Ke Baifanshi Woods, 10 grams of atoleines, under the conditions of 70 DEG C, stirs, and be gradually added into step 1）In prepared product；

3）By step 2）Products therefrom, is cooled to 40 DEG C under conditions of stirring, adds 2 samples 5, stirs, and drop To room temperature, the heel cracking cream containing papain, bromelain is derived from.

Embodiment 14：The effectiveness study of lysozyme, staphylococcus lysozyme hydrophobic sol in mastadenitis of cow

Test group：Infection of staphylococcus aureus china holstein cowses（Secrete the milk phase）10, medicine is that embodiment 6 is made Standby sample 6, sample 6 is filled into the syringe of 5ml, each medication 1 time after milking to each newborn area's injection 5ml, daily early, evening, It is used in conjunction 4 days.

Blank control group：Infection of staphylococcus aureus china holstein cowses（Secrete the milk phase）6, any controlling is not received Treat.

Test method：

Somatic number is as performance assessment criteria with milk, by comparing administration the previous day, and body in the 14th day milk after administration Cell number changes, and study sample 6 is to mastitis treatment effect.Result as shown in table 7, is dredged by lysozyme, staphylococcus lysozyme Water solution treatment, milk cow somatic number is remarkably decreased, and shows splendid therapeutic effect of this product to mastadenitis of cow.

Table 7：Influence × 10 of the sample 4 to milk cow somatic number⁴Individual/ml

	Administration the previous day	14 days after administration
			Test group	97.30±41.68	37.52±9.11
Blank control group	92.74±39.03	86.17±36.74

The following examples 15-20 is that the hydrophobic sol containing albumen or polypeptide of the invention is made into showing for various formulations Example.

Embodiment 15：The preparation of soft capsule preparation

By the prescription of embodiment 1,10kg samples 1 are prepared, select 4# olive-type moulds, pelleting is carried out in automatic encapsulating machine, done It is dry to gelatin rubber water content below 10%（Percentage by weight）, obtain final product insulin hydrophobic sol soft capsule.

Embodiment 16：The preparation of nano particle

0.2 gram of Poly(D,L-lactide-co-glycolide PLGA (75/25) is taken, 10ml acetone is added, stirred completely molten to PLGA Solution, adds the sample 2 prepared by 0.1 gram of embodiment 2, stirs transparent limpid to solution, and 25ml is added in the case of stirring Mass percent concentration be 2% PVA（Polyvinyl alcohol）Solution, the ultrasound 30s under condition of ice bath, being removed under reduced pressure has After machine is molten, 10min is centrifuged under 20000g centrifugal force, precipitation is obtained eventually and is the nano particle containing PTH1-34 hydrophobic sols.

Embodiment 17：The preparation of oral liquid

By the prescription of embodiment 3, prepare 5kg samples 3, by micropore it is degerming after, be distributed into every bottle in automatic liquid filling machine The specification of 1.5ml, after sealing, obtains final product GLP-1 hydrophobic sol oral liquids.

Embodiment 18：The preparation of emulsion

By the prescription of embodiment 4,70g samples 4 are prepared, under 300rpm stirring price adjustments, addition 8g Emulsifier EL-60s, 30g water, after forming colostrum, the homogeneous under 700bar pressure is circulated 3 times, and products obtained therefrom is emulsion.

Embodiment 19：The preparation of parenteral solution

By the prescription of embodiment 6, after preparation 5kg 6,0.22 miillpore filters of sample are degerming, in the filling mechanical irrigation of precharging type syringe Dress, every filling 5ml of syringe obtains final product hydrophobic sol parenteral solution.

Embodiment 20：It is prepared by Nano capsule

Take N-Lock starch（National of the United States's starch）10g, is added in 25ml water, and stirring is added to after forming settled solution Sample 7 prepared by 3g embodiments 7,0.5g Solutol HS15（Germany, BASF）, 5000rpm stirs 5mi n, dry through spraying After dry, nano-microcapsule is obtained.

Although specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change in guarantor of the invention Within the scope of shield.Four corner of the invention is given by appended claims and its any equivalent.

Claims

1. a kind of hydrophobic sol containing albumen or polypeptide, its component and content such as following 1) -7) any one group of institute in group Show：

1)

2)

3)

4)

5)

6)

7)

2. a kind of hydrophobic sol containing albumen or polypeptide, any one group of institute in its component and content such as following (1)-(7) group Show：

(1)

(2)

(3)

(4)

(5)

(6)

(7)