JP2006199589A - Nanoparticle containing physiologically active protein or peptide, method for producing the same and external preparation comprising the nanoparticle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a nanoparticle containing a physiologically active protein or peptide that has excellent absorbability in vivo, exerts high bioavailability and has an effect comparable with that of injection administration by an administration method through the skin or mucosa with regard to a physiologically active protein or peptide. <P>SOLUTION: The nanoparticle containing a physiologically active protein or peptide is obtained by making a physiologically active protein or peptide insoluble in water, preparing a primary nanoparticle by using the water-insoluble matter, a medium/long-chain organic compound containing both a hydrophobic group and an anionic residue and a surfactant and bringing the primary nanoparticle into contact successively with a divalent or trivalent metal salt and a divalent or trivalent basic salt. The external preparation comprises the nanoparticle as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nanoparticle containing a physiologically active protein or peptide, and more particularly to a nanoparticle containing a physiologically active protein or peptide, a method for producing the same, and an external preparation comprising the nanoparticle.

  With the advancement of biotechnologies and human genome analysis, bioactive proteins are becoming increasingly important as pharmaceuticals. Since bioactive proteins are inactivated by peptidases present in the gastrointestinal tract, they cannot be administered orally, and are usually administered in vivo by injection. However, such a method is unfavorable for the patient because it gives pain at the injection site. In addition, when administration is performed at regular intervals, the patient is extremely painful. Therefore, it is desired to develop a non-injection administration method that is simple and self-administerable for safe and frequent administration.

  In recent years, the development of technology that absorbs biologically active proteins at high concentrations from the skin, mucous membranes, etc., in the active body, regardless of injection, is an important theme in therapeutic research, especially DDS (drug delivery system) research. It has become one of the. However, in human absorbability and bioavailability (bioavailability), satisfactory results have not yet been obtained, and administration methods for physiologically active proteins via the skin and mucous membranes, particularly transdermal absorption administration. The method is almost unsuccessful.

  In fact, as one of the physiologically active proteins, various studies have been conducted on administration methods through the skin and mucous membranes using relatively low molecular weight and chemically stable insulin. Although it is higher in the order of the digestive mucosa and the respiratory mucosa, the absorption rate after administration through the mucosa is still only a few percent in the reliable data, and is hardly absorbed by the administration through the skin. (Non-Patent Document 1).

  Also proposed are preparations in which physiologically active proteins are encapsulated in calcium-containing poorly water-soluble inorganic particles (Patent Document 1), water-insoluble sustained-release compositions by precipitation of physiologically active proteins or peptides and zinc ions (Patent Document 2), etc. Has been. However, these preparations are not sufficient in terms of drug absorbability or local irritation, and have not yet been put into practical use. In addition, there is no known technique for in vivo absorption through the skin or mucous membrane using nanoparticles containing the physiologically active protein or peptide targeted by the present invention.

International Publication WO 02/096396 Japanese Patent Laid-Open No. 2003-081865 DRUG DELIVERY SYSTEM Today's DDS Drug Delivery System (Pharmaceutical Journal) 325-331, 1999 Clinical Pharmacology (Jpn. J. Clin. Pharmacol. Ther.) 26 (1), p.127-128 (1995) Yakugaku Zasshi, 121 (12), p.929-948 (2001) J. Controlled Release, 79, p.81-91 (2002)

  As described above, physiologically active proteins cannot exert their medicinal effects when administered orally, but are mainly due to injection. Therefore, in order to ease the pain caused by injection to the patient and simplify the administration method capable of self-administration, the administration method via the skin and mucous membrane is effective as a parenteral administration means instead of injection administration. However, there is still nothing satisfactory in absorbability and bioavailability, and the present situation is that there is a strong desire in the medical field for the emergence of drugs with high in vivo absorbability and bioavailability.

  Therefore, the present invention has not been able to exert its medicinal effects by oral administration until now, and the bioactive protein or peptide which has been mainly administered by injection can be absorbed into the living body by the administration method through the skin and mucous membrane. The objective is to provide a technology that excels in high bioavailability.

  As a result of diligent studies, the present inventors have applied nanotechnology and succeeded in incorporating so-called nanoparticles that are much smaller than erythrocytes and containing bioactive proteins or peptides therein. However, when the nanoparticles thus obtained are administered via the skin and mucous membrane, the bioactive protein or peptide contained in the nanoparticles is highly absorbed into the living body and excellent in bioavailability. As a result, the present invention has been completed.

Accordingly, the present invention provides nanoparticles containing bioactive proteins or peptides that have superior absorbability and bioavailability upon dermal or mucosal administration.
More specifically, the present invention provides:
(1) A bioactive protein or peptide is made into a water-insoluble material, primary nanoparticles are prepared using the water-insoluble material, a medium-long chain organic compound having a hydrophobic group and an anion residue, and a surfactant. Nanoparticles containing a bioactive protein or peptide obtained by sequentially contacting the nanoparticles with a divalent or trivalent metal salt and a divalent or trivalent basic salt,
(2) The primary nanoparticle is a water-insoluble material, a medium-long chain organic compound having a hydrophobic group and an anion residue, and a surfactant are dissolved in an organic solvent or a water-containing organic solvent, and this is stirred in a large amount of water. Nanoparticles according to (1), which are produced by adding with shaking,
(3) The nanoparticle according to (1) or (2), wherein the particle has a diameter of 5 to 150 nm,
(4) The nanoparticle according to (1) or (2), wherein the particle has a diameter of 10 to 80 nm,
(5) The bioactive protein or peptide is insulin, interferon-α, interferon-β, interferon-γ, growth hormone, G-CSF, GM-CSF, erythropoietin, thrombopoietin, urokinase, t-PA, IL-11, etanercept Infliximab, SOD, FGF, FGF, HGF, NGF, BDNF, leptin, NT-3, an antigen, an antibody and an enzyme according to (4),
(6) The means for making the physiologically active protein or peptide water-insoluble is any one of contact with a divalent or trivalent metal ion, contact with an acidic or basic polysaccharide, adjustment of pH, or change in ionic strength. The nanoparticle according to (1), which is a means of
(7) The bivalent or trivalent metal ion to be contacted to make the physiologically active protein or peptide insoluble in water is selected from zinc ion, calcium ion, iron ion and copper ion. The described nanoparticles,
(8) The medium-to-long chain organic compound having both a hydrophobic group and an anion residue is selected from myristic acid, oleic acid, lauric acid, palmitic acid and salts thereof (1) to (4) The described nanoparticles,
(9) The divalent or trivalent metal salt brought into contact with the primary nanoparticles is a calcium salt, zinc salt, iron salt or copper salt, and the divalent or trivalent basic salt is a carbonate, phosphate, or salt. Nanoparticles according to (1) to (4), which are acid salts, lactates or uric acid salts,
(10) An external preparation for skin or mucous membrane, comprising the nanoparticles according to any one of (1) to (9) above,
(11) The external preparation according to (10), wherein the external preparation is selected from ointments, gels, nasal drops, eye drops, sprays, suction agents, suspension agents, cataplasms, and patches. ,
(12) A physiologically active protein or peptide is made into a water-insoluble substance, and the water-insoluble substance, a medium-long chain organic compound having a hydrophobic group and an anion residue, and a surfactant are dissolved in an organic solvent or a water-containing organic solvent. A primary nanoparticle is prepared by adding the solution to a large amount of water while stirring and shaking, and a divalent or trivalent metal salt and a divalent or trivalent basic salt are sequentially added to the primary nanoparticle-containing solution. A method for producing nanoparticles containing a physiologically active protein or peptide,
(13) The means for making the physiologically active protein or peptide water-insoluble is contact with a divalent or trivalent metal ion, and the divalent or trivalent metal salt to be brought into contact with the primary nanoparticles is a calcium salt, The method for producing nanoparticles according to (12), wherein the divalent or trivalent basic salt is a carbonate,
(14) The method for producing nanoparticles according to (13), wherein the metal ion is zinc ion,
Is to provide.

  The nanoparticle provided by the present invention absorbs a physiologically active protein or peptide contained therein via skin or mucous membrane, and exhibits in vivo absorbability that can be substituted for injection administration. Therefore, it has an epoch-making effect that allows bioactive protein or peptide to be absorbed in vivo by transcutaneous or transmucosal, which has not been achieved so far, and contains a highly absorbable bioactive protein and peptide. It has an excellent effect of creating an agent.

  As described above, the present invention uses primary nanoparticles using a physiologically active protein or peptide as a water-insoluble substance, the water-insoluble substance, a medium-long chain organic compound having both a hydrophobic group and an anion residue, and a surfactant. And then, a nanoparticle containing a bioactive protein or peptide obtained by sequentially contacting the primary nanoparticle with a divalent or trivalent metal salt and a divalent or trivalent basic salt, and a method for producing the nanoparticle And an external preparation composed of the nanoparticles.

  The diameter of the nanoparticles of the present invention is about 5 to 150 nm, preferably about 10 to 80 nm. The particle diameter can be adjusted by the blending ratio of the water-insoluble substance of the physiologically active protein or peptide to be contained and the medium-long chain organic compound, the amount of the solvent to be used, the strength of stirring, and the diameter is about 5 to 500 nm. Particles can be made. In addition, it became clear that a particle diameter will become small when the compounding quantity of a medium-long chain organic compound is increased. The particle diameter can be measured by a light scattering method or an electron microscope.

  The physiologically active protein or peptide contained in the nanoparticles provided by the present invention is not particularly limited, but those that form a precipitate with a divalent or trivalent metal salt are preferred. Such bioactive proteins or peptides include insulin, interferon-α, interferon-β, interferon-γ, growth hormone, G-CSF, GM-CSF, erythropoietin, thrombopoietin, urokinase, t-PA, IL-11, Examples include etanercept, infliximab, SOD, FGF, EFG, HGF, NGF, BDNF, leptin, NT-3, antigens, antibodies, and various enzymes. Among these, insulin, interferon-α, interferon-β, interferon-γ, human growth hormone, and erythropoietin are particularly preferable.

  In the production of the nanoparticles of the present invention, the physiologically active protein or peptide is first made into a water-insoluble substance. The most preferable means for making this insoluble in water is to use a divalent or trivalent metal ion that forms a precipitate with a physiologically active protein or peptide. Such divalent or trivalent metal ions include zinc ions from zinc salts such as zinc chloride and zinc sulfate; calcium ions from calcium salts such as calcium chloride, calcium sulfate and calcium carbonate; iron chloride and iron sulfide. Examples thereof include iron ions due to iron salts; copper ions due to copper salts such as copper chloride and copper sulfate. Among them, zinc ions can be preferably used.

  In this case, the compounding ratio of the physiologically active protein or peptide and the divalent or trivalent metal ion is not particularly limited, as long as it is a ratio sufficient to generate a water-insoluble material by combining both substances. For example, in the case of zinc ions, the physiologically active protein or peptide and the zinc salt are preferably about 10: 1 to 1: 2 by weight. Other water insolubilization methods include contacting with acidic or basic polysaccharides such as chondroitin sulfate sodium, hyaluronic acid, chitosan, etc., or adjusting the pH of a solution in which a physiologically active protein or peptide is dissolved, changing ionic strength, etc. Is also possible. In addition, when the physiologically active protein or peptide itself is water-insoluble, it can be used as it is.

  In order to produce the nanoparticles of the present invention, it is necessary to blend a compound having both a hydrophobic group and an anion residue such as a carboxyl group, a phosphate group, and a sulfate group. Any organic compound having both a hydrophobic group and an anion residue can be used, but a medium-long chain organic compound having a carboxyl group is particularly preferred. As such a medium-long chain organic compound, myristic acid, oleic acid, lauric acid, and palmitic acid are preferable. The medium-long chain organic compound can be added as it is when it is a powder, but it is preferably used by dissolving in an organic solvent or a water-containing organic solvent. As such an organic solvent, acetone; lower alcohols such as methanol, ethanol, propanol, and butanol can be used. Of these, acetone and ethanol are preferable. The blending weight ratio between the physiologically active protein or peptide and the medium-long chain organic compound is preferably about 1: 0.5 to 1: 0.03.

  In producing the nanoparticles of the present invention, it is preferable to add an appropriate amount of a surfactant in order to avoid aggregation between the generated nanoparticles, and the amount of the surfactant is appropriately selected so that the nanoparticles do not aggregate with each other. It is good to use about 0.3-0.03 by molar ratio with respect to a medium-long chain organic compound. Such surfactants include polyoxyethylene hydrogenated castor oil (Tween 80), polyethylene glycol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, sorbitan fatty acid ester, glycerin fatty acid Nonionic surfactants such as esters and polyoxyethylene polyoxypropylene glycol can be used. Among them, polyoxyethylene hydrogenated castor oil (Tween 80) is preferable.

  The most important feature of the present invention is that a bioactive protein or peptide is made into a water-insoluble substance, and the primary nanoparticle is prepared by using the water-insoluble substance, a medium-long chain organic compound and a surfactant. By sequentially bringing the particles into contact with a divalent or trivalent metal salt and a divalent or trivalent basic salt, the metal salt and the basic salt are sequentially bonded, resulting in water around the primary nanoparticles. It is to make a conjugate having a state in which an insoluble metal salt is covered.

  The thus obtained conjugate itself is the nanoparticle intended by the present invention. Surprisingly, when the nanoparticle is transdermally or transmucosally administered, the nanoparticle acts as a carrier, The bioactive protein or peptide contained in the product is well absorbed in vivo, and the in vivo absorbability can be substituted for injection administration.

  Examples of the divalent or trivalent metal salt used include calcium salts such as calcium chloride, calcium acetate and calcium sulfate; zinc salts such as zinc acetate, zinc chloride and zinc sulfate; iron salts such as iron chloride and iron sulfide; Or it is copper salts, such as copper chloride and copper sulfide, and calcium salt, especially calcium chloride is preferable. Although the compounding quantity of a metal salt cannot be limited generally, it is preferable that it is about 3-0.01 by weight ratio with respect to the bioactive protein or peptide used as an active ingredient.

  Examples of the divalent or trivalent basic salt include carbonates such as sodium carbonate, potassium carbonate, and calcium carbonate; phosphates such as sodium phosphate, potassium phosphate, and calcium phosphate; sodium oxalate, potassium oxalate, Oxalates such as calcium oxalate; lactates such as sodium lactate, potassium lactate and calcium lactate; urates such as sodium urate, potassium urate and calcium urate. Among them, carbonates, particularly sodium charcoal are preferable. Although the compounding quantity of a basic salt cannot be generally limited, it is preferable that it is about 1.0-0.05 by molar ratio with respect to said metal salt.

Below, the manufacturing method of the nanoparticle which this invention provides is demonstrated.
First, a physiologically active protein or peptide is dissolved in acidic, basic or neutral water, and a divalent or trivalent metal ion is added to the solution to make the physiologically active protein or peptide insoluble in water. To this suspension is added a solution obtained by dissolving a medium-long chain organic compound and a surfactant in an organic solvent or a water-containing organic solvent, completely dissolved, and this solution is added to a large amount of water while stirring and shaking. By stirring this solution for about 1 to 30 minutes, primary nanoparticles are produced. Add a divalent or trivalent metal salt to the solution containing the primary nanoparticles thus prepared, stir for 1 to 30 minutes, then add a divalent or trivalent base salt and stir for 1 to 30 minutes Thus, the nanoparticles of the present invention can be produced.

  The solution of the nanoparticles containing the bioactive protein or peptide of the present invention thus produced is lyophilized, dried under reduced pressure, spray dried, etc., to remove the solvent, and appropriately as a pharmaceutical composition, a formulation base and an additive Etc. can be used to prepare a desired external preparation.

  The present invention also provides an external preparation for skin and mucous membranes containing nanoparticles containing such physiologically active protein or peptide as an active ingredient. As such an external preparation, it can be applied in a form that can be administered in the form of local application, sticking, dripping, spraying, etc. for the purpose of systemic and local administration / treatment, specifically, an ointment, Gels, nasal drops, eye drops, sprays, suction agents, suspensions, cataplasms, patches and the like can be mentioned. Effective dosage forms include application to the skin or mucous membrane and spraying to the upper respiratory tract.

  Examples of bases and other additive components used in the preparation of these external preparations include bases and components that are pharmaceutically used in the preparation of external preparations. Specifically, oily bases such as petrolatum, platis base, paraffin, liquid paraffin, light liquid paraffin, honey beeswax, silicone oil; solvents such as water, macrogol, ethanol, methyl ethyl ketone, cottonseed oil, olive oil, peanut oil; Nonionic surfactants such as oxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, sorbitan fatty acid ester, polyoxyethylene polyoxpropylene glycol; polyvinyl Thickeners such as pyrrolidone, carboxymethylcellulose (CMC), xanthan gum, tragacanth gum, gelatin, gum arabic, and albumin; Moisturizers such as glycerin, 1,3-butylene glycol, propylene glycol, urea, sucrose, erythritol sorbitol; preservatives such as methyl paraoxybenzoate, butyl paraoxybenzoate, sodium dehydroacetate, p-cresol, etc. And can be appropriately selected and used depending on the dosage form.

  For example, in the case of an ointment containing the nanoparticles of the present invention as an active ingredient, petrolatum is used as a component such as a base, and 0.05 to 0.5% carboxymethylcellulose ( CMC) may be used together.

  The present invention will be described in more detail with reference to the following examples and test examples, but the present invention is not limited thereto.

Example 1:
1 mg (21 IU) of insulin was dissolved in 1 μL of 1M hydrochloric acid and 99 μL of distilled water. To this solution, 0.4 mg of zinc chloride was added, and then 10 μL of a solution obtained by dissolving 6.5 mg of myristic acid in 100 μL of acetone and further adding 30 μL of water was added. 2 μL of Tween 80 was added to this solution, and 98 μL of distilled water was added and stirred. The above mixed solution was added to 5 mL of distilled water and stirred well to produce primary nanoparticles. 15 μL of 5M calcium chloride aqueous solution was added to this solution and stirred for 30 minutes. Further, 15 μL of 1M aqueous sodium carbonate solution was added, stirred for 5 minutes, and allowed to stand for 30 minutes to prepare nanoparticles (nanoparticles of the present invention). The solution containing the nanoparticles was lyophilized overnight. When the particle size of the obtained nanoparticles was observed with a light scattering method and an electron microscope, most of the particles had a diameter of 10 to 90 nm, and most of them had a diameter of 60 nm.

Using these nanoparticles, a predetermined amount of insulin was redispersed in petrolatum or 1% CMC solution and mixed to obtain an ointment or sol.
The ointment or sol produced as described above (hereinafter sometimes referred to as “the present preparation”) was used in the following animal experiments.

Example 2:
Nanoparticles of the present invention were produced in the same manner as in Example 1 except that 15 μL of 5M aqueous solution of zinc acetate was used instead of 15 μL of 5M aqueous solution of potassium chloride in Example 1.

Test Example 1: Skin permeability test (congenital diabetic mice)
Nine mice with congenital diabetes mellitus, KKAy / ta male, 5 weeks old (CLEA Japan), 9 animals, pre-bred for 1 week, and after 1 week, blood glucose level was measured with Glucocard Diameter (Arkray Factory) After confirming that diabetes was developed, it was subjected to an experiment.
The backs of three mice were shaved with an electric clipper so as not to damage the skin surface, and this preparation (insulin amount: 3 μg) was applied to the back.
The other three mice were subcutaneously injected with a suspension (insulin amount: 2 μg) in which paste-like nanoparticles after lyophilization in Example 1 were redispersed in physiological saline.
Furthermore, another group of 3 animals was untreated and used as a control.

The mice started the test 2 hours after fasting, and blood was collected from the tail vein before insulin administration and every 1, 2, 3, 4, 6 and 24 hours after administration, and blood glucose levels were measured using the Glucocard Diameter (Arkray Factory). I).
The mice were allowed to freely eat food 6 hours after the start of the test, and stopped food intake after 22 hours.
The results are shown in Table 1, and the transition of the blood glucose level is shown in FIG.

Test Example 2: Skin permeability test (diabetic rat)
Streptozotocin (60 mg / kg) was administered continuously for 3 weeks using 3 wistar male rats (8 weeks old) with normal blood glucose levels and physical condition, and the onset of diabetes was confirmed by measuring blood glucose levels. Used for testing.
The back hairs of two rats were shaved in the same manner as in Test Example 1, and this preparation (insulin amounts: 20 μg and 5 μg) was applied to the back skin of each rat.
The remaining one rat served as a non-administration control.

Rats started the test 2 hours after fasting, blood was collected from the tail vein before insulin administration, every 30, 60, 90 minutes, 2, 3, 4, 6, 24, and 48 hours after administration. Measurement was performed using a Glucocard Diameter (Arkray Factory Co.).
Rats were allowed to freely eat food 6 hours after the start of the test, and stopped food intake after 22 and 46 hours.
The results are shown in Table 2, and the transition of blood glucose level is shown in FIG.

Test Example 3:
About the mouse | mouth and rat used in Test Example 1 and 2, the skin of the part which apply | coated this preparation was extract | collected and the sample was produced. Calcium was stained with alizarin S, and the transdermal absorbability of this preparation was examined using calcium as a marker.
As a result, both of the mice used in Test Example 1 (Animal Nos. 1 to 3) and the rats used in Test Example 2 (Animal Nos. 1 and 2) have a transdermal absorbability of the present formulation on the skin applied with the present formulation. A positive calcium staining was confirmed.

As is apparent from Tables 1 and 2 showing the results of Test Example 1 and Test Example 2 and FIGS. 1 and 2, this preparation was applied to the skin in congenital diabetes-onset mice and streptozotocin-onset diabetic rats. As a result, a significant decrease in blood glucose level was observed, and the blood glucose level lowering action was sustained. Furthermore, the blood glucose level lowering effect was comparable to subcutaneous injection administration.
This shows that insulin is satisfactorily absorbed into the living body and the blood glucose level is lowered by administering this preparation containing nanoparticles containing insulin as an active ingredient. This point was also supported by the results of Test Example 3.
Therefore, the present preparation comprising insulin-containing nanoparticles as an active ingredient is significantly beneficial to patients compared with conventional insulin administration preparations by injection administration, and is extremely effective clinically.

Example 3:
1 mg of human growth hormone was dissolved in 100 μL of distilled water, 100 μg of zinc chloride was added to this solution, and the mixture was allowed to stand after stirring. Next, 100 μL of 2% tween 80 aqueous solution was added and stirred, and then a solution in which 0.1 to 0.5 mg of myristic acid was dissolved in 100 μL of acetone was added, and further stirred for 5 minutes to produce primary nanoparticles. Next, 30 μL of 0.5 M calcium chloride aqueous solution was added and stirred for 30 minutes. Thereafter, 30 μL of a 0.1 M sodium carbonate aqueous solution was added and stirred for 30 minutes to produce nanoparticles of the present invention containing human growth hormone.
When the particle size of the nanoparticles contained in this solution was measured by the light scattering method, most of the nanoparticles had a diameter of 10 to 90 nm. In particular, when 0.3 mg of myristic acid was added, those having a particle size of 50 nm were most often present.

Formulation Example 1: Ointment / Hydrogel Take an appropriate amount of the nanoparticles of the present invention obtained in Example 1, white petrolatum, carboxymethylcellulose and methyl parahydroxybenzoate, mix until the total amount is homogeneous, It was set as the hydrogel agent.

Formulation Example 2: External patch (aqueous patch)
Nanoparticles of Example 1 0.1 parts by weight polyacrylic acid 2.0 parts by weight sodium polyacrylate 5.0 parts by weight sodium carboxymethylcellulose 2.0 parts by weight gelatin 2.0 parts by weight polyvinyl alcohol 0.5 parts by weight glycerin 25.0 parts by weight kaolin 1.0 part by weight aluminum hydroxide 0.6 part by weight tartaric acid 0.4 part by weight EDTA-2-sodium 0.1 part by weight purified water balance A patch (aqueous patch) was obtained.

  As described above, the present invention makes primary nanoparticles by using a physiologically active protein or peptide as a water-insoluble substance, the water-insoluble substance, a medium-long chain organic compound having both a hydrophobic group and an anion residue, and a surfactant. And then, a nanoparticle containing a physiologically active protein or peptide obtained by sequentially contacting the primary nanoparticle with a divalent or trivalent metal salt and a divalent or trivalent basic salt. The nanoparticle of the present invention absorbs a physiologically active protein or peptide contained therein via skin or mucous membrane, and exhibits in vivo absorbability that can be substituted for administration by injection. Therefore, it has an epoch-making effect that allows bioactive protein or peptide to be absorbed in vivo by transdermal or transmucosal, which has not been achieved so far, and highly absorbable external use containing bioactive protein and peptide Drugs have been created and have great medical value.

It is a figure which shows transition of the blood glucose level of the mouse | mouth in the test example 1. FIG. It is a figure which shows transition of the blood glucose level of the rat in Test Example 2.

Claims (14)

  Using a bioactive protein or peptide as a water-insoluble substance, a primary nanoparticle is prepared using the water-insoluble substance, a medium-to-long chain organic compound having a hydrophobic group and an anion residue, and a surfactant. Nanoparticles containing a bioactive protein or peptide obtained by sequentially contacting a divalent or trivalent metal salt and a divalent or trivalent basic salt.   Primary nanoparticles dissolve water-insoluble matter, medium-long chain organic compounds having both hydrophobic groups and anionic residues, and surfactants in an organic solvent or water-containing organic solvent, and stir and shake it in a large amount of water. The nanoparticle according to claim 1, wherein the nanoparticle is produced by adding while adding.   The nanoparticle according to claim 1 or 2, wherein the diameter of the particle is 5 to 150 nm.   The nanoparticle according to claim 1 or 2, wherein the particle has a diameter of 10 to 80 nm.   The biologically active protein or peptide is insulin, interferon-α, interferon-β, interferon-γ, growth hormone, G-CSF, GM-CSF, erythropoietin, thrombopoietin, urokinase, t-PA, IL-11, etanercept, infliximab, The nanoparticle according to any one of claims 1 to 4, which is selected from SOD, FGF, EFG, HGF, NGF, BDNF, leptin, NT-3, an antigen, an antibody and an enzyme.   The means for making a physiologically active protein or peptide insoluble in water is any means of contact with a divalent or trivalent metal ion, contact with an acidic or basic polysaccharide, adjustment of pH, or change in ionic strength. The nanoparticle according to claim 1.   The nano of claim 6, wherein the divalent or trivalent metal ion that is contacted to make the physiologically active protein or peptide insoluble in water is selected from zinc ion, calcium ion, iron ion and copper ion. particle.   The medium-long chain organic compound having both a hydrophobic group and an anion residue is selected from myristic acid, oleic acid, lauric acid, palmitic acid and salts thereof. The described nanoparticles.   The divalent or trivalent metal salt brought into contact with the primary nanoparticles is a calcium salt, zinc salt, iron salt or copper salt, and the divalent or trivalent basic salt is carbonate, phosphate, oxalate, The nanoparticle according to any one of claims 1 to 4, which is lactate or urate.   An external preparation for skin or mucous membrane, comprising the nanoparticles according to any one of claims 1 to 9.   The external preparation according to claim 10, wherein the external preparation is selected from ointments, gels, nasal drops, eye drops, sprays, suction agents, suspension agents, cataplasms, and patches.   A physiologically active protein or peptide is made into a water-insoluble substance, and the water-insoluble substance, a medium-long chain organic compound having both a hydrophobic group and an anionic residue, and a surfactant are dissolved in an organic solvent or a water-containing organic solvent. The primary nanoparticles are prepared by adding to the water with stirring and shaking, and the divalent or trivalent metal salt and the divalent or trivalent basic salt are sequentially added to the primary nanoparticle-containing solution. A method for producing nanoparticles containing a physiologically active protein or peptide.   The means for making the physiologically active protein or peptide water-insoluble is contact with a divalent or trivalent metal ion, and the divalent or trivalent metal salt to be brought into contact with the primary nanoparticles is a calcium salt. The method for producing nanoparticles according to claim 12, wherein the trivalent basic salt is a carbonate. The method for producing nanoparticles according to claim 13, wherein the metal ions are zinc ions.

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