JPH01163199A - Water-insoluble substance - Google Patents

Abstract

NEW MATERIAL:A water-insoluble cytokinin substance. EXAMPLE:Interleukine 2. USE:A sustained release injection. PREPARATION:A biocompatible protein (e.g., human blood serum albumin) or high-polymer organic acid (e.g., alginic acid) having about >=5000 molecular weight or salt thereof (e.g., sodium salt) is reacted with cytokinin in an aqueous medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a water-insoluble form of cytolytic acid useful as a sustained-release injection, an injectable composition containing the same, and a method for producing the water-insoluble form.

Insulin lead aqueous suspension injection and protamine insulin zinc aqueous suspension injection (Japanese Pharmacopoeia 10th revision) are known as drugs that can prolong the action of Yame Δ Bunri pharmaceuticals.

However, although these are insolubilized insulin, which has low water solubility, there are no insolubilized cytokines that are easily water soluble.

In the development of physiologically active substances as pharmaceuticals, there are various problems based on their properties. For example, interleukin-2 ([L-2), a type of Zytoin, is highly expected to be applied as a pharmaceutical, but the half-life of the serum concentration after administration is approximately 0.6 hours when administered intravenously. Intramuscular administration is as short as about 1.3 hours, and in order to more effectively express its medicinal effects, it was necessary to develop a formulation that was endowed with sustained release properties and target directivity.

Therefore, the present inventors thought that if IL-2 is made into a water-insoluble form, it will take time to become water-soluble, so sustained release properties can be expected. Based on this, the present inventors conducted further research and found that common salt was found.

Inorganic salts such as calcium chloride, zinc chloride, and phosphoric acid, low-molecular organic acids (salts) such as ammonium acetate and sodium citrate, and amino acids such as glycine are added to the IL-2 aqueous solution so that the osmotic pressure is at least isotonic. When the solution was added to water and shaken, it was found that although water-insoluble rL-2 was produced, the physiological activity of the water-soluble IL-2 was reduced to about half.

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors conducted intensive research with the aim of obtaining a water-insoluble substance that retains physiological activity, and found that a protein that is biocompatible with cytokines or has a molecular weight of approximately 5,000 or more. We discovered that a water-insoluble form possessing the physiological activity of Cytolyne could be obtained only by reacting it with a high-molecular organic acid or its salt in an aqueous medium, and as a result of further research,
The invention has been completed.

The present invention provides (1) a water-insoluble form of cytokin that has physiological activity, (2) a composition for injection containing a water-insoluble form of cytokin that has physiological activity, and (3) a water-insoluble form of cytokin that has physiological activity. This is a method for producing a side-in water-insoluble material possessing physiological activity, which is characterized by reacting a biocompatible protein, a high-molecular organic acid having a molecular weight of about 5,000 or more, or a salt thereof in an aqueous medium.

Examples of cytokines include monokine.

Examples include lymphokines.

The monolayer may include, for example, tumor necrosis factor (tumor necrosis factor).
or necrosis factor, TNF)
, interleukin (IL-1), differentiation-inducing factor (DA
F) etc.

Examples of the lymphokine include T cell growth factors (interleukin-2, IL-2), interferon-
α, -β, -γ, interleukin-3° lymphotoxin (LT), macrophage fugue inhibition factor (MIF),
Macrophage activating factor (MAF), macrophage chemoattractant factor (MCF), B cell growth factor (BCGF),
B cell differentiation factor (BCDF), neutrophil chemoattractant factor (NC
F), leukocyte fugue inhibition factor (Ll''F), and the like.

In addition, in this specification, interleukin-2
are IL-2 and interferon-α.

-β and =γ are abbreviated as IFN-α, -β, and -γ, respectively.

The IL-2 may be a substance that has the same activity as IL-2, that is, a substance that has the ability to maintain T cells through generations while maintaining their functions. Specifically, for example, polypeptide (I) (human IL-2) having the amino acid sequence shown in FIG. It may also be a fragment consisting of a partial amino acid sequence.

The above fragment includes, for example, polypeptide (1)
One amino acid from the amino terminus of (EPC Publication 9153
9) or a fragment lacking four amino acids (
Examples include JP-A-60-126088) and fragments lacking several amino acids at the carboxyl terminal. Furthermore, polypeptide (1) having the amino acid sequence shown in FIG. 1 has some of its constituent amino acids deleted or substituted with other amino acids, for example, the cysteine residue at position 125 is substituted with a serine residue. What was done [Unexamined Japanese Patent Publication No. 5]
9-93093] Publication.

Further, the above-mentioned IL-2 active substance may be chemically modified with a polyethylene glycol derivative or the like [for example, as disclosed in JP-A-60-226821.

In particular, in the present invention, human Il having the amino acid sequence shown in FIG. 1 of JP-A-61-78799
, -2 is preferably used, and in this case, a mixture of those with and without a methionine residue (Ivlet) at the amino terminal [JP-A-60-115528, JP-A-61-78799] No. Publication] No. Publication- is also good, and alanine (Ala) is also used without adding Met to the amino terminus.
[JP-A No. 61-78799] starting with [JP-A-61-78799].

Moreover, it may have a sugar chain.

The IFN-α may be any substance that exhibits IF'N-α activity, that is, an antiviral effect, and natural type I
Even with FN-α, IF obtained by genetic engineering methods
It may be N-α. IFN- obtained by genetic engineering method
As α, for example, rl FN-αA, B, C, D,
E, F, G, I-1, I, J [Unexamined Japanese Patent Publication No. 57-798
No. 97, European Patent Publication No. 43980].

The IFN-γ may be any substance that has IFN-γ activity, that is, a substance that has antiviral activity and has the effect of activating the immune system. IFN-γ obtained in .

Examples of IFN-γ obtained by genetic engineering methods include those obtained by the method described in JP-A No. 58-90514, and those obtained by the method described in JP-A-59-186995. can be mentioned.

Alternatively, it may be a fragment lacking X or several amino acids from the amino terminal and/or carboxyl terminal of IFN-γ. Examples of such fragments include those described in JP-A-60-202899;
Examples include those described in JP-A-61-5096.

The cytokines used in the present invention described above are all readily soluble in water, for example, their solubility is o, 5 m
g/- or more.

Examples of biocompatible proteins used in producing the water-insoluble material of the present invention include human serum albumin, human serum globulin, and fibrinogen.

Examples include those derived from living organisms such as collagen and casein.

Examples of biocompatible high-molecular organic acids with a molecular weight of about 5,000 or more used in producing the water-insoluble material of the present invention include alginic acid, hyaluronic acid, chondroitin sulfate, pectic acid, pectinic acid, heparic acid, acrylic acid,
Examples include pecudin and carrageenan, which have these as main ingredients.

Examples of the salts of the polymeric organic acids include sodium salts, calcium salts, magnesium salts, and zinc salts.

Examples include ammonium salt, arginine salt, N-methylglucamine salt, and the like.

Examples of the aqueous medium used in the method of the present invention include those containing water as a main component.

In order to cause a biocompatible protein to act on a cytokine in an aqueous medium, the cytokine and the biocompatible protein are present in the aqueous medium and a physical stimulus is applied.

Examples of the physical stimulation include shaking, stirring, and the like. The concentration of the cytokine in the aqueous solution containing the cytokine and the protein during the above operation is preferably about 0.1 mg/min or more, particularly 0.1 mg/min.
, 5 mg/J or more is desirable. The protein concentration is preferably about 0.5 times or more, particularly about 1 time or more, the cytokine intensity.

The upper limit is preferably about 500 times or less, more preferably about 100 times or less.

The pH of the aqueous solution is adjusted to about 3 to 12 by buffering agents such as inorganic acids such as hydrochloric acid, sodium hydroxide, or phosphoric acid, organic acids such as acetic acid and citric acid, and amino acids such as glycine. More preferably I)H about 4 to 11
It is best to adjust it to

In addition to the protein, this aqueous solution contains inorganic salts such as common salt, calcium chloride, and zinc chloride, and organic acids (salts) such as ammonium acetate and sodium citrate in order to promote water insolubilization.
Alternatively, amino acids such as glycine may be added.

The shaker, the protein-containing protein
This is carried out by shaking a container containing an aqueous solution of H114 using a portex mixer or a shaker. The required shaking strength and time vary depending on the composition of the aqueous solution and its liquid m, but basically, in order to increase the production rate of water-insoluble substances, it is preferable to shake strongly and for a certain length of time. Specifically, the shaking time is preferably about 5 seconds or more and 30 minutes or less, and more preferably about 30 seconds or more and 10 minutes or less.

The agitation increases the pH containing the cytoplasmic acid and the protein.
This is carried out by stirring the aqueous solution of the knotted Ul',] using a propeller stirrer, a magnetic cooler, a homogenizer, or the like. The strength and time of stirring required will vary depending on the composition of the aqueous solution and its volume, as in the case of shaking, but basically, to increase the production rate of water-insoluble substances, stirring should be strong and for a certain length of time. Specifically, it is preferably about 5 seconds or more and 30 minutes or less, and more preferably about 30 seconds or more and 10 minutes or less.

The action of a biocompatible high-molecular organic acid or a salt thereof having a molecular weight of 5,000 or more on a cytokine in an aqueous medium is carried out by adding the high-molecular organic acid or a salt thereof to an aqueous solution of cytotoxin.

The preferable conditions for the concentration of cytotoin, the concentration of the polymeric organic acid, and the pH in the aqueous solution containing the cytokine and the polymeric organic acid when performing the above operation are the same as in the case of adding the biocompatible protein described above. be.

This aqueous solution also contains salt, calcium chloride, magnesium chloride, and zinc chloride to promote water insolubilization.

An inorganic acid such as common salt, an organic acid (salt) such as ammonium acetate or sodium citrate, or an amino acid such as glycine may be added. Furthermore, calcium chloride or magnesium chloride may be added for the purpose of gelling the added high-molecular organic acid rather than promoting insolubilization. Note that when using the polymeric organic acid, unlike when adding biocompatible proteins, shaking or stirring is not particularly necessary, but in order to promote water insolubilization, the same steps as above are necessary. Shake or stir.

The amount of polymeric acid or its salt added is preferably about 0.5 times or more (by weight) relative to the cytokine mound, and more preferably about 1 to 100 times m (weight) or less. It is preferable that

Furthermore, the water-insoluble substance of the present invention can be efficiently produced by allowing a biocompatible protein to act on a cytokine in an aqueous medium in the coexistence of a water-soluble organic solvent.

Examples of the water-soluble solvent include acetone, methanol, and ethanol.

The coexistence of the water-soluble organic solvent is carried out by adding it to an aqueous solution containing the cytokine and the protein. The amount of water-soluble organic solvent required varies depending on the composition of the aqueous solution and its volume, but basically it is preferably as large as possible in order to increase the production rate of water-insoluble substances. Specifically, it is about 4 volumes per 1 volume of the aqueous solution. The above water-soluble organic solvents are preferred. The organic solvent is preferably as large as possible, but practically it is about I
Preferably, amounts up to O capacity are used. If desired, stirring may be performed after adding the organic solvent.

In this way, a water-insoluble cytokine that retains physiological activity is obtained.

The level of physiological activity possessed by the water-insoluble body is about 60% or more, more preferably about 80% or more, compared to the original cytokine.

The physiologically active water-insoluble form of Cytolyne thus obtained can be used as a sustained-release injection.

Moreover, by formulating the water-insoluble cytokine having physiological activity with various carriers, it can be used as a sustained-release injection composition.

That is, for example, even if the water-insoluble material of the present invention is suspended in water or various dispersion media in the form of a suspension and is directly administered intramuscularly or subcutaneously, it is solubilized at the administration site and exhibits sustained release. Therefore, it can be used as a sustained-release injection. In addition, for example, albumin, collagen, fibrinogen, alginic acid.

Bio-derived polymeric substances such as pectic acid, pectin, carrageenan and their salts, polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, ethylcellulose, polydimethylchloroxane polymer, polyethylene, polyhydroxymethacrylate Rate-ethylene glycol methacrylate copolymer, polyvinyl alcohol, poly2-hydroquinoethyl methacrylate, ethylene-vinyl acetate copolymer. Using a biocompatible synthetic polymer material such as a polyoquineethylene-polyoquinopropylene copolymer, solutions, sol, gel, jelly, etc.
Its sustained release properties can be further enhanced by incorporating it into a matrix, microcapsules, etc. Among these polymeric substances, polylactic acid, polyglycolic acid, or copolymers thereof are particularly used for uJll.

Furthermore, the water-insoluble cytolyte of the present invention can also be incorporated into liposomes using phosphatinorcholine or the like by a conventional method by treating the suspension liquid as an aqueous phase.

Furthermore, the water-insoluble cytokine of the present invention is prepared by adding the dry powder to the oil phase of vegetable oil such as soybean oil or safflower oil, and then using a surfactant such as natural lecithin or sorbitan fatty acid ester as an emulsifier. , by the usual method, 0
/W-type emulsion, or as a W10/W-type emulsion in which the suspension of the water-insoluble material of the present invention is treated as an aqueous phase, dispersed in an oil phase, and then dispersed in another aqueous phase. You can also use it.

In the injectable composition of the present invention, an isotonizing agent (
e.g., salt, glucose, mannitol, sorbitol)
, p I (n-moderating agents (e.g., hydrochloric acid, sodium hydroxide).

Stabilizers (e.g., sodium pyrosulfite, Rongalite,
(sodium metabisulfite), @turbidity agent (e.g., polyethylene glycol, polysorbate 80, glycerin, carboxymethyl cellulose, polyvinyl alcohol, sodium alginate, pectin, casein, gelatin)
, analgesic agents (e.g., xylocaine hydrochloride, chlorobutanol, procaine hydrochloride), and preservatives (e.g., benzyl alcohol, phenol, paraoxyamne omega, methyl frate, propyl paraoxybenzoate), etc., and have properties as an injection. It is also possible to increase

The water-insoluble cytokine of the present invention and the injectable composition containing the same gradually solubilize the cytokine and exhibit sustained release when administered by injection, and also retain the physiological activity of cytotoxicity.

Thus, the water-insoluble cytokine of the present invention possesses the physiological activity of cytotoxicity. Moreover, since a biocompatible substance is used as an insolubilization aid, the insoluble material of the present invention has extremely low toxicity.

Therefore, the water-insoluble cytokine of the present invention can be used for the same purpose as known cytokines.

Since the cytokine water-insoluble form of the present invention exhibits sustained release properties, when administering the water-insoluble form as a sustained-release injection, the amount corresponding to the number of days of the sustained-release period, that is, one day of the sustained-release period. The daily dose is the known daily dose of the cytokine, plus the number of days during the sustained release period. If necessary, the amount may be reduced slightly.

IL-2 used in the Examples described below was obtained from the transformant Escherichia coli (Escherichia coli).
li) DHI/II) TF4 (IFO14299°FE
RM BP-628) or Escherichia coli N
4830/pTB285 (IFO14437, FFRM
BP-852), JP-A-60-115528
It is produced by a method similar to that described in Japanese Patent Application Laid-Open No. 61-78799.

The above transformant Escherichia colt
D H+ /1) T F 4 is November 2, 1988
From the 5th, the Institute for Fermentation Foundation (IFO) received the accession number IF.
It was deposited as O14299, and this microorganism was
From April 6, 2009, the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Research Institute (FRI) received accession number FERM I'-757.
The deposit was transferred to a deposit under the Budapest Treaty and is kept at the same research institute (FRI) under accession number FERMBP-628.

The above transformant Escherichia colt
N4830/p'l'B285 was released on April 25, 1985.
The microorganism was deposited with the IFO under the accession number IFO14437 from April 30, 1985, and the microorganism
Deposited with accession number FERM P-8199,
The deposit was transferred to a deposit under the Budapest Treaty and is deposited at Frtl under accession number FERM BP-852.

The activity of IL-2 in the Examples described below was measured by the method described in JP-A-60-115528.

IFN-α used in the examples described below was disclosed in Japanese Patent Application Laid-Open No. 1986-
It is manufactured by a method similar to that described in Japanese Patent No. 79897.

IFN-γ used in the examples described below was disclosed in Japanese Patent Application Laid-Open No. 1986-
It is manufactured by a method similar to that described in Japanese Patent No. 186995.

The present invention will be explained in more detail with reference to Examples below, but the present invention should not be limited thereto.

Example 1 IL-2 stock solution (approx. 1 mg/mg, pH 5, 0) was placed in each of six glass spitz rolls with IO rotation capacity.
Add 0.5 mg of human serum albumin lyophilized powder, dissolve the human serum albumin, and then
Add 0.05mM ammonium acetate buffer to pH
is 2.5, 3.5, 5.4, 7.9, 9.9 or 12
．． Six types of IL-2 aqueous solutions were prepared. The aqueous solution in these spitz rolls was mixed with a Portex mixer (Micro Thermo Mixer Model T, manufactured by Thermonics Co., Ltd.).
M-1ot) was used to strongly stir for 3 minutes.

As a result, 1) water-insoluble substances are generated from the aqueous solutions of H5, 4, 7, 9, and 9.9, and the water-insoluble substances are contained in the insoluble substances.
The ratio of IL-2 to the charged amount was pH 5.

It was 86% at pH 4, 72% at pH 7 and 9, and 62% at pH 9 and 9.

This water-insoluble material was washed with 1-component distilled water for injection, dissolved in a pH 3 buffer, and analyzed by gel permeation chromatography.
-2 was found to coexist with human serum albumin.

Example 2 IL-2 raw material in a glass spitz roll with a capacity of 10
10 mg of lyophilized human serum albumin powder was added to L (approximately 1 mg/d, pH 5.0) and shaken for 3 minutes using a portex mixer to make IL-2 insoluble in water. Next, this water-insoluble material was centrifuged at 3500 rpm for 10 minutes, and the supernatant was removed. After adding the solution and redispersing it, it was centrifuged again under the same conditions and the supernatant was removed to obtain a water-insoluble IL-2.

Next, 1) Two volumes of 0.025M glycine buffer of 83.5 were added to the obtained water-insoluble material to dissolve it. When an aqueous solution of IL-2 containing 10 mg of albumin and 1 d of 0.025M glycine buffer with a pi-r of 3.5 was measured as a control, the aqueous solution in which the water-insoluble form of IL-2 was dissolved was 12,500 U/d.

For the control aqueous solution, a result of 17,300 U/d was obtained. Therefore, considering the yield (78%) of the water-insoluble IL-2, it is judged that there is almost no decrease in the titer of IL-2 due to insolubilization.

Example 3 IL-2 stock solution (
Approximately 1 mg/mg, pH 5, 0) was mixed with 31n1 and human serum albumin was shaken for 5 minutes using a portex mixer to generate a water-insoluble form of [L-2] that coexists with human serum albumin.

This water-insoluble material was centrifuged, the supernatant was removed, freeze-dried, sieved through a 350-mesh sieve, 2.4 mg of it was dissolved in 1-component physiological saline, and the muscle was injected into the thigh of a domestic rabbit. I noted it. 4 hours and 1 day after administration.

After 4 days, blood was collected from the auricular vein and 11. The −2a degree was measured by enzyme immunoassay using the Sand-Deutsch method using an anti-recombinant IL-2 goat antibody as the primary antibody and an anti-recombinant rabbit antibody conjugated with veru-o-quintase as the secondary antibody. As a result, serum [L-26 degrees] was 44 ng after 4 hours, 1.1 ng after 1 day, and 0.4 ng after 4 days.
The water-insoluble form of IL-2 is solubilized in vivo and becomes IL-2.
It was confirmed that 2 was being released in a sustained manner.

Example 4 10 IL-2 stock solution (concentration 1 mg/goose, pl-I 5.0) was placed in a 30 J capacity glass spitz roll.
J and 0.5 d of human serum albumin aqueous solution with a concentration of 200 mg/kura. 6 added. The IL-2 aqueous solution in the spitz roll was shaken for 10 minutes using a portex mixer to generate a water-insoluble IL-2 coexisting with human serum albumin.

This suspension of water-insoluble matter was centrifuged at 300 rpm for 10 minutes, the supernatant liquid was removed, and then distilled water was added to wash the water-insoluble matter, and the water-insoluble matter was washed out again.
The supernatant was removed by centrifugation at pm for 10 minutes. Next, the concentrated slurry of the water-insoluble substance was freeze-dried by a conventional method, and then sieved using a 350-mesh sieve. Next, this sieved IL-
2. One part of 6.4 mg of water-insoluble powder was mixed with 0.5 g of polylactic acid-polyglycolic acid copolymer (PLGA).
After uniformly dispersing the solution in which 75d of methylene chlorite was dissolved using a portex mixer, this solution was mixed with 0.
Gradually add it to 25% 5% polyvinyl alcohol aqueous solution and use an ultra high speed homogenizer (Kinematica).
(manufactured by Germany) to obtain an O/W emulsion in which PLGA was encapsulated and dispersed in an aqueous phase. Next, this O/W
The emulsion was stirred with a propeller stirrer to dry the methylene chloride in the microcapsules in water. After centrifuging the obtained O/W emulsion and removing the supernatant, the microcapsules were freeze-dried again in small fractions with an aqueous solution containing 100 mg of mannitol to obtain 387 mg of a mixed powder of microcapsules and mannitol.

Next, this mixed powder was dispersed in 1 d of an aqueous solution containing 1 mg of polysorbate 80 and 50 mg of mannitol so that the dose as IL-2 was 180 mcg, and the mixture was injected intramuscularly into the thigh of a domestic rabbit, one day after administration.

After 3, 14, and 20 days, blood was collected from the auricular vein, and the IL-26 degree in the serum was measured by enzyme immunoassay in the same manner as in Example 3. As a result, the IL-25 degree in serum 1 was 1.5 degrees after 1 day. Ing, 1.0 ng after 3 days.

After 14 days, 7 ng, after 20 days, 0,6 ng,
It was confirmed that IL-2 was sustainedly released from the microcapsules.

Example 5 IL-2 stock solution (approximately 1 mg/form, pH 5, 0), I
F N -α stock solution (approximately 1 mg/1nf1. pH 5, 0
) and rFN-7 stock solution (approximately 2 mg/ml, pl(6,0
) was dispensed into two vials each and lyophilized. For each of the 6 vials of 3 types of lyophilized protein, 55% sodium alginate was added to each protein.
When 0.3mg of water was added and 0.3mg of water was added, IL-2 water-insoluble and IF'N coexisted with sodium alginate.
-α water-insoluble material and IFN-γ water-insoluble material were produced.

Each liter vial of these water-insoluble liquids was directly freeze-dried and then sieved through a 100-mesh sieve to obtain IL-2
, a lyophilized powder of a water-insoluble form of IF N-α or IFN-γ was obtained. Further, an aqueous solution of O and ld containing 5 mg of calcium chloride was added to each of the suspensions of the water-insoluble substances in the remaining one vial to gel the excess sodium alginate, and this gel was freeze-dried to 100
Passed through a mesh sieve to obtain IL-2 and IFN-α, respectively.
Alternatively, a freeze-dried alginate gel powder containing a water-insoluble form of IFN-γ was obtained.

Example 6 IL-2 stock solution (concentration approximately 1 mg/ha, I'lH5) in a 30 volume glass spitz roll 5 rn
50 mg of sodium alginate was added and dissolved in 1 ml. The IL-2 aqueous solution in the spitz roll was shaken for 10 minutes using a portex mixer to generate an IL-2-containing water-insoluble substance coexisting with sodium alginate.

This water-insoluble suspension was centrifuged and the supernatant was removed, and the obtained water-insoluble material was washed with distilled water, freeze-dried, and then sieved using a 350-mesh sieve. Next, 1 of this sieved powder
0.5 g of I'LGA in the same manner as in Example 4.
After uniformly dispersing the microcapsules in a solution containing the following, 363 mg of a mixed powder of microcapsules and mannitol was obtained by the same procedure as in Example 4 (containing 100 mg of mannitol). A total of q of this mixed powder was dispersed in 1 d of an aqueous solution containing 80 1 mg of polysorbate and 50 mg of mannitol, and then intramuscularly injected into the thigh of a domestic rabbit, 1 day, 3 days, and 7 days after administration.

After 14 and 20 days, blood was collected from the auricular vein, and the serum I
The L-2 concentration was measured by enzyme immunoassay in the same manner as in Example 3. As a result, the IL-2 concentration in serum ld was 0.3 after 1 day.
ng, 0.5 ng after 3 days, 0°4 ng after 7 days,
The amount was 0.5 ng after 141 days, and 0.2 ng after 20 days, confirming that IL-2 was sustainedly released from the microcapsules.

Example 7 IL-2 stock solution (concentration approximately 1 mg/m9. pH 5, 0).

IFN-α stock solution (concentration approximately 1 mg/Ml, pH 5, 0)
or IFN-7 stock solution (concentration approximately 2 mg/form, I) H6
, 0) was added with freeze-dried powder of human serum albumin (I (SA)) at the concentrations shown in Table 1 and shaken to obtain a water-insoluble material.

A glass spitz roll with a capacity of 1 Od was used as the container, and shaking was performed for 3 minutes using a portex mixer.

The precipitated insoluble material was centrifuged at 3000 rpm for 10 minutes, the supernatant was removed, and one portion of 0.04M phosphate buffer (I) H7,4 was added to investigate its solubility. The results are shown in Table 1.

As is clear from Table 1, the produced water-insoluble material was not dissolved in the pH 7.4 buffer in any case.

Comparative example 1 to? IL- in a glass spitz roll of nfl capacity
2. Add 18mg of salt to the stock solution (approx. 1mg/d, pH 5.0)
After addition, shake for 3 minutes using a portex mixer.
L-2 was made insoluble in water. Next, 0.1 M glycine buffer (I)83,0 was added once to this suspension solution of IL-2 water-insoluble body to dissolve the IL-2 water-insoluble body, and then this T L
The titer of the -2 solution (sample A) was determined as 1, and the IL-2 solution (sample B) of the same composition without the water insolubilization treatment of -2 was measured as a control. The results are shown in Table 2. Furthermore, IL-2
The titer was measured using the proliferation of mouse natural killer cells, which depends on the amount of IL-2, as an index.

In this way, when common salt is used, the water-insoluble I
It was found that the titer of L-2 was reduced to less than 60%.

Example 8 The entire amount of the concentrated slurry of IL-2 water insoluble obtained by the method described in Example 4 was added to soybean oil tO, Og and uniformly dispersed using an ultra-high speed homogenizer (manufactured by Kinematica) to form a W10 emulsion. I got it. Using this soybean oil and 1.2 g of egg yolk lecithin as an emulsifier, it was mixed at 2°5 w/w%.
A W10/W emulsion was prepared by dispersing it in 100ml of an aqueous glycerol solution using a homogenizer. Dispense this emulsion into two vials aseptically and use a rubber stopper.
The cap was closed and sealed to obtain a sustained-release intramuscular injection of IL-2.

Example 9 After uniformly dispersing 5 mg of the water-insoluble freeze-dried powder obtained by the method described in Example 4 using an ultra-high-speed homogenizer, the soybean oil was mixed with 2 g of egg yolk lecithin as an emulsifier. The mixture was uniformly dispersed in 100 d of a .5 w/w% aqueous glycerol solution using a homomixer to obtain an O/W emulsion. This emulsion was aseptically dispensed into two vials, sealed and sealed with rubber stoppers and caps, and IL-
A sustained release injection for intramuscular injection of No. 2 was obtained.

Example IO The lyophilized powder of IL-2 water-insoluble obtained by the method described in Example 4 was sieved through a 350-mesh sieve, and 5 mg thereof was added to a 2-mesh aqueous solution containing 200 mg of sodium alginate. After uniformly dispersing 100 mg of C
a Alginic acid was gradually added to a diluted aqueous solution containing CQ ffi to gel it. After freeze-drying this gel,
The mixture was sieved through a 0 mesh sieve to obtain an alginate gel powder containing a water-insoluble IL-2. 50 mg of this alginate gel powder was aseptically filled into a vial and sealed with a rubber stopper and cap to obtain a sustained release injection of IL-2.

This injection is suspended in one volume of physiological saline containing 2 mg of polysorbate 80 and administered intramuscularly to humans.

Example 11 The IL-2 water-insoluble material obtained by the method described in Example 4 was freeze-dried and then sieved through a 350-mesh sieve, and 5 mg of the IL-2 water-insoluble material was sieved with 200 mg of human serum albumin and 50 mg of reduced geltadeone. Human serum albumin was uniformly dispersed in a one-turn aqueous solution, and the aqueous solution was kept at 110°C for 15 hours to gelatinize human serum albumin with reduced glutathione. Next, this gel was freeze-dried and then sieved through a 100-mesh sieve to obtain an albumin gel powder containing II, -2 water-insoluble material. This albumin gel powder 50
mg and 9 mg of sodium chloride were aseptically filled into vials and sealed and sealed with rubber stoppers and caps to obtain a sustained-release injection of IL-2. This injection contains 22% of polysorbate 80.
It is suspended in 1 g of distilled water for injection containing mg and administered intramuscularly to humans.

Example 12 Example 1] L instead of 50 mg of Nobugen type glutathione
- Using 40 mg of cysteine hydrochloride, an albumin gel powder containing water-insoluble TL-2 was obtained in the same manner as in Example 11, and a sustained-release injection using the albumin gel was obtained. This injection is polysorbate 80
1 d of distilled water for injection containing 2 mg of fI! ! ! It is administered intramuscularly to humans.

Example 13 After sieving the lyophilized powder of IL-2 water-insoluble obtained by the method described in Example 4 through a 350-mesh sieve,
After uniformly dispersing 5 mg of it in an aqueous solution of TY containing 200 mg of human serum albumin, the reduced glutathione 2
0 mg was added and dissolved.

Gradually add this aqueous solution to 5 volumes of ethanol and use an ultra high-speed homogenizer (manufactured by Kinematica).
A dispersion of human serum albumin spherules was obtained by emulsification.

This dispersion was kept at 40° C. for 15 hours, and human serum albumin was gelatinized by the coexisting reduced geltadeone. The gelled microspheres were centrifuged, the supernatant was removed, and then lyophilized to obtain a powder of albumin microspheres containing water-insoluble IL-2. 30 mg of these albumin spherules were aseptically filled into a vial together with 50 mg of mannitol, and the vial was closed and sealed with a rubber stopper and cap to obtain a sustained-release injection of IL-2. This injection contains 2m of polysorbate 80.
It is suspended in 1 yd of distilled water for injection containing 1 yd of water for injection and administered intramuscularly to humans.

Example 14 A powder of albumin spherules containing water-insoluble rL-2 was obtained in the same manner as in Example 13, except that 20 mg of L-cysteine hydrochloride was used in place of 20 mg of glutathione. 30mg of these albumin globules and 9g of salt
mg was filled into a vial aseptically, and the vial was closed and sealed with a rubber stopper and cap to obtain a sustained-release injection of IL-2. This injection is suspended in distilled water for injection containing 802 mg of polysorbate and administered intramuscularly to humans.

Example I5 IL- produced by the method described in Example 5
A mixed powder of mannitol and microcapsules containing the lyophilized water-insoluble IL-2 was obtained by the method described in Example 4 using 5 mg of the lyophilized water-insoluble powder of IL-2. Next, put this mixed powder into a 50 mg vial.
Fill the container gently, and close and seal using a rubber stopper and cap to obtain a sustained-release injection of IL-2. This injection contains 2 mg of polysorbate 80 and 50 mg of mannitol.
It is dispersed in an aqueous solution containing 1 volume and administered intramuscularly to humans.

Example 16 5 m of lyophilized powder of alginate gel containing water-insoluble fL-2 produced by the method described in Example 5
Obtain a sustained-release injection of IL-2 in the same manner as in Example 15 using G.g.

Example 17 Instead of 10 d of IL-2 stock solution, [PN-α stock solution (concentration of about 1 mg/bj2.pL (5,0)I OyJ was used, obtained by the same method as described in Example 4). I
Example 4 using 5 mg of freeze-dried powder of FN-α water-insoluble substance
A mixed powder of Mic[l capsules containing IFN-α water-insoluble material and mannitol] is obtained in the same manner as above. Next, 50 mg of this mixed powder is aseptically filled into vials and sealed with rubber stoppers and caps to obtain a sustained-release injection of IF''N-α.The administration method is the same as in Example 15. .

Example 18 IFN-α produced by the method described in Example 5
A sustained-release injection of IFN-α is obtained in the same manner as in Example 15 using 5 mg of the water-insoluble freeze-dried powder.

Example I9 rFN-α produced by the method described in Example 5
5 m freeze-drying bed of alginate gel containing water-insoluble matter
A sustained-release injection of IFN-α is obtained in the same manner as in Example 15 using G.

Example 20 IPN-γ stock solution (e4 degree: about 2 mg/y4.1) H6,0) I0rnl was used instead of IL-2 stock solution 10 precepts, and obtained by the same method as described in Example 4. A mixed powder of microcapsules containing IFN-γ water-insoluble and mannitol was obtained in the same manner as in Example 4 using 5 mg of the freeze-dried powder of IFN-γ water-insoluble.

Next, somg of this mixed powder is aseptically filled into a vial, and the vial is closed and sealed with a rubber stopper and cap to obtain a sustained-release injection of IFN-γ. The administration method is the same as in Example 15.

Example 21 IFN-γ produced by the method described in Example 5
A sustained release injection of IFN-γ is obtained in the same manner as in Example 15 using 5 mg of the water-insoluble freeze-dried bed.

Example 22 IFN-γ produced by the method described in Example 5
5 m freeze-drying bed of alginate gel containing water-insoluble matter
A sustained-release injection of [FN-γ] was obtained in the same manner as in Example 15 using [FN-γ].

Example a3 IL-2 stock solution (
After dissolving 100 mg of human serum albumin by simmering the solution (concentration: about 1 mg/goose, pH 5.0), methanol was added to form water-insoluble substances of 1 and -2 that coexisted with serum albumin.

This water-insoluble substance was separated and dried, and 40 mg was subcutaneously administered to rats. Blood was collected from the rat tail vein 3 hours, 1 day, and 3 days after administration, and IL-2a levels in the serum were measured using an anti-recombinant IL-2 goat antibody as the primary antibody and a peroxidase-conjugated anti-recombinant antibody as the secondary antibody. It was measured by enzyme immunoassay using the Sand-Deutsch method using a recombinant rabbit antibody.

As a result, the IL-26 degree in one drop of serum was 12 degrees after 3 hours.
Qng.

11.8 ng after 11 days, 1.11 ng after 3 days, and IL
It was confirmed that the water-insoluble form of -2 was solubilized in vivo and IL-2 was released in a sustained manner.

Example 24 IL-2 stock solution (
Concentration 1 mg/J, ptr 5, O) at 10
After sterilizing and dissolving 250 mg of human serum albumin,
Add acetone 20, blood 11! A water-insoluble form of L-2 coexisting with albumin was produced.

+2 for this water-insoluble substance! The suspension was centrifuged at 300 Orpm for 10 minutes, and the supernatant was removed. Next, the water-insoluble material was dried and ground in a mortar. This was uniformly dispersed in a solution of 4.5 g of polylactic acid-polyglycolic acid copolymer (PLGA) dissolved in 5.6 layers of methylene chloride using a vortex mixer. was gradually added to a 5% polyvinyl alcohol aqueous solution (1000ml) and then emulsified using an ultra-high speed homogenizer to obtain an O/W emulsion in which PLGΔ was encapsulated and dispersed in the aqueous phase. Next, this O/W emulsion was stirred with a propeller stirrer to dry the methylene chloride in the microcapsules in water. After centrifuging the obtained O/W emulsion and removing the supernatant, the microcapsules were freeze-dried in small fractions with an aqueous solution containing 100 mg of mannitol.

Next, microcapsules were subcutaneously administered to rats at a dose of 250 mcg as IL-2, and one day later,
After 3 and 7 days, blood was collected from the tail vein and serum II, -2
The concentration was measured by enzyme immunoassay. As a result, serum l w&
The z, -2et degree in the middle was 1.26 ng after 1 day and 0.
321 ng and 0.425 ng after 17 days, confirming that IL-2 was being released in a sustained manner from the microcapsules.

Effects of the Invention The water-insoluble form of the cytokine of the present invention has sufficient cytolytic physiological activity, and when administered in vivo, the insoluble form is gradually solubilized, and the cytolytic physiological activity is maintained for a long period of time. Therefore, the water-insoluble cytokine can be advantageously used as a pharmaceutical preparation, particularly as a sustained-release injection.

Agent Patent Attorney Iwa 1) Hiroshi

Claims (4)

[Claims] (1) Water-insoluble cytokines possessing physiological activity. (2) An injection composition containing a water-insoluble cytokine having physiological activity. (3) A method for producing a water-insoluble cytokine having physiological activity, which comprises reacting the cytokine with a biocompatible protein or a polymeric organic acid having a molecular weight of about 5,000 or more or a salt thereof in an aqueous medium. (4) The production method according to claim 3, wherein a biocompatible protein acts on the cytokine in the coexistence of a water-soluble organic solvent.