JPH11236336A - Chondroitinase composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition having an extremely slight reduction in enzyme activity even by long-term preservation in pharmaceutical preparation, useful for treating hernia of the intervertebral disk, etc., by including chondroitinase and a medicine carrier specifying a reducing impurity. SOLUTION: This composition contains (A) chondroitinase and (B) a medicine carrier having a reducing impurity in an amount of <=0.4 mL calculated as a titer by a titration using 0.01 N ammonium cerium nitrate contained in 1 g of the component B. Preferably the component A is chondroitinase ABC and has >=300 U/mg protein of specific activity and the component B is a saccharose and/or a polyethylene glycol, has <=20 ppm peroxide content and has been treated with activated carbon.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a pharmaceutical composition containing chondroitinase, and an agent for treating a herniated disc comprising the pharmaceutical composition.

[0002]

2. Description of the Related Art Chondroitinase ABC (Chondroitin)
ase ABC) (EC 4.2.2.4) has the effect of degrading glycosaminoglycans into unsaturated oligosaccharides and unsaturated disaccharides,
An enzyme that strongly catalyzes the degradation of chondroitin sulfate A from mammalian cartilage, chondroitin sulfate C from shark cartilage, and chondroitin sulfate B (dermatan sulfate) from mammalian skin, and weakly catalyzes the degradation of hyaluronic acid. is there.

This enzyme is produced by bacteria such as Proteus vulgaris as a research reagent for removing glycosaminoglycans from animal tissues and for identifying glycosaminoglycans in tissues. Enzymes have been commercialized.

On the other hand, for the treatment of herniated disc, which is classified as the etiology of low back pain in humans, a proteolytic enzyme derived from plant papaya, for example, chymopapain or collagenase derived from bacteria is injected into the disc of herniated patients. Discolysis (ID therapy), which dissolves the nucleus, was developed. In Europe and the United States, chymopapain is a drug (trade name,
It is commercially available as chymodiactin).

[0005] However, the ID therapy using the above protease is not only for the hernia of the spine and intervertebral disc, but also for the ID therapy.
It has the disadvantage that it also degrades the protein part of the surrounding structural tissues and easily causes side effects such as nerve palsy and allergy.

In recent years, attempts have been made to treat disc herniation by directly administering chondroitinase ABC or chondroitinase AC to the intervertebral disc, and is expected to be used as a therapeutic agent for herniated disc [US Patent No.
4696816 specification, ClinicalOrthopaedics, 253,301-30
8 (1990)].

[0007] Compositions containing chondroitinase are disclosed in JP-A-4-330280 and JP-A-6-1.
No. 35851, JP-A-6-153947, JP-A-7-67642 and the like. For example, JP-A-4-330280 discloses dextrans, saccharose, lactose, maltose, mannitol, xylitol , Dry and stabilized chondroitinase ABC containing at least one additional component selected from the group consisting of sorbitol and serum albumin. JP-A-6-153947 describes a purified chondroitinase ABC having a very high degree of purification and a high stability, and a pharmaceutical composition containing the same. Further, although WO93 / 00807 does not specifically describe chondroitinase, it is suitable for freezing and drying biomaterials (including enzymes), such as biomaterials, polyethylene glycol and sugar (saccharose). (A.) are disclosed.

[0008]

[0009] Chondroitinase is an unstable substance, and according to the method described in the above-mentioned literature and the like,
When formulated into a form such as a lyophilized product or a liquid preparation using an ordinary pharmaceutical carrier, there is a problem that the enzyme activity gradually decreases during the formulation or due to long-term storage.

[0009] An object of the present invention is to develop a pharmaceutical composition containing chondroitinase with a very small decrease in enzyme activity even during preparation or storage for a long period of time.

[0010]

Means for Solving the Problems The present inventors have conducted various studies on factors that cause a decrease in enzyme activity in a pharmaceutical composition prepared from chondroitinase and a pharmaceutical carrier. The inventors have found that the reducing impurities have a large effect, and have completed the present invention.

Thus, the present invention comprises a chondroitinase and a pharmaceutical carrier, wherein the amount of reducing impurities contained per gram of the pharmaceutical carrier is determined by titration using 0.01N ammonium cerium nitrate. As 0.
4 mL or less of a pharmaceutical composition (hereinafter, referred to as
"The pharmaceutical composition of the present invention").

The present invention also provides an agent for treating a herniated disc comprising the pharmaceutical composition (hereinafter also referred to as “the agent for treating the present invention”).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The pharmaceutical composition and the treating agent of the present invention will be described below in more detail.

Chondroitinase: The chondroitinase used in the pharmaceutical composition of the present invention is not particularly limited as long as it is an enzyme that degrades chondroitin sulfate. Specific examples of chondroitinase include chondroitinase ABC (derived from Proteus vulgaris;
No. 947, T. Yamagata, H. Saito, O. Habuchi,
S. Suzuki, J. Biol. Chem., 243, 1523 (1968), S. Suzu
ki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y. Ka
wai, T. Furuhashi, J. Biol. Chem., 243, 1543 (196
8)), chondroitinase AC (Flavobacterium heparin
um; T. Yamagata, H. Saito, O. Habuchi, S. Suzu
ki, J. Biol. Chem., 243, 1523 (1968)), chondroitinase ACII (from Arthrobacter aurescens; K. Hiyam)
a, S. Okada, J. Biol. Chem., 250, 1824 (1975), K. H.
iyama, S. Okada, J. Biochem. (Tokyo), 80, 1201 (197
6)), chondroitinase ACIII (Flavobacterium sp.
From Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa,
Kiyochika Tokuyasu, Biochemistry, 61, 1023 (1989)), Chondroitinase B (from Flavobacterium heparinum; YM Michela
cci, CP Dietrich, Biochem. Biophys. Res. Commu
n., 56, 973 (1974), YM Michelacci, CPDietrich,
Biochem. J., 151, 121 (1975), Kenichi Maeyama, Akira Tawada,
Akiko Ueno, Keiichi Yoshida, Biochemistry, 57, 1189 (1985)), Chondroitinase C (from Flavobacterium sp.Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa, Kiyochika Tokuyasu, Biochemistry, 61, 1023 ( 1989)), and any of these chondroitinases can be used.

Also, chondroitinase having the following physicochemical properties can be used (Biochemistry, 67,737 (1)
995).

Action: Hydrolyzes N-acetylhexosaminide bonds of glycosaminoglycans.

As a result of acting on chondroitin sulfate, saturated chondroitin sulfate oligosaccharides of mainly 12 to 16 saccharides are produced.

Substrate specificity: At pH 5, it acts on chondroitin sulfate and does not act on hyaluronic acid, keratan sulfate, heparan sulfate and heparin.

It acts on chondroitin sulfate and hyaluronic acid at pH 3.5.

Optimum reaction pH: around pH 5 (substrate: chondroitin sulfate derived from shark cartilage (average molecular weight 4400)
0), buffer: 50 mM citric acid-Na2HP containing 0.15 M NaCl
Isoelectric point: around pH 5 Molecular weight: sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis using a polyacrylamide gel obtained by copolymerizing a chondroitin sulfate chain having an allyl group at the end (Zymography; Anal. Bi
ochem. 225, 333-340 (1995)), about 36 kDa.
It is.

An endo-type enzyme.

It is of human origin.

This enzyme can be obtained, for example, from human gastric cancer peripheral tissues by a conventional method of extracting and purifying the enzyme. Specific examples of the extraction method include, for example, fragmentation with scissors, homogenization, sonication, osmotic shock, extraction by cell crushing and extraction methods such as freeze-thaw method, surfactant extraction, and combinations thereof. And the like, but a method of fragmenting a tissue with scissors is particularly preferable. Specific examples of the purification method include salting out with ammonium sulfate (ammonium sulfate) and sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography,
Ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, zymography and the like, and processing operations such as a combination thereof, Zymography (Anal. Biochem. 225, 333-340 (1995)) is particularly preferred.

Since this enzyme can be adsorbed on Heparin-Sepharose (Pharmacia), it can be purified by chromatography using a carrier containing heparin as a ligand.

Furthermore, the chondroitinase having the above-mentioned physicochemical properties can be obtained by cloning the gene of this enzyme according to a method known per se, introducing the gene into an appropriate host, and expressing it. For example, using the specific chondroitin sulfate-degrading activity of the enzyme as an indicator, isolating a DNA encoding the enzyme from a human DNA library, inserting the DNA into a vector by genetic recombination technology, and introducing into a host cell for expression. By this, this enzyme can be obtained. In addition, cloning can be performed by preparing an antibody specific to this enzyme and using the antibody. Alternatively, the amino acid sequence on the N-terminal side of this enzyme is determined, and cloning can be performed using a DNA or oligonucleotide having a nucleotide sequence deduced from this sequence as a probe. The expressed enzyme can be recovered using the extraction / purification method as described above.

Further, chondroitinase having the following physicochemical properties can also be used (J. Biol. Che.
m., 272, 9123-9130 (1997)).

Action: (1) It decomposes the N-acetylhexosaminide bond of glycosaminoglycan and, when completely decomposed, produces substantially only unsaturated glycosaminoglycan disaccharide.

(2) An exo-type enzyme.

Substrate specificity: chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, chondroitin 6-sulfate hexasaccharide, chondroitin 6
-It acts on both tetrasaccharide sulfate and the chondroitin sulfate chain portion of chondroitin sulfate proteoglycan.

It has no effect on keratan sulfate, heparin and heparan sulfate.

Molecular weight: about 105 kD in SDS-polyacrylamide gel electrophoresis under reducing conditions
a.

Amino acid composition: Cystine is detected by amino acid analysis of the acid hydrolyzate of this chondroitinase. This indicates that the chondroitinase contains cysteine residues and / or cystine residues.

N-terminal amino acid: The N-terminal amino acid is leucine.

Isoelectric point: pH about 8.45 (isoelectric focusing method) Optimum temperature: around 40 ° C. (substrate: chondroitin 6-sulfate, buffer: Tris-HCl buffer, pH :
8.0) Optimum reaction pH: around pH 8 (substrate: chondroitin 6)
-Sulfuric acid, buffer: Tris-HCl buffer, temperature: 37 ° C) This enzyme is known as chondroitin sulfate ABC exolyase or exochondroitinase ABC, and its production method. Are also known (A. H
amai, N. Hashimoto, H. Mochizuki, F. Kato, Y. Maki
guchi, K. Horie and S. Suzuki, J. Biol. Chem., 272,
9123-9130 (1997)).

These various chondroitinases are only examples, and the present invention is not limited to such chondroitinases. The chondroitinase used in the present invention may be one kind of chondroitinase, or may be a combination of a plurality of kinds of chondroitinase. Includes both of these meanings.

In the present invention, it is particularly preferable to use chondroitinase ABC. Further, among the chondroitinase ABCs, it is particularly preferable to use chondroitinase ABC derived from Proteus vulgaris.

The chondroitinase is preferably purified to such an extent that it can be used as a medicament, and is substantially free from substances that are unacceptable as a medicament. That is, chondroitinase is preferably a purified chondroitinase having an enzyme specific activity of 300 U / mg protein or more, having an enzyme specific activity of 300 U / mg protein or more,
A purified chondroitinase substantially free of endotoxin and having a nucleic acid and protease content below the detection limit is more preferred, and a purified chondroitinase ABC having such properties is particularly preferred. In the present specification, one unit (U) of chondroitinase is necessary to release 1 micromol of a reaction product per minute from chondroitin sulfate under conditions of optimum pH and optimum temperature. It is the amount of enzyme. For example, in the case of chondroitinase ABC, this is the amount of enzyme required to release 1 micromol of unsaturated disaccharide from chondroitin sulfate at pH 8.0 and 37 ° C for 1 minute. Enzyme specific activity is 300U / mg
By using chondroitinase, which is a protein or more, without affecting surrounding tissues when administered to a living body as an injectable drug, a target site (for example, a herniated mammal, preferably a human intervertebral disc) can be used. Proteoglycans can be appropriately degraded, and can be a drug with high safety and efficacy.

The most preferred chondroitinase used in the pharmaceutical composition of the present invention is a purified chondroitinase ABC having the following properties.

(I) When the molecular weight is measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (in both reduction and non-reduction) and by gel filtration,
100,000 daltons.

(Ii) The isoelectric point is about pH 8.2 and about pH 8.5.

(Iii) The optimum pH is 8.0 to 8.2 (substrate: chondroitin sulfate C, buffer: Tris-HCl buffer), and the optimum temperature is 37 ° C.

(Iv) The activity is inhibited by Zn ²⁺ , Ni ²⁺ , Fe ³⁺ and Cu ²⁺ .

(V) the N-terminal amino acid is alanine;
The terminal amino acid is proline.

(Vi) SDS-PAGE shows a single band, and also shows a single peak in high performance liquid chromatography (gel filtration and cation exchange).

(Vii) substantially free of endotoxin;
The nucleic acid and protease contents are below the detection limit.

(Viii) It can be crystallized.

(Ix) The specific activity is 300 U / mg or more.

Such a chondroitinase ABC comprises
For example, it can be obtained by the method described in JP-A-6-153947.

Pharmaceutical carrier : As the pharmaceutical carrier in the pharmaceutical composition of the present invention, the amount of reducing impurities contained in 1 g of the pharmaceutical carrier is determined by titration using 0.01N ammonium cerium nitrate as 0. 0.4 mL or less, preferably 0.36 mL or less, and further having a peroxide content of 20 ppm or less, particularly 1 ppm or less.
More preferably, the content is 8.5 ppm or less. The pharmaceutical carrier used in the pharmaceutical composition of the present invention is preferably one that has been treated with activated carbon.

Here, the “titration method using 0.01N ammonium cerium nitrate” is a method in which 2 g of a pharmaceutical carrier is added to 25 g of
Dissolve in warm water of 25 mL, dilute sulfuric acid 25 mL and ferroin (tris (1,10-phenanthroline) iron (II) complex:
0.1 mL of [Fe (C ₁₂ H ₈ N ₂ ) ₃ ] ²⁺ ) is added, and the color is changed from red to blue-green with 0.01 N ammonium cerium nitrate, and titration is performed until the temperature is maintained for 30 seconds. The volume of 0.01N ammonium cerium nitrate required for the titration is determined, and this is converted into a titer per 1 g of the pharmaceutical carrier, whereby the amount of reducing impurities can be determined.

[0051] Pharmaceutical carriers include conventional excipients, binders, lubricants, coloring agents, disintegrants, buffers, isotonic agents, preservatives, soothing agents, and other additives usually used in medicine. Is exemplified.

The pharmaceutical carrier used in the pharmaceutical composition of the present invention preferably has such a purity that it can be used as a medicament, and does not substantially contain a substance that is not allowed to be mixed as a medicament. Specifically as such a pharmaceutical carrier, for example, dextran, saccharose, lactose, maltose,
Xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin, gelatin, creatinine, polyethylene glycol, nonionic surfactants (eg, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, sucrose fatty acid ester, Polyoxyethylene polyoxypropylene glycol) and the like.

The above polyoxyethylene sorbitan fatty acid ester (polysorbate) includes:
Monolaurate, monopalmitate, monooleate, monostearate, trioleate and the like of polyoxyethylene sorbitan (degree of polymerization of ethylene oxide: about 20) can be mentioned.
(Polyoxyethylene sorbitan monooleate (20 E.
O.)), polysorbate 60 (polyoxyethylene sorbitan monostearate (20 EO)), polysorbate 40 (polyoxyethylene sorbitan monopalmitate (20 E.
O.)), polysorbate 20 (polyoxyethylene sorbitan monolaurate (20 EO)), polysorbate 21, 81,
65, 85, etc. (here, 20 EO means that the degree of polymerization of ethylene oxide in the polyoxyethylene portion is about 20). As polyoxyethylene hydrogenated castor oil, commercially available HCO-10, HCO
-50, HCO-60 (Nikko Chemicals) and the like. Examples of the sucrose fatty acid ester include commercially available DK ester F-160 (Daiichi Kogyo Seiyaku Co., Ltd.), ryotoshijugar ester (Mitsubishi Chemical Foods), and the like. As polyoxyethylene polyoxypropylene glycol (poloxamer), commercially available Pluronic F-68 (Asahi Denka Kogyo Co., Ltd.)
And the like.

The buffer is not particularly limited as long as it is physiologically acceptable. For example, hydrochloric acid, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, phosphoric acid, dihydrogen phosphate Potassium, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, aminoacetic acid, sodium benzoate, citric acid, sodium citrate, acetic acid, sodium acetate, tartaric acid, sodium tartrate, lactic acid, sodium lactate, ethanol Buffers containing one or more of amine, arginine, ethylenediamine and the like are exemplified.

In the pharmaceutical composition of the present invention, these pharmaceutical carriers can be used in appropriate combination. Among them, polyethylene glycol and / or saccharose,
Or polyoxyethylene sorbitan fatty acid ester,
In particular, polyoxyethylene sorbitan monooleate (20
EO) is preferred.

In particular, polyethylene glycol, when providing the pharmaceutical composition of the present invention in a lyophilized form, can suppress the decrease in the enzyme activity of chondroitinase after lyophilization and keep the water content in the lyophilized product low. When the lyophilized product is redissolved, it is clear and free of foreign substances, the cake shape after lyophilization is good, and the enzyme activity of chondroitinase due to long-term storage of the lyophilized product is relatively low. It is suitable because it has the effect of being small.

Further, when a mixture of polyethylene glycol and saccharose is used to provide the pharmaceutical composition of the present invention in a freeze-dried form, the decrease in the enzyme activity of chondroitinase after freeze-drying is suppressed, and the water content in the freeze-dried product is reduced. It can be kept low, when the lyophilized product is redissolved, it is clear and free of foreign matter, the cake shape after lyophilization is good,
In addition, it is particularly preferable since it has an effect that the enzyme activity of chondroitinase is extremely reduced due to long-term storage of the lyophilized product.

The polyethylene glycol which can be used in the above case is such that the amount of reducing impurities contained per gram of the polyethylene glycol is 0.4 mL or less by titration using 0.01 N ammonium cerium nitrate. It is not particularly limited as long as it is preferably 0.36 mL or less, but it is more preferable to use one having a peroxide content of 20 ppm or less, particularly 18.5 ppm or less.

The polyethylene glycol preferably has an average molecular weight of 200 to 25,000, and more preferably is a solid at room temperature, for example, having an average molecular weight of 200.
More preferably, the number is from 2000 to 4,000.
000 is particularly preferable, and 3000 to 4000
Are highly preferred. Average molecular weight 3000-
Examples of polyethylene glycol having a molecular weight of 4000 include those having an average molecular weight of 3250, 3350, and 4000.

Saccharose is also 1 g of the saccharose.
The amount of the reducing impurity contained in the solution is 0.4 mL or less, preferably 0.36 mL or less, as a titer determined by titration using 0.01 N ammonium cerium nitrate. Further, it is more preferable to use a saccharose having a peroxide content of 20 ppm or less, particularly 18.5 ppm or less.

It has been confirmed by the present inventors that commercially available saccharose generally has a high endotoxin content. Therefore, the concentration of endotoxin in a 10% (w / w) saccharose solution by activated carbon treatment is 0.03E.
The treatment is preferably performed so as to be U / mL or less, desirably 0.01 EU / mL or less, and more desirably 0.0006 EU / mL or less.

The endotoxin concentration in saccharose is
Although it can be measured using a method for measuring endotoxin known per se, a Limulus test using a horseshoe crab, amebosite and lysate component is preferred. As the Limulus reagent used in the Limulus test, it is preferable to use an endotoxin-specific Limulus reagent. As the Limulus reagent, for example, the following commercially available Limulus reagents can be used; Toxicolor System LS-6
LS-20, LS-200, Endspecy ES-6
Endspecy ES-200 (sold by Seikagaku Corporation), Limulus ES-II Test Wako, Limulus E
S-II Single Test Wako, Limulus ES-III Test Wako, Limulus ES-J Test Wako (all sold by Wako Pure Chemical Industries, Ltd.).

When polyethylene glycol and / or saccharose is used as a pharmaceutical carrier, the weight ratio of polyethylene glycol / saccharose is generally 0/1 to 10 /
It is preferable to mix them so as to be about 1. In addition, when a mixture of polyethylene glycol and saccharose is used as a pharmaceutical carrier, the weight ratio of polyethylene glycol / sucrose is about 1/10 to 10/1. It is preferable to mix them so as to be about 1.5 / 1 to 3/1, and it is particularly preferable to mix them so as to be about 2/1.

In this case, the amount of reducing impurities in the mixture of polyethylene glycol and saccharose was 0.0
It is important that the titration by a titration method using 1N ammonium cerium nitrate be 0.4 mL or less, preferably 0.36 mL or less, per 1 g of the mixture. Has a peroxide content of 20 ppm or less,
In particular, it is more preferable to be 18.5 ppm or less.

When a polyoxyethylene sorbitan fatty acid ester is used as a pharmaceutical carrier, the amount of reducing impurities contained in 1 g of the ester is 0.4 mL or less as a titer by a titration method using 0.01 N ammonium cerium nitrate. Preferably, a polyoxyethylene sorbitan fatty acid ester having a concentration of 0.36 mL or less is used. Further, the peroxide content in the ester is 20 ppm
Hereinafter, it is more preferable to use those having a concentration of 18.5 ppm or less.

The peroxide content in the pharmaceutical carrier is determined by measuring the content of the pharmaceutical carrier 1
g accurately, add distilled water to make 10 mL,
That is, a 10% (w / v) aqueous solution is used,
0.25 mL of 20% (v / v) sulfuric acid and 1 M TiO
₄ Add 0.15 mL (manufactured by BDH), measure the ultraviolet absorption at 408 nm, and then measure the H ₂ O ₂ concentration based on a calibration curve created using a known concentration of H ₂ O _2. can do.

The amount of reducing impurities and / or peroxide content in the pharmaceutical carrier desired to be used in the pharmaceutical composition of the present invention can be reduced, for example, by treating the pharmaceutical carrier with activated carbon in a conventional manner. it can. The peroxide content can also be reduced by heat treating the pharmaceutical carrier.

The mixing ratio of chondroitinase to the pharmaceutical carrier in the pharmaceutical composition of the present invention is not particularly limited.
Those skilled in the art can appropriately determine the dose according to the dosage and the form of the pharmaceutical composition of the present invention. For example, when the composition of the present invention is provided (preserved) in a lyophilized state, the content of chondroitinase in the composition of the present invention is preferably such that the shape of the lyophilized cake can be maintained.

The preparation of the pharmaceutical composition of the present invention can be carried out using chondroitinase and the above-mentioned pharmaceutical carrier by a method known per se. The pharmaceutical composition of the present invention may be in any of a solution state, a frozen state, and a dried state.

When the pharmaceutical composition of the present invention is provided in the form of a solution, for example, a pH-adjusted buffer is prepared, and the above-mentioned chondroitinase and a pharmaceutical carrier are added thereto.
5 U / mL or more chondroitinase, preferably 10 to 40
It can be produced by making a solution containing 0 U / mL and sterilizing by filtration as needed.

When the pharmaceutical composition of the present invention is provided in a frozen state, it can be produced, for example, by freezing the pharmaceutical composition of the present invention in the above-mentioned solution state at, for example, -80 to -18 ° C.

When the pharmaceutical composition of the present invention is provided in a dry state, it can be produced, for example, by subjecting the pharmaceutical composition of the present invention in a solution state to a drying treatment under non-heating conditions such as a freeze-drying method. .

Among these, the preferred state of the pharmaceutical composition of the present invention is a dried state, more preferably a lyophilized state, that is, a lyophilized substance.

The pharmaceutical composition of the present invention is in a solution state (when the pharmaceutical composition of the present invention is in a frozen state, it is in a solution state before freezing and after thawing, and when the pharmaceutical composition of the present invention is a lyophilized composition, it is in a frozen state. It is usually desirable to adjust the pH to 5 to 9, preferably 6 to 8 before drying and after re-dissolving by adding a solvent. For this purpose, the composition of the present invention is generally blended with a buffer capable of maintaining the pH range. The buffer is not particularly limited as long as it is physiologically acceptable. For example, hydrochloric acid, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, phosphoric acid,
Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, aminoacetic acid, sodium benzoate, citric acid, sodium citrate, acetic acid, sodium acetate, tartaric acid, sodium tartrate, lactic acid, Examples include sodium lactate, ethanolamine, arginine, ethylenediamine or mixtures thereof. Particularly, a phosphate buffer (agent) is preferable. The pH of the pharmaceutical composition of the present invention in a solution state is increased by these buffers.
The area can be adjusted and maintained between 5 and 9, preferably between 6 and 8. If the pH is lower than 5 or higher than 9, chondroitinase may be inactivated or insolubles may be generated in a solution state. The concentration of the buffer in the pharmaceutical composition of the present invention is 1 mM or more, preferably 10 to 50.
It can be mM. The pharmaceutical composition of the present invention may contain components necessary for isotonicity (salts such as sodium chloride, saccharides and the like), a preservative, a soothing agent, and the like, in addition to the buffer.

The pharmaceutical composition of the present invention can be used as a final dosage form for administration as a pharmaceutical as it is, or can be used as a raw material for other final dosage forms of pharmaceuticals, for example, liquids, freeze-dried agents and the like. .

The pharmaceutical composition of the present invention is mainly used as an injectable preparation containing chondroitinase as an active ingredient. When the pharmaceutical composition of the present invention is provided as an injectable preparation in the form of a solution, the pharmaceutical composition of the present invention in the form of a solution prepared by the above method is filled and sealed in an appropriate container such as an ampoule, a vial, or a syringe for injection. Then, it can be distributed or stored as it is, and provided for administration as an injection.

When the pharmaceutical composition of the present invention is provided as a frozen preparation for injection, the frozen pharmaceutical composition of the present invention produced by the above-mentioned method is placed in a suitable container such as an ampoule, a vial, or a syringe for injection. Can be kept in a sealed state, distributed or stored, melted before administration, and provided for administration as an injection.

The distribution and storage are preferably performed at a temperature of -80 to -25 ° C.

When the pharmaceutical composition of the present invention is provided as a preparation for injection in a dry state, the pharmaceutical composition of the present invention in a dry state produced by the above method in an appropriate container such as an ampoule, a vial, or a syringe for injection is provided. Keep things in a sealed state,
It can be distributed or stored, dissolved in distilled water for injection, physiological saline, aqueous glucose solution or aqueous sorbitol solution before administration, and used as an injection for administration. The pharmaceutical composition of the present invention in a dry state may be provided together with a solvent for dissolution.

Of the above-mentioned preparations for injection, those in a dry state are preferable, and those in a lyophilized state are more preferable. That is, it is particularly preferable that the pharmaceutical composition of the present invention is in the form of a freeze-dried composition for injection.

When filling or sealing the composition of the present invention in a suitable container such as an ampoule, a vial, or a syringe for injection,
In order to prevent the chemical reaction of the pharmaceutical composition of the present invention, particularly oxidation,
It is preferable to fill or seal together with an inert gas such as a nitrogen gas or a rare gas.

The material of containers such as ampules, vials, syringes for injection, etc., which can be filled and sealed with the pharmaceutical composition of the present invention, should not affect the pharmaceutical composition of the present invention and should be pharmaceutically acceptable. It is not particularly limited as long as it is glass.

The pharmaceutical composition of the present invention provided as a freeze-dried composition for injection is characterized in that the enzyme activity of chondroitinase is significantly reduced before and after freeze-drying. Specifically, the pharmaceutical composition of the present invention can maintain the enzyme activity of chondroitinase after freeze-drying at 90% or more of the enzyme activity of chondroitinase before freeze-drying. As a method for measuring the enzyme activity of chondroitinase, for example, using a chondroitin sulfate as a substrate, an unsaturated disaccharide having a pronounced ultraviolet absorption generated when chondroitinase is allowed to act at 37 ° C. is determined spectrophotometrically. (E.g. 2
(Absorbance at 32 nm).

In the case of the pharmaceutical composition of the present invention provided in the form of a freeze-dried composition for injection, it is generally considered that the lower the water content, the higher the stability of the freeze-dried preparation. 3% (w /
Since the setting of w) or less is usually performed in pharmacology, the water content of the freeze-dried composition for injection of the present invention is also preferably 3% (w / w) or less. . The moisture content is determined, for example, by heating the sample under the following conditions, measuring the weight before and after the heating with a microbalance, and determining the reduced weight as the water content (heating condition: the sample is heated from 25 ° C. to 105 ° C., 2.5 ° C. / Min, hold at 105 ° C for 20 minutes when the temperature reaches 105 ° C) Dry loss method (TG method), or stir the sample in methanol for 3 minutes to extract water, and coulometrically titrate the extracted water And a Karl Fischer method for converting the amount of water from the amount of electricity (coulomb) required.

The pharmaceutical composition of the present invention provided in the form of a freeze-dried composition for injection is preferably clear and free of foreign substances when the freeze-dried composition is dissolved in physiological saline. . The clearness and the absence of foreign matter can be easily determined by visual observation.

The pharmaceutical composition of the present invention, which is provided in the form of a freeze-dried composition for injection, has the advantage that the enzyme activity of chondroitinase is extremely small even when stored at room temperature for a long time. For example, when the pharmaceutical composition of the present invention is stored at 40 ° C. for 30 days in a glass container filled with nitrogen, it is possible to retain 90% or more of the enzyme activity of chondroitinase at the start of storage.

The pharmaceutical composition of the present invention can be used, for example, for treating herniated discs, thereby providing the therapeutic agent of the present invention. The therapeutic agent of the present invention can be injected into a disc of a mammal with a hernia, preferably a human, as an injection, and can be used in a disc lysis therapy for dissolving and treating a nucleus pulposus. The dose is to be set individually according to symptoms, age, etc., and is not particularly limited.When using chondroitinase ABC as chondroitinase, it is usually possible to inject about 0.1 to 100 U once. it can.

[0088]

EXAMPLES Next, the present invention will be described in more detail with reference to examples relating to the pharmaceutical composition of the present invention containing chondroitinase ABC, but these examples are merely illustrative of the present invention, and The scope is not limited to these.

Chondroitinase ABC used in Examples
Is manufactured by the method described in JP-A-6-153947.

This chondroitinase ABC was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (S
DS-PAGE) showed a single band, and high performance liquid chromatography (gel filtration and cation exchange) showed a single peak. From these results, it was confirmed that the chondroitinase ABC was completely purified.

The endotoxin content per 100 U of this chondroitinase ABC was measured using a Toxicolor system (manufactured by Seikagaku Corporation).
(0.00986EU; EU means endotoxin unit, 1pg =
(0.0029 EU), which is very small and substantially free of endotoxin.

The amount of nucleic acid (DNA) in the chondroitinase ABC was measured by a threshold method (DNA measuring apparatus: threshold (manufactured by Molecular Devices)), but no DNA was detected.

The protease in the chondroitinase ABC was converted to FITC (fluorescein isothiocyanate).
As a result of measurement using casein as a substrate, no protease was detected.

The enzyme activity of chondroitinase ABC was measured by the following method.

Using 1.2 mg of chondroitin sulfate C as a substrate, an enzyme sample (chondroitinase ABC) was added to 50 mM Tris-HCl buffer (pH 8) containing 50 mM sodium acetate and 10 μg of casein, and reacted at 37 ° C. for 10 minutes. PH 1.8
The reaction was stopped by adding 0.05 M hydrochloric acid, and the absorbance at ultraviolet (232 nm) was measured. On the other hand, a heat-denatured enzyme sample was held in a substrate solution having the above composition as a control, and the same treatment was performed to measure the absorbance at 232 nm. The amount of unsaturated disaccharide produced by the action of chondroitinase ABC was calculated from the increase in absorption relative to the control. In addition, 2-acetamide
2-unit deoxy-3-0- (β-D-gluco-4-enpyranosyluronic acid) -6-0-sulfo-D-galactose with a molar absorption coefficient of 5.5 and 1 unit (U) of enzyme The amount was defined as the amount of enzyme that catalyzes a reaction that releases 1 micromole of unsaturated disaccharide per minute under the above reaction conditions.

As a result, this chondroitinase ABC
Has a specific activity of 300 U / mg protein or more.

Example 1 Preparation of a Pharmaceutical Composition of the Invention Consisting of Chondroitinase ABC and Polysorbate 80 Polysorbate 80 was obtained from a number of sources, and the amount of reducing impurities in 801 g of polysorbate and peroxidation were determined by the method described above. The substance concentration was measured.

Thereafter, sodium chloride (0.75%) and the above-mentioned highly purified chondroitinase ABC (specific activity 350 U / mg; final concentration 25 U / mL) were added to a 20 mM phosphate buffer (pH 7).
And polysorbate 80 (0.15% (w / w)) were dissolved to prepare a composition (solution), and a membrane filter (pore size 0.22 μm; Myrex GV, MILLIPOR) was prepared.
E)), sterilized by filtration, filled into sterilized glass ampules by 2 mL, and sealed.

The sealed glass ampule was stored at 25 ° C. for one month and evaluated by the following method.

Evaluation method: (1) Properties: White and black paper were used as backgrounds, and observed with the naked eye at a position of about 1,000 lux under a white light source.

(2) Measurement of enzyme activity of chondroitinase ABC: Measurement was performed using a solution before storage or a solution after storage as an “enzyme sample” in the above-described method for measuring enzyme activity.

The ratio of the enzyme activity of the solution after storage (Post solution) to the enzyme activity of the solution before storage (Pre solution) was calculated by the following equation, and the change in the enzyme activity after storage at 25 ° C. for one month was compared. .

[Enzyme activity of Post solution / enzyme activity of Pre solution] × 100 (%) The results were regarded as “stability” and the mass of reducing impurities and the peroxide concentration in 1 g of a pharmaceutical carrier (polysorbate 80) were determined. The results are shown in Table 1 together with the results. In Table 1, "N /
"T" indicates that no measurement was performed.

[0104]

[Table 1]

As a result, the pharmaceutical carrier (polysorbate 80)
The amount of reducing impurities in 1 g was determined to be 0.01 by titration using 0.01 N ammonium cerium nitrate.
Pharmaceutical compositions of 4 mL or less (A to G in Table 1) were liquid and had an activity of 60% or more even after storage at 25 ° C for 1 month.

On the other hand, a pharmaceutical carrier (polysorbate 8)
0) A pharmaceutical composition (H to L in Table 1) in which the amount of reducing impurities in 1 g is 0.4 mL or more by titration using 0.01 N ammonium cerium nitrate,
The activity of the liquid after storage at 25 ° C. for one month was 40% or less.

After storage at 25 ° C. for one month, all the samples were colorless and transparent, and generation of insoluble foreign matter was not recognized.

Example 2 Effect of Reduction of Reducing Impurities and Peroxide Concentration by Activated Carbon Treatment Using Polysorbate 80 of Product B and Polysorbate 80 of Product L in Table 1 above, Effect of Activated Carbon Treatment of Polysorbate 80 Was examined.

After dissolving 10 g of each of polysorbate 80 of product B and product L in water to make 100 mL,
After adding 10 g of activated carbon heated for 5 hours, the mixture was stirred at room temperature for 30 minutes, and then the activated carbon was removed by filtration to obtain a polysorbate 80-treated activated carbon.

With respect to the polysorbate 80 thus obtained, the content of reducing impurities and the content of peroxide before and after the treatment with activated carbon were measured (all measured by the same method as described above), and the composition having the composition described in Example 1 was used. The stability of chondroitinase ABC after preparation and storage at 25 ° C. for one month was examined. Table 2 shows the results.

[0111]

[Table 2]

The results show that the activated carbon treatment reduces the amount of reducing impurities and the peroxide content. In particular, the reduction of the amount of reducing impurities due to the activated carbon treatment was remarkable.

The pharmaceutical composition prepared using polysorbate 80 treated with activated carbon has a more stable chondroitinase ABC activity than the pharmaceutical composition prepared using polysorbate 80 not treated with activated carbon. Indicated.

Example 3 Preparation of Pharmaceutical Composition of the Present Invention (Lyophilized Product) Consisting of Chondroitinase ABC and Polyethylene Glycol (1) Polyethylene Glycol used in this Example Polyethylene Glycol 4000 (PEG 4000, average molecular weight 2600 to 3800) ; First grade, manufactured by Wako Pure Chemical Industries, Ltd., Lot. No. CAE0369) is dissolved in endotoxin-free distilled water (water for injection), and the aqueous solution is treated with activated carbon to reduce the amount of reducing impurities and the peroxide content. Was measured by the method described above, and the amount of reducing impurities in the 4% (w / v) polyethylene glycol solution was 0 mL by titration, and the peroxide content in the 6.66% (w / v) polyethylene glycol solution was determined. Was below the detection limit (0.0017% (w / v) or less).

(2) Test method (Stability test) Chondroitinase ABC (final concentration 20 U / mL) and polyethylene glycol 4000 (final concentration 1% (w / w)) were added to 10 mM phosphate buffer (pH 7). Dissolve,
1 mL was dispensed per vial (20 U / vial as chondroitinase ABC) and freeze-dried. Freeze-drying is performed by freezing from room temperature to -45 ° C,
(Torr) for 12 hours and then heated to 25 ° C (12 hours)
Then, it was secondarily dried at 25 ° C. for 10 hours. After drying, the pressure was restored with nitrogen gas and stoppered.

Next, according to the following method, the “lyophilized product” was subjected to “enzyme activity of the solution before freeze-drying” at 40 ° C. for 3 hours.
The ratio of the "enzyme activity of the solution redissolved after storage for 0 days" was determined. After the freeze-drying, the enzyme activity of chondroitinase (hereinafter, "enzyme activity of chondroitinase" may be simply referred to as "enzyme activity"), cake shape, resolubility and water content were evaluated.

The enzyme activity was measured according to the method for measuring the enzyme activity described above, using a solution before freeze-drying or a solution redissolved in physiological saline after freeze-drying as an enzyme sample.

The ratio of the enzyme activity of the solution (Post-FD solution) redissolved after freeze-drying to the enzyme activity of the solution before freeze-drying (Pre-FD solution) was calculated by the following equation, and the ratio was calculated during freeze-drying and storage thereafter. The changes in enzyme activity at the time were compared.

[Enzyme activity of Post-FD / enzyme activity of Pre-FD] × 100 (%) The cake shape was a tablet-like cake having a good dry cake shape after freeze-drying, as observed with the naked eye. The cake which was not a good tablet-like cake after freeze-drying was regarded as "poor".

The resolubility was determined by freeze-drying and adding
Judgment was made by observing the solubility when redissolved in mL and the properties of the solution after dissolution. Specifically, after confirming that the solution was dissolved within 1 minute after the addition of physiological saline, the solution after dissolution was visually observed with the naked eye and found to be insoluble foreign matter with “+”, and clear Yes, no foreign matter was recognized as "-". The water content was measured by the Karl Fischer method described above.

(3) Results As a result of a stability test (preserved at 40 ° C. for 30 days) of a freeze-dried product comprising the combination of chondroitinase ABC and polyethylene glycol 4000, the freeze-drying comprising the combination of chondroitinase ABC and polyethylene glycol 4000 was carried out. Even when the dried composition is stored at 40 ° C. for 30 days, the enzyme activity is little reduced (the ratio of the enzyme activity of Post-FD to the enzyme activity of Pre-FD solution is 76.1%), and it can be maintained extremely stably. Indicated.

The moisture content of the freeze-dried product was 3% (w / w)
The cake shape was "good", and the resolubility was "-".

Example 4 Chondroitinase ABC,
Preparation of the pharmaceutical composition of the present invention (freeze-dried product) comprising polyethylene glycol and saccharose (1) Polyethylene glycol used in this example As in Example 3, polyethylene glycol 4000 treated with activated carbon was used.

(2) Saccharose saccharose (purified sucrose; Dainippon Meiji Seika Co., Ltd.)
Lot. No. 910403) was dissolved in endotoxin-free distilled water (water for injection) so as to have a concentration of 10% (w / w), and the endotoxin concentration in the aqueous solution was measured using a Toxicolor system (manufactured by Seikagaku Corporation). As a result, 6.66
EU / mL (2296.49 pg / mL). This aqueous solution was treated with activated carbon, and the endotoxin concentration was measured in the same manner. As a result, it was 0.001 EU / mL (0.43 pg / mL). The peroxide content and the reducing impurity content in the activated carbon-treated saccharose were measured by the above-mentioned method. As a result, the amount of the reducing impurity in the 4% (w / v) sucrose solution was 0 mL by titration. Yes, no peroxide was detected in the 6.66% (w / v) sucrose solution.

(3) Test method (3-1) Preliminary test As a preliminary test, first, a sample containing only a pharmaceutical carrier without the addition of chondroitinase ABC was prepared, and the shape of the freeze-dried product was examined. Specifically, a 10 mM phosphate buffer (pH
In 7), saccharose and polyethylene glycol 4000 were mixed and dissolved as the pharmaceutical carriers at the ratios shown in Table 3, and freeze-dried samples were used to evaluate the cake shape after freeze-drying.

[0126]

[Table 3]

For freeze-drying, after cooling and freezing from room temperature to −45 ° C., primary drying is performed under reduced pressure (60 mTorr) for 12 hours, then temperature is raised to 25 ° C. (12 hours), and secondary drying is performed at 25 ° C. for 10 hours. It was performed by drying. After freeze-drying, the pressure was restored with nitrogen gas, and stoppering was performed.

As a result, when the mixing ratio of saccharose to polyethylene glycol 4000 was 1/2, no melting back (melt back), disintegration or shrinkage occurred.

(3-2) Main test (stability test) Chondroitinase ABC was added to 10 mM phosphate buffer (pH 7).
(Final concentration 20 U / mL), saccharose (final concentration 1% (w /
w)) and polyethylene glycol 4000 (final concentration 2)
% (W / w)), dispensed at 0.5 mL per vial (10 U / vial as chondroitinase ABC), and lyophilized. For freeze-drying, cool and freeze from room temperature to -45 ° C, first dry under reduced pressure (60 mTorr) for 12 hours, then raise the temperature to 25 ° C (12 hours), and second dry at 25 ° C for 10 hours. Was performed. After drying, the pressure was restored with nitrogen gas and stoppered.

The subsequent tests were performed and evaluated in the same manner as the “stability test” described in Example 3.

(4) Results As a result of a stability test (preserved at 40 ° C. for 30 days) of a freeze-dried product comprising a combination of chondroitinase ABC, saccharose and polyethylene glycol 4000, chondroitinase ABC, saccharose and polyethylene glycol 4000 were tested. The freeze-dried composition comprising the combination showed almost no decrease in enzyme activity even after storage at 40 ° C. for 30 days (90.9% of the enzyme activity of Post-FD to the enzyme activity of Pre-FD solution). It was shown to be stably retained.

The moisture content of the freeze-dried product was 1.5% (w / w)
The cake shape was "good", and the resolubility was "-".

[0133]

As described above, the pharmaceutical composition of the present invention comprises chondroitinase, preferably chondroitinase having high specific activity and high purity, and 0.01N ammonium cerium nitrate having a reducing impurity concentration of 0.01N. And a pharmaceutical carrier having a titer of 0.4 mL or less per g and desirably a peroxide concentration of 20 ppm or less.

According to the present invention, by selecting such a composition, it is possible to provide a pharmaceutical composition in which the enzyme activity of chondroitinase after storage for a long period of time is extremely low. Furthermore, by using the pharmaceutical composition of the present invention, it is possible to provide a safe and effective drug that can be stored for a long time, is easy to handle, and is particularly effective in treating a herniated disc.

Claims (12)

[Claims] 1. The method according to claim 1, which comprises a chondroitinase and a pharmaceutical carrier, wherein the amount of reducing impurities contained in 1 g of the pharmaceutical carrier is 0.4 mL as a titer by a titration method using 0.01 N ammonium cerium nitrate. A pharmaceutical composition comprising: 2. The pharmaceutical composition according to claim 1, wherein the peroxide content of the pharmaceutical carrier is 20 ppm or less. 3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical carrier has been treated with activated carbon. 4. The method according to claim 1, wherein the pharmaceutical carrier is saccharose and / or polyethylene glycol.
4. The pharmaceutical composition according to any one of 3. 5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical carrier is polyethylene glycol. 6. The pharmaceutical carrier according to claim 4, wherein the pharmaceutical carrier is a mixture of saccharose and polyethylene glycol.
The pharmaceutical composition according to any one of the preceding claims. 7. The pharmaceutical composition according to claim 4, wherein the mixing ratio of polyethylene glycol to sucrose is 0/1 to 10/1 by weight ratio of polyethylene glycol / sucrose. 8. The pharmaceutical composition according to claim 1, which is in the form of a lyophilized product. 9. The pharmaceutical carrier according to claim 1, wherein the pharmaceutical carrier is a polyoxyethylene sorbitan fatty acid ester.
The pharmaceutical composition according to any one of the above. 10. The pharmaceutical composition according to claim 1, wherein the chondroitinase is chondroitinase ABC. 11. The pharmaceutical composition according to claim 10, wherein the specific activity of chondroitinase ABC is 300 U / mg protein or more. 12. An agent for treating a herniated disc, comprising the pharmaceutical composition according to any one of claims 1 to 11.