JPH11193245A - Chondroitinase abc composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition containing chondroitinase ABC and a contrast medium, capable of reducing the content of the chondroitinase ABC, holding the nucleus pulposus liquefaction effect of an animal having a herniated intervertebral disk and useful for treating various kinds of the herniated intervertebral disks and for confirming the effects of the treatments. SOLUTION: This chondroitinase ABC composition comprises chondroitinase ABC and a contrast medium. The concentration of the chondroitinase ABC in the composition is about 250 units/ml, and the chondroitinase ABC composition is useful as an agent for treating the hernia of the intervertebral disk. The concentration of the chondroitinase ABC is preferably 250±100 units/ml.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a composition containing chondroitinase ABC and a contrast agent, and a therapeutic agent for a herniated disc comprising the composition.

[0002]

2. Description of the Related Art Chondroitinase ABC (Chondroitin)
ase ABC) [EC 4.2.2.4] acts on chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate B (dermatan sulfate), chondroitin and hyaluronic acid, decomposing them into unsaturated oligosaccharides and unsaturated disaccharides. It is an enzyme that does.

[0003] In recent years, attempts have been made to treat a herniated disc by administering chondroitinase ABC or the like to the disc to melt the nucleus pulposus and to be expected to be used as a therapeutic drug for the herniated disc [US Patent No. 4696816, Clinical Orthopaedics, 253, 301-308 (1990)].

Attempts have also been made to mix chondroitinase ABC with a contrast agent and administer the mixture to the disc.
(Spine, Vol. 21, No. 21, pp. 2405-2411 (1996)). Spine, Vol. 21, No. 21, pp. 2405-2411 (1996) describes a technique closest to the present invention, and describes a composition containing chondroitinase ABC and a contrast agent. There is. By performing X-ray imaging using such a composition, it can be confirmed that chondroitinase ABC has been injected into a desired site in a desired amount, and the nucleus pulposus melting effect can also be confirmed.

[0005] This composition comprises chondroitinase ABC.
Concentration of 4 units / 10μL (400 units / ml)
It is described that it has an effect of melting the nucleus pulposus of a normal rabbit disc. However, a composition comprising chondroitinase ABC and a contrast agent, wherein the concentration of chondroitinase ABC is about 250 units / ml
And there is no mention or suggestion that the composition has the effect of melting the nucleus pulposus of an animal with herniated disc.

[0006]

SUMMARY OF THE INVENTION Chondroitinase A
Since BC is a substance artificially administered to the body,
The chondroitinase ABC content in the composition containing chondroitinase ABC should be as small as possible, but the effect of reliably melting the nucleus pulposus of an animal with herniated disc and treating herniated disc is maintained. There must be.

That is, an object of the present invention is to provide a composition containing chondroitinase ABC and a contrast agent, which reduces the chondroitinase ABC content, maintains the nucleus pulposus melting effect of an animal with herniated disc, and has various effects. It is an object of the present invention to provide a composition useful for treating a type of herniated disc and confirming the effect of the treatment.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on a composition containing chondroitinase ABC and a contrast agent. As a result, the known chondroitinase ABC composition (chondroitinase ABC) was used. Concentration 40
0 units / ml) compared to chondroitinase ABC
The composition having the content reduced by about 1.6 times has a nucleus pulposus melting effect in an animal with herniated disc and is useful for treating various types of herniated disc and confirming the therapeutic effect. We have found and completed the present invention.

That is, the present invention relates to chondroitinase A
A composition comprising BC and a contrast agent, wherein the concentration of chondroitinase ABC in the composition is about 250 units /
ml of chondroitinase AB
C composition (hereinafter, referred to as “the composition of the present invention”).

[0010] The present invention also provides a therapeutic agent for herniated disc comprising the composition (hereinafter, also referred to as "the therapeutic agent of the present invention").

[0011]

Embodiments of the present invention will be described below.

<1> Composition of the present invention (1) Chondroitinase ABC The origin of chondroitinase ABC used in the composition of the present invention is not limited as long as it has an action as chondroitinase ABC, but it is not limited to Proteus bulgari. Chondroitinase ABC from Proteus vulgaris
Is preferred.

[0013] Chondroitinase ABC from Proteus vulgaris is described in JP-A-6-153947, T. Yamagata, H. Saito, O. Habuchi, S. Suzuk.
i, J. Biol. Chem., 243, 1523 (1968), S. Suzuki, H. S.
aito, T. Yamagata, K. Anno, N. Seno, Y. Kawai, T.
Furuhashi, J. Biol. Chem., 243, 1543 (1968), J. Bio
l. Chem., 272, 9123-9130 (1997).
The enzyme described in J. Biol. Chem., 272, 9123-9130 (1997) is known as chondroitin sulfate ABC exolyase or exochondroitinase ABC. , Its production method and the like are also described in the same document.

As the chondroitinase ABC, those extracted and purified from the cells of Proteus vulgaris may be used, and cDNA encoding chondroitinase ABC may be used.
And the like, and may be produced by a genetic engineering technique.

[0015] The chondroitinase ABC is preferably an enzyme which is purified to such an extent that it can be used as a medicament and contains substantially no substances which are not allowed to be contaminated as medicament.
That is, chondroitinase ABC is 300 units /
It is preferably a purified chondroitinase ABC having an enzymatic activity of at least mg protein, having an enzymatic activity of at least 300 units / mg protein, containing substantially no endotoxin, and having a nucleic acid and protease content below the detection limit. More preferred is the purified chondroitinase ABC. It should be noted that, in the present specification, “1” of chondroitinase ABC is used.
The “unit” means an amount of an enzyme that releases 1 micromol of unsaturated disaccharide from chondroitin sulfate C at pH 8.0 and 37 ° C. for 1 minute. By using chondroitinase ABC having an enzymatic activity of 300 units / mg protein or more, it can be administered to a target site (eg, an animal with herniated disc) without affecting surrounding tissues when administered to a living body as an injectable drug. The proteoglycan of the intervertebral disc can be appropriately degraded, and can be a drug with high safety and efficacy. Such chondroitinase ABC
Can be obtained, for example, by the method described in JP-A-6-153947.

(2) Contrast agent The contrast agent used in the composition of the present invention is chondroitinase A
BC can be dissolved and chondroitinase AB
There is no particular limitation as long as it does not affect the enzyme activity of C. More specifically, it is preferably a positive contrast agent, more preferably an aqueous iodine contrast agent, particularly preferably an aqueous nonionic iodine contrast agent, and an aqueous non-iodine contrast agent containing iohexol. Very particular preference is given to ionic iodine contrast agents.

When an aqueous iodine contrast agent is used, the concentration of iodine in the contrast agent is not particularly limited as long as X-ray imaging is possible, but is preferably about 140 mg / ml to 350 mg / ml. When a contrast agent having a relatively high iodine concentration is used, X
This is preferable because the line contrast image becomes clear and the administration volume of the composition of the present invention can be reduced. Specifically, the iodine concentration in the contrast agent is more preferably about 300 mg / ml to 350 mg / ml, particularly preferably about 350 mg / ml.

For example, when a contrast agent containing iohexol is used as an aqueous nonionic iodine contrast agent, the concentration of iohexol in the contrast agent is 300 mg / ml to 755 m
It is preferably about g / ml, more preferably about 647 mg / ml to 755 mg / ml, and particularly preferably about 755 mg / ml.

(3) Concentration of chondroitinase ABC in the composition The composition of the present invention comprises chondroitinase ABC in the composition.
Is about 250 units / ml.

The most preferable concentration of chondroitinase ABC in the composition of the present invention is 250 units / ml, but it does not deviate significantly from the concentration and the effect of the composition of the present invention is not lost. To the extent possible, it should be understood that slight differences in the concentrations are included in the compositions of the present invention.

Examples of such a slight difference in the concentration include, for example, (1) errors in weighing and mixing of chondroitinase ABC and a contrast agent during production of the composition of the present invention, and (2) chondroitin used. Consideration may be given to differences in lots between specific lots of the specific activity of ABCA, (3) slight changes in the concentration depending on the type of animal to be administered, sex, age / week, type of herniated disc, degree of progression of herniated disc, etc. Can be

Specifically, the concentration of chondroitinase ABC is preferably 250 units / ml ± 100 units / ml, more preferably 250 units / ml ± 5.
It should be understood that a range of about 0 units / ml is included in the composition of the present invention.

[0023] The composition of the present invention may further contain a pharmaceutical carrier used in the production of ordinary medicaments. Pharmaceutical carriers include conventional excipients, binders, lubricants, coloring agents, disintegrants, buffers, isotonic agents, preservatives, soothing agents,
Examples of additives usually used in medicine are exemplified.

The composition of the present invention may be in any of a solution state, a frozen state and a lyophilized state. When the composition of the present invention is provided in the form of a solution, for example, chondroitinase ABC and, if necessary, a pharmaceutical carrier or the like are added to a contrast agent so that the concentration of chondroitinase ABC is about 2%.
It can be manufactured by adjusting to 50 units / ml and performing sterilization by filtration as needed. Also, a composition containing a desired amount of chondroitinase ABC can be prepared by dissolving and mixing the contrast agent, chondroitinase ABC, and, if necessary, a pharmaceutical carrier and the like in an appropriate solvent.

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

When the composition of the present invention is provided in a freeze-dried state, for example, the composition of the present invention in a solution state described above can be produced by freeze-drying. In this case, the chondroitinase AB defined in the present invention
The concentration of C indicates the concentration when the composition in the lyophilized state is dissolved in an appropriate solvent at the time of use.

Among these, the preferred state of the composition of the present invention is a frozen state, and when provided at a temperature of about -18 ° C., the chondroitinase ABC activity can be stably maintained for 9 months or more. Is preferred. The composition of the present invention can be used as a final dosage form to be administered as a pharmaceutical as it is, or can be used as a raw material for other final dosage pharmaceuticals, for example, a liquid preparation, a lyophilized agent and the like.

The composition of the present invention is mainly used as a preparation for injection. When the composition of the present invention is provided as an injectable preparation in a solution state, the composition of the present invention in a solution state produced by the method described above is filled and sealed in a suitable container such as an ampoule, a vial, or an injection syringe, It can be distributed or stored as it is and can be used as an injection for administration.

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

It should be noted that distribution and storage are carried out at -80 to -18 ° C, especially-
It is preferable to carry out at a temperature of about 18 ° C. Further, when the composition of the present invention is provided as a freeze-dried preparation for injection, the dried composition of the present invention produced by the above-mentioned method is sealed in an appropriate container such as an ampoule, a vial or an injection syringe. And dissolved or dissolved in distilled water for injection, physiological saline, aqueous glucose solution or aqueous sorbitol solution before administration, and can be administered as an injection before administration. The composition of the present invention in a lyophilized state,
It may be provided as a set together with a solvent for dissolution.

Also, a freeze-dried product of chondroitinase ABC and a contrast agent can be provided as a set, and the freeze-dried product of chondroitinase ABC can be dissolved with a contrast agent before administration, and used as an injection. .

When filling or sealing the container of the present invention in a suitable container such as an ampoule, a vial, or a syringe for injection, a nitrogen gas, a rare gas or the like is used in order to prevent a chemical reaction of the composition of the present invention, especially oxidation. The inert gas may be filled or sealed together.

The material of containers such as ampules, vials, injection syringes and the like which can be filled and sealed with the composition of the present invention is not particularly limited as long as it does not affect the composition of the present invention and is pharmaceutically acceptable. Not limited.

The enzyme activity of chondroitinase ABC is
For example, using chondroitin sulfate C as a substrate, chondroitinase ABC is reacted at pH 8.0 and at 37 ° C. to produce an unsaturated disaccharide having remarkable absorption in the ultraviolet region, which is detected by a spectrophotometric method (for example, 232 nm). Absorbance).

<2> Therapeutic agent of the present invention The composition of the present invention can be used, for example, for treatment of herniated disc, thereby providing the therapeutic agent of the present invention.
The therapeutic agent of the present invention can be used as an injection in disc lysis therapy in which the nucleus pulposus is dissolved and injected by injecting it into the disc of an herniated animal.

The dose of the composition of the present invention or the therapeutic agent of the present invention should be individually determined according to the kind, sex, age / week, type of herniated disc, degree of progression of herniated disc, etc. Although not particularly limited, for example, once per disc of dogs, usually once to 2 times
About 0 units, preferably about 10 to 15 units,
More preferably, a therapeutic agent in an amount corresponding to about 12.5 units of chondroitinase ABC can be injected.

The composition of the present invention or the therapeutic agent of the present invention can be administered to animals including humans, specifically, mammals. Among mammals, mammals other than humans are preferable,
Mammals raised as domestic animals are more preferred, and dogs are particularly preferred. The present invention also encompasses a method for treating a herniated disc, which comprises administering the composition of the present invention or the therapeutic agent of the present invention to a mammal (particularly a dog) other than a human with herniated disc.

The safety of the composition of the present invention and the therapeutic agent of the present invention can also be confirmed from the results of Examples described later.

[0039]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, which are illustrative of the present invention and are not intended to limit the present invention.

<1> Materials and methods used in this example (1) Chondroitinase ABC Chondroitinase ABC used in this example was produced by the method described in JP-A-6-153947. is there.

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 units of this chondroitinase ABC was measured using a Toxicolor system (manufactured by Seikagaku Corporation).
g (0.00986EU; EU means endotoxin unit, 1pg
= 0.0029 EU) and contained substantially no endotoxin.

The 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, 50 mM Tris-containing 50 mM sodium acetate was used.
An enzyme sample (chondroitinase ABC) was added to hydrochloric acid buffer (pH 8) and 10 μg of casein, and the mixture was reacted at 37 ° C. for 10 minutes.
2 nm). 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. The molar extinction coefficient of 2-acetamido-2-deoxy-3-0- (β-D-gluco-4-enpyranosyluronic acid) -6-0-sulfo-D-galactose was 5.5 and 1 unit. Under the above reaction conditions, the amount of the enzyme of
It was defined as the amount of enzyme that catalyzes the reaction to release micromoles.

As a result, the above chondroitinase AB
C was confirmed to have a specific activity of 300 units / mg protein or more.

The chondroitinase ABC is mixed with an aqueous nonionic iodine contrast agent containing iohexol (Omnipark, Daiichi Pharmaceutical, Tokyo) so that the concentration of chondroitinase ABC is 50 units / 200 μL. And dispensed into vials. This composition was stored frozen at -18 ° C and thawed immediately before use. still,
This composition was stable at -18 ° C for more than 9 months. Hereinafter, this composition is simply referred to as “chondroitinase ABC composition”.

(2) Test Animals Fifty-nine dogs showing clinical symptoms of herniated disc were used as test animals. These dogs exhibited various symptoms ranging from simple pain in the neck or lower back to paralysis of the forelimbs, hindlimbs, limbs, or the entire trunk. The hernia was diagnosed by first listening to the petitioner from the animal owner, observing the posture and gait, and then performing X-ray and neurological examinations. Radiographically, simple radiographs of the spine (cervical vertebra or chest, lumbar vertebra depending on the case) and dorsoventral, and spinal radiographs in the same direction were taken using an iodine contrast medium. . A herniated disc was diagnosed when it was determined that the symptoms indicated by the dog could result in a spinal cord compression image on myelography. Also. On myelography radiographs,
Ansen's method (Acta Orthop Scand, 1952, 20:28
Disc types were identified according to 0). In some cases, osteogenesis was seen on the ventral side between two vertebral bodies facing each other across the disc, and the vertebral bodies fused through the new bone, indicating osteoporosis. Also. In many cases, myelography radiographs showed spinal cord compression at multiple disc sites.

(3) Method of administering the chondroitinase ABC composition to the test animal The test animal is anesthetized by inhalation of halothane (1 to 4%, Takeda Pharmaceutical Co., Ltd.), and depending on the case, the lower back or side neck or The skin of the abdominal neck was shaved. Thereafter, the shaved skin was disinfected with 70% ethanol and iodine (Isodine, Meiji Seika, Tokyo). Keeping the dog in the lateral position on the X-ray examination table,
Under fluoroscopy, a 23 gauge spinal needle with mandolin was inserted into the herniated disc either laterally or dorsally. At the stage when the tip of the spinal needle reached the annulus fibrosus, the mandolin was removed, and a 31-gauge inner needle approximately 7 mm longer than the mandolin was inserted into the spinal needle, and the inner needle was passed through the annulus fibrosus. Then, using a microsyringe, chondroitinase AB
50 μL of the C composition (chondroitinase ABC content was 12.5 units) was injected into the disc. In a case of cervical disc herniation with spondyloarthropathy, a needle was inserted through the ventral midline. In cases where spinal cord compression was observed in multiple discs on myelography radiographs, the cause of the manifestation was 1
It was not possible to identify the discs at multiple locations. In such a case, the chondroitinase ABC composition was administered to all the discs at the site where the spinal cord compression image was found. In addition,
In a possible case, intra-disc injection of chondroitinase ABC was performed after a period of rest in cage cages.

(4) Observation of clinical symptoms As clinical disc herniation symptoms, observation of posture and gait, skin pain area, skin and muscle during body movement, pain occurrence site of facet joint, trunk or limb paralysis. The site of occurrence was identified. Also. Dogs can use Chrisman's method (Ch
risman CL, Thephysical and neurologic examination
s. In: Chrisman CL, ed. Problems in Small Animal Ne
urology. 2nd ed. Philadelphia: Lea & Febiger, 199
1: 41-72), the following neurological examinations were performed.

Bounce and step response under blind and unblind conditions, proprioception, flexion and extension, deep pain, cross-extension, patella tendon reflex, anal reflex,
Locomotion of the tail, urination.

The above findings were scored as follows. -: Disappeared, +: attenuated, ++: normal, +++: excessive reaction

Based on the scores of the individual test items and general observations, the degree of herniated disc in dogs was classified into 1 to 5 as shown in Table 1. The degree of the herniated disc was determined at least once before the administration of the chondroitinase ABC composition, and once a week after the administration as much as possible once every 1 to 3 days regardless of the dog's condition. 1 week after administration, once every 1 to 3 weeks, especially if the dog is in good condition,
The judgment was made up to 60 days after the administration. After 60 days after administration,
Only gait observation was performed. The evaluation of the therapeutic effect was performed using the difference in the degree of herniated disc symptoms before and after administration as an index. Table 2 shows the evaluation categories.

[0054]

[Table 1]

[0055]

[Table 2] Table 2 Evaluation method of degree of clinical symptom improvement 程度 Improvement degree Improvement of hernia symptoms ─著 Notable Hernia symptoms disappeared 7 days after administration Degree of symptoms before and 7 days after administration Difference of 3 or more Moderate Difference in severity of symptoms before administration and 7 days after administration Mild Difference in severity of symptoms before administration and 7 days after administration 1 Invariable Severity of symptoms before administration and 7 days after administration The difference is 0 ─────────────────────────────

(5) X-ray Examination of the Intervertebral Disc A lateral X-ray photograph of the spine was taken before, immediately after, and 7 days after the administration of the chondroitinase ABC composition. On these radiographs, Nakayama et al.'S method (Masunari Nakayama et al., “Chymopapai
Intradisc Injection Therapy with n ”The Japanese Society of Clinical Veterinary Medicine (Kinki)
The disc height (the ratio of the height of the disc to the width of the disc; DHR) was measured in the abstract of the lecture: 126-127, 1989).
That is, on a lateral plain radiograph, two tangents are drawn on the dorsal and ventral edges of the vertebral body adjacent to the intervertebral disc, and a straight line connecting the dorsal and ventral contacts of this tangent is drawn. The intersection of the caudal dorsum of the caudal vertebral body is A, the intersection of the caudal dorsum of the caudal vertebral body is B, the intersection of the caudal dorsum of the caudal vertebral body is C, and the caudal / ventral margin of the caudal vertebral body Is defined as D, the lengths of AB, AC, and BD were measured, and a numerical value was obtained from the following equation. Disc height = (AC + BD) / 2AB DHR reduction was calculated as the percentage of DHR 7 days post-dose relative to pre-dose DHR.

<2> Results (1) Occurrence of canine disc herniation Using all the animals to which the chondroitinase ABC composition was administered, the occurrence of herniated disc in dogs was examined. The sexes of the searched cases were 59% for males, 39% for females, and 3% for unknown. The distribution of these ages and varieties is shown in Table 3.

[0058]

[Table 3] Table 3 年 齢 Age varieties ────── １ 1 Shiba Inu 1 Coffee 1 2 2 1 3 3 4 1 Coffee 1 Sea 1 Homer 2 Yorkshire Terrier 1 Maltose 1 Hackle 1 Tax Hund 2 5 Miniature Hook Shell 1 Hack 1 1 Maltose 1 Hookle 2 Tax Hund 1 6 Toy Foot 1 Yorkshire Terrier 1 Hook 2 Tax Hound 1 7 Shetland Sea 1 Thick 1 Sheep 1 Hook 2 Yorkshire Terrier 2 Maltose 1 Peak 2 Tax Hund 4 9 Raffle Retriever 1 Mini New Hellshell 1 Shetland Seafight 1 Sea Shear 1 Maltose 1 Head 1 110 Hexenice 1 Homelanian 2 Hybrid 1 Maltose 1 Tax Hund 2 11 Hybrid 1 Maltose 1 12 Hybrid 1 13 14 Yorkshire Terrier 1 Unknown Tax Hunt ───────────────────────────

The ages are between 1 and 14 years and 4-10
The oldest age was the most. On the other hand, the most common varieties were dachshunds (22%), followed by beagles (1%).
5%). Maltese (10%) occurred in that order. The incidence was high in the Dachshund and Beagle breeds, which are considered to be chondrodystrophic dogs, and these two breeds accounted for 37% of the total.

FIG. 1 shows the distribution of intervertebral discs diagnosed as hernia.
It was shown to. A total of 219 discs were injected with the chondroitinase ABC composition. These discs were diagnosed as compressing the spinal cord, and the average number of discs compressed per head was 3.7. The incidence of hernia in the intervertebral disc of each individual was highest in the lower thoracic vertebra to lumbar disc, with the incidence in the disc between T11 / 12 and L4 / 5 being 83.6%. Also. Hernia incidence in the cervical disc was 10%.

(2) Changes in herniated disc herniation In 7 out of 59 cases to which the chondroitinase ABC composition was administered, surgery such as hemi-laminectomy or fenestration was performed simultaneously with the injection of the chondroitinase ABC composition. Surgery was also given. The change in symptoms following treatment in these cases was due to the effects of surgery and chondroitinase A
Since the effect of the BC composition could not be distinguished, it was excluded from the evaluation of the effect of the chondroitinase ABC composition. Furthermore, the other four cases were excluded from the evaluation because the test after administration of the chondroitinase ABC composition was not performed or was insufficient. The remaining 48 cases were used to evaluate the effects of nucleus pulposus fusion therapy with the chondroitinase ABC composition.

FIG. 2 shows the time course of hernia symptoms up to 60 days after administration of the chondroitinase ABC composition once. The number and ratio of animals having a degree of symptom of 5, 4, 3, 2, and 1 before administration of the chondroitinase ABC composition were 0 (0%), 7 (14.6%), and 15 (31.3), respectively.
%), 8 (16.7%), and 18 (37.5%). Of these, 24 cases (50 days after administration)
%), But 36 days (75%) were normal 59 days after administration.

For 11 cases chondroitinase AB
It was possible to follow up more than one year after administration of the C composition. 10 of these
In the example, no recurrence of the herniated disc was observed, and the therapeutic effect of the chondroitinase ABC composition was maintained for one year or more. From the above results, it was shown that the treatment with the chondroitinase ABC composition is also effective for cases that exhibited hernia symptoms for a long time before administration of the chondroitinase ABC composition. This is thought to be due to the fact that not only the escaped portion modified by inflammation but also the remaining nucleus pulposus that pressurizes the escaped portion from below plays an important role in the compression of the nerve tissue by the hernia mass. However, in one case, 8 months after administration of the chondroitinase ABC composition,
Disc herniation recurred in one (L1 / 2) of the six discs to which the chondroitinase ABC composition was administered. No adverse effects due to the chondroitinase ABC composition were observed over the entire follow-up period.

The duration of herniated disc symptoms before administration of the chondroitinase ABC composition ranged from 1 to 635 days, averaging 54.4 days. FIG. 3 shows chondroitinase ABC.
The relationship between the onset period of the herniated disc before administration of the composition and the change in the degree of hernia symptoms after administration was shown. In general, cases with longer onset tended to have lower symptoms. This may reflect that less symptomatic patients have been receiving conservative therapy for an extended period of time. Irrespective of the length of the onset of herniated disc, the degree of herniated symptoms was definitely improved after a single administration of the chondroitinase ABC composition to the disc.

Of the 48 cases, 9 were diagnosed with Hansen type I herniated disc. The remaining 39 cases were diagnosed with only Hansen type II hernia. Since the chondroitinase ABC composition was considered to be safe even when it comes into contact with the nerve tissue around the hernia lesion or the capillaries of the pia mater, nucleus pulposus lysis by the chondroitinase ABC composition was also performed in such cases. Tried therapy.

FIG. 4 shows changes in the degree of hernia symptoms after administration of the chondroitinase ABC composition for each of these two hernia types. In any hernia type,
The degree of improvement in hernia symptoms was the same. Hansen
In the type I hernia cases, the degree of hernia symptoms before administration of the chondroitinase ABC composition was higher than in the Hansen type II hernia cases. this is,
It is considered that Hansen type I hernia has a large nucleus pulposus volume that escapes and presses against nerve tissue. However, the chondroitinase ABC composition was also effective in such cases. The disc height (DHR) of the administered disc 7 days after administration of the chondroitinase ABC composition was as follows:
As shown in FIG. 5, it was between 76.0 and 87.2% before administration.

7 after administration of the chondroitinase ABC composition
On the day, slight improvement was seen in 44 (91.7%) of the 48 cases. FIG. 6 shows the degree of improvement for each degree of Hernia symptoms before administration 7 days after administration. Seven days after the administration of the chondroitinase ABC composition, the degree of symptoms before administration was 94.7% of the cases where the degree was 1, and the degree of symptoms before administration was 2
75.0% of the cases which were markedly improved. 80 of 15 patients with hernia symptoms of 3 prior to administration.
0% improved to "significant" and 20.0% improved to "moderate". In addition, 50.0% of the administered cases improved to show no clinical symptoms. On the other hand, in 7 cases in which the degree of hernia was 4 before administration of the chondroitinase ABC composition, only 28.6% improved "markably".
However, including the "moderate" and "mild" improvements, 85.7% of the cases with a degree 4 hernia symptom before administration showed some improvement. The above indicates that chondroitinase A
It is believed that the nucleus pulposus thawing process with the BC composition is almost complete within 7 days after administration.

In 17 cases, cage rest was performed for 5 days to 1 month, and after confirming that no improvement in symptoms was observed, administration of the chondroitinase ABC composition was performed. Therefore, it is considered that the effect of the administration of the chondroitinase ABC composition and the effect of the cage rest could be distinguished. In all cases, including the case of combined use of surgery, searched in this case, no side effects considered to be caused by administration of the chondroitinase ABC composition were observed.

(3) Transition of X-ray image of intervertebral disc DHR of the intervertebral disc could be measured before and after administration of the chondroitinase ABC composition in 128 intervertebral discs. Among them, DHR decreased in 115 intervertebral discs (89.
8%). 13 discs showed an increase in DHR after administration, of which 2 discs (all in the same case)
After a further lapse of time after the administration, X-ray photography was again performed and the results were measured. As a result, a decrease in DHR was observed as compared to before the administration.

After administration of the chondroitinase ABC composition, spinal imaging was performed again in 10 cases. Of these, the spinal cord compression image was legible in 7 cases. Improvement of the spinal cord compression image was confirmed in 2 cases (1
In the example, there were only two discs, in other examples only three discs).
In addition, an improvement in the spinal cord compression image was also observed in one of the combined surgery cases (two intervertebral discs) (Table 4).

[0071]

Table 4 Spinal cord imaging findings after administration of the chondroitinase ABC composition ──── Case number ────────────────────────── 9 12 13 22 24 25 29 30 32 33 52 ^* ──────日 Date of imaging after administration 7 7 7 8 22 10 7 7 7 7 54 Interpretation of spinal cord compression image No Yes No Yes Yes Yes Yes No No No Yes Yes Number of discs to be administered 5 3 4 2 6 3 4 Number of discs with improved compression image 2 0 0 0 0 3 2 Number of intervertebral discs with compression image 3 3 4 2 6 0 2 Symptoms before administration Degree 3 3 3 3 3 1 4 Degree of symptoms after administration 0 0 0 0 0 0 4 ───────────────────────────── ─────── *: Surgery combined use case

[0072]

The composition of the present invention comprises chondroitinase A
In the composition containing BC and the contrast agent, the content of chondroitinase ABC is reduced by about 1.6 times as compared with the known composition, which is expected to further enhance the safety. . In addition, since the amount of chondroitinase ABC used for producing the composition of the present invention can be reduced, it can be provided at a lower cost than known compositions. The therapeutic agent of the present invention has the same effect as the composition of the present invention. Furthermore, the therapeutic agent of the present invention can effectively thaw the nucleus pulposus of an animal with herniated disc and is effective for treating various types of herniated disc. In addition, by performing X-ray imaging, the administration position can be confirmed, and it can be confirmed that the therapeutic agent of the present invention has been accurately injected into a desired site in a desired amount. Furthermore, the nucleus pulposus melting effect can be confirmed,
Thereby, the therapeutic effect of the herniated disc can be confirmed.

[Brief description of the drawings]

FIG. 1 is a graph showing the distribution of herniated discs.

FIG. 2 is a graph showing the time course of hernia symptoms up to 60 days after a single administration of the chondroitinase ABC composition.

FIG. 3 is a graph showing the relationship between the onset period of herniated discs before administration of the chondroitinase ABC composition and the change in the degree of hernia symptoms after administration. Before "PRE" administration,
“D” represents a day and “w” represents a week. "*" Mark is from 1st
The significant difference by the t test of the student of the group of 3 days and the group of 3 weeks or more is shown (P <0.01).

FIG. 4 is a graph showing changes in the degree of hernia cases in Hansen type I and Hansen type II after administration of the chondroitinase ABC composition. "PRE" before administration, "d" represents days, and "w" represents weeks. The “**” mark and the “##” mark indicate a significant difference by the Student's (**) or Aspin-Weich (##) t-test of each group (P <0.01). .

FIG. 5 is a graph showing the relationship between the degree of symptom improvement and the rate of change in intervertebral disc height (DHR) 7 days after administration of the chondroitinase ABC composition.

FIG. 6 is a graph showing the degree of improvement for each degree of Hernia symptoms before administration on day 7 after administration of the chondroitinase ABC composition.

Claims (3)

[Claims] 1. A composition comprising chondroitinase ABC and a contrast agent, wherein the concentration of chondroitinase ABC relative to the total amount of the composition is about 250 units / ml. Stuff. 2. The method according to claim 1, wherein the chondroitinase ABC has a concentration of 25.
2. The composition according to claim 1, wherein the composition is 0 ± 100 units / ml. 3. A composition comprising the composition according to claim 1 or 2,
Treatment for herniated disc.