JPH07316071A - Therapeutic agent for muscular dystrophy - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for muscular dystrophy containing insulin-like growth factor-I, etc., as an active ingredient and capable of exhibiting excellent effect on suppression of crisis of muscular dystrophy and delay of progress of muscular dystrophy. CONSTITUTION:This muscular dystrophy contains e.g. an insulin-like growth factor-I having an amino acid sequence of the formula or its biologically active fragment as an active ingredient. Furthermore, the therapeutic agent contains preferably a pharmaceutically permissible carrier such as distilled water, physiological saline or an aqueous solution of glucose and daily dose of the active ingredient is preferably in the range of 10mug to 10mg/kg.adult.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic drug for muscular dystrophy.

[0002]

2. Description of the Related Art Muscular dystrophy is a general term for a group of progressive hereditary diseases that cause temporary degeneration of skeletal muscle, and is one of extremely serious muscle diseases. Muscular dystrophy (progressive muscular dystrophy)
It is classified into the following three typical types according to its inheritance form. 1) Duchenn with sex-linked recessive inheritance
e Muscular Dystrophy (hereinafter,
"DMD") and Becker Muscul
ar Dystrophy (hereinafter abbreviated as "BMD"), 2) Limb-Gird with autosomal recessive inheritance
le, 3) Facio-Sca with autosomal dominant inheritance
pulo-Humeral.

Of these, DMD is the fastest and the most serious. The clinical features of DMD include a delay in virgin walking around 3 years old, stair climbing, standing, and running, and a very high creatine kinase (CK) level, proximal muscle contraction and weakness, Characterized by pseudohypertrophy of the leg muscles. Histologically, muscle fiber degeneration and necrosis proceed. EMG and muscle biopsy findings show characteristic patterns. Progress is fast, 7-
By the age of 11, he was unable to walk, and by the age of 20 he died of respiratory failure and heart failure.

The incidence of DMD is approximately 30 for male offspring.
It is a ratio of 1 in 00. Mutation locus is Xp21
In 1987, Kunkel et al. Of Harvard Medical School identified this causative gene (Cel
1, 51 , 919 (1987)). This causative gene product has a molecular weight of 427 kd, which accounts for about 0.002% of muscle protein.
Protein named dystrophin. Dystrophin is a membrane cytoskeletal protein that lines the cytoplasmic side of muscle cell membranes. It is not detected by immunohistochemical techniques in DMD, but is detected in BMD, but in 2/3 cases, the size and amount of the molecule are abnormal. In the dystrophin gene of DMD patients, partial deletion was found in 60 to 70%, duplication was found in 6 to 10%, and a point mutation in the rest caused a structural defect in dystrophin. Conceivable. It seems more doubtful that dystrophin abnormalities are the cause of DMD, but no clear answer has been found as to why it induces muscle fiber collapse.

By the way, there is no fundamental cure for muscular dystrophy. For example, transplantation of normal muscle cells, gene therapy and the like are theoretically possible, but it is considered that it will take a considerable amount of time to realize them. The current symptomatic treatment is
Preventive gait training and respiratory function training require muscle contracture and joint deformity prevention, especially in the scoliosis. When the patient becomes unable to walk at the end of life and general muscle atrophy progresses, respiratory failure becomes a major disorder, and long-term respiratory management and drug therapy for heart failure are performed.

[0006] That is, the present situation is that no effective therapeutic method for delaying the progression of muscular dystrophy patients has been found. In order to estimate the therapeutic effect in DMD patients, for example, a gene mutation equivalent to this,
The mdx mouse, which is a model animal in which muscle fiber collapse progresses, is used. In mdx mouse, the 3185th base C of mouse dystrophin cDNA is replaced with T,
The codon that should be CAA (Gln) becomes TAA (stop codon), and it has been revealed that protein synthesis is stopped up to about 1/3 of the rod structure of dystrophin (Science, 244 , 1578 (198).
9)). However, although the mdx mouse has a gene mutation equivalent to that of a human patient, the mdx mouse usually does not exhibit a behavioral disorder. The reason is m
Basic fibroblast growth factor (bFG) is present in dx mouse muscle.
F) is abundantly contained, mdx muscle satellite cells proliferate by reacting more strongly than normal cells, mice have a higher blood vessel density in muscle tissue than humans, and have a good nutritional supply for muscle fiber maintenance. Mice are quadrupedal animals, and because the load per muscle fiber applied to the extremities is small, it is considered that the muscle fiber collapses slowly and muscle regeneration is in time.

[0007] Therefore, it is not possible to adequately evaluate the inhibition or delay of the onset of muscular dystrophy, and it is not easy to quantitatively evaluate the degeneration and necrosis of muscle fibers in terms of pathology. The means by which the protective effect of drugs on muscle fibers is clearly established has not yet been established.
On the other hand, insulin-like growth factor-I (hereinafter referred to as "IGF-
Abbreviated as "I") is a single-chain peptide consisting of 70 amino acids (J. Biol. Chem., 253 , 276).
9 (1978); FEBS Lett. , 89 , 283
(1978)) and produced in the liver and other peripheral tissues. IGF-I is an effect of promoting uptake of sugars and amino acids into cells, an effect of promoting protein synthesis, an effect of promoting cell growth,
It has a differentiation promoting action, anabolic promoting action, etc., and targets many organs such as muscles, bones, and nerves.

Administration of IGF-I to growth hormone deficient mice has been shown to increase kidney and spleen weights, as well as weight and length (Endocrin).
LOGY, 124 , 2519 (1989)). Similar effects have been observed in pituitary-deprived rats (J. Endocrinol., 112 , 123 (19).
87), etc.), the therapeutic effect of IGF-I in scoliosis has been clinically investigated from these findings. In addition, the therapeutic effect of IGF-I has also been investigated in type A diabetes that exhibits marked insulin resistance (N.
Engl. J. Med. , 317 , 137 (1987)
etc). However, regarding the effect of IGF-I on muscular dystrophy that causes severe and fatal muscle tissue destruction,
It has never been reported so far.

[0009]

[Means for Solving the Problems] In the present situation, the present inventors suppress the collapse of muscle fibers, necrosis, and promote the assimilation of muscle tissue in a situation in which a muscular dystrophy patient falls, thereby promoting the progression of muscular dystrophy pathology. It has been studied for the purpose of providing a means for delaying. As a result, mdx
The present invention was completed by studying a novel method for quantitatively evaluating the progression of muscular dystrophy using mice, and by discovering that IGF-I can be an effective therapeutic drug for muscular dystrophy, the present invention was completed.

That is, the gist of the present invention resides in a therapeutic agent for muscular dystrophy containing IGF-I or a biologically active fragment thereof as an active ingredient. Hereinafter, the present invention will be described in detail. IGF-I is a known substance as described above. Human IGF-I was isolated from human serum by Rinderknecht et al. (Proc. Natl.
Acad. Sci, USA, 73 , 4379 (197).
6)), and then its amino acid sequence was determined (J. B.
iol. Chem. , 253 , 2769 (1978);
FEBS Lett. , 89 , 283 (1978)
etc). Therefore, IGF-I can be prepared as a natural protein from serum according to the method described in the above literature. IGF-I can also be produced using recombinant DNA technology. European Patent Publication 123228
No., European Patent Publication 128733, European Patent Publication 219
Recombinant IGF-I can be obtained according to the method described in each publication of No. 814 or the like, or according thereto. Specifically, a gene encoding IGF-I or a precursor gene thereof is introduced into an appropriate expression vector, and using such an expression vector, bacteria such as Escherichia coli and Bacillus subtilis (prokaryotic cell),
(Mammalian) animals such as yeasts such as baker's yeast, insect cells such as ovary-derived cells of Spodoptera litura (Sf9 cell line), ovary-derived cells of Chinese hamsters (CHO cells), kidney-derived cells of African green monkeys (COS cells), etc. A target recombinant protein can be obtained by transforming a cell or the like and culturing the transformant. further,
IGF-I may be chemically synthesized according to a known peptide synthesis method (Proc. Natl.
Acad. Sci, USA, 80 , 2216 (198).
3) etc.) As the IGF-I used in the present invention, any IGF-I can be used as long as it has a biologically equivalent activity to that of IGF-I. Considering the treatment of human patients, it is preferable that IGF-I also be of human origin, but the IGF-I sequence of other mammals, birds, amphibians, reptiles, and fishes is included in the amino acid sequence. May be For the purpose of increasing activity and stability, it is also possible to add a few amino acids from the N-terminal, or conversely add some amino acids to the N-terminal. In the present invention, IGF-I is also defined as having a modification such as removal, insertion, modification, substitution or addition of a part of the amino acid sequence within the range not impairing the biological activity. It IGF
As a modified form and an analogue of -I, European Patent Publication 1588
92, JP-A-62-169733, JP-A-1-50.
899, JP-A-1-63597, JP-A-1-661.
98, JP-A-1-66199, European Patent Publication 346.
No. 429, Proc. Natl. Acad. S
ci, USA, 83 , 4904 (1986), Bioc.
hem. Biophys. Res. Commun. , 1
49 , 398 (1987), Biochem. Biop
hys. Res. Commun. , 149 , 672 (1
987), Endocrinology, 123 , 37.
3 (1988), J. Biol. Chem. , 263 ,
6233 (1988), Biochem. Biophy
s. Res. Commun. , 165 , 766 (198
9) etc.

As a method for quantitatively evaluating the degeneration and necrosis of muscle fibers, for example, a muscle-staining pigment such as Evans blue is used in an amount of 10 to 10.
Phosphate buf at a dose of 250 mg / kg
It is dissolved in a ferred saline solution (hereinafter abbreviated as "PBS") and administered into the tail vein of mdx mouse.
One to several days later, the mouse is sacrificed with ether or the like, and the skin is peeled off to expose the muscle tissue. The muscle-staining pigment is observed to be specifically taken up by the disintegrated muscle fibers, and the muscle fibers are stained (stained blue with Evans blue). This makes it possible to semi-quantitatively estimate the number of muscle fiber collapses. Furthermore, when a pathological section of this muscle fiber was created and observed using a fluorescence microscope, the number of muscle-disintegrated fibers was increased because the dye-incorporated fiber fluoresces (when Evans blue is used, it fluoresces red). Can be quantitatively measured.

The therapeutic agent for muscular dystrophy of the present invention comprises the above-mentioned IGF-I as an active ingredient, and is preferably used in the form of a suitable formulation (form) together with a suitable diluent or other additive. It As a dosage form, a dosage form suitable for parenteral administration is generally used. Preferred examples include an ampoule for injection and a freeze-dried powder for injection (vial).

The above-mentioned various dosage forms can be prepared by a conventional method commonly used in this technical field. As a pharmaceutical carrier used for formulation, diluents, additives and the like which are commonly used for preparing various dosage forms are used. For example, a lyophilized powder for injection is a purified I
An effective amount of GF-I is dissolved in, for example, a diluent such as distilled water, physiological saline or glucose aqueous solution, and if necessary, an excipient such as carboxymethylcellulose or sodium alginate,
Add benzyl alcohol, benzalkonium chloride, preservatives such as phenol, soothing agents such as glucose, calcium gluconate, procaine hydrochloride, pH adjusting agents such as hydrochloric acid, acetic acid, citric acid, sodium hydroxide, etc. It is prepared by freeze-drying.

The ampoule for injection is prepared by dissolving an effective amount of IGF-I in a diluent such as distilled water, physiological saline or Ringer's solution, and if necessary, a solubilizing agent such as sodium salicylate or mannitol and citric acid. Sodium, glycerin and other buffering agents, glucose, invert sugar and other isotonic agents, polyethylene glycol and other stabilizing agents, the above preservatives, the above soothing agents, and the above pH adjusting agents and other additives are added. It is sterilized by heat sterilization, sterile filtration, etc.

The therapeutic agent for muscular dystrophy of the present invention, when treating muscular dystrophy, is given to a patient in need thereof parenterally, generally by subcutaneous, intramuscular or intravenous injection in a predetermined amount in a single dose. , Or divided into multiple doses or administered continuously. The dose of the therapeutic agent for muscular dystrophy of the present invention is such that, in the case of single or multiple administration, it is usually used as an active ingredient (IGF-I) in adults.
It is preferable to administer this in a range of about 10 μg to 10 mg / kg daily, intravenously, subcutaneously or intramuscularly to a patient to be administered, depending on the patient's pathological condition, nutritional status, age, weight, concomitant drug, etc. It can be increased or decreased as appropriate. In the case of continuous administration, it is about 0.1 to 50 μm per adult hour.
An amount corresponding to the above-mentioned daily dose can be administered in the range of g / kg, preferably 1 to 30 μg / kg.

As an administration method, in the case of an ampoule for injection, it can be directly administered subcutaneously, intramuscularly or intravenously, but in the case of intravenous administration, a minipump for infusion is used if necessary. You can also do it. In the case of a freeze-dried powder for injection, the powder is dissolved in a diluent such as distilled water, physiological saline or Ringer's solution at the time of use, and then administered in the same manner as in the case of ampoule.

Further, the therapeutic agent for muscular dystrophy of the present invention is
When treating muscular dystrophy, a predetermined amount of glucose infusion such as glucose may be mixed in advance and administered, or at the same time as administration of the glucose infusion, an effective amount thereof may be administered alone from a peripheral vein or the like.

[0018]

EXAMPLES The present invention will be described in detail below with reference to examples, but the invention is not limited to the following without departing from the gist thereof. Example 1 Recombinant human IGF-I (SEQ ID NO: 1 in the sequence listing) was produced and purified by a gene recombination method using Escherichia coli as a host cell according to the method described in European Patent Publication 128733.

Experiments show that muscle fiber collapse is beginning 3
Three week-old mdx mice (male and female, both delivered by the Central Institute for Experimental Animals) and B without gene mutation
Three 10 mice were used. The mouse was bred in a normal cage. IGF-I was administered twice daily at 10 am and 8 pm at a dose of 4 mg / kg / day.
It was administered subcutaneously for a day. The control group (5 mdx mice, 4 B10 mice) was administered with PB in the same administration form.
S was administered.

IGF at 8 o'clock on the 6th day after the start of administration
After administration of -I, Evans blue dissolved in PBS was intravenously administered at a dose of 100 mg / kg under ether anesthesia. The next day, after slaughtered with ether, the skin was peeled off and the mouse was fixed in 5% formalin. Two days later, the rectus femoris and the diaphragm were isolated, embedded in paraffin, sliced and stained with hematoxylin and eosin, observed by an optical microscope and a fluorescent microscope, and photographed. In the rectus femoris specimen, the area of the region in which Evans blue was taken in by the collapse of the muscle fiber and emitted red fluorescence was measured with a digitizer on the photograph. The ratio of the cross-sectional area of the region occupied by the collapsed muscle fibers to the rectus femoris cross-sectional area is shown in FIG. 1 and Table 1.

[0021]

[Table 1]

As is clear from FIG. 1 and Table 1, IGF
26.2% in the -I non-administered group, whereas IGF-I
In the administration group, a clear inhibitory effect on muscle fiber collapse due to IGF-I administration was observed, which was 15.7%. And in the diaphragm,
More severe muscle fiber collapse was observed in the non-IGF-I administered group.

[0023]

INDUSTRIAL APPLICABILITY The therapeutic drug for muscular dystrophy of the present invention is expected as a drug that exerts excellent effects in suppressing the onset of muscular dystrophy and delaying its progression.

[0024]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 70 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 1 5 10 15 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 20 25 30 Ser Ser Ser Ar Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 35 40 45 Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 50 55 60 Lys Pro Ala Lys Ser Ala 65 70

[Brief description of drawings]

FIG. 1 is a drawing showing the ratio of muscle fiber disintegration regions after drug (PBS and IGF-I) treatment in mdx mice and B10 mice.

Claims (2)

[Claims] 1. A therapeutic agent for muscular dystrophy comprising insulin-like growth factor-I or a biologically active fragment thereof as an active ingredient. 2. The therapeutic agent for muscular dystrophy according to claim 1, which comprises a pharmaceutically acceptable carrier.