JP2003113116A - Medicine for achondroplasia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new medicine for achondroplasia caused by the mutation of fibroblast growth factor 3 (FGFR3). SOLUTION: This medicine for the achondroplasia caused by cartilage growth suppression due to the mutation of FGFR3 gene contains a guanylyl cyclase B (GC-B)-activating substance as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent and a therapeutic method for achondroplasia.

[0002]

2. Description of the Related Art Achondroplasia is the most common congenital disease that causes limb-shortening dwarfism, in which the limbs are shorter than the trunk. In addition to impaired long-term growth of tubular bones of the limbs, the skull largely projects to the frontal region,
Diagnosis is based on physical features such as a low nose and a radiograph. The onset is said to be one in 10,000 to 25,000. This disease is an autosomal dominant genetic disease, but 80 to 90% of cases are sporadically observed. Treatment includes orthopedic surgery, such as hip joint replacement and leg extension, and the administration of growth hormone. In leg extension, the bones are cut after 10 years old by a special machine (leg extender) to gradually increase the height over about half a year, but this operation causes great pain to the patient. In addition, growth hormone therapy improves growth in height by injecting growth hormone regularly from infancy, but growth stops when injection is stopped. None of the treatments cures the disease and is considered not ideal from the perspective of patient quality of life (American Jounal of Med
ical Genetics 72: 71-76, 1997; European Jounal of
Endocrinology 138: 275-280, 1998), development of a therapeutic agent for achondroplasia based on a new mechanism is desired.

In recent years, it has been reported that a mutation in fibroblast growth factor receptor 3 (FGFR3) present on chromosome 4p16.3 is recognized in patients with achondroplasia, and at present two types of mutations have been reported. Mutations are known. Of these mutations, 97% were G1138A (1138th G was mutated to A), 2.5% were G1138C (1138th G was mutated to C), and as a result, the 380th amino acid was Gly. From Arg
Has been replaced with (G380R) (Nature 371: 252-254, 199)
4; Cell 78: 335-342, 1994). In order to investigate the relationship between this mutation and achondroplasia, a G380R-type mutant FGFR3 (below, FGFR3
⁽ Also called ^ach ) transgenic mice were produced, and shortening of limbs and hypoplasia of craniofacial bone were observed (Development. 125: 4977-4988, 1998).

On the other hand, natriuretic peptides (NPs) consist of three types, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), and CNP (C-type natriuretic peptide). It is thought that biological activity is shown by increasing intracellular cGMP concentration through anilyl cyclase coupled receptors (GC-A receptor for ANP and BNP, GC-B receptor for CNP) (Annu. Rev. Biochem. 60: 229-255, 199
1). NPs have been reported to play important roles in the regulation of fluid homeostasis and blood pressure (J. Clin. Invest.
93: 1911-1921, 1987; J. Clin. Invest. 87: 1402-1412,
1994), its expression in various tissues other than cardiovascular system and its physiological activity are also known (Endocrinology. 129: 1104-11).
06, 1991; Annu. Rev. Biochem. 60: 553-575, 1991).
One of them has a role as a bone growth factor. CNP significantly promotes long bone growth in mouse embryo tibial organ culture (J. Biol. Chem. 273: 11695-11700, 199).
8). CNP is also used in organ cultures of mouse fetus tibia, cartilage culture cells, and osteoblast culture cells, and is more cG than ANP or BNP.
High MP productivity (J. Biol. Chem. 269: 10729-10733, 19
94; Biochem. Biophys. Res. Commun. 223: 1-6, 1996; B
iochem. Biophys. Res. Commun. 215: 1104-1110, 199
Five). Furthermore, CNP and its receptor, GC-B, are expressed in bone growth plates (J. Biol. Chem. 273: 11695-11700, 1
998; Proc. Natl. Acad. Sci. USA 95: 2337-2342,
1998). In addition, transgenic mice expressing CNP in a cartilage-specific manner have been found to have a growth plate cartilage layer-enhancing action of CNP (Yatsuda et al., The 72nd Annual Meeting of the Japanese Society of Endocrinology, 1999 abstract).

Since CNP knockout mice become dwarfism, a relationship between CNP and dwarfism has been pointed out (Proc. Natl. Acad. Sci. USA 98: 4016-4021, 2).
001) has not been described in relation to achondroplasia due to FGFR3 mutation, and there is no conclusive evidence that CNP is effective in achondroplasia due to FGFR3 mutation. Absent. That is, it is known that mutations in FGFR3 are associated with cartilage amorphism, and that CNP is involved in chondrogenesis. However, the relationship between the two, particularly FGFR3 and CNP, are both regulatory pathways for endochondral ossification. It is not known to date whether it is located upstream or not and whether CNP has a therapeutic effect on achondroplasia.

[0006]

The object of the present invention is to provide FGFR.
(EN) It is intended to provide a new therapeutic agent and therapeutic method for achondroplasia caused by mutation of 3.

[0007]

[Means for Solving the Problems] The present inventors have established a substance (eg, CNP) that activates guanylyl cyclase B (GC-B).
Based on the expectation that they could be applied to diseases associated with chondrogenesis, we searched for an appropriate cartilage deficiency model and created a double transgenic mouse obtained by mating that animal model with a CNP-transgenic mouse. I decided to verify whether the symptoms of cartilage failure could be recovered. As described above, as an animal model of human achondroplasia, a transgenic mouse of G380R type FGFR3 (FGFR3 ^ach ) was produced, and shortening of limbs and craniofacial bone hypoplasia were observed (Development. 125: 4977-4
988, 1998). Therefore, when the above FGFR3 ^{ach -transgenic} mouse was obtained and the CNP-transgenic mouse prepared by the present inventors was crossed to prepare a CNP / FGFR3 ^{ach -double} transgenic mouse, CNP was F
It was found that GFR3 ^ach can restore the inhibition of bone growth, and completed the present invention relating to a therapeutic agent and a treatment method for achondroplasia by CNP.

That is, the present invention relates to guanylyl cyclase B.
Contains a substance that activates (GC-B) as an active ingredient,
Constituting a therapeutic agent for achondroplasia caused by cartilage growth inhibition by mutation of fibroblast growth factor receptor 3 (FGFR3) gene and a substance that activates guanylyl cyclase B (GC-B) A method for treating achondroplasia is provided.

In the present invention, the term "chondrodysplasia caused by the inhibition of cartilage growth due to the mutation of fibroblast growth factor receptor 3 (FGFR3) gene" refers to the enhancement of FGFR3 function and the inhibition of function thereof due to the mutation of FGFR3 gene. Failure or F
It refers to achondroplasia caused by increased expression of the GFR3 gene, and achondroplasia is used as a synonym for achondroplasia. Also, in this specification, FG
FR3 ^ach shows fibroblast growth factor receptor 3 (FGFR3) having a mutation (G380R), which is an amino acid at position 380 of FGFR, Gly to Arg, and it is known that this mutation leads to hyperfunction of FGFR3. (Development. 125: 4977
-4988, 1998).

In the present invention, "guanylyl cyclase"
The "substance that activates B" is a substance (peptide or low-molecular compound) that binds to and activates GC-B, which is known as a CNP (C-type natriuretic peptide) receptor, and is preferable. Substances that have CNP (C-type natriuretic peptide) -like activity (peptides or low-molecular compounds)
Is. For example, mammalian CNP (CNP-22: Biochem.
ophys. Res. Commun. 168, 863-870, 1990, WO91 / 1634
2, CNP-53 (Biochem. Biophys. Res. Commun. 170, 973
-979, 1990, JP4-74198, JP4-139199), CNP derived from birds (JP4-120094), CNP derived from amphibians (JP4-1992)
120095) and CNP analog peptides (Japanese Patent Laid-Open No. 6-9688), and the like, preferably CNP derived from mammals, and more preferably CNP-22. In addition, "guanylyl cyclase
As a method for identifying a "substance that activates B", for example, the GC-B receptor is expressed in cultured cells such as COS-7, and the target substance (peptide or low-molecular compound) is added to the medium. After a certain temperature and after a certain time (eg 37
After 5 minutes at ℃, there is a method (Science 252, 120-123, 1991) for measuring the concentration of cGMP in the cell extract.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The CNP-transgenic mouse produced by the present inventors had a long body length due to overgrowth of bone in the long axis direction due to endochondral ossification. As a result of further analysis of this CNP-transgenic mouse, by histochemical analysis of the growth plate, 1) the thickness of the growth plate was increased by the extension of both the proliferating chondrocyte layer and the hypertrophic chondrocyte layer,
2) The extracellular matrix of the proliferating chondrocyte layer was increased, and 3) the size of mature hypertrophied chondrocytes was increased. These facts, combined with the fact that no apparent changes in chondrocyte proliferation, as shown by BrdUrd staining, were observed in the hypertrophic chondrocyte layer of the growth plate of CNP-transgenic mice, CNP was found to It is shown that it promotes the expression of chondrocyte differentiation traits at each differentiation stage of the growth plate, rather than contributing to the commitment of differentiation or proliferation. This is because although the expression of type X collagen mRNA of hypertrophic chondrocytes in the growth plate of CNP-transgenic mice was larger than that of wild-type littermate mice, the region of cells expressing them was expanded. It is supported by the fact that it was of similar intensity. Incidentally, the width of the skull due to membranous ossification is not changed in CNP-transgenic mice. This is CNP
Is not expressed in the skull or is not involved in the process of membranous ossification.

Ex vivo organ culture experiments provided further information on the mechanism of action of growth plate CNP. CNP-
The extent of cartilage primordium elongation with expanded extracellular matrix in the cultured tibia of transgenic mice,
The degree of enlargement of hypertrophic chondrocyte size was similar to the addition of CNP at a concentration of 10 ⁻⁷ M to the cultured tibia of wild-type littermate mice. This histological change is due to the non-peptidic NP receptor antagonist HS-142-1 (Circ. Res. 78: 606-61).
4, 1996), but it was completely suppressed by the addition of HS-142-1 when CNP was added to the cultured tibia of wild-type littermate mice at a concentration of 10 ^-7 M and when HS-142-1 was added. Met. These results indicate that in cultured tibia from CNP-transgenic mice, cNP is the second messenger of CNP.
Coupled with the fact that GMP production is increased, it is possible that Col may alter the in vivo phenotype of growth plate cartilage.
II-CNP transgene (mouse CNP cDN described in Example 1
A fragment of mouse procollagen a1 type II (Col 2a1)
It shows that the gene inserted in the promoter region DNA fragment) is fully functional. Increased extracellular matrix synthesis, represented by increased uptake of ³⁵ S in cultured tibia of CNP-transgenic mice, is consistent with increased extracellular matrix of CNP-transgenic mouse growth plates. Therefore, the mechanism of expansion of the growth plate of CNP-transgenic mice can be explained. The elongation of the epiphyseal cancellous bone observed in CNP-transgenic mice indicates that the replacement of cartilage with calcified bone is done smoothly. These experiments revealed the importance of CNP in endochondral ossification.

Next, a G380R type mutant FGFR3 (FGFR3^ach)
Obtained transgenic mice (Da University of Washington Da
vid M. Ornitz) and CNP-Transgeny
Mock Mice and CNP / FGFR3^ach -Double transformer
A transgenic mouse was created. CNP / FGFR3^ach -Doublet
In transgenic mice, the CNP-Tg gene is also FGFR3 ^ach
-Tg gene also grows in the resting chondrocyte layer of growth plate and proliferating chondrocytes
Expressed in a layer, FGFR3^ach-Transgenic Mau
The symptoms of dwarfism in Sus were visibly recovered. By the way
In addition, the endogenous CNP, GC-B and FGFR3 were
It was expressed in the hypertrophic chondrocyte layer.

The effect of the present invention is most clearly shown in FIG. FIG. 6A shows three-month-old wild type littermate mice, CNP-transgenic mice, FGFR3 ^ach − from the top.
Fig. 6D shows the overall appearance of the transgenic mouse, CNP / FGFR3 ^{ach -double} transgenic mouse, and Fig. 6D.
Shows an overview of their skeleton. The nasal-anal length of CNP / FGFR3 ^ach -double transgenic mice was similar to that of wild-type littermates, indicating that the limb shortening observed in FGFR3 ^ach- transgenic mice can be restored by CNP overexpression. There is.

Since CNP restores the symptoms of dwarfism in FGFR3 ^{ach -transgenic} mice, CNP is, at least in large part, in the regulatory pathway of endochondral ossification.
I think it is not located upstream. FG
FR3 ^ach- transgenic mouse shortened growth plate C
Overexpression of NP extended both proliferating and hypertrophic chondrocyte layers, but partial histological features were different from those of wild-type littermate mice. The extracellular matrix of both the proliferating chondrocyte layer and the hypertrophic chondrocyte layer was increased, the arrangement structure of the hypertrophic chondrocytes was disturbed, and the size of the hypertrophic chondrocytes was increased. CNP, like FGFR3, is not involved in the commitment of chondrocyte differentiation, as overexpressed CNP does not affect the delayed formation of secondary ossification centers in FGFR3 ^ach- transgenic mice. It promotes gene expression in chondrocytes at each stage of differentiation. Thus, the pathway by which CNP regulates endochondral ossification does not appear to be the same as that of FGFR3.

Further, using a mouse chondrocyte cell line, CNP and F
When the interaction with GFR3 was examined in vitro, CNP / GC
It was revealed that the -B system and the basic FGF / FGFR3 system (basic FGF is a ligand of FGFR3) mutually influences intracellular signal transduction in chondrocytes.

The present inventors are not bound by the above-mentioned specific theory, but from the above results, although the regulation mechanism of endochondral ossification of CNP and FGFR3 is different, FGFR3 ^{ach -transgenic} mouse It was confirmed that the growth retardation was restored by the overexpression of CNP. Therefore, it was suggested that CNP has a therapeutic effect as a pharmaceutical for promoting the growth of long bones for the purpose of treating patients with achondroplasia, and the present invention has been completed.
The main cause of known achondroplasia is FGFR3 gene hyperactivity due to mutation of FGFR3 gene, but there is also a possibility of achondroplasia symptom caused by FGFR3 dysregulation or FGFR3 gene hyperexpression. Conceivable. For these achondroplasia, activating GC-B and promoting the gene expression, protein expression, and protein function of its ligand, CNP, can be a new therapeutic agent. Regarding the promotion of CNP gene expression, there are cases where the expression of the endogenous CNP gene is enhanced, and gene therapy by introducing an exogenous CNP gene into the living body is also considered.

The therapeutic agent for achondroplasia of the present invention contains a substance that activates GC-B as an active ingredient, and further contains a carrier, an excipient, and other additives used in ordinary formulation. Is prepared using.

Examples of carriers and excipients for the formulation include
Lactose, magnesium stearate, starch, talc,
Examples thereof include gelatin, agar, pectin, acacia, olive oil, sesame oil, cocoa butter, ethylene glycol and the like, and other commonly used ones.

As the solid composition for oral administration, tablets, pills, capsules, powders, granules and the like are used. In such solid compositions, at least one active ingredient is at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, aluminomethasilicate. Mixed with magnesium etc. The composition is an additive other than an inert diluent according to a conventional method, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium fibrin glycolate, a solubilizing agent such as glutamic acid or aspartic acid. May be included. If necessary, the tablets or pills may be coated with sugar coating such as sucrose, gelatin, hydroxypropylmethylcellulose phthalate or a film of a gastric or enteric substance, or with two or more layers. Further included are capsules of absorbable material such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, commonly used inert diluents, For example, it may contain purified water, ethanol or the like. The composition may contain, in addition to an inert diluent, a wetting agent, an auxiliary agent such as a suspending agent, a sweetening agent, a flavoring agent, an aromatic agent, a preservative and the like.

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solutions and suspensions include, for example, water for injection and physiological saline for injection. Examples of non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, Polysorbate 80 (registered trademark) and the like. Such a composition may further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg lactose) and solubilizing agents (eg glutamic acid, aspartic acid). . They are,
For example, it is possible to sterilize by filtration sterilization with a microfiltration membrane, heat sterilization such as high-pressure steam sterilization, or an ordinary sterilization method such as blending of a bactericide. The injection may be a solution formulation or a lyophilized solution for reconstitution before use. As the excipient for freeze-drying, sugar alcohols and sugars such as mannitol and glucose can be used.

When the therapeutic agent of the present invention is used for gene therapy, a viral vector, preferably a lentiviral vector, an adeno-associated viral vector, more preferably an adenoviral vector, or a chemically synthesized liposome, viral envelope, or viral envelope. And a known liposome suitable for gene therapy such as a complex of liposome, a promoter sequence capable of functioning in a host cell, for example, cytomegalovirus promoter (CMV p
romoter), etc., downstream of the substance that activates GC-B, such as
The thing which integrated the nucleic acid which concerns on CNP can be used.

The therapeutic agent for achondroplasia of the present invention is preferably administered by an administration method generally used in medicine, for example, an oral administration method or a parenteral administration method. When the active ingredient is a GC-B agonist antibody, it is usually administered parenterally, for example by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, nasal, transpulmonary. Oral administration is also possible.

The active ingredient contained in the formulation of the present invention
The amount of substance that activates GC-B depends on the type of disease to be treated,
It can be determined according to the severity of the disease, the age of the patient, etc.
Generally, it can be administered in the range of 0.005 μg / kg to 100 mg / kg, but it is preferably administered in the range of 0.025 μg / kg to 5 mg / kg.

The therapeutic agent for achondroplasia of the present invention can be used in combination with a conventional therapeutic agent such as growth hormone, or in combination with orthopedic surgery such as artificial joint replacement of the hip joint or leg extension.

The present invention can include, but is not limited to, the following items. (1) Chondrodysplasia caused by the suppression of cartilage growth due to a mutation in the fibroblast growth factor receptor 3 (FGFR3) gene, which contains a substance that activates guanylyl cyclase B (GC-B) as an active ingredient Therapeutic agent. (2) The therapeutic agent according to (1) above, which restores the inhibition of cartilage growth by enlarging hypertrophic chondrocytes and increasing the extracellular matrix of the proliferating chondrocyte layer. (3) The therapeutic agent according to (1) or (2) above, wherein the substance that activates GC-B is a peptide. (4) The peptide is a C-type natriuretic peptide (CN
The therapeutic agent according to (3) above, which is P). (5) The therapeutic agent according to (4) above, wherein CNP is CNP-22 or CNP-53. (6) The therapeutic agent according to (1) or (2) above, wherein the substance that activates GC-B is a low molecular weight compound. (7) A therapeutic agent containing a substance that activates GC-B as an active ingredient is used for gene expression, protein expression of a substance that activates GC-B,
The therapeutic agent according to (1) or (2) above, which promotes the function of a protein. (8) The therapeutic agent according to (1) or (2) above, wherein the therapeutic agent containing a substance that activates GC-B as an active ingredient promotes CNP gene expression, CNP protein expression, and CNP protein function. (9) Chondrodysplasia due to cartilage growth suppression by mutation of fibroblast growth factor receptor 3 (FGFR3) gene, which comprises administering a substance that activates guanylyl cyclase B (GC-B) How to treat. (10) The treatment method according to the above (9), wherein the growth inhibition of cartilage is restored by enlarging hypertrophic chondrocytes and increasing the extracellular matrix of the proliferating chondrocyte layer. (11) The treatment method according to the above (9) or (10), wherein the substance that activates GC-B is a peptide. (12) The peptide is a C-type natriuretic peptide (C
The treatment method according to (11) above, which is NP). (13) The above (12), wherein the CNP is CNP-22 or CNP-53.
How to treat. (14) The above (9) or (10), wherein the substance that activates GC-B is a gene (for example, DNA) encoding a peptide
The described method of treatment. (15) The peptide is a C-type natriuretic peptide (C
The treatment method according to (14) above, which is NP). (16) The above (15), wherein CNP is CNP-22 or CNP-53.
How to treat. (17) The treatment according to the above (14) to (16), which comprises directly introducing the gene encoding the peptide or incorporating it into a vector suitable for gene therapy (for example, an adenovirus-derived vector) or a liposome. Method. (18) A substance described in (3) to (6) above for producing a therapeutic agent for achondroplasia caused by growth inhibition of cartilage due to mutation of fibroblast growth factor receptor 3 (FGFR3) gene use. Hereinafter, the present invention will be described in more detail with reference to Examples.

[0028]

EXAMPLES Example 1 Preparation of Recombinant Gene for CNP-Transgenic Mouse Production As shown in FIG. 1A, a mouse encoding 1-127 amino acids.
CNP cDNA fragment (489bp; FEBS Lett. 276: 209-213, 199
0) to the mouse procollagen a1 type II (Col2a1) promoter region DNA fragment (6.5 kb; Dev. Dyn. 204: 202-21
0, 1995). This promoter region DNA fragment is a B. de Crombrugg from Anderson Cancer Center in Huston.
he. Provided by MD. Also, this promoter region
The DNA fragment contained a promoter, exon 1, intron 1, and an artificial splice acceptor site, and was ligated to a CNP cDNA fragment downstream. Furthermore, exon 1 of this promoter region DNA fragment
The translation initiation codon therein is inactivated by a point mutation. A DNA fragment (0.3 kb) containing the bovine growth hormone polyadenylation signal is ligated downstream of CNP-cDNA. The NotI / NotI DNA fragment (7.3 kb) in FIG. 1A was purified for injection into fertilized eggs and used as a col-CNP DNA solution.

Example 2 Preparation of CNP-Transgenic Mouse A mouse for obtaining a fertilized egg (a mouse for egg collection) injected with a col-CNP DNA solution (hereinafter referred to as a DNA solution for injection) was C57BL / 6J pure strain mouse was purchased and used.
Females over 8 weeks old are superovulated and bred with males over 8 weeks old, a large number of fertilized eggs are collected, transferred to M2 medium and incubated at 37 ° C for 5
The cells were cultured in a carbon dioxide gas incubator. Then DNA
2 pL of the DNA solution for injection was injected into the male pronucleus of the fertilized egg by a microinjection method using an injection pipette. The fertilized egg into which the DNA solution for injection was injected was transferred to M16 medium and cultured overnight at 37 ° C. in a 5% carbon dioxide gas incubator. ICR inbred mice were purchased from CLEA Japan Co., Ltd. and used as female mice (temporary parent mice) for pregnancy, delivery and rearing of fertilized eggs into which the DNA solution for injection was injected and male mice to be mated with them. 8 vas deferens surgery was performed
Male mice over the age of a week were mated with female mice over the age of 8 weeks and those with vaginal plugs were used as foster mothers. The temporary parent was 0.01 mL of anesthetic prepared by diluting Nembutal injection solution (Dynabot Co., Ltd., sodium pentobarbital, 50 mg / mL) with a dilution solution (mixture of 20 mL of propylene glycol, 10 mL of ethanol, and 70 mL of sterilized water). / g body weight was injected intraperitoneally and the left and right oviducts were exposed outside the body by surgical operation. Of the fertilized eggs that had been cultured overnight, those that developed in the 2-cell stage embryo were selected and inserted into the left and right oviducts by 10 to 15 pieces, and then the surgical site was sutured. The foster mother was bred for 3 weeks, and after delivery, the pups were cut 5 weeks later, the tails were cut by about 1 cm, and the chromosomal DNA was extracted and purified using Easy-DNA Kit (manufactured by Invitrogen). The presence of the introduced gene was confirmed by PCR using this tail DNA. The mouse in which the presence of the transgene was confirmed reached the age of 7 weeks as an ancestor transgenic mouse, and then naturally mated with wild type C57BL / 6J at the age of 7 weeks or more to obtain a progeny transgenic mouse.

Mouse C57B for egg collection by gene injection experiment
A total of 336 L / 6J eggs yielded 5278 eggs, of which 228
Since 0 eggs were confirmed to be fertilized eggs, the DNA solution for injection was injected. The next day, 1600 (70%) developed in 2-cell stage embryos, of which 14
76 were transplanted into the oviducts of a total of 60 foster mothers. Thirty-seven foster parents gave birth, giving a total of 108 (7%) offspring mice. tail
A total of 4 animals (4
%) Founder transgenic mice (2 males, 2 females)
Was obtained. These founder transgenic mice were naturally mated with wild-type C57BL / 6J to obtain progeny.
Lines (Tg-1055♂, Tg-1077♀) transmitted the transgene to the offspring.

Example 3: Gene analysis of CNP-transgenic mice 3-1 Gene transfer by PCR of transgenic mice
Confirmation Confirmation of the introduced gene was performed by Southern hybridization using the extracted and purified tail DNA. Tail DNA
Digested with the restriction enzyme SacI and subjected to Southern hybridization using a ³² P-labeled CNP cDNA fragment (526 bp). The transgene had a 2.1 kb band and the endogenous gene had a 3.0 kb band.
A kb band was given (Fig. 1B). Regarding the copy number, the density of the 2.1 kb band was compared with the density of the endogenous 3.0 kb band, and the strain Tg-1055♂ strain judged to have 10 copies was used for the subsequent analysis.

3-2 Expression Analysis of IskD77N Gene by PCR Method Expression analysis of the transgene was performed by Real Time-PCR method. From newborn pups of wild type mice and transgenic mice, cartilage and other organs were rapidly excised from the lower vertebra and tail, and stored in liquid nitrogen. Physcotoron homogeni
After homogenizing with zer (NITION Medical Supply, Chiba, Japan), total RNA was isolated and purified using ISOGEN reagent. Superscript first strand synthesis kit
(GIBCO / BRL, Gaithersburg, MD), after synthesizing cDNA with Oligo-dT primer, upstream primer (in exon 1) and downstream primer (in cDNA) as shown in Fig. 1C.
Was used to perform PCR. PCR reaction at 95 ° C for 30 seconds, 58 ° C
The three-step reaction was carried out 45 times for 30 seconds at 72 ° C. for 1 minute. PCR
After the reaction, 10 μL was dispensed and assayed by 1% agarose electrophoresis. A positive band of 450 bp was confirmed only in cartilage, but not in brain, heart, lung, liver, kidney, small intestine, or muscle. In wild type littermate mice, a positive band of 450 bp was not detected in cartilage and other organs.

Example 4 Measurement of Growth Curve of CNP-Transgenic Mouse The length between the nose and anus (hereinafter, nose-anal length) was measured every week to prepare a growth curve of the mouse. CN during the weekly production period
P-transgenic mice were indistinguishable from wild-type littermates. On the first day after birth, Alizar the bone and cartilage
When stained with in red S and Alcian blue, long-term overgrowth of long bones, vertebrae, skulls and cartilage of limbs of CNP-transgenic mice was observed (Fig. 2A). No delayed ossification of the epiphysis of the extremities at this time was observed. In the CNP-transgenic mouse, the ossification center of the phalanx had already appeared, similar to the wild-type littermate mouse. As they grew, CNP-transgenic mice had a markedly increased nasal-anal length (Fig. 2B). 10-week-old CNP-transgenic female mice were 19% longer than wild-type litter female mice (n = 7). CNP-transgenic male mice were longer than wild type littermate males (n = 7), but to a lesser extent than females (10%). Homo CNP-
Transgenic male mice were longer than heterozygous CNP-transgenic male mice (6% female, 4% male, n =
7). 6-month-old CNP-transgenic mice had soft X
According to the line analysis, the limb length is longer than that of the wild type littermate mouse,
The length of the vertebrae and the length of the long axis of the skull were greatly increased. All of these bones were formed by endochondral ossification, but the width of the skull was not increased (Fig. 2).
C). The vertebrae and proximal long bones (upper tibia and femur) are particularly prominent, with 28%, 25% of each length of wild type littermate mice,
It was 23% (n = 6).

Example 5: Histological analysis of CNP-transgenic mice For light microscopy, the tibia and vertebrae were removed and 10% fo
It was fixed with rmalin / PBS (pH 7.4). 20% ED for calcified bone
It was decalcified in 10% formalin / PBS (pH 7.4) containing TA. Paraffin blocks were prepared by conventional histochemical methods. Sections (5-6um) were prepared at multiple sites, and Alcian blue
After staining with (pH 2.5), counterstaining was performed with hematoxylin / eosin. The length of each layer of the growth plate, the diameter of growing hypertrophic chondrocytes,
The BrdUrd labeling index of the proliferating chondrocyte layer was analyzed using the NIH image program on a Macintosh computer. BrdUrd staining was performed on 2-week-old mice.
BrdUrd (100 ug / g body weight) was injected intraperitoneally and killed 1 hour later. Immunohistochemical staining in which BrdUrd was incorporated into cells of the tibial growth plate was performed by a standard method. Von using undecalcified sections to assess the degree of calcification of the specimen.
Kossa staining was performed.

For in situ hybridization analysis, digoxigenin-labeled sense and antisense riboprobes were prepared from digoxigenin RNA from rat pro-a1 (X) collagen cDNA fragment and mouse pro-a1 (II) collagen cDNA fragment. labeling kit (Roche Diagnostics, Ind
ianapolis, IN).

In the prenatal CNP-transgenic mouse, typical histological changes were not observed in the cartilage of the epiphysis, but with growth, at least 3 weeks of age and after, CNP-
The height of the growth plate of the long bones of the vertebrae of the transgenic mice was remarkable (Fig. 3A, B). Among the cartilage layers of the growth plate of the tibia of 3-week-old mice, the hypertrophic chondrocyte layer (234 ± 12
μm vs. 207 ± 14 μm, n = 4, p <0.05), also for the proliferating cartilage layer (215
± 3 μm vs. 193 ± 16 μm, n = 4, p <0.05) was also longer in CNP-transgenic mice than in wild-type littermate mice. Hypertrophic chondrocyte layer and proliferating cartilage layer are in situ
Hybridization analysis reveals type X collagen or type II collagen (Fig. 3E-H). Chondrocyte size (24.3 ± 1.2 μm vs. 21.2 ± 1.
3 μm, n = 6, p <0.05) are enlarged (Fig. 3C, D). The length of the resting chondrocyte layer was unchanged in CNP-transgenic mice. The band of BrdUrd-positive chondrocytes was wider in CNP-transgenic mice than in wild-type littermates, but the number of BrdUrd-positive chondrocytes changed (13.3 ± 3% vs 12.5 ± 2.9%, n = 4) Was absent (Fig. 3K, L). Von Kos of tibial growth plate of 3 week old mice
From the sa staining, the trabecular bone at the epiphysis formed from the adjacent hypertrophic cartilage layer was clearly longer in the CNP-transgenic mouse than in the wild-type littermate mouse, and the volume of the trabecular bone was larger ( (Fig. 3I, J).

Example 6: Effect of cartilage-specific CNP expression in cultured cells of fetal tibia of CNP-transgenic mice Fetal tibia of CNP-transgenic mice or wild type littermate mice was excised at 16.5 days after mating, Suspension culture was carried out for 4 days in an artificial medium. In order to inhibit the effects of endogenous CNP, tibial cultures were treated with a non-peptidic NP receptor antagonist, HS-142-1 (Komatsu et al., Circ Res. 78: 606).
-614, 1996) was added to the medium at a concentration of 50 mg / L. At the end of the culture period, the cultured tibia was measured for its major axis length, fixedly embedded and used for histological analysis. Alcian blue (pH2.5) is a 5 μm thick slice from the embedded specimen.
Staining and counterstaining with hematoxylin / eosin were performed. CGM of cultured tibia
The amount of P was measured by RIA after 4 days of culture. Glycosaminoglycan synthesis in cultured tibia was assessed by measuring the uptake of ³⁵ SO ₄ (Mericq et a., Pediatr Res 4
7: 189-193, 2000). That is, the cultured tibia of CNP-transgenic mice and wild-type littermate mice contained 5 μCi / ml of N.
Labeling was carried out with a ₂ ³⁵ SO ₄ (Amersham, specific activity 100 mCi / mmol) for 1 hour. Cultured tibia is packed with Pack's saline (Sigma Chemica
(Co. St. Louis, MO), rinsed 3 times for 10 minutes, and digested in 1.5 ml of a fresh medium containing 0.3% papain at 60 ° C. for 24 hours. Next, 0.5 ml of 10% cetylpyridinium chloride (Sig
ma Chemicl Co.)-0.2M NaCl was added and the mixture was kept warm at room temperature for 18 hours to precipitate glycosaminoglycan. 1 ml of sediment
0.1% cetylpyridinium chloride (Sigma Chemicl Co.)-
After washing 3 times with 0.2M NaCl and dissolving in 1 ml of 23N formic acid
^The amount of ³⁵ SO ₄ was measured with a liquid scintillation counter.

Before culture, the cultured organ pieces of the tibia of CNP-transgenic mice were significantly longer than those of wild-type littermates (FIG. 4A). During the culture, the length of the major axis of the cultured organ pieces of the tibia of the CNP-transgenic mouse was significantly increased, and after 4 days of culture, it was about 35% longer than that of the wild-type littermate mouse.
% Longer (n = 6, Fig. 4A). There was a significant increase in cartilage primordia (40% increase) compared to the other parts of the tibial culture organ pieces. HS-142-1 inhibits the effects of cartilage endogenous CNP but inhibits the natural growth of cultured tibial organ pieces from wild-type littermate mice (Fig. 4A). Furthermore, the increase in the length of the cultured organ pieces of tibia derived from CNP-transgenic mice was completely inhibited by HS-142-1 (50 mg / L), and
2-1 The length was the same as that of the cultured organ piece of the tibia of the wild-type littermate mouse that had been treated (FIG. 4A). The amount of cGMP in the tibial cultured organ pieces of CNP-transgenic mice was approximately 9 times that of wild-type littermates (18.7 ± 1.2 fmol / mg protein vs 2.1 ± 0.2 fmol / mg protein, n = 5). , FIG. 4B). Glycosaminoglycan synthesis was increased by 25% in CNP-transgenic mice compared to wild-type littermates (2300 ± 170 cpm).
/ Tibia vs. 1840 ± 140 cpm / Tibia, n = 6, Figure 4C). Histologically, the proliferating cartilage layer (369 ± 26 μm vs. 287 ± 26 μm) of the epiphyseal cartilage of the cultured organ pieces of the tibia of CNP-transgenic mice.
14 μm, n = 4, p <0.05) hypertrophic chondrocyte layer (450 ± 29 μm)
294 ± 16 μm, n = 4, p <0.05) also increased in height, and the cartilage matrix of the proliferating chondrocyte layer was treated with Alcian b
The extracellular region stained with lue was also increased (Fig. 5A, B).
The hypertrophic chondrocyte layer was also enlarged (17.8 ± 0.8 μm vs. 1
5.4 ± 1.4 μm, n = 6, p <0.05, Figure D). At the same concentration in CNP-transgenic mice, HS-142-1 also abolished the change of epiphyseal cartilage of cultured tibia.

Example 7: Analysis of CNP / FGFR3 ^ach -double transgenic mouse Female CNP-transgenic mouse and male FGFR3 ^{ach -transgenic} mouse (University of Washington, David M.
(Obtained from Professor Ornitz)). Since FGFR3 ^ach- transgenic mice are FVB / N background, double transgenic mice use only F1, and control CNP, FGFR3 ^ach , and wild type mice
Littermate mice were used.

At 3 months of age, CNP-transgenic mice were longer than wild-type littermates, and FGFR3 ^{ach -transgenic} mice were shorter than wild-type littermates (FIG. 6A). The nose-anal length of CNP / FGFR3 ^{ach -transgenic} mice was almost the same as that of wild-type littermate mice. At this time, the expression level of CNP in the cartilage of the CNP / FGFR3 ^{ach -transgenic} mouse was equivalent to that of the CNP-transgenic mouse (Fig. 6).
C). CNP / FGFR3 ^ach- Transgenic mouse, FGFR3
^The growth curves of ^ach- transgenic mice and wild-type littermate mice with nasal-anal length revealed that the growth retardation of FGFR3 ^ach- transgenic mice was ^rescued by CNP overexpression in growth plate cartilage. 1
At 0 weeks of age, CNP / FGFR3 ^{ach -transgenic} mice had a nose-anal length of 94.7 ± 4.0 mm, FGFR3 ^{ach -transgenic} mice had 87.0 ± 2.6 mm, which was 8% longer than wild-type littermates (97.0 ± 4.2 mm). )) (Fig. 6)
B). According to soft X-ray analysis, the shortened bone length observed in the FGFR3 ^{a ch} -transgenic mouse is
The length of the parietal bone, the length of the humerus, and the length of the vertebra (L1-7) are CNP /
It was partially ^{rescued in} FGFR3 ^{ach -transgenic} mice. Width skull, FGFR3 ^ach - Transgenic mice CNP / FGFR3 ^ach - Transgenic mice were unchanged (Fig. 6D). 2-week-old CNP / FGFR3 ^ach-
Transgenic mice, FGFR3 ^ach- transgenic mice, wild-type litter ^{mice.Microscopic} analysis of proximal tibial growth plate cartilage showed that the height of hypertrophic chondrocytes in FGFR3 ^ach- transgenic mice was higher than that of wild-type littermate mice. Decreased (169 ± 15 μm vs. 220 ± 15 μm). CNP / FGFR
It was recovered in 3 ^ach- transgenic mice (229
± 21 μm, Fig.7A-C). Compared with FGFR3 ^{ach -transgenic} mice and wild-type littermate mice, CNP / FGFR3 ^{ach -transgenic} mice showed abnormal columnar arrangement of hypertrophic chondrocytes and extracellular matrix of prehypertrophic cartilage and upper hypertrophic chondrocyte layers. Dilation was observed (Fig. 7D-F). CNP / FGFR3
^ach - size of hypertrophic chondrocytes of transgenic mice, FGFR3 ^ach - was significantly greater than the transgenic mice and wild type littermates (20.1 ± 1.5 μm, 18.4
± 1.2 μm, 19.0 ± 0.2 μm, n = 6, p <0.05, FIG. 7D-F).
In the proximal tibia of 10 week old mice, the secondary ossification centers are
It was well formed in wild-type littermate mice, but not in FGFR3 ^{ach -transgenic} mice or CNP / FGFR3 ^{ach -transgenic} mice (Fig. 7A-C).

Example 8: CNP using mouse chondrocyte cell line
Of interaction between FGFR3 and FGFR3 mouse chondrocyte cell line ATDC cells (J. Bone. Miner. Res., 1
2, 1174-1188, 1997, obtained from Kyoto University Research Institute for Frontier Medical Sciences, Assistant Professor Chichishuku Minami, and Professor Yuji Kai) were pretreated with 1-10 ng / ml of basic FGF (SIGMA product), a ligand for FGFR3. . Then, the cells were stimulated with CNP 10 ^-9 to 10 ^-7 M,
The intracellular cGMP production was measured by the RIA method (cyclic GMP Assay Kit, product of Yamasa Shoyu Co., Ltd.). Also phosphorylation
MAP-K antibody and MAP-K antibody (both Cell Signaling Techn
Phosphorylation of p44 and p42 MAP kinase (ERK1 / 2) after basic FGF stimulation by MAP, MAP: mitogen-activated protein, mitogen-activated protein, MAP kinase (MEK) and p44
The expression of MAP kinase (ERK1) was measured.

As a result, intracellular CGMP production after CNP stimulation was 70% of that of the control after pretreatment with 1 ng / ml of basic FGF for 1 hour.
Fell to. In addition, phosphorylation of ERK1 / 2 by basic FGF was significantly suppressed by pretreatment with CNP 10 ^-7 M for 1 hour.

From this, the CNP / GC-B system and basic FGF / FG
It was revealed that the FR3 system mutually influences intracellular signal transduction in chondrocytes.

[0044]

EFFECTS OF THE INVENTION The therapeutic agent for achondroplasia provided by the present invention acts as a low-molecular substance that activates CNP gene, CNP protein, or GC-B at an action point different from that of growth hormone. It is possible to treat achondroplasia. The therapeutic agent for achondroplasia of the present invention has less burden and pain on the patient as compared with the conventional orthopedic surgery such as artificial joint replacement of the hip joint and leg extension, and can be an excellent therapeutic agent in consideration of the QOL of the patient. . Furthermore, the transgenic animal according to the present invention can be used for verifying its effectiveness against achondroplasia caused by mutations other than the G380R mutation in FRFR3.

[Brief description of drawings]

FIG. 1 is a diagram showing the production of a transgenic mouse that overexpresses CNP in a cartilage-specific manner. A. A schematic diagram showing the structure of a recombinant gene for producing CNP-transgenic mouse; B. CNP-transgenic mouse Showing the results of Southern hybridization using tail DNA of C .; C. Col I of each organ derived from CNP-transgenic mouse
It is a photograph showing a result of analysis by I-CNP expression RT-PCR.

FIG. 2 is a view showing an overview of CNP-transgenic mice, A. A photograph showing the skeletons of 1-day-old wild-type mice (top) and CNP-transgenic transgenic mice (bottom); B. CNP- FIG. 3 is a graph showing the growth curves of male (left) and female (right) transgenic mice, where the black circles (●) are hetero, the black squares (■) are homo, and the white circles (◯) are wild-type littermate mice; C. Left side is a 6 month old wild-type litter female mouse (left) and CNP.
-Soft X-rays of the skull (top) and legs (bottom) of the transgenic female mouse (right), the right side is wild type litter female mouse (open bar) and CNP-transgenic female mouse (black bar). 4) is a graph showing a comparison of some bone lengths measured from the left side photograph of FIG.

FIG. 3 is a view showing histological analysis of growth plates of CNP-transgenic mice, where A to D are Alcian blue and hemat.
Fig. 2 is a photograph showing oxylin / eosin staining (3 weeks old), A. Tibial growth plate of wild-type litter mouse (x50); B. Tibial growth plate of CNP-transgenic mouse (x5).
0); C. Tibial growth plate of wild-type litter mouse (x200); D. Tibial growth plate of CNP-transgenic mouse (x20)
0), and E to H are in
Fig. 2 is a photograph showing in situ hybridization (2 weeks old), E. tibial growth plate of wild-type litter mouse (Type II collagen, x200); F. Tibial growth plate of CNP-transgenic mouse (Type)
II Collagen, x200); G. Tibial growth plate of wild-type litter mouse (Type X Collagen, x200); H. Tibial growth plate of CNP-transgenic mouse (Type)
X collagen, x200), and I to J are photographs showing Von Kossa staining (3 weeks old), I. Trabecular bone of the epiphysis of wild-type littermate mice (x50); J. CNP-transgenic mice Epiphyseal trabecular bone (x5
0), K to L are photographs showing BrdUrd staining (2 weeks old), K. wild type litter mouse tibial growth plate (x50); L. CNP-transgenic mouse tibial growth plate (x50).
Is.

Fig. 4 is a diagram showing an organ culture of the tibia of a CNP-transgenic mouse, A. The left side is a photograph showing the appearance of the tibia of a 16.5-day-old mouse fetus after 4 days of culture (upper left) Type littermate mouse; (top right) CNP-transgenic mouse; (bottom left)
Wild type littermate mouse (HS-142-1 (50 mg / L) added to the medium); (lower right) CNP-transgenic mouse (HS-142
-1 (50 mg / L) was added to the medium), and the right side is a graph showing the time course of tibia length at the start of tibial organ culture and after 4 days of culture. White circles are wild-type littermate mice, n =
6, open squares are CNP-transgenic mice, n = 6, filled circles are wild-type littermate mice (HS-142-1), n = 6, filled squares are CNP-
Transgenic mouse (HS-142-1), n = 6). * Is
P <0.05 CNP-transgenic mouse vs. wild type litter mouse, ** indicates P <0.05 HS-142-1 treated wild type litter mouse vs. untreated wild type litter mouse, *** indicates P <0.01 HS-142 -1 processing
CNP-transgenic mice versus untreated CNP-transgenic mice; B. CNP-transgenic mouse embryonic culture tibial cGM
It is a graph which shows the quantity (n = 5) of P. * Indicates P <0.01 CNP-transgenic mice vs. wild-type littermates; C. CNP-transgenic mice embryos in cultured tibia
It is a graph which shows the amount of incorporation of ³⁵ SO ₄ (n = 6). * Is P
<0.05 CNP-transgenic versus wild type littermate.

[Fig. 5] Histochemical analysis of cultured tibia of CNP-transgenic mice (Alcian blue and hematoxylin / eosin staining)
A. Wild type littermate mouse (x25); B. CNP-transgenic mouse (x25); C. CNP-transgenic mouse (HS-142-1 treated) (x2
5); D. Wild type littermate mouse (x200); E. CNP-transgenic mouse (x200); F. CNP-transgenic mouse (HS-142-1 treated) (x2
00).

FIG. 6 is a diagram showing the overall phenotype of CNP-transgenic mice, FGFR3 ^{ach -transgenic} mice, and CNP / FGFR3 ^{ach -double} transgenic mice. A. From the top, a 3-month-old wild-type littermate Mouse, CNP-transgenic mouse, FGFR3 ^{ach -double} transgenic mouse, CNP / FGFR3 ^{ach -double} transgenic mouse showing the overall appearance; B. FGFR3 ^{ach -transgenic} female mouse (black triangle),
CNP / FGFR3 ^ach- transgenic female mouse ( ^open square), wild-type littermate mouse (black circle) showing growth curve (n = 7) of nasal-anal length; C. Cartilage-derived total RNA by RT-PCR Col II-C used
Fig. 2 is a photograph showing detection of NP expression, Lane 1, wild type littermate mouse; lane 2, CNP-transgenic mouse; lane.
3, FGFR3 ^{ach -Transgenic} mouse; D. Left side is from the top, 3 month old wild type litter mouse, CNP-
It is a photograph showing the appearance of the skeleton of a transgenic mouse, FGFR3 ^{ach -double} transgenic mouse, CNP / FGFR3 ^{ach -double} transgenic mouse, and the right side is
Comparison of bone lengths in wild type littermate mice (white), CNP-transgenic mice (black), FGFR3 ^{ach -transgenic} mice (shaded), CNP / FGFR3 ^{ach -double} transgenic mice (shadow) (n = 4) is a graph showing. * Is p <0.05. The skull (anterior-posterior length), the skull (horizontal length), the humerus, the femur, and the vertebrae are shown.

FIG. 7 is a photograph showing histochemical analysis (Alcian blue and hematoxylin / eosin staining) of tibia of growth plate of 2-week-old mouse, A. Wild type littermate mouse (x50); B. FGFR3 ^ach -transgenic Mouse (x50); C. CNP / FGFR3 ^ach- Transgenic mouse (x50); D. Wild type litter mouse (x100); E. FGFR3 ^ach- Transgenic mouse (x100); F. CNP / FGFR3 ^ach- Transgenic Mouse (x100).

Claims (5)

[Claims] 1. A cartilage-free tissue which contains a substance that activates guanylyl cyclase B (GC-B) as an active ingredient and suppresses the growth of cartilage by mutation of the fibroblast growth factor receptor 3 (FGFR3) gene. Plastic treatment agent. 2. The therapeutic agent according to claim 1, which restores growth inhibition of cartilage by enlarging hypertrophic chondrocytes and increasing extracellular matrix of proliferating chondrocyte layer. 3. The therapeutic agent according to claim 1 or 2, wherein the substance that activates GC-B is a peptide. 4. The therapeutic agent according to claim 3, wherein the peptide is C-type natriuretic peptide (CNP). 5. The therapeutic agent according to claim 4, wherein CNP is CNP-22 or CNP-53.