KR19990036081A - Human MP52 aripiprazole protein - Google Patents

Abstract

The present invention relates to pharmaceutical medicinal compositions for promoting cartilage and tibia formation comprising human MP52 Arg and especially human MP52 Arg. In particular, the pharmaceutical composition is useful for the treatment of tibial diseases caused by abnormal tibial metabolism such as osteoporosis, tibial fracture treatment and orthopedic reconstruction, tibial transplantation, plastic surgery and dental treatment. It is also useful for treating cartilage disease.

Description

Human MP52 aripiprazole protein

The present invention relates to a pharmaceutical composition for promoting cartilage and tibia formation comprising a novel compound, human MP52Arg and in particular human MP52Arg. In particular, the present pharmaceutical composition is useful for the treatment of tibial diseases caused by abnormal tibial metabolism such as osteoporosis, fracture treatment and orthopedic reconstruction, tibial transplantation, plastic surgery and dental treatment. This is particularly useful for the treatment of connective diseases.

Pharmaceutical compositions comprising vitamin D3, calcitonin, estrogen and bisphosphonate derivatives have been used clinically to treat tibial disorders. However, its therapeutic results have not been entirely satisfactory and a much better pharmaceutical composition is highly needed.

It has been reported that TGF-beta growth factor groups including BMP, TGF, and an inhibin-related protein are useful for wound healing and tissue treatment. The tibolytic activity of some of these proteins is also known. PCT Patent Applications Nos. WO 93/16099 and WO 95/04819 describe DNA sequences encoding human TGF-? -Like proteins and describe human MP52 as a preferred protein.

EE Storm et al., In Nature, (1994, vol. 368, p. 639-643), found that a mutation of the TGF-β phase and the member of the mouse growth / differentiation factor 5 (GDF5) gene is responsible for the brachypodism of the mouse . Mouse GDF5 has the same amino acid sequence of the expected mature form as human MP52, except for one amino acid. However, there is no suggestion of using these proteins in the treatment of tibial diseases.

It is therefore an object of the present invention to provide additional growth factors useful as stimulants for tibial or cartilage formation. Thus, one embodiment of the present invention is a protein human MP52 Arg comprising amino acids 1 to 121 of SEQ ID NO: 1.

In fact, it has surprisingly been found that, by the present invention, when expressing a suitable DNA sequence, there is a cell line forming a mixture of human MP52 Arg or human MP52 and human MP52 Arg. For the first time, the present invention succeeded in providing a human MP52 Arg having tibolytic activity, which is useful for preventing and / or treating tibial diseases.

Also, human MP52 Arg induces cartilage formation from undifferentiated hepatocyte tissue cells and stimulates osteoblast differentiation and maturation. Thus, human MP52 Arg is effective in preventing and / or treating tibial disorders caused by abnormal tibial metabolism such as osteoporosis. It also promotes the treatment of tibial fractures. Moreover, it is useful for orthopedic reconstruction, tibial transplantation and dental treatment due to its tibolytic activity. In addition, MP52 Arg is effective in preventing and / or treating cartilage disorders caused by abnormal cartilage metabolism.

A second object of the present invention is to introduce at least a portion of the DNA sequence as shown in SEQ ID NO: 1 into a suitable host cell under conditions that favor protein formation and expression of the DNA sequence, And separating from other proteins to be produced.

Within the framework of the present invention, the DNA sequence set forth in SEQ ID NO: 1 may be used to prepare human MP52Arg, or a portion thereof may also include a DNA sequence in a suitable vector / host cell system if they still encode human MP52Arg Can be expressed. Appropriate expression systems are readily known by those skilled in the art and can be readily determined by routine experimentation with minimal requirements for the DNA sequence of SEQ ID NO:

After protein formation, the protein is recovered from the host cell by means of methods known per se and eventually separates human MP52 Arg from it. In particular, highly precise differentiation methods known to those skilled in the art can be used to separate human MP52 Arg and MP52 Arg different from each other on a single amino acid basis. One example is MP52, which is performed according to the method of Davis (Ann. NY Acad. Sci., 121, 404-427, 1964) with slight modification, such as adding 0.1% Nonidet P-40 and 6 M urea. Arg electrophoretic separation. After electrophoresis, isolated MP52 Arg is electroeluted from the gel in the same buffer.

A third object of the present invention is a pharmaceutical composition containing human MP52 Arg. Optionally, such compositions may also comprise conventional carrier materials, auxiliary substances, diluents and / or fillers. The pharmaceutical composition of the present invention is useful for treatment or prevention of tibial formation, tibia, cartilage, connective tissue, skin, mucous membrane, epithelium or dental damage, and for application to dental implants and for wound-healing and tissue regeneration processes.

For the treatment of tibial disorders caused by abnormal tibial metabolism, human MP52 Arg is administered to the system by intravenous, intramuscular and intraperitoneal injections such as injections, oral administration, parenteral administration such as suppositories, and other convenient and convenient methods .

For the treatment of tibial fractures, it is administered locally to the system by injection, oral and parenteral administration. Also a matrix containing human MP52 Arg is also preferably implanted close to the fractured tibia. Suitable matrices are natural polymers such as collagen and fibrin clots and artificial polymers that can be biodegraded in vivo, such as polylactated glycolic acid.

In the case of orthopedic reconstruction, plastic surgery, tibial implantation and dental implantation, for example, the human MP52 Arg can be coated on the surface of the tibia and teeth that can be implanted using collagen paste, fibrin glue and other sticky materials have. It can also be applied to the alveolar bone, tibia, or tissue around which the tibia and teeth are implanted. For tibial implants, this can be used for natural and artificial tibia. As artificial tibia and tooth material, conventional materials such as metals, ceramics, glass and other natural or artificial inorganic substances are used. Hydroxyapatite is the preferred artificial material. The artificial tibia can be composed of a thick material inside and a porous material in the other part. For example, thick steel covered with porous steel can be mentioned. Porous hydroxyapatite is one of the materials for making artificial tibia. When such a porous material is used, human MP52 Arg can penetrate into it. The artificial tibia surface can also be roughened to maintain human MP52 Arg on the surface.

In order to promote the reconstruction of the tibia, it is advantageous to apply human MP52 Arg to the portion of the tibia where the cancer is removed.

The capacity of human MP52 Arg that can be obtained is determined according to the application purpose and method. Generally, doses of 1 - 100 μg / kg are given to the system and the preferred dose is 30 μg - 30 mg per site.

The purified human MP52 Arg can be formulated into conventional forms such as injection solutions, pills, capsules and suppositories. For local administration, human MP52 Arg may be included in a matrix such as collagen, fibrin glue, and polylactated glycolic acid. For implantation, this is applied to the porous part of the tibia and teeth or to the surface.

The present invention is illustrated by the following examples.

Example 1

Construction of expression vector for MP52 Arg

The plasmid pSK52s (WO 95/04819) was digested with HindIII and extracted from 0.8% low melting agarose gel to isolate the DNA fragment containing the cDNA encoding the complete coding region for MP52 and ligated to the pABstop vector (Behringwerke AG; ≪ / RTI > Gerd Zettlmeissl feed). The structure of the thus obtained MP52 expression vector, pMSS99 (5.0 kb) was confirmed by DNA sequencing and restriction enzyme mapping. The genetic elements of pMSS99 are shown diagrammatically in FIG. The MP52 sequence of pMSS99 includes nucleotide 576-2279 of Sequence Listing No. 1.

Example 2

Identification of CHO to generate MP52 Arg

CHO-DUKX-B11, which is a dhfr-deficient CHO cell as an expression plasmid of pSVOAdhfr (Zettlmeissl, G. et al., (1987) Bio / Technology 5, 720-725) and MP52 (pMSS99) by calcium phosphate- (Urlaub, G. Chasin, LA (1980) Proc. Natl. Acad. Sci. USA, 77, pp.4216-4220) were simultaneously transfected. After that, a clone that produces a large amount of MP52 Arg by gene amplification protocol using methotrexate (MTX) was identified.

In summary, 10 μg of pMSS99 and 2 μg of pSVOAdhfr were dissolved in 1 ml of 25 mM HEPES - 140 mM NaCl - 0.75 mM Na ₂ HPO ₄ (pH 7.05) and then mixed with 50 μl of 2.5 M CaCl ₂ . The precipitate thus obtained was superimposed on CHO-DUKX-B11 cells and incubated at room temperature for 30 minutes. Ribo-and deoxyribonucleoside (MBM alpha ⁺ ) and MEM ALPHA media containing 10% fetal bovine serum (FBS) were then added to the cell layer and incubated in a CO ₂ incubator for 4-6 hours. Containing 10% FBS in MEMα ^* MEMα containing FBS for after treatment for 3 minutes at room temperature in 10% glycerol ⁺ 10% and cultured for 2 days in the cell. After ribonucleic containing dialyzed FBS was of 10% and deoxyribonucleic - by placing the cells in MEM ALPHA medium (MEMα ^_) without a nucleoside selected the elastic element. Modified clones were isolated and the expression of MP52 Arg was analyzed by Western blot analysis as described in the next section.

A high producer clone of MP52 Arg was identified by a gene amplification protocol using methotrexate (MTX). The MP52 gene was amplified according to the pSVOAdhfr gene by sequentially cloning the clones producing MP52 Arg by increasing the concentration of methotrexate (MTX) stepwise. Several clones were obtained (at 10 ⁶ cells / 400 nM) producing 1-3 μg of mature MP52 Arg. 24 hours

Example 3

Detection of MP52 Arg in the culture supernatant

Clones were examined for expression of MP52 Arg by Western blot analysis as follows: The culture supernatant (1-15 μl) was subjected to SDS-PAGE (15-25% polyacrylamide gradient gel, Daiichi Pure Chemicals) And the protein was transferred to a PVDF membrane (Clear Blot Membrane-P, ATTO). Membranes were blocked with Block Ace (Dai-Nihon Seiyaku) and rinsed with Tris-buffered saline (TBS) and then treated with 10 μl / ml chicken antibody in MP52 Arg in 10-fold diluted Block Ace. Membranes were washed with 0.1% Tween 20 in TBS (TTBS) and the membranes were treated with rabbit anti-chicken IgG-ALP conjugate (Sigma A 9171) in 10-fold diluted Block Ace for 1 hour. The membrane was washed with TTBS and reacted with an alkaline phosphatase conjugate substrate kit (BIO-RAD) to visualize the binding to MP52 Arg.

Example 4

Cell culture of MP52 Arg-producing CHO cell line

A CHO cell line with a high degree of production of MP52 Arg, MP52, MC-2 (deposited with No. FERM BP-5142, Japan National Institute for Biomedical Science and Human Technology) was diluted with 10% FBS, 400 nM MTX, 100 U / ml of penicillin and 100 μg / ml streptomycin supplemented with a MEMα ^- were grown in roller bottles containing. After incubation with MC-2 cells, they were washed with serum-free MEMα-n and incubated with 10 mM HEPES (pH 7.3), 10 KIE / ml aprotinin, 1 mM sodium butyrate, 6 μg / In a serum-free DME / F12 medium supplemented with 5 μg / ml transferrin, 18 μg / ml ethanolamine, 9 μg / ml insulin, 100 U / ml phenylsiline and 100 μg / ml streptomycin Lt; / RTI > Conditioned medium was collected daily for one week.

Example 5

refine

One liter of the culture supernatant was mixed with 0.1 volume of 0.2 M sodium phosphate buffer (pH 6.0) and applied to a POROS HS column (10 ml, PerSeptive Biosystems). And eluted with linear slant 0.3 - 2 M NaCl. The eluate containing MP52 Arg was applied to a reversed-phase column (RESOURC, Pharmacia). Eluted by linear gradient of acetonitrile containing 0.05% TFA and MP52 Arg eluted at approximately 35% acetonitrile. N-terminal amino acid sequence analysis was performed on MP52 Arg purified using a pulsed liquid phase sequencer (Applied Biosystems model 476). The results are shown in Table 1. MP52 Arg shows proteolysis from its precursor at Arg (380) -Arg (381) (amino acid position sequence No. 1 -1 and +1). However, the precursor can also proceed in Arg (381) -Ala (382) (+1 and +2 in amino acid position sequence No. 1).

Example 6

Biological activity

Bone-producing ROB-C26 cells (Calcif. Tissue Int. Vol. 49, p. 221-225, 1991) were plated on a 48-well multi-well plate (Coaster) at a density of 1.5 x 10 ⁴ cells / And preincubated for 3 days in MEM < RTI ID = 0.0 > ^_ < / RTI > containing 10% hepatitis B liver serum (FBS). After removal of the culture medium, fresh MEMα ^_ containing 10% FBS and MP52 Arg (2 μl / ml) in 10 mM HCl successively diluted were added to the culture and incubated for 6 days with medium and additives varied every 3 days . Phosphate-washing the cell layer with a buffered saline and extracted with 0.2% Nonidet in containing 1 mM MgCl _2. The activity of alkaline phosphate (ALP) was measured according to the procedure of Takuwa et al. (Am. J. Physiol. Vol.257, p. E797-E803, 1989). As shown in Table 2, treatment of ROB-C26 with MP52 Arg resulted in a concentration-dependent increase in total ALP activity per well.

Effect of CHO cell-induced MP52 Arg on ALP activity in ROB-C26 cell line The concentration of MP52 Arg (ng / ml) ALP activity (nmol / min / well) The vehicle (10 mM HCl) -treated control 5.22 ± 0.19 3 5.79 ± 0.62 10 7.10 ± 0.75 ^* 30 8.36 ± 0.77 ^* 100 9.35 ± 0.67 ^* 300 10.34 + - 1.06 ^* 1000 16.09 + - 1.26 ^*

The figure means ± SD of 4 cultures. ^* p < 0.01

Compared to vehicle-treated control (Dunnett's test).

Sequence List

(1) General information:

(i) Applicant:

(A) Name: Biopharm Gesellschaft zur biotechnologischen Entwicklung von Pharmaka mhH

(B) Distance: Czernyring 22

(C) City: Heidelberg

(E) Country: Germany

(F) Postal code (ZIP): 69115

(ii) Title of invention: Novel protein human MP52 Arg

(iii) SEQ ID NO: 2

(iv) Computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC Compatibility

(C) Operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30 (EPO)

(v) Current application data:

Application No. EP 95112241.5

(2) Sequence No. 1:

(i) Sequence properties:

(A) Length: 2703 base pairs

(B) Form: hexane

(C) Chain property: Double chain

(D) Topology: Linear

(vi) source of source

(A) Organism: Homo sapiens

(ix) Features:

(A) Name / Key: CDS

(B) Location: 640..2142

(ix) Features:

(A) name / key: sig-peptide

(B) Location: 640..720

(ix) Features:

(A) Name / Key: mat-peptide

(B) Location: 1780..2142

(xi) Description of Sequence 1:

(2) Information on sequence 2

(i) Sequence properties:

(A) Length: 501 amino acids

(B) Form: Amino acid

(D) Topology: Linear

(ii) Molecular form: protein

(xi) description of sequence 2

International format

[Budapest Treaty on the International Recognition of Microorganism Deposit under Patent Procedure]

Deposits on the original deposits issued under Rule 7.1 of the following international deposit

Name (title) Heiesto Japan Co., Ltd. International R & D Headquarters

Address 350-11) Kawagoe City, Saitama Prefecture

I. Marking of microorganisms (Corresponding number given by donor MC-2 (Accession number) FERM BF-5142 II. Scientific properties and taxonomic location The microorganisms of I are classified under the following names: ■ Scientific properties ■ Taxonomic location III. submission The depositary authority of the home country consigned the microorganism of I received on August 21, 2007 (original deposit day) IV. Receipt of Escalation Claim The International Depositary of the host country receives the microorganism I on the first day of the month (original deposit day) and is subsequently transferred on the day of the month in accordance with the Budapest Treaty on Deposit V. Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology Name: Michio, Director, Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (AIST) Address: 1-2, Gamagi Higashi, Tsukuba City, Ibaraki, Japan (1995) June 21

Claims (6)

A protein comprising amino acids 1 to 121 of SEQ ID NO: 1 of the included sequence listing. 4. The method of claim 1, wherein at least a portion of the DNA-sequence as set forth in SEQ ID NO: 1 is introduced into a suitable host cell under conditions that permit expression of the DNA-sequence and protein formation, &Lt; / RTI &gt; and isolating the protein from other proteins produced by the method. A pharmaceutical composition containing the protein of claim 1 as an active substance together with conventional carrier substances, adjuvants, diluents and fillers, if desired. The pharmaceutical composition of claim 3 is used for the treatment or prevention of tibialisation promotion, tibia, cartilage, connective tissue, skin, mucous membrane, epithelium or tooth injury, application to dental implantation and wound- Purpose. The use of the pharmaceutical composition of claim 3 for the treatment of osteoporosis or tibial fractures. 4. A pharmaceutical composition according to claim 3 for use in orthopedic reconstitution, tibial implantation, plastic surgery or dental implantation