WO1992015615A1 - Serum calcium depressing factor - Google Patents
Serum calcium depressing factor Download PDFInfo
- Publication number
- WO1992015615A1 WO1992015615A1 PCT/JP1992/000251 JP9200251W WO9215615A1 WO 1992015615 A1 WO1992015615 A1 WO 1992015615A1 JP 9200251 W JP9200251 W JP 9200251W WO 9215615 A1 WO9215615 A1 WO 9215615A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- serum calcium
- lowering
- factor
- lowering factor
- calcium
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
- A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a serum calcium-lowering factor derived from pig, a method for producing the factor, and a medicament containing the factor. Background technology
- an object of the present invention is to provide a novel isolated serum calcium-lowering factor derived from pig.
- the present inventors have succeeded in isolating and purifying the desired serum calcium-lowering factor from swine spleen, elucidating its characteristics as a substance, and further clarifying some biological characteristics.
- the present invention has been completed. That is, the present invention has the following properties:
- N The following amino acid sequence at or near the terminal (SEQ ID NO: 1): V al — V al — G ly — G ly— G in— A sn — A la — I 1 e — P ro — H having is-Ser-Trp-Pro-Trp-Gln-I1e-Arg-Leu-;
- Protease activity is measured by the protease inhibitor phenylmethansulfonyl fluoride (PMSF) (1 mM), chymostatin (100 ⁇ M) and trypsin inhibitor (100 g / ml). Harmful, but (4-amidinofenyl) methanesulfonyl fluoride (APMSF) (50 ⁇ ) and ⁇ —C ⁇ - (L-13—trans-carboxyxylane-2-carbonyl) ) 1 L 1 bit isl] Agmatine ( ⁇ -64) (2.5 g ml) is not inhibited;
- the present invention further provides a method for producing the serum calcium-lowering factor, which comprises isolating the serum calcium-lowering factor from porcine excreta. .
- the present invention further provides a medicament comprising the serum calcium lowering factor.
- Figure 1 shows the elution profile of Q-Sepharose Fast Fast Flow Chromatography. Only Beak III Has serum calcium lowering effect.
- FIG. 2 A shows the state of elution in gel filtration chromatography using Superdex 75 HR.
- A shows inhibition of calcium release (by PTH) in Lewis bone culture system by several aspects corresponding to B. Only the main peak had an inhibitory effect on calcium release.
- Figure 3 shows the elution of ion-exchanged chromatographic liquid with a Mono Q column. Only the main beak has a serum calcium lowering effect.
- Figure 4 shows the appearance of elution in reverse-phase liquid chromatography using Wakoshi15C-18 columns.
- FIG. 5 is a graph showing that serum calcium-lowering factor (open circles) and serum calcium-lowering factor (black circles) treated with protease inhibitor PMSF lower serum calcium in a dose-dependent manner.
- acetate was added to this extract at a concentration of 30% to form a precipitate. After removing the solid, add acetone to the supernatant to a concentration of 60% to obtain the formed solid.
- the 30-60% acetone fraction thus obtained was filtered through a suitable buffer solution, for example, deionized water, to remove the acetate, and then added with ammonium sulfate. 4 Remove the deposits generated after 5% saturation. Next, ammonium sulfate is added to the supernatant to make it 60% saturated, and the resulting precipitate is obtained.
- a suitable buffer for example, 5 mM acetate buffer (pH 5.5), and the solution is dialyzed to remove ammonium sulfate to obtain a crude serum calcium-lowering factor extract.
- the serum calcium-lowering factor of the present invention can be obtained from this extract by the method described in detail in Example 1. Briefly, the crude extract was first applied to a Q-Sepharose Fast Flow column, and a 5 OmM acetate buffer (pH 5.5) and 0.1 M Na After passing the same acetate buffer containing C 1 through the column, 0.2 M
- the main beak obtained by the gel filtration chromatography was subjected to reverse-phase liquid chromatography using Wakosi 15 C-18 column to obtain a solution in 0.1% trifluoroacetic acid (TFA).
- TFA trifluoroacetic acid
- the elution profile shown in Fig. 4 was obtained by elution with a linear gradient of tonitrile concentration, indicating that the serum calcium-lowering factor of the present invention was purified as a single protein by purification with a M0 no QHR column. It is confirmed.
- serum calcium-lowering factor of the present invention was isolated and purified as described above, it was once isolated and purified, and after its properties were elucidated as described below, the properties of the protein It is clear that the serum calcium-lowering factor of the present invention can be isolated and purified using any conventional method used for isolation and purification. You.
- the serum calcium lowering factor of the present invention can be produced by genetic engineering means.
- mRNA can be isolated from pig kidney according to a conventional method, and a cDNA library can be prepared based on the mRNA according to a conventional method.
- a DNA probe for screening this cDNA library can be designed, for example, based on the amino acid sequence at or near the N-terminus of serum calcium-lowering factor identified according to the present invention. .
- a DNA probe can be designed based on the amino acid sequence. .
- the cDNA encoding the serum calcium-lowering factor thus obtained is inserted into an appropriate expression vector, and then a host is transformed with the expression vector and the transformant is cultured.
- the serum calcium-lowering factor of the present invention can be produced.
- Conventional hosts such as prokaryotic cells such as Escherichia coli, lower eukaryotic cells such as yeast, and higher eukaryotic cells such as cultured mammalian cells can be used.
- the molecular weight of the serum calcium-lowering factor of the present invention When the molecular weight of the serum calcium-lowering factor of the present invention is measured by SDS-acrylamide gel electrophoresis, it shows a single molecular weight of about 26,500 to 28,000 D. (2) m ⁇
- the serum calcium-lowering factor of the present invention exhibits a single isoelectric point of about 4.5.
- V a 1-V a 1-G 1-G 1 y-G 1 n— A sn— A la — I 1 e — P ro — H is — Ser — T rp — P rp — T rp — G in— It has I le —A rg—L eu—.
- This amino acid sequence is composed of porcine plasminogen, human apoprotein 8, porcine elastase 2, ⁇ ⁇ ⁇ ⁇ ⁇ A ⁇ ⁇ A, ⁇ ⁇ ⁇ ⁇ ⁇ B B B, and ⁇ ⁇ ⁇ ⁇
- the serum calcium-lowering factor of the present invention is a novel protein that is homologous to, but not identical to, the amino acid sequence of human coagulation factor (Pre) and human coagulation factor XI (Pre). is there.
- the serum calcium-lowering factor of the present invention has protease activity, and the behavior of the serum calcium-lowering factor of the present invention with respect to a protease inhibitor is described in detail in Example 3 and Table 2.
- the serum calcium-lowering factor of the present invention has a serum calcium-lowering effect in vivo, it has various bone diseases, for example, osteoporosis, primary hyperparathyroidism, hypercalcemia associated with malignant tumors. Active ingredients of medicines for treatment and prevention of etc. It is expected to be useful as.
- the serum calcium-lowering factor of the present invention can be formulated parenterally with a conventional pharmaceutical carrier and administered parenterally, for example, by intravenous administration, subcutaneous administration, intramuscular administration, or enteral or oral administration. it can.
- Example 1 Purification of serum calcium-lowering porcine derived pig swine Acetone powder of swine spleen was prepared and extracted with 0.1 M Tris-HCl buffer containing 2% NaC1. Then, an acetone sedimentation fraction was obtained at an acetate concentration of 30 to 60%. This fraction was filtered through deionized water, and then subjected to ammonium sulfate salt to obtain a precipitate fraction at 45 to 60% saturation with ammonium sulfate.
- mice were examined for serum calcium lowering effect in mice by the following method (in vivo measurement).
- PTH (1 0 - 7 M ) is to promote about 80% the release of calcium, fractions corresponding to the main Lee Nbiku completely inhibited the calcium release promoting. On the other hand, the fractions corresponding to the three minor beaks and the areas not corresponding to any of the peaks did not show any inhibitory action.
- This main beak was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of sodium dodecyl sulfate, and the molecular weight standards (albumin 67 KD, ovalbumin 43 KD, force-carbonic anhydrase 30%) were determined. KD, trypsin inhibitor The main band showed a molecular weight of about 26,500 D when compared with 20 KD and 14 KD of n-lactalbumin.
- the main beak obtained by the gel filtration chromatography was subjected to ion exchange chromatography using a M0n0QHR5Z5 column.
- Buffer A 50 mM sodium acetate (PH5.5)
- Buffer B 50 mM sodium acetate (pH 5.5) containing 0.5 MNaCl
- the mixture was eluted at a flow rate of 0.5 mlZ with a linear gradient of NaCl concentration using, and fractionated 1 ml each.
- Figure 3 shows the results.
- a main beak was obtained at a NaC1 concentration of about 0.2 M, and this beak had a serum calcium lowering effect in in vivo measurement.
- the purity of the active main beak obtained by ion exchange chromatography using the M0n0Q column was examined by reversed-phase liquid chromatography. W akosi 1 5 C
- the active beak obtained by the Mono Q ion exchange chromatography and Wak 0 si 15 C-18 reverse When the molecular weight of the peak obtained by phase liquid chromatography was determined by non-reducing SDS-PAGE according to the method described above, the molecular weight of serum calcium-lowering factor was calculated to be about 28,000 D.
- V a 1-V a 1-G 1 y— G l -G ln -A sn -A la-I 1 e— P ro— H is— S er— T rp— P rp— T rp— G 1 n— I 1 e— A rg— L eu—
- Phenylmethansulfonyl fluoride which is a serine protease inhibitor; and (41-amidinophenyl) methansulfonyl fluoride (APMSF), which is a triscine-type serine protease inhibitor; Leupeptin, a tribosine-type serine proteinase thiol protease inhibitor; chymostatin, a chymotribine-type serine proteinase inhibitor (chymostatin), a thiol protease inhibitor N-C-(L 13-trans-lipoxyxanlan 2-carbonyl) 1 L-lysyl] agmatine (E-64), an acidic protease inhibitor Pepstatin; trypsin inhibitor, an inhibitor of trypsin, factor Xa, plasmin and plasmacaline
- serum calcium-lowering factor inhibits the release of calcium by PTH or vitamin D in a Louis bone culture system.
- serum calcium-lowering factor inhibited the effect of PTH at a concentration as low as 1 ng / ml, but did not inhibit the effect of vitamin D at a concentration of 10 ng / ml.
- PMSF does not lower serum calcium, but untreated serum Calcium-lowering factors reduced serum calcium in a dose-dependent manner. PMSF-treated serum calcium-lowering factor also reduced serum calcium in a dose-dependent manner, with the curve shifting to a much lower concentration. This result suggests that serum calcium-lowering factor, whose protease activity was inhibited, may have a serum calcium-lowering effect.
- the serum calcium-lowering factor of the present invention has a serum calcium-lowering effect in vivo, it is used for treatment of various bone diseases, for example, osteoporosis, primary hyperparathyroidism, hypercalcemia associated with malignant tumors, and the like. And it is expected to be useful as an active ingredient of a medicine for prevention.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Diabetes (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP92906236A EP0641858A1 (en) | 1991-03-06 | 1992-03-03 | Serum calcium depressing factor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3/40073 | 1991-03-06 | ||
JP3040073A JPH04279598A (ja) | 1991-03-06 | 1991-03-06 | 血清カルシウム降下因子 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992015615A1 true WO1992015615A1 (en) | 1992-09-17 |
Family
ID=12570754
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1992/000251 WO1992015615A1 (en) | 1991-03-06 | 1992-03-03 | Serum calcium depressing factor |
Country Status (4)
Country | Link |
---|---|
JP (1) | JPH04279598A (ja) |
AU (1) | AU1338292A (ja) |
OA (1) | OA09712A (ja) |
WO (1) | WO1992015615A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6352973B1 (en) | 1995-06-07 | 2002-03-05 | Osteopharm Inc. | Bone stimulating factor |
US6596498B1 (en) | 1993-03-12 | 2003-07-22 | Osteopharm Inc. | Bone stimulating factor |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2753395A (en) * | 1994-06-24 | 1996-01-19 | Chugai Seiyaku Kabushiki Kaisha | Dna coding for caldecrin and process for producing caldecrin by using the dna |
US20090053788A1 (en) | 2006-12-28 | 2009-02-26 | Aspion Co., Ltd. | Muscular dystrophy drug |
-
1991
- 1991-03-06 JP JP3040073A patent/JPH04279598A/ja active Pending
-
1992
- 1992-03-03 AU AU13382/92A patent/AU1338292A/en not_active Abandoned
- 1992-03-03 WO PCT/JP1992/000251 patent/WO1992015615A1/ja not_active Application Discontinuation
- 1992-11-06 OA OA60299A patent/OA09712A/en unknown
Non-Patent Citations (3)
Title |
---|
"Term Calcitonin Biochemical Dictionary", April 10, 1984 (10.04.84), Tokyo Kagaku Dojin (Tokyo), p. 276-277. * |
Proc. Natl. Acad. Sci. USA, Vol. 86, No. 15 (1989), NISHI-M- et al., "Conservation of the Sequence of Islet Amyloid Polypeptide in Five Mammals is Consistent with its Putative Role as an Islet Hormone", p. 5738-5742. * |
See also references of EP0641858A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6596498B1 (en) | 1993-03-12 | 2003-07-22 | Osteopharm Inc. | Bone stimulating factor |
US6352973B1 (en) | 1995-06-07 | 2002-03-05 | Osteopharm Inc. | Bone stimulating factor |
Also Published As
Publication number | Publication date |
---|---|
AU1338292A (en) | 1992-10-06 |
JPH04279598A (ja) | 1992-10-05 |
OA09712A (en) | 1993-08-30 |
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