Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-11-30
2002-10-01
Benzion, Gary (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024330, C435S006120, C435S091100, C435S091200, C435S243000, C435S320100, C435S325000, C435S410000
Reexamination Certificate
active
06458944
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a 5′ upstream region DNA containing a promoter of a human bone morphogenetic protein (BMP, hereafter-4). Further, the present invention relates to a method for exploring a low molecular weight compound positively or negatively which regulates the expression of human BMP-4 by using a mass of animal or yeast cells that are introduced with a 5′ upstream region DNA containing the human BMP-4 promoter and a recombinant expression vector integrated into a suitable reporter gene, and by using a reporter activity as an indicator.
(2) Description of the Related Art
At present, a bone morphogenetic activity has been reported for a bone morphogenetic factor, BMP, belonging to TGF (transforming growth factor) -&bgr; superfamily (Science 150, 893-897, 1965; Science 242: 1528-1534, 1988). Known species of BMP are BMP-1 to BMP-14. Among them, the members from BMP-2 to BMP-14 have been known as showing the bone morphogenetic activity. BMPs ranging from BMP-2 to BMP-14 are considered as effective to therapeutic and preventive treatment for various bone dysfunction and bone diseases, however, they exist in very small amount in nature. Therefore, an available large quantity from BMP-2 to BMP-14 used for these treatments requires production of recombinant protein. The production of the recombinant protein generally is very expensive compared with a low molecular weight compound. On the other hand, there are many restrictions as a medical drug in terms of physical properties and administration due to its proteinic characteristics. Considering these points, a small molecular organic compound having the equal activity to that of said BMP protein, if any, will become a very promising medical drug. The substance obtained by the exploring method provided by the present invention has the activity to induce the expression of human BMP-4, a bone morphogenetic protein, and also has the same activity as that of human BMP-4 to be a very useful application. On the contrary, there is a report (New Engi. J. Med., Vol. 335, p. 555-561, 1996) suggesting that human BMP-4 causes bone and cartilage hyperplasia. In this case, inhibiting the expression of human BMP-4 may prevent osteohyperplasia. The present invention is able to detect the inhibition of the expression of human BMP-4 and provides a method for exploring a substance which prevents such hyperplasia.
For such an exploring method, an example was so far only reported using a murine BMP-2 promoter (WO97/15308) ; there is no example of using a human BMP-4 promoter. The region of the murine BMP-4 promoter has been already cloned (Biochem. Biophys. Acta, Vol. 1218, p. 221-224, 1994), but no detailed sequence data of the human BMP-4 promoter has been elucidated. The present invention first disclosed the sequence of the human BMP-4 promoter. The homology of the entire sequence of 5′ upstream region DNA between human and murine BMP-4 (J. Biol. Chem., Vol. 270, p. 28364-28373, 1995) is 52.2%. The materials for the exploring method provided by the present invention are-all derived from human sources so that the substance obtained by the exploration would be a clinical application practically. It is expected to obtain a substance capable of more accurate regulation of the human BMP-4 expression by transfecting two promoters (promoter
1
and promoter
2
) separately or simultaneously in the recombinant expression vector into the host cells.
SUMMARY OF THE INVENTION
The present invention provides a 5′ upstream region DNA containing a promoter of human BMP-4. By using 5′ upstream region gene containing the human BMP-4 promoter and an animal cell introduced with a recombinant expression vector which has been integrated in an appropriate reporter gene, the low molecular weight compounds which regulate positively or negatively the expression of human BMP-4 can be explored with reference to a reporter activity. The low molecular weight compounds and their derivatives have morphogenetic activity and inhibiting activity for bone and cartilage through the expression of human BMP-4 and are useful as preventive or therapeutic agents for cartilage and bone diseases, remedies for osteometastasis, or therapeutic and preventive agents for osteohyperplasia.
REFERENCES:
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Wozney et al. GenBank Accession No.: I62859.*
XP002062064 pp. 789-793 Genomic . . . Multiple Transcripts.
XP002121984 pp. 559-560 Finge Mapping . . . Hybridization.
XP002029032 pp. 1049-1056, Murine Bone . . . Region vol. 192, No. 3, 1993.
XP002029031 pp. 28364-28373 The Mouse . . . Gene, vol. 270, No. 47 1995.
XP002120993 pp., 221-229 The Human . . . Regulation 1998, Shore et al.
Kawai Shinji
Sugiura Takeyuki
Aventis Pharma S.A.
Benzion Gary
Bierman, Muserlian and Lucas
Spiegler Alexander H.
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