Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-12-10
2004-04-06
Achutamurthy, P. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C435S071100, C536S023200
Reexamination Certificate
active
06716613
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel metalloprotease having an aggrecanase activity and causing joint diseases (to be referred to as “joint disease aggrecanase” hereinafter), a gene coding for this “joint disease aggrecanase”, a method for producing the “joint disease aggrecanase”, a method for screening a substance capable of inhibiting the aggrecanase activity with the use of the “joint disease aggrecanase”, a pharmaceutical composition for inhibiting degradation of proteoglycans, which comprises the substance capable of inhibiting the aggrecanase activity as the active ingredient, and a promoter gene of the “joint disease aggrecanase”.
BACKGROUND ART
Joint diseases are diseases which show damage and degeneration of joint cartilage as the main morbid states. Though a disease having the most frequent number of patients among joint diseases is osteoarthritis (OA), analgesic anti-inflammatory drugs and hyaluronic acid preparations are used in the current therapeutic method merely as a symptomatic therapy for the purpose of alleviating pains accompanied by the degeneration of cartilage and the destruction of bone under cartilage, so that it cannot be said that they are exerting sufficient therapeutic effects.
Joint cartilage is a tissue mainly composed of type II collagen and aggrecan which is a cartilage-specific proteoglycan, and degradation and degeneration of both of them are observed in the joint diseases. Because of this, it has been considered for a long time that control of the degradation and degeneration of these extracellular matrix components would lead to the treatment of joint diseases, so that attempts have been positively made to identify degradation-concerned proteases (collagenase and aggrecanase) and to screen their inhibitors and develop them as medicaments.
As proteases having collagenase activities, matrix metalloproteases (MMP1, MMP8, MMP13, MMP14 and the like) have been identified, and their selective inhibitors have been discovered. However, in spite of the attempts to develop a large number of MMP inhibitors having collagenase inhibition activities as therapeutic drugs for joint diseases including OA and rheumatic arthritis (RA), MMP inhibitors to be used in these diseases as the indication have not been put on the market. Under such circumstances, attention has been directed toward aggrecanase which selectively degrades aggrecan which is another main constituting component of joint cartilage.
A joint disease-related role of an enzyme aggrecanase which cleaves aggrecan at the site between Glu
373
-Ala
374
has been revealed by the reports of Sandy et al. and Lohmander et al. stating that all of the main digested aggrecan fragments found in the synovial fluid of human arthritis patients were generated by cleaving at the aggrecanase digestion site (Sandy J. D. et al.,
J. Clin. Invest.,
89, 1512-1516, 1992; Lohmander L. S. et al.,
Arthritis Rheum.,
36, 1214-1222, 1993). On the other hand, it has been known that, in an in vitro explant culture system of joint cartilage, degradation of aggrecan firstly occurs by IL-1 induction and then degradation of type II collagen is accelerated (Dingle L. T. et al.,
Ann. Rheum. Dis.,
34, 303-311, 1975; Cawston T. E. et al.,
Biochem. Biophys. Res. Comm.,
215, 377-385, 1995; Kozaci L. D. et al.,
Arthritis Rheum.,
40, 164-174, 1997). It has been reported that the aggrecan degradation takes the precedence of the type II collagen degradation in a mouse arthritis model too (van Meurs J. B. et al.,
Arthritis Rheum.,
42, 1128-1139, 1999). These reports suggest a possibility that the type II collagen degradation can be controlled by inhibiting the preceding aggrecan degradation.
However, the entity of the aggrecanase which causes joint diseases (“joint disease aggrecanase”) has been unclear for long time, though its biochemical properties had been elucidated, namely it is a metalloprotease, it exists in outside of cells, a glycosaminoglycan side chain is concerned in its substrate recognition, its activity is induced by IL-1, TNF and retinoic acid, and the like. Recently, ADAMTS4 (aggrecanase-1: Tortorella M. D. et al., Science, 284, 1664-1666, 1999) and ADAMTS11 (aggrecanase-2: Abbaszade I. et al.,
J. Biol. Chem.,
274, 23443-23450, 1999) have been reported as proteases having an aggrecanase activity. However, it was revealed that they are not the “joint disease aggrecanase”, because their gene expression in human OA cartilage is not increased, and their gene expression in an in vitro explant culture system of human knee joint cartilage is not induced by IL-1, TNF and retinoic acid which induce the aggrecanase activity that causes joint diseases (Flannery C. R. et al.,
Biochem. Biophys. Res. Commun.,
260, 318-322, 1999). As described above, the “joint disease aggrecanase” has not been obtained.
DISCLOSURE OF THE INVENTION
Under such circumstances, the present inventors have conducted intensive studies and, as a result, succeeded in isolating a gene coding for a novel metalloprotease having the aggrecanase activity, which is the “joint disease aggrecanase”, determining its full-length ORF sequence and thereby achieved production of a recombinant protein.
Also, a vector comprising this gene, a host cell comprising this vector and a method for producing the novel protein using this host cell were established.
Also, the inventors have succeeded in providing a screening method which uses this protein and found that a compound selected by carrying out this screening method significantly inhibits the “aggrecanase activity” (namely, the activity of this protein to cleave the extracellular substrate aggrecan selectively at the site between Glu
373
-Ala
374
) and can become a medicament useful in preventing and/or treating joint diseases.
In addition, a promoter gene of the protein, which is useful in screening a medicament for preventing and/or treating joint diseases was isolated, resulting in accomplishment of the present invention.
Accordingly, the invention relates to:
[1] a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease,
[2] a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease,
[3] a metalloprotease having an aggrecanase activity, which consists of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 687th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 950th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 687th position of the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease,
[4] a gene which encodes the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an amino acid sequence of an equivalent of the metalloprotease,
[5] a vector which comprises the gene described in [4],
[6] a host cell which comprises the vector described in [5],
[7] a method for producing the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an equivalent of the metalloprotease, which comprises using the host cell described in [6],
[8] an antibody against the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an equivalent of t
Abe Kunitake
Nagase Takahiro
Nishimura Kouichi
Nomura Nobuo
Ohara Osamu
Achutamurthy P.
Pak Yong D
Sughrue & Mion, PLLC
Yamanouchi Pharmaceutical Co. Ltd.
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