Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 8 to 10 amino acid residues in defined sequence
Reexamination Certificate
1999-06-02
2004-06-01
Romeo, David S. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
8 to 10 amino acid residues in defined sequence
Reexamination Certificate
active
06743895
ABSTRACT:
The present invention relates to polypeptides which stimulate bone growth.
Understanding of issues related to bone growth and strength has progressed over the years, a summary being provided in international patent application No. PCT/CA 94/00144, published under international publication No. WO 94/20615 on Sep. 15, 1994.
Various approaches to treatment of diseases involving reduction of bone mass and accompanying disorders are exemplified in the patent literature. For example, U.S. Pat. No. 4,877,864, issued Oct. 31, 1989 describes human and bovine “bone inductive factors.” International patent application published Sep. 17, 1992 under No. 92/15815 describes a protein derived from a porcine pancreas which acts to depress serum calcium levels for treatment of bone disorders that cause elevation of serum calcium levels. European Patent Application No. 504 938 published Sep. 23, 1992 describes the use of di- or tripeptides which inhibit cysteine protease in the treatment of bone diseases. International patent application published Sep. 3, 1992 under No. 92/14481 discloses a composition for inducing bone grow the composition containing activin and bone morphogenic protein. European Patent Application No. 499 242 published Aug. 19, 1992 describes the use of cell growth factor compositions thought to be useful in bone diseases involving bone mass reduction because they cause osteoblast proliferation. International patent application published Jun. 25, 1992 under No. 92/10515 1992 describes a drug containing the human N-terminal parathyroid hormone (PTH) fragment 1-37. European Patent Application No. 451 867 published Sep. 16, 1991 describes parathyroid hormone peptide antagonists for treating dysbolism associated with calcium or phosphoric acid, such as osteoporosis. U.S. Pat. No. 5,461,034 issued Oct. 24, 1995 to Yissum Research Development Company of the Hebrew University of Jerusalem describes osteogenic growth polypeptides identified from regenerating bone marrow.
A relatively short half life of PTH in the blood serum and the positive effect of intermittent PTH injection on bone volume led the present investigator to the hypothesis that PTH may in some way lead to induction of a second factor into the circulatory system. The presence of such a second factor in blood serum of rats and of humans has thus been investigated.
It has been found possible to isolate from rat blood serum a polypeptide substance which, upon administration to rats incapable of producing PTH (parathyroidectomized rats), produces an increase in the observed bone mineral apposition rate. A nucleic acid probe, based on the amino acid sequence of the rat peptide was synthesized and used to screen a human liver cDNA fetal library in order to isolate a human nucleic acid sequence coding for a human bone apposition polypeptide. A polypeptide derived from the nucleic acid sequence was thus chemically synthesized according to the derived sequence Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Pro (SEQ ID NO:1). It has been observed that the bone apposition rate in intact rats increases in a dose dependent fashion upon administration of this chemically synthesized compound. Reduced bone growth, normally observed for ovariectomized rats, was observed not to occur in rats after being administered with the polypeptide over a four week period beginning two weeks after ovariectomization. Bone calcium density was found to be maintained in ovariectomized rats administered with the polypeptide over an eight week period beginning eight weeks after ovariectomization.
It is thought possible that the active polypeptide is a dimer of the foregoing sequence, there being evidence of significant dimer formation, presumably due to a disulfide bridge between two polypeptides having the sequence shown.
A modified form of the polypeptide containing a cys→ala substitution was thus synthesized: Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Ala Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln Pro (SEQ ID NO:3). Some of the bone stimulatory effects of the “normal” polypeptide (SEQ ID NO:1) were found for the modified polypeptide.
In other experiments, the bone mineral apposition rate in rats administered with rabbit antibodies to the normal polypeptide (SEQ ID NO:1) was found to be suppressed. The suppression was found to be attenuated in rats administered with both the normal polypeptide and antibodies to same.
Further, certain polypeptide fragments of the normal polypeptide (SEQ ID NO:1) have been synthesized and each has been found to have bone stimulatory effects:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val SEQ ID NO:4:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala SEQ ID NO:5:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu SEQ ID NO:6:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile SEQ ID NO:7:
Gly Ile Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys SEQ ID NO:8:
Arg Thr Asn Glu His Thr Ala Asp Cys Lys SEQ ID NO:9:
Further, the polypeptide identified as SEQ ID NO: 7 has been found to increase bone calcium content of ovariectomized rats when administered over a period of eight or twelve weeks.
Other polypeptide fragments of the normal polypeptide (SEQ ID NO: 1) have also been synthesized and have been found to lack the bone stimulatory effect found for the normal polypeptide:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn Gln SEQ ID NO:10:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn SEQ ID NO:11:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln SEQ ID NO:12:
Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp SEQ ID NO:13:
Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp SEQ ID NO:14:
Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile Lys Pro Asn Thr Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gln Asn SEQ ID NO:15:
Arg Thr Asn Glu His Thr Ala Asp Cys Lys Ile SEQ ID NO:16:
The polypeptide identified as SEQ ID NO:9 was modified to include a protecting group at each end. The N-terminus was thus acetylated and C-terminus was amidated. The activity of this protected polypeptide, identified as SEQ ID NO:24, was found to increase the bone mineral apposition rate in rats beyond that observed for each of the polypeptides identified as SEQ ID NOs: 1,7 and 9.
It has been reported that histidine and cysteine residues can effect degradation of asparaginyl- and aspartyl-containing polypeptides in the absence of catalytic enzymes [Int. J. Peptide Protein Res. 45, 1995, 547,553]. The following analogues of the polypeptide identified as SEQ ID NO:9 were tested, for stability and for effects on bone mineral apposition rate:
CH
3
CO-Arg Thr Asn Glu His Thr Ala
Glu
Cys Lys-NH
2
SEQ ID NO:25
CH
3
CO-Arg Thr
Gln
Glu His Thr Ala
Glu
Cys Lys-NH
2
SEQ ID NO:26
CH
3
CO-Arg Thr
Gln
Glu His Thr Ala Asp Cys Lys-NH
2
SEQ ID NO:27
In terms of stability under the various conditions tested, the polypeptides identified as SEQ ID NOs:25 and 26 were found to be more stable than those identified as SEQ ID NOs:9, 7 and 24. The polypeptide identified as SEQ ID NO:27 was found to be less stable than any of SEQ ID NOs: 7, 9, 24, 25 and 26.
Each of the polypeptides identified as SEQ ID NOs:24, 25, 26 and 27 were found to increase the bone apposition rate over that observed for control rats.
Polypeptide sequences corresponding to SEQ ID NOs:25, 26 and 27 in which the terminal amino acid residues are not protected are referred to herein as SEQ ID NOs:28, 29 and 30, respectively.
It has further been found, by a series of substitutions that the general charge pattern, based on the side ch
Hunt John C.
Osteopharm Inc.
Romeo David S.
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