Patent US4963150B1 successfully refreshed from Google Patents.
US5786327A
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Bone stimulating factor, methods of isolating same, and methods of increasing bone growth comprising administering same
Assignee: Gensci Regeneration Sciences Inc
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Filing: 1994-09-12
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Granted: 1998-07-28
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Expires: 2014-09-12
Abstract
Abstract A polypepide substance isolated from rat serum which, upon administration to rats incapable of producing PTH (parathyroidectomized rats), produces an increase in the observed bone mineral apposition rate. The substance bas been isolated in two forms, a first larger polypeptide having a molecular weight about twice that of a second smaller polypeptide. The first eleven amino acids of the sequence of the smaller polypepeptide have been determined to be Gly Pro Gly Gly Ala Gly Glu Thr Lys Pro Ile (SEQ ID NO:3). The first seven amino acids of the larger polypeptide have been determined to be Gly Pro Gly Gly Ala Gly Glu (SEQ ID NO:2). The larger polypeptide might be the dimer of the smaller peptide. A nucleic acid probe, based on the amino acid sequence of the rat peptide was used to screen a human liver cDNA fetal library. A polypeptide was thus chemically synthesized according to the 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: 11). The bone apposition rate in rats increases in a dose dependent fashion upon administration of this chemically synthesized compound. A modified polypeptide containing a cysteine→alanine substitution was carried synthesized: Glr 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 Gin Asn Gin Pro (SEQ ID NO: 13). Some of the bone stimulatory effects of the normal polypeptide were found for the modified polypeptide.
Inventors
- 👤 Cherk Shing Tam
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 12:26
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/521; C07K14/522; A; A61; A61P; A61P19/00; A61P19/08; A61P43/00; C07K14/475; C07K14/51; A61K; A61K38/00
Claims Analysis
Total Claims: 67
Claim 1
Independent Claim
28 words
An isolated polypeptide having the amino acid sequence (SEQ ID NO:11) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 2
Dependent Claim
28 words
An isolated polypeptide having the amino acid sequence (SEQ ID NO:11) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 3
Dependent Claim
15 words
A chimeric bone stimulating factor comprising the amino acid sequence set forth in claim 1.
Claim 4
Dependent Claim
18 words
A polypeptide of claim 1 wherein the polypeptide has at least about 50% homology with SEQ ID NO:11.
Claim 5
Dependent Claim
18 words
A polypeptide of claim 1 wherein the polypeptide has at least about 60% homology with SEQ ID NO:11.
Claim 6
Dependent Claim
18 words
A polypeptide of claim 1 wherein the polypeptide has at least about 70% homology with SEQ ID NO:11.
Claim 7
Dependent Claim
18 words
A polypeptide of claim 1 wherein the polypeptide has at least about 80% homology with SEQ ID NO:11.
Claim 8
Dependent Claim
18 words
A polypeptide of claim 1 wherein the polypeptide has at least about 90% homology with SEQ ID NO:11.
Claim 9
Dependent Claim
28 words
A polypeptide of claim 1 wherein at least one non-polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different non-polar aliphatic neutral amino acid.
Claim 10
Dependent Claim
28 words
A polypeptide of claim 1 wherein at least one polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different polar aliphatic neutral amino acid.
Claim 11
Dependent Claim
26 words
A polypeptide of claim 1 wherein at least one charged acidic amino acid of SEQ ID NO:11 is substituted by a different charged acidic amino acid.
Claim 12
Dependent Claim
26 words
A polypeptide of claim 1 wherein at least one charged basic amino acid of SEQ ID NO:11 is substituted by a different charged basic amino acid.
Claim 13
Dependent Claim
56 words
An isolated dimeric polypeptide wherein each monomer of polypeptide contains the following amino acid sequence (SEQ ID NO:11) NH 2 -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-CO 2 H; wherein the monomers are linked to each other by a disulfide bridge between the cysteine residues of the respective sequences, or a conservatively substituted variant thereof exhibiting mammalian bone stimulatory activity.
Claim 14
Dependent Claim
56 words
An isolated dimeric polypeptide wherein each monomer of polypeptide contains the following amino acid sequence (SEQ ID NO:11) NH 2 -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-CO 2 H; wherein the monomers are linked to each other by a disulfide bridge between the cysteine residues of the respective sequences, or a conservatively substituted variant thereof exhibiting mammalian bone stimulatory activity.
Claim 15
Dependent Claim
18 words
A polypeptide of claim 12 wherein the polypeptide has at least about 50% homology with SEQ ID NO:11.
Claim 16
Dependent Claim
18 words
A polypeptide of claim 12 wherein the polypeptide has at least about 60% homology with SEQ ID NO:11.
Claim 17
Dependent Claim
18 words
A polypeptide of claim 12 wherein the polypeptide has at least about 70% homology with SEQ ID NO:11.
Claim 18
Dependent Claim
18 words
A polypeptide of claim 12 wherein the polypeptide has at least about 80% homology with SEQ ID NO:11.
Claim 19
Dependent Claim
18 words
A polypeptide of claim 12 wherein the polypeptide has at least about 90% homology with SEQ ID NO:11.
Claim 20
Dependent Claim
28 words
A polypeptide of claim 12 wherein at least one non-polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different non-polar aliphatic neutral amino acid.
Claim 21
Dependent Claim
28 words
A polypeptide of claim 12 wherein at least one polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different polar aliphatic neutral amino acid.
Claim 22
Dependent Claim
26 words
A polypeptide of claim 12 wherein at least one charged acidic amino acid of SEQ ID NO:11 is substituted by a different charged acidic amino acid.
Claim 23
Dependent Claim
26 words
A polypeptide of claim 12 wherein at least one charged basic amino acid of SEQ ID NO:11 is substituted by a different charged basic amino acid.
Claim 24
Dependent Claim
60 words
An isolated polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide comprising a monomer having the following amino acid sequence (SEQ ID NO:11) N-2Gly-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-CO.sub.2 H and dimers thereof; wherein the monomers are linked to each other by a disulfide bridge between the cysteine residues of the respective sequences, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 25
Dependent Claim
60 words
An isolated polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide comprising a monomer having the following amino acid sequence (SEQ ID NO:11) N-2Gly-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-CO.sub.2 H and dimers thereof; wherein the monomers are linked to each other by a disulfide bridge between the cysteine residues of the respective sequences, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 26
Dependent Claim
18 words
A polypeptide of claim 22 wherein the polypeptide has at least about 50% homology with SEQ ID NO:11.
Claim 27
Dependent Claim
18 words
A polypeptide of claim 22 wherein the polypeptide has at least about 60% homology with SEQ ID NO:11.
Claim 28
Dependent Claim
18 words
A polypeptide of claim 22 wherein the polypeptide has at least about 70% homology with SEQ ID NO:11.
Claim 29
Dependent Claim
18 words
A polypeptide of claim 22 wherein the polypeptide has at least about 80% homology with SEQ ID NO:11.
Claim 30
Dependent Claim
18 words
A polypeptide of claim 22 wherein the polypeptide has at least about 90% homology with SEQ ID NO:11.
Claim 31
Dependent Claim
28 words
A polypeptide of claim 22 wherein at least one non-polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different non-polar aliphatic neutral amino acid.
Claim 32
Dependent Claim
28 words
A polypeptide of claim 22 wherein at least one polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different polar aliphatic neutral amino acid.
Claim 33
Dependent Claim
26 words
A polypeptide of claim 22 wherein at least one charged acidic amino acid of SEQ ID NO:11 is substituted by a different charged acidic amino acid.
Claim 34
Dependent Claim
26 words
A polypeptide of claim 22 wherein at least one charged basic amino acid of SEQ ID NO:11 is substituted by a different charged basic amino acid.
Claim 35
Dependent Claim
27 words
A polypeptide having the amino acid sequence (SEQ ID NO:13) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 36
Dependent Claim
27 words
A polypeptide having the amino acid sequence (SEQ ID NO:13) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 37
Dependent Claim
15 words
A chimeric bone stimulating factor comprising the amino acid sequence set forth in claim 32.
Claim 38
Dependent Claim
18 words
A polypeptide of claim 32 wherein the polypeptide has at least about 60% homology with SEQ ID NO:11.
Claim 39
Dependent Claim
18 words
A polypeptide of claim 32 wherein the polypeptide has at least about 70% homology with SEQ ID NO:11.
Claim 40
Dependent Claim
18 words
A polypeptide of claim 32 wherein the polypeptide has at least about 80% homology with SEQ ID NO:11.
Claim 41
Dependent Claim
18 words
A polypeptide of claim 32 wherein the polypeptide has at least about 90% homology with SEQ ID NO:11.
Claim 42
Dependent Claim
28 words
A polypeptide of claim 32 wherein at least one non-polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different non-polar aliphatic neutral amino acid.
Claim 43
Dependent Claim
28 words
A polypeptide of claim 32 wherein at least one polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different polar aliphatic neutral amino acid.
Claim 44
Dependent Claim
26 words
A polypeptide of claim 32 wherein at least one charged acidic amino acid of SEQ ID NO:11 is substituted by a different charged acidic amino acid.
Claim 45
Dependent Claim
26 words
A polypeptide of claim 32 wherein at least one charged basic amino acid of SEQ ID NO:11 is substituted by a different charged basic amino acid.
Claim 46
Independent Claim
42 words
A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having the amino sequence (SEQ ID NO:11) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 47
Independent Claim
42 words
A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having the amino sequence (SEQ ID NO:11) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulators activity.
Claim 48
Independent Claim
42 words
A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having the amino sequence (SEQ ID NO:13) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulatory activity.
Claim 49
Independent Claim
42 words
A method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having the amino sequence (SEQ ID NO:13) NH 2 -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-CO 2 H, or a conservatively substituted variant thereof exhibiting mammalian bone stimulatory activity.
Claim 50
Dependent Claim
31 words
A substantially pure circulatory nolypeptide isolated from blood serum of a rat, which polypeptide induces an increased bone apposition rate and has the following N-terminal amino acid sequence (SEQ ID NO:1):Gly-Pro-Gly-Gly-Ala-Gly-Thr-Lys-Pro-Ile.
Claim 51
Dependent Claim
31 words
A substantially pure circulatory nolypeptide isolated from blood serum of a rat, which polypeptide induces an increased bone apposition rate and has the following N-terminal amino acid sequence (SEQ ID NO:1):Gly-Pro-Gly-Gly-Ala-Gly-Thr-Lys-Pro-Ile.
Claim 52
Independent Claim
51 words
A method of producing the polypeptide having an N-terminal amino acid sequence of (SEQ ID NO:1) Gly-Pro-Gly-Gly-Ala-Gly-Glu-Thr-Lys-Pro-Ile from rat blood serum, comprising the steps of: obtaining a protein fraction of the blood serum; removing from the fraction proteins having a molecular weight greater than about 30,000 daltons; and isolating the polypeptide.
Claim 53
Independent Claim
51 words
A method of producing the polypeptide having an N-terminal amino acid sequence of (SEQ ID NO:1) Gly-Pro-Gly-Gly-Ala-Gly-Glu-Thr-Lys-Pro-Ile from rat blood serum, comprising the steps of: obtaining a protein fraction of the blood serum; removing from the fraction proteins having a molecular weight greater than about 30,000 daltons; and isolating the polypeptide.
Claim 54
Dependent Claim
22 words
The method of claim 45 wherein isolating the polypeptide comprises collecting the polypeptide from a reverse phase high performance liquid chromatography column.
Claim 55
Dependent Claim
47 words
The method of claim 46 wherein collecting the polypeptide includes eluting the polypeptide from a reverse phase high performance liquid chromatography column packed with silica gel having three carbon chain side groups attached thereto using an elution solvent comprising at least about 52 to 53 percent acetonitrile.
Claim 56
Independent Claim
75 words
A method of obtaining a polypeptide from a mammal which stimulates bone growth in a mammal, comprising the steps of: (a) feeding the mammal a calcium deficient diet to increase the level of the polypeptide in the blood serum of the mammal; (b) isolating a sample of the blood serum of the mammal; and (c) collecting from the sample in substantially pure form a polypeptide having the N-terminal amino acid sequence (SEQ ID NO:1) Gly-Pro-Gly-Gly-Ala-Gly-Glu-Thr-Lys-Pro-Ile.
Claim 57
Independent Claim
75 words
A method of obtaining a polypeptide from a mammal which stimulates bone growth in a mammal, comprising the steps of: (a) feeding the mammal a calcium deficient diet to increase the level of the polypeptide in the blood serum of the mammal; (b) isolating a sample of the blood serum of the mammal; and (c) collecting from the sample in substantially pure form a polypeptide having the N-terminal amino acid sequence (SEQ ID NO:1) Gly-Pro-Gly-Gly-Ala-Gly-Glu-Thr-Lys-Pro-Ile.
Claim 58
Dependent Claim
40 words
An isolated polypeptide having the amino acid sequence (SEQ ID NO:11) NH 2 -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-CO 2 H, or a conservatively substituted variant having at least about 50% homology with SEQ ID NO:11 which stimulates bone growth in a mammal.
Claim 59
Dependent Claim
40 words
An isolated polypeptide having the amino acid sequence (SEQ ID NO:11) NH 2 -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-CO 2 H, or a conservatively substituted variant having at least about 50% homology with SEQ ID NO:11 which stimulates bone growth in a mammal.
Claim 60
Dependent Claim
18 words
A polypeptide of claim 49 wherein the polypeptide has at least about 60% homology with SEQ ID NO:11.
Claim 61
Dependent Claim
18 words
A polypeptide of claim 49 wherein the polypeptide has at least about 70% homology with SEQ ID NO:11.
Claim 62
Dependent Claim
18 words
A polypeptide of claim 49 wherein the polypeptide has at least about 80% homology with SEQ ID NO:11.
Claim 63
Dependent Claim
18 words
A polypeptide of claim 49 wherein the polypeptide has at least about 90% homology with SEQ ID NQ:11.
Claim 64
Dependent Claim
28 words
A polypeptide of claim 49 wherein at least one non-polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different non-polar aliphatic neutral amino acid.
Claim 65
Dependent Claim
28 words
A polypeptide of claim 49 wherein at least one polar aliphatic neutral amino acid of SEQ ID NO:11 is substituted by a different polar aliphatic neutral amino acid.
Claim 66
Dependent Claim
26 words
A polypeptide of claim 49 wherein at least one charged acidic amino acid of SEQ ID NO:11 is substituted by a different charged acidic amino acid.
Claim 67
Dependent Claim
26 words
A polypeptide of claim 49 wherein at least one charged basic amino acid of SEQ ID NO:11 is substituted by a different charged basic amino acid.
References Cited (Backward Citations)
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