Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-01-13
2004-02-17
Romeo, David S. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S328000, C530S345000, C530S380000, C530S402000
Reexamination Certificate
active
06693081
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, for example, international patent application No. PCT/CA 94/00144, published on Sep. 15, 1994 under WO 94/20615, U.S. Pat. No. 5,320,970 and European patent application No. 92 302 446, published under 505 210 on Sep. 23, 1992, the contents of which applications are incorporated herein by reference.
By way of background to the present invention, described below, neutrophil-activating peptide (NAP-2; SEQ ID NO:1) and a variant of NAP-2, here termed “NAP-2V” (SEQ ID NO:2) have been known for some time (Walz, A., and M. Baggiolini, (1989)
Biochem. Biophys. Res. Commun.
159:969). British Patent No.2 231 872 (British Patent No. 2 231 872. Inventors: M. Baggiolini, K. J. Clemetson, and A. Walz. Published Jun. 14, 1990.), describes the amino acid sequence of NAP-2 and three apparently naturally occurring variants, including NAP-2V. The other two variants have an additional four (SEQ ID NO:3) and three (SEQ ID NO:4) amino acids at the N-terminal of the NAP-2 sequence. NAP-2 is a subsequence of &bgr;-thromboglobulin (&bgr;-TG; SEQ ID NO:5) which has an additional eleven amino acids at the N-terminal end. &bgr;-TG is itself a subsequence of connective tissue-activating peptide (CTAP-III; SEQ ID NO:6) which has an additional four amino acids at the N-terminal. CTAP-III is a subsequence of platelet basic protein (PBP; SEQ ID NO:7) which has an additional nine amino acids at the N-terminal.
NAP-2 along with interleukin-8 (human IL-8; SEQ ID NO:8; porcine IL-8 SEQ ID NO:9) and melanoma growth-stimulating activity (MGSA) have been assigned to a subfamily known as the &agr;-chemokines. The &agr;-chemokines have in common with each other four cysteine residues at highly conserved positions, which enclose the core region of the molecules, as described by Brandt et al (Ehlert, J. E., F. Peterson, M. H. G. Kubbuta, J. Gerdes, H.-D. Flad, and E. Brandt, (1995)
J. Biol. Chem.
270:6338). Brandt et al. found an apparently naturally occurring C-terminus truncated variant of NAP-2, lacking the last four amino acids of NAP-2, that displays enhanced increase in potency to stimulate neutrophil degranulation. Brandt et al. also synthesized variants lacking the final one, two, three, five and six amino acids of the C-terminus of NAP-2. All of these C-truncated polypeptides exhibited a moderate increase in potency over NAP-2 with the exception of the sequence having only the first sixty-four amino acids of NAP-2. Brandt et al. discussed the possible significance of the sequence modifications with respect to the structure of NAP-2 and its function.
Platelet factor 4 (PF4; SEQ ID NO:10) is a seventy amino acid polypeptide (Hermodson, M., G. Schmer and K. Kurachi, (1977)
J. Biol. Chem.
252:6276; Morgan, F. J., G. S. Begg, C. N. Chesterman, (1979)
Thromb. Haemost
42:1652). PF4 has been shown to inhibit proliferation of two osteoblastic osteosarcoma cell lines, Saos-2 and G-292 (U.S. Pat. No. 5,304,542. Inventor: D. M. Tatakis. Issued Apr. 19, 1994). Indomethacin apparently did not affect PF4-induced inhibition of the cell proliferation. Particular fragments, PF4(58-70), PF4(47-70) and monomeric low-affinity PF4 (LAPF4), which is 50% homologous to PF4 and contains an &agr;-helical C-terminus were also suggested as being useful. PF4 and such related polypeptides were thus described as being useful in a method for inhibiting proliferation of osteoblasts, in among other things, humans suffering from osteoporosis.
The first 70 amino acids of NAP-2V and the sequence of PF4 are about 51% homologous and the positions of the four cysteine residues are conserved between the two polypeptides.
It has now been shown that NAP-2, NAP-2V, as well as subsequences of NAP-2V also show bone stimulatory effects, while certain subsequences do not display bone stimulatory activity. NAP-2V-(1-26) (SEQ ID NO:11) and NAP-2V-(13-26; gln
25
→glu
25
) (SEQ ID NO:12) were found to increase the observed bone apposition rate, the latter of the two being more potent than the former. NAP-2V-(10-26) (SEQ ID NO:13) appeared to cause a small increase in the observed bone apposition rate, although the statistical significance of the observed increase was questionable. Protected versions of NAP-2V-(13-26; gln
25
→glu
25
) (SEQ ID NO:17) and of NAP-2V-(15-22) (SEQ ID NO:18) were also found to have bond stimulatory activity. NAP-2V-(11-26) (SEQ ID NO:14) and NAP-2V-(12-26) (SEQ ID NO:15) were found to have no effect on observed bone mineral apposition rate.
The invention thus includes a compound which promotes bone growth in mammals, comprising an amino acid sequence which consists essentially of:
(i) up to 69 consecutive amino acids of the sequence corresponding to SEQ ID NO:2 with (a) from 6 to about 14 amino acids deleted from the N-terminus of SEQ ID NO:2, or (b) 7 to about 53 amino acids deleted from the C-terminus of SEQ ID NO:2, or both (a) and (b) and wherein the sequence includes no cysteine residues or at least two cysteine residues;
(ii) a variant of a polypeptide of (i) containing a plurality of said amino acid sequences;
(iii) a conservatively substituted variant of a polypeptide of (i) or (ii); or
(iv) a functionally equivalent homologue of (i), (ii) or (iii).
In another aspect, the invention includes a polypeptide in which the amino acid sequence consists essentially of an amino acid sequence corresponding to SEQ ID NO:11, or corresponding to SEQ ID NO:11 with from 6 to about 14 amino acids deleted from the N-terminus of SEQ ID NO:11, or corresponding to SEQ ID NO:11 with from 6 to about 12 amino acids deleted from the N-terminus of SEQ ID NO:11; or corresponding to SEQ ID NO:11 with from 6 to about 9 amino acids deleted from the N-terminus of SEQ ID NO:11; or a functionally equivalent homologue any of the foregoing polypeptides.
A polypeptide of the present invention can have an amino acid sequence which consists essentially of an amino acid sequence corresponding to SEQ ID NO:12, or SEQ ID NO:13, or SEQ ID NO:18, or a functionally equivalent homologue of any of these.
The present invention includes a compound “derived from” any of the preceding polypeptides.
The present invention thus includes a polypeptide wherein the amino acid sequence consists essentially of:
(v) an amino acid sequence corresponding to SEQ ID NO:12;
(vi) an amino acid sequence corresponding to SEQ ID NO:13;
(vii) an amino acid sequence corresponding to SEQ ID NO:18;
(viii) a variant of a polypeptide of (v), (vi) or (vii) containing a plurality of said amino acid sequences; or
(ix) a conservatively substituted variant of a polypeptide of (v), (vi), (vii) or (viii).
In preferred embodiments, a polypeptide of the present invention that cysteine residues, includes two cysteine residues that are located at positions corresponding to the tenth and twelfth positions of SEQ ID NO:2.
A polypeptide of the present invention can have, if desired, or necessary, one or the other or both of the N-terminal amino acid and the C-terminal amino acid protected by a protecting group.
The polypeptide can be synthetic and the amino acid sequence can have a molecular weight in the range of from about 1000 to 4000 daltons; or from about 1000 to about 3000; or from about 1000 to about 2000; or more preferably from about 1200 to about 1800.
In another aspect, the invention is a first polypeptide which promotes bone growth in mammals comprising a sequence of amino acids which consists essentially of up to 69 amino acids and is sufficiently duplicative of a second polypeptide such that the first polypeptide is encoded by a DNA that selectively hybridizes under stringent conditions with DNA encoding the second polypeptide. In this instance, the second polypeptide comprises an amino acid sequence which consists essentially of:
(i) up to 69 consecutive amino acids of the sequence corresponding to SEQ ID NO:2 with (a) from 6 to about 14 amino acids deleted from the N-terminus of SEQ ID NO
Hunt John C.
Osteopharm Inc.
Romeo David S.
LandOfFree
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