WO1997012036A2 - Bone stimulating factor - Google Patents
Bone stimulating factor Download PDFInfo
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- WO1997012036A2 WO1997012036A2 PCT/CA1996/000653 CA9600653W WO9712036A2 WO 1997012036 A2 WO1997012036 A2 WO 1997012036A2 CA 9600653 W CA9600653 W CA 9600653W WO 9712036 A2 WO9712036 A2 WO 9712036A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- 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 September 15, 1994 under WO 94/20615, United States Patent No 5,320,970 and European patent application No 92 302 446, published under 505 210 on September 23, 1992 the contents of which applications are incorporated herein by reference
- neutrophil- activating peptide NAP-2, SEQ ID NO 1
- NAP-2V SEQ ID NO 2
- British Patent No 2 231 872 British Patent No 2 231 872 Inventors M Baggiohni.
- NAP-2 is a subsequence of ⁇ -thromboglobulm ( ⁇ -TG, SEQ ID NO 5) which has an additional eleven ammo acids at the N-terminal end ⁇ -TG is itself a subsequence of connective tissue- activating peptide (CTAP-III, SEQ ID NO 6) which has an additional four ammo acids at the N- terminal CTAP-III is a subsequence of platelet basic protein (PBP, SEQ ID NO 7) which has an additional nine am o acids at the N-termmal
- NAP-2 along with ⁇ nterleuk ⁇ n-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 ⁇ -chemokines
- the ⁇ -chemok ⁇ nes 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 a/ (Ehlert, J E , F Peterson, M H G Kubbuta, J Gerdes, H -D Flad, and E Brandt, (1995) J Biol Chem 2706338) Brandt et al found an apparently naturally occurring C-termmus truncated variant of NAP-2, lacking the last four ammo 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 ammo acids of the C-term
- Platelet factor 4 (PF4, SEQ ID NO 10) is a seventy ammo 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 (United States Patent No 5,304,542 Inventor D M Tatakis Issued April 19, 1994) Indomethacin apparently did not affect PF4- ⁇ nduced 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 ⁇ - helical C-termmus 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
- NAP-2V The first 70 ammo 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 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 polypeptide which promotes bone growth in mammals, where the polypeptide includes an ammo acid sequence corresponding to SEQ ID NO 2 with (a) from 6 to about 12 ammo acids deleted from the N-termmus of SEQ ID NO 2, (b) 7 to about 49 ammo acids deleted from the C-terminus of SEQ ID NO 2, or both (a) and (b), wherein the sequence includes no cysteine residues or at least two cysteine residues or a functionally equivalent homologue
- a polypeptide of the present invention is an ammo acid sequence corresponding to SEQ ID NO 11 up to 69 am o acids in length, or corresponding to SEQ ID NO 11 with from 6 to about 12 ammo acids deleted from the N-terminus of SEQ ID NO 11 , or corresponding to SEQ ID NO 11 with from 6 to about 9 ammo acids deleted from the N-termmus of SEQ ID NO 11 , wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue
- the invention is a polypeptide which promotes bone growth in mammals where the polypeptide has an ammo acid sequence corresponding to SEQ ID NO 12 up to 69 ammo acids in length or a functionally equivalent homologue, or having an ammo acid sequence consisting essentially of the ammo acid sequence corresponding to SEQ ID NO 11 , or a conservatively substituted variant thereof or a polypeptide having an ammo acid sequence consisting essentially of the ammo acid sequence
- a polypeptide of the present invention can have at least two cysteine residues which 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-termmal ammo acid and the C-termmal ammo acid protected by a protecting group
- the polypeptide can be synthetic and the am o acid sequence can have a molecular weight in the range of from about 1000 to 4000, or from about 1500 to about 3000 or more preferably from about 1500 to about 1800
- the invention is a first polypeptide comprising a sequence of ammo acids sufficiently dup cative of a second polypeptide having an am o acid sequence corresponding to SEQ ID NO 2 with (a) from 6 to about 12 am o acids deleted from the N- terminus of SEQ ID NO 2 (b) 7 to about 49 am o acids deleted from the C-terminus of SEQ ID NO 2, or both (a) and (b), wherein the sequence includes no cysteine residues or at least two cysteine residues, or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide
- the DNA sequence of NAP-2V disclosed by Walz ef al (British Patent No 2 231 872 Inventors M Baggiohni, K J Clemetson, and A Walz Published June 14, 1990
- nucleic acid sequence identified as SEQ ID NO 16 corresponds to sequences coding for the polypeptides identified as SEQ ID NO 1 , NO 11 , NO 12 (Glu-GIn) or NO 13
- nucleic acids 37 through 78 of SEQ ID NO 16 encode the am o acid subsequence identified as SEQ ID NO 12 in which the penultimate ammo acid of the subsequence is glutamine
- Stringency conditions takes on its common meaning to a person skilled in the art here Appropriate stringency conditions which promote nucleic acid hybridization, for example, 6x sodium chloride/sodium citrate (SSC) at about 45°C are known to those skilled in the art The following examples are found in Current Protocols in Molecular Biology John Wiley & Sons NY (1989), 6 3 1-6 3 6
- SSC sodium chloride/sodium citrate
- a second suitable hybridization solution can be 1% crystalline BSA (fraction V) 1 mM EDTA 0 5 M Na 2 HPO ⁇ pH 7 2, 7% SDS
- the salt concentration in the wash step can be selected from a low stringency of about 2x
- the first polypeptide can have a sequence of am o acids sufficiently duplicative of a second polypeptide having an ammo acid sequence corresponding to SEQ ID NO 12 up to 69 ammo acids in length, or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide
- the first polypeptide can have a sequence of ammo acids sufficiently duplicative of a second polypeptide having an am o acid sequence corresponding to to SEQ ID NO 11 , or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide, or it can have a sequence of ammo acids sufficiently duplicative of a second polypeptide comprising an ammo acid sequence corresponding to SEQ ID NO 12, or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide, or it can have a sequence of ammo acids sufficiently duplicative of a second polypeptide having an ammo acid sequence corresponding to SEQ ID NO 13, or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide A bone growth polypeptid
- the invention includes any number of chimeric bone stimulating factors made having an ammo acid sequence of polypeptides of the present invention
- the invention is an agent for use in prevention and treatment of a bone reduction related disease which includes any polypeptide or polypeptides of the present invention as an active ingredient
- the invention is, alternatively, a pharmaceutical composition for promoting bone growth, having a therapeutically effective amount of a polypeptide or polypeptides of the present invention
- the invention includes use of a polypeptide or polypeptides of the present invention for the treatment of osteoporosis
- the use of a polypeptide or polypeptides can be to promote bone growth in a mammal
- the invention includes use of the polypeptide or polypeptides in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis
- the invention includes a diagnostic kit for determining the presence of a polypeptide or polypeptides of the present invention
- the kit can includes an antibody to a said polypept ⁇ de(s) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypept ⁇ de(s) and the antibody are bound together
- the invention includes an antibody synthesized using a polypeptide consisting of an ammo acid sequence identified as SEQ ID NO 11 , SEQ ID NO 12. or SEQ ID NO 13 or a conservatively substituted variant thereof
- the invention includes an antibody which binds to a polypeptide or polypeptides of the present invention, synthesized using the polypept ⁇ de(s)
- the invention includes an isolated DNA fragment which encodes the expression of any of the polypeptides of the present invention, and DNA which differs from the fragment due to the degeneracy of the genetic code
- the invention includes a vector comprising a DNA sequence which encodes the expression of any of any of the polypeptides of the present invention
- the invention includes a process for producing a polypeptide of the invention which includes the steps of a) preparing a DNA fragment containing a nucleotide sequence which encodes the polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant
- the invention is a synthetic polypeptide up to 65 ammo acids in length, having in vivo bone stimulatory activity in mammals, having an ammo acid sequence which is at least about 19% conserved in relation to the ammo acid sequence identified as SEQ ID NO 2, a conservatively substituted variant thereof, or a functionally equivalent homologue
- the synthetic polypeptide can also be at least about 22%, 25%, 28%, 31%.
- Such a synthetic polypeptide can have at least 49 ammo acids deleted from the sequence
- the polypeptide can have no cysteine residues or at least two cysteine residues
- the polypeptide can have a molecular weight in the range of from about 1000 to 4000
- the present invention is a first polypeptide having a sequence of ammo acids sufficiently duplicative of a second polypeptide which includes an ammo acid sequence of any of the synthetic polypeptides, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide
- the invention includes a chimeric bone stimulating factor comprising an ammo acid sequence of any of the synthetic polypeptides
- An agent of the present invention for use in prevention and treatment of a bone reduction related disease can include one or more of the synthetic polypeptides
- the invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of one or more of the synthetic polypeptides
- a synthetic polypeptide can be used for the treatment of osteoporosis or to promote bone growth in a mammal
- Such a synthetic polypeptide can be used in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis
- the invention includes a diagnostic kit for determining the presence of one or more the synthetic polypeptides, the kit including ant ⁇ body( ⁇ es) to a the polypept ⁇ de(s) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypept ⁇ de(s) and the ant ⁇ body( ⁇ es) are bound together
- the invention includes an antibody which binds to one or more of the synthetic polypeptides, synthesized using the polypeptide
- the invention includes an isolated DNA fragment which encodes the expression of any of the synthetic polypeptides, and DNA which differs from the fragment due to the degeneracy of the genetic code
- the invention includes a vector which includes such a DNA sequence
- the invention includes a process for producing any of the synthetic polypeptides, which includes a) preparing a DNA fragment containing a nucleotide sequence which encodes a polypeptide, b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication, c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide, and d) culturmg the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture
- the invention includes a polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide being up to 65 ammo acids in length and havmg the sequence identified as SEQ ID NO 11 , SEQ ID NO 12, or SEQ ID NO 13, analogues thereof wherein the am o acids in the sequence may be substituted, deleted or added, so long as the bone stimulatory activity in mammals derived the three dimensional structure of the sequence is preserved and conjugates of each of the polypeptides or analogues thereof, wherein if the polypeptide sequence contains a cysteine residue, there are at least two cysteine residues
- Such a polypeptide can be substantially pure and the ammo acid sequence can have a molecular weight in the range of from about 1000 to about 4000, or from about 1500 to about 3000 or from about 1500 to about 1800
- the invention is also a first polypeptide that includes a sequence of am o acids sufficiently duplicative of a second polypeptide having an ammo acid sequence corresponding such a polypeptide or a
- the invention includes an isolated DNA sequence encoding the ammo acid sequence of any of the polypeptides of the invention, or an analogue thereof, wherein the ammo acids in the sequence may be substituted, deleted or added, so long as bone stimulatory activity in mammals derived from the three dimensional conformation of the sequence is preserved in a polypeptide comprising the ammo acid sequence, sequences which hybridize to the DNA and encode an ammo acid sequence of a polypeptide which displays bone stimulatory activity in mammals and DNA which differs from the sequence due to the degeneracy of the genetic code DESCRIPTION OF THE DRAWINGS
- the error bars are ⁇ 1 S D
- Figure 2 graphically illustrates the observed bone mineral apposition rate ( ⁇ m per day) for rats injected with chemically synthesized polypeptides of A, NAP-2V-(1-26) (SEQ ID NO 11 ), B, NAP-2V-( 13-26, gln 25 -glu 25 ) (SEQ ID NO 12, ), C, NAP-2V-( 10-26) (SEQ ID NO 13), D, NAP-2V-(11-26) (SEQ ID NO 14), and E, NAP-2V-( 12-26) (SEQ ID NO 15)
- the first bar in the graph represents the control
- the number of rats used for the determinations were 4, 4, 3, 4, 4, and 4, respectively
- the error bars are ⁇ 1 S D
- Figure 3 illustrates the ammo acid sequences of polypeptides tested, corresponding ammo acids aligned with each other, the active peptides being shown above the line and sequences which were not found to stimulate bone growth being below the line Approximate molecular weights are shown below the sequence identification numbers
- Polypeptides having the sequences of NAP-2 (SEQ ID NO 1 ) and NAP-2V (SEQ ID NO 2) were chemically synthesized directly according to standard methods and experiments were conducted to determine whether the chemically synthesized polypeptides displayed activity
- Each rat of the first group was given, by subcutaneous injection into the left gluteus maximus region, 200 ⁇ l of a 1% aqueous acetic acid solution containing 25 nmol (about 191 ⁇ g) of NAP-2 (SEQ ID NO 1 )
- Each rat of the second group was similarly given 200 ⁇ l of a 1% aqueous acetic acid solution containing 25 nmol (about 207 ⁇ g) of NAP-2V (SEQ ID NO 2)
- Each rat of the third group, the control group was similarly given 200 ⁇ l of 1% acetic acid solution
- Polypeptides having the sequence corresponding to either SEQ ID NO 1 or SEQ ID NO:2 have thus been found to stimulate bone growth.
- each rat was injected in the right gluteus maximus with 200 ⁇ l of a 1M tetracycline hydrochloride (Sigma Chemical) solution This dosage of tetracycline is about 16 mg per kg of animal body weight About 48 hours later, each rat was injected in the left gluteus maximus with the same dosage of tetracycline Twenty-four hours later the rats were sacrificed by C0 2 narcosis and the right femur taken for bone mineral apposition rate determination One rat died during the course of the experiments
- Each cured block was cut into 400 ⁇ m thick sections using a Leitz saw microtome equipped with a diamond charged blade
- the relatively thick sections were ground down between two ground glass plates pre-roughened with carborundum powder to a final thickness of about 10 ⁇ m, water being used as the grinding lubricant
- the thin sections were dried and mounted unstained in Permount (Fisher)
- Table TWO Comparison of Group Arithmetic Means of Bone Apposition Rates ( ⁇ m per day) shown in Figure 2.
- NAP-2V-(1-26) SEQ ID NO 11
- NAP-2V-( 13-26, gln 25 -glu 25 ) SEQ ID NO 12
- NAP-2V-( 10-26) (SEQ ID NO 13) appeared to cause a small increase in the observed bone apposition rate although the significance of the observed increase was questionable 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 sequence of NAP-2V- (10-26) retains both the cys 10 and cys 12 residues
- the sequences of NAP-2V-(11-26) and NAP-2V-( 12-26) each retain the cys 12 residue
- the sequence of NAP-2V-(13-26, gln 25 -glu 25 ) retains neither of the cys 10 and cys 12 residues All of these NAP-2V subsequences lack the cys 36 and cys 52 residues present in the parent NAP-2V It may be that the reduced activity of NAP-2V-( 10-26), NAP
- deletions of one or more ammo acids are concerned, it is likely that deletions of a small number of ammo acids from each end of either sequence might be possible, bearing in mind the observation that the deletions to obtain SEQ ID NOs 14 and 15 yield polypeptides which do not appear to enhance bone growth Further, symmetrical, or nearly symmetrical deletions would likely be the most possible to be made while retaining the three-dimensional configuration Internal deletions, although likely to be possible to some limited extent, should be few Additions of ammo acids could very likely be made at the ends of the sequence, and as with deletions, symmetrical or nearly symmetrical additions to the carboxy and ammo terminals are likely to be possible Internal additions, although likely to be possible to some limited extent, should be few
- terminal additions are most likely to be most useful, as such a modification can serve a variety of functions an identifying group as for use in a radioimmunoassay, or a linking group, as examples
- a polypeptide of the present invention can be improved with respect to possible degradation, as might occur in the body in the presence of protease, for instance, by protection of the C-terminus, the N-termmus, or both the C-termmus and N-termmus of the polypeptide
- protected terminal am o group refers to a terminal ammo group (N-termmus) coupled with any of various amino-terminal protecting groups that can be employed in peptide synthesis
- suitable groups include acyl protecting groups, for example, formyl, acetyl, benzoyi, t ⁇ fluoroacetyl, succinyl, and methoxysuccinyl, aromatic urethane protecting groups, for example benzyloxycarbonyl, and aliphatic urethane protecting groups, for example f-butoxycarbonyl or adamantyloxycarbonyl (Gross and Mienhofer eds ,
- protected terminal carboxyl group refers to a terminal carboxyl group (C-terminus) coupled with any of various carboxy-terminal protecting groups
- suitable groups include f-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond
- Compounds may also be synthesized using manual or automatic techniques, for example, an Applied BioSystems 430A Peptide Synthesizer (Foster City, California) or a Biosearch SAM 11 automatic peptide synthesizer (Biosearch, Inc , San Rafael, California)
- Compounds of the present invention and compositions containing them find use in numerous therapeutic and prophylactic applications in the prevention and treatment of bone reduction related to a disease
- Compounds can thus be used as treatments to promote bone growth, in the treatment of osteoporosis, for example, by any suitable route
- the preferred routes are suitable for delivery of polypeptide-type compounds to the bloodstream of a subject, bearing in mind proper storage and handling conditions required for polypeptides such as those described herein
- compositions containing an effective amount of compounds of the present invention including the nontoxic addition salts, amides and esters thereof, which may, alone, serve to provide the treatment benefits described above
- Such compositions can also be provided together with physiologically tolerable liquid, gel or solid diluents adjuvants and excipients
- the daily dosage may well be between 0 01 and 300 mg or more per kg of bodyweight More preferably, the dosage would be in the neighbourhood of from about 0 1 to about 30 mg per kg of bodyweight It may be that the preferred frequency of administration would be greater or less than once per day, depending upon the route of administration, convenience, and the variation of effectiveness of treatment with frequency of and amount used per administration
- the dosage administered also depends on the subject and to which effect such administration is to give
- the dosage of any one or more of the compounds will depend on many factors including the specific compound or combination of compounds being utilized, the mode of administration, and the mammal being treated Dosages of a particular compound or combination of compounds can be determined using conventional considerations, for example, by customary comparison of the differential activities of the subject compounds and that of a known agent that is by means of an appropriate pharmacological protocol in which for example, bone density of subjects
- compositions include the employment of the compounds in admixture with conventional excipients, that is, pharmaceutically acceptable organic or inorganic carrier substances which do not deleteriously react with the compounds, and which possibly enhance the storage and handling stability of the compounds
- the preparative procedure may include the sterilization of the pharmaceutical preparations
- the compounds may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, salts for influencing osmotic pressure, etc , which do not react deleteriously with the compounds
- compositions are conventionally administered parenterally, by injection, for example either subcutaneously or intravenously
- Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and, in some cases, oral formulations
- suppositories traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides, such suppositories may be formed from mixtures containing the active ingredient in the range of 0 5% to 10%, preferably 1%-2%
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like
- These compositions take the form of solutions, suspensions, tablets, pills capsules, sustained release formulations, or powders, and contain 10% - 95% of active ingredient, preferably 25% - 70%
- These oral formulations include formulations designed to protect the peptide
- a DNA sequence encoding a desired polypeptide of the present invention is synthesized using standard automated techniques, or the coding sequences or portions thereof are retrieved from cDNA or genomic libraries This DNA is ligated into suitable expression vectors and these vectors are transformed into appropriate hosts
- suitable expression vectors include both procaryotic and eukaryotic culture systems Procaryotes most frequently are represented by various strains of E coli
- procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta- lactamase (penicillmase), lactose (1nc) promoter systems (Chang et al , (1977) Nature 198 1056), the tryptophan (trp) promoters system (Goeddel et al , (1990
- the expression systems useful in the eukaryotic systems of the invention comprise promoters derived from appropriate eukaryotic genes
- a class of promoters useful in yeast include promoters for synthesis of glycolytic enzymes, including alcohol dehydrogenase promoters, glyceraldehyde-3-phosphate dehydrogenase promoter (Holland & Holland, (1980) J Biol Chem 25 2596), alpha-factor promoter (Bitter et al , (1984) Proc Natl Acad Sci 81 5330), the gal promoter (Johnston & David, (1984) Mol Cell Biol 4 1440) those for 3-phosphoglycerate kinase (Hitzeman et al , (1980) J Biol Chem 256 1385) or the Leu2 gene obtained from YEp13 (Broach J , et al , (1978) Gene 8 121 )
- Suitable mammalian promoters include the early and late promoters from SV40 (Fiers et al , (1978) Nature 273 113) or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses Suitable viral and mammalian enhancers are cited above In the event plant cells are used as an expression system the nopaline synthesis promoter is appropriate (Depicker, A et al (1982) J Mol Appl Gen 1 56) The expression systems are included on replication vectors or are caused to integrate into the chromosome of a recombinant host For systems wherein the vectors include a replication system, these may be low or high copy number, usually having copy numbers of fewer than about 1000, although in certain situations, runaway vectors may be employed.
- the sequence encoding a polypeptide of the invention may be ligated in tandem with an amplifying gene such as dihydrofolate reductase, metallothioneins, thymidine kinase, or the like
- an amplifying gene such as dihydrofolate reductase, metallothioneins, thymidine kinase, or the like
- both the amplifying gene and the target gene can be under the regulation of the same transcnptional and translational regulatory regions
- the vector will include a marker which allows for selection of host cells containing the expression system; the nature of these markers depends on the host and is understood in the art.
- additional sequences such as enhancers can also be employed to enhance the level of transcription.
- an upstream sequence encoding signal peptides such as those described in U.S Pat. Nos 4,336,336, 4,338,397; and 4,546,082 may be employed
- the signal sequence is enzymatically cleaved as the polypeptide product is secreted
- transformation is done using standard techniques appropriate to such cells.
- the calcium treatment employing calcium chloride, as described by Cohen, S.N., (1972) Proc Natl Acad Sci USA 69:2110; or the RbCI method described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring
- the system is transfected into the appropriate host and successful transformants are selected by markers contained on the expression vectors. Successfully transformed colonies are then cultured in order to produce the desired polypeptide.
- a promoter which can be controlled by regulating conditions in the environment be used so that the cells can be grown under conditions where the gene encoding the desired polypeptide of the invention is not expressed, and then production of the polypeptide induced by appropriate manipulation of conditions
- the trp promoter is used in E coli, the cells are grown in the presence of tryptophan and expression is then induced by diminution of tryptophan concentration or by addition of a tryptophan analogue such as indolylacetic acid.
- the cells are grown at relatively low temperature such as at about 35°C , to a suitable cell density, and the temperature is then elevated to activate this promoter
- the N-terminal methionine may or may not be cleaved
- the use of the metallothionein promoter permits induction by addition of heavy metals or glucocorticoids This protocol is preferred to prevent premature accumulation of the polypeptide which might be harmful to the growth of the cell
- polypeptide can be produced mtracellularly, or in secreted form by construction of vectors in which the peptide is preceded by a signal peptide workable in the appropriate host
- the polypeptide is recovered from the medium or from the cells using suitable techniques generally known in the art, and purified by, for example, ion exchange chromatography, ammonium sulfate precipitation, gel permeation chromatography, and so forth
- the polypeptide made available by the invention disclosed herein can be used to obtain antisera thereto (Stites, D P and A I Terr 1991 In Basic & Clinical Immunology, 7th Ed Appleton and Lange, Norwalk, Connecticut and San Matea California)
- Methodology and products can be developed using an antibody to a polypeptide for use in detecting the polypeptide with which the antibody binds This apparently having been accomplished at least for the polypeptide having the sequence of CTAP-III (SEQ ID NO 3) (Baggiohni, M , Clemetson, K J , Walz, A Internationai Patent Appication No PCT/EP89/01389, published June 14, 1990 under WO90/06321 )
- Methodology and products can be developed using an antibody
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96931700A EP0853667A2 (en) | 1995-09-26 | 1996-09-26 | Bone stimulating factor |
AU70811/96A AU7081196A (en) | 1995-09-26 | 1996-09-26 | Bone stimulating factor |
JP9513029A JPH11514858A (en) | 1995-09-26 | 1996-09-26 | Bone stimulating factor |
US09/229,304 US6693081B2 (en) | 1995-09-26 | 1999-01-13 | Bone stimulating factor |
US10/718,526 US20040147450A1 (en) | 1995-09-26 | 2003-11-24 | Bone stimulating factor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US431495P | 1995-09-26 | 1995-09-26 | |
US60/004,314 | 1995-09-26 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US4805898A Continuation | 1995-09-26 | 1998-03-26 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1997012036A2 true WO1997012036A2 (en) | 1997-04-03 |
WO1997012036A3 WO1997012036A3 (en) | 1997-06-05 |
Family
ID=21710157
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA1996/000653 WO1997012036A2 (en) | 1995-09-26 | 1996-09-26 | Bone stimulating factor |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0853667A2 (en) |
JP (1) | JPH11514858A (en) |
AU (1) | AU7081196A (en) |
WO (1) | WO1997012036A2 (en) |
ZA (1) | ZA968062B (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000042069A1 (en) * | 1999-01-13 | 2000-07-20 | Osteopharm Inc. | Bone stimulating factor |
WO2000075185A1 (en) * | 1999-06-02 | 2000-12-14 | Osteopharm Inc. | Bone stimulating factor |
WO2002006300A2 (en) * | 2000-07-19 | 2002-01-24 | Universite De Bordeaux I | Mutated pf-4, its fragments and mutated fusion peptides, their analogues, corresponding dna, cdna and mrna sequences and their use for inhibiting angiogenesis |
US7399826B1 (en) | 2003-10-02 | 2008-07-15 | Ali Sadat M | Peptide for promoting healing of fractures |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102019207859A1 (en) * | 2018-12-21 | 2020-06-25 | Gelita Ag | Synthetic and recombinantly produced collagen peptides with biological effectiveness |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994005309A1 (en) * | 1992-08-28 | 1994-03-17 | University Of Louisville Research Foundation, Inc. | Use of platelet factor 4 to inhibit osteoblast proliferation |
WO1995028172A1 (en) * | 1994-04-18 | 1995-10-26 | Osteopharm Limited | Human bone stimulating factor |
-
1996
- 1996-09-25 ZA ZA968062A patent/ZA968062B/en unknown
- 1996-09-26 WO PCT/CA1996/000653 patent/WO1997012036A2/en not_active Application Discontinuation
- 1996-09-26 EP EP96931700A patent/EP0853667A2/en not_active Withdrawn
- 1996-09-26 AU AU70811/96A patent/AU7081196A/en not_active Abandoned
- 1996-09-26 JP JP9513029A patent/JPH11514858A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994005309A1 (en) * | 1992-08-28 | 1994-03-17 | University Of Louisville Research Foundation, Inc. | Use of platelet factor 4 to inhibit osteoblast proliferation |
WO1995028172A1 (en) * | 1994-04-18 | 1995-10-26 | Osteopharm Limited | Human bone stimulating factor |
Non-Patent Citations (1)
Title |
---|
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 11, 17 March 1995, MD US, pages 6338-6344, XP002022825 JAN E. EHLERT ET AL.: "Limited and defined truncation at the C terminus enhances receptor binding and degranulation activity of the neutrophil-activating peptide 2 (NAP-2)" cited in the application * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6693081B2 (en) | 1995-09-26 | 2004-02-17 | Osteopharm Inc. | Bone stimulating factor |
WO2000042069A1 (en) * | 1999-01-13 | 2000-07-20 | Osteopharm Inc. | Bone stimulating factor |
JP2002538775A (en) * | 1999-01-13 | 2002-11-19 | オステオファーム インコーポレイテッド | Bone stimulating factor |
AU779261B2 (en) * | 1999-01-13 | 2005-01-13 | Osteopharm Inc. | Bone stimulating factor |
WO2000075185A1 (en) * | 1999-06-02 | 2000-12-14 | Osteopharm Inc. | Bone stimulating factor |
WO2002006300A2 (en) * | 2000-07-19 | 2002-01-24 | Universite De Bordeaux I | Mutated pf-4, its fragments and mutated fusion peptides, their analogues, corresponding dna, cdna and mrna sequences and their use for inhibiting angiogenesis |
FR2811991A1 (en) * | 2000-07-19 | 2002-01-25 | Univ Bordeaux 1 | PF-4 MUTE, ITS FRAGMENTS AND MERGED MELTING PEPTIDES, THEIR ANALOGS, THE CORRESPONDING DNA, DNA AND RNA SEQUENCES AND THEIR USE IN THE INHIBITION OF ANGIOGENESIS |
WO2002006300A3 (en) * | 2000-07-19 | 2002-04-11 | Univ Bordeaux 1 | Mutated pf-4, its fragments and mutated fusion peptides, their analogues, corresponding dna, cdna and mrna sequences and their use for inhibiting angiogenesis |
US7399826B1 (en) | 2003-10-02 | 2008-07-15 | Ali Sadat M | Peptide for promoting healing of fractures |
Also Published As
Publication number | Publication date |
---|---|
EP0853667A2 (en) | 1998-07-22 |
JPH11514858A (en) | 1999-12-21 |
AU7081196A (en) | 1997-04-17 |
ZA968062B (en) | 1997-06-18 |
WO1997012036A3 (en) | 1997-06-05 |
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