WO2000075185A1 - Facteur de stimulation des os - Google Patents

Facteur de stimulation des os Download PDF

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Publication number
WO2000075185A1
WO2000075185A1 PCT/CA2000/000673 CA0000673W WO0075185A1 WO 2000075185 A1 WO2000075185 A1 WO 2000075185A1 CA 0000673 W CA0000673 W CA 0000673W WO 0075185 A1 WO0075185 A1 WO 0075185A1
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Prior art keywords
seq
polypeptide
group
amino acid
bone
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PCT/CA2000/000673
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English (en)
Inventor
Cherk Shing Tam
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Osteopharm Inc.
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Publication date
Priority claimed from US09/323,854 external-priority patent/US6743895B1/en
Application filed by Osteopharm Inc. filed Critical Osteopharm Inc.
Priority to AU53792/00A priority Critical patent/AU5379200A/en
Publication of WO2000075185A1 publication Critical patent/WO2000075185A1/fr

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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

Definitions

  • the invention includes a synthetic polypeptide having in vivo bone stimulatory activity in mammals and which increases mineral content in bones of mammals, having an amino acid sequence which is at least about 28% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 and having at least one amino acid deleted therefrom.
  • the invention includes any of the foregoing synthetic polypeptides in which at least six amino acids deleted from the polypeptide sequence; or in which at least eleven amino acids deleted from the sequence; or in which at least sixteen amino acids deleted from the sequence; or in which at least twenty-one amino acids deleted from the sequence; or in which at least twenty-six amino acids deleted from the sequence.
  • the invention is a polypeptide that includes an amino acid sequence that is between 19% and 90% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 ; or an amino acid sequence that is between 19% and 86% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 ; or an amino acid sequence that is between 19% and 69% conserved in relation to the amino acid sequence identified as SEQ ID NO:1 ; or an amino acid sequence that is between 19% and 56% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 42% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 39% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 19% and 28% conserved in relation to the amino acid sequence identified as SEQ ID NO:1; or an amino acid sequence that is between 28% and 90% conserved in relation to the amino acid sequence identified as SEQ ID
  • q can be 10, 5 or 0.
  • the remaining amino acids can be selected from the group consisting of giycine, alanine, valine, isoleucine, serine, threonine, methionine, asparagine and glutamine.
  • the invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide (or a pharmaceutical composition including the polypeptide) described above as part of the invention.
  • the invention includes the treatment of osteoporosis, promotion of bone growth in a mammal or treatment of a human of a bone reduction related disease.
  • the invention includes a diagnostic kit for determining the presence of a polypeptide of the invention, in which the kit includes an antibody to a polypeptide (or polypeptides) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide (or polypeptides) and the antibody become bound together.
  • the invention includes an isolated DNA sequence encoding any amino acid sequence of the invention, or an analogue thereof, wherein the amino 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 having the amino acid sequence; sequences which hybridize to the DNA and encode an amino 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.
  • Figure 5 graphically depicts the bone mineral apposition rate of intact rats as determined by tetracycline labelling.
  • Group A rats were treated with rabbit antibodies to the chemically synthesized normal polypeptide (SEQ ID NO:1).
  • Group B rats were treated with the same antibodies and the polypeptide itself.
  • Group C is the control group.
  • the error bars indicate ⁇ 1 standard deviation (S.D.).
  • Figure 9 graphically depicts the bone mineral apposition rate ( ⁇ m per day) in rats injection with chemically synthesized polypeptides: SEQ ID NO:8 (Group F); SEQ ID NO:9 (Group G).
  • Figure 11 is a DEXA image of a right femur of a rat showing scanned neck area.
  • the error bars indicate ⁇ 1 standard deviation (S.D.).
  • Figure 15 graphically depicts the bone mineral apposition rate ( ⁇ m per day) in rats injected with chemically synthesized polypeptide fragments SEQ ID NO:26 (Group LL); SEQ ID NO:44 (Group MM); SEQ ID NO:45 (Group NN); and SEQ ID NO:46 (Group PP).
  • the error bars indicate ⁇ 1 standard deviation (S.E.). P ⁇ 0.001 , 0.05, 0.0025 and 0.01 for Groups LL, MM, NN and PP, respectively.
  • Figure 17 graphically depicts dosage dependence of the bone mineral apposition rate ( ⁇ m per day) in rats injected with chemically synthesized polypeptides SEQ ID NO:34 (Group BB, ( ⁇ )); SEQ ID SEQ ID NO:35 (Group CC, (A)); SEQ ID NO:36 (Group DD, (T)); SEQ ID NO:37 (Group EE, ( ⁇ )); and SEQ ID NO:38 (Group FF, (hexagons)).
  • the error bars indicate ⁇ 1 S.E.
  • a miniosmotic pump (Alzet) was loaded with about 1.5 ml of the chemically synthesized peptide having an N-terminal acetyl group (SEQ ID NO:2) in 0.1% acetic acid so as to give a calculated daily delivery of about 25 ⁇ g per day.
  • a pump was implanted under the subcutaneous fascia of the dorsal aspect of the left side of the thorax of five rats which had been parathyroidectomized seven days earlier. Five similarly parathyroidectomized rats received similar implants containing only 0.1% acetic acid. Five intact rats were also used as controls.
  • a second group of six ovariectomized rats was similarly treated with a 0.1% acetic acid solution containing no peptide over the same 28 day period.
  • a fourth group of six intact rats was similarly treated with a 0.1% acetic acid solution containing no peptide over the same 28 day period.
  • each of the right femurs was dissected out from its soft tissue, fixed for two days, and X-rays taken at 70 kV for 1 min., 2 min., and 3 min. The 3 minute exposures gave the most satisfactory results.
  • the right femur of each rat was decalcified separately.
  • the decalcification fluid consisted of 10% formic acid (v/v) and 5% sodium citrate (w/v) at pH 3.0. Each bone was placed in 6 ml of the decalcification fluid.
  • the bone mineral apposition rate was determined, as described previously, by measurement of the lower metaphysis of the left femur. The results are tabulated in Table Three and graphically depicted in Figure 3.
  • a second group of seven ovariectomized rats was similarly treated with a 0.1% acetic acid solution containing no peptide over the same period.
  • a fourth group of five intact rats was similarly treated with a 0.1% acetic acid solution containing no peptide over the same 8 week period. Two rats of the second group became ill during the 8 week period and were sacrificed prematurely. Post-mortem blood was taken by cardiac puncture and serum frozen until analyzed. An autopsy was performed on each rat. No obvious pathology was observed in the rats except for surgical scars and atrophy of the uterus and vagina of ovariectomized rats.
  • Peptide and KLH solutions were prepared as described in the preceding section.
  • To 1.25 ml KLH solution were added 1.25 ml peptide solution.
  • To the resultant solution were added 2.5 mg of EDC.
  • the solution was stirred constantly at room temperature for 4 hours and then dialysed against 1 litre of PBS. The PBS was changed three times.
  • the gel was blotted overnight at 30V and blocked with 3% milk in PBS. The gel was incubated overnight with rabbit serum diluted 1:250 in 1% milk/PBS followed by incubation with goat anti-rabbit-alkaline phosphatase diluted 1 :1000 for 1 hour. The gel was then developed with substrate. The synthetic peptide was seen by comasie blue staining. The peptide was detected by the second bleed of each rabbit and was not detected by the preimmune serum of either rabbit.
  • EXPERIMENTS INVOLVING RATS AND ANTIBODIES TO THE CHEMICALLY SYNTHESIZED PEPTIDE Antibody serum was prepared in 10 mM Tris.CI at pH 7.4. Each of five rats received 100 ⁇ l of the solution by injection into the left gluteus maximus. Each rat of a second group of five rats was treated similarly, but with an additional injection of solution containing 45 ⁇ g of the polypeptide (SEQ ID NO:1) into the right gluteus maximus. Each rat of a third group of five rats received an injection of 100 ⁇ l of 10 mM Tris.CI at pH 7.0.
  • Each of the fifteen rats was then injected as before with tetracycline hydrochloride, in the amount of 24 mg per Kg of body weight.
  • a second dose of tetracycline hydrochloride was injected about 48 hours later. The rats were sacrificed after about another 24 hours.
  • the bone mineral apposition rate was determined by measurements, described above, of the lower right femoral metaphysis. The results are given in Table Five and Figure 5.
  • an assay relying upon fluorescence of a substance bound by the enzyme being assayed could be used. It will be appreciated that there are a number of reporter systems which may be used, according to the present invention, to detect the presence of a particular polypeptide. With standardized sample collection and treatment, polypeptide presence above a threshold amount in blood serum could well be determined. Such a method based on antigenic response to the chemically synthesized human polypeptide (SEQ ID NO:1) could be developed and variants of the polypeptide obtained, as described above for amino acid substitution, deletion and addition, (and conjugates) could then be pre-screened as potential bone stimulating factors. Those that react positively with the antibody to the already known peptide could then be tested for bone stimulatory effects in vivo using the system described herein for rats, for example.
  • SEQ ID NO:1 chemically synthesized human polypeptide
  • Such an antibody-linked reporter system could be used in a method for determining whether blood serum of a subject contains a deficient amount of the polypeptide. Given a normal threshold concentration of such a polypeptide in blood serum of a given type of subject, test kits could thus be developed.
  • a modified sequence (SEQ ID NO:3) of the chemically synthesized peptide (SEQ ID NO:1) obtained by substitution of the cysteine residue at position 13 by alanine was prepared by standard chemical procedures.
  • An alanine residue is sterically similar to a reduced cysteine residue while rendering the polypeptide incapable of spontaneous dimerization.
  • a tricine SDS electrophoretic gel of the modified and unmodified (normal) peptides is shown in Figure 6.
  • Polypeptides having the amino acid sequences identified as SEQ ID NOs:4, 5, 6, 7, 8 and 9 were synthesized according to well known chemical procedures.
  • Gly lie Gly Lys Arg Thr Asn Glu His Thr Ala Asp Cys Lys lie
  • Bone mineral apposition rates were determined by measurements of the lower metaphysis of the right femur, as described previously. Results obtained in the two sets of experiments are summarized in Tables Seven and Eight and graphically depicted in
  • Ovariectomies were performed on rats as described above. A 0.1% acetic acid solution containing 25 nmoles of the polypeptide was administered subcutaneously to each rat each day for the duration of the experiment. One group of rats was treated for 12 weeks beginning 100 days after ovariectomization. Another group of rats was treated for eight weeks beginning eight weeks after ovariectomization. Rats were sacrificed at the end of the treatment period and dissected and post mortem assessment of bone mineral content was carried out.
  • the lumbar spines L1 - L4 were cleaned with a power nylon brush to remove the attached muscle. They were placed ventral side down under 3 cm of distilled water in a polypropylene container and scanned by a dual energy x-ray absorptometer (DEXA), Hoiogic 100, to determine the calcium content in grams.
  • DEXA dual energy x-ray absorptometer
  • the right femur of each rat was also dissected out intact and cleared of the attached muscles with a power nylon brush. It was scanned dorsal side down under 3 cm of distilled water by DEXA.
  • Four regions of the femur were scanned, as indicated in Figures 10 and 11: A, proximal end; B, diaphysis; C, distal end; and D, neck.
  • the bone mineral (i.e., calcium) content in grams was estimated in the four zones of the femur based on absorption and using an internal standard of the machine. Results are tabulated
  • Group H SEQ ID NO:1 :
  • Group L SEQ ID NOs: 10,11 ,12 & 13 (mixture): Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gin Asn Gin Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gin Asn Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gin Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp Gin Leu His Lys Lys Ala Ala Glu Thr Leu Met Val Leu Asp
  • a polypeptide having the ten amino acid sequence of SEQ ID NO:9, but protected at both ends was synthesized and tested for bone stimulatory activity in comparison to polypeptides identified as SEQ ID NOs: 1 , 7 and 9.
  • the protected polypeptide was acetylated at the amino terminus and amidated at the carboxy terminus and is identified herein as SEQ ID NO:24. Results obtained according to experimental procedures described above, using about 125 nmoles of polypeptide per Kg of body weight of animal are presented in Table Twenty and Figure 13.
  • Each of the polypeptides having the sequences identified as SEQ ID NO: 7 and 24 was dissolved in 5 mM acetic acid to a final concentration of 1 mg/ml and incubated at 37°C.
  • the peptide compositions were analyzed weekly by capillary electrophoresis on a P/ACE 6000 system (Beckman) using 50 mM sodium phosphate pH 2.5 as the running buffer on a 57 cm long x 75 ⁇ m internal diameter capillary.
  • the incubated peptide (20 ⁇ l) was diluted with 80 ⁇ l of running buffer, placed in a 500 ⁇ l vial prior to injection onto the P/ACE using pressure for 20 seconds. Following electrophoresis of the incubated peptide at 30 kV for 15 min a second run was carried out using freshly dissolved peptide as a control.
  • Each of the polypeptides was dissolved in 20 mM sodium phosphate pH 3.0, 20 mM ammonium acetate pH 4.0, 20 mM ammonium acetate pH 5.0, 20 mM MES pH 6.0, 20 mM sodium phosphate pH 7.0, 20 mM sodium phosphate pH 7.5, 20 mM sodium phosphate pH 8.0, 20 mM ammonium acetate pH 8.5 or 20 mM ammonium acetate pH 9.5 to a final concentration of 1 mg/ml.
  • the peptide was incubated at 37°C and the peptide assayed weekly by separation on P/ACE as described above. Some samples were separated by RP- HPLC and the isolated peaks subjected to mass spectroscopic analysis.
  • polypeptides having amino acid sequences identified as SEQ ID NOs are amino acid sequences identified as SEQ ID NOs.
  • SEQ ID NOs: 7 and 24 degraded overtime when dissolved in dilute acids. These peptides were found to be most stable when dissolved in pH 4.5 buffer. Analogues, SEQ ID NOs: 25 and 26 were found to be more stable than SEQ ID NOs: 7 or 24, while SEQ ID NO:27 was found to be significantly less stable.
  • each side chain indicated by an "X” the side chain would not bear a full ionic charge under physiological conditions.
  • the side chain of threonine includes a hydroxyl group, which is polar.
  • Asparagine the third amino acid, is also polar.
  • Alanine the seventh amino acid is considered to be relatively non-polar.
  • Cysteine the ninth amino acid, is considered polar, but the polypeptide is also thought to spontaneously dimerize by formation of an intermolecular disulfide bridge, as described above.
  • a second dosage of tetracycline was administered 48 hours later and the animals sacrificed 24 hours after that.
  • the lower metaphysis of the right femur was examined to determine the bone mineral apposition rate.
  • experiments were performed using a control in which no compound was present in the acetic acid solution and using a polypeptide having the sequence identified as SEQ ID NO:24. In all cases the N-terminus of the test compound was acetylated and C-terminus was amidated. The results obtained are shown in Figure 15.
  • a polypeptide having the ninth amino acid, cysteine, replaced by the amino acid tyrosine, SEQ ID NO:43 was synthesized and tested for bone stimulatory activity.
  • Four rats (about 300 grams) were each administered with 100 nmoles of material n 400 ⁇ l 20 mM acetic acid solution) by subcutaneous injection along with tetracycline (5 mg per animal in 400 ⁇ l water), as described above.
  • a second dosage of tetracycline was administered 48 hours later and the animals sacrificed 24 hours after that.
  • Such a polypeptide can have up to or be based on 30, 25, 20, 15 or 10 consecutive amino acids from the amino acid sequence identified as SEQ ID NO:1.
  • a compound "derived from" a polypeptide having a particular amino acid sequence is any molecular entity which is identical, substantially homologous, or otherwise functionally or structurally equivalent to that polypeptide.
  • a molecule derived from a particular polypeptide may encompass the amino acid sequence of the polypeptide, any portion of that polypeptide, or other molecular entity that functions to stimulate bone growth.
  • a molecule derived from such a binding domain will mimic the polypeptide from which it is derived.
  • Such molecular entities may include peptide mimetics and the like.
  • Peptides mimetics are structures which serve as substitutes for peptides in interactions with acceptor molecules (see Morgan et al. (1989) Ann. Reports Med. Chem. 24:243-252 for a review of peptide mimetics). Peptide mimetics, as used herein, include synthetic structures which may or may not contain amino acids and/or peptide bonds, but retain structural and functional features of a peptide from which they are derived. The term, "peptide mimetics” also includes peptoid and oligopeptoids, which are peptides or oligomers of N-substituted amino acids (Simon et al. (1972) Proc. Natl. Acad. Sci USA 89:9367-9371). Further included as peptide mimetics are peptide libraries, which are collections of peptides designed to be a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto.
  • Two polypeptide sequences are "substantially homologous" when at least about 85% (preferably at least about 85% to 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the polypeptide.
  • substantially homologous also refers to sequences showing identity to the specified polypeptide sequence.
  • analogues containing amide bond surrogates may be used to investigate aspects of peptide structure and function, such as rotational freedom in the backbone, intra and intermolecular hydrogen-bond patterns, modifications of local and total polarity and hydrophobicity, and oral unavailability.
  • Local conformational constraints can also be introduced to determine conformational requirements for activity of a potential peptide mimetic having bone stimulatory activity.
  • ⁇ , ⁇ -distributed amino acids may be used to examine the effects of conformational constraints on peptide activity (see, e.g. Manning et al. (1982) J. Med. Chem. 25:408-414; Mosberg et al. (1983) Proc. Natl. Acad. Sci. USA 106:506-512; Pelton et al. (1985) Proc. Natl. Acad. Sci. USA 82:236-239).
  • the synthetic molecules can also include D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule from enzymatic degradation (see, e.g.
  • cyclic amino acids such as 1-aminocyclopentanccarboxylic acid (cycloleucine) and ⁇ , ⁇ -cyclopentamethlyene- ⁇ - mercaptopropionic acid (see Hruby et al (1990), supra).
  • Peptoids will find use herein. Peptoids are oligomers of N-substituted amino acids (Simon et al (1972), supra), and can be used as motifs for the generation of chemically diverse libraries of novel molecules, which can then be tested for binding and bone stimulatory activity.
  • the monomers may incorporate t-butyl-based side-chain and 9- fluorenylmethoxy-carbonyl ⁇ -amine protection.
  • Oligomerization of the peptoid monomers may be performed by for example, in situ activation by either benzotriazol-l- yloxytris(pyrrolidino)phosphonium hexafluo ⁇ hosphate or bromotris(pyrrolidino)phosphonium hexafluo ⁇ hosphate. Other steps are identical to conventional peptide synthesis using ⁇ -(9- fluorenylmethoxycarbonyl)amino acids. Oligopeptoids may be identified which have affinities comparable to the corresponding polypeptides and, thus, are potentially useful as bone stimulatory agents.
  • a compound or polypeptide having the "charge pattern" of a particular polypeptide has the number and distribution (i.e., same order) of the charges of the side chains of the amino acids of the sequence of the particular polypeptide.
  • the charge of each side chain is based on the predominant charge that is present under physiological conditions. Spacing of the charges would also be similar to that provided by the polypeptide. In preferred instances, the spacing would be substantially the same as that provided by the polyamide backbone of the particular polypeptide, and so the compound is said in such instances to have substantially the same "charge pattern and spacing" of the polypeptide.
  • the polypeptide identified as SEQ ID NO:24 (and the corresponding unprotected polypeptide, SEQ ID NO:9) has a sequence of 10 amino acids contained in the 36 amino acid sequence of the polypeptide identified as SEQ ID NO:1.
  • SEQ ID NO:24 the protected version of the 10-amino acid polypeptide sequence, SEQ ID NO:24, has a bone stimulatory effect which exceeds that of either SEQ ID NO:1 or the 10-amino acid unprotected version, SEQ ID NO:9.
  • polypeptide identified as SEQ ID NO:27 (and the corresponding unprotected polypeptide, SEQ ID NO:30) has a sequence of 10 amino acids, only nine of which are the same as those contained in SEQ ID NO:1.
  • the protected version of this polypeptide was found to have the bone stimulatory effect also, but it was not as great as that of SEQ ID NO:26.
  • the polypeptides vary from each other according to the conditions to which the polypeptides are exposed. It is generally desirable that an active polypeptide not be degraded to an inactive or less active moiety when stored or during administration. Information about the stability of an active fragment is useful in formulating preparations for storage and administration. Stability information might also be useful in selecting a fragment that is longer lived once administered to an individual.
  • polypeptides which provide similar activity are generally related by having the same or similar three-dimensional portion(s) which interacts with another agent, such as a receptor with which the polypeptide binds in some way. This is why it is possible to have several polypeptides that are related to each other that display similar bone-stimulating activity.
  • the polypeptide of the present invention can be described as a polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide having the sequence identified as SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; analogues thereof wherein the amino acids in the sequence may be substituted, deleted or added, so long as the bone stimulatory activity in mammals derived from the three dimensional structure of the sequence is preserved; and conjugates of each of the polypeptides or analogues thereof, wherein if the polypeptide sequence has that identified as SEQ ID NO: 1, then there is at least one amino acid deleted therefrom.
  • a polypeptide of the present invention would include such a sequence which sequence would have a molecular weight in the range of from about 1000 to 4000. It is to be understood however that the sequence might be added to by conjugation or other technique, which could increase the molecular weight of the overall compound beyond 4000. It will also be understood, without the intention of being limited thereby, that a variety of substitutions of amino acids is possible while "preserving" the three-dimensional structure responsible for the bone stimulatory effect of the polypeptides disclosed herein. It is thus expected, for example, that interchange among non-polar aliphatic neutral amino acids, giycine, alanine, proline, valine and isoleucine, would be possible.
  • substitutions among the charged acidic amino acids, aspartic acid and glutamic acid can be made, as shown above, and substitutions among the charged basic amino acids, lysine and arginine are also likely to be possible. Substitutions can be made alone or in combination. These sorts of substitutions and interchanges are well known to those skilled in the art. United States Patent Nos. 5,487,983 and 5,512,548, for instance, describes other possible substitutions including substitutions involving amino acids not encoded by the gene. Other substitutions might well be possible.
  • polypeptides SEQ ID NOs:9, 24, 25, 26, 27, 28, 29 and 30
  • amino acids 5 to 14 of SEQ ID NO:1 displays bone stimulatory activity while polypeptides lacking the first nine N-terminus amino acids, but having amino acids 10 to 32 (SEQ ID NO: 14) or amino acids 20 to 35 (SEQ ID NO: 10) do not display bone stimulatory activity. It may be that it is possible to delete more amino acids from either end of the polypeptide identified as SEQ ID NO:9 while retaining the three-dimensional configuration of the subsequence of the polypeptide responsible for bone stimulatory activity. Internal deletions, although they might be possible to some limited extent, should be few.
  • polypeptide having the sequence identified as SEQ ID NO:16 which differs by only one amino acid residue from the amino acid sequence identified as SEQ ID NO: 9.
  • the former does not display activity while the latter does display bone stimulatory activity. It is possible using the experimental methods disclosed herein to distinguish between sequences which do and do not stimulate bone growth and which do and do not increase calcium bone content.
  • a polypeptide of the present invention would usually be synthetic, whether prepared by techniques of conventional "chemistry" or by recombinant techniques.
  • a polypeptide so produced is referred to as being substantially pure or biochemically pure when it is generally free of polypeptides or proteins with which it would occur if found directly in nature, as in blood serum from an animal, for example.
  • Nucleic acid (DNA) sequences coding for the active portions of the normal polypeptide would be as follows: SEQ ID NO:17 (corresponding to SEQ ID NO:4):
  • SEQ ID NO:18 (corresponding to SEQ ID NO:5):
  • SEQ ID NO:20 (corresponding to SEQ ID NO:7): GGG ATC GGA AAA CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA ATT
  • SEQ ID NO:21 (corresponding to SEQ ID NO:8):
  • SEQ ID NO:22 (corresponding to SEQ ID NO:9): CGA ACA AAT GAA CAT ACG GCA GAT TGT AAA
  • SEQ ID NO:31 (corresponding to SEQ ID NO:28): CGA ACA AAT GAA CAT ACG GCA GAA TGT AAA
  • SEQ ID NO:32 (corresponding to SEQ ID NO:29): CGA ACA CAA GAA CAT ACG GCA GAA TGT AAA
  • SEQ ID NO:33 (corresponding to SEQ ID NO:30): CGA ACA CAA GAA CAT ACG GCA GAT TGT AAA
  • a vector inco ⁇ orating such a DNA sequence could be constructed for use in synthesizing a polypeptide, as described previously, and particularly in international patent application No. PCT/CA 94/00144.
  • the DNA sequence coding for the polypeptide identified as SEQ ID NO:1 is given as SEQ ID NO:23 in the sequence listing of this specification.
  • a DNA sequence or fragment of the present invention may be any fragment that contains a nucleotide sequence which encodes a polypeptide of the present invention.
  • the DNA fragment can have an appropriate promoter and an SD sequence (or a suitable ribosome binding site) at its 5'-end, and if necessary, a nucleotide sequence containing a translation initiation codon at the 5'-end and a nucleotide sequence containing a termination codon at the 3'-end.
  • the genetic code is "degenerate".
  • a nucleotide in a gene sequence can thus be replaced by another nucleotide in accordance with the degeneracy of a particular codon (coding triplet), without changing the amino acid sequence of the polypeptide coded for by the gene.
  • a DNA fragment of the present invention can thus be derived from any of the above sequences (and DNA sequences corresponding to substituted polypeptide or other analogues not explicitly illustrated), and such replacement might be done in such a way that the resulting codon(s) shows a high utilization frequency in a specific host cell when producing a polypeptide of the present invention using genetic engineering techniques.
  • protected terminal amino group refers to a terminal amino group (N-terminus) 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, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups, for example benzyloxycarbonyl; and aliphatic urethane protecting groups, for example t-butoxycarbonyl or adamantyloxycarbonyl (Gross and Mienhofer, eds., The Peptides, vol 3, pp.
  • protected terminal carboxyl group refers to a terminal carboxyl group (C-terminus) coupled with any of various carboxy-terminal protecting groups.
  • suitable groups include t-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.
  • Compounds within the scope of this invention can be synthesized chemically by means well known in the art such, for example, solid phase peptide synthesis. The synthesis is commenced from the carboxy-terminal end of the peptide using an ⁇ -amino protected amino acid.
  • 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.
  • the present invention also provides 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.
  • compositions can also be provided together with physiologically tolerable liquid, gel or solid diluents, adjuvants and excipients.
  • physiologically tolerable liquid, gel or solid diluents, adjuvants and excipients In the above examples involving subsequences of about 125 nmol of polypeptide per kg of bodyweight of animal was used per administration.
  • 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 neighborhood 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.
  • 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 is measured over time.
  • Pharmaceutical preparations include any of the compounds prepared as an injectable solution, including an injectable solution prepared just prior to use, for promoting bone growth and/or treatment of osteoporosis.
  • An injectable can be either a liquid solution or suspension; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active polypeptide is often mixed with diluents and excipients which are physiologically tolerable and compatible with the polypeptide. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the compositions can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents, and the like.
  • compositions include the employment of the compounds in admixture with conventional excipients, that is, pharmaceutically acceptable organic or inorganic carrier substances which do not deieteriously 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 deieteriously 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.
  • 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%.
  • oral formulations include formulations designed to protect the peptide until it can be absorbed.
  • the peptide compounds may be formulated into the compositions as neutral or salt forms.
  • Pharmaceutically acceptable non-toxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the compounds of the invention can be homopolymerized to themselves (i.e., (peptide)n) or, heteropolymerized to one another.
  • the compounds can also be conjugated to biocompatible polymeric compounds, such as BIOPOLTM (WR Grace & Co..-Conn.).
  • 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 (penicillinase), lactose (1 nc) promoter systems (Chang et al., (1977) Nature 198:1056), the tryptophan (trp) promoters system (Goeddel et al., (1990) Nucleic Acids Res 8:4057), and the lambda-derived P promoter and N-gene ribosome binding site (Shimatake et al., (1981) Nature 292:128).
  • promoters as the beta- lactamase (penicillinase), lactose (1 nc) 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 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.
  • Transformations into yeast are carried out, for example, according to the method of Van Solingen, P., et al., (1977) J Bacter 130:946; and Hsiao, C.L., et al., (1979) Proc Natl Acad Sci USA 76:3829.
  • 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.
  • the polypeptide can be produced intracellularly, 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.
  • an enzyme-linked immunosorbent assay would have in common with RIAs and IRMAs a relatively high degree of sensitivity, but would generally not rely upon the use of radioisotopes.
  • a visually detectable substance may be produced or at least one detectable in a spectrophotometer.
  • An assay relying upon fluorescence of a substance bound by the enzyme being assayed could be used. It will be appreciated that there are a number of reporter systems which may be used, according to the present invention, to detect the presence of a particular polypeptide. With standardized sample collection and treatment, polypeptide presence above a threshold amount in blood serum could well be determined.
  • Such an antibody-linked reporter system could be used in a method for determining whether blood serum of a subject contains a deficient amount of the polypeptide. Given a normal threshold concentration of such a polypeptide in blood serum of a given type of subject, test kits could thus be developed.

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Abstract

L'invention concerne les polypeptides stimulant la croissance des os: Arg-Thr-Gln-Glu-His-Thr-Ala-Glu-Ala-Lys ; Arg-Thr-Gln-Glu-His-Thr-Ala-Glu-Tyr-Lys; et Arg-Thr-Gln-Glu-His-Thr-Ala-Glu-Ser-Lys. L'invention concerne également les séquences des nucléotides associés, ainsi que les méthodes de préparation et l'utilisation.
PCT/CA2000/000673 1999-06-02 2000-06-02 Facteur de stimulation des os WO2000075185A1 (fr)

Priority Applications (1)

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AU53792/00A AU5379200A (en) 1999-06-02 2000-06-02 Bone stimulating factor

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US09/323,854 1999-06-02
US09/323,854 US6743895B1 (en) 1995-06-07 1999-06-02 Bone stimulating factor

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050701A1 (fr) * 2002-12-05 2004-06-17 Osteopharm Inc. Facteur de croissance osseuse
US6884598B2 (en) 2000-09-22 2005-04-26 Immunex Corporation Screening assays for agonists and antagonists of receptor activator of NF-κB
WO2006007682A1 (fr) * 2004-07-19 2006-01-26 Osteopharm Inc. Polypeptides destines a attenuer les effets du vieillissement chez les mammiferes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020615A1 (fr) * 1993-03-12 1994-09-15 Osteopharm Limited Facteur de simulation de la croissance osseuse
WO1997012036A2 (fr) * 1995-09-26 1997-04-03 Gensci Regeneration Sciences Inc. Facteur de stimulation de la croissance osseuse
WO1998026070A1 (fr) * 1996-12-11 1998-06-18 Gensci Regeneration Sciences Inc. Facteur de stimulation osseuse
WO2000042069A1 (fr) * 1999-01-13 2000-07-20 Osteopharm Inc. Facteur de stimulation de la croissance osseuse

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020615A1 (fr) * 1993-03-12 1994-09-15 Osteopharm Limited Facteur de simulation de la croissance osseuse
WO1997012036A2 (fr) * 1995-09-26 1997-04-03 Gensci Regeneration Sciences Inc. Facteur de stimulation de la croissance osseuse
WO1998026070A1 (fr) * 1996-12-11 1998-06-18 Gensci Regeneration Sciences Inc. Facteur de stimulation osseuse
WO2000042069A1 (fr) * 1999-01-13 2000-07-20 Osteopharm Inc. Facteur de stimulation de la croissance osseuse

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6884598B2 (en) 2000-09-22 2005-04-26 Immunex Corporation Screening assays for agonists and antagonists of receptor activator of NF-κB
US7572594B2 (en) 2000-09-22 2009-08-11 Immunex Corporation Screening assays for agonists or antagonists or receptor activator of NF-κB
WO2004050701A1 (fr) * 2002-12-05 2004-06-17 Osteopharm Inc. Facteur de croissance osseuse
WO2006007682A1 (fr) * 2004-07-19 2006-01-26 Osteopharm Inc. Polypeptides destines a attenuer les effets du vieillissement chez les mammiferes

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