AU2012201490B2 - Method of drug delivery for bone anabolic protein - Google Patents

Abstract

C.\NRPonbl\DCC\REC\41II3X_I DOC-lW3/20I(12 The present invention provides a storage-stable composition containing a parathyroid hormone-related protein (PTHrP) analogue and methods of using a PTHrP analogue and the PTHrP compositions described herein to treat osteoporosis, to increase bone mass or to increase bone quality. The composition is storage stable, in sterile form, and in general may be stored at room temperature for at least several weeks to allow convenient parenteral administration to human patients.

Description

Australian Patents Act 1990- Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title Method of drug delivery for bone anabolic protein" The following statement is a full description of this invention, including the best method of performing it known to us: P/00/011 C:\NRPorth\DCC\REC\4183199_1 DOC - 13/3/12 CNRPorb1\DCCREC4IK1flN_ .DOC-11,112012 METHOD OF DRUG DELIVERY FOR BONE ANABOLIC PROTEIN RELATED APPLICATION This application is a divisional of Australian Patent Application No. 2007322334, the entire content of which is incorporated herein by reference. This application claims the benefit of U.S. Provisional Application No. 60/848,960, filed on October 3, 2006. The entire teachings of the above application is incorporated herein by reference. BACKGROUND OF THE INVENTION Parathyroid hormone-related protein ("PTHrP") is a 139 to 173 amino acid-protein. PTHrP and certain analogs are known to be useful to improve bone mass and quality in the treatment of osteoporosis and related disorders. However, the commercial use of these proteins as pharmaceutical agents requires the development of a formulation that is acceptable in terms of storage stability and ease of preparation. Furthermore, currently available osteoporosis drugs have limitations on suitable dosage ranges due to the unwanted side-effects, such as hypercalcemia and increased stimulation of bone resorption. These unwanted side-effects and resulting dose limitations reduce the beneficial effects which can be achieved from these drugs. Thus a need exists for compounds which can be administered at a dose which will increase the beneficial effects without an increase in the unwanted side-effects. SUMMARY OF THE INVENTION The present invention provides a storage-stable composition containing a parathyroid hormone-related protein (PTHrP) analogue and methods of using those analogues and compositions containing those analogues as described herein to treat osteoporosis, to increase bone mass or to increase bone quality. The composition is storage stable, in sterile form, and in general may be stored at room temperature for at least several weeks to allow convenient parenteral administration to human patients. In one embodiment, the present invention provides a storage-stable composition suitable for administration to a subject (e.g., a human). The -2 composition comprises a PTHrP analogue and an effective amount of buffer to maintain the pH of the composition between 2 and 7. In a particular embodiment, the PTHrP is [Glu 22

.

25 , Leu 2 3

'

3 1 , Aib 29 , Lys 2630 JhPTHrP(1 -34)NH2 (SEQ ID NO.: 2). In another embodiment, the present invention provides a sealed container containing a storage-stable composition suitable for administration to a subject. The composition comprises PTHrP or an analog thereof and an effective amount of buffer to maintain the pH of the composition between 2 and 7. In a particular embodiment, the PTHrP analogue is (Glu 22 25 , Leu 23

'

28 31 , Aib 29 , Lys 2 63 0 ]hPTHrP(1 34)NH 2 (SEQ ID NO.: 2). In another embodiment, the present invention provides a drug delivery device comprising one or more than one single-use container which comprises a storage stable composition comprising PTHrP or an analog thereof and an effective amount of buffer to maintain the pH of the composition between 2 and 7. In a 22.25 23,28,31 . 9 26,30 particular embodiment, the PTHrP analogue is [Glu2, Leu ', Aib2, Lys ]hPTHrP(1-34)NH2 (SEQ ID NO.:2). In another embodiment, the present invention provides a drug delivery device comprising one or more than one multi-use container, which comprises a storage stable composition comprising PTHrP or an analog thereof and an effective amount of buffer to maintain the pH of the composition between 2 and 7. In a particular embodiment, the PTHrP analogue is [Glu 22 25 , Leu 23 28 3 1 , Aib 29 , Lys 26 30 ]hPTHrP(1-34)NH2(SEQ ID NO.: 2). In another embodiment the present invention provides a method of treating osteoporosis in a subject in need thereof comprising administering to the subject a single daily subcutaneous dose of [Glu -2, Leu 2 ', Aib 2, Lys 26 _3 ]hPTHrP(1 34)NH2 (SEQ ID NO.: 2) in an amount between 40 and 160 pg for a duration of time sufficient to treat the subject, typically between about 3 months to 36 months. In some embodiments, the treatment period is between about 3 months to 18 months. In another embodiment the present invention provides a method of increasing bone mass or increasing bone quality in a subject in need thereof comprising administering to the subject a single daily subcutaneous dose of [Glu 2 22 5 , Leu 23 2831 , Aib 29 , Lys 2 63 0 ]hPTHrP(I -34)NH 2 (SEQ ID NO.: 2) in an C.\NRPorbPlDCOREC46 I K 142 1 DOC. 19/09/2012 -3 amount between 40 and 160 pg for a duration of time sufficient to treat the subject, typically between 3 months and 36 months. In some embodiments, the treatment period is between about 3 months to 18 months. In one aspect the present invention provides the use of a storage stable composition in the manufacture of a medicament for stimulating bone growth in a subject in need thereof, said storage-stable composition comprising: a) a PTHrP analogue whose sequence is [Glu' 2 , Leu 2 3 28

,

3 1 Aib 29 Lys26' 3 0 ]hPTHrP(1-34)NH 2 (SEQ ID NO.2); and b) an effective amount of a pH buffer to maintain the pH in a range of about 4.5 to about 5.6. In one aspect the present invention provides a method of stimulating bone growth in a subject in need thereof comprising the administration to the subject of a storage-stable composition comprising: 22,25 23,2,31 2 a) a PTHrP analogue whose sequence is [Glu' 2 , Leu ' , Aib Lys26, 3 0 ]hPTHrP(l-34)NH 2 (SEQ ID NO.2); and b) an effective amount of a pH buffer to maintain the pH in a range of about 4.5 to about 5.6 In one aspect the present invention provides the use of a composition comprising 80 pg of [Glu 22

'

2 , Leu 23

,

28

,

3 1 , Aib 2 9 Lys 26

,

30 ]hPTHrP(1-34)NH 2 in the manufacture of a medicament for treating osteoporosis wherein the composition is formulated for subcutaneous administration to a human in need thereof In one aspect the present invention provides a method of treating osteoporosis comprising administering subcutaneously a composition comprising 80 pg of [Glu , Leu 23

,

28

,

31 , Aib 2 9 , Lys 2 6

,'

3 0 ]hPTHrP(I-34)NI-H 2 to a human in need thereof. The PTHrP and analogue compositions of the invention exhibit storage stability in terms of hormone composition and activity. Furthermore, these compositions can be administered, in general, in higher dosages than currently available osteoporosis drugs, with the reduction or elimination of unwanted side-effects, such as, hypercalcemia or stimulation of bone resorption. This has the advantage of an increase in beneficial physiological effects due to the increased dosages and can result in a reduction in the length of treatment time.

C:\RPonbl\DCC\REC'461942_.DOC-19/9/2012 -3A BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the stability of SEQ ID NO. 2 over 24 months at 5 'C and 25 *C without any chemical stabilizer. FIG. 2 is a graph showing the stability of lyophilized SEQ ID NO. 2 over 24 months at 5 'C 25 *C and 40 'C. DETAILED DESCRIPTION OF THE INVENTION The sequence of native hPTHrP (1-34) is as follows: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser lie GIn Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala (SEQ ID NO: 1). In a particular embodiment, the PTHrP analogue is [Glu 22 ' , Leu3 , Aib2, Lys 26

,

3 0 ]hPTHrP(1-34)NH 2 (SEQ ID NO:2). Other PTHrP analogues are described in 6,921,750, 5,955,574, 6,544,949, 5,723,577 and 5,696,095 the entire contents of each of which are incorporated herein by reference. A "buffer" as used herein is any acid or salt combination which is pharmaceutically acceptable and capable of maintaining the composition of the present invention within a desired pH range. Buffers in the disclosed compositions maintain the pH in a range of about 2 to about 7, about 3 to about 6, about 4 to about 6, about 4.5 to about 5.6, or about 5.1. Suitable buffers include, any pharmaceutical acceptable buffer capable of maintaining the above pH ranges, such as, for example, -4. acetate, tartrate phosphate or citrate buffers. In one embodiment, the buffer is an acetate or tartrate buffer. In another embodiment the buffer is an acetate buffer. In one embodiment the buffer is acetic acid and sodium acetate. In the disclosed compositions the concentration of buffer is typically in the range of about 0.1 mM to about 1000 mM, about 0.2 mM to about 200 mM, about 0.5 mM to about 50 mM, about 1 mM to about 10 mM or about 6 mM. As used herein, an anti-microbial agent is a pharmaceutically acceptable preservative, suitable for administration to a subject, which inhibits, prevents or delays the growth or micro organisms including, for example bacteria, viruses and fungi in the compositions of the present invention. Suitable anti-microbial agents for use in the compositions and methods of the present invention include, but are not limited to, cresols, benzyl alcohol, phenol, benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol, methyl paraben, propyl paraben, thiomersal and phenylmercuric nitrate and acetate. In one embodiment the anti microbial agents is m-cresol, chlorocresol or phenol, In another embodiment the anti-microbial agents is chlorocresol or phenol. In another embodiment the anti microbial agents is phenol. As used herein an effective amount of an anti-microbial agent is an amount effective to inhibits, prevents or delays the growth or micro organisms including, for example bacteria, viruses and fungi in the compositions of the present invention. In the compositions of the present invention, the amount of anti-microbial agent is typically in the range from about 0.1 to about 20 mg/ml, about 0.2 to about 30 mg/ml, about 0.2 to about 10 mg/ml, about 0.25 to about 5 mg/ml, about 0.5 to about 50 mg/ml, about I to about 10 mg/ml, about 3 mg/ml or about 5 mg/ml. The compositions of the present invention typically are ready to administer, aqueous solutions which are sterile, storage-stable and pharmaceutically acceptable without the need for reconstitution prior to administration. The compositions of the present invention are suitable for administration to a subject which means that they are pharmaceutically acceptable, non-toxic, do not contain any components which would adversely affect the biological or hormonal effects of the peptide. The compositions of the present invention do not, for example, comprise any cells.

-5 As used herein a composition of the present invention is storage-stable if the amount, purity of the PTHrP remains above about 95% of the original amount under one of the following conditions: (1) storage for over 2 years at 5 "C; or (2) storage for over 30 days at 25 *C. The compositions are typically stored in a sealed container, vial or cartridge which is typically suitable for long term storage. "Suitable for long-term storage" means that the vial, container or cartridge does not allow for the escape of components of the compositions of the present invention or the ingress of external components, such as, micro organisms when kept for at least 3 months at 25 *C. The compositions of the present invention are preferably administered by injection, typically subcutaneous injection. The compositions of the present invention, can be stored in single-dose or multi-dose sealed containers, vials or cartridges. The sealed container, vial or cartridge is typically suitable for use with a single or multi-dose injection pen or drug delivery device, which typically allows the patient to administer the peptide themselves. The sealed container can comprise one or more doses of the peptide of the present invention, wherein each dose comprises an effective amount of the peptide as described herein. A single-dose injection pen, or drug delivery device is typically a disposable device which uses a sealed container which comprises a single dose of an effective amount of a PTHrP in the compositions described herein. A multi-dose injection pen or drug delivery device typically contains more than one dose of an effective amount of a PTHrP thereof in the compositions described herein. The multi-dose pen can typically be adjusted to administer the desired volume of the storage stable compositions described herein. In certain embodiment the multi-dose injection pen prevents the ingress of microbial contaminants from entering the container or cartridge which can occur through multiple uses of one needle. Injection pens, as used herein, can also comprise two containers one of which contains a PTHrP, as described herein, in a lyophilized powder, as described below, and the second container contains a liquid for reconstitution of the lyophilized powder. The contents of the two containers can be mixed prior to administration.

-6 As discussed above the compositions of the present invention can be administered by injection. Suitable volumes of the compositions of the present invention for injection include about 0.5 to about 1 ml, about 0.1 to about I ml, about 0.02- to about 0.04 ml, about 0.1- to about 5.0 pl, or about 0.1- to about 1.0 ±l. In the compositions of the present invention the concentration of the peptides is from about 20 mg/ml to about 20,000 mg/ml, from about 100 mg/ml to about 10,000 mg/ml, from about 300 mg/ml to about 300 mg/ml, from about 500 mg/ml to about 2000 mg/ml and about 2 mg/ml. The compositions of the present invention can also be lyophilized using lyophilization techniques known in the art and stored as a powder which can be reconstituted prior to administration. The term "lyophilization" as used herein is a freeze drying or dehydration technique which involves removing a solvent, preferably a water miscible solvent, more preferably water from a composition or the present invention, typically by sublimation under high vacuum when the composition is in a frozen state. Typically, lyophilization is carried out in lyophilization equipment (a lyophilizer), which comprises a drying chamber with variable temperature controls, a condenser to collect water, and a vacuum system to reduce the pressure in the drying chamber. The terms "lyophilized composition", as used herein mean the solid residue or powder which is produced or which remains after the lyophilization procedure as defined above. The lyophilized composition of the present invention typically further comprise a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" as used herein refers to a substance which is added to a solution prior to lyophilization to enhance characteristics such as the color, texture, strength, and volume of the lyophilized cake. Pharmaceutically acceptable excipients may be, for example, buffers and pH adjusters, crystalline bulking excipients, stabilizers, and tonicity raising agents. In certain preferred embodiments the pharmaceutically acceptable excipient is a crystalline bulking excipient. The terms "crystalline bulking excipient" or "crystalline bulking agent" as used herein means an excipient which provides bulk and structure to the lyophilization cake. These crystalline bulking agents are inert -7 and do not react with the peptide. In addition, the crystalline bulking agents are capable of crystallizing under lyophilization conditions. Examples of suitable crystalline bulking agents include hydrophilic excipients, such as, water soluble polymers; sugars, such as mannitol, sorbitol, xylitol, glucitol, ducitol, inositiol, arabinitol, arabitol, galactitol, iditol, allitol, maltitol, fructose, sorbose, glucose, xylose, trehalose, allose, dextrose, altrose, lactose, glucose, fructose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose, lactulose, fucose, rhamnose, melezitose, maltotriose, raffinose, altritol, their optically active forms (D- or L-forms) as well as the corresponding racemates; inorganic salts, both mineral and mineral organic, such as, calcium salts, such as the lactate, gluconate, glycerylphosphate, citrate, phosphate monobasic and dibasic, succinate, sulfate and tartrate, as well as the same salts of aluminum and magnesium; carbohydrates, such as, the conventional mono and di-saccharides as well as the corresponding polyhydric alcohols; proteins, such as, albumin; amino acids, such as glycine; emulsifiable fats and polyvinylpyrrolidone. Preferred crystalline bulking agents are selected from the group consisting of glycine, mannitol, dextran, dextrose, lactose, sucrose, polyvinylpyrrolidone, trehalose, glucose and combinations thereof. Particularly useful bulking agents include dextran. As used herein a stabilizer is a composition which maintains the chemical, biological or hormonal stability of the peptide. Examples of stabilizing agent include polyols which includes a saccharide, preferably a monosaccharide or disaccharide, e.g., glucose, trehalose, raffinose, or sucrose; a sugar alcohol such as, for example, mannitol, sorbitol or inositol, a polyhydric alcohol such as glycerine or propylene glycol or mixtures thereof and albumin. The compositions described herein can be used to stimulate bone growth in a subject. Thus-they are useful in the treatment of diseases or disorders associated with deficiency in bone growth such as osteoporosis and bone fractures. In one embodiment, the present invention is a method of treating osteoporosis in a subject comprising administering to the subject an effective amount of composition described herein.

-8.

As used herein, "treating" can include both prophylactic, and therapeutic treatment. For example, therapeutic treatment can include delaying inhibiting or preventing the progression of osteoporosis, the reduction or elimination of symptoms associated with osteoporosis. Prophylactic treatment can include preventing, inhibiting or delaying the onset of osteoporosis. As used herein, an effective amount refers to an amount sufficient to elicit the desired response. In the present invention, the desired biological response is an decrease in the rate of bone loss and/or an increase in the bone mass or bone quality of a subject. Suitable dosage for use in the compositions and methods of the present invention include from about 40 to about 160 pg, about 80 to about 120 pg about 80 to about 100 pg; or from about 40 to about 50 pg, about 50 to about 60 pg, about 60 to about 70 Mg, about 70 to about 80 pg, about 80 to about 90 pg, about 90 to about 100 Mg, about 100 to about 110 pg, about 110 to about 120 pg, about 120 to about 130 pg, about 130 to about 140 Mg, about 140 to about 150 pg, about 150 to about 160 pg; or from 40 to about 45 Mg, about 45 to about 50 Mg, about 50 to about 55 pg, about 55 to about 60 Mg, about 60 to about 65 Mg, about 65 to about 70 pg, about 70 to about 75 Mg, about 75 to about 80 pg, about 80 to about 85 pg, about 85 to about 90 pg, about 90 to about 95 Mg, about 95 to about 100 Mg, about 100 to about 105 "g, about 105 to about 1 10 pg, about I10 to about 115 pg, about 115 to about 120 pg, about 120 to about 125 Mg, about 125 to about 130 pg, about 130 to about 135 Mg, about 135 to about 140 pg, about 140 to about 145 Mg, about 145 to about 150 Mg, about 150 to about 155 Mg, about 155 to about 160 pg administered once per day, once every other day, twice per week once per week, once every two weeks, once per month. The doses can be a pulsatile injection, for example, once per month which causes pulsatile release of singles doses of the composition described herein. When the dosages described above are administered once per day, once per week etc., typically the dosages are of equal amounts. The subject as used herein can be an animal, for example, a mammal, such as a human. A pharmaceutically acceptable salt is a salt which is suitable for administration to a subject, such as, a human. The peptides of the present invention -9 can have one or more sufficiently acidic proton that can react with a suitable organic or inorganic base to form a base addition salt. Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases such as alkoxides,.alkyl aides, alky) and aryl amines, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like. The peptides of the present invention having a sufficiently basic group, such as an amine can react with an organic or inorganic acid to form an acid addition salt. Acids commonly employed to form acid addition salts from compounds with basic groups are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromo'phenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-I,4-dioate, hexyne- 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- I -sulfonate, naphthalene-2-sulfonate, mandelate, and the like. The compositions of the present invention typically do not show any or show reduced side-effects such as hypercalcemia and typically do not increase the stimulation of bone resorption at the dosage listed above. This reduction in side effects allows for-administration of higher doses than commercially available osteoporosis drugs. The compositions of the present invention can be administered by injection as described herein.

-10 The compositions of the present invention may be administered alone or in combination with an additional therapeutic agent, such as an antiresorptive therapy, for example, bisphonsphonates and calcitonin. EXEMPLIFICATION EXAMPLE I demonstrates Glu , Leu 23 8 ', Aib 9 , Lys ' 3 ]hPTHrP(l-34)NH 2 (SEQ ID NO.: 2) stability at low acetate concentration (1mM), without stabilizer TABLE I Material Supplier Unitary Formula (per cartridge) (SEQ ID NO.: 2) Ipsen Ireland 0.140 mg (free base) Tri-hydrate sodium acetate 0.1 N Prolabo 14.6 mg Acetic acid 0.1 N Prolabo .9 mg qs pH 5.1 Water for Injection Meram qs 1.4 g Type I clear glass Cartridge 1.5 ml, Bunderglass via I washed, siliconised and sterilised Vetter Grey PTFE bromobutyl cartridge rubber DaYkyo I stopper Chlorobutyl rubber-metal cartridge crimp West I Pharmaceutical qs = quantity sufficient to achieve The formulation delivered 100 mcg of (SEQ ID NO.: 2) per 0.1 ml. (SEQ ID NO.: 2) was dissolved in Water for Injection containing dilute acetate buffer to give pH 5.1 was used.

- 11 Results confirm excellent chemical stability over 24 months, at 5 *C as shown in FIG. 1. This solution contains no stabilizer or preservative and only 6 mM acetate buffer. In summation for (SEQ ID NO.: 2), stabilizer is not needed to give good stability in solution. EXAMPLE 2: Use of citric acid buffer in lyophilised form of (SEQ ID NO.: 2). TABLE 2 Material Supplier Unitary Formula (per vial) (SEQ ID NO.: 2) Ipsen Ireland 0.1 mg (free base) Dextran 70 Interchemical 50 mg Citric acid 0.25 % (w/v) Prolabo qs pH 4.5* Water for injections** Meram qs I g Type I clear glass vial, 11-13 ml Verretubex Grey chlorobutyl PTFE stopper, 20 mm DaYkyo Flip-off metal crimp West Pharma **to get pH 5 - 5.5 after lyophilisation removed after freeze-drying step. The solutions in TABLE 2 were reconstituted with NaCl 0.9 %, to give: ONE vial of 2 ml (=50 pg/ml) providing 10 to 80 pg/d doses (with injections of 200 pl to 1.6 ml), or ONE vial of 5 ml (=20 pg/ml solution) providing 5 to 40 4g/d doses (with injections of 250 pl - 2 ml). Citric acid was used to adjust pH and Dextran was used to provide a bulking agent to aid cake formation during lyophilization. The solutions described were lyophilized in glass vials, and stored at various temperatures for up to 24 months. The content of [Glu , Leu2, Aib9, Lys 26

.

30 ]hPTHrP(1 -34)NH 2 (SEQ ID NO.: 2), purity and physical tests were conducted on samples removed from storage at different times. Results are presented in FIG. 2, for peptide concentration, as percent remaining.. The data in FIG. 2 shows excellent stability over 24 months at 2-8"C.

- 12 EXAMPLE 3: Screening of formulations for Glu22.25, Leu2'83, Aib29, Lys2630 JhPTHrP(1-34)NH 2 (SEQ ID NO.: 2) to Compare Different Preservatives TABLE 3 below shows Methyparaben and Benzyl Alcohol are not suitable preservatives for use with Glu 22

.

2 , Leu 23, Aib , Lys 263 0 ]hPTHrP(I-34)NH 2 (SEQ ID NO.: 2), as precipitation and/or inactivity in preservative activity was seen. TABLE3 Example 3a Example 3b Example 3c Example 3d Example 3e Methylparaben 1.5 mg/ mL 1.35 mg/ mL - - Propylparaben - 0. 15 mg/ mL - - Phenol - - 5mg/ mL - Chlorocresol - 3 mg/ mL Benzyl alcohol - - - 10 mg/ mL Preservative Failed Pass Pass Pass Pass effectiveness test Observationo or Precipitation ssues observed Preservative Not Tested effectiveness as Pass Pass Pass Fail test after storage precipitated .5 months at 5"C initially Solutions were prepared containing [Glu 2 2 n, Leu 2 3

'

2 3 1 , Aib 29 , Lys 6

'

3 ]hPTHrP(I -34)NH 2 (SEQ ID NO.: 2) 2mg/ml, acetate buffer 6mM and water for injection, with various different preservatives added at concentrations recommended for effective antimicrobial activity. Solutions were prepared at room temperature, by dissolution of the various ingredients in water for injection, with stirring over < 30 minutes to ensure complete dissolution, Solutions were filtered through 0.2 micron filter and filled into glass vials, to which a rubber stopper was applied and crimped in place to ensure complete closure.

- 13 The solution with methylparaben was less acceptable due to precipitation and inactivity immediately after manufacture of the solution. The solutions were then stored for up to 3 months at 25"C, and up to 4.5 months at 5*C and the preservative effectiveness test repeated. as described in Example 5. EXAMPLE 4: Evaluation of Anti-microbial Preservative Effectiveness of Various concentrations of Glu 22 25 , Leu 2'., Aib 29 , Lys 26

.

30 ]hPTHrP(l -34)NH 2 (SEQ ID NO.:. 2) Compositions (stability study) TABLE4 P87228 P87229 P87230 P87231 (SEQ ID NO.: 2) 2 mg/ mL 2 mg/ mL 2 mg/ mL 2 mg/ mL Anti-microbial Phenol Chlorocresol Chlorocresol Benzyl alcohol 5 mg/ mL 3 mg/ mL 2 mg/ mL 10 mg/ mL cetate buffer pH 5.1 pH 5.1 pH 5.1 PH 5.1 The solutions were tested according to European Pharmacopoeia, Chapter 5.1.3 "Efficacitd de la conservation anti-microbienne" (Anti-microbial effectiveness test) to prove the effectiveness of the preservative.

- 14 TABLE 5: Preservative effectiveness test after manufacturing Initial Nb of cfu present / preparation . organism (plate-count method) Organisms concentration . Test P87228 P87229 P87230 P87231 coctentration interval _____ in cfu / mL Phenol 1hlorocreso Chlorocresol Benzyl alc. -5mg/ml 3mg/ml 2mg/ml 10mg/ml Staphylococcus 3.8 x 10' T 0 3 <5 <5 4.7x 10' aureus T+6 hrs <5 <5 <5 6.8 x 10' T+24 <5 <5 <5 <5 hrs T+28 5 () <5 <5 <5 days Pseudomonas 1.3 x 10" T 0 5 <5 <5 .5x 0 eruginosa T+6 hrs <5 <5 <5 <5 T+24 <5 <5 <5 <5 T+28 <5 <5 <5 <5 days E.coli 6.7 x 10' T 0 7.2 x V07 <5 <5 I.i x 101 T+6 hrs <5 <5 <5 <5 T+24 <5 <5 <5 <5 T+28 <5 <5 <5 <5 days <5 <5 *) Bacillus Gram +, different from St. Aureus -> result conform Initial Nb of cfu present / preparation Organism: organism Test (plate-count method) Yeast and concentration e P87228 P87229 P87230 I P87231I j old in fu / L interval________ nold in c / mL Phenol chlorocresol chlorocresol Benzyl alc. 5mg/ml 3mg/ml 2mg/ml I 10mg/ml \spergillus 3.4 x 10' T 0 4.0 x 10 <5 <5 4.1 x 10' niger T+7 <55<5< days I < <5 <5 T+28 <5 <5 <5 <5 ________days I _ __ _ I_____ ______ ____ Candida TO9 x 10 T 0 4.4 x 10 5 <5 <5 3.8 x 10 5 albicans T+7 <5 5 days T+28 <5 <5 days Results: Conform - Conform Conform Conform Conform Nb of cfu= number of colony forming units -15 TABLE 5 Cont'd.: Preservative effectiveness test results after 3 months storage at 25 0 C Initial Nb of cfu present / preparation (plate-count . organism Test method) Organisms concentration interval P87228 | P87229 P87230 P87231 actena in cfu / mL (days) Phenol Ichlrocresol chlorocresol Benzyl alc. 5mg/ml 3mg/ml 2mg/ml 10mg/mi Staphylococcu 2.7 x 10 Ohr 1.9 x 10' <5 <5 | 3.8 x 10' s aureus (P87228, 6hr 30 | <5 <5 |5.9 x 1d' P87229, P87231 = <5 <5 <5 <5 5.2 x 10' (P87230) 28day <5 <5 <5 <5 Pseudomonas 9.9 x 105 Ohr <5 <5 <5 <5 aeruginosa (P87228, 6hr <5 <5 <5 <5 P87229, F8723 l)24hr <5 <5 <5 <5 8.5 x 10 5 28day <5 <5 <5 <5 (P87230) E.coli 68 r 1.7x10 <5 <5 8.0x10 (P87228, 6hr <5 <5 <5 5 P87229, P87231) ' <5 <5 <5 <5 9.5 x 10' 28day <5 <5 <5 <5 (P87230) Initial Nb of efu present / preparation (plate-count Organism: organism Test method) Yeast and concentration terval P87228 | P87229 | P872301 P87231| moldin fu / m terva0 old in c / mL Phenol Chlorocresol Chlorocresol Benzyl alc. 5m/ml 3mg/mi 2mg/ml 10mg/ml Aspergillus 3.3 x 10 Ohr 3.8 x 10' 55 70 4.1 x 10' niger (P87228, P87229, P87231) 7day <5 <5 <5 <5 4.1 x 10' 28day <5 <5 <5 <5 (P87230) Candida 2.7 x 105 Ohr 4.0 x 10' <5 <5 3.8 x 10 5 albicans (P87228, P87229, P87231) 7day <5 <5 <5 <5 3.7 x 10 5 28day <5 <5 <5 <5 (P87230) Resutr - C m CNot Results. Conform - Conform IConform Conform Con for - 16 TABLE 5 Cont'd.: Preservative effectiveness test results after 4.5 months storage at 5*C Initial Nb of cfu present / preparation (plate-count Organisms: organism Test method) I 3ras: concentration interval P87228 P87229 P87230 I P87231 Bacteia in cfu / mL (days) Phenol chlorocresol chlorocresol Benzyl alc. 5mg/ml 3mg/ml 2mg/m 10mg/mi Staphylococcus 5.4 x 105 Ohr 4.1 x 10' <5 . <5.. 0 aureus 6hr <5< <5 '7.2 x 10' 24hr <5 <5 <5 <5 28day <5 <5 <5 <5 seudomonas 9 .7 x 0 Ohr <5 <5 <5 <5 eruginosa 6hr <5 <5 <5 <5 24hr <5 <5 <5 <5 28day <5 <5 <5 <5 .coli 6.1 x 10' Ohr 7.0 x 104 5 5 4.2 x 104 6hr <5 <5 <5 <5 24hr <5 <5 <5 <5 28day <5 <5 <5 <5 Initial Nb of cfu present / preparation (plate-count Organism: organism Test method) Yeast and concentration test P87228 P87229 P87230 P87231 old in cfu / m iterval nold in cfu / mL Phenol Chlorocresol Chlorocresol Benzyl alc. 5mg/ml 3mg/ml 2mg/ml 10mg/ml Aspergillus 5.3 x 10' Ohr 3.7 x 10 1.8 x 10' 7.5 x 103 4.1 x 10' niger 7day <5 <5 <5 <5 28day <5m <5 <5 <5 Candida 4.1 x 10' Ohr 4.5 x 10' <5 5 4.5 x 10' albicans 7day <5 <5 <5 <5 28day <5 <5 <5 <5 1 CoformI Coform1 I Not Results : Conform Conform Conform Conform TABLE 5 shows Phenol, Chlorocresol and Benzyl Alcohol all produce compliant -results immediately after manufacture for both Bacteria and Yeasts/moulds. After 3 and 4.5 months storage, the preservative efficacy is maintained for Phenol and Chlorocresol, for both Bacteria and Yeasts/moulds.

-17 However, for Benzyl Alcohol, the efficacy against Bacteria is not compliant, as the data shows insufficient rate of kill against S Aureus (TABLE 5). EXAMPLE 5: Chemical stability of different formulations TABLE 6 details the chemical stability of the formulations described in.Example 4. TABLE 6: Glu 22

,

25 , Leu 23

'

28

'

31 , Aib, 29 Lys 26

.

30 ]hPTHrP(1-34)NH 2 (SEQ ID NO.: 2) stability results Storage conditions: 25 0 C, 60% RH (SEQ ID NO.: 2) content in mg/mL (% initial concentration at t=0) Batch Composition 0 month I month 3 months ,(SEQ ID NO.: 2) 1.90 1.88 1.83 P87228 (2mg/ml) / Phenol (100%) (98.9%) (96.3%) (5mg/ml) (SEQ ID NO.: 2) 1.98 1.96 1.94 P87229 (2mg/ml) / Chlorocresol (100%) (99.0%) (98.0%) (3mg/ml) (SEQ ID NO.: 2)1.93 1.89 1.86 P87231 (2mg/ml) / Benzyl (100%) (9.9%) (96.4%) Alcohol (10mg/ml) Storage conditions :5C (SEQ ID NO.: 2) content in mg/mL (% initial concentration at t=0) Batch Composition 0 month 3 month 4.5 month (SEQ ID NO.: 2) 1.90 1.91 1.89 P87228 (2mg/ml) / Phenol (100%) (100.5%) (99.5%) ____ (5mg/ml) (SEQ ID NO.: 2) 1.98 1.96 1.97 P87229 (2mg/ml) / Chlorocresol (100%) (99.0%) (99.5%) (3mg/ml) (SEQ ID NO.: 2) 1.93 - 1.94 1.92 P87231 (2mg/ml) / Benzyl (100%) (100.5%) (99.5%) 1 Alcohol (10mg/mi) As can be seen from TABLE 6 and [Glu 2 2 2 5 , Leu 23 2 8

.

3 , Aib 2 9 Lys 26

',

30 ]hPTHrP(l-34)NH 2 (SEQ ID NO.: 2) solution stability is not significantly influenced by the preservative selected.. TABLE 7 details the content of each preservative for the same formulations.

-18 TABLE 7: Preservative stability results Storage conditions: 25"C, 60% RH Preservative content in mg/ml (% initial concentration at t=0) Batch Composition 0 month 1 month 3 month (SEQ ID NO.: 2) 4.86 4.82 4.79 P87228 (2mg/mI) / Phenol (100%) (99.2%) (98.6%) (5mg/ml) (SEQ ID NO.: 2) 2.78 2.70 2.56 P87229 (2mg/ml) / Chlorocresol (100%) (97.1%) (92.1%) (3mg/mi) (SEQ ID NO.: 2) 9.92 9.83 9.82 P87231 (2mg/ml) / Benzyl (100%) (99.1%) (99.0%) Alcohol (10mg/ml) Storage conditions 5 0 C Preservative content in mg/mL (% initial concentration at t=) Batch Composition 0 month 3 month 4.5 month .(SEQ ID NO.: 2) 4.86 4.83 4.84 .82228_ 2mg/m4)L. henol -- (T4 -(99.430) (99.60) (5mg/ml) (SEQ ID NO.: 2)2.78 2.73 2.74 P87229 (2mg/ml) / Chlorocresol 100%) (98.2%) (98.6%) (3mg/ml) (SEQ ID NO.: 2) 9.92 9.89 9.94 P87231 (2mg/ml) / Benzyl (100%) (99.7%) (100.2%) -_______Alcohol (10mg/ml) ( ( ( As can be seen from TABLE 7 chlorocresol is the preservative which has the lower stability, with greater loss in preservative content under both 5 and 25*C storage. While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

C:\NRfrtblDCC\REcWiX330A_1.DOC-11M3/2112 - 18A Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and 'comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (7)

1. Use of a storage stable composition in the manufacture of a medicament for stimulating bone growth in a subject in need thereof, said storage-stable composition comprising: 22,25 23,28,1 2 a) a PTHrP analogue whose sequence is [Glu ' , Leu , Aib 29 Lys 2,30]hPTHrP(1-34)NH 2 (SEQ ID NO.2); and b) an effective amount of a pH buffer to maintain the pH in a range of about

4.5 to about 5.6. 2. A method of stimulating bone growth in a subject in need thereof comprising the administration to the subject of a storage-stable composition comprising: 22,25 23,28,1 2 a) a PTHrP analogue whose sequence is [Glu', Leu , Aib 29 Lys 6 , 3 0 ]hPTHrP(l-34)NH 2 (SEQ ID NO.2); and b) an effective amount of a pH buffer to maintain the pH in a range of about 4.5 to about 5.6 3. The use or method according to claim I or claim 2 wherein said subject is administered the medicament by single daily subcutaneous injection of an amount of said composition containing from about 75 to about 80 pg of [Glu 2 , Leu '3, Aib 2 9 , Lys 26 , 30 ]hPTHrP(1-34)NH 2 (SEQ I D NO.2). 4. The use or method according to claim I or claim 2 wherein said subject is administered the medicament by single daily subcutaneous injection of an amount of said composition containing from about 40 to about 45 pg of [Glu', Leu ' ' , Aib 2 9 , Lys 26 ' 3 0 ]hPTHrP(1-34)NH 2 (SEQ ID NO.2).

5. The use or method according to any one of claims 1 to 4 wherein said storage stable composition further comprises phenol in a concentration from 0.25 to 5 mg/mL. II REC\hilcrwoven,\NRPonbl\DC\REC525566HI doc-24/W-/2013 -20

6. The use or method according to any one of claims I to 5 wherein said pH buffer is an acetate buffer.

7. The use or method according to any one of claims 1 to 5 wherein said pH buffer is a citrate buffer.

8. The use or method according to any one of claims 1 to 6 wherein said subject has a bone fracture.

9. Use according to claim 1 or method according to claim 2 substantially as hereinbefore described with reference to any one of the examples.