A PHARMACEUTICAL FORMULATION COMPRISING A GROWTH HORMONE AND LEUCINE

Abstract: A pharmaceutical formulation comprising a growth hormone and leucine shows a very high stability against deamidation, oxidation and cleavage of peptide bonds. The stability of the product allows for the storing and shipment thereof in a lyophilized state or in the form of a dissolved or re-dissolved formulation at ambient temperature.

Description: TITLE A PHARMACEUTICAL FORMULATION COMPRISING A GROWTH HORMONE AND LEUCINE FIELD OF THE INVENTION The present invention relates to a stabilized pharmaceutical formulation comprising growth hormone, to a method of making such formulation, and the use of leucine for stabilizing a formulation of growth hormone. BACKGROUND OF THE INVENTION The growth hormones from man and from the common domestic animals are proteins of approximately 191 amino acids, syn¬ thesized and secreted from the anterior lope of the pituitary gland. Human growth hormone consists of 191 amino acids. Growth hormone is a key hormone involved in the regulation of not only somatic growth, but also in the regulation of meta- bolism of proteins, carbohydrates and lipids. The major ef¬ fect of growth hormone is to promote growth. The organ systems affected by growth hormone include the skeleton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys. Until the development of the recombinant technology and the cloning of the growth hormone gene now giving rise to produc¬ tion of e.g. human growth hormone (hGH) and Met-hGH in indu¬ strial scale, human growth hormone could only be obtained by extraction from the pituitary glands of human cadavers. The very limited supplies of growth hormone restricted the use thereof to longitudinal growth promotion in childhood and puberty for treatment of dwarfism, even though it has been proposed for inter alia treatment of short stature (due to growth hormone deficiency, normal short stature and Turner syndrome) , growth hormone deficiency in adults, infertility, treatment of burns, wound healing, dystrophy, bone knitting, osteoporosis, diffuse gastric bleeding, and pseudoarthrosis as well as for decreasing the proportion of fat in animals to be slaughtered for human consumption. Pharmaceutical formulations of growth hormone tend to be un- stable. Degradation products such as deamidated or sulfoxy- dated products and dimer or polymer forms are generated - especially in solutions of growth hormone. The predominant degradation reactions of hGH are 1) deamidation by direct hydrolysis or via a cyclic succinimide intermeadiate to form various amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH, and D-iso-asp-hGH (Y.-C.J. Wang and M.A. Hanson. Parenteral For¬ mulations of Proteins and Peptides: stability and Stabilizers. J. Parenteral Science and Technology 42 (Suppl.) (1988) 53-525; M.C. Manning, K. Patel, R.T. Borchardt. Stability of Protein Pharmaceuticals. Pharmaceutical Research 6 (11) (1989) 903-918; and B.A. Johnson, J.M. Shirokawa, W.S. Hancock, M.W. Spellman, L.J. Basa and D.W. Asward. J.Biol.Chem. 264, 1462-71 (1989)) and 2) oxidation of the methionine residues in positions 14 and 125 (L.C. Teh et al., J.Biol.Chem. , 262, 785-794 (1987); G.W. Becker et al., Biotech.Appl.Biochem. , 10, 326-337 (1988); R.A. Houghten et al., Arch.Biochem.Biophys. , 178, 350-355 (1977); R.M. Riggin et al., Anal.Biochem. , 167, 199-209 (1987); P. Gellerfors et al., Acta Paediatr.Scand (suppl), 370, 93-100 (1990); M.J. Kaufman, Pharm.Res., 7 (3) 289-292 (1990)). The major degradation product of hGH in lyophilized state as well as in solution is deamidated hGH. Deamidation especially takes place at the Asn in position 149 and to a minor extent in position 152. hGH is also rather easily oxidized in positions 14 and 125, especially in solution (L.C. Teh et al., J.Biol.Chem., 262, 785-794 (1987); G.W. Becker et al., Biotech.Appl.Biochem. , 10, 326-337 (1988); R.A. Houghten et al., Arch.Biochem.Biophys. , 178, 350-355 (1977); R.M. Riggin et al., Anal.Biochem. , 167, 199-209 (1987); P. Gellerfors et al., Acta Paediatr.Scand (suppl), 370, 93-100 (1990)). The oxidation of hGH in solution forming sulfoxides is nor¬ mally due to the oxygen dissolved in the formulation. The solubility of oxygen in distilled water is about 200 μM (M.J. Kaufman, Pharm.Res., 7 (3) 289-292 (1990)). As the concentration of hGH in a formulation comprising 4 IU/ml is 1.3 mg/ml corresponding to 60 nM hGH, oxygen will, at normal storing conditions, be present in an excess of about 3000 times the stoichiometric amount for oxidation of hGH. It is not feasible to try to solve the problem by degassing of buffers before tapping and packing the formulations. At present, it is not believed that these deamidated forms and oxidized forms of hGH should have toxic or altered biolo¬ gical activity or receptor binding properties. However, there are indications that the conformation stability of the sulfoxides is reduced as compared to native hGH. For the development of a stable, dissolved formulation com¬ prising hGH it is important to know the rate of deamidation and formation of sulfoxides as well as means to control the reactions. The kinetics of degradation depend on temperature, pH and various additives or adjuvants in the hGH formulation. Due to its instability, growth hormone is, at present, lyophilized and stored in the lyophilized form at 4*C until it is reconstituted for use in order to minimize the degradation. The lyophilized pharmaceutical formulations comprising hGH are, at present, reconstituted by the patient and then stored as a solution during the use for a period of up to 14 days at 4*C, during which some degradation will take place. Further¬ more, the process of reconstitution of the lyophilized growth hormone tends to provide difficulties for the patient. There¬ fore, it is at present preferred to reconstitute the growth hormone as late as possible before use and to store and ship the formulation in a lyophilized state. The links in the distribution process form the manufacturer to the pharmacy are equipped for handling the formulations at a controlled low temperature of e.g. 4°C which allows for a long shelf life of up to two years. However, the extended use of pen systems for self-medication and the expanded field of use calls for a formulation which is stable for a sufficiently long time with the end user under conditions where "sufficient" cooling is not always available. Preferably, a formulation should be stable with the end user in a lyophilized state for about one month and additionally for one month in a reconstituted state in a pen device for the intended period of use of a cartridge. Thus, there is a need for more stable formulations of growth hormone being stable in a lyophilized state at a relatively high temperature for at least about one month. Such stabilization is of very great importance when moving the administration of the growth hormone from clinics to the ho¬ mes of the individuals to be treated where optimal storage may not be available as indicated above. Furthermore, the shift in pattern of administration of growth hormone to the use of pen devices calls for a stable dissol¬ ved formulation comprising growth hormone in order to facili¬ tate the handling to be performed by the patient. A stable dissolved formulation comprising growth hormone may be pro- duced ready to use in the form of cartridges fitting into the pen device used by the patient who may then avoid the recon- stitution of the formulation and, hence, will not have to be in the possession of a lyophilized formulation, a suitable vehicle for reconstitution as well as the necessary skill and sterile equipment for sterile reconstitution of the formula¬ tion. For safety reasons it will also be desirable to avoid the reconstitution of a lyophilized formulation just before the use of the formulation. Furthermore, it would also be an advantage to avoid the lyo- philization step in the production of growth hormone formula¬ tions. Lyophilization is a time consuming and costly process and is also often a "bottleneck" in the production due to the limited capacity of the freeze drier. Thus, there is a need to reduce the rate of the degradation processes in order to allow for dissolved hGH formulations to be stable during shelf life and during the period of use of up to one month. Prior attempts to stabilize hGH have not fully succeeded in preventing the formation of dimers. The problems associated with diroer formation is e.g. noted in Becker, G.W. , Biotech- noloqy and Applied Biochemistry 9, 478 (1987). International Patent Publication No. WO 89/09614 and Austra¬ lian patent application No. 30771/89 disclose a stable phar¬ maceutical formulation containing human growth hormone, gly- cine, and mannitol. Such a formulation shows improved stabi- lity during normal processing and storage in a lyophilized state as well as in the period of use after the reconstitu¬ tion. Published European patent application No. 303 746 discloses that animal growth hormone may be stabilized with various stabilizers to give decreased formation of insolubles and preservation of the soluble activity in aqueous environments, such stabilizers including certain polyols, amino acids, polymers of amino acids having a charged side group at physi¬ ological pH, and choline salts. Polyols are selected from the group consisting of non-reducing sugars, sugar alcohols, sugar acids, pentaerythritol, lactose, water-soluble dextrans and Ficoll; amino acids are selected from the group consist¬ ing of glycine, sarcosine, lysine or salts thereof, serine, arginine or salts thereof, betaine, N,N,-dimethyl-glycine, aspartic acid or salts thereof, glutamic acid or salts there¬ of; a polymer of an amino acid having a charged side group at physiological pH may be selected from polylysine, polyaspar- tic acid, polyglutamic acid, polyarginine, polyhistidine, polyornithine and salts thereof; and choline derivatives are selected from the group consisting of choline chloride, choline dihydrogen citrate, choline bitartrate, choline bi¬ carbonate, tricholine citrate, choline ascorbate, choline borate, choline gluconate, choline phosphate, di(choline)sul¬ phate and dicholine mucate. US patent specification No. 4,917,685 discloses a delivery device designed to be implanted comprising growth hormone stabilized using the same stabilizers as mentioned in EP 303746. Published European patent application No. 374,120 discloses a stabilized formulation comprising hGH and a polyol having three hydroxy groups. Glycerol and tris(hydroxyEmethyl)amino- methane are mentioned. Furthermore, the presence of histidine hydrochloride as a buffer together with the polyol is disclo¬ sed. International Patent Publication No. WO 93/12811 discloses stabilized formulations of growth hormone in the form of a lyophilized powder or an aqueous solution - comprising asparagine. International Patent Publication No. WO 93/12812 discloses stabilized formulations of growth hormone in the form of a lyophilized powder or an aqueous solution comprising histidi¬ ne. In such formulations the deamidation is reduced by 25-30% as compared to a corresponding formulation of growth hormone comprising phosphate buffer. International Patent Publication No. WO 93/19776 discloses protein formulations comprising growth hormone comprising citrate as buffer substance being more stable than formula¬ tions comprising phosphate buffer. The formulations may also comprise amino acids such as glycine and alanine and/or man- nitol or other sugar alcohols and/or glycerol and/or other carbohydrates and optionally a preservative such as benzyl alcohol. International Patent Publication No. WO 94/03198 discloses a stable aqueous formulation containing human growth hormone, a buffer, a non-ionic surfactant, and, optionally, a neutral salt, mannitol, or, a preservative. SUMMARY OF THE INVENTION It has now surprisingly been found that a formulation of hu- man growth hormone comprising leucine as additive shows a very high stability against deamidation. The stability of the product allows for the storing and shipment thereof in a lyo¬ philized state or in the form of a dissolved or re-dissolved formulation. The invention s related to such a formulation. Additioanlly, invention is related to a method form making such a formulation comprising adding the growth hormone to a solution comprising leucine. The invention is also directed to a method 'for treating a disorder associated with growth hormone deficiency, comprising administering the pharmaceuti- cal formulation of claim 7 in an amount effective to treat said deficiency. DETAILED DESCRIPTION OF THE INVENTION The pharmaceutical formulations of the invention may be for¬ mulated for administration in any suitable way, e.g. by parenteral or oral administration or administration to a mucosal membrane, e.g. nasal administration. The pharmaceuti¬ cal formulation may be presented in the form of a dose in a vial or cartridge or any other suitable container such as a prefiled syringe or a pen device. Thus, the formulation of the invention may be in the form of a lyophilized powder to be reconstituted later using conven¬ tional vehicles such as distilled water or water for injec¬ tion or in the form of a solution comprising growth hormone. Such vehicles may comprise conventional preservatives such as m-cresol and benzyl alcohol. A preferred embodiment of the invention is in the form of a pharmaceutical formulation of human growth hormone comprising leucine and further comprising a carrier in the form of a buffered aqueous solution of growth hormone. Such a formula- tion is in a ready-to-use form and may be stored and shipped as an aqueous solution without any considerable degradation. The pharmaceutical formulation of the invention may further¬ more comprise salts for adjusting the tonicity and/or an excipient in order to facilitate the processing thereof, e.g. lyophilization and the rapid and complete dissolution of a lyophilized formulation when reconstituting the formulation before use. An excipient may be selected from disaccharides such as lactose, trehalose, and sucrose, sugar alcohols such as sorbitol or mannitol, polysaccharides such as the polymers commercialized as Dextran ® products such as Dextran ® 40, β Dextran ® 70 or Dextran ® 75, and Ficoll and polyvalent alcohols such as polyethylene glycol or polyvinyl alcohol or a combination of two or more of these. The pharmaceutical formulations of the invention may be prepared by adding growth hormone to a solution comprising leucine, preferably in an amount from about 0.1 mM up to 100 mM, more preferred in an amount of about 1-10 mM, preferably 2-6 mM, most preferred about 3-5 mM. The growth hormone may be in solid form or may be in a buffer solution, e.g. histidine, citrate, tartrate or phosphate buffer. The leucine solution may be obtained by dissolving leucine in deionized water optionally containing benzyl alcohol. The pH of said formulation may be adjusted from about 2 to about 8 more pre¬ ferred to pH from 5 to 7, especially to about 6.8 by adding an acid which has no adverse effect on the growth hormone, preferably a physiologically acceptable acid e.g. a mineral acid such as hydrochloric acid, sulpuric acid or nitric acid or an organic acid such as acetic acid. In one embodiment of the method of the invention, salts and/or an excipient may be added. In yet another embodiment, the solution is filled into a container and lyophilized. Still another aspect of the invention relates to the use of leucine for the formulation of a stabilized formulation of growth hormone. In the present context "growth hormone" may be growth hormone from any origin such as avian, bovine, equine, human, ovine, porcine, salmon, trout or tuna growth hormone, preferably bovine, human or porcine growth hormone, human growth hormone being most preferred. The growth hormone used in accordance with the invention may be native growth hormone isolated from a natural source, e.g. by extracting pituitary glands in a conventional manner, or a growth hormone produced by recombi- nant techniques, e.g as described in E.B. Jensen and S. Carlsen in Biotech and Bioeng. 3_6, 1-11 (1990) . The "growth hormone" may also be a truncated form of growth hormone wherein one or more amino acid residues has (have) been deleted; an analogue thereof wherein one or more amino acid residues in the native molecule has (have) been substituted by another amino acid residue, preferably a natural amino acid residue, as long as the substitution does not have any adverse effect such as antigenicity or reduced action; or a derivative thereof, e.g having an N- or C-terminal extension such as Met-hGH. The preferred growth hormone is human growth hormone (hGH) . The term "dose" of growth hormone refers to that amount that provides therapeutic effect in an administration regimen. The formulations hereof are prepared containing amounts of hGH at least about 0.1 mg/ml, preferably upwards of about 10 mg/ml, preferably from about 1 mg/ml to about 40 mg/ml, more prefer¬ ably from about 1 mg/ml to about 25 mg/ml, e.g. from 1 mg/ml to about 5 mg/ml, calculated on the ready-to-use formulation. For use of these compositions in administration to human beings suffering from hypopituitary dwarfism, for example, these formulations contain from about 0.1 mg/ml to about 10 mg/ml, corresponding to the currently contemplated dosage regimen for the intended treatment. The concentration range is not critical to the invention and may be varied by the physician supervising the administration. Leucine to be used in accordance with the present invention is preferably naturally occurring alpha leucine. The amino acid(s) may be 1 or d amino acid(s) or a mixture thereof. In the present context "high stability" is obtained when the formulation is more stable than the conventional formulation comprising phosphate buffer and preferably as stable as a corresponding formulation comprising histidine as stabilizer in which the de-amidation of hGH is reduced by approximately 20-30% as compared with phosphate buffer as disclosed in WO 93/12812. The solvent used in the method of the invention may be water, alcohols such as ethyl, n-propyl or isopropyl, butyl alcohol or mixtures thereof. The solvent may comprise a preservative such as m-cresol or benzyl alcohol. EXPERIMENTAL PART EXAMPLE 1 Reduction of the deamidation. The rate of deamidation was examined at 37 * C for hGH formula- tions comprising 4 mg/ml hGH at pH 6.8 in the presence of 5 mM L-leucine or D-leucine as compared to histidine at pH 6.8. The hGH formulations were prepared by dissolving 8 mg hGH in 2 ml of 10 mM solution of the amino acid. Thus, 2 ml 3.0% of benzyl alcohol was added to give a final formulation of 4 mg/ml hGH, 5 mM D- or L-leucine or histidine, 1.5% benzyl alcohol, pH 6.8 (adjusted adding HC1 or NaOH) . The hGH formulations stated in the below table were stored at 37*C for 7 days and analysed for the content af deamidated hGH by IE-HPLC using urea as an eluent. The results appear from the below table. Table 1 Formulation pH start/pH Corrected Contents of end/% content of desamido hGH desamido deamidated as compared hGH* with His L-leucine 6.7/6.6/15.3 16.8 97 D-leucine 6.7/6.7/16.6 17.6 102 Histidine 6.7/6.8/16.8 17.3 100 * Desamido corrected by 1% per 0.1 pH unit deviation from 6.8 The contents of deamidated hGH in the starting material was: 2.0% From the above table it appears that the deamidation of hGH is reduced by 25-30% by addition of D-leucine or L-leucine as compared with phosphate buffer (at least as stable as com¬ pared to histidine, cf. WO 93/12812 above) . The above results show that the rate of deamidation is redu¬ ced by adding L-leucine or D-leucine in a low concentration of up to 100 mM, preferably about 5mM. The rate of deamida¬ tion may be reduced by more than 30% by substituting the phosphate buffer with L-leucine or D-leucine. The use of benzyl alcohol as preservative seems to have no influence on the rate of deamidation. EXAMPLE 2 Reduction of the deamidation. The rate of deamidation was examined at 37"C for hGH formula¬ tions comprising 4 mg/ml hGH at pH 6.8 in the presence of 5 mM L-leucine as compared to histidine at pH 6.8. The hGH formulations were prepared by dissolving 8 mg hGH in 2 ml of 10 mM solution of the amino acid. Thus, 2 ml 3.0% of benzyl alcohol was added to give a final formulation of 4 mg/ml hGH, 5 mM L-leucine or histidine, 1.5% benzyl alcohol, pH 6.8 (adjusted adding HC1 or NaOH) . The hGH formulations stated in the below table were stored at 37"C for 7 days and analysed for the content af deamidated hGH by IE-HPLC using acetate as an eluent. The results appear from the below table. Table 2 Formulation pH start/pH Corrected Contents of end content of desamido hGH deamidated as compared hGH* with His 5 mM L-leucine 6.8/7.0 11.8 95 5 mM L-histidi- 6.9/6.9 12.4 100 ne Na ? HPO t 6.9/6.9 16.5 133 * Desamido corrected by 1% per 0.1 pH unit deviation from 6.8 The contents of deamidated hGH in the starting material was: 2.0% From the above table it appears that the deamidation of hGH is reduced by 25-30% by addition of leucine as compared with phosphate buffer (at least as stable as compared to histidine, cf. WO 93/12812 above). The above results show that the rate of deamidation is redu¬ ced by adding leucine in a low concentration of up to 100 mM, preferably about 5mM. The rate of deamidation may be reduced by more than 30% by substituting the phosphate buffer with leucine. The use of benzyl alcohol as preservative seems to have no influence on the rate of deamidation.

Claims: CLAIMS 1. A pharmaceutical formulation comprising a growth hormone and leucine. 2. A pharmaceutical formulation according to claim 1, in 5 which said formulation is in the form of a buffered aqueous solution. 3. A pharmaceutical formulation according to claim 1 or 2, wherein the pH value is adjusted to a value in the interval from about 2 to about 8. 104. A pharmaceutical formulation according to any of the pre¬ ceding claims, wherein the concentration of leucine is up to 100 mM. 5. A pharmaceutical formulation according to any of the pre¬ ceding claims, further comprising salts and/or an excipient. 156. A pharmaceutical formulation according to any of the pre¬ ceding claims, wherein the growth hormone is hGH. 7. A method of preparing the pharmaceutical formulation of claim 1 comprising adding growth hormone to a solution comprising leucine. 208. A method for stabilizing a formulation of growth hormone comprising adding an amount of leucine effective to stabilize said formulation. 9. The method according to claim 7 or 8, in which the solution comprising leucine is obtained by dissolving leucine 25 in deionized water. 10. The method according to any of the preceding claims, in which the solution comprising leucine also comprises benzyl alcohol. 11. The method according to any of the preceding claims, 5 which further comprises adjusting the pH value of said formulation to from about 2 to about 8. 12. The method according to any of the preceding claims, which further comprises adding salts. 13. The method according to any of the preceding claims, 10 which further comprises adding an excipient. 14. The method according to any of the preceding claims, which further comprises filling a solution comprising growth hormone and leucine into a container and lyophilizing said solution. 1515. A method for treating a disorder in a patient affectable by growth hormone, comprising treating the patient with an amount of the pharmaceutical formulation of claim 1-6 effective to treat said disorder. 16. Any novel feature or combination of features disclosed 20 herein.

Inventors: SOERENSEN HANS HOLMEGAARD, SOERENSEN, HANS, HOLMEGAARD
Applicants: NOVONORDISK AS, SOERENSEN HANS HOLMEGAARD, NOVO NORDISK A/S, SOERENSEN, HANS, HOLMEGAARD
Application year: 1995
Main classification: A61K38
IPC codes: A61K9, A61K38, A61K47