A STABILIZED PHARMACEUTICAL FORMULATION COMPRISING GROWTH HORMONE AND ASPARAGINE

Abstract: A pharmaceutical preparation comprising a growth hormone and asparagine as additive or buffering substance 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 preparation at ambient temperature.

Description: TITLE . A stabilized pharmaceutical formulation com¬ prising growth hormone and asparagine. 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 asparagine 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 a ino 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 ske¬ leton, 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 syndro ) , growth hormone deficiency in adults, infertility , treatment of burns, wound healing, dystrophy, bone knitting, osteoporosis, diffuse gastric bleeding, and pseudoarthrosis. Furthermore, growth hormone has been proposed for increasing the rate of growth of domestic animals or for decreasing the proportion of fat in animals to be slaughtered for human con¬ sumption. Pharmaceutical preparations of growth hormone tend to be un¬ stable. Degradation products such as deamidated or sulfoxy- dated products and di er or polymer forms are generated - especially in solutions of growth hormone. The predominant degradation reactions of hGH are 1) deamida¬ tion by direct hydrolysis or via a cyclic succinimide inter- meadiate to form various amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH, and D-iso-asp-hGH (ref 1-3) , 2) oxidation of the methionine residues in positions 14 and 125 (ref 4-9) , and 3) cleavage of peptide bonds. Deamidation especially takes place at the Asn in position 149. hGH is rather easily oxidized in positions 14 and 125, espe¬ cially in solution (4-8) . The oxidation of hGH in solution forming sulfoxides is nor¬ mally due to the oxygen dissolved in the preparation. The solubility of oxygen in distilled water is about 200 μM (9) . As the concentration of hGH in a preparation comprising 4 IU/ml is 1.3 g/ml corresponding to 60nM hGH, oxygen will, at normal storing conditions, be present in an excess of about 3000 times the stoichio etric amount for oxidation of hGH. It is not feasible to try to solve the problem by degassing of buffers before tapping and packing the preparations. At present, it is not believed that these degradation pro¬ ducts should have toxic or altered biological activity or receptor binding properties, but there is indication to the effect that the conformation stability of the sulfoxides is reduced as compared to native hGH. For the development of a stable, dissolved preparation com¬ prising hGH it is of importance to know the rate of formation of sulfoxides as well as means to control the oxidation. The kinetics of degradation depend on temperature, pH and various additives or adjuvants in the hGH formulation. Due to the 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 preparations 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. Furthermore, the process of reconstitution of the lyophilized growth hormone tends to provide difficulties for the patient. Thus, it is at present preferred to reconstitute the growth hormone as late as possible before use and to store and ship the preparation in a lyophilized state. The chain from the manufactorer to the pharmacy is apt for handling the prepara- tions at a controlled low temperature of e.g. 4°C which al¬ lows 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 preparation which is stable for a sufficient long time with the end user under conditions where "sufficient" cooling is not always avai¬ lable. Preferably, a preparation 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 preparations of growth hormone being stable in a lyophilized state at a relative high temperature for a period and additionally for a period of use at a relatively high temperature in solution. 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 preparation comprising growth hormone in order to facili¬ tate the handling to be performed by the patient. A stable dissolved preparation 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 preparation and, hence, will not have to be in the possession of a lyophilized preparation, a suitable vehicle for reconstitution as well as the necessary skill and sterile equipment for sterile reconstitution of the prepara¬ tion. For safety reasons it will also be desirable to avoid the reconstitution of a lyophilized preparation just before the use of the preparation. Furthermore, it would also be an advantage to avoid the lyo- philization step in the production of growth hormone prepara¬ 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 preparations being stable during shelf life and during the period of use of up to one month. Prior attempts to stabilize hGH has not fully succeded in preventing the formation of dimer. The problems associated with dimer formation is e.g noted in Becker, G.W. , Biotech- nology 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 preparation 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 stabilized with various stabi- lizers to give decreased formation of insolubles and preser¬ vation of the soluble activity in aqueous environments, such stabilizers including certain polyolε, amino acids, polymers of amino acids having a charged side group at physiological pH, and choline salts. Polyols are selected from the group consisting of non-reducing sugars, sugar alcohols, sugar aci¬ ds, pentaerythritol, lactose, water-soluble dextrans and Fi- coll; amino acids are selected from the group consisting 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 thereof; a po¬ lymer of an amino acid having a charged side group at physio¬ logical pH may be selected from polylysine, polyaspartic acid, polyglutamic acid, polyarginine, polyhistidine, polyor- nithine and salts thereof; and choline derivatives are selec¬ ted from the group consisting of choline chloride, choline dihydrogen citrate, choline bitartrate, choline bicarbonate, tricholine citrate, choline ascorbate, choline borate, choli- ne gluconate, choline phosphate, di(choline)sulphate and di- choline mucate. BRIEF DESCRIPTION OF THE INVENTION It has now surprisingly been found that a preparation of hu¬ man growth hormone comprising asparagin as additive or buf- fering substance shows a very high stability against deamida¬ tion, 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-dis¬ solved preparation. Thus, the preparation of the invention may be in the form of a lyophilized powder to be reconstituted later using conven¬ tional vehicles such as distilled water br 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 preparation of human growth hormone comprising asparagine in the form of a buffered aqueuos solution of growth hormone buffered with asparagine buffer. Such prepara- tion is in a ready-to-use form and may be stored and shipped as an aqueous solution without any considerable degradation. For stability reasons the pH of a solution is preferably ad¬ justed to a value in the interval from 2-8. Preparations ha¬ ving a pH from 5 to 8 are more preferred and most preferred are preparations having a pH from 6 to 7.5. In order to obtain the stabilizing effect aparagine is pre¬ ferably added in an amount of up to 100 mM. The concentration of asparagine is preferably on the interval from 2 to 20 mM, most preferred from 2 to 10 mM The pharmaceutical preparation of the invention may also be in the form of a lyophilized preparation. The pharmaceutical preparation og the invention may further¬ more comprise salts and saccharides in order to facilitate the processing thereof, e.g. lyophilization. Conventionally mannitol is used as a bulking agent for lyophilization. Still another aspect of the invention relates to the use of asparagine for the preparation of a stabilized preparation 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. 2 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, Met-Glu-Ala-Glu-hGH or Ala-Glu-hGH. The pre¬ ferred growth hormone is hGH. In the present context "high stability" is obtained when the preparation is more stable than the conventional formulation comprising phosphate buffer. The term "salts" used in this specification to designate ad- ditional constituents of a pharmaceutical preparation compri¬ ses conventional such salts such as alkaline metal, alkaline earth metal or ammonium salts of organic acids such as citric acid, tartaric acid, or acetic acid, e.g. sodium citrate, sodium tartrate or sodium acetate, or of mineral acids such as hydrochloric acid, e.g. sodium chloride. "Saccharides" may be monosaccharides, disaccharides or sugar alcohols which are conventionally used for pharmaceutical preparations such as lactose and mannitol. The solvent used in the preparations of the invention may be water, alcohols such as ethyl, n-propyl or isopropyl, butyl alcohol or mixtures thereof. The solvent may comprise a pre¬ servative such as m-cresol or benzyl alcohol. DETAILED DESCRIPTION OF THE INVENTION The invention is explained more in detail in the below Exam- pies which illustrate the invention. They are not to be con¬ sidered as limiting the scope of the invention being defined by the appended claims. EXPERIMENTAL PART EXAMPLE Reduction of the deamidation. The rate of deamidation was examined at 25"C for hGH pre¬ parations comprising 6 IU hGH at pH 6.5 in the presence of 10 mM Asn as compared to 8 mM phosphate buffer at the same pH and pH 7.3. The hGH preparations were prepared by dissolving 20 mg hGH in 10 ml of 10 mM asparagine solution prepared by dissolving 13.2 mg, of asparagine in 10 ml deionized water containing 0.9% (v/v) of benzyl alcohol and adding 0.1 N hydrochloric acid to the stated pH. The hGH formulations stated in the below Table were stored at 25°C and analyzed for the desamido contents after 14 and 30 days by IE-HPLC. The results appear from he below Table. Table. Contents of desamido hGH as determined by IE-HPLC as a func¬ tion of the formulation and the time in solution at 25°C: Formulation (*) Formation of desamido compound at 25"C 14 days ( « ) 30 days 8 mM di-Na-Phosfat 7.8 10.8 pH 6.5 8 mM di-Na-Phosfat 15.2 20.3 pH 7.3 8 mM di-Na-phosfat 9.4 13.2 pH 6.5, 0.3% m-cresol 10 mM Asp, pH 6.5 21.7 ' nd 10 mM Asn, pH 6.5 6.5 8.3 10 mM Glu, pH 6.5 14.8 nd *: Comprises 0.9% benzyl alcohol except formulation # 3. The contents of desamido-hGH in starting material was: 2.1 % From the above Table it appears that the de-amidation of hGH is reduced by approximately 25% by the addition of asparagine as compared with phosphate buffer at ph 6.5. Addition of Asp or Glu increases the rate of deamidation as compare to phosphate at pH 6.5. The above results show that the rate of de-amidation is redu¬ ced by lowering the pH and by adding asparagine in a low con- centration of up to 100 mM, preferably about 5mM. The rate of de-amidation may be reduced by more than 50% by lowering the pH and substituting the phosphate buffer with asparagine. The use of m-cresol or benzyl alcohol as preservative seems to have no influence on the rate of de-amidation. Split-formation (hydrolysis of peptide bonds) is reduced by asparagine at pH 6.5 in comparison with phosphate. REFERENCES 1) Y.-C.J. Wang and M.A. Hanson. Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers. J. Parenteral Science and Technology 42 (Suppl.) (1988) 53-525. 2) M.C. Manning, K. Patel, R.T. Borchardt. Stability of Protein Pharmaceuticals. Pharmaceutical Research 6 (11) (1989) 903-918. 3) 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). 4) L.C. Teh et al. , J.Biol.Chem., 262, 785-794 (1987). 5) G.W. Becker et al., Biotech.Appl.Biochem. , 10, 326-337 (1988) . 6) R.A. Houghten et al., Arch.Biochem.Biophys. , 178, 350- 355 (1977) . 7) R.M. Riggin et al., Anal.Biochem. , 167, 199-209 (1987). 8)- P. Gellerfors et al., Acta Paediatr.Scand (suppl), 370, 93-100 (1990) . 9) M.J. Kaufman, Pharm.Res., 7 (3) 289-292 (1990).

Claims: CLAIMS 1. A pharmaceutical preparation comprising a growth hormone and asparagine. 2. A pharmaceutical preparation as claimed in claim 1 in the 5 form of a buffered aqueuos solution of growth hormone contai¬ ning asparagine. 3. A pharmaceutical preparation as claimed in claim 1 or 2 wherein the pH is adjusted to a value in the interval 2-8. 4. A pharmaceutical preparation as claimed in claim any of 10 the preceeding claims wherein the concentration of asparagine is up to 100 mM. 5. A pharmaceutical preparation as claimed in claim any of the preceeding claims further comprising salts and sacchari- des. 156. A pharmaceutical preparation as claimed in any of claims 1-6 wherein the growth hormone is hGH. 7. Use of asparagine for the preparation of a stabilized pre¬ paration of growth hormone.

Inventors: SOERENSEN HANS HOLMEGAARD, SOERENSEN, HANS, HOLMEGAARD
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: A61K37
IPC codes: A61K9, A61K31, A61K38, A61K47