MXPA98000358A - Stabilized pharmaceutical formulation comprising a pretracted growth hormone with zincy optionally lysine or ions cal - Google Patents

Abstract

A pharmaceutical formulation is provided which comprises a growth hormone pretreated with zinc and optionally lysine or calcium ions which shows very high stability during deamination, oxidation and cleavage of peptide bonds. The stability of the product allows storage and shipment thereof in a lyophilized state or in the form of a dissolved or redissolved formulation at room temperature.

Description

HORMONE OF THE EC-I-MIENTQ PRETRA-TADA WITH ZTMT and OPC-TCMAI-MEM * - LISINA Q IONES CA CTO FIELD OF THE -P1VENCT-ÓN The present invention relates to a stabilized pharmaceutical formulation comprising growth hormone, with a method of producing such a formulation, with the use of zinc to stabilize a growth hormone formulation, and with a method of treating a hormone-affected disorder. of growth. ftTTTEQgDKNTES OF THE INVENTION Growth hormones for man and for common pets are proteins of approximately 191 amino acids, synthesized and secreted in the anterior lobe of the pituitary gland or pituitary gland. Human growth hormone consists of 191 amino acids. Growth hormone is a key hormone involved in the regulation not only of somatic growth, but also in the regulation of the metabolism of REF: 26543 proteins, carbohydrates and lipids. The main effect of growth hormone is to promote growth. The systems of organs affected by growth hormone include the skeleton, connective tissue, muscles and visas such as liver, intestines and kidneys. Until the development of recombinant technology and the cloning of the gene for growth hormone that now leads to the production of, for example, human growth hormone (hGH) and Met-hGH on an industrial scale, human growth hormone could only obtained by extraction of pituitary or pituitary glands from human cadavers. Very limited supplies of growth hormone restricted its use to a promotion of longitudinal growth in childhood and puberty for the treatment of dwarfism, although it had been proposed, for example, for the treatment of short statue (due to deficiency in growth hormone, normal short statue and Turner syndrome), growth hormone deficiency in adults, infertility, treatment of burns, wound healing, dystrophy, bone repair, osteoporosis, diffuse gastric bleeding and pseudoarthrosis. In addition, growth hormone has been proposed to increase the growth rate of domestic animals or to decrease the proportion of fats in animals that are to be slaughtered for human consumption. The pharmaceutical formulations of growth hormone tend to be unstable. The degradation products such as deamidated or sulfoxidated products and dimeric or polymeric forms are especially generated in growth hormone solutions. The predominant reactions of degradation of hGH are: 1) deamidation by direct hydrolysis or via a cyclic succinimide intermediate to form varying amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH and D-iso- asp-hGH (references 1-3); and 2) oxidation of methionine residues at positions 14 and 125 (references 4-9). The main degradation product of hGH in the lyophilized state as well as in solution is deamidated hGH. Deamidation occurs especially in Asn at position 149, and to a lesser extent at position 152. The hGH hormone is also easily oxidized at positions 14 and 125. The oxidation of hGH in solution to form sulfoxides is usually due to dissolved oxygen in the formulation. The solubility of oxygen in distilled water is approximately 200 μM (9). As the concentration of hGH in a formulation comprising 4 IU / ml is 1.3 mg / ml corresponding to 60 nM hGH, oxygen, under normal storage conditions, will be present in an excess of approximately 3000 times the stoichiometric amount of oxidation of hGH. It is not feasible to try to solve the problem by degassing shock absorbers before packing and packing the formulations. To date, these deaminated forms and oxidized forms of hGH are not considered to have altered toxic or biological activity or receptor binding properties., but it is an indication that the conformational stability effect of the sulfoxides is reduced compared to native hGH. For the development of a stable and dissolved formulation consisting of hGH it is of importance to know the speed of deamidation and formation of sulfoxides as well as means to control the reactions. The degradation kinetics depends on the temperature, pH and various additives and adjuvants in the hGH formulation. Due to the instability, to date the growth hormone is freeze-dried and stored in lyophilized form at 4 ° C until it is reconstituted for use in order to minimize degradation. The lyophilized pharmaceutical formulations comprising hGH, to date, are reconstituted by the patient and then stored as a solution during the use of a period of up to 14 days at 4 ° C, during which time some degradation takes place. In addition, the process of reconstitution of lyophilized growth hormone tends to provide difficulties for the patient. Therefore, to date it is preferred to reconstitute the growth hormone as late as possible before use and store and ship the formulation in a lyophilized state. The chain from the manufacturer to the pharmacy must be suitable for handling the formulations at a controlled low temperature, for example, 4 ° C which allows a shelf life of up to two years. However, the widespread use of pen-type systems for self-medication and the expanded field of use requires a formulation which is stable for a sufficiently long time with the end user under conditions where cooling is not always available " enough". Preferably, a formulation must be stable with the end user in a lyophilized state for about a month, and additionally for one month in a reconstituted state in a pen-type device for the proposed period of use of a cartridge.

Therefore, there is a need for more stable formulations of growth hormone which are stable in a lyophilized state at a relatively high temperature for a certain period, and additionally during an additional period of use at a relatively high temperature, in solution. Such stabilization is of great importance when the administration of growth hormone is shifted from clinics or hospitals to the homes of people who are going to be treated where optimal storage is not always available, as indicated above. In addition, the displacement in the pattern of administration of growth hormone to the use of pen type devices requires a stable dissolved formulation constituted of growth hormone in order to facilitate the handling that can be carried out by the patient. A stable dissolved formulation constituted of growth hormone can be produced easily for use in the form of cartridges that fit into a pen type device used by the patient who can then prevent the reconstitution of the formulation and, therefore, will not be position of a lyophilized formulation, a suitable vehicle for reconstitution as well as the sterile tool and equipment necessary for the sterile reconstitution of the formulation.

For safety reasons, it will also be desirable to avoid reconstitution of a lyophilized formulation just prior to the use of the formulation. In addition, it would also be advantageous to avoid the lyophilization step in the production of growth hormone formulations. Freeze-drying is a time-consuming and expensive process and is often a "bottleneck" in production due to the limited capacity of the freeze dryer. Therefore, there is a need to reduce the speed of the degradation process in order to allow the dissolved hGH formulations to be stable during the shelf life and during the period of use of up to one month. Previous attempts to stabilize hGH have not been completely successful in preventing the formation of a dimer.

The problems associated with the formation of dimers are indicated, for example, in Bec er, G.W., Biotechnology and Applied Bioc emistry 9, 478 (1987). The publication for international patent number WO 89/09614 for Australian patent number 30771/89 describe a stable pharmaceutical formulation containing human growth hormone, glycine and mannitol. Such formulations show improved stability during normal processing and storage in a lyophilized state as well as in the period of use after reconstitution. European patent application number 303 746 discloses that animal growth hormone can be stabilized with various stabilizers to provide decreased formation of insoluble substances and preservation of soluble activity in aqueous environments, such stabilizers include certain polyols, amino acids, amino acid polymers and they have a side group loaded at physiological pH and choline salts. The polyols are selected from the group consisting of non-reducing sugars, sugar alcohols, sugar acids, pentaerythritol, lactose, water-soluble dextrans and Ficoll; the amino acids are selected from the group consisting of glycine, sarcosine, lysine or salts thereof, serine, arginine or salts thereof, betaine, N, N-dimethylglycine, aspartic acid or salts thereof, glutamic acid or salts thereof. same; a polymer of an amino acid having a side group charged at physiological pH will. may be selected from polylysine, polyaspartic acid, polyglutamic acid, polyarginine, polyhistidine, polyornithine, and salts thereof, and the choline derivatives are selected from the group consisting of choline chloride, choline diacid, choline bitartrate, bicarbonate hill, tricholine citrate, choline ascorbate, choline borate, choline gluconate, choline phosphate, di (choline) sulfate and dicoline mucate. US Patent Specification No. 4,917,685 discloses a delivery device designed to be implanted, comprising stabilized growth hormone using the same stabilizers 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 (hydroxymethyl) aminomethane are mentioned. In addition, the presence of histidine hydrochloride is described as a buffer together with the polyol. International patent publication number WO 92/00998 discloses that crystals can be formed by crystallizing growth hormone with zinc. International patent publication number WO 93/12811 describes stabilized formulations of growth hormone in the form of a lyophilized powder or an aqueous solution, which comprises asparagine. Publication for international patent number WO 93/12812 discloses stabilized formulations of growth hormone in the form of a lyophilized powder or an aqueous solution comprising histidine. In such formulations deamidation is reduced by 25-30% as compared to a corresponding formulation of growth hormone comprising phosphate buffer. In addition, it is disclosed that crystals that crystallize growth hormone with zinc can be formed in the presence of histidine. In International Patent Publication No. 93/19776 describe protein formulations comprising growth hormone constituted of citrate as a buffer substance which are more stable than formulations comprising phosphate buffer. The formulations may also be comprised of amino acids such as glycine and alanine and / or mannitol or other sugar alcohols and / or glycerol and / or other carbohydrates and optionally a preservative or preservative such as benzyl alcohol. International patent publication number WO 94/03198 describes a stable aqueous formulation containing human growth hormone, a buffer, a nonionic surfactant and, optionally a neutral salt, mannitol or a preservative. The published European patent application number 177,478 describes a prolonged release composition constituted of zinc and somatotropin in a continuous phase with a biocompatible oil.

The published European patent application number 216,485 describes a sustained release composition constituted of zinc and somatotropin in a vehicle comprising vegetable oil and an adjuvant, especially beeswax, aluminum monostearate, near carnauba or paraffin. Published European Patent Publication No. 277,043 describes recovery of somatotropin from an aqueous solution by adding a salt of a transition metal and precipitation of an insoluble complex. The publication for international patent number ' WO 93/13792 discloses an implantable delayed release device comprising a somatotropin and zinc, and optionally a basic side group containing a solubilizing amino acid of Zn-somatotropin, arginine, alinin, histidine and glutamic acid are mentioned as amino acid examples. International patent publication number WO 92/17200 discloses growth hormone and metal ion formulations which are stable in the form of dimers which exhibit denaturing stability. The formulations may be constituted of glycine and optionally additionally of alanine, glutamine, asparagine, arginine or lysine, such amino acids are particularly advantageous when lyophilizing the formulation to generate a sufficient mass to form a stable formulation, in the form of a dry cake.

BRIEF DESCRIPTION OF THE IMMEDITION It has now been surprisingly found that a formulation of human growth hormone with zinc and optionally with lysine or calcium ions before the preparation of the final formulation shows a very high stability against deamination in aqueous solution and can be easily dissolved in solvents aqueous when lyophilized. The stability of the product allows the storage and transport thereof in a lyophilized state or in the form of a dissolved or redissolved formulation. The pharmaceutical formulations of the invention can be formulated for administration in any suitable route, for example, by parenteral or oral administration or by administration to a mucosal membrane, for example, by nasal administration. The pharmaceutical formulation can be presented, in the form of a dose constituted in a bottle or cartridge or in any other suitable container such as a pre-filled syringe or a pen type device. Therefore, the formulation of the invention may be in the form of a lyophilized powder to be reconstituted subsequently using conventional vehicles such as distilled water or water for injection, or in the form of a solution that includes growth hormones. Such vehicles may comprise conventional preservatives such as phenol, m-cresol and benzyl alcohol. A preferred embodiment of the invention is in the form of a pharmaceutical formulation of human growth hormone pretreated with zinc and optionally with lysine or calcium ions and further comprising a carrier in the form of a buffered aqueous solution of growth hormone. Such a formulation is in ready-to-use form and can be stored and sent as an aqueous solution without any considerable degradation. A buffer for use in a pretreated growth hormone solution may be, for example, histidine, citrate, tartrate or phosphate buffer. Preferably, the buffer is histidine buffer. The pretreatment solution is preferably adjusted to a value in the range of from about 2 to about 9, more preferably to a pH of from 6 to 8, especially from about 7-7.3. For reasons of stability, the pH in the final formulation of the pretreated growth hormone is preferably adjusted to a value from about 2 to about 9, more preferably at a value from about 6 to about 8, especially at a value of about 6.0 to approximately 6.8. In order to obtain the stabilizing effect of zinc and optionally of lysine or calcium ions, zinc is preferably added in an amount of up to 2 mM, preferably from about 1 to about 4 moles of Zn per mole of growth hormone. , preferably, from about 1 to 2 moles of Zn per mole of growth hormone, much more preferably about 1 mole of Zn per mole of growth hormone. Zinc is preferably added in the form of a physiologically acceptable soluble salt such as chloride. When calcium ions are present, they are preferably added in the form of a physiologically acceptable soluble salt such as chloride. The pharmaceutical formulation of the invention may additionally comprise sugar salts or alcohols for adjusting the tonicity and optionally an excipient in order to facilitate the processing thereof, for example, lyophilization and rapid and complete dissolution of a lyophilized formulation when reconstituted. the formulation before use. A disaccharide excipient such as lactose, trehalose and sucrose, sugar alcohols such as sorbitol or mannitol, polysaccharides such as polymers, marketed as Dextran "* products such as Dextran" * 40, Dextran "* 70 or Dextran can be selected. "* 75 and Ficoll" *, and polyvalent alcohols such as polyethylene glycol or polyvinyl alcohol, or a combination of two or more of these In a further aspect, the invention relates to a method for preparing a pharmaceutical formulation comprising a growth pretreated with zinc and optionally lysine or calcium ions, wherein the growth hormone is dissolved in a solution comprising zinc and optionally lysine or calcium ions by dissolving zinc chloride in deionized water which optionally contains lysine or calcium ions, leaving the solution stand for a certain time, add the growth hormone and optionally adjust the pH from about 2 to about ximately 9. The pH can be adjusted by adding an acid which has no adverse effects on growth hormone, preferably a physiologically acceptable acid, for example, a mineral acid, such as hydrochloric acid, sulfuric acid, nitric acid or an organic acid such as acetic acid. In one embodiment of the method of the invention, salts and an excipient are optionally added, after which the solution is applied as a filler in a container and lyophilized. Another additional aspect of the invention relates to the use of zinc and optionally lysine or calcium ions for the pretreatment of growth hormone for the formulation of a stabilized formulation of growth hormone. In another additional aspect, the invention relates to a method for treating a disorder that can be altered by growth hormone, which comprises administering a formulation which comprises a growth hormone pretreated with zinc and optionally lysine or calcium ions. In the present context, "growth hormone" can be growth hormone of any origin such as growth hormone of poultry, bovine, equine, human, ovine, porcine, salmon, trout or tuna, preferably human growth hormone. bovine, human or porcine growth, with human growth hormone being additionally preferred. The growth hormone used according to the invention can be native growth hormone isolated from a natural source, for example, by extraction of pituitary gland or pituitary gland in a conventional manner, or a growth hormone produced by recombinant techniques, for example, as is described in EB Jensen and S. Carlsen in Biotech and Biogen. 1 £, ll-ll (1990). The "growth hormone" may also be a truncated or incomplete form of growth hormone in which one or more amino acid residues have been deleted; an analogue thereof wherein one or more amino acid residues in the native molecule have been replaced by another amino acid residue, preferably a natural amino acid residue, insofar as the substitution does not have some adverse affect such as antigenicity or reduced action; or a derivative thereof, for example, having an N or C terminal extension such as Met-hGH. The preferred growth hormone is hGH. The term "dose" of growth hormone refers to that amount which provides therapeutic effect in a regimen of administration. The formulations therein are prepared to contain amounts of hGH of at least about 0.1 mg / ml, preferably a greater amount of about 10 mg / ml, preferably from about 1 mg / ml to about 40 mg /. ml, more preferably from about 1 mg / ml to about 25 mg / ml, for example from 1 mg / ml to about 5 mg / ml, calculated in a ready-to-use formulation. For the use of these compositions for administration in humans suffering from hypopituitary dwarfism, for example, these formulations contain from about 0.1 mg / ml to about 10 mg / ml, which corresponds to the dosage regimen currently contemplated for the proposed treatment. The concentration range is not critical to the invention and can be changed by the physician supervising the administration. The lysine to be used according to the present invention is preferably the alpha amino acid that occurs naturally. The lysine can be 1 or d lysine, 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 is as stable as a corresponding formulation comprising histidine as a stabilizer in which the deamidation of hGH by approximately 20%, compared to the phosphate buffer as described 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 phenol, m-crésol or benzyl alcohol. The term "pretreated" is used in the present context in connection with the formulations of growth hormone to designate a growth hormone which is treated with zinc and optionally with lysine or calcium ions before the addition of or for the additional components for the preparation of a growth hormone formulation.

The invention is explained in more detail in the following examples, which illustrate the invention. They should not be considered as limiting the scope of the invention that is defined by the appended claims.

EXPERIMENTAL PART EXAMPLE Processing deamidation.

The rate of desaturation is examined after pretreatments other than 37 ° C for formulations of hGH containing 4 mg / ml hGH, ZnCl2 0.18 M, histidine 3 mM, 1.5% benzyl alcohol, pH 6.8 compared to histidine and phosphate buffer at pH 6.8. The hGH formulations are prepared by dissolving 24 mg of hGH in 2.5 ml of: a) 2 mM CaCl2 + ZnCl. 0.36 mM, pH 7-7.3, b) 2 mM lysine + 0.36 mM ZnCl2. pH 7-7.3, or c) ZnCl2 0.36 mM, pH 7-7.3.

After storage for 1 h at 4 ° C (to allow Zn ** to form complexes with hGH), preparations a) -c) are formulated using a PD10 desalting column (Pharmacia) in 3 ml of 6 mM histidine, ZnCl2 0.36 mM, hGH 8 mg / ml. Subsequently 3 ml of 3% benzyl alcohol are added resulting in a final formulation of hGH 4 mg / ml, 3 mM histidine, 0.18 mM ZnCl2, 1.5% benzyl alcohol (the pH is adjusted to 6.8 by adding HCl / NaOH) . The following reference formulations were used: a) hGH, 4 mg / ml, 3 mM histidine, 0.18 mM ZnCl 2, 1.5% benzyl alcohol, pH 6.8; b) hGH, 4 mg / ml, 3 mM histidine, 1.5% benzyl alcohol, pH 6.8; and c) hGH, 4 mg / ml, 3 mM Na2HP04, 1.5% benzyl alcohol, pH 6.8. The reference formulations are not treated with Zn **. The formulations of hGH set forth in the following table are stored at 37 ° C for 7 days and analyzed for deaminated hGH content by IE-CLAP. The results appear in the following table.

# The deamid content is corrected by 1% per 0.1 pH unit of deviation from 6.8. It is evident from the above table that the rate of deamidation of hGH at 84% relative to histidine (68% relative to the phosphate formulation) is reduced when the hGH solution is treated with Zn ** in the presence of Ca ** or lysine before formulation in the final histidine formulation.

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. Borchard. Stability of Protein Pharmaceuticals. Pharmaceutical Research 6 (11) (1989) 903-918. 3) B.A. Johnson, J.M. Shiro a a, W.S. Hancock, M.W. Spell an, 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).

) 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) It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. Having described the invention as above, the contents of the following are claimed as property or property:

Claims (10)

REHIEVEMENTS

1. A pharmaceutical formulation characterized in that it comprises a growth hormone pretreated with zinc and optionally lysine or calcium ions.

2. The pharmaceutical formulation according to claim 1, characterized in that it additionally comprises a carrier in the form of a buffered aqueous solution of growth hormone pretreated with zinc and optionally lysine or calcium ions.

3. The pharmaceutical formulation according to claim 1 or 2, characterized in that the pH is adjusted to a value in the range from about 2 to about 9.

4. The pharmaceutical formulation according to any of the preceding claims, characterized in that the concentration of zinc is up to about 2 mM.

5. The pharmaceutical formulation according to any of the preceding claims, characterized in that it additionally comprises salts and / or saccharides and / or alcohols and sugar.

6. The pharmaceutical formulation according to any of claims 1 to 5, characterized in that the growth hormone is hGH.

7. A method for preparing a pharmaceutical formulation comprising a growth hormone pretreated with zinc and optionally lysine or calcium ions, the method is characterized in that the growth hormone is dissolved in a solution comprising zinc and optionally lysine or calcium ions upon dissolving chloride. zinc in deionized water that optionally contains lysine or calcium ions, let the solution sit for some time, add growth hormone, optionally add a condom or preservative, and optionally adjust the pH from about 2 to about 9.

8. The method according to claim 7, characterized in that sugar salts and / or alcohols and an excipient are optionally added, after which the solution is supplied as a filling in a container and freeze-dried.

9. The use of zinc and optionally lysine or calcium ions for treatment of growth hormone for the formulation of a stabilized formulation of growth hormone.

10. A method for treating a disorder or disorder that can be altered by growth hormone deficiency, characterized in that it comprises administering the formulation according to claim 1.