MXPA98000309A - Stabilized pharmaceutical formulation comprising a hormone of growth and a peptide that comprises at least one residue of basic amino acid and at least one waste of aminoacido ac - Google Patents

Abstract

A pharmaceutical formulation comprising a growth hormone and a peptide comprising at least one basic amino acid residue and at least one amino acid residue as an additive or buffer is disclosed, which shows a very high stability against deamidation, oxidation and cleavage of peptide bonds. The product stability allows the storage and shipment thereof in a lyophilized state or in the form of a dissolved or redissolved formulation at room temperature.

Description

STABILIZED PHARMACEUTICAL FORMULATION COMPRISING ONE HORMONE OF GROWTH AND A PEPTIDE THAT COMPRISES THE LESS A RESIDUE OF BASIC AMINO ACID AND AT LEAST ONE WASTE OF ACID AMINO ACID FIELD OF THE INVENTION The present invention relates to a stabilized pharmaceutical formulation comprising growth hormone, to a method for making such a formulation, to the use of a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue, to stabilize a growth hormone formulation, and to a method for the treatment of an aggravating disorder by growth hormone.

BACKGROUND OF THE INVENTION The growth hormones of humans and common domestic animals are proteins of approximately 191 amino acids, synthesized and secreted from the anterior lobe of the pituitary gland. The human growth hormone consists of 191 amino acids.

REF: 26523 Growth hormone is a key hormone involved in the regulation not only of somatic growth, but also in the regulation of the metabolism of proteins, carbohydrates and lipids. The greatest effect of growth hormone is to promote growth. The organ systems affected by growth hormone include the skeleton, connective tissue, muscles, and viscera such as the liver, intestines, and kidneys. Until the development of the recombinant technology and the cloning of the growth hormone gene, which gives rise to the production of, for example, the human growth hormone (hGH) and Met-hGH on an industrial scale, the human growth hormone it could only be obtained by extraction through the pituitary glands of human corpses. Very limited supplies of growth hormone restricted its use to the promotion of longitudinal growth in childhood and puberty for the treatment of dwarfism, even though the treatment of short stature (among other things) has been proposed. to deficiency of growth hormone, to normal short stature and to Turner's syndrome), growth hormone deficiency in adults, infertility, treatment of burns, wound healing, dystrophy, soldering bones, osteoporosis, diffuse gastric bleeding, and pseudoarthrosis. In addition, growth hormone has been proposed to increase the rate or growth rate of domestic animals or to decrease the proportion of fat in animals to be slaughtered for human consumption. The pharmaceutical formulations of growth hormone tend to be unstable. The degradation products such as the deaminated or sulfoxidated products and the dimeric or polymeric forms are generated especially in growth hormone solutions. The predominant degradation reactions of hGH are 1) deamination by direct hydrolysis or via a cyclic succinimide intermediate to form various amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH D-iso- asp-hGH (ref 1-3), and 2) oxidation of the methionine residues at positions 14 and 125 (ref 4-9). The best degradation product of hGH in the lyophilized state as well as in solution is deamidated hGH.

The deamidation takes place especially in the sn at position 149 and to a lesser degree at position 152. hGH is also rather easily oxidized at positions 14 and 125, especially in solution (4-8). The oxidation of hGH in solution forming sulfoxides, is usually due to dissolved oxygen in the formulation. The solubility of oxygen in distilled water is approximately 200 μM (9). Since the concentration of hGH in a formulation comprising 4 IU / ml is 1.3 mg / ml corresponding to 60 nM hGH, oxygen, at normal storage conditions will be present in an excess of approximately 3000 times the stoichiometric amount for the oxidation of hGH . It is not feasible to try to solve the problem by degassing the dampers before covering and packaging the formulations. To the date, it is not believed that those forms of health and oxidized forms of hGH should have toxic or altered activity or altered binding properties, but there is indication for the effect that the stability of the conformation of sulfoxides is reduced compared to native hGH .

For the development of a stable dissolved formulation, comprising hGH, it is of importance to know the rate or proportion of the deamidation and the formation of the sulfoxides, as well as the means to control the reactions. The kinetics of degradation depend on the temperature of the pH and various additives or adjuvants of the hGH formulation. Due to the instability, the growth hormone is currently lyophilized and stored in the lyophilized form at 4 ° C until it is reconstituted with the use in order to minimize degradation. The lyophilized pharmaceutical formulations comprising hGH are, to date, reconstituted by the patient, and then stored as a solution during use for a period of up to 14 days at 4 ° C, during which some degradation will take place. In addition, the process of reconstitution of lyophilized growth hormone tends to provide difficulties with the patient. Thus, 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 is able to handle the formulations at a controlled low temperature, for example, 4 ° C, which allows a prolonged shelf life of up to two years. However, the intended use of storage systems for self-medication and the field of extended use, that a formulation that is stable for a sufficiently long time with the end user under conditions where "sufficient" cooling is not always available. Preferably, a formulation must be stable in a freeze-dried state of about one month with the end user, and additionally for one month in a reconstituted state in a storage device for the intended period of use of a cartridge. Thus, there is a need for more stable formulations of growth hormone that are stable in a lyophilized state at a relatively high temperature for a period and also for a period of use at a relatively high temperature in solution. Such stabilization is of great importance when transferring the administration of growth hormone, from the clinic to the homes of the individuals being treated, where optimal storage may not be available as indicated above. In addition, the displacement in the pattern of the administration of growth hormone for the use of the deposit devices represents a dissolved, stable formulation, comprising growth hormone, in order to facilitate the handling to be performed by the patient. patient. A stable dissolved formulation comprising the growth hormone can be produced ready for use in the form of cartridges that fit within the reservoir device used by the patient, who can then avoid the reconstitution of the formulation and, therefore, they will not have to be in possession of a lyophilized formulation, an appropriate vehicle for reconstruction as well as the necessary experience and sterile equipment 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 could also be an advantage to avoid the lyophilization step in the production of the growth hormone formulations. Freeze-drying is a costly and time-consuming process, and is also often a "bottleneck" in production, due to the limited capacity of the freeze dryer. Thus, there is a need to reduce the rate or proportion of the degradation processes, in order to allow the dissolved formulations of hGH 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 the dimer. Problems associated with dimer formation are noted, for example, in Becker, G.W., Biotechnology and Applied Biochemistry 9, 478 (1987). International Patent Publication No. WO 89/09614 and Australian Patent Application No. 30771/89 describe a stable pharmaceutical formulation containing the human growth hormone, glycine and mannitol. Such formulation shows improved stability during normal processing and storage, in a lyophilized state, as well as in the period of use after reconstitution. Published European Patent Application No. 303 746 discloses that animal growth hormone can be stabilized with various stabilizers to give decreased formation of insoluble products and preservation of soluble activity in aqueous environments, including such stabilizers, certain polyols, amino acids, polymers of amino acids that 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 Fi-col; the amino acids are selected from the group consisting of glycine, sarcosine, usina or salts thereof, serine, arginine or salts thereof, betaine, N, N-dimethyl-glycine, aspartic acid or salts thereof, glutamic acid or salts thereof; a polymer of an amino acid having a side group charged at physiological pH, can be selected from polyglycine, polyaspartic acid, polyglutamic acid, polyarginine, polyhistidine, polyornithine and salts thereof; and choline derivatives are selected from the group consisting of choline chloride, choline diacid citrate, choline bitartrate, choline bicarbonate, tricholine citrate, choline ascorbate, choline borate, choline gluconate, choline phosphate, di (choline) and dicoline mucate.

US Patent Specification No. 4,917,685 discloses a dispensing device designed to be implanted, comprising growth hormone stabilized using the same stabilizers as mentioned in European Patent 303746. Published European Patent Application No. 374,120 describes a formulation stabilized comprising hGH and a polyol having three hydroxyl groups. It is mentioned in glycerol and tris (hydroxymethyl) aminomethane. In addition, the presence of histidine hydrochloride as a buffer together with the polyol is also disclosed. International Patent Publication No. WO 93/12811 describes stabilizing 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 the stabilized formulations of growth hormone in the form of a lyophilized powder or an aqueous solution comprising histidine. In such formulations deamidation is produced by 25-30% as compared to a corresponding formulation of growth hormone comprising phosphate buffer. The International Patent Publication No. WO 93/19776 describes protein formulations comprising growth hormone that includes citrate as a buffer, which is more stable than formulations comprising phosphate buffer. The formulations may also comprise amino acids such as glycine or alanine and / or mannitol or other sugar alcohols and / or glycerol and / or other carbohydrates, and optionally a preservative such as benzyl alcohol. The International Patent Publication No. WO 94/03198 describes a stable aqueous formulation containing human growth hormone, a buffer, a non-ionic surfactant and, optionally, a neutral salt, mannitol or a preservative.

BRIEF DESCRIPTION OF THE INVENTION It has now surprisingly been found that a formulation of the human growth hormone comprising a peptide including at least one basic amino acid residue and at least one acidic amino acid residue as an additive, exhibits a very high stability against deamination and oxidation. The stability of the product allows the storage and shipment thereof in a lyophilized state and in the form of a dissolved and redissolved formulation. The peptide including at least one basic amino acid residue and at least one acidic amino acid residue to be used according to the present invention, may be a peptide that includes from 10 to 20 amino acid residues, preferably from 3 to 10 amino acid residues, more preferably from 3 to 6 amino acid residues, such as 3 or 4 amino acid residues. According to one aspect of the invention, the acidic basic amino acid residues of the shorter peptides are separated by 1 or 2 amino acid residues. The pharmaceutical formulations of the invention can be formulated for administration in any appropriate manner, for example, by parenteral or oral administration or administration to the mucous membrane, for example nasal administration. The pharmaceutical formulation may be presented in the form of a dose comprised in a bottle or cartridge or any other suitable container such as a prefilled syringe or a reservoir device.

Thus, 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 comprising growth hormone. Such carriers may comprise conventional preservatives such as benzyl alcohol and phenols, for example, phenol or m-cresol or a mixture thereof. A preferred embodiment of the invention is in the form of a pharmaceutical formulation of human growth hormone comprising a peptide having at least one basic amino acid residue and at least one acidic amino acid residue, and further including a carrier in the form of a buffered aqueous solution of growth hormone. Such formulation is a ready-to-use form and can be stored and shipped as an aqueous solution without any considerable degradation. A buffer to be used in a growth hormone solution can be for example histidine, citrate, tartrate or phosphate buffer. For reasons of stability the pH of a solution is preferably adjusted to a value in the range of from about 2 to about 8, preferably from about 5 to about 7, more preferably from about 6.0 to about 7.0, and even more preferably from about 6.0 to about approximately 6.8. In order to obtain the stabilizing effect, a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue is preferably added in an amount of about up to 100 mM, more preferably in an amount of about 1. -10 mM, preferably about 2-6 mM, more preferably about 3-5 mM. The pharmaceutical formulation of the invention can further comprise salts 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 the formulation is reconstituted before of use. An excipient may be selected from disaccharides such as lactose, trehalose and sucrose, sugar alcohols such as sorbitol or mannitol, polysaccharides such as commercialized polymers such as Dextran products such as Dextran ® ® 40, Dextran 70 or Dextran 75, and Ficoll and polyvalent alcohols such as polyethylene glycol or polyvinyl alcohol or a combination of two or more thereof. In a further aspect, the present invention relates to a method for the preparation of a pharmaceutical formulation comprising a growth hormone and a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue, wherein the Growth hormone is dissolved in a solution comprising a peptide including at least one basic amino acid residue and at least one acidic amino acid residue by dissolving a peptide comprising at least one basic amino acid residue and at least one residue of acidic amino acid in deionized water optionally comprising benzyl alcohol, adding growth hormone and optionally adjusting the pH from about 2 to about 8. The pH can be adjusted by the addition of an acid which has no adverse effect on growth hormone , preferably a physiologically acceptable acid, for example an acid mineral such as hydrochloric acid, sulfuric acid or 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 filled into a container and lyophilized. Yet another aspect of the invention relates to the use of a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue for the formation of a stabilized formulation of growth hormone. In another aspect more, the invention relates to a method for treating a growth hormone-disruptable disorder, comprising administering a formulation including a growth hormone and a peptide comprising at least one basic amino acid residue and at least one amino acid residue. acid In the present context the "growth hormone" can be the growth hormone from any origin such as avian, bovine, equine, human, ovine, porcine, salmon, trout or tuna, preferably growth hormone, bovine , human or porcine, with human growth hormone being more preferred. The growth hormone used according to the invention can be the native growth hormone isolated from a natural source, for example, by extracting pituitary glands 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 Bioeng, 3_6, 1-11 (1990). The "growth hormone" can also be a truncated form of growth hormone, where one or more amino acid residues have (have) been deleted; an analogue thereof wherein one or more of the amino acid residues in the native molecule has been replaced by another amino acid residue, preferably a natural amino acid residue, so long as the substitution does not have an adverse effect 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 the therapeutic effect in a regimen of administration. The formulations herein are prepared, containing amounts of hGH at least about 00.1 mg / ml, preferably up to about 10 mg / ml, preferably from about 1 mg / ml to about 40 mg / ml, more preferably from about 1 mg / ml to about 40 mg / ml, even 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 the ready-to-use formulation. For the use of these compositions for administration to humans suffering from hypopituitary dwarfism, for example, these formulations contain from about 0.1 mg / ml to about 10 mg / ml, corresponding to the dosage regimen currently contemplated for the intended treatment. The concentration range is not critical to the invention and can be varied for the physician supervising the administration. A peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue to be used according to the present invention, is that which preferably comprises the alpha-amino acid residues of natural origin. The amino acid (s) may be the amino acid (s) in the form 1 or d or a mixture thereof. "Residues of acidic amino acids" are for example Glu or Asp, and "basic amino acid residues" are for example Lys or Arg.

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 a stabilizer in which the deamidation of hGH is reduced by about 20%, compared to the phosphate buffer as described in WO 93/12812. The solvent used in the method of the invention can be water, alcohols such as n-propyl alcohol or isopropyl, butyl alcohol or mixtures thereof. The solvent may comprise a preservative such as benzyl alcohol and phenols, for example, phenol or m-cresol or a mixture thereof.

DETAILED DESCRIPTION OF THE INVENTION The invention is explained in more detail in the following examples, which illustrate the invention. These are not considered to be limiting of the scope of the invention, as defined by the appended claims.

EXPERIMENTAL PART EXAMPLE Reduction of the deamidation.

The rate or rate of deamidation was examined at 37 ° C for formulations of hGH comprising 4 mg / ml hGH at pH 6.8 in the presence of 5 mM peptide comprising at least one basic amino acid residue and at least one amino acid residue acid (Lys-Gly-Asp-Ser) compared to histidine at pH 6.8. The hGH formulations were prepared by dissolving 8 mg of hGH in 2 ml of 10 mM solution of Lys-Gly-Asp-Ser or histidine. In this way, 2 ml of 3.0% benzyl alcohol was added to give a final formulation of 4 mg / ml of hGH, Lys-Gly-Asp-Ser or 5 mM histidine, 1.5% of benzyl alcohol, pH 6.8 (adjusted by adding HCl or NaOH). The formulations of hGH established in the above table were stored at 37 ° C for 7 days, and analyzed for the content of deamidated hGH by IE-HPLC. The results appear in the following table.

Table * Deamido corrected by 1% by a deviation of 0.1 pH unit of 6.8.

The content of deamidated hGH in the initial material was: 2.0%. From the previous table, it seems that the deamidation of hGH is reduced by. the addition of Lys-Gly-Asp-Ser at least at the same level as that obtained by the addition of histidine (25-30% in comparison to the phosphate buffer, see WO 93/12812 above). The above results show that the deamidation rate or rate is reduced to a very high degree by the addition of Lys-Gly-Asp-Ser at a low concentration of up to 100 mM, preferably 1-10 mM, more preferably 2-6 mM and more preferably about 3-5 mM. The rate of deamination can thus be reduced by more than 30% by replacing the phosphate buffer with Lys-Gly-Asp-Ser. The use of benzyl alcohol as a preservative seems to have no influence on the rate of deamination.

REFERENCES 1) Y.-C.J. Wang and M.A. Hn. Parenteral Formulations of Preoteins and Peptides: Stability and Stabilizers, J. Parenteral Science and Technology 42 (Suppl.) (1998) 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. Shiroka a, 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).

) G.W. Becker et al., Biotech. ppl. 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 to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (10)

1. A pharmaceutical formulation, characterized in that it comprises a growth hormone and a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue.

2. A pharmaceutical formulation according to claim 1, characterized in that it further comprises a carrier in the form of a buffered aqueous solution of growth hormone, which contains a peptide comprising at least one basic amino acid residue and at least one amino acid residue acid.

3. A pharmaceutical formulation according to claim 1 or 2, characterized in that the pH is adjusted to a value in the range of about 2 to about 8.

4. A pharmaceutical formulation according to any of the preceding claims, characterized in that the concentration of a peptide comprising at least one basic amino acid residue and at least one acidic amino acid residue is up to about 100 mM.

5. A pharmaceutical formulation according to any of the preceding claims, characterized in that it also comprises salts and / or saccharides.

6. A 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 and a peptide including at least one basic amino acid residue and at least one acidic amino acid residue, characterized in that it comprises (a) dissolving the peptide in deionized water, which optionally contains a preservative to form a solution, and (b) the addition of the growth hormone to the solution, and (c) the optional adjustment of the pH of pH 2 to about pH 8.

8. A method according to claim 7, characterized in that salts and / or an excipient are optionally added, after which the solution is filled into a container and lyophilized.

9. The use of a peptide, characterized in that it comprises at least one basic amino acid residue and at least one acidic amino acid residue by the formation of a stabilized formulation of growth hormone.

10. A method for treatment of a disorder that can be affected by growth hormone, characterized in that it comprises administering the formulation according to any of claims 1-6.