KR19990028908A - Stabilized pharmaceutical formulation consisting of a growth hormone, a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue - Google Patents

Abstract

Pharmaceutical formulations comprising growth hormone and peptides consisting of at least one basic amino acid residue and at least one acidic amino acid residue as additives or buffers exhibit very high stability against deamidation, oxidation and degradation of peptide bonds. The stability of this product permits its storage and transport in the form of dissolved or re-dissolved preparations, either lyophilized or at ambient temperature.

Description

Stabilized pharmaceutical preparation consisting of a peptide consisting of growth hormone, at least one basic amino acid residue and at least one acidic amino acid residue

Growth hormone from human and general livestock is a protein of about 191 amino acids synthesized and secreted from the anterior pituitary gland. Human growth hormone is composed of 191 amino acids.

Growth hormone is a major hormone involved in the regulation of protein, carbohydrate and lipid metabolism as well as the regulation of somatic cell growth. The main effect of growth hormone is to promote growth.

Organ systems affected by growth hormones include the intestines such as skeletal, connective tissue, muscle and liver, intestines and kidneys.

For example, up to the development of recombinant technology and cloning of growth hormone genes that now lead to the production of human growth hormone (hGH) and Met-hGH on an industrial scale, human growth hormone can only be obtained by extracting from the pituitary gland of the human body. there was. A very limited supply of growth hormone may be due to the fact that this is particularly short (due to growth hormone deficiency, normal shortness and Turner syndrome), growth hormone deficiency in adults, treatment of infertility, burns, wound healing, malnutrition, osteoadhesion, osteoporosis, diffuse gastric bleeding. And its use in promoting length growth in childhood and adolescence for the treatment of subauthentication, even though it has been proposed for the treatment of gastric joints.

Moreover, growth hormone has been proposed to increase the growth rate of livestock or to reduce fat distribution in animals to be slaughtered for human consumption.

Pharmaceutical formulations of growth hormone tend to be unstable. Degradation products such as deamidation or sulfoxylation products and dimer or polymer forms occur in particular in growth hormone solutions.

The main degradation reactions of hGH were: 1) various amounts of L-asp-hGH, L-iso-asp-hGH, D-asp-hGH and D-iso-asp- either by direct hydrolysis or via cyclic succinimide intermediates. deamidation (ref 1-3) to form hGH, and 2) oxidation of methionine residues at positions 14 and 125 (ref 4-9). The main degradation product of hGH in solution as well as in lyophilized state is deamidated hGH.

Deamidation occurs in particular to a low extent at Asn at position 149 and at position 152.

hGH is also more easily oxidized, particularly at positions 14 and 125 in solution (4-8).

Oxidation of hGH in sulfoxide forming solutions is usually due to oxygen dissolved in the formulation. The solubility of oxygen in distilled water is about 200 μM (9). Since the concentration of hGH in the formulation of 4 IU / ml is 1.3 mg / ml, corresponding to 60 nM hGH, under normal storage conditions, it is present in excess of about 3000 times the stoichiometric amount for hGH oxidation. Attempts to solve the problem by degassing the buffer prior to tapping and packing the formulation are not possible.

Currently, the deamidated and oxidized forms of these hGH are not believed to have toxic or modified biological activity or receptor binding properties, but the morphological stability of sulfoxides shows a reduced effect compared to native hGH. .

It is important to know how to control the reaction as well as the deamidation rate and sulfoxide formation rate for the development of stable, dissolved formulations consisting of hGH.

The degradation reaction depends on the temperature, pH and various additives or adjuvants of the hGH preparation.

Because of instability, growth hormone is currently lyophilized and stored in lyophilized form at 4 ° C. until reconstituted for use to minimize degradation.

Lyophilized pharmaceutical formulations consisting of hGH are currently stored as a solution during use over a period of up to 14 days at 4 ° C. after reconstitution by the patient, during which some degradation will occur.

Moreover, the reconstitution process of lyophilized growth hormone tends to present difficulties for patients. Therefore, it is presently desirable to reconstitute growth hormone as soon as possible before use and to store and transport the formulation in a lyophilized state. The chain from the manufacturer to the pharmacy, for example, is appropriate to handle the formulation at a controlled low temperature of 4 ° C. which allows for a long shelf life up to 2 years.

However, prolonged use and extended use of the pen system for self-treatment requires a formulation that is stable for a sufficiently long time to the end user under conditions where "sufficient" cooling is not always available.

Preferably, the formulation should be stable for one month in a lyophilized state for up to one month to the end user and further in a reconstituted state in the pen device for the intended use of the cartridge.

Accordingly, there is a need for more stable formulations of growth hormone that are stable during use, at relatively high temperatures for a period of time and further at relatively high temperatures in solution. This stabilization is very important when moving the administration of growth hormone from the clinic to the home of the individual to be treated if optimal storage is not available as indicated above.

Moreover, replacement of growth hormone dosages in the use of pen devices requires stable dissolved formulations of growth hormone to facilitate manipulations to be performed by the patient. Stable dissolved formulations of growth hormone can be prepared for immediate use in the form of cartridges suitable for pen devices used by patients so that patients can avoid reconstitution of the formulation and therefore only lyophilized formulations, suitable excipients for reconstitution But it would not be necessary to possess the necessary techniques and sterilization devices for sterile reconstitution of the formulation.

It would also be desirable to avoid reconstitution of the lyophilized formulation immediately prior to use of the formulation for stability reasons.

Moreover, it would also be advantageous to avoid the lyophilization step in the preparation of growth hormone preparations. Lyophilization is a waste of time and an expensive process and is also often a "hinder" in manufacturing due to the limited capacity of the lyophilizer.

Thus, it is necessary to reduce the degradation process rate to allow stable dissolved hGH formulations for shelf life and up to one month of use.

Previous attempts to stabilize hGH have not been successful enough to prevent the formation of dimers. Problems related to dimer formation are pointed out, for example, in Becker, GW, Biotechnology and Applied Biochemistry 9, 478 (1987).

International Publication No. WO 89/09614 and Australian Patent Application No. 30771/89 disclose stable pharmaceutical preparations containing human growth hormone, glycine and mannitol. Such formulations exhibit improved stability during storage as well as during normal processing and in lyophilized conditions as well as during post-reconstitution use.

Published European Patent Application No. 303 746 can be stabilized with a variety of stabilizers that provide for the formation of reduced insoluble substances and preservation of soluble activity in aqueous environments, such as polyols, amino acids, physiological pH. And polymers of amino acids having side groups charged at and choline salts. The polyol is selected from the group consisting of non-reducing sugars, sugar alcohols, sugar acids, pentaerythritol, lactose, water soluble dextran and Ficoll; The amino acid is 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 Become; The polymer of amino acids having side groups charged at physiological pH may be selected from polylysine, polyaspartic acid, polyglutamic acid, polyarginine, polyhistidine, polyornithine and salts thereof; And choline derivatives include choline chloride, choline dihydrogen citrate, choline bitartrate, choline bicarbonate, tricholine citrate, choline ascorbate, choline borate, choline gluconate, choline phosphate, di (choline) sulfate and Dicholine mucate.

US Pat. No. 4,917,685 discloses a delivery device designed for injection consisting of growth hormone stabilized using the same stabilizer as mentioned in EP 303746.

Published European Patent Application No. 374,120 discloses stabilized preparations consisting of hGH and a polyol with three hydroxy groups. Glycerol and tris (hydroxymethyl) aminomethane are mentioned. Moreover, the presence of histidine hydrochloride as a buffer with the polyol is disclosed.

International Publication No. WO 93/12811 discloses stabilized preparations of growth hormone in the form of lyophilized powder or in the form of an aqueous solution consisting of asparagine.

International Publication No. WO 93/12812 discloses stabilized preparations of growth hormone in the form of lyophilized powder or in the form of an aqueous solution consisting of histidine. Deamidation in these formulations is reduced by 25-30% compared to the formulation of the corresponding growth hormone consisting of phosphate buffer.

International Publication No. WO 93/19776 discloses a protein preparation consisting of growth hormone consisting of citrate as a buffer material more stable than a preparation consisting of phosphate buffer. The formulation may also consist of amino acids such as glycine and alanine and / or mannitol or other sugar alcohols and / or glycerol and / or other carbohydrates and optionally preservatives such as benzyl alcohol.

International Publication No. WO 94/03198 discloses stable aqueous formulations containing human growth hormone, buffers, nonionic surfactants and optionally neutral salts, mannitol, or preservatives.

The present invention relates to a stabilized pharmaceutical formulation consisting of growth hormone, a method for preparing the formulation, the use of a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue to stabilize the growth hormone preparation, and a growth hormone. A method of treating a disease that may be affected.

It has now been surprisingly found that the preparation of human growth hormone consisting of peptides consisting of at least one basic amino acid residue and at least one acidic amino acid residue as an additive shows very high stability against deamidation and oxidation. The stability of this product allows its storage and transport in lyophilized state or in the form of dissolved or redissolved preparations.

Peptides consisting of at least one basic amino acid residue and at least one acidic amino acid residue to be used according to the present invention are more preferably 10 to 20 amino acid residues, preferably 3 to 10 amino acid residues, 3 or 4 amino acid residues. Preferably a peptide consisting of 3 to 6 amino acid residues. According to one aspect of the invention the basic and acidic amino acid residues of the short peptides are separated into one or two amino acid residues.

The pharmaceutical preparations of the present invention may be formulated for administration in any suitable manner, eg, parenteral or oral or by mucosal administration, eg, nasal administration. The pharmaceutical preparation may be provided in dosage form consisting of a vial or cartridge, or any other suitable container, such as a prefilled syringe or pen device.

Thus, the formulations of the present invention may be in the form of lyophilized powders or solutions which will later be reconstituted using conventional excipients such as distilled or injectable water consisting of growth hormone. Such excipients may consist of conventional preservatives such as benzyl alcohol and phenols such as phenol or m-cresol or mixtures thereof.

Preferred embodiments of the present invention are in the form of a pharmaceutical preparation of human growth hormone which consists of a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue and further contains a carrier in the form of a buffered aqueous solution of growth hormone. Such formulations are in ready-to-use form and can be stored and transported as aqueous solutions without any significant degradation.

The buffer used for the solution of growth hormone may be, for example, histidine, citrate, tartrate or phosphate buffer.

For stability reasons the pH of the solution is preferably adjusted to a value of an interval of about 2 to about 8, preferably about 5 to about 7, more preferably about 6.0 to about 7.0, even more preferably about 6.0 to about 6.8.

To obtain a stabilizing effect, the peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue is preferably in an amount up to 100 mM, more preferably about 1-10 mM, preferably about 2-6 mM, most Preferably in an amount of about 3-5 mM.

The pharmaceutical formulations of the present invention further contain salts for adjusting osmoticity and optionally excipients to facilitate rapid and complete dissolution of the lyophilized formulations when reconstituting the formulations prior to processing thereof, eg lyophilization and use. can do.

Excipients include lactose, trehalose, and sugar alcohols such as disaccharides, sorbitol or mannitol, such as sucrose, Dextran ^R 40, Dextran ^R 70 or a polysaccharide and polyethylene glycol, or poly, such as a polymer that is commercially available as Dextran ^R products and Ficoll ^R, such as Dextran ^R 75 Polyhydric alcohols such as vinyl alcohol or combinations of two or more thereof.

In a further aspect, the present invention relates to a process for the preparation of a pharmaceutical formulation comprising a growth hormone, a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue, wherein the growth hormone is a desorption agent, optionally containing benzyl alcohol. At least one basic amino acid residue and at least one acidic acid by dissolving a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue in ionized water, adding growth hormone and optionally adjusting the pH to about 2 to about 8 Soluble in a solution consisting of a peptide consisting of amino acid residues.

The pH can be adjusted by adding acids which do not adversely affect growth hormone, preferably physiologically acceptable acids such as inorganic acids such as hydrochloric acid, sulfuric acid or nitric acid or organic acids such as acetic acid.

In an embodiment of the method of the invention, after the salt and excipients are optionally added, the solution is filled into a container and lyophilized.

Another aspect of the invention relates to the use of a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue for the preparation of a stabilized preparation of growth hormone.

In still another aspect the present invention relates to a method of treating a disorder that may be affected by growth hormone consisting of administering a formulation consisting of a growth hormone and a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue.

A "growth hormone" in this context may be a growth hormone from any origin, preferably a bovine, human or pig growth hormone, such as algae, bovine, horse, human, sheep, pig, salmon, trout or tuna growth hormone, Growth hormone is most preferred. Growth hormones used in accordance with the present invention are natural growth hormones isolated from natural raw materials, for example, by extracting the pituitary gland in a conventional manner, or for example EB Jensen and S. Carlsen in Biotech and Bioeng. 36 , 1-11 (1990), may be growth hormone produced by recombinant technology. “Growth hormone” also includes truncated growth hormones in which one or more amino acid residues are deleted; Unless substitution has adverse effects such as antigenicity or reduced action, analogs in which one or more amino acid residues in a natural molecule are substituted with other amino acid residues, preferably with natural amino acid residues; Or a derivative thereof having, for example, an N- or C-terminal extension, such as Met-hGH. Preferred growth hormone is hGH.

The term "dosage" of growth hormone refers to the amount that provides a therapeutic effect in the dosing schedule. Its formulation is at least about 0.1 mg / ml, preferably at least about 10 mg / ml, preferably from about 1 mg / ml to about 40 mg / ml, more preferably about 1 mg, as calculated for ready-to-use formulations. / ml to about 25 mg / ml, for example, 1 mg / ml to about 5 mg / ml. For the use of these compositions in administration to a person suffering from pituitary insufficiency subcertification, for example, these formulations range from about 0.1 mg / ml to about 10 mg / ml, corresponding to a dosage plan generally considered for the intended treatment. contains ml. Concentration ranges are not critical to the invention and may be varied by the physician administering the administration.

Peptides consisting of at least one basic amino acid residue and at least one acidic amino acid residue to be used according to the invention preferably consist of naturally occurring alpha amino acid residues. The amino acid (s) may be l or d amino acids or mixtures thereof.

"Acid amino acid residue" is for example Glu or Asp, and "basic amino acid residue" is for example Lys or Arg.

In this context, “high stability” is histidine as a stabilizer in which the formulation is more stable than conventional formulations consisting of phosphate buffers and preferably the deamidation of hGH is reduced by about 20% compared to the phosphate buffers disclosed in WO 93/12812. It is obtained when it is as stable as the said preparation containing.

The solvent used in the process of the invention may be water, an alcohol such as ethyl, n-propyl or isopropyl, butyl alcohol or mixtures thereof. The solvent may consist of preservatives such as benzyl alcohol and phenols such as phenol or m-cresol or mixtures thereof.

The invention is illustrated in more detail by the following examples which illustrate the invention. They are not to be considered as limiting the scope of the invention as defined by the appended claims.

Reduction of deamidation

The deamidation rate was compared to histidine at pH 6.8 at 4 mg / ml hGH at pH 6.8 in the presence of a 5 mM peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue (Lys-Gly-Asp-Ser). The hGH formulations made were investigated at 37 ° C.

hGH preparations were prepared by dissolving 8 mg hGH in 2 ml of a 10 mM solution of Lys-Gly-Asp-Ser or histidine. Then, 2 ml of 3.0% benzyl alcohol was added to obtain a final formulation of 4 mg / ml hGH, 5 mM Lys-Gly-Asp-Ser or histidine, 1.5% benzyl alcohol, pH 6.8 (adjusted by adding HCl or NaOH). .

The hGH formulations mentioned in Table 1 below were stored at 37 ° C. for 7 days and analyzed for content of deamidated hGH by IE-HPLC. The results are shown in Table 1 below.

Preparation pH start / pH end /% desamido Corrected content of deamidated hGH ^＊ Desamido hGH content in comparison with His Lys-Gly-Asp-Ser 6.8 / 6.8 / 13.6 13.6 83 Histidine 6.8 / 6.8 / 16.4 16.4 100 ^* The des amino corrected by 1% per 0.1 pH unit deviation from 6.8

The content of deamidated hGH in the starting material was 2.0%.

From the table it is shown that deamidation of hGH is reduced by the addition of Lys-Gly-Asp-Ser to at least the same level as obtained by the addition of histidine (25-30% when compared to phosphate buffer, WO 93 / 12812).

The results show a very high degree of deamidation by adding Lys-Gly-Asp-Ser at low concentrations of up to 100 mM, preferably 1-10 mM, more preferably 2-6 mM and most preferably about 3-5 mM. Prove to be reduced. The deamidation rate can thus be reduced by at least 30% by replacing phosphate buffer with Lys-Gly-Asp-Ser.

The use of benzyl alcohol as a preservative does not appear to affect the deamidation rate.

(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 (10)

A pharmaceutical preparation comprising a growth hormone, a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue. The pharmaceutical preparation according to claim 1, further comprising a carrier in the form of a buffered aqueous solution of growth hormone containing a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue. The pharmaceutical formulation according to claim 1 or 2, wherein the pH is adjusted to a value at intervals of about 2 to about 8. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the concentration of the peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue is up to about 100 mM. The pharmaceutical preparation according to any one of claims 1 to 4, which further contains salts and / or sugars. The pharmaceutical preparation according to any one of claims 1 to 5, wherein the growth hormone is hGH. In the preparation of a pharmaceutical preparation consisting of a peptide consisting of growth hormone, at least one basic amino acid residue and at least one acidic amino acid residue, (a) optionally dissolving the peptide in deionized water containing a preservative to form a solution; and (b) adding growth hormone to the solution, and (c) optionally adjusting the pH to pH 2 to about pH 8. 8. A method according to claim 7, characterized in that after the addition of the salt and / or excipient optionally, the solution is filled into a container and lyophilized. Use of a peptide consisting of at least one basic amino acid residue and at least one acidic amino acid residue for the preparation of a stabilized preparation of growth hormone. A method for treating a disease that may be affected by growth hormone, comprising administering the agent of any one of claims 1 to 6.