PL152974B1 - Preparation for stabilizing the growth stimulating hormones - Google Patents

Description

A STABILIZED PREPARATION WE STIMULATE GROWTH

The present invention relates to a stabilized growth promoting preparation containing an effective amount of a stabilized growth promoting hormone.

The stabilizers reduce the formation of the insoluble forms and protect the bloactivity of the soluble form of the growth stimulating hormone in aquatic environments. One of the major problems with the administration of growth-promoting hormones is denaturation of the native globular structure due to aggregation of growth hormone in excised forms, which reduces the amount of active growth hormone available. Damage to the system almost always results from the formation of these insoluble forms In addition to the formation of insoluble forms, another problem of ^^ m when administering growth-promoting phosphonates is to maintain the bioactivity of the hormone as soluble. , the stabilizer should reduce the formation of insoluble forms and inhibit the bioactivity of the soluble growth hormone

Various stabilizers have been disclosed in the art that avoid degradation in protein structure. For example, glycerin is used to stabilize the activity of various proteins. Gekko et al. ΒίοοίιβιηΐβίΓγ, 20, 4666-4676 / 1981 / Examples of proteins that are described in this article, include chymorΓypskiogji A / from bovine pancreas /, ribonuclease A / from bovine pancreas /, β-lactoglobulin / from milk /, bovine serum albumin, insulin / isustal lysoyym from egg white and oo -chymotrypsin

US Patent No. 4,179,337 discloses a method of conjugating a polypeptide, such as enzymes and insulin, with a polyethylene glycol or polyprocyanin glycolate with a molecular weight of 500-20,000 daltons. Polyhydroxy glycol or glycol

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Polypropylene has been described as anti-loss of activity polypeptide, and the agent can be injected without any immuno-enzyme response.

U.S. Patent No. 4,439,181 discloses a method of preventing protein precipitation in drug suction systems which relies on the fluidity of the infusion to function properly. The method comprises mixing a polyol with a protein solution prior to introducing the solution into the release system. Drug · Examples of the polyols described include glycerin and the biocompatible β-polyols. Examples of these types of polyols are:

erythritol. arlbinlzrel _ł keylose, ribose, adonitol, arabi.tol, rhamnose, inositol, fructose, galactose, glucose, mannose, sorbose, maize, mesitose, sucrose and raffinose first as an aqueous solution and mixed with insulin to provide a final polyol concentration in the solution of about 10 to 90% / w / v / protein complemented Other proteins that have been described to be subject to the same precipitation test include: growth hormone, glucagon, etc.

While the prior art has considered a number of different stabilizers for specific proteins, unfortunately the fact that a particular stabilizer is effective for a particular protein does not necessarily mean that that particular stabilizer is suitable for stabilizing a growth promoting hormone. the need for a method of stabilizing the growth promoting hormone, reducing the formation of insoluble forms and protecting the bioactivity of the hormone.

Wynnlazek relates to a stabilized growth promoting preparation which contains a growth promoting hormone and a stabilizer in an amount that stabilizes the hormone.

The invention enhances the formation of insoluble forms and protects the bioactivity of the soluble form of the growth promoting hormone in an aqueous environment. Stable <stabilized growth hormone is produced by acting on the growth promoting hormone of the stabilizer when used in an effective amount, such as one or more of the following: a / polyol such as non-reducing sugars, sugar alcohols, sugar-derived acids, pentaeeithrite, lactose, water-soluble dextrans and Ficoll, b) amino acid such as glycine, sarcosine, lysine or its salts, betaine, N6-dimethylglycine, aspartic acid or its salts, gluaamic acid or its salts, c) an amino acid polymer with a pendant group at a pH physooriizet, and d) a choline derivative such as choline chloride, choline dihydrocytate, choline bitartrate, choline bicarbonate, tri (choline) citrate, ascorbate choline, choline borate, choline gluconate, choline phosphate, si di (choline) arate and the di (choline) salt of mucic acid.

The figure shows a graph showing the average amount of porcine growth hormone in ng / ml of blood serum depending on the time of taking a blood serum sample after implantation

The stabilizers used in the preparations according to the invention not only promote the formation of insoluble forms, but also protect the bioactivity of the soluble part of the growth hormone. stimulating animal growth · In this application, the stabilized growth hormones are found in an aqueous solution containing dissolved growth hormone and dissolved stabilizer.

According to another variant, the stabilized growth hormones are administered via an implantable device. In this variant, the stabilized growth hormones may be liquid or dry. growth is in the form of a dry mixture containing solid growth hormone and a solid stabilizer, which after implantation in an aqueous environment become moist and begin to dissolve.

The stabilizers are particularly suitable for animal growth-promoting hormones. Examples of these types of hormones include bovine, human, equine, avian, sheep, and porcine growth-promoting hormones.

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The preferred hormone is bovine or porcine growth promoting hormone While the concentration of the growth promoting hormone may vary depending on the dosage desired and the particular growth promoting hormone, the hormone is generally present in an amount of from about 1 to 97.5% by weight of the total weight of the stimulant preparation. increase. Preferably, the growth promoting hormone is present in an amount from about 5 to about 50% of the total weight of the growth promoting preparation. It may be appreciated by one skilled in the art that growth hormones may "comprise a high percentage by weight" of the total amount of a growth promoting preparation when the preparation is in a solid state, since water and other suitable aqueous solvents are not required to create a growth promoting preparation. the preparation was suitable for injection ·

The growth hormone to be used in the preparation according to the invention can be obtained by extracting methods followed by concentration from the pituitary glands of various animals. Likewise, there are also growth hormones produced by recombinant DNA methods. The amino acid sequences of the various hormones used in the method according to the invention are known. For example, the amino acid sequence of the human growth promoting hormone is described in the article by C.H. Li in: Kirk-Othmer Encyclopedia of Chemical Technologyi, 3rd Edition, Vol. 12, pp. 549-552. The amino acid sequence of bovine growth hormone is described in the article by R.P. Woychik in: Nucleic Acid Rs ··, 10 «7197/1982 / · The amino acid sequence of sheep's growth hormone is described in the article by C.H. Liu 1 in ·, Arch. Bloch. Biophys ·, 156, 493-608 / 1973 /. The amino acid sequence of porcine growth hormone is described in the article by P.H. Seeburg et al., In DNA, 2.37, 45 (1983).

In addition to the above, growth hormones modified by cutting up to 12 amino acid residues from the amino terminus of the amino acid sequence may also be used.

The stabilizers used in the formulation according to the invention generally have two characteristics. First, they are generally highly polar. Second, the stabilizers can be soluble in aqueous solutions. According to one application, the stabilizers are preferably used in sufficient amounts in the sense that when the growth stabilizer preparation is in an aqueous medium or solution, the solution is saturated with the stabilizer. In fact, different stabilizers have their own solubility and may be present in different amounts. When the growth promoting formulation is dispersed or dissolved in an aqueous solution, the concentration of the stabilizer should be from about 5% by weight to full saturation in the growth hormone containing aqueous formulation. When a dry formulation is selected, the stabilizer may correspond to a higher percentage of the total formulation since the main ingredient, i.e. water or an aqueous solvent, is eliminated.

Accordingly, the weight percentages of the specific stabilizers listed below include both aqueous and solid formulations containing a stabilizer and a growth promoting hormone.

Stabilizers that can be used in the formulation of the present invention are polyols such as non-reducing sugars, sugar alcohols, sugar derived acids, lactose, pennaerythrite, water soluble dextrans, and Ficoll. An example of a non-reducing sugar is sucrose. Examples of sugar derived alcohols include mannitol, xylitol, erythritol, threitol, glucitol, and glycerin. An exemplary sugar product acid is L-glioonic acid and its mr 2 salts. [0035] Preferably, the polyol is xylitol. Contrary to what you may believe from reading many known publications, in particular US Pat. No. 4,439,181, not all polyols are suitable for the stabilization of growth-promoting hormones. While many polyols can provide additional formation of insoluble forms, some of them do not protect the bioactivity of the resilient part of the growth promoting hormone. Examples of such polyols are reducing sugars, e.g., fructose, mannose and maltose. Lactose, being a reducing sugar, appears to be exceptional among the other reducing sugars. The skilled person should be aware that the polyols which are used in the process according to the invention have various solubilities in water. When the polyol is present in an amount in a stabilizing amount, it can be present in an amount from about 2.5 to about 99% by weight of the total amount of the complete formulation prior to incorporation. the aquatic environment. Preferably, the polyol is present in an amount from about 15 to about 60% by weight.

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Other stabilizers used in the formulation according to the invention are certain amino acids. Examples of amino acids that can be used are glycine, sarcosine, lysine or its salts, serine, arginine or its salts, betaine, N, N-dimethylglycine, aspartic acid or its salts, and glutamic acid or its salts. Mixtures of these amino acids can also be used. The preferred amino acid is sarcosine. When the amino acid is present in a stabilizing amount, it is generally present in an amount of from about 2.5 to about 99% by weight of the total amount of the complete preparation before being incorporated into it. in an aqueous medium Preferably, the amino acid is present in an amount from about 15 to about 60% by weight.

Certain amino acid polymers have been found to be useful growth hormone stabilizers · These polyamino acids or their salts can be characterized by having charged side groups at physooggy pH · The charge may be either "+ * or Examples of an amino acid field that provide a stabilizing effect are polyisine, poly aspartic acid, polyglutamic acid, polyarginine, polyhistidine, polyornithine, and salts of these amino acid fields Preferably, the polasmino acid salt is poly-DL-lysine hydrobromide Generally, these specific stabilizers are present in an amount from about 2.5 to about 99% by weight of total vitamin amount of the full preparation · Preferably, a polyamino acid derivative is present. in an amount of about 15 to about 60% by weight

Another class of stabilizers that can be used are choline derivatives. Examples of such stabilizers are choline chloride, choline dihndrocytrate, choline hydrogen carbonate, choline bicarbonate, tricholine citrate, choline ascorbate, choline borate, choline mononate, phosphate. choline, di / choline sulfate and mucic acid salt with di / choline. The preferred choline derivative is choline chloride. Generally, these stabilizers are present in an amount of from about 2.5 to about 99% by weight of the complete formulation prior to incorporation into the aqueous medium. Preferably, the choline derivative is present in an amount of from about 15 to about 60%.

The preparation of stabilized growth promoting preparations containing the stabilizer and the growth hormone can be carried out by ordinary mechanical mixing. When a stabilized growth promoting preparation in an injectable form is desired, the stabilizer is first dispersed in an aqueous solution which can be stirred or shaken to bring about a faster After the stabilizer dissolves, the growth promoter hormone is added after the stabilizer has dissolved · The weight percentage of the aqueous solution or water should only be sufficient to dissolve the stabilizer and the growth hormone and allow the preparation to be administered by injection. about 90% by weight is considered sufficient. While the above procedure is described as being advantageous, the order of addition can be changed and should in no way be considered as limiting the scope of the invention. In another variant, the stabilizer and the growth hormone may be mixed wet or dry to obtain a solid preparation which is particularly suitable for implantation. After the growth hormone and stabilizer have been mixed, any other additives such as buffers, salts, adjuvants etc. may be added. Since the growth hormone preparations according to the invention are intended for administration to an animal, the pH value must be physiologically acceptable for the animal and not contribute to the destabiiization of the growth promoting hormone. Generally, the pH value of the stabilized growth promoting preparations is in the range of about 4 to about 10. preferably from about 6.5 to about 8.0. The pH value of the growth promoter formulation can be adjusted with an effective amount of a pharmaceutically acceptable base or acid to obtain the desired value. Acids and bases are known to those skilled in the art.

The formulation according to the invention is for carrying out a method of promoting growth in an animal comprising administering to the animal an effective amount of a stable growth promoting preparation containing a growth promoting amount of a growth promoting hormone and a stabilizing amount of a stabilizer such as one or more of the following: a / polyol such as non-reducing sugars, sugar alcohols, sugar-derived acids, lactose, pentaerythrite, Ficoll, and water-soluble dextrans, b) an amino acid such as glycine, sarcosine, lysine or its salts, serine, arginine or its salts, betaine

N, N-dimethylglycine, aspartic acid or its salts, glutamic acid or its salts, c / poer

152,974 amino acid with side groups charged at physiological pH, and d) choline derivatives such as choline chloride, choline dihydrocitrate, choline bitartrate, choline bicarbonate, tri (choline) citrate, choline ascorbate, choline borate, choline gluconate, choline phosphate , di (choline) sulfate and di (choline) mucic acid salt.

The growth promoting preparations are intended for administration to animals, preferably pigs and cows. These preparations can be administered in various ways. 1 / In one application, the growth promoter may be in liquid form or in a solution that is administered by subcutaneous injection, or from a fluid reservoir of an implanted release device.

In another application, the stabilized growth hormone preparation is compressed into tablets or microtablets prior to placing in the reservoir of the release device. Various binders are preferably used to formulate the microtablets from the stabilized growth promoting preparation. Exemplary suitable binders include sodium bentonite, ethyl cellulose, stearic acid, calcium stearate, adipic acid, fumaric acid, glycol, polyethylenear, deacetylated chitlone, and cellulose acetate. Generally the binder is present in an amount from about 0.5 to about 10% by weight of the total amount of the solid growth promoting preparation. Preferably the binder is present in an amount of from 1 to 5% by weight.

lubricants are preferably added to the microtablet as an aid to achieving a sustained αaiα model and as a vialing aid. Such agents are, for example, the usual water-insoluble lubricants used in tabletting, such as mmgnesium stearate, sodium stearate, calcium stearate, powdered steayic acid, talc, paraffin, cocoa butter, greflt and lycopodium and their comings. Preferably, the lubricant is a stearic acid derivative such as stearates, magnesium magnesium stearate, sodium stearate and calcium stearate. Although the amount of the lubricant may vary, it is generally present in an amount from about 0.5 to about 10% by weight, and preferably in an amount from about 1 to about 5% by weight of the formulation.

The liberated growth hormone preparation, preferably micro-tabletted, can be placed in the reservoir of the liberation device. The reservoir is defined and surrounded by a wall, at least part of which is porous ^ ^ InUiέln ^ · Maeros suitable for pore formation of this part of the outer wall are generally those through which the stabilizer and the growth hormone can pass the pores. The pore size in the porous measure may be up to about 5 µm to about 250 µm, with 10 to 100 µm preferably being preferred. Suitable malerials to form these walls are naturally occurring or synthetic materials that are biologically compatible with body fluids, tissues, and organs and are substantially insoluble in the body fluids with which the device comes into contact. The use of body fluid-soluble umlerals is undesirable because the dissolution of the device wall would affect both growth hormone bleaching and the ability of the system to leave it in place for extended periods of time. It is also desirable that the mmlerota has a constant chewiness. If the porosity changes with drunkenness with time, the rate of GH release will also change with time. The materials suitable for forming the porous wall portion of the device are known in the art. techniques as shrinking multiple conjugated particles, which provides a supporting structure with interconnecting pores of microscopic size. Such malerials are known and can be produced by methods known in the art, including nuclear particle vylation, leaching, polyelectrolytic processes, ion exchange polymer reactions, and other techniques.

See, e.g., Synthetic Polymer Hembranes, R.E. Kesting, chapters 4 and 5, published by McGaw-Hill (1971), and Chembcal Reviews: Ultrafiltratkon 16, 363-455 (1934).

Microporous polyamides useful in the processing of these device parts according to the invention include microporous polyalkylenes, soft polyethylene polyethylene, microporous polycarbonates, microporous polyamides, microporous modacrylic acid copolymers or alkyl esterification * esterified esters. phenolic strained polymers, solid polyols, polyurethanes, polyamides or polybenzimidazoles. A preferred microporous malerialum is microporous polyethylene.

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The rate of diffusion of the contents of the reservoir depends on the specific material, the components in the reservoir, the size of the pores of the porous material and the dimensions of the porous material, i.e. thickness and surface area. Whilst the invention is not limited to any specific dimensions of the porous portion of the device, excellent results are achieved with a microporous polyethylene disc having a pore size of 10 to 70 µm, a diameter of 2 to 4 mm and a thickness of 1.5 to 3 mm. .

In devices which can advantageously be used in connection with the invention, usually only a part of the outer wall comprises the porous material. The remainder of the wall consists of a material that is substantially impenetrable to the growth hormone and stabilizer (s) contained in the reservoir and to body fluids in contact with the implanted or inserted device. It is expedient to characterize this outer part as much as the porous part of the wall has been described above. The material should be compatible with body fluids, tissues, organs and may contain substances that are commercially available or can be produced by methods known in the art. Suitable impenetrable materials include steel and other suitable acetates, acyl-substituted celluloses and their ralkyl derivatives, partially and fully hydrolyzed alkene-vinyl acetate copolymers, unplasticized polyvinyl acetate, cross-linked polyacrylate and polyvinyl ester alkyl polyalkrylate homopolymers and copolymers, polyvinyl ester alkyl ester copolymers and copolymers of polyvinyl ester alkyl esters. , polyvinyl fluoride, silkkon, polycarbonate, polyurethane, polyamide, polysulfones, polyimides, polyolefins, polybenzimide, ole, styrene-acrylonitrile copolymers, cross-linked polyoxyethylene, polyalkylenes, polyvinylimidazole, polyesters, chlorosyl esters and copolyethylene copolymers r vinyl, such as ethylene vinyl acetate. A preferred materiaieil is silkkon.

The invention is illustrated by the following examples.

Example I. An isotonic, physiological phosphate buffer (IPPB) with a pH value of 7.3 from sodium hydrogenphosphate / 0> 1 F phosphates / + 0.2% NaN ^ · Using this buffer, the additive solutions are prepared as shown in Table 1. About 40 mg aliquots of bovine growth hormone in 50 ml jute centrifuge tubes are then moistened at ³⁷ ° C. ^P · u ^p ływie period Tempe ratu sculpted 37 ° C / for periods ^p sector to Table 1 / with each 50 ml tried / ka oostęouje typically as nastąpaj C was first added to each tube 40 ml of IPPB / as determined the beginning of the dissolution period /. With the use of an Oaniα ultrasound device, it is effectively suspended (dissolved in a test tube). The suspension formed is then vortexed while being kept at room temperature. The corresponding tube is centrifuged for about 5 minutes at 1900 xg (defined as end of time dissolved). Almost all of the supernatant is removed with a pipette and the undissolved residue is resuspended in 20 ml of added water.

After centrifugation and removal of the supernatant, the residue in the tube is dried at room temperature with the Savant Speed Vac Concealer to a final chamber pressure of about 0.200 mbar. The deposited dry residue is removed from the tube and its weight is determined by difference with the weight of the tube.

The tables below show the results of the rrdioecepiol tests (RRA) and the hypox growth test for a number of preparations. + Sign indicates substantially 10% of the activity compared to the white standard NATO ⁿ bovine growth hormone / bGH / - * - indicates negligible activity or no activity, and * indicates an intermediate level of activity. Two symbols indicate the result in between.

In the following examples, bovine growth hormones identified as lot 7500/1 /, lot 7237C, lot 8096C and 7500 were Parlow-purified pituitary growth hormones derived from A.F. Parlow from the University of Califomia School of Mdr (^ ioe,

Harbor General Hc ^ pta !, Torrance, Celi ^ rnia.

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T a b 1 i c a 1

Γ -1 - - - - - Pre- .BGH used ¹ Appendix ¹ Quantity ,Days 1 Time 1 Characters 1 RRA Test bye bye Series 1 1 addition in , 37 ^O C 1 dissolve - and not- 1 growth 1 1 1 solution 1 waking ! admissible 1 rat 1 | 1 /% by weight / 1 / hours /% by weight 1 / hypox / 1 1 1 output 0 k - 1 - - - - - 1 1 1 «1 and. . '.bSHH ___ 1 1 1 1 ¹ 7500/1 / , Sucrose ¹ 5 and 22 1 1.6 1 39 | ♦ 2 . 7500/1 / ¹ Sucrose and 16 l 21 1 1.5 1 20 1 3 and 7500/1 / Saccharose and 50 1 twenty 1 1.3 1 (6 1 4 '7237C and Sucrose ΐ 50 1 1 15 1 l 2.5 and 5 ♦ 5 , 7237C ¹ Mannitol and 15 1 19 1 1.7 1 20 '+ / </ 6 and 7500/1 / Mannitol 1 and 15 and twenty 1 2.3 and 21 1 1 7 '7500/1 / and Glucitol '50 ' twenty Ł and 1.3 and 6 1+ 8 '7237C and Glucitol J 50 | 19 V | 1.7 and 9 l + + 9 , 7237C ¹ Glucitol and 41 AND twenty 1 1.5 ¹ 5 1 </ ♦ / 10 and 7237C ¹ Lactose 1 15 and 19 1 1.5 ¹ 12 I 1 ♦ + 11 7237C Lactose 1 52 1 twenty 1 1.5 . 5 . ♦ / </, 12 ¹ 7500/1 / , Ficoll ¹ 24 ' 21 1 | 1.5 and 26 1 13 '7237C | • Ficoll and 19 1 19 1 2.6 and 23 1 ♦ 14 . 7500 / Ί / 1 Glycerin and 51 and 19 1 0.75 1 17 + | 15 1 7237C Glycerine and 51 and 19 1 1.5 and ¹⁰ + ♦ 16 ¹ 7500/1 / , Glycine 1 5 1 22 1 1.6 . 35 1 17 '7500/1 / and Glycine '15 ' 1 22 1 | 3.0 and 19 1 18 . 7500/1 / ¹ Glycine _| 15 1 17 1 2.3 1 13 1 19 and 7500/1 / Glycine and 15 and 17 1 2.3 '14 | 1 | twenty '7237C Glycine ¹ 15 1 19 1 1.5 1 16 . + + 21 '7237C and Glycine '15 1 1 twenty 1 1 1.5 and 16 1 22 '7237C • Sarcosine e 5 1 twenty 1 1 1.7 '46 1 23 , 7237C ¹ Sarcosine and 15 and twenty 1 1.7 ¹ 31 1 24 and 7237C They are rcosine and 30 and twenty 1 1.7 1 8 1 1 25 ¹ 7237C , Sarcosine 1 30 1 twenty 1 1.7 and 12 1 26 '7237C | and Sarcosine '40 1 | twenty 1 vol 1.7 and 11 1 27 , 7237C 1 1Bro / Ipoli-DL-lysine hydride and / mass of frequent and 29 and 1 19 | 1 1.5 ¹ 16 1 1 1 1 1 1 , kowa 37 kilodal tones / 1 1 1 1 28 and 7237C and Betaine • 45 1 1 twenty 1 1 1.7 and 20 ¹ + 29 , 7237C 1 Poly-L-asparagus and 1 28 and 19 1 1.5 1 12 Ł + 1 Sodium 1nate / 14000 / 1 1 1 1 thirty 1 7237C Choline chloride 50 and 21 1 1.5 ¹ 10 | 1 | 31 ¹ 7237C 1 , L-a rg inine hydrochloride - 45 1 1 twenty 1 1 1.5 _and e | . + | 32 and 7237C Sodium D-gluconate 33 and twenty 1 1.5 7 | ♦ | 33 '8096C 1 , Sodium L-Aspartate AND ¹ 45 1 1 19 1 1 1.5 1 14 | ♦ l 34 ¹ 8096C 1 , L-arginine, L-aspartate 1 39 1 1 twenty 1 1 1.5 1 13 1 1 35 '8096C 1 1 iL-lysine and iL-aspartate and 50 1 and 1 19 1 | 1 1.5 '11 1 1+ 1 36 , 7237C 1 N, Ν-di / ethyl- 1 30 1 twenty 1 1.7 1 1 1 glycine ¹ NaCl _and 17 1 1 1 1 1 1 1 1 37 and 7237C 1 Poly-L-guuka wound ¹ sodium i / 60000 / and 17 and AND | 19 1 AND | 2.0 '16 i 1 1 + and 1 38 f 7237C »Poly-L-ornithine and ³⁰ and 19 1 1.5 '18 • + 39 , 7237C 1Poly-L-glute / and ¹ sodium nanate and 17 and AND 19 1 1 2.0 '18 1 + 1 L. i / 60000 / L - - _ - Ł 1 1

and

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Example II · Control experiments · A series of control experiments are made by repeating the procedure described in Example I · The following table 2 shows the relevant data from these control experiments · 3 As can be seen from the table 2 below, a 1PPB solution alone yields from about 30 to about 52% by weight of insoluble forms Thus, any reduction in the amount of insoluble forms below this range is assessed as an improvement. In the table below, many of the compounds used, while providing a significant reduction in the formation of insoluble forms, do not maintain the bioactivity at a level suitable for acceptable · Unexpectedly, lots of polyols! described in the prior art as being suitable to be considered as stabilizers in keeping a variety of proteins flowing, not suitable for use with growth promoting hormones.

Arrays · 2

Pre- ¹ Used parate ¹ bGH ¹ Series 1 I 1 T ......- and - --- " Appendix .Hośc, Days Godatku at 37 ° C 1 cubicle i i% b wt / j I 1 1 1 1 1 Time (Dissolving forms 1) ^and / 9 ^o dziny / weighting of ictic output i, bGH / 1 _ 1 1 1 1 1 RRA 1 . Growth test 1 rat 1 / hypox / 1 1 1 , 7503/1 / 1 1 , ΙΡΡΒ, - 1 1 15 1 ₁ ² 8 1 1 52 1 1 1 1 2 and 7237C iIPPB i - and 19 and 2,3 and 39 1 ♦ 3 • 7500/1 / Glucose 1 15 23 3.2 «14 1 < 1 < 4 1 7237C Gtotosis ¹ 51 ¹ 15 1 2.3 1 2 1 - / </ ¹ < 5 , 7237C (Hannoza 51 '19 l ^1.7 '3 1 | - 1 6 and 7237C and Fructose | 50 and 19 and li7 and 10 1 - 1 7 and 7237C Maatosis and 28 19 and 2.6 and 10 1 < 1 8 1 7237C 1 1 1 Poly-1 glycol 20 • tyenic 1 / part weight and stoczkowa 400, / ¹ 18 1 1 1 '2.5 1 1 1 1 36 1 1 1 1 1 1 1 ♦ 1 ♦ 1 1 1 9 1 75 (03/1 / 1 Ethyl-1 glycol 50 1 new i 1 21 1 1 1 * 5 1 • 83 1 1 1 1 1 10 1 7237C 1 'Glycol et ^ e- ^{1 50} 1 new 1 1 19 1 1 2.6 1 1 80 1 1 1 1 1 w- -1 - - - - X ----- - ♦ I- - - - - u - - - - - J.

Example III. Two experiments are carried out using Pearl bGH dry mixed with a stabilizer to simulate the implant conditions Wetting experiments are carried out as follows · About 50 ml tubes are filled with dry hormone and IPPB additive are incubated at 37 ° C for 19 days. After incubation, the samples are analyzed as in Example 1. Table 3 below shows the corresponding data from these experiments with dry additive. The insoluble forms are not as distinctly different as in Example 1, where the additive was first dissolved in the solution. Thus, simulated implant experiments demonstrate the effectiveness of the method of the invention.

Pre- ' parat 1 1 Used bGH Series 1 Appendix 1 1 1 1 1 1 Quantity bGH / mg / 1 Hość 1 and in addition and in development (creation · ^ -8 ^- Quantity IPPB / ^ 1 / 1 Days ^r 37 ^O C 1 1 1 Characters and insoluble, in ^^, / output bUd / r ------- and RRA 1 1 1 r - - t - - - - - Test growth 1 rat and / liypoM / 1 1 1 7237C • Pentai erythritol and and 40 1 1 47 1 1 100 19 1 and 27 1 1 * 1 1 1 2 ' 7237C L-serine 40 1 41 J. 150 • 19 • 19 1 * 1 1 . - - J . L -___ 1 1-. - - - J L - - L - - _ - - - - L - - 1

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Example IV. The following experiments are carried out to demonstrate the effectiveness of the porcine growth hormone i implant device. the stabilizer, compared to the implant device, contains only porcine growth hormone. A Teflon disk is inserted into each of the 20 hollow silicon tubes measuring 2 x 0.7 cm in order that each tube has two chambers of equal size. To each compartment of 10 tubes, hereinafter referred to as the tubes A, introduced into 20 mg of porcine growth hormone Parlow / Series 4556 /. · The tubes are sealed at each end mikrlporooatym disk pllietylenowym a pore size of 70 pm ₉ remaining 10 tubes, hereinafter referred to as tubes B , is prepared in the same way except that Parlov's porcine growth hormone is a mixture of series 4654 and 4673. No significant difference was detected between growth hormone in tubes A and B. In addition to growth hormones, tubes B additionally contain 80 mg of sucrose which homogeneously oymiθszanl with growth hormone before insertion into the tubes. Each tube is implanted subcutaneously behind the pig's ear. Then, the presence of growth hormone in each pig is determined by analyzing its level in the blood serum. Growth hormone is measured in units expressed as ng / ml.

Table 4 below shows the mean amount of porcine growth hormone in ng / ml of blood serum versus the time / implaanaaja / in which the blood serum sample was collected

The figure shows the data from Table 2 in the form of a graph. Additionally, a line was drawn showing the minimum desired level of growth hormone / 8 ng / ml /

These experiments show that stable growth hormone preparations provide higher serum levels of growth hormone than growth hormone alone.

Table 4

Effect of release systems on the level of growth hormone in the blood serum.

1 1 1 l_ d Day I ¹ , Layout for i ¹ A ___________ 1 B ¹ 1 1 ; ^{2 * * * * *} . ⁸ 1 1 35.8 t 1 1 2 , 3.4 1 45.0 i 1 3 and 3.8 1 | 23.2 i 1 5 ¹ 5.1 1 25.4 ¹ 1 7 '4.6 1 7 ^, 0 ' 1 | 9 ¹ 6 ^, 9 1 9.3, 1 11 and 4.4 1 AND 2.4 and 1 13 ¹ 2.7 1 1 2.0 ¹ The above mean values were corrected with using the co-warranty with the Intermediate hormone levels growth on the day before implantation and on 0. Reservations p a t e o w e

1. Stabililowaoy growth stimulating preparation, characterized in that

Claims (15)

1. A stabilized growth promoting preparation, characterized in that it contains a direct stimulating hormone and a stabilizer in a stabilizing amount, such as one or more of the following compounds: a) polyol such as non-reducing sugars, sugar-derived alcohols, sugar-derived acids, lactose, pentaerythrite, water-soluble dextrans and Ficoll, b) amino acid such as glycine, sarcosine, lysine or its salts, serine, arginine or its salts, betaine, N, N-dimmtyllicine, aspartic acid or its salts, glutamic acid or its salts, c) polymer a ^ ^ noko ^ sj with a pendant group at a physiological pH, and d) a choline derivative such as choline chloride. 152 S74 choline dihydrogen citrate, choline bitartrate, choline bicarbonate, tri (choline) citrate, choline ascorbate, choline borate, choline gluconate, choline phosphate, di / choline sulphate / and mucic acid salt with di / choline / 2 Stabilized growth promoting preparation according to shot, characterized in that the growth promoting hormone comprises a hormone such as sheep, porcine, equine, avian, human and bovine growth stimulating hormone 3 A stabilized growth promoting preparation according to claim 1, characterized in that the growth promoting hormone is bovine growth promoting hormone. 4. A stabilized growth promoting preparation according to claim 1, characterized in that the polyol is present in an amount from about 2.5 to about 99% by weight of the stabilized growth promoting preparation. 5. A stabilized growth promoting formulation according to claim 1, characterized in that the polyol is xylitol. 6 A stabilized growth promoting preparation according to claim 1, characterized by having an amino acid in an amount of about 2.5 to about 99% by weight of the stabilized growth promoting preparation. 7. A stabilized growth promoting preparation according to claim 1, characterized in that the amino acid is sarcosine 8 · Stabilized. the growth promoting preparation according to Claim 1, wherein the amino acid polymer is present in an amount from about 2.5 to about 99% by weight of the stabilized growth promoting preparation. 9. A stable growth promoting preparation according to Claim 1, characterized in that the amino acid polymer is a polyamino acid such as polysine, poly aspartic acid, poly glutamic acid, polyarginine, polyhistidine, polyormitin and salts of these amino acid fields. 10. SiabiiZzoother growth promoting preparation according to claim 9, characterized in that the polyamino acid salt is poly-DL-lysine hydrobromide. 11 · The stable growth promoting preparation according to Claim 1, characterized in that the choline derivative is present in an amount from about 2.5 to about 99% by weight of the stabilized growth promoting preparation. 12. A stabilized growth promoting preparation according to Claim 1, characterized in that the choline derivative is choline chloride. 13. A stabilized growth promoting preparation according to Claim 1, characterized in that the preparation is in a dry state. 14. Stabilizowain growth promoting preparation according to claim 1, characterized in that the preparation is dispersed in an aqueous solution. 15. A stabilized growth promoting formulation as claimed in Claim 1 wherein the formulation is dissolved in an aqueous solution.