CN1056537C - A pharmaceutical formulation - Google Patents

Abstract

A pharmaceutical preparation comprising a growth hormone and histidine or a derivative of histidine as additive or buffering substance shows a very high stability against deamidation, oxidation and cleavage of peptide bonds. The stability of the product allows for the storing and shipment thereof in a lyophilized state or in the form of a dissolved or re-dissolved preparation at ambient temperature. Crystallization of growth hormone in the presence of histidine or a derivative thereof gives rise to a higher yield of crystals having a higher purity than known methods.

Description

A kind of pharmaceutical preparation

The present invention relates to a kind of stabilised pharmaceutical that contains growth hormone, prepare the method for said preparation, contain histidine or each derivant of one the growth hormone crystal, prepare the purposes of this crystalline method and histidine or derivatives thereof stable growth hormone preparation.

The growth hormone (GH) that comes from people and common domestic animal is about 191 amino acid whose protein, and synthetic justacrine is in the frontal lobe of hypophysis.The human growth hormone is made up of 191 aminoacid.

Growth hormone is a kind of important hormone that not only relates to the adjusting of body growth but also relate to protein, saccharide and lipid metabolism adjusting.The main effect of growth hormone is to promote growth.

The airframe systems that influenced by growth hormone comprises skeleton, connective tissue, muscle and internal organs such as liver, intestinal and kidney.

Caused in present recombinant technique and growth hormone gene clone's development before the industrial-scale production as human growth hormone (hGH) and Met-hGH, the human growth hormone can only obtain by extracting in people's corpse hypophysis.This very limited supply of growth hormone has limited in childhood and adolescence and has promoted longitudinal growth with it and treat dwarfism, although even ad hoc proposal it is used for the treatment of treatment, wound healing, malnutrition, knitting, osteoporosis, dispersivity gastrorrhagia and the pseudarthrosis of short-and slight in figure (because growth hormone deficiency, normal short-and slight in figure and Turner's syndrome), the shortage that becomes the human growth hormone, infertility, burn.

And growth hormone has been recommended to be used to increase the speed of growth of domestic animal or reduction and to butcher ratio for human consumption's animal body fat.

The pharmaceutical preparation of growth hormone is unsettled often.Particularly in growth hormone solution, can produce the form of catabolite such as deacylated tRNA amine or sulphoxylic acid product and dimer or polymer.

The main degradation reaction of hGH is 1) direct hydrolysis or form not commensurability L-asp-hGH through ring-type succinamide intermediate deamidate, L-iso-asp-hGH, D-asp-hGH and D-iso-asp-hGH, (list of references 1-3), 2) methionine residue (list of references 4-9) on oxidation 14 and 125, and 3) the cracking peptide bond.

Deacylated tRNA amine is easy to occur on 149 the Asn especially.

HGH is oxidized quite easily on 14 and 125, particularly in solution (4-8).

HGH is oxidized to sulfoxide in solution generally be owing to be dissolved with oxygen in the said preparation.The dissolubility of distillation water oxygen is about 200 μ M (9).Because the concentration of hGH is that 1.3mg/ml is equivalent to 60nm hGH in containing the 4IU/ml preparation, under general holding conditions, oxygen will exist to surpass the about 3000 times amount of oxidation hGH chemistry consumption.It is very difficult attempting to address this problem to the method for solution degasification before being used in gland and packing said preparation.

At present, do not think that these catabolites have toxicity or changed physiologically active or had the characteristic of bind receptor.But there are indications to compare the effect that reduces the sulfoxide conformational stability is arranged with original hGH.

In order to develop the preparation of a kind of stable dissolved hGH, it is very important understanding the method that sulfoxide forms speed and controlled oxidation.

Degradation kinetics depends on temperature, PH and various additives or excipient in the hGH preparation.

Because the unstability of growth hormone is prepared so that make degraded reduce to minimum again its lyophilizing and when storing up to use with lyophilized form under 4 ℃ of temperature at present again.

Again prepare freeze dried hGH pharmaceutical preparation by the patient at present, nearly storing at low temperatures with solution in operating period of 14 days, in about 4 ℃ refrigerator, during this period some degraded will take place usually then.

And, concerning the patient the process for preparation again of lyophilizing growth hormone often the difficulty.

Therefore, preferably prepare growth hormone at present before use as far as possible lately again and preferably store and transport said preparation with lyophilised state, from the manufacturer to the pharmacy, be easy at controllable low temperature it to be had reach the expiry date in 2 years as 4 ℃ of operation management said preparations.

Yet the expansion with the said preparation application of promoting the use of of pen container system (pen system) self treatment requires can not arrive under " enough " freezing conditions at those have stable formulation in the sufficiently long time concerning the end user.

Preferably in cartridge case uses effect duration, with lyophilised state place about one month again in the pen container device form with preparation again placed one month, said preparation should be still stable concerning the end user.

Thereby, needing a kind of more stable somatotropin preparation, it can stablize a period of time and stablize another section service time with the solution form under higher temperature with lyophilised state under the higher temperature.When the use of growth hormone being moved on to from the clinic when can not get the individual family that the aforesaid suitable quilt of depositing treats, this stability is very important.

And the change of using the pen container device to take the mode of growth hormone requires a kind ofly to stablize dissolved somatotropin preparation so that the patient is easy to operate.Can produce and a kind ofly stablize dissolved somatotropin preparation and be convenient to use in the cartridge case mode that is installed in the pen container device that patient uses, the patient can avoid preparing again said preparation and thereby not need lyophilized formulations, prepares suitable carrier and sterilize the again necessary technology of formulated and the sterilizing installation of usefulness again.

For reasons of safety, also need to avoid before using said preparation, to prepare this lyophilized formulations again.

And it also is useful avoiding step of freeze drying in the production of somatotropin preparation.Lyophilizing be one consuming time and the consumption money step and because of the limited capacity of freezer dryer also often be a link that influences production procedure aborning.

So, in order to make dissolved hGH preparation, in storage period with reach in operating period of one month stablely, just need to reduce the speed of degradation process.

The trial of stablizing hGH is in the past preventing not success fully aspect the dimeric formation, and the relevant issues that form with dimer are as at Becker, G.W. (record to some extent among the Biotechnology and Applied Bio-chemistry 9,478 (1987).

The stabilised pharmaceutical preparation of a kind of human growth hormone of containing, glycine and mannitol is disclosed in open WO89/09614 of international monopoly and the big Leah patent application 30771/89 difficult to understand.This preparation shows good stable in conventional preparation process neutralization with lyophilizing attitude lay up period and in the operating period after preparing again.

Disclosed european patent application 303746 discloses with various stabilizing agents stablizes animal growth hormone with the formation that reduces insoluble matter and remain on dissolubility in the aqueous environment, and this class stabilizing agent comprises specific polyhydric alcohol, aminoacid, has the aminoacid polymer and the choline salt of charged side group when the physiology pH value.Polyhydric alcohol is selected from non-reducing saccharide, sugar alcohols, sugar-acids, tetramethylolmethane, lactose, water solublity dextran class and Ficoll; Aminoacid is selected from glycine, sarcosine, lysine or its salt, serine, arginine or its salt, betanin, N, N-dimethyl-glycine, Radix Asparagi ammonia or its salt, glutamic acid or its salt; The aminoacid polymer that has charged side group at the physiology pH value is selected from polylysine, poly-aspartate, polyglutamic acid, poly arginine, polyhistidyl, poly ornithine and salt thereof; And the choline derivant is selected from choline chloride, dihydrogen citrate choline, hydrogen tartrate choline, bicarbonate choline, citric acid three cholines, ascorbic acid choline, choline borate, choline gluconate, phosphocholine, sulphuric acid two cholines and glactaric acid two alkali (dicholine mucate).

EP 374120 discloses a kind of stable somatotropin preparation that contains resiliency polyhydric alcohol excipient, and this excipient contains the polyhydric alcohol with three hydroxyls and can make pH reach the buffer agent that keeps the scope of growth hormone biological activity at long enough in the time.Mention that histidine is as the polyhydric alcohol buffer agent with three hydroxyls.

Be surprised to find now the histidine or derivatives thereof that only contains that adds 0.1 to 12mg histidine or derivatives thereof with every mg growth hormone and had high resistance deacylated tRNA amine, the cracked stability of oxidation and peptide bond as the human growth hormone's of additives or cushion preparation.The stability of this product allows with lyophilised state or has dissolved or dissolved dosage form to preserve and transport.

EP303746 has mentioned polyhistidyl as the potential stabilizing agent of animal growth hormone, can not stablize animal growth hormone or human growth hormone but indicate.

The growth hormone that EP374120 has instructed histidine monohydrochloride to can be used as buffer agent buffering to have the polyhydric alcohol of three hydroxyls to improve to contain high concentration and a kind of polyhydric alcohol are as the stability of the somatotropin preparation of the solution form of stabilizing agent.Must add histidine monohydrochloride with about 3% (solution weight) solution of 0.15M histidine monohydrochloride concentration (be equivalent to～) consumption.EP374120 has pointed out that also independent histidine can not bring somatotropin preparation chemistry and physical stability.

Preparation of the present invention can be the form of freeze-dried powder, prepares again with conventional carrier such as distilled water or water for injection then, and perhaps can be to contain crystalline solution of growth hormone or form of suspension.This class is carried part can comprise conventional antiseptic such as metacresol and benzyl alcohol.

The preferred embodiment of the invention is the form with the human growth hormone's pharmaceutical preparation that contains the histidine or derivatives thereof of the buffered resiliency growth hormone of histidine buffer aqueous solution form.This preparation is that a kind of backup form and form that can aqueous solution are stored and transported and without any significant degraded.L-histidine pKA is 6.0 thereby suits as buffer agent at PH6.5.

Think that the histidine preparation of PH6.5 approximately stablized 50 days under 25 ℃ of temperature.

Another embodiment preferred of the present invention is the human growth hormone's pharmaceutical dosage forms that contains the histidine or derivatives thereof with the crystalline water slurry form of the buffered resiliency growth hormone of histidine buffer.Said preparation be very stable and store with standby form with transport during growth hormone can be remained on crystal mutually in and produce a lower degraded trend.Said preparation is similar a kind of dissolving preparation when injection, that is, the human growth hormone continues to discharge.

For stable, the pH value of solution or suspension preparation preferably is transferred between the 2-9.PH5 to 8 particularly the preparation of PH6 to 7 for more preferably.

For reaching stable effect, the concentration of histidine is preferably from 1mM to 100mM.More preferably, added histidine concentrations is 2 to 20mM, is preferably 5 to 15mM.

Adding 10% ethanol or 5% methanol can make deacylated tRNA amine reduce more than 20%.

Preparation of the present invention also can be freeze dried powdery or pie form, it contains growth hormone or growth hormone derivatives and every mg growth hormone or growth hormone derivatives is the histidine or derivatives thereof of 0.1 to 12mg histidine or derivatives thereof consumption and the extender that lyophilizing is used, and this extender is selected from sugar alcohols and disaccharide and composition thereof.The preferred mannitol of sugar alcohol.

The lyophilized formulations of the present invention that contains sucrose is preferred, this is because it has a very high stability, and the preparation that contains sucrose and mannitol is particularly preferred, it very high stability and extraordinary processing characteristics are combined and obtain easily molten and the dissolving back in solution over a long time in highly stable firm freeze-drying prods.Further the preferred preparation of the present invention is to contain mannitol and the trehalose preparation as freezing extender.The preparation of the present invention that contains mannitol and disaccharide contains two kinds of components (by weight) of about equivalent.

Cane sugar content can change within a large range in the preparation of the present invention.The ratio of growth hormone and sucrose can change (by weight) between 0.005 to 1.5.Therefore, the sucrose consumption can be every mg growth hormone 0.67 to 200mg, and preferred every mg growth hormone is between 1.1 to 50mg.

Freeze dried hGH can not produce any problem in histidine buffer.The dissolving back is compared with phosphoric acid buffer agent again, and deacylated tRNA amine speed still reduces by 20%.

Pharmaceutical preparation of the present invention can further contain salt commonly used to be convenient to its preparation (as lyophilizing or preparation again).

According to the present invention, another method of stable growth hormone is that the crystal that forms growth hormone resists degraded to obtain the better protection effect.The somatotropin preparation that has been surprised to find that the crystal form that contains histidine can satisfy above mentioned requirement.Again the GH preparation of preparing with conventional method before the crystal of dried forms can directly be used as and use.

So, the present invention also relates to contain the crystal of histidine or derivatives thereof and cationic growth hormone of a kind of organic or inorganic or growth hormone derivatives.Shown that this crystalline performance is better than the performance that reaches with above-mentioned preparation.

Although can easily obtain the crystallization of capacity, the not successful GH crystallization of report up to now.Reported crystallite in various sources, or amorphous material: Jones etc., Biotechnology (1987) 5,499-500; Wilhelmi etc., J.Biol..Chem. (1984) 176,735-745; Clarkson etc., J.Mol.Biol. (1988) 208, and 719,721; With Bell etc., J.Biol.Chem. (1985) 260; 8520-8525.

The drop method of dangling is to attempt the used prevailing method of crystallization GH.Obviously owing to the inhomogeneities of somatotropin preparation, crystalline size of this that reported and shape also are changed significantly.Reported maximum crystal by (1987) such as Jones.They have used the Polyethylene Glycol 3500 and the β-octyl group glycoside mixture of neutral pH value in the test of their success.Clarkson etc. (1989) have reported that use lower alcohol and acetone can produce 0.001 to 0.005mm ³Difform crystal.Yet do not have a kind of known method to be suitable for commercially producing the GH crystallization, this is because need several thoughtful 1 year production cycles.

In the mixture of divalent ion and a kind of oil, prepared the bovine growth hormone of using for the veterinary (EP343696).In the presence of lipoid with ZnCL ₂Be added to and be prepared into uncertain granule in the growth hormone of cattle or pig and form a kind of long-acting delivery formulations.With this method this growth hormone is dispersed in and makes each growth hormone molecule surround 1 to 4 Zn molecule in the carrier.This solution is to prepare under degeneration solute (1 to 4M carbamide) at variable concentrations and high pH value (9.5) condition.Repeat this process with hGH and shown that this method can not produce crystal.

From document, known and in the crystallization process of insulin, exist bivalent cation not only fabulous orientation can be arranged in analysis, and can improve crystalline physical condition (as referring to United States Patent (USP) 2174862).But growth hormone is bigger more than three times and diverse conformation arranged than insulin.Now be surprised to find that adding cation in the solution that contains hGH and histidine or derivatives thereof comes the stable uniform growth hormone crystallization of high productivity production to become possibility.And the generation high-quality hGH crystal required time cycle shortens relatively.

Another aspect of the present invention is preparation growth hormone and histidine or the crystalline method of histidine derivative, and the step that it comprises has:

A) be made into the solution of growth hormone or growth hormone derivatives and add the histidine or derivatives thereof in a kind of solvent, it is 5 to 8 that reuse hydrochloric acid is suitably regulated pH value.

B) add the organic or inorganic cation.

C) about 0 ℃ make under about 30 ℃ of temperature solution crystallization and

D) separate formed crystal with known method.

Have been found that when histidine or its a kind of derivant exist crystallization hGH obtains the crystal than the high yield of the bigger purer more uniform crystal form of crystallization when preparation hGH preparation phosphate buffer commonly used exists.Therefore, this crystalline separation and purification become more convenient.

When in the presence of histidine, carrying out crystallization crystal yield than before the preparation crystallization yields increase about 20%.

Raw material (growth hormone) can be the concentrate that directly directly obtains or be dissolved in the solvent and be adjusted to certain density lyophilized formulations commonly used that this concentration is preferably greater than 0.1mg/ml from fermentation liquid, preferred concentration is 4 to 7mg/ml, and optium concentration is 6mg/ml.The solvent that is suitable in the step a) is a kind of water-containing buffering liquid such as phosphate buffer or histidine buffering liquid.

Allow crystallization to carry out under a certain temperature 1 to 120 hour, be preferably 5 to 72 hours, the best is 20 to 48 hours.Preferred 4 to 25 ℃ of this temperature.

PH value in the step a) is generally 5.0 to 7.5, and is preferred 5.0 to 6.8, and more preferably 5.8 to 6.5, be preferably 6.0 to 6.3.

The concentration of histidine or histidine derivative can change between 5-25mM in the step a), and preferred 5-15mM is so that form the crystal with aforesaid suitable size and performance.

Bivalent cation is preferred and has found inorganic cation such as Zn ⁺⁺Be particularly suitable for forming fast stable GH crystal.Also can use cationic mixture.

Should add this cation so that the defined crystalline amount that fast and effeciently formation is good to be provided.Add the cation amount the upper limit be to cause the non-specific sedimentary amount of a large amount of noncrystalline things.

Use Zn ⁺⁺The time, suitable concentration generally is about 0.2 to 10mol Zn ⁺⁺/ molGH.Yet, if this crystallization reaction mixture contains buffer agent or other chemical compound that can combine (as with form complexed) with this cation, for compensating this cation that needs to add higher concentration in the crystallization process that is combined in.

Preferably can cause that the crystal formation consumption of GH uses Zn ⁺⁺, this crystalline Zn ⁺⁺Be about 0.2 to 10 with the GH mol ratio, more preferably about being 0.5 to 5, the best is about 0.5 to about 2.

When using other inorganic cations, concentration can 0.5 and 10mol cation/molGH between change.

The preferred embodiment of the invention is the mixture that adds a kind of organic solvent or organic solvent in step a).

The suitable organic solvent that adds for crystallization can be selected from aliphatic series, alicyclic ring or the aromatic alcohol of short chain and ketone such as methanol, ethanol, 1-and 2-propanol, Hexalin, acetone and phenol or metacresol.Preferred organic is ethanol and acetone, and the best is an ethanol.

This solution can be by adding less measured defined hexagonal or needle-like hGH crystal is put into crystal seed, but will add crystal seed preferably.

The concentration of this organic solvent can be from 0.1 to 50%V/V, and preferably from 0.1 to 30%, be preferably 0.1 to 20%, more preferably 5 to 15%, the best is 6 to 12%V/V.

Owing to form crystallization in the solution of larger volume, this method can be used as described growth hormone following current technology fast and effectively.

When using ethanol as organic solvent, its suitable concn is 0.1 to 20%, and is preferred 5 to 15%, and the best is 6 to 12% (V/V).

Formed crystallization can separate with conventional method, and as centrifugal or filtration, the organic solvent of trace is removed in washing and any lyophilizing.

Crystalline big young pathbreaker is depended on Zn used in this process ⁺⁺Ratio and the choice of Solvent of using and consumption with GH.

HGH crystal of the present invention shows to have the biological effect that is similar to classical dissolubility hGH in vitro tests.So new GH crystal can be applicable to and the commercial identical symptom of hGH preparation of buying.

Another aspect of the present invention relates to the application of histidine or derivatives thereof in preparation stable growth hormone preparation.

Pharmaceutical preparation of the present invention is preferably the form of unit dose, and each unit dose contains 4IU to the 100IU growth hormone.

" growth hormone " can be the growth hormone that derives from as any biology of bird, cattle, horse, people, sheep, pig, salmon, true trout or tuna in this article, the growth hormone of preferred cattle, people or pig, and the human growth hormone is best.According to the used growth hormone of the present invention can be isolating spontaneous growth hormone from natural resources, as, obtain with conventional method extraction hypophysis, or the growth hormone of producing with recombinant technique, at Biotech and Bioeng.36,1-11 (1990) is described as E.B.Jensen and S.Carlsen." growth hormone derivatives " can be a kind of growth hormone of clipped form, and wherein one or more amino acid residues are deleted; A kind of analog of growth hormone, wherein the one or more amino acid residues in the natural molecule are replaced by another amino acid residue (amino acid residue of preferred natural generation), as long as this replacement acts on as antigenicity or reduction effect without any paying; Or a kind of derivant of growth hormone, as the deacylated tRNA amine or the sulfoxide form of this growth hormone or have N or C-is terminal prolongs as Met-hGH the form of Met-Glu-Ala-Glu-hGH or Ala-Glu-hGH.Preferred growth hormone is hGH.

Be the object of the invention, term " histidine derivative " is meant amide-type and esters such as the methyl or the ethyl ester of histidine, dipeptides such as His-Gly, His-Ala, His-Leu, His-Lys, His-Ser and His-Phe, and the analog of histidine or derivant such as imidazoles, deaminizating-histidine or polyhistidyl.Cause for convenience, the content of histidine or derivatives thereof is calculated and is used the molal weight of histidine itself in the preparation of the present invention.

Use term " salt " to be expressed as the processing that makes things convenient for pharmaceutical preparation or to prepare added additives again, it comprises alkali metal, alkaline-earth metal or the ammonium salt of conventional additives such as organic acid such as citric acid, tartaric acid or acetic acid, as alkali metal, alkaline-earth metal or the ammonium salt of sodium citrate, sodium tartrate or sodium acetate or mineral acid example hydrochloric acid, as sodium chloride.

This " high stability " herein is to approve when preparation is stablized than the conventional formulation that contains phosphate buffer.

" sugar alcohol " can be mannitol, xylitol, red bright alcohol, threitol, sorbitol or glycerol.

" disaccharide " is meant the disaccharide of natural generation herein, as, sucrose, trehalose, maltose, lactose, agarose, turanose, laminaribiose, dextrinose, gentiobiose or close disaccharide.

Used solvent can be water, alcohols such as ethanol, normal propyl alcohol or isopropyl alcohol, butanols or its mixture in the preparation of the present invention.This solvent can contain antiseptic such as metacresol or benzyl alcohol.

With reference to the accompanying drawings the present invention can be described in more detail:

Fig. 1. shown not add the prepared crystalline photo of histidine,

Fig. 2. shown the crystalline photo of hGH of the present invention.

In following explanation embodiments of the invention, explained the present invention in more detail.They are not considered to limit the scope of the present invention by the claims definition.Embodiment 1

The reduction effect of deacylated tRNA amine

Compare the deacylated tRNA amine speed that detects with identical hGH preparation in the phosphate buffer at identical pH value containing the hGH preparation of 4IU and 12IU under 37 ℃ of temperature in to histidine buffering liquid at PH6.5.

The 15.5mg histidine being dissolved in the deionized water that contains 0.9% benzyl alcohol of 10ml and adding 0.1N hydrochloric acid is 6.5 to make the 10mM histidine buffering liquid to PH, again 13.3mg hGH is dissolved in the hGH preparation that contains 4IU for preparing compositions A in the 10mM histidine buffering liquid of 10ml.40mg hGH is dissolved in making in the aforesaid same buffer contains the 12IU preparation.

The phosphoric acid that is dissolved in the 17.8mg sodium hydrogen phosphate in the deionized water that 10ml contains 0.9% (V/V) benzyl alcohol and adds 0.1N is 6.5 to make the sodium hydrogen phosphate buffer of 10mM to pH value, and dissolving 13.3mg hGH makes the hGH preparation that contains 4IU of compositions B in this buffer of 10ml.40mg hGH is dissolved in makes the preparation that contains 12IU in the aforesaid same buffer.

Compositions A:

10mM histidine (His)

0.9% benzyl alcohol

It is 6.5 that HCl adds to PH

Compositions B:

The 10mM sodium hydrogen phosphate

0.9% benzyl alcohol

It is 6.5 that phosphoric acid adds to PH

In preparation back again at once and after 37 ℃ are placed 7 days, detect the content of deacylated tRNA amine-hGH in the said preparation with IE-HPLG.The results are shown in the following table 1.

Table 1. deacylated tRNA amine

Preparation deacylated tRNA amine % buffer A 4IU/ml 1.7 beginning 12IU/ml 2.1 buffer A 4IU/ml are (37 ℃) 12IU/ml 10.4 buffer B 4IU/ml 1.8 beginning 12IU/ml 2.3 buffer B 4IU/ml (37 ℃) 12IU/ml 14.9 after 16.97 days after 10.17 days

Last table shows at 37 ℃ to be compared with phosphate buffer, and the hGH desamidation significantly reduces in the histidine buffering liquid.

Embodiment 2

The reduction effect of deacylated tRNA amine in the presence of the histidine or derivatives thereof

The hGH preparation deacylated tRNA amine speed that when down detecting 5mM, 10mM and 100mM histidine buffering liquid PH7.3 and PH6.5 for 25 ℃, contains 6IU hGH, and compare with identical PH 8mM phosphate buffer.And, tested histidine derivative His-Gly, His-Ala, His-Leu, His-Lys, His-Phe, His-Ser, histidine methyl ester, histidinol, imidazoles, imidazoles-4-7 acid and histamine.

Hydrochloric acid to the described pH value that is dissolved in the histidine of 7.8mg, 15.5mg and 155.2mg in the deionized water of benzyl alcohol that 10ml contains 0.9% (V/V) respectively and adds 0.1N makes the histidine buffering liquid of desired concn, 20mg hGH is dissolved in this histidine buffering liquid of 10ml making the hGH preparation again.

Depositing following table 2 described hGH preparations under 25 ℃ and after 14 and 30 days, analyzing the content of deacylated tRNA amine in the said preparation with IE-HPLC.The results are shown in the following table 2.

Table 2.

Measure with IE-HPLC by the performance of preparation with in the time that 25 ℃ of following solution are placed

The content of deacylated tRNA amine hGH:

Preparation ( ^*) the desamidization compound of 25 ℃ of formation

14 days 30 days 5mM His PH6.5 6.5 9.15mM His PH7.3 11.0 17.410mM His PH6.5 6.8 9.710mM His PH7.3 11.3 16.6100mM His PH6.5 9.8 15.2100mM His PH7.3 19.3 28.88mM sodium hydrogen phosphate PH6.5 7.8 10.88mM sodium hydrogen phosphate PH7.3 15.2 20.38mM sodium hydrogen phosphate PH6.5 9.4 13.20.3% metacresol 10mM Asp; PH6.5 21.7 nd10mM Glu; PH6.5 14.8 nd10mM His-Gly, PH6.2 5.6 8.110mM His-Ala PH6.5 6.2 8.510mM His-Leu PH6.5 8.8 12.310mM His-Lys PH6.5 8.6 12.010mM His-Phe PH6.5 7.5 11.310mM His-Ser PH6.3 22.0 nd10mM histidine methyl ester PH6.5 4.6 5.210mM histidinol PH6.5 27.4 nd10mM imidazoles PH6.5 9.2 12.210mM imidazoles-4-acetic acid PH6.5 10.3 14.210mM histamine PH6.5 9.8 12.2*: except prescription 9, all contain 0.9% phenmethylol

The content of deacylated tRNA amine-hGH is 2.1% in the starting material

Last table 2 shows that pH value 6.5 compares with adding phosphate buffer with 7.3 times, and the desamidation of hGH has descended about 20% when adding histidine.And pH value is reduced to pH value 6.5 from the conventional pH value 7.3 of commercial hGH preparation itself can make deacylated tRNA amine speed descend 50%.

As if under experiment condition, histidinol does not make preparation stabilization, and the histidine that adds than big consumption does not only have increase to reduce required effect on the contrary.

Use histidine analog such as imidazoles, histamine and imidazoles-4-acetic acid and histidine methyl ester can obtain similar result, promptly after placing 30 days under 25 ℃, only have 3.1% deacylated tRNA amine-hGH to form, make the effect duration of said preparation can reach 3-4 month.

Compare with phosphoric acid when PH6.5, adding Asp or Glu has increased deacylated tRNA amine speed.

The dipeptides that adds the His-X type demonstrates His-Gly and His-Ala is played extremely long-pending effect, and His-Ser reduces the effect of stablizing deacylated tRNA amine.

Above result shows the speed that reduces pH value and can reduce deacylated tRNA amine with low concentration (preferably about 5mM-10mM) adding histidine.Reducing pH value and substituting phosphate buffer with histidine to make deacylated tRNA amine speed reduce more than 50%.

Use metacresol or benzyl alcohol as if deacylated tRNA amine speed not to be influenced as antiseptic.

Compare with phosphoric acid, histidine can reduce cracked generation (hydrolysis of peptide bond) when PH6.5.

Embodiment 3

Reduce the effect that sulfoxide forms

Detection is to the dependency of pH value and buffer type.

The dependency of pH value:

Method:

The commercial hGH preparation that contains bicarbonate, glycine and mannitol+0.9% benzyl alcohol (Norditropin, ^12IU/ml) with the hydrochloric acid of 0.1N with PH be adjusted to 8.3,8.07.5,7.0,6.5 and 6.0, and place samples at 37 ℃.After 0,7 and 14 day, analyze with RP-HPLC.The results are shown in the following table 3.

Table 3

The formation of sulfoxide

Sample temperature (℃) placement natural law sulfoxide (%)

PH 8.37 - 0 1.0

PH 8.37 37 7 9.0

PH 8.04 37 7 8.7

PH 7.52 37 7 8.3

PH 7.01 27 7 7.7

PH 6.52 37 7 6.5

PH 6.02 37 7 4.8

PH 8.37 37 14 14.9

PH 8.04 37 14 14.5

PH 7.52 37 14 14.0

PH 7.01 37 14 12.9

PH 6.52 37 14 11.1

PH 6.02 37 14 7.7

To recognize, when pH value when 8.4 drop to 6.0 the hGH formation of sulfoxide also be lowered.

The type of buffer, PH:

The ratio of the B-hGH preparation that contains the 12mg/ml distilled water with 1+10 is the concentration of 15mM and can adds or not add other additives with various buffer dilutions.Place sample down at 25 ℃, and after 10 and 34 days, analyze with RP-HPLC.The analysis result of RP-HPLC and dispensable additives are shown in the following table 4.

Table 4

The formation of sulfoxide

Buffer solution ph additives sulfoxideization

10 days 34 days

％

Phosphate 7.3-1.9 5.5

Histidine 7.3-0.9 2.4

Histidine 6.9-0.9 2.0

Histidine 6.5-0.8 1.9

Histidine 7.3 18mM Met 0.8 2.0

Histidine 7.3 18mM Cys 2.4 2.9

Histidine 7.3 0.42mM toc.1.1 3.0

Histidine 7.3 9% ethanol 1.3 4.2

Histidine 7.3 18mM asc. 41 nd

Histidine 7.3 0.8%NaCl 1.3 3.5Toc: vitamin E, asc: ascorbic acid.Compare with phosphate buffer, in histidine buffering liquid (PH 7.3), can be observed sulfoxide B-hGH and form remarkable decline.In histidine buffering liquid, can be observed with pH value decline sulfoxide and form also decline.

Add antioxidant or other additives and do not obtain further effect.

Embodiment 4

The crystallization of hGH in the presence of phosphate or histidine buffering liquid

Will by Dalboege etc. (Biotechnology (1987), 5, the aliquot of the hGH of method preparation 161-164) is carried out crystal growth with the concentration of 6mg/ml in the 10mM of PH6.2 phosphate or 10mM histidine buffering liquid.Ethanol is added to make ultimate density in each sample be 7.5% (V/V), add ultimate density that acetic acid zinc solution makes zinc in phosphate buffer afterwards again and be 1.34mol Zn/mol hGH and in histidine buffering liquid the ultimate density of zinc be 5.5mol Zn/mol hGH.

In suspension, make crystal growth 16 hours and monitor crystallization with phase contrast microscope.Formed crystal has good hexagon outward appearance defined of the same size and contains seldom or do not have an amorphous impurities (Fig. 1) in histidine buffering liquid.On the contrary, under the really identical condition in phosphate buffer formed hGH crystal have significantly many inhomogeneous profiles and contain the noncrystalline thing (Fig. 2) of obvious amount.

Allow crystal regrowth 5 days.Be dissolved in the 7M carbamide subsequent analysis hGH by centrifugal collection histidine and the formed crystal of phosphate buffer and crystal.

Buffer crystal (%) hGH (%) that dissociates

Histidine 65 35

Phosphate 55 45

Thereby with regard to crystalline output and quality, histidine buffering liquid provides better condition for the hGH crystallization.

Embodiment 5

The lyophilizing hGH stability of formulation that will contain histidine and sucrose or mannitol compares with the conventional hGH preparation that contains phosphate, glycine and mannitol.

The desalination of hGH solution is made down series preparation 1-8 in described histidine buffering liquid.With various histidine buffering liquids the concentration adjustment of hGH after 6IU/ml, dissolve in the mannitol and the sucrose of described consumption.

Preparation 9 is consistent with conventional hGH preparation and be used as reference.

The hGH solution of all 1-9 is filled in the vial of 1ml and lyophilizing.

After preparing again, carry out the analysis of hGH with 0.9% benzyl alcohol solution.

1.hGH 6IU/ml

Be transferred to PH6.5 with HCl

Mannitol 33mg/ml

2.hGH 6IU/ml

Be transferred to PH6.5 with HCl

Sucrose 62mg/ml

3.hGH 6IU/ml

Be transferred to PH7.0 with HCl

Mannitol 33mg/ml

4.hGH 6IU/ml

Be adjusted to PH7.0 with HCl

Sucrose 62mg/ml

5.hGH 6IU/ml

Be transferred to PH6.5 with HCl

Mannitol 33mg/ml

6.hGH 6IU/ml

Slide into PH6.5 with HCl

Sucrose 62mg/ml

7.hGH 6IU/ml

Be transferred to PH7.0 with HCl

Mannitol 33mg/ml

8.hGH 6IU/ml

Be transferred to PH7.0 with HCl

Sucrose 62mg/ml

9.hGH 6IU/ml

Na ₂ HPO ₄·2H ₂O 0.59mg/ml

NaH ₂ PO ₄·2H ₂O 0.53mg/ml

Mannitol 20.5mg/ml

Be transferred to PH7.0. with phosphoric acid

Freeze dried product is easy to dissolving and forms transparent aqueous solution.

Listed in the table 5 with % represent before the lyophilizing behind (BL) and lyophilizing immediately, 4 ℃ after 7 months, 4 ℃ add after 7 months 37 ℃ 4 months and 4 ℃ of amounts that add 25 ℃ of polymers of 4 months after 7 months.

Listed the amount of the dimer of representing with % in the table 6.

Listed the amount of the deacylated tRNA amine hGH that represents with % in the table 7, and

Listed the amount of the sulfoxide of representing with % in the table 8.

The method of pressing embodiment 1-4 is measured the amount of deacylated tRNA amine hGH and sulfoxide.

Measure the amount of dimer and polymer with gp-HPLC.

Table 5

Buffer	The amount of polymer (%)
						BL	T＝0	7 months (4 ℃)	7 months (4 ℃)+4 months (37 ℃)	7 months (4 ℃)+4 months (25 ℃)
	NO.1	0.2	1.5 1.5	2.0	6.0						-
NO.2						-	0.2 0.2	0.2	0.2	0.2
	NO.3	0.2	0.8 1.0	1.8	4.0						2.6
NO.4						-	0.2 ＜0.2	0.2	＜0.2	＜0.2
	NO.5	＜0.2	0.8 0.7	1.6	3.1						3.0
NO.6						-	0.2 0.2	0.2	0.2	＜0.2
	NO.7	0.2	1.3 1.4	2.0	3.1						2.3
NO.8						-	0.2 0.2	0.2	＜0.2	＜0.2
	NO.9	0.2	1.2 1.4	2.3	2.9						2.2

The amount of polymer is obviously lower in containing the sample of sucrose.

Table 6

Buffer	The amount of dimer (%)
						BL	T＝0	7 months (4 ℃)	7 months (4 ℃)+4 months (37 ℃)	7 months (4 ℃)+4 months (25 ℃)
	No.1	0.4	0.8 0.9	1.8	3.6						-
No.2						-	0.4 0.4	0.3	2.8	0.4
	No.3	0.4	1.1 1.1	2.2	5.6						4.7
No.4						-	0.4 0.4	0.4	3.3	0.4
	No.5	0.3	0.6 0.6	1.1	3.4						2.2
No.6						-	0.4 0.4	0.3	3.1	0.3
	No.7	0.3	0.9 0.9	1.4	4.7						3.3
No.8						-	0.4 0.5	0.4	3.0	0.4
	No.9	0.5	0.6 0.7	1.0	4.3						2.2

The amount of dimer is obviously lower in containing the sample of sucrose.

Table 7

Buffer	The amount (%) of deacylated tRNA amine hGH
						BL	T＝0	7 months (4 ℃)	7 months (4 ℃)+4 months (37 ℃)	7 months (4 ℃)+4 months (25 ℃)
	NO.1	2.0	1.8 1.4	1.1	3.9						-
NO.2						-	1.4 1.5	3.1	22.8	1.3
	NO.3	2.1	1.2 1.3	2.1	5.6						1.5
NO.4						-	1.6 1.6	2.1	23.6	1.0
	NO.5	1.6	1.3 0.9	1.4	12.1						1.0
NO.6						-	1.2 1.3	1.6	23.0	1.4
	NO.7	2.0	1.4 1.6	1.0	4.8						4.6
NO.8						-	1.5 1.4	1.9	20.2	2.9
	NO.9	2.1	1.7 1.5	2.0	9.9						3.6

The amount of 4 ℃ 7 months+25 ℃ deacylated tRNA amine-hGH after 4 months is containing histidine

Very low in the compositions.

Table 8

Buffer	The amount % of sulfoxide
						BL	T＝0	7 months (4 ℃)	7 months (4 ℃)+4 months (37 ℃)	7 months (4 ℃)+4 months (25 ℃)
	NO.1
NO.2										1.0
	NO.3										4.8
NO.4										1.1
	NO.5										-
NO.6										1.0
	NO.7										1.8
NO.8										1.4
	NO.9										2.4

The amount of sulfoxide is obviously lower in containing the sample of sucrose.Embodiment 6

The stability that contains the lyophilized formulations of histidine, mannitol and disaccharide

Prepare series preparation down with embodiment 5 described same procedure.

10.hGH 6IU/ml

Be transferred to PH6.5 with HCl

Sucrose 21mg/ml

Mannitol 22mg/ml

11.hGH 6IU/ml

Be transferred to PH7.0 with HCl

Sucrose 21mg/ml

Mannitol 22mg/ml

12.hGH 6IU/ml

Be transferred to PH7.0 with HCl

Trehalose 20mg/ml

Mannitol 22mg/ml

Before lyophilizing (BL), t=0,40 ℃ after three months and 25 ℃ of amounts (%) of measuring deacylated tRNA amine hGH, polymer and dimer after 6 months.

The result is presented among the following table 9-11.

Clearly, the sample that contains mannitol and sucrose or trehalose has better stability than only containing the sample of mannitol as the lyophilizing extender.

Table 9

Buffer	The amount of deacylated tRNA amine-hGH (%)
					BL	T＝0	3 months (40 ℃)	6 months (25 ℃)
	NO.1		0.8	3.0					3.1
NO.10					1.3	0.9	1.7	2.4
	NO.3		0.9	2.4					3.0
NO.11					0.9	1.4	1.2	2.0
	NO.12		1.0	1.8					1.9
NO.9					0.7	0.7	2.8	3.4

Table 10

Buffer	The amount of polymer (%)
					BL	T＝0	3 months (40 ℃)	6 months (25 ℃)
	NO.1		1.1	5.0					4.1
NO.10					0.4	0.6	1.8	1.3
	NO.3		0.9	5.2					3.2
NO.11					0.5	0.6	1.5	1.1
	NO.12		0.8	1.3					1.1
NO.9					0.4	0.6	1.9	1.6

Table 11

Buffer	The amount of dimer (%)
					BL	T＝0	3 months (40 ℃)	6 months (25 ℃)
	NO.1		1.3	4.3					3.8
NO.10					0.5	0.9	1.8	1.6
	NO.3		1.3	5.0					4.3
NO.11					0.6	1.2	2.2	1.8
	NO.12		1.1	1.6					1.4
NO.9					0.6	0.9	1.0	2.3

Embodiment 7

Contain the preparation of the crystalline pharmaceutical preparation of hGH

Method grown crystal as described in example 4 above and 4 ℃ of storages.Also remove mother solution subsequently by the centrifugalize crystal then.Again the crystal freeze overnight is obtained not having the dried crystals of residue organic solvent.Drug suspension according to following prescription preparation dried crystals:

HGH crystal 1.3mg/ml

Histidine 1.6mg/ml

Zn(Ac) ₂·H ₂O 0.1mg/ml

Benzyl alcohol 0.9% (V/V) is transferred to 6.5 to pH value with HCl.

Embodiment 8

Remove and omit Zn (Ac) ₂H ₂O repeats embodiment 7 outward, obtains the outstanding of following prescription

Supernatant liquid:

HGH crystal 1.3mg/ml

Histidine 1.6mg/ml

Benzyl alcohol 0.9% (V/V) is transferred to 6.2 with PH.

Embodiment 9

Handle crystal and prepare following suspension with embodiment 7 same procedure:

HGH crystal 1.3mg/ml

Histidine 1.33mg/ml

NaCl 5.7mg/ml

Benzyl alcohol 0.9% (V/V) is transferred to 6.2 to PH.

Embodiment 10:

Handle crystal and prepare following solution with embodiment 7 same procedure:

HGH crystal 1.3mg/ml

Histidine 1.14mg/ml

NaCl 9.0mg/ml is transferred to 6.1 to PH.

Embodiment 11

With the similarity method described in the embodiment 1, compound concentration is the biosynthetic human growth hormone of 6IU/ml in 0.9% benzyl alcohol of PH6.5 with the histidine of variable concentrations (0,1,2,5,10,20,30,50 or 100mM).Under 37 ℃, sample was deposited 7 days and was carried out with aforesaid same procedure the content analysis of deacylated tRNA amine, oxidised form and dimer and polymer.The results are shown in the following table 12, wherein the content of deacylated tRNA amine-hGH, dimer and polymer and oxidised form is measured by IE-HPLC, GP-HPLC and RP-HPLC, and the content of the hGH of cracking form records with IE-HPLC.

When the concentration of histidine be 1mM or 1mM when above the amount of dimer lower, obtain satisfied result when the concentration of histidine is up to 30mM concerning forming the desamidization compound, and concerning forming oxidised form histidine concentrations to be lower than 20mM be preferred.Generally speaking histidine concentrations is preferably 5mM.

Table 2

The concentration of histidine (mM)	The amount of deacylated tRNA amine (%)	The amount of dimer (%)	The amount of polymer (%)	The amount of oxidised form (%)	The amount of cracking form (%)	PH
							0	7.7	0.7	＜0.2	1.5	1.4	6.3
1	7.9	0.4	＜0.2	1.5	1.2	6.4
							2	8.7	0.3	＜0.2	1.6	1.2	6.5
5	9.6	0.4	＜0.2	1.7	n.d.	6.6
							10	10.8	0.4	＜0.2	1.7	n.d.	6.6
20	11.6	0.4	＜0.2	2.0	n.d.	6.6
							30	12.0	0.3	＜0.2	2.1	n.d.	6.6
50	14.4	0.5	＜0.2	3.8	n.d.	6.6
							100	17.0	0.3	＜0.2	2.6	n.d.	6.6

N.d. can not detect

List of 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，E.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.Gellerforset al.，Acta Paediatr.Scand(suppl)，

370，93-100(1990).9) M.J.Kaufman，Pharm.Res.，7(3)，289-292(1990).

Claims (9)

1. pharmaceutical preparation, it contains a kind of growth hormone or a kind of growth hormone derivatives and histidine or a kind of histidine derivative, and its consumption is that every mg growth hormone＞0.1 is to 12mg histidine or derivatives thereof.

2. according to the pharmaceutical preparation of claim 1, it is to the buffered resiliency growth hormone of 100mM concentrations of histidine buffer aqueous solution form with 1mM.

3. according to the pharmaceutical preparation of claim 1, it is with the crystalline water slurry form of the buffered resiliency growth hormone of histidine buffering liquid.

4. according to arbitrary pharmaceutical preparation of claim 1-3, wherein pH value is transferred between the 2-9.

5. according to the pharmaceutical preparation of claim 4, wherein pH value is transferred between the 5-8.

6. according to the arbitrary pharmaceutical preparation among the claim 1-3, can also contain sugar alcohol or disaccharide or its mixture.

7. according to the arbitrary pharmaceutical preparation among the claim 1-3, it contains mannitol or disaccharide or its mixture.

8. according to the pharmaceutical preparation of claim 7, disaccharide wherein is sucrose or trehalose.

9. according to the arbitrary pharmaceutical preparation among the claim 1-3, growth hormone wherein is hGH.

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