CN101491681B

CN101491681B - Medicine containing PEG human growth hormone conjugate and use thereof

Info

Publication number: CN101491681B
Application number: CN200810051562A
Authority: CN
Inventors: 金磊; 裴瑾; 王俊才; 牛俊
Original assignee: Changchun Genscience Pharmaceuticals Co Ltd
Current assignee: Jinsai Drug Co., Ltd., Changchun
Priority date: 2008-12-09
Filing date: 2008-12-09
Publication date: 2012-10-10
Anticipated expiration: 2028-12-09
Also published as: CN101491681A

Abstract

The invention relates to a medicine composition and a medicine containing low-content pegylation (PEG) human growth hormone conjugate, and medical application of the low-dosage PEG human growth hormone conjugate to a human patient (in particular to a human patient suffering from endogenous growth hormone deficiency). The low-dosage medicine has the advantages of smaller toxic and side effect, less adverse effect, lower production cost and the like. Furthermore, the invention also relates to a high-purity pegylation human growth hormone conjugate suitable for medical application with the purity not less than 96 percent, and relates to a medicine preparation containing the conjugate, a preparation method of the conjugate and medical application thereof.

Description

Contain the medicine and the application thereof of PEGization human growth hormone conjugate

Invention field

The invention belongs to field of biological pharmacy; Particularly, the Polyethylene Glycol (PEG) that the present invention relates to contain low content is changed the PEGization human growth hormone conjugate of pharmaceutical composition and low dosage of human growth hormone conjugate to people patient's (especially endogenous growth hormone shortage and morbific people patient) medicinal usage.This low-dose drugs has advantages such as toxic and side effects is littler, production cost is lower.

Technical background

Human growth hormone (hGH) can be used for treating because of not enough dwarfism or the Turner syndrome that causes of hypophysis cerebri function, also can be used for promoting children growth in addition, treatment chronic renal insufficiency, AIDS depletion and aging etc.Natural hGH is by the excretory protein of people's hypophysis, and it is made up of 191 amino acid residues, and molecular weight is 22kDa.The main biological function of hGH is to promote the growth of immature body of mammals tissue and maintain old mammiferous tissue, and wherein related tissue comprises skeleton, connective tissue, muscle and such as the tissue of organs such as liver, intestinal and kidney.The human growth hormone can also obtain through sophisticated gene recombination technology, as can be referring to the work of (Gene, 39:247,1985) such as (Gene, 55:189,1987), Goeddel etc. (Nature, 281:544,1979) such as Chang and Gray.Current had multiple reorganization hGH to buy from market, like Genotropin, Nutropin and Somatonorm.

Protein such as direct administration hGH will influence its stability owing to making its pharmacological activity shorter action time to proteinic high clearance rate in the health.For this reason; The retardance proteolytic enzyme directly contacts with protein main chain people through come effectively with these protein of water-soluble polymer chemical modification; Prevent its degraded, thereby effectively reduce clearance rate in its body, improve its stability; Increase its circulation time in vivo, thereby can reduce administration number of times.Polyethylene Glycol (PEG) is one of water-soluble polymer that is most commonly used to chemically modified protein matter.PCT application WO9300109A has just reported with PEG and has modified the human growth hormone.Yet; According to (Journal of Biological Chemistry such as Clark; Researching and analysing 271:21969-21977,1996), the hGH conjugate that is not all PEGization can both keep original hGH active; The PEGization form of some hGH even will reduce its external activity greatly is to such an extent as to can't have the application of practicality.Thus, people attempt in a large number developing and on specific site, carry out the hGH conjugate that special PEG modifies, and in the hope of keeping on the active basis of hGH, reduce clearance rate in inhomogeneity and the body, increase its circulation time, raising stability in vivo.For example; PCT application WO9711178A discloses through carry out PEGization on the lysine sites in hGH; But this need introduce new lysine (like the replacement of K168A and K172R) in the natural human growth hormone, other researchs are recorded in the documents such as Chinese patent (application) CN100369953C and CN10109080A.The N end that PCT application WO2003044056A and one Chinese patent application CN1477126A have disclosed respectively at hGH carries out the conjugate that PEG modifies; Yet can only making, wherein disclosed preparation process have only about 90% PEG to be conjugated on the N end of hGH in the final conjugation product; Even add the wherein disclosed step that is further purified; The hGH conjugate of the final PEGization that produces also can only reach the purity about 95%, comprising unconjugated PEG, hGH and their catabolite.Although claiming, one Chinese patent application CN1565624A can obtain purity greater than 98% conjugate; But the process detail of its preparation (purification) is painstakingly covered; Even the technical scheme that reaches the purity about 95% that does not have PCT application WO2003044056A and one Chinese patent application CN1477126A to be disclosed is detailed; Do not have to confirm the existence of this purity product yet, can't expect and to obtain corresponding purity effect through the detection means of generally acknowledging.And the inventor is through arduous, secular effort; Also optimize the method for preparing of the hGH conjugate of the covalently bound PEGization that forms of single free amine group that a kind of and human growth hormone surprisingly; It can be under the condition that does not increase production cost; Through specific optimum organization of puting together with purification step; The purity of the conjugate that is obtained can be considerably beyond 95%, even can reach to detect with general detection means such as efficient size exclusion chromatography (SEC-HPLC) and RPHPLC (RP-HPLC) and reach the purity level more than 99%.In having prior art the advantage of the hGH conjugate of PEGization; More and more cause at current adverse effect under the situation of patient and the attention of medicine supervision department; This highly purified hGH conjugate has very important meaning for the high-quality pharmaceutical preparation of preparation; Not only can reduce the adverse effect that causes owing to heterogeneity, and be convenient to the quality management in the pharmaceutical preparation production.The autograph that this highly purified hGH conjugate is recorded on May 30th, 2008 application is in No. the 200810050760.8th, the one Chinese patent application of " the PEGization human growth hormone conjugate and the pharmaceutical preparation thereof of purification ", and it includes this paper reference in full in.

But, in order can actual human body being used, to need to confirm effectively and people's amount of application of safety, and to prepare the pharmaceutical composition (or pharmaceutical preparation) of respective unit doses in view of the above.Yet; Prior art is short of for the research of the drug dose aspect that the people of hGH conjugate uses very much; Mostly be to utilize animal models such as Mus to calculate that medicine reaches the dosage of drug effect; And people patient's dosage does not also not solve, and people's the build particularity of accepting the hGH treatment of PEGization has further increased the complexity that drug dose is studied.For example; Under situation about using with the hGH of human growth hormone's the covalently bound PEGization that forms of single free amine group, the rat dosage of hGH with PEGization of drug effect (but not being used for drug metabolism study) is proved and will reaches 1.8mg/kg body weight (referring to Chinese patent (application) CN1608079A and CN1929857A); And (can be referring to the instruction of medicine administrative organs such as FDA, SFDA according to waiting (medicine) to imitate the conversion rule of dosage; Concrete convert " Huang Jihan etc. in the pharmacological testing between animal and the dose,equivalent between the animals and human beings body convert. Chinese clinical pharmacology and therapeutics, 2004 Sep; 9 (9): 1069-1072 " on the books in, the adult is 1: 6.17 with the dosage ratio of rat), the adult's of SBW dosage should be the 0.288mg/kg body weight; And accept people's (because retarded growth just need treatment) of hGH for needs; Its build is less (to be child or child's build; Body weight is 10 to 40kg), body surface area is relatively large, so the dosage of the respective patient of calculating according to corresponding correction formula will be greater than the 0.288mg/kg body weight.Yet; The inventor, has found based on full and accurate achievement in research through long-term and arduous combination clinical research surprisingly; For people patient; Especially the people patient who lacks for endogenous growth hormone, the hGH conjugate with the covalently bound PEGization that forms of the single free amine group human growth hormone more low dosage has had excellent drug effect, even can be superior to the drug effect of existing medicine; And because required dosage is lower, have correspondingly that toxic and side effects is littler, advantage such as less adverse effect, production cost are lower.Current, more and more receive under the situation of government and popular attention at adverse drug reaction and health care cost, it is very positive and important to believe that these advantages can play a part.

Summary of the invention

The object of the present invention is to provide the invention of two aspects; One is to provide the Polyethylene Glycol (PEG) of low dosage to change human growth hormone conjugate at application or product aspect the treatment people, and two for providing highly purified Polyethylene Glycol (PEG) to change human growth hormone conjugate and compositions, application and method for preparing.The technical scheme of these two aspects can combine, and the technical characterictic that mutual alternative is corresponding also can not combine and constitutes two inventions separately.

On first aspect of invention; The PEGization human growth hormone conjugate that the object of the present invention is to provide the Polyethylene Glycol (PEG) that contains low content to change pharmaceutical composition, medicine and the low dosage of human growth hormone conjugate is directed against people patient's medicinal usage; Its dosage is lower than the dosage that prior art converts and comes from the effective dosage of animal; But still can reach good therapeutic effect, can obtain as ratifying other therapeutic effect of marketed drug level people patient especially really.Advantage such as such low dosage has that toxic and side effects is littler, less adverse effect, production cost are lower.Following summary of the invention partly will specifically be set forth the technical scheme of first aspect.

Particularly, aspect first, the hGH conjugate that the invention provides low dosage and the covalently bound PEGization that forms of the single free amine group human growth hormone is used for treating the application of the medicine of the people's disease of patient that need accept the hGH treatment in preparation.Wherein, said low dosage refers to and is lower than the people's dosage that converts and get through existing animal effective dose, promptly is lower than the people's dosage that converts and get through the 1.8mg/kg rat body weight, as is lower than the 0.288mg/kg body weight.The inventor finds through precisian's clinical experiment; Use than prior art (as; CN1608079A and CN1929857A) the few dosed administration of people's dosage calculated, even corresponding dosage lacks 1/3rd even over half than what prior art was calculated, not only can reduce the probability that untoward reaction is taken place by the people patient of administration; Reduced production cost, and still obtained excellent therapeutic effect people patient.Aspect first, preferred wherein said low dosage is the 0.01-0.26mg/kg body weight, preferably the 0.03-0.25mg/kg body weight; Be more preferably the 0.05-0.24mg/kg body weight; Be more preferably the 0.07-0.23mg/kg body weight, be more preferably the 0.08-0.22mg/kg body weight, be more preferably the 0.09-0.21mg/kg body weight; Especially preferably 0.1-0.20mg/kg body weight, especially especially preferably 0.2mg/kg body weight, 0.15mg/kg body weight or 0.1mg/kg body weight.In the specific embodiment of the present invention, the dosage that is used for people patient is 0.1mg/kg body weight and 0.2mg/kg body weight.According to required dosage, manufacturer of drugs can prepare corresponding medicine according to the behave patient colony of patient individual or same body weight of the market demand, as producing the medicine that the medicine that is applicable to 15kg body weight people patient or production are applicable to 25kg body weight people patient.

In this article (promptly; Not only in aspect of the present invention; And can be applicable to other aspects and the aspect of this description; Down with), if do not specialize, with human growth hormone's the covalently bound PEGization that forms of single free amine group the hGH conjugate be the single free amine group of hGH and the product that PEG puts together.Such hGH conjugate is well known in the prior art, for example those disclosed among WO2003044056A, CN1477126A, CN1565624A, CN1608079A and/or the CN1929857A.In this article; With human growth hormone's the covalently bound PEGization that forms of single free amine group preferred 60% the single free amine group of wherein surpassing of hGH conjugate be the free amine group of the N terminal amino acid of hGH; More preferably wherein surpassing 70% single free amine group is the free amine group of the N terminal amino acid of hGH; More preferably wherein surpassing 80% single free amine group is the free amine group of the N terminal amino acid of hGH, and the hGH conjugate of preferred especially and human growth hormone's the covalently bound PEGization that forms of single free amine group is the highly purified hGH conjugate that the present invention's second aspect provides.In addition; In this article; If do not specialize, the dosage of conjugate is the dosage in effective active composition in the conjugate, promptly; With the dosage of the hGH conjugate of human growth hormone's the covalently bound PEGization that forms of single free amine group be with the dosage of hGH protein refractometer wherein, refer to that like the dosage of 0.2mg/kg body weight hGH albumen is the dosage of 0.2mg/kg body weight in the conjugate.The route of administration of the conjugate of corresponding dosage and the administration frequency preferably specific embodiment of the invention are described.

Aspect second; The invention provides the pharmaceutical composition that is used to treat the people's disease of patient that to accept the hGH treatment; It comprises hGH conjugate and pharmaceutically acceptable carrier with human growth hormone's the covalently bound PEGization that forms of single free amine group, and the content of the hGH conjugate of said and human growth hormone's the covalently bound PEGization that forms of single free amine group is low content.Wherein, Refer to said the low content described low dosage in above-mentioned first aspect and people's weight in patients multiply each other and weight; Promptly this low content is the use amount of people patient in single administration, and just the effective active of relative medicine unit dosage forms (like a, pin) becomes component.Need accept the hGH treatment and promote people's weight in patients of growth to be lower than the normal person; Therefore its body weight can go out in the relative medicine compositions content with the hGH conjugate of human growth hormone's the covalently bound PEGization that forms of single free amine group according to the described low dosage range computation of first aspect between 10-40kg.Therefore, aspect this second, preferred wherein said low content is 0.1-10.4mg; Preferably 0.3-10mg is more preferably 0.5-9.6mg, is more preferably 0.7-9.2mg; Be more preferably 0.8-8.8mg; Be more preferably 0.9-8.4mg, especially preferably 1-8mg, more especially preferably 2-8mg, 1.5-6mg or 1-4mg.Medical personnel or patient or its family members use when administration and do not carry out medicine and merge for ease; Preferred low content should satisfy most patients' needs and make wastage few as far as possible; Therefore also preferably 11mg, 10.5mg, 10mg, 9.5mg, 9mg, 8.5mg or 8mg of above-mentioned low content; According to the distribution situation of the inventor for the weight in patients that occurs in the clinical trial, most preferably low content is 9mg.The carrying out that the administering mode of this pharmaceutical composition and administration frequency can be put down in writing according to prior art, the clinical treatment described in the specific embodiment most preferably of the present invention carries out.

Corresponding with this second aspect, aspect the 3rd, the present invention also provides the pharmaceutical composition of second aspect of the present invention to be used for treating the application of the medicine of the people's disease of patient that need accept the hGH treatment in preparation.

In this article, medicine has identical implication with pharmaceutical composition, all is well known to those of ordinary skill in the art, all refers to have the medicinal chemical products of treatment or preventive effect, works through the composition that treatment or prophylactic activity are wherein arranged.In addition; If do not specialize; The content of conjugate is the content in effective active composition in the conjugate in the compositions; That is, be with the content of hGH protein refractometer wherein with the content of the hGH conjugate of human growth hormone's the covalently bound PEGization that forms of single free amine group in the compositions, refer to that like the content of 2-8mg/kg hGH albumen weight is 2-8mg in the conjugate contained in the compositions.

Aspect the 4th, the invention provides the medicine that is used to treat the people's disease of patient that need accept hGH treatment, it comprises,

(1) container, it is equipped with and the hGH conjugate of human growth hormone's the covalently bound PEGization that forms of single free amine group or the pharmaceutical composition of second aspect of the present invention; With

(2) be used to indicate label with the hGH conjugate administration of low dosage and the covalently bound PEGization that forms of the single free amine group human growth hormone; Preferred wherein said low dosage is the 0.01-0.26mg/kg body weight; Preferably the 0.03-0.25mg/kg body weight is more preferably the 0.05-0.24mg/kg body weight, is more preferably the 0.07-0.23mg/kg body weight; Be more preferably the 0.08-0.22mg/kg body weight; Be more preferably the 0.09-0.21mg/kg body weight, especially preferably 0.1-0.20mg/kg body weight, especially especially preferably 0.2mg/kg body weight, 0.15mg/kg body weight or 0.1mg/kg body weight.

Medicine be for masses common product, can obtain in places such as pharmacy, hospitals.Wherein, container can be that bottle, box, syringe etc. can hold above-mentioned conjugate, pharmaceutical composition or be used for the container commonly used of the adjuvant of interim compounding pharmaceutical.Medicine can include only a container, also can comprise a plurality of containers, and different vessels can be equipped with identical chemical products, also the different chemical product can be housed.Label can be attached on the said vesse, perhaps directly prints on the said vesse, also can exist with form independently, as as loading onto the medicine box of stating container or as the description that provides separately.Label is that this product of medicine is necessary, because according to relevant pharmaceuticals administration rules, should include the description of indicating dosage in the medicine, therefore said label has played this description just, meets Compulsory Feature in order to make medicine.Certainly, on the label that is used to indicate with the hGH conjugate administration of low dosage and the covalently bound PEGization that forms of the single free amine group human growth hormone, also can indicate other essential conditions, like effective composition, administering mode etc.According to the clinical trial in content, the especially embodiment of the invention such as the effective ingredient of this paper record, administering mode, those skilled in the art can prepare (as, print) go out these labels.The label indication is with hGH conjugate administration low dosage and the covalently bound PEGization that forms of the single free amine group human growth hormone; Wherein, The indication of label specifically can be represented with " dosage/per weight "; Also can represent,, at this moment need simply convert according to the body weight situation like " 15kg body weight people patient's consumption ", " 25kg body weight people patient's consumption " with the absolute dosage of specific crowd.If what adorn in the container is pharmaceutical composition; Then can according in the unit dosage forms (like, a pin) with the content of the hGH conjugate of human growth hormone's the covalently bound PEGization that forms of single free amine group; And low dosage is converted into the quantity of unit dosage forms, indicate with label.These calculate this area and brush personnel is easily.Usually the same forms of pharmaceutical compositions of only pretending in the different vessels of a preferred medicine.Although not preferred, the container of different dosage form is included in a situation in the medicine and is also included within the scope of the present invention.As required, as conveniently transporting, depositing, medicine can further be packed in the into bigger packing, and such product also within the scope of the invention.

Preferably in the various aspects of first aspect of the present invention, said people patient is the people of child or child's body weight.Wherein, the child is the age more than or equal to 3 years old and people before adolescence, and its body weight is between 10-40kg, its male or female; It is big than the child that the people of child's body weight refers to the age, but the body weight people between 10-40kg still.These people need promote normal growth, maybe need receive treatment to lag behind normal growth to remedy, and therefore are preferably included in the various aspects of first aspect of the present invention.

Preferably in the various aspects of first aspect of the present invention, except promoting normal growth, said disease is because of lacking the disease that growth hormone causes.Include but not limited to because of lacking the disease that growth hormone can cause; Short stature, ELBW, dwarfism (growth hormone deficiency), metabolism syndrome and Noonan syndrome, burn (especially severe burn), adult's GHD and child Te Fa property are short and small; Growth hormone deficiency preferably; Be more preferably the endogenous growth hormone deficiency disease, the children growth that especially causes because of the endogenous growth hormone deficiency disease is slow.

Preferably in the various aspects of first aspect of the present invention; The hGH conjugate of said and human growth hormone's the covalently bound PEGization that forms of single free amine group is the highly purified hGH conjugate of second aspect of the present invention; Promptly pure basically conjugate, this pure basically conjugate is by single free amine group covalently bound form of Polyethylene Glycol through linking group and hGH, and the purity of said conjugate is not less than 96%; Preferably be not less than 97%; Preferably be not less than 98%, more preferably be not less than 99%, most preferably detect purity and reach more than 99% with efficient size exclusion chromatography and/or RPHPLC.More preferably wherein the aminoacid sequence of hGH shown in Seq ID NO:1; And/or wherein linking group is the N-hydroxy-succinamide base; And/or wherein the molecular weight of Polyethylene Glycol is about 40kDa.

In this article, " pharmaceutically acceptable carrier " refers to nontoxic solid-state, semisolid or liquid filler, diluent, buffer agent, protective agent, antiseptic, lapping or other pharmaceutical adjuncts.Preferably in the various aspects of first aspect of the present invention, come administration through injecting pathway, subcutaneous injection especially, more preferably weekly injection once, therefore preferably injectable powder (like lyophilized injectable powder) and liquid preparation of said pharmaceutical composition.Especially liquid preparation need not to prepare before use temporarily, and convenient the use is therefore preferred in the various aspects of first aspect of the present invention; Said pharmaceutical composition is a liquid preparation; More preferably injection in a specific embodiment, most preferably is the subcutaneous injection agent.

On second aspect of the present invention; The present invention also aims to provide a kind of hGH conjugate of highly purified PEGization; It gets through specific combination of puting together with purification step, can under the condition that does not increase cost, increase the purity of products therefrom; Thereby can more help it is used as effective ingredient; Can reduce the adverse effect that causes owing to heterogeneity on the one hand, be more convenient in pharmaceutical preparation is produced, tightening quality control, have good practical significance aborning.The present invention also aims to provide the method for preparing of above-mentioned hGH conjugate, comprise specific puting together and purification step.In addition, the present invention also aims to comprise pharmaceutical composition or the pharmaceutical preparation and the medicinal application of above-mentioned hGH conjugate.Summary of the invention on second aspect of the present invention can be preferably applied in application (purposes), pharmaceutical composition and/or the medicine of first aspect of the present invention; But also can be applied in application (purposes), pharmaceutical composition and/or the medicine etc. of non-first aspect of the present invention; For example; Highly purified hGH conjugate on second aspect of the present invention or contain the reliability of its pharmaceutical composition because of its pharmaceutical quality aspect, and can substitute hGH or its conjugate in prior art or the technology newly developed.Following summary of the invention partly will specifically be set forth the technical scheme of second aspect.

Particularly, in first aspect, the invention provides pure basically conjugate, it is characterized in that, said conjugate is by single free amine group covalently bound form of Polyethylene Glycol through linking group and human growth hormone, and the purity of said conjugate is not less than 96%.That is to say that used " pure basically " is not less than 96% with purity among this paper has identical implication.Human growth hormone's (abbreviating hGH among this paper as) is the protein that promotes the growth of child's torso tissue and maintain adult's tissue.The human growth hormone extracts to obtain, but preferably obtain through gene recombination technology.From existing state of development, obtaining hGH through gene recombination technology is mature technique, and commercially available product has also been arranged at present.In the present invention, preferred hGH is natural hGH, and its aminoacid sequence is shown in Seq ID NO:1.The single free amine group of hGH can be the α amino of its N end or the free amine group of its side chain (side chain like lysine is amino), and preferably the α of the N of hGH end is amino.Under preferred situation, Polyethylene Glycol is connected through the α amino covalence of linking group with human growth hormone N end phenylalanine.The present invention also preferably is suitable for the hGH that obtains at expression in escherichia coli, and its aminoacid sequence can be to add a methionine again at the N of the aminoacid sequence shown in Seq ID NO:1 end.Like this, Polyethylene Glycol is connected through the α amino covalence of linking group with human growth hormone N end methionine.

In this article, term " purity " has the conventional implication of understanding of albumen pharmaceutical field technical staff, and its implication is equivalent to homogeneity.Because during the preparation protein conjugate; Because proteinic degraded or effectively do not put together or put together the difference in site; Cause except the purpose product; Also can produce protein degradation products or its conjugate, not have the product of puting together, the by-products such as product of puting together the site difference or puting together in a plurality of sites simultaneously, causing the protein conjugate of preparing thus is the mixture of purpose product and by-product, is not the purpose product of complete and homogeneous (or pure).This inhomogeneity (or impure) causes drug product quality to be difficult to control.Purity can be represented with the mass percent or the molar percentage of purpose product in the protein conjugate of preparing and protein conjugate; But in practice, use the peak of representing the purpose product in the conventional detecting instrument to represent usually with the percent value of the area of representing the peak that comprises the heterogeneity product.In the present invention, if no special instructions, all adopt the described percentage of the latter recently to represent purity.

In the present invention, the inventor gropes through optimization, adopts the described method for preparing of third aspect present invention, has obtained 96% conjugate that purity is not less than.The mensuration of purity is with instrument known in those skilled in the art and detects step and carry out that these assay methods all come from regulation and the standard that corresponding medicine detects usually, preferably carry out through authentication method listed in the specific embodiment of the invention.The purity of the preferred conjugate of the present invention is not less than 97%, preferably is not less than 98%, more preferably is not less than 99%, most preferably uses efficient size exclusion chromatography (SEC-HPLC) and/or RPHPLC (RP-HPLC) to detect purity and reaches more than 99%.Because the present invention can obtain the high product of purity, therefore,, just can realize the product that corresponding purity is lower easily like the harmless composition of the trace that can characterize rule of origin as long as suitably be added into the heterogeneity composition.

In first aspect of the present invention, Polyethylene Glycol and linking group are covalently bound, form activated polyglycol (that is, the product that Polyethylene Glycol and linking group are covalently bound), are connected with hGH through the linking group in the activated polyglycol again.Wherein, linking group can be covalently bound with the single free amine group of hGH, preferably can be connected with the α amino covalence of N end, and like N-hydroxy-succinamide (NHS), butanimide propionic ester (SPA) or aldehyde etc., preferred N-hydroxy-succinamide base.PEG can be straight chain PEG, also can be side chain PEG, preferably side chain PEG.The molecular weight of PEG is 10kDa ~ 120kDa, is preferably 30 ~ 90kDa, more preferably about 40kDa or about 80kDa.Wherein, numerical value shown in " pact " expression is limited to up and down ± 10%, 40kDa is 36 ~ 44kDa according to appointment.Except preparing the activated polyglycol through covalently bound Polyethylene Glycol of chemical reaction and linking group; Also can directly adopt commercial activated polyglycol; Like methoxyl group PEG propionic aldehyde (mPEG-ALD), bi-methoxy PEG N-hydroxy-succinamide base ester (mPEG2-NHS) and the methoxyl group PEG butanimide propionic ester (PEG-SPA) etc. that Shearwater company provides, put together in order to direct and hGH.

In second aspect, the invention provides pharmaceutical composition, it comprises described conjugate of first aspect present invention and pharmaceutically acceptable carrier.Adopt highly purified conjugate feedstock production pharmaceutical composition, can improve the quality of the product of generation.Known technology according to this area; Can pharmaceutical composition be processed various dosage forms according to the needs of therapeutic purposes, route of administration; Preferred said composition is a unit dosage form; Like solid preparation (like tablet, membrane, pill, capsule, powder, injectable powder or granule etc.), liquid preparation (firmly penetrating with solution or suspension, aerosol or liquid spray, drop or injection etc.) and automated injection device or suppository, be more preferably injectable powder and liquid preparation like syrup and emulsion agent, disinfectant.Especially liquid preparation need not to prepare before use temporarily, and conveniently use, so pharmaceutical composition of the present invention is liquid preparation preferably.

Yet,, therefore develop a large amount of experiments of the hGH conjugate needs that contain PEGization and grope because with different in the solid environment, in liquid environment, protein or its conjugate receive hydrolysis more easily and degrade.PH value has very big influence to the stable and dissolution properties of protein conjugate, and it is about 6.0 that the pH of pharmaceutical composition of the present invention is preferably, and is 5.4 ~ 6.6, most preferably is 6.0.The buffer agent that pharmaceutical composition of the present invention comprises is preferably selected from citrate, histidine salt and maleate; Discovering wherein through the inventor; When adopting citrate as buffer agent, can keep pharmaceutical preparation of the present invention stable substantially, therefore pharmaceutical composition of the present invention preferably includes the citrate as buffer agent; More preferably the concentration of citrate is 3-7mM, and most preferably the concentration of citrate is 5mM.The surfactant that pharmaceutical composition of the present invention comprises is preferably selected from poloxamer 188, Tween 80 and polysorbas20; Discovering wherein through the inventor; When adopting poloxamer 188 as surfactant, can effectively prevent the hGH conjugate degradation of PEGization, therefore pharmaceutical composition of the present invention preferably includes the poloxamer 188 as surfactant; More preferably the concentration of poloxamer 188 is 0.5 ~ 3mg/ml, and most preferably the concentration of poloxamer 188 is 1mg/ml.In a specific embodiment of the present invention, liquid preparation of the present invention comprises the 5mM trisodium citrate, 1mg/ml poloxamer 188, and 2.5mg/ml phenol, the hGH conjugate of 9mg/ml sodium chloride and 9.0mg/ml PEGization, this liquid preparation pH is 6.0.

In the third aspect, the invention provides the method for preparing the described conjugate of first aspect present invention, it comprises:

(a) human growth hormone's solution is mixed with activated polyglycol, make human growth hormone and Polyethylene Glycol generation conjugation reaction; With

(b) the reaction mixture dilution that step (a) is obtained in order to the equilibrated SephadexG-25 chromatographic column of the level pad of pH 6.5-8.0 purification, is collected the eluent of first eluting peak; With

(c) eluent that step (b) is obtained is splined on the equilibrated Q-Sepharose chromatographic column of the level pad of pH 6.5-8.0, with the level pad gradient elution that contains 10mM NaCl, then with containing the level pad eluting of 50mM NaCl and collecting eluent.Wherein, step (a) is preferably: human growth hormone's solution is mixed with activated polyglycol, stirred 16～18 hours with 50-100rpm in 4 ℃, make human growth hormone and Polyethylene Glycol generation conjugation reaction.Wherein, the pH of level pad is preferably pH7.0-7.8 for for pH6.5-8.0, and more preferably pH7.2-7.6 most preferably is pH7.4, and in the specific embodiment of the present invention, level pad is Tris-HCl.Wherein, activated polyglycol is the covalently bound product of Polyethylene Glycol and linking group, bi-methoxy PEG N-hydroxy-succinamide base ester preferably, and the molecular weight of the mPEG in the specific embodiment of the present invention among the mPEG2-NHS is about 40kDa.Through the method for third aspect present invention, can obtain first aspect present invention purity and be not less than 96% conjugate.According in the method described in the specific embodiment of the present invention, the conjugate that obtains detects purity more than 99% with efficient size exclusion chromatography (SEC-HPLC) and/or RPHPLC (RP-HPLC).

In fourth aspect, the invention provides the application in the medicine that preparation promotes to grow of described conjugate of first aspect present invention or the described pharmaceutical composition of second aspect present invention.Medicine of the present invention can carry out administration through the administering mode that one of ordinary skill in the art knew, promote experimenter (especially people's) growth.The for example injection of available administering mode, oral, rectum, Sublingual, pulmonary, transdermal, ion penetrate, vagina and intranasal administration, and preferred gastrointestinal tract external administration is like subcutaneous, intramuscular or intravenous injection.Dosage changes according to the situation of action time of dosage form and expectation and treatment target to some extent; The required amount of actual therapeutic can by the clinician according to experimenter's practical situation (as, patient's the state of an illness, body weight, age, sex etc.) and confirm easily.For to child's drug administration by injection; Drug dose of the present invention; In the hGH conjugate of PEGization, can be the 0.05-0.5mg/kg body weight, preferred dosage is the 0.1-0.3mg/kg body weight; Most preferably described like first aspect of the present invention, be 0.2mg/kg body weight, 0.15mg/kg body weight or 0.1mg/kg body weight.

For the ease of understanding, below will describe in detail the present invention through concrete accompanying drawing and embodiment.What need particularly point out is that instantiation and accompanying drawing only are in order to explain, not constitute limitation of the scope of the invention.Obviously those of ordinary skill in the art can explain according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.In addition, the present invention has quoted open source literature, and these documents also are in order more clearly to describe the present invention, and their full text content is all included the present invention in and carried out reference, just look like they full text in description of the present invention repeated description the same excessively.

Description of drawings

Fig. 1. the SEC-HPLC collection of illustrative plates of the hGH conjugate of high-purity PEGization of the present invention

Fig. 2. the RP-HPLC collection of illustrative plates of the hGH conjugate of high-purity PEGization of the present invention

Fig. 3, the SDS-PAGE electrophoretogram of the hGH conjugate of high-purity PEGization of the present invention, wherein first road is PEG-GH, second road is the molecular weight of albumen labelling

The protease hydrolysis mass spectrum of the hGH conjugate of Fig. 4 A high-purity of the present invention PEGization

Fig. 4 B is as the protease hydrolysis mass spectrum of the hGH of contrast

Substance assistant laser desorpted ionizing time of flight mass spectrometry (MALDI-TOF-MS) figure of the hGH conjugate of Fig. 4 C high-purity of the present invention PEGization

The specific embodiment

Following this paper will describe invention through concrete embodiment.As do not specialize part; Can be according to " molecular cloning experiment guide " (third edition) (Cold Spring Harbor laboratory Press), " cell experiment guide " (Science Press that those skilled in the art were familiar with; Beijing; China, calendar year 2001), listed method is implemented in " protein technical manual " handbooks such as (Science Press, 2000) and related drugs rules, regulation and the list of references that this paper quoted.In addition, employed material all can be bought from market through commercial sources except that special instruction is arranged among the embodiment.

Puting together of [embodiment 1] human growth hormone and Polyethylene Glycol

1, human growth hormone's solution (human growth hormone's concentration is 10mg/ml, is dissolved in the 50mM phosphate buffer pH to 6.5) is placed aseptic triangular flask, with the speed stirring of 50-100rpm.Continuing under the stirring condition; In adding reaction vessel, add bi-methoxy PEG-N-HOSu NHS base ester (mPEG2-NHS in batches; Molecular weight is 42.5kDa, available from Shearwater company), the adding gross weight of bi-methoxy PEG N-hydroxy-succinamide base ester is 4 times of the human growth hormone (weight ratio that is itself and rhGH is 4: 1); Bi-methoxy PEG N-hydroxy-succinamide base ester to adding all dissolves; Stirred 16～18 hours with 50-100rpm in 4 ℃, make human growth hormone and Polyethylene Glycol generation conjugation reaction, get reaction mixture.

2, human growth hormone's solution (human growth hormone's concentration is 10mg/ml, is dissolved in the 50mM phosphate buffer pH to 6.5) is placed aseptic triangular flask, with the speed stirring of 50-100rpm.Continuing under the stirring condition; In adding reaction vessel, add bi-methoxy PEG-N-HOSu NHS base ester (mPEG2-NHS in batches; Molecular weight is 42.5kDa, available from Shearwater company), the adding gross weight of bi-methoxy PEG N-hydroxy-succinamide base ester is 6 times of the human growth hormone (weight ratio that is itself and rhGH is 6: 1); Bi-methoxy PEGN-HOSu NHS base ester to adding all dissolves; Stirred 16～18 hours with 50-100rpm in 4 ℃, make human growth hormone and Polyethylene Glycol generation conjugation reaction, get reaction mixture.

The purification of the hGH conjugate of [embodiment 2] PEGization

With 10mM sodium hydroxide washing Sephadex G-25 chromatographic column (available from magnificent company), extremely neutral with the water for injection flushing then, follow this chromatographic column of Tris-HCl buffer balance with 20mM, pH 7.4.After the balance; The 5 times of volume waters for injection of reaction mixture adding that obtain to the embodiment 1-1 of 1 times of volume of note work dilute, and go up appearance then, wherein go up an appearance volume and are no more than 15% of column volume; Buffer solution elution with 20mM Tris-Hcl pH7.4; The flow velocity of last appearance and eluting detects the absorbance value of eluted product at wavelength 280nm place between 150～250cm/h, collect the eluent of first eluting peak.

With 10mM sodium hydroxide washing Q-Sepharose chromatographic column (available from magnificent company), extremely neutral with the water for injection flushing then, then with level pad (being the Tris-HCl buffer of 20mM, pH 7.4) this chromatographic column of balance.After the balance; Appearance on the eluent of above-mentioned first eluting peak is to the Q-Sepharose chromatographic column, steady with level pad washing chromatographic column to baseline then, then with the level pad gradient elution impurity that contains 10mM NaCl; Then with the level pad eluting that contains 50mM NaCl; Wherein go up appearance, wash to the flow velocity of baseline and eluting all between 90～150cm/h, detect wavelength 280nm, collect the absworption peak that absorption is arranged with the 280nm place of the level pad eluting that contains 50mM NaCl; Get the hGH conjugate solution of PEGization, wherein the single free amine group on PEG and the hGH is puted together.

The hGH conjugate of [embodiment 3] PEGization evaluation

Get the hGH conjugate solution of the PEGization of embodiment 2 gained, identify as follows:

1, size exclusion high performance liquid chroma-tography (SEC-HPLC)

Adopt SEC-HPLC that the product of embodiment 2 gained is estimated.According to manufacturer's explanation, adopting Protein Pak 300SW post (7.8mm * 300mm is available from Waters company) is mobile phase with the 50mM phosphate buffer of pH6.5, among the 0.1%SDS, with flow velocity 0.6mL/ minute, carries out SEC-HPLC and analyzes, and detects wavelength 214nm.As shown in Figure 1, purity 99.14%.

2, RPHPLC (RP-HPLC)

Adopt RP-HPLC that the product of embodiment 2 gained is estimated.According to manufacturer explanation, (the RP-HPLC post (available from U.S. GRACE VYDAC company) of 250mm * 4.6mm) carries out RP-HPLC to adopt Vydac.Experiment is carried out at room temperature, and typical volume containing the sample is 10mg protein/sample.A is the phosphate buffer of pH6.5,50mM mutually; B is acetonitrile mutually, gradient elution, and B increases to 20% by 0% in 20 minutes, flow velocity 0.6ml/min, the detection wavelength is 214nm.As shown in Figure 2, the result is unimodal, purity 99.51%.

3，SDS-PAGE

Concentrate glue and 10% separation gel with 4.5% the product of embodiment 2 gained is carried out SDS-PAGE.As shown in Figure 3, it is the single band about 64kDa that the result is shown as molecular weight.

4, peptide figure analysis

Get embodiment 2 gained PEGization hGH conjugate (being designated hereinafter simply as PEG-GH) and as the hGH of not PEGization of contrast, carry out peptide figure analysis.Get hGH and 0.5mgPEG-GH desalination, the lyophilizing of 0.5mg; Be divided into 20ug/ part; Respectively get in pH8.5 that portion is dissolved in 10ul, the 25mM Tris buffer and process specimen, in specimen, add Lys-C endo protease (0.5mg/mL) 2ul respectively, 37 ℃ of insulations 16.5 hours.BioAge Pharmaceuticals Inc. carries out mass spectral analysis in the U.S..The result with shown in the 4B, has only a N-terminal fragment in the mass spectrum of hGH, to occur like following table 3.1 and Fig. 4 A, but this fragment do not occur in the mass spectrum of PEG-GH, is illustrated on the α amino that the N of hGH among the PEG-GH holds to have puted together PEG equably.

Get in pH8.5 that 20ug PEG-GH lyophilizing sample is dissolved in 10ul, the 25mM Tris buffer; Carry out substance assistant laser desorpted ionizing time of flight mass spectrometry (MALDI-TOF-MS) in U.S. BioAgePharmaceuticals Inc; The result is shown in Fig. 4 C; The molecular weight that shows PEG-GH is 64918, shows that a PEG only is coupled on the growth hormone molecule.

Table 3.1 MASS SPECTRAL DATA ANALYSIS

P1(N)

P2

P3

P4

P5

P6

P7

P8

P9

P10

hGH

√

MW

4580

403

3360

5126

2791

625

1489

1276

507

2130

PEG-GH

√

[embodiment 4] contain the liquid preparation research of the hGH conjugate of PEGization

1, the liquid preparation prescription:

The 5mM trisodium citrate, 1mg/ml poloxamer 188,2.5mg/ml phenol, 9mg/ml sodium chloride, the hGH conjugate of 9.0mg/ml PEGization, pH6.0.

2, method for preparing:

Accurately take by weighing trisodium citrate, poloxamer 188 (hereinafter referred PoL188), phenol, sodium chloride, be mixed with 0.1M lemon acid three sodium solutions, 50mg/ml poloxamer solution, 20mg/ml phenol solution, 4M sodium chloride solution with water for injection.The hGH conjugate of PEGization is mixed with above-mentioned solution and water for injection; With hydrochloric acid adjust pH to 6.0, make that final concentration is trisodium citrate 5mM, 1mg/ml poloxamer 188; Phenol 2.5mg/ml, sodium chloride 9mg/ml and Polyethylene Glycol recombinant human somatropin 9.0mg/ml.

Also can in said method, use other reagent or concentration instead, carry out following contrast test.

3, the screening of liquid preparation

Buffer agent, surfactant, concentration and pH in the prescription are studied, carry out accelerated test at 40 ℃.Result such as following table 4.1～4.4 are said.

The various buffer agents of table 4.1 are to the protective effect of PEG-GH

The result shows that trisodium citrate is compared with other buffer, has the better protection effect for PEG-GH.

Table 4.2 trisodium citrate buffer concentration is to the influence of PEG-GH stability

The result shows, and is best to the PEG-GH protective effect when trisodium citrate buffer concentration is 5mM.

Table 4.3pH is to the influence of PEG-GH stability

The result shows, when PEG-GH liquid preparation pH stability 6.0 time best.

Confirming of table 4.4 surfactant and concentration thereof

The result shows that 1mg/ml Pol188 has best protective effect for PEG-GH.

3, the liquid preparation prescription relatively

According to above-mentioned research, be combined into the PEG-GH liquid preparation of following different formulations:

Prescription 1:5mM trisodium citrate, 1mg/ml poloxamer 188,2.5mg/ml phenol, 9mg/ml sodium chloride, the hGH conjugate of 9.0mg/mlPEGization, pH6.0;

Prescription 2:5mM histidine, 1mg/ml Tween 80,2.0mg/ml phenol, 5mg/ml sodium chloride, the hGH conjugate of 9.0mg/ml PEGization, pH6.0;

Prescription 3:5mM maleic acid, 1mg/ml poloxamer 188,1.5mg/ml phenol, 9mg/ml sodium chloride, the hGH conjugate of 9.0mg/ml PEGization, pH6.0.

Respectively hGH liquid preparation and PEG-GH liquid preparation are placed 4 ℃ of placements, whenever detect at a distance from sampling in 2 months, compare their stability, the result is shown in following table 5.5.

4 ℃ of stability of table 4.5hGH liquid preparation and PEG-GH liquid preparation relatively

The result shows: the PEG-GH liquid preparation all has better stability than the hGH liquid preparation.

The clinical research of the hGH conjugate of [embodiment 5] PEGization

Below experiment is all carried out according to State Food and Drug Administration's corresponding requirements, and the weight in patients of wherein receiving treatment is between 10-40kg.

1, human tolerance's experimental study result

In the health volunteer, the safety and the toleration of researching human body single subcutaneous injection PEG-GH injection.

The single-dose dose groups is made as 0.01mg.kg ^-1, 0.06mg.kg ^-1, 0.2mg.kg ^-1, 0.5mg.kg ^-1, 0.8mg.kg ^-1Five dose groups, the 34 routine men and women experimenters that Pass Test requires get into each dose groups at random.0.5-0.8mg.kg ^-1Dose groups has 1 example and 3 routine experimenters to cause that during medication blood glucose slightly raises respectively, recovers normal after the drug withdrawal, 0.8mg.kg ^-1Dose groups has 1 routine experimenter the edema symptom to occur, estimates the above-mentioned untoward reaction that is likely that unusually the Polyethylene Glycol recombinant human somatropin causes through relatedness.0.8mg.kg ^-1Diarrhoea and fecal occult blood inspection (+) took place in 168h after dose groups had 1 routine experimenter's administration, estimated the untoward reaction that above-mentioned detected value is likely that unusually the Polyethylene Glycol recombinant human somatropin causes through relatedness.

2, clinical pharmacokinetics experimental study result

Through the test of the pharmacokinetics behind experimenter's single subcutaneous injection PEG-GH injection injection, understand this medicine in the intravital absorption of people, distribution, metabolic rule, for formulating rational dosage regimen foundation is provided.

Research method: the clinical pharmacokinetics test of single-dose: establish 0.1mg.kg ^-1, 0.2mg.kg ^-1, 0.4mg.kg ^-1Three dose groups; Each dose groups experimenter is 10 examples; Single subcutaneous injection PEG-GH injection.

● each dose groups 10 routine male receives property person.

● adopt ELISA (ELISA method) to measure the concentration of PEG-GH, calculate each main pharmacokinetic parameter according to gained concentration-time data.

0.1,0.2,0.4mg kg qualitative investigation result: ^-1The t of the PEG-GH of three dose groups _1/2Close, C _Max, AUC _0-t, AUC _{0 → ∞}Increase along with the increase of dosage, at 0.1-0.4mg kg ^-1Single subcutaneous injection PEG-GH injection is at the intravital process basic symbols of people zygonema property dynamic characteristic in the scope.

3, PEG-GH injection for treating children growth hormone deficiency at random, opening, positive drug parallel control, multicenter II phase clinical research result

This test be at random, multicenter, opening, with the parallel control II clinical trial phase of the positive contrast medicine of reorganization hGH injection (available from Changchun Jinsai Medicine Co.,Ltd).Carrying out randomization according to statistical method divides into groups; Be divided into three groups: low dosage test group (subcutaneous injection PEG-GH 0.1mg/ (kg week); This group is hereinafter to be referred as low dose group), high dose test group (subcutaneous injection PEG-GH 0.2mg/ (kg week), this organize hereinafter to be referred as high dose group) and matched group (subcutaneous injection hGH0.25mg/ (kg is all)).Respectively organize study subject example number according to the pro rate that contrasts 1: 1: 1 at random, every group 36 example amounts to 108 examples, carries out simultaneously in six tame hospitals.Be 6 months total course of treatment, carries out 4 times altogether and make a house call.The main evaluation carried out in treatment in back 3 months and 6 months, like discontented 6 months of the course of treatment, then when treatment finishes, carried out.Treat when carrying out in 6 months adding up final period, six tame hospitals accomplish the treatment of several 101 examples of MethodsThe cases enrolled altogether, and the case load that the actual FAS of including in collection (complete analysis collection) is analyzed is 97 examples, low dose group 32 examples, male 23 examples, women 9 examples; High dose group 31 examples, male 25 examples, women 6 examples; Matched group 34 examples, male 23 examples, women 11 examples.The case load of including PPS collection (effectively analysis of cases collection) analysis in is 95 examples, low dose group 31 examples, high dose group 30 examples, matched group 34 examples.The case load of including SS collection (safety analysis collection) analysis in is 98 examples, low dose group 33 examples, high dose group 31 examples, matched group 34 examples.The equal no difference of science of statistics of ordinary circumstance (p＞0.05) such as sex, age, body weight, height between three groups, the preceding health basic condition of treatment is not statistically significant (p＞0.05) more also.Three groups of experimenters are during participating in this clinical trial, and the situation of not having serious adverse events takes place.

The result: after three groups of experimenters accept different pharmaceutical, various dose and treated for 4 weeks, before and after each group test relatively the growth in stature amount and height year growth rate all significantly increase, significant difference (p＜0.001) is arranged on the statistics.After the growth in stature amount is carried out statistical comparisons between three groups, FAS collection and the equal no difference of science of statistics of PPS collection (p＞0.05) after 4 weeks of treatment.After height year, growth rate was carried out statistical comparisons between three groups, FAS collection and PPS collection were equally at 4 week of treatment back no difference of science of statistics (p＞0.05).

4, PEG-GH injection for treating children growth hormone deficiency at random, opening, positive drug parallel control, multicenter III phase clinical research result

This test is at random, multicenter, opening, serve as the III clinical trial phase that contrasts the parallel control of medicine with reorganization hGH injection (available from Changchun Jinsai Medicine Co.,Ltd).Select test group (subcutaneous injection PEG-GH 0.2mg/ (kg week)) and matched group (subcutaneous injection hGH 0.25mg/ (kg week)), enlarge and respectively organize experimenter's example number, further observe effectiveness and the safety of PEG-GH.Test group 240 examples, matched group 120 examples amount to 360 examples, carry out simultaneously at six tame centers.Be 6 months total course of treatment, carries out 4 times altogether and make a house call.The main evaluation carried out in treatment in back 3 months and 6 months, like discontented 6 months of the course of treatment, then when treatment finishes, carried out.Treat when carrying out in 6 months adding up final period, six tame hospitals accomplish the treatment of several 346 examples of MethodsThe cases enrolled altogether, and the case load of the actual FAS of including in set analysis is 343 examples, test group 228 examples, male 187 examples, women 41 examples; Matched group 115 examples, male 93 examples, women 22 examples.The case load of including the PPS set analysis in is 331 examples, test group 218 examples, matched group 113 examples.The case load of including the SS set analysis in is 343 examples, test group 228 examples, matched group 115 examples.The equal no difference of science of statistics of ordinary circumstance (p＞0.05) such as sex, age, body weight, height before the treatment between two groups; The preceding growth promoter situation of treatment is not statistically significant (p＞0.05) more also, and curative effect index such as height, year growth rate, IGF-1, IGFBP-3, height standard deviation integration compare not statistically significant (p＞0.05) between two groups before treatment.Two groups of experimenters are during participating in this clinical trial, and the situation of not having serious adverse events takes place.

The result: two groups of experimenters are after 4 weeks of treatment, 13 weeks and 25 weeks, and respectively relatively growth in stature amount and all significantly increases of height year growth rate before and after the group test has significant difference (p＜0.001) on the statistics.After the growth in stature amount is carried out statistical comparisons between two groups, FAS collection and the equal no difference of science of statistics of PPS collection (p＞0.05) after 4 weeks of treatment, FAS collection test group is 1.23 ± 0.59cm, matched group is 1.09 ± 0.57cm; The PPS collection is respectively 1.23 ± 0.59cm and 1.09 ± 0.57cm.And significant difference (p＜0.05) is all arranged at treatment 13 all back FAS collection and PPS collection, and FAS collection and PPS collection all are that the test group increment is higher than matched group, FAS collection test group and matched group growth in stature amount are respectively 3.68 ± 1.07cm and 3.39 ± 0.91cm; The PPS collection is respectively 3.74 ± 1.04cm and 3.40 ± 0.92cm for two groups.No difference of science of statistics (p＞0.05) between two groups of treatment 25 week back FAS collection, test group is 6.71 ± 1.86cm, matched group is 6.28 ± 1.49cm; And the PPS collection has significant difference (p＜0.05), and two groups are respectively 6.88 ± 1.69cm and 6.30 ± 1.47cm, and the test group increment is higher than matched group.After height year, growth rate was carried out statistical comparisons between two groups, FAS collection and PPS collection had significant difference (p＜0.05) at 4 weeks of treatment, 13 weeks and 25 Zhou Houjun, and test group is higher than matched group.Height year growth rate FAS collection 2.26 is increased to 14.71 ± 7.04cm/yr for ± 0.87cm/yr before treatment 4 when week test group is by treatment; Matched group 2.25 before by treatment is increased to 13.11 ± 6.83cm/yr for ± 0.82cm/yr.Two groups of recruitments are respectively 12.45 ± 7.16cm/yr and 10.86 ± 6.95cm/yr.Two groups are increased to 14.72 ± 4.29cm/yr and 13.56 ± 3.66cm/yr respectively during 13 weeks, and recruitment is respectively 12.47 ± 4.48cm/yr and 11.31 ± 3.77cm/yr.Two groups are increased to 13.41 ± 3.72cm/yr and 12.55 ± 2.99cm/yr respectively during 25 weeks, and recruitment is respectively 11.15 ± 3.87cm/yr and 10.31 ± 3.14cm/yr.The PPS collection 2.26 is increased to 14.76 ± 7.04cm/yr for ± 0.86cm/yr before treatment 4 when week test group is by treatment; Matched group 2.26 before by treatment is increased to 13.08 ± 6.88cm/yr for ± 0.82cm/yr.Two groups of recruitments are respectively 12.50 ± 7.17cm/yr and 10.82 ± 7.00cm/yr.Two groups are increased to 14.97 ± 4.15cm/yr and 13.58 ± 3.69cm/yr respectively during 13 weeks, and recruitment is respectively 12.70 ± 4.37cm/yr and 11.32 ± 3.80cm/yr.Two groups are increased to 13.76 ± 3.38cm/yr and 12.60 ± 2.94cm/yr respectively during 25 weeks, and recruitment is respectively 11.50 ± 3.58cm/yr and 10.34 ± 3.10cm/yr.The osseous maturation degree relatively has notable statistics difference (p＜0.0001) in the group before and after treatment; And between two groups at FAS and the equal no difference of science of statistics of PPS collection (p＞0.05), more equal not statistically significant (p＞0.05) of osseous maturation degree between two groups of the masculinity and femininities.Stone age in year after the treatment of male FAS collection test group is changed to 1.02 ± 0.77yr, and matched group is 1.20 ± 0.79yr; Stone age in year after the treatment of PPS collection test group is changed to 1.07 ± 0.76yr, and matched group is 1.20 ± 0.79yr.Stone age in year after the treatment of women FAS collection test group is changed to 1.26 ± 1.06yr, and matched group is 1.08 ± 0.74yr; Stone age in year after the treatment of PPS collection test group is changed to 1.26 ± 1.06yr, and matched group is 1.08 ± 0.74yr.Explanation does not promote the faster growth of stone age behind growth hormone therapy.

Sequence table

< 110>Changchun Jinsai Medicine Co.,Ltd

< 120>contain the medicine and the application thereof of PEGization human growth hormone conjugate

<160>1

<210>1

<211>191

<212>PRT

< 213>people (homo sapiens)

<400>1

Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asp Ala Met Leu Arg

1 5 10 15

Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu

20 25 30

Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asp Pro

35 40 45

Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asp Arg

50 55 60

Glu Glu Thr Gln Gln Lys Ser Asp Leu Glu Leu Leu Arg Ile Ser Leu

65 70 75 80

Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Ser Leu Arg Ser Val

85 90 95

Phe Ala Asp Ser Leu Val Tyr Gly Ala Ser Asp Ser Asp Val Tyr Asp

100 105 110

Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu

115 120 125

Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

130 135 140

Lys Phe Asp Thr Asp Ser His Asp Asp Asp Ala Leu Leu Lys Asp Tyr

145 150 155 160

Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

165 170 175

Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe

180 185 190

Claims

1. low dosage and the covalently bound PEGization hGH conjugate that forms of the single free amine group human growth hormone are used for treating the application of the short and small medicine of severe burn or child Te Fa property in preparation, wherein,

Said low dosage is the 0.01-0.26mg/kg body weight;

Said conjugate is by single free amine group covalently bound form of Polyethylene Glycol through N-hydroxy-succinamide base and human growth hormone; Said conjugate detects purity with efficient size exclusion chromatography or RPHPLC and reaches more than 99%; Wherein human growth hormone's aminoacid sequence is Seq ID NO:1, and the molecular weight of Polyethylene Glycol is 36～44kDa; Said conjugate is by being prepared as follows the method preparation, and it comprises:

(b) the reaction mixture dilution that step (a) is obtained in order to the equilibrated Sephadex G-25 of the level pad of pH 7.4 chromatographic column purification, is collected the eluent of first eluting peak; With

(c) eluent that step (b) is obtained is splined on the equilibrated Q-Sepharose chromatographic column of the level pad of pH 7.4, with the level pad eluting that contains 10mMNaCl, then with containing the level pad eluting of 50mM NaCl and collecting eluent.

2. the described application of claim 1, wherein said low dosage are the 0.03-0.25mg/kg body weight.

3. the described application of claim 1, wherein said low dosage are the 0.05-0.24mg/kg body weight.

4. the described application of claim 1, wherein said low dosage are the 0.07-0.23mg/kg body weight.

5. the described application of claim 1, wherein said low dosage are the 0.08-0.22mg/kg body weight.

6. the described application of claim 1, wherein said low dosage are the 0.09-0.21mg/kg body weight.

7. the described application of claim 1, wherein said low dosage are the 0.1-0.20mg/kg body weight.

8. the described application of claim 1, wherein said low dosage are the 0.2mg/kg body weight.

9. the described application of claim 1, wherein said low dosage are 0.15mg/kg body weight or 0.1mg/kg body weight.

10. each described application of claim 1-9, wherein said people patient is the people of child or child's body weight.

11. the described application of each of claim 1-9, wherein said medicine are liquid preparation.

12. the described application of each of claim 1-9, wherein said medicine are injection.

13. the described application of each of claim 1-9, wherein said medicine are the subcutaneous injection agent.

14. be used to treat severe burn or the short and small pharmaceutical composition of child Te Fa property, it comprises low content and covalently bound PEGization hGH conjugate that forms of the single free amine group human growth hormone and pharmaceutically acceptable carrier, wherein,

Said low content is 0.1-10.4mg;

15. the described pharmaceutical composition of claim 14, wherein said low content is 0.3-10mg.

16. the described pharmaceutical composition of claim 14, wherein said low content is 0.5-9.6mg.

17. the described pharmaceutical composition of claim 14, wherein said low content is 0.7-9.2mg.

18. the described pharmaceutical composition of claim 14, wherein said low content is 0.8-8.8mg.

19. the described pharmaceutical composition of claim 14, wherein said low content is 0.9-8.4mg.

20. the described pharmaceutical composition of claim 14, wherein said low content is 1-8mg.

21. the described pharmaceutical composition of claim 14, wherein said low content is 2-8mg.

22. the described pharmaceutical composition of claim 14, wherein said low content is 1.5-6mg.

23. the described pharmaceutical composition of claim 14, wherein said low content is 1-4mg.

24. the described pharmaceutical composition of claim 14, wherein said low content is 9mg.

25. be used to treat severe burn or the short and small medicine of child Te Fa property, it comprises,

(1) container, it is equipped with and the hGH conjugate of human growth hormone's the covalently bound PEGization that forms of single free amine group or each described pharmaceutical composition of claim 14-24, wherein,

(c) eluent that step (b) is obtained is splined on the equilibrated Q-Sepharose chromatographic column of the level pad of pH 7.4, with the level pad eluting that contains 10mMNaCl, then with containing the level pad eluting of 50mM NaCl and collecting eluent; With

(2) be used to indicate label with the hGH conjugate administration of low dosage and the covalently bound PEGization that forms of the single free amine group human growth hormone, wherein,

Said low dosage is the 0.01-0.26mg/kg body weight.

26. the described medicine of claim 25, wherein said low dosage are the 0.03-0.25mg/kg body weight.

27. the described medicine of claim 25, wherein said low dosage are the 0.05-0.24mg/kg body weight.

28. the described medicine of claim 25, wherein said low dosage are the 0.07-0.23mg/kg body weight.

29. the described medicine of claim 25, wherein said low dosage are the 0.08-0.22mg/kg body weight.

30. the described medicine of claim 25, wherein said low dosage are the 0.09-0.21mg/kg body weight.

31. the described medicine of claim 25, wherein said low dosage are the 0.1-0.20mg/kg body weight.

32. the described medicine of claim 25, wherein said low dosage are 0.2mg/kg body weight, 0.15mg/kg body weight or 0.1mg/kg body weight.

33. the described pharmaceutical composition of each of claim 14-24 is used for treating the application of the short and small medicine of severe burn or child Te Fa property in preparation.