CN101010105A - Glycerol branched polyethylene glycol human growth hormone conjugates, process for their preparation, and methods of use thereof - Google Patents
Glycerol branched polyethylene glycol human growth hormone conjugates, process for their preparation, and methods of use thereof Download PDFInfo
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- CN101010105A CN101010105A CNA2005800291171A CN200580029117A CN101010105A CN 101010105 A CN101010105 A CN 101010105A CN A2005800291171 A CNA2005800291171 A CN A2005800291171A CN 200580029117 A CN200580029117 A CN 200580029117A CN 101010105 A CN101010105 A CN 101010105A
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- hgh
- peg
- growth hormone
- conjugate
- pegylation
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Abstract
The present invention relates to PEGylation of human Growth Hormone (hGH) using a glycerol branched PEG. The present invention also relates to processes for the PEGylation of hGH. In addition, the present invention relates to pharmaceutical compositions comprising the PEGylated hGH. A further embodiment is the use of the PEGylated hGH for the treatment of growth and development disorders.
Description
The application requires the U.S. Provisional Application serial number US60/605 that submitted on August 31st, 2004 according to title 35 U.S. code § 119, and 945 priority intactly is incorporated herein by reference the document, as writing this paper.
Invention field
The present invention relates to the Pegylation of human growth hormone (hGH), can change chemistry and/or the physiological property of hGH by this method.Pegylation hGH conjugate can have the blood plasma demurrage of increase, the clearance rate of reduction, the stability of improvement, the antigenicity of reduction, Pegylation inhomogeneities or its combination of minimizing.The invention still further relates to the method for modifying hGH.In addition, the present invention relates to comprise the pharmaceutical composition of modified hGH.HGH the application in treatment g and D obstacle of another embodiment for modifying.
Background of invention
Natural human growth hormone (hGH) is a kind of protein, and it comprises by two 191 amino acid whose strands that disulphide bridges is crosslinked, and this monomeric form has the molecular weight of 22kDa.People GH is by the pituitary gland secretion and can pass through the generation of genetic recombination engineering.HGH can cause growth in all bodily tissues that can grow.HGH is not only aspect the interim promotion growth of human growth, but also keeping play an important role aspect normal health composition, anabolism and the lipid metabolism (K.Bameis. and U.Keller, Baillieres Clin.Endocrinlo.Metab.10:337 (1996)).
Commercially available several years of reorganization hGH.The reorganization hGH goods that have two types treatment application in the market: real a kind of for example Genotropin
TMOr Nutropin
TMAnd on the N-end, have the analog of extra methionine residues, for example a Somatonorm
TMHGH is used for stimulating the patient's who suffers from hypopituitary dwarfism (being also referred to as growth hormone deficiency (GHD)) or Turner syndrome linear growth, also pointed out other indication, comprised that long-term treatment when birth suffer from handkerchief-syndrome Wei (PWS), chronic renal insufficiency (CRI), AIDS less than the child's of gestational age (SGA) growth deficiency, treatment and consume and aged patient.Adult GH lacks (aGHD) patient and has variety of issue, and the characteristic of forming such as health changes, and comprises that fat mass increase, lean body mass and extracellular fluid reduce and bone mineral density decline, abnormalities of sugar/lipid metabolism and cardiovascular function obstacle.Many in those problems are improved (J.Verhelst J and R.Abs.Drugs. by the hGH alternative medicine; 62:2399 (2002).
The main biological action of growth hormone (GH) is to promote immature mammal growth and keep older mammiferous tissue.Affected tract comprises skeleton, connective tissue, muscle and internal organs, such as liver, intestinal and kidney.Growth hormone by with target cell membrane on the interaction of specific receptor bring into play its effect.HGH is the member (Nicoll, C.S. etc. (1986) Endocrine Reviews 7:169) who comprises other heredity of galactagogin, prolactin antagonist and growth hormone and plant the homology hormone family of variant.The uncommon reason of hGH is that it shows species specificity widely and in conjunction with clone's physicogenic (Leung, D.W. etc. [1987] Nature 330 in them; 537) or hprl receptor (Boutin, J.M. etc. [1988] Ce11; 53:69).In bacillus coli (Escherichia coli), expressed the cloned genes (Chang, C.N. etc. [1987] Gene 55:189) of hGH and reported its DNA and aminoacid sequence (Goeddel etc. [1979] Nature 281:544 with secreted form; Gray etc. [1985] Gene 39:247).
Human growth hormone (hGH) participates in the great majority of normal person's bulk-growth and growth are regulated.This pituitary hormone shows numerous biological actions, comprises linear growth (body constitution formation), lactogenic, macrophage activation, Insulin-Like and causes (Chawla, R, K. (1983) Ann.Rev.Med.34,519 such as diabetes effect; Edwards, C.K. etc. (1988) Science 239,769; Thomer, M.O. etc. (1988) J.CHn.Inves are t.81:745).Children growth hormonoprivia causes dwarfism, and successfully having treated by the exogenous hGH of giving should disease more than 10 years.
In adult and child, hGH stores by increase nitrogen and stimulates Skeletal Muscle Growth and keep normal health composition by the mobilization of body fat.Visceral adipose tissue reacts to hGH especially.Except that the lipolysis of strengthening, hGH has reduced triglyceride and has taken in the body fat storage.The serum-concentration of IGF-I (insulin like growth factor-1) and IGFBP3 (insulin-like growth factor binding protein 3) increases because of hGH.
HGH is effective anabolic agent, particularly because storing nitrogen, phosphorus, potassium and calcium.Use GH to treat at least a portion growth rate that HPX rat can recover rat.Endocrinology 122:2920-2926 (1988) such as Moore.Its one of the most significant effect in hypopituitarism (GH-shortage) object is the acceleration linear growth of bone-growth plate-cartilage, thereby causes height to increase.Kaplan,Growth Disorders inChildren and Adolescents(Springfield,IL:Charles C.Thomas,1964).
HGH produces different physiology and metabolism in various animal models, comprise rectilinearity osteogenesis, lactogenic, macrophage activation, Insulin-Like and cause (Annu.Rev.Med.34:519 (1983) such as R.K.Chawla such as diabetes effect; Annu.Rev.Physiol.47 such as O.G.P.lsaksson, 483 (1985); Science such as C.K.Edwards 239,769 (1988); M.O.Thomer and M.L.Vance, J.Clin.Invest.82:745 (1988); J.P.Hughes and H.G.Friesen, Ann.Rev.Physiol.47:469 (1985)).Reported especially in postclimacteric women, the GH secretion descended with the age.Neurobiol.Aging such as Millard, 11:229-235 (1990); NeuroendocrinologyM such as Takahashi, L6-137-142 (1987).In addition, referring to J.CHn.Invest such as Rudman, 67:1361-1369 (1981) and Blackman, Endocrinology and Aging, 16:981 (1987).In addition, report has proposed some aged performance, comprises that lean body mass descends, adipose tissue mass expands and thinning of skin, can by weekly 3 times with the GH treatment and alleviate.For example, referring to N.Eng.J.Med such as Rudman, the article (pp.52-54) that 323:1-6 (1990) and Dr.Vance follow in identical first phase magazine.These biological actions derive from the interaction between hGH and the specific cell receptor.Two kinds of different people's receptors have been cloned, hGH liver receptor (Nature330:537 (1987) such as D.W.Leung) and people's hprl receptor (MoI.Endocrinology.3:1455 (1989) such as J.M.Boutin).Yet, may there be other receptor, comprise human placental lactogen receptor (M.Freemark, M.Comer, G.Komer, and S.Handwerger, Endocrinol.120:1865 (1987)).These homology receptors contain glycosylation born of the same parents pheromone binding structural domain, single membrane spaning domain and cytoplasm domain, its sequence with big or small aspect obviously different.Inferring one or more receptors is playing a decisive role aspect the physiological reaction of hGH.
Generally observedly health is gone in the physiologically active protein administration may only show the pharmacologically active in time limit short period, this is because of due to its high clearance rate in vivo.In addition, these proteinic relative hydrophobicitys can limit its stability and/or dissolubility.
In order to reduce clearance rate, improve stability or to eliminate the proteic antigenic purpose of treatment, certain methods has been proposed, wherein use water-soluble polymer that protein is carried out chemical modification.The such chemical modification effectively physical property of blocks protein hydrolytic enzyme and protein skeleton self contacts, and prevents degraded thus.The chemical bond of some water-soluble polymer can increase because of the hydrodynamics volume of molecule and effectively reduce renal clearance.In some cases, extra advantage comprises the increase proteic stability of treatment and circulation time, increase dissolubility and reduces immunogenicity.Polyalkylene oxide, particularly poly-(ethylene glycol) are that a kind of this class is used to prepare the chemical part (verb " Pegylation " means additional at least one PEG molecule) for the treatment of protein product (PEG).The additional of verified poly-(ethylene glycol) can prevent Proteolytic enzyme, Sada etc., and J.Fermentation Bioengineering 71:137-139 (1991), and can utilize in conjunction with some poly-(ethylene glycol) method partly.Referring to the U.S. Pat 4,179,337 of December in 1979 mandate on the 18th, Davis etc., " non-immunogenic polypeptide class "; With the U.S. Pat 4,002,531 of authorizing on January 11st, 1977, Royer, " using the product of polyethyleneglycol modified enzyme and production thereof ".With regard to summary, referring to Abuchowski etc., EnzymesasDrugs. (J.S.Holcerberg and J.Roberts, eds.pp.367-383 (1981)).
Used other water-soluble polymer, such as ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, glucosan, poly-(vinyl alcohol), poly-(vinylpyrrolidone), poly-(1, the 3-dioxolanes), poly-(1,3,6-three alkane), ethylene/copolymer-maleic anhydride, poly--aminoacid (homopolymer or randomcopolymer).
With regard to poly-(ethylene glycol), used multiple mode to make poly-(ethylene glycol) molecule and protein bound.Generally speaking, poly-(ethylene glycol) molecule is connected with this protein by the reactive group of finding on the protein.Such as those on lysine residue or at the amino of N-end for this class in conjunction with being easily.For example, Royer (above-mentioned U.S. Pat 4,002,531) has described and has been used to make poly-(ethylene glycol) molecule to combine with enzyme reductive alkylation.BioconjugateChem.5:133-140 such as Chamow (1994) have reported by reductive alkylation and have modified the CD4 immune conglutinin with poly-(ethylene glycol) aldehyde of a methoxyl group.U.S. Pat 5,824 has confirmed pegylated G-CSF under the reductive alkylation condition (being included in the N-end) in 784.
Relate to the method for the reactive tissue of GH that stimulates mammal or birds among the WO 93/00109, comprise continuous effective blood plasma GH concentration is kept the time limit more than 3 days or 3 days.Described a kind of mode that obtains this class plasma concentration and be that use and macromolecular substances are such as poly-(ethylene glycol) link coupled GH.Describing with the macromolecular substances coupling to cause the half-life to improve.The human growth hormone who has reported use mPEG aldehyde-5000 and mPEG N-hydroxy-succinamide base ester (mPEG-NHS-5000) Pegylation among the WO 93/00109 with obtain as described greater than the filtering 70K weight shutoff of kidney value (Knauf, M.J etc., J.Biol.Chem.263:15064-15070,1988) hydrodynamics volume.The application of mPEG-NHS has produced the heterogeneous mixture of the multiple Pegylation form of hGH.Also disclosed the application of mPEG-maleimide in making cysteine hGH variant Pegylation among the WO 93/00109.
Disclosed the cysteine variant growth hormone of Pegylation among the WO 99/03887.It is named as BT-005, it is believed that the comparable hGH of this conjugate more effectively stimulating growth hormonoprivia rat weight increase and have the longer half-life.
The succinimido ester of use carboxy methylation PEG such as Clark has also been reported the human growth hormone (Journal of Biological Chemistry271:21969-21977,1996) of Pegylation.Clark etc. have described the hGH derivant of the increase size of using selectivity to be conjugated to the mPEG-NHS-5000 of primary amine class and obtaining.The level increase that PEG modifies reduced it to the affinity of receptor and in based on the test of cell with EC
50Increase is up to 1500 times.Olson etc. are at Polymer Preprints 38:568-569, have disclosed N-hydroxy-succinamide (NHS) PEG and succinyl phosphorons amino propyl acid ester (SPA) PEG in 1997 in the application that obtains multiple Pegylation hGH kind apoplexy due to endogenous wind.
Disclosed to prophesy property Pegylation hGH among the WO 94/20069 as the ingredient of pulmonary delivery with preparation.
US 4,179, disclosed enzyme and the hormone Pegylation method with the water soluble polypeptide conjugate of the non-immunogenic that obtains physiologically active that makes in 337.GH is mentioned as an example that remains in the hormone of Pegylation.
Disclosed among EP 458064 A2 and to have introduced in the growth hormone or the Pegylation of naturally occurring cysteine residues.Further mentioned among EP 458064 A2 two cysteine residues have been introduced in the ring that is called ω ring (it is said at residue 102-112 place) in the wild type bovine somatotropin, more particularly, 102 and 112 the residue of being numbered that has disclosed among EP 458064 A2 respectively bovine somatotropin is replaced into Cys and is replaced into Cys by Tyr by Ser.
The thio group of the cysteine residues of having introduced in having pointed out PEG among the WO 95/11987 and being present in parent molecule or by direct mutagenesis combines.The Pegylation that relates to protease-nexin-1 among the WO 95/11987 but, has also been pointed out hGH and other proteic Pegylation general.
Disclosed among the WO 99/03887 and for example combined the growth hormone of modifying with the cysteine residues of introducing by serine and PEG on replacing 25 with cysteine residues.
Relate to preparation among the WO 00/42175 and contain the proteic method that is useful in conjunction with the free cysteine residues of PEG.The following mutain of the hGH that discloses among the WO 00/42175: T3C, S144C and T148C and cysteine Pegylation thereof.
Relate to of the application of GH variant among the WO 97/11178 (and US 5849535, US 6004931 and US 6022711) as agonist or the antagonist of hGH.Also disclose the Pegylation of hGH among the WO 97/11178, comprised the introducing or the replacement (for example K168A and K172R) of lysine Pegylation and lysine.Also disclosed displacement G120K among the WO 9711178.
Disclose multiple Pegylation hGH kind among the WO 03/044056, comprised the 40K PEG aldehyde hGH conjugate of lysine branching.
Disclosed the PEG butyraldehyde hGH conjugate of lysine branching among the US 2004/0127417.
Have reactive group on the primary carbon on the 1-position that has disclosed at glycerol backbone among WO 04/46222, US 2005/0058620, JP 08-059818, JP 11-228685 and the JP 2000-001541 and on 2-and 3-position, have the polylalkylene glycol derivatives of polyalkylene glycol chain.
Giving at present rhGH every day, to need for a long time time limit and less thus administration frequency be very ideal.HGH molecule with longer circulating half-life can reduce necessary administration number of times and the treatment hGH level of more optimizing may be provided and follow enhanced therapeutical effect.
Although the hGH that has carried out researching and developing long-acting form comprises a large amount of trials of Pegylation hGH, the demand of Pegylation hGH molecule with the proper characteristics that becomes successfully commodity still is not met.The invention provides the PEG-hGH conjugate with the single PEG on the terminal phenylalanine of the N-that mainly is connected hGH, this provides the advantage that surpasses other PEG-hGH conjugate.(Journal of Biological Chemistry271:21969-21977 such as WO 93/00109, Clark, 1996 and Olson etc. (Polymer Preprints 38:568-569,1997) described use mPEG aldehyde-5000 or mPEG N-hydroxy-succinamide base ester (mPEG-NHS-5000) make a plurality of low-molecular-weights (5Kd) PEG be combined in α-or epsilon-amino site (terminal and 9 lysines of the N-on the hGH) on.This has produced inhomogenous colony.As an illustration, have that some molecule can have 10 bonded PEG among the hGH of 9 lysines, some has 9, and some has 8, some has 7, and some has 6, and some has 5, and some has 4, some has 3, and some has 2, and some has 1 and some and has 0.In addition, in having several molecules, PEG can not be combined on the same position of different molecular.There is defective in the inhomogeneities of this generation when research and development treatments product, thus make put together, purification and sign difficulty, cost is high and height can not reproduce.Another kind of means (WO 00/42175) have been used and have been contained the hGH variant that is useful in conjunction with the free cysteine residues of PEG.Yet this means produce non-natural hGH variant and also can cause having the incorrect folding protein of incorrect paired disulfide bond, thereby produce the product that PEG is combined in the inhomogeneous Pegylation on some or all cysteine.A plurality of PEG combine with a plurality of sites and can cause molecule to have key so unstable between PEG and the different loci, and it can dissociate with different rates.This makes and causes the pharmacokinetics that is difficult to accurately predict product quantitatively inaccurate.Also there is disadvantageous problem in acquisition administration section in inhomogeneous product aspect the approval of treatment product.
Therefore, need have Pegylation hGH molecule with the single PEG that is combined on the single site.The present invention has solved this demand in many ways.
Summary of the invention
The present invention relates to use the Pegylation hGH of glycerol branched (branched) poly-(ethylene glycol) part, it can have the chemistry or the physiological property of at least a improvement, and this characteristic is selected from but the clearance rate that is not limited to reduce, the blood plasma demurrage of increase, the stability of increase, the dissolubility of improvement and the antigenicity of minimizing.Therefore, as what hereinafter more specifically describe, the present invention has a plurality of aspects of using glycerol branched poly-(ethylene glycol) part chemical modification hGH that relate to.
The present invention can also have one or more and the characteristic of comparing improvement based on the PEG human growth hormone conjugate of the branching of lysine, includes, but are not limited to: a) the glycerol backbone stability of Zeng Jiaing; B) receptors bind of Zeng Jiaing; C) cost of Jiang Diing; D) the N-end of Zeng Jiaing is in conjunction with selectivity; E) dissolubility of Zeng Jiaing; F) immunogenicity of Jian Shaoing; G) the conjugate stability of Zeng Jiaing; H) manufacturability of Zeng Jiaing; And i) Proteolytic enzyme reduces.
The invention still further relates to the method for producing Pegylation hGH.Especially, the present invention relates to use glycerol branched PEG to produce the method for Pegylation hGH.
The invention still further relates to comprise independent or with the compositions of the Pegylation hGH of another kind of therapeutic agent combination.The invention still further relates to independent Pegylation hGH of the present invention or itself and being combined in of another kind of therapeutic agent and prevent and/or treat useful disease of GH treatment therein and/or the application in the disease.
The accompanying drawing summary
Accompanying drawing 3 shows human growth hormone's aminoacid sequence (SEQ ID NO:1).
Accompanying drawing 7 shows that 6 days dosage of glycerol branched 43K PEG aldehyde hGH increase efficacy study.According to described in the accompanying drawing 4 similarly mode carry out this increment study, but the glycerol branched 43K PEG aldehyde hGH of the single dose that only changed and this research carried out 6 days at the 0th day.Matched group was accepted 1 subcutaneous injection 0.3mg/kg hGH (◆) or PBS carrier (o) every day continuous 6 days.Glycerol branched 43K PEG aldehyde hGH test group was accepted the glycerol branched 43K PEG aldehyde hGH of single dose in the time of the 0th day.Glycerol branched 43K PEG aldehyde hGH dosage is 1.8mg/kg (■), 0.6mg/kg (X), 0.2mg/kg (+), 0.067mg/kg (▲).6 animals are arranged in every group.
Accompanying drawing 8 shows the serum I GF-1 level of 6 days efficacy studies.Described in accompanying drawing 7, treat animal.Measure serum I GF-1 level at the different time points blood sampling of drawing and by ELISA.To organize (n=6) meansigma methods is used to use one way analysis of variance that the value of measuring is calculated the IGF-1 response, and to 1.8,0.6,0.2 and 0.067mg/kg administration group measured 37,839,28,1292,22 respectively, 958 and 20,040 AUC d0-6 (ng/mL*24h) value.
Accompanying drawing 9 shows that the PK/PD that HPX female rats is given behind the glycerol branched 43KPEG aldehyde hGH of single dose estimates.1.8mg/kg the single dose administration of the glycerol branched 43K PEG aldehyde hGH of SC is to the effect of drug plasma level (a) or blood plasma IGF-1 response (b).
Detailed Description Of The Invention
The present invention relates to glycerol branched polyethylene glycol-human growth hormone (HGH) conjugate. In a specific embodiment, glycerol branched polyethyleneglycol derivative with between the reactive functional groups at the primary carbon place on aldehyde reaction base and the optional polyethylene glycol that exists and the glycerol backbone 1-position be connected base and described in WO 04/46222 or US 2005/0058620 (being incorporated herein by reference) on 2-and 3-position with polyalkylene glycol chain, so that generation hGH conjugate. To the basic not special restriction of described connection, as long as it is covalent bond, but preferably include alkylidene and the alkylidene that contains ester bond, urethane key, amido link, ehter bond, carbonic acid ester bond or secondary amino group. Preferred alkylidene comprises methylene, ethylidene, 1,3-propylidene, propylidene, inferior isopropyl, Isosorbide-5-Nitrae-butylidene, butylidene, isobutylidene, 1,5-pentylidene and 1,6-hexylidene.
A specific embodiment of the present invention is human growth hormone (HGH) with following formula-PEG conjugate:
Wherein:
N is the integer of 60-75;
M is the integer of 450-460; And
R is people's growth hormone polypeptides.
In a specific embodiment, n is about 64-about 72.
In a specific embodiment, (CH
2CH
2O)
nMean molecule quantity and particularly mean molecule quantity that part has the about 3.5Kd of about 2.6-are about 3Kd.
In a specific embodiment, each (CH
2CH
2O)
mMean molecule quantity and particularly mean molecule quantity that part has the about 22Kd of about 17.6-are about 20Kd.
In a specific embodiment, (CH
2CH
2O)
nPart has mean molecule quantity and the each (CH of about 3Kd
2CH
2O)
mPart has the mean molecule quantity of about 20Kd.
Term " pact " means in the Polyethylene Glycol goods with the molecular weight coupling of peg moiety the time, and the weight of some molecule is higher than, some is lower than described molecular weight, and described molecular weight means mean molecule quantity.Should understand existence to a certain degree with polymer such as poly-(ethylene glycol) relevant polydispersity.The preferred PEG that uses with low polydispersity.In a specific embodiment, one of end polymer hydroxyl end groups is transformed or by methyl blocking.Term used herein " mPEG " means PEG, and it is at one end by methyl blocking.MPEG structurally can be represented as:
CH
3O-(CH
2CH
2O)
n-H
Term " human growth hormone's polypeptide ", " hGH polypeptide " or " hGH albumen " comprise all hGH polypeptide classes when using in this article, it is characterized in that promoting growth and keeping normal body composition, anabolism and lipid metabolism at trophophase.Preferred term " hGH polypeptide " means the hGH polypeptide of SEQ ID NO:1.
Can prepare hGH polypeptide class of the present invention according to the mode of any appropriate.This class hGH polypeptide class and its fragment can be used and well known to a person skilled in the art that the technology purification is from natural origin, by chemosynthesis, produce by recombinant technique, comprise external translation technology or in the reconstitution cell that can express hGH cDNA, express, or the combination of these methods is (for example, for being used for the whole bag of tricks of protein purification, referring to " Methods in Enzymology; AcademicPress, 1993 "; Creighton, (1983) Proteins:Structures andMolecular Principles, W.H.Freeman﹠amp; Co.2nd Ed., T.E., NewYork; With with regard to chemosynthesis protein, referring to (1984) Nature.310 (5973): 105-11 such as Hunkapiller; And with regard to recombinant technique, referring to (1986) BasicMethods in Molecular Biology such as Davis, ed., Elsevier Press, NY is incorporated herein by reference the content intact that these documents are disclosed).Preferably providing polypeptide class of the present invention and they with isolating form can be part or preferred purification basically.
A specific embodiment of the present invention is human growth hormone-PEG conjugate, wherein greater than 80%, more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably amino-terminal phenylalanine of the human growth hormone of 97% and more preferably 98% Polyethylene Glycol and SEQ ID NO:1 is puted together.
Another embodiment of the invention is to choose the terminal Pegylation hGH of the N-goods of the homogeneous basically in pharmaceutically acceptable diluent, carrier or adjuvant wantonly, is substantially free of the hGH of Pegylation on the site of non-N-end in the described goods.Term " goods of homogeneous basically " means a kind of goods, wherein greater than 80%, and more preferably 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, more preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97% and more preferably 98% is (monoPEGylated) of a Pegylation.
In one embodiment of the invention, as Bioconjugate Chem.5:133-140 (1994), U.S. Pat 4,002 such as Chamow, 531, WO 90/05534 and U.S. Pat 5,824 are described in 784, use appropriate reductant, such as NaCNBH
3, NaBH
3, pyridine borine etc., between the N-of hGH polypeptide terminal primary alpha-amido and glycerol branched chain PEG aldehyde, form the secondary amine key by reductive alkylation.Glycerol branched PEG aldehyde with hGH polypeptide incubation, is caused by schiff bases formation peg moiety being added on the amino.By using the Reducing agent reduction that these keys are changed into stable secondary amine class.The reductive alkylation reaction process is (from Chamow etc.) as shown in following scheme.
The conjugation reaction that is called " pegylation reaction " is used the polymer of molar excess to carry out in history in solution and is not considered that polymer will be attached to protein wherein.Yet, the common verified enough biological activitys that is not enough to when biological activity protein and nonantigenic polymer are puted together, keep of this class general technology.The bioactive mode of a kind of hGH of keeping is to avoid basically puting together those hGH reactive groups relevant with receptor binding site in the polymer coupling process.Another aspect of the present invention is to provide the method that makes poly-(ethylene glycol) and hGH put together and keep high-level retentive activity.
Can under the condition of any appropriate that generally is used for bioactive substance and activatory poly-(ethylene glycol) reaction, carry out chemical modification by covalent bond.Carrying out conjugation reaction to avoid making the hGH inactivation under the temperate condition relatively.Gentle condition comprises that the pH with reaction solution maintains the scope of 3-10 and reaction temperature is maintained in about 0 ℃-37 ℃ scope.With regard to the situation that the reactive amino acid residues among the hGH has free amine group, above-mentioned modification preferably at 4 ℃-37 ℃ following and non-limiting suitable buffer of enumerating (pH4-10), comprises and carrying out among phosphate, MES, citrate, acetate, succinate or the HEPES 1-48 hour.Use reagent such as PEG aldehyde targeting-terminal amino acid in, preferably keep pH4-8.Can be doubly with about 0.01-100, the preferably about 0.01-2.5 times of mole to the free amine group number of hGH used activatory poly-(ethylene glycol).
Although reaction condition as herein described can produce the hGH of a large amount of unmodifieds, be easy to make the hGH of unmodified to be recirculated into following batch, so that carry out extra conjugation reaction.Method of the present invention has produced unexpectedly seldom, promptly is lower than about 20% and contain the kind of one or more polymer chain more preferably less than about 10% high molecular weight species and each hGH.These reaction conditions form contrast with those conditions that the typical case is used for the polymer conjugation reaction, and activatory polymer is to exist with respect to target several times molar excess among the latter.
Conjugation reaction of the present invention provides reactant mixture or the storehouse of containing one-PEG-hGH conjugate, unreacted hGH, unreacted polymer and being lower than about 20% high molecular weight species at first.High molecular weight species comprises conjugate and/or the polymeric PEG-hGH kind that contains one or more polymer chain.After unreacted kind and high molecular weight species are removed, reclaimed the compositions of the hGH conjugate that mainly contains one-Pegylation.In view of the conjugate major part comprises this fact of single polymer chain, so conjugate is essentially homogeneous.It is active at least about 0.1% that the hGH of these modifications has the external biological relevant with the hGH of natural or unmodified, as using standard FDC-P1 cell proliferation test (Journal of BiologicalChemistry 271:21969-21977 such as Clark, 1996), (US 5 for receptor binding assays, 057,417) or the growth of pituitectomy rat Journal of Biological Chemistry271:21969-21977 such as (, 1996) Clark measure.Yet, in preferred aspects of the invention, the hGH that modifies has about 25% external biological activity, more preferably the hGH of Xiu Shiing has about 50% external biological activity, more preferably the hGH of Xiu Shiing has about 75% external biological activity, and the hGH that most preferably modifies has the external biological activity that is equal to or improves.
Method of the present invention preferably includes the ratio of quite limited polymer and hGH.Therefore, have been found that the hGH conjugate mainly is limited to the kind that only contains a polymer chain.In addition, polymer significantly is lower than the situation of using high molar excess polymer to be connected base with the bonded randomness of hGH reactive group.After stopping conjugation reaction, can use ion exchange or size exclusion chromatography or similar isolation technics to make the hGH that is present in the unmodified in the reaction gleanings be recirculated into following reaction.
Can be by being used for the conventional method of protein purification, such as dialyse, saltout, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatograph (HIC), gel chromatography and electrophoresis, the hGH of purification poly-(ethylene glycol) from reactant mixture-modify.Ion exchange chromatography can be removed unreacted poly-(ethylene glycol) and hGH especially effectively.In another embodiment of the invention, from reactant mixture, separate the hGH kind of a Pegylation so that remove high molecular weight species and the hGH of unmodified.By blended kind being placed the buffer solution that contains the 0.5-10mg/mL hGH-polymer conjugate of having an appointment separate.Suitable solution has the pH of about 4-about 10.These solution preferably contain one or more buffer salts, and they are selected from KCl, NaCl, K
2HPO
4, KH
2PO
4, Na
2HPO
4, NaH
2PO
4, NaHCO
3, NaBO
4, CH
3CO
2H and NaOH.
According to the difference of reaction buffer, hGH polymer conjugate solution may at first must carry out buffer-exchanged/ultrafiltration so that remove any unreacted polymer.For example, can be with the membrane ultrafiltration of PEG-hGH conjugate solution by low-molecular-weight cutoff (10,000-30,000 dalton) so that remove most of unwanted material, such as unreacted polymer, show activating agent (if existence) etc.
The preferred gleanings (pool) that uses the ion exchange chromatography medium conjugate fractionated to be gone into to contain the expectation kind.This class medium can be by the charge differences selective binding PEG-hGH conjugate that changes in to a certain degree predictable mode.For example, determine the surface charge of hGH according to the number of the charged groups utilized on the protein surface.These charged groups generally are used as the possible attachment point of polyalkylene oxide hydrocarbon polymer.Therefore, the hGH conjugate can have be different from other kind electric charge to allow to carry out Selective Separation.
Strong polarity anion or cation exchange resin such as quaternary ammonium or sulfopropyl resin, are used for method of the present invention respectively.Especially preferred anionic exchanger resin.Comprise and be applicable to that the non-limiting tabulation that is purchased cation exchange resin of the present invention is SP-hitrap , SP SepharoseHP and SP Sepharose fast flow.Can also use other suitable cation exchange resin, for example S and CM resin.Be applicable to that anion exchange resin of the present invention comprises that the non-limiting tabulation that is purchased anion exchange resin is Q-hitrap , Q Sepharose HP and Qsepharose fast flow.Can also use other suitable anion exchange resin, for example the DEAE resin.
For example, preferably with anion or cation exchange resin is filled in the post and balance by conventional methods.Use has the identical pH and the buffer of osmolality with the hGH solution that polymer is puted together.Elution buffer preferably contains one or more salt, and they are selected from KCl, NaCl, K
2HPO
4, KH
2PO
4, Na
2HPO
4, NaH
2PO
4, NaHCO
3, NaBO
4(NH
4)
2CO
3Make the solution absorbs that contains conjugate then to the post of not detention unreacted polymer and some high molecular weight species.When end of the sample, will have the elution buffer gradient current upper prop of salinity of increase so that the eluting polyalkylene oxide-the required fraction of the hGH that puts together.After cation or anion exchange separation step, the collection fraction of eluting preferably is limited to the homogeneous polymer conjugate.Then can be by routine techniques any unconjugated hGH kind of backwash from the post.If desired, can be further by extra ion exchange chromatography or size exclusion chromatography with one and the hGH kind of poly ethylene glycolization separated from one another.
Can also use the technology of the degree steps such as many that use to increase salinity or pH.The many isocratic elutions step that increases concentration makes two-and be one-hGH-polymer conjugate eluting successively then.
The temperature range that is used for eluting is at about 4 ℃-Yue 25 ℃.Preferably under about 4 ℃-Yue 22 ℃ temperature, carry out eluting.For example, by eluting at the UV at 280nm place absorption detecting PEG-hGH fraction.Can carry out fraction by simple time eluting spectrogram collects.
Surfactant can be used for method that poly-(ethylene glycol) polymer and hGH are partly puted together.Suitable surfactant comprises ion-type reagent, such as sodium lauryl sulphate (SDS).Can also use other ionic surfactant, such as lithium dodecyl sulfate, quaternary ammonium compound, taurocholic acid, sad, last of the ten Heavenly stems sulfonic acid etc.Also can use nonionic surfactant.For example, can use such as poly-(oxygen ethylene) anhydrosorbitol class (Tweens), poly-this class material of (oxygen ethylene) ethers (Tritons).In addition, referring to Neugebauer, A Guide to theProperties and Uses of Detergents in Biology andBiochemistry (1992) Calbiochem Corp..To unique restriction of the surfactant that is used for the inventive method is that they are in the substantive irreversible denaturation that can not cause hGH with not exclusively suppress to use under condition that polymer puts together and the concentration.The about 0.01-0.5% of the amount of surfactant in reactant mixture; Preferred 0.05-0.5%; And 0.075-0.25% most preferably from about.Also pay close attention to surfactant mixtures.
Think that surfactant puts together the reversible protection system that provides temporary transient in the process at polymer.The verification table surface-active agent effectively selectivity hinder polymer and put together, allow simultaneously based on lysine or based on carrying out aminoterminal puting together.
The hGH of poly-(ethylene glycol) of the present invention-modification has more persistent pharmacological action, and this may be owing to the half-life in the body of its prolongation.
Another embodiment of the invention relates to being used to prevent and/or treat wherein uses the useful disease of the preferred hGH of GH or the method for disease, comprise hGH or its agonist variant of the patient that these needs are arranged being treated the present invention's poly-(ethylene glycol) of effective dose-modify, independent or with another kind of therapeutic agent combination.The invention still further relates to the hGH of the present invention's poly-(ethylene glycol)-modification or its agonist variant is used for preventing and/or treating the medicine that wherein uses useful disease of the preferred hGH of GH or disease in preparation application.In addition, the invention still further relates to be used to prevent and/or treat and wherein use the hGH that comprises the present invention's poly-(ethylene glycol)-modify of useful disease of the preferred hGH of GH or disease or the pharmaceutical composition of its agonist variant.
Wherein use useful disease of GH or disease to include but not limited to growth hormone deficiency (GHD), adult's growth hormone deficiency (aGHD), Turner syndrome, during birth less than the child's of gestational age (SGA) growth deficiency, handkerchief-syndrome Wei (PWS), chronic renal insufficiency (CRI), AIDS consumes, old and feeble, whole latter stage renal failure, cystic fibrosis, erectile dysfunction, the HIV lipodystrophy, fibromyalgia, osteoporosis, dysmnesia, depression, Crohn disease, skeleton development is unusual, traumatic brain injury, subarachnoid hemorrhage, Noonan syndrome, mongolism, the special property sent out short stature (ISS), end stagerenaldisease (ESRD), extremely low birth weight (VLBW), the bone marrow stem cell rescue, metabolism syndrome, the glucocorticoid myopathy, short stature that causes because of the glucocorticoid among child treatment and short and small premature infant's growth lag behind.
In more particular embodiment of the present invention, the hGH of poly-(ethylene glycol) of the present invention-modification or its agonist variant are used to prevent and/or treat the disease or the disease of the group that is selected from GHD, aGHD, SGA, PWS, Turner syndrome and CRI composition.
In another more particular embodiment of the present invention, the hGH of poly-(ethylene glycol) of the present invention-modification or its agonist variant are used to prevent and/or treat the disease or the disease of the group that is selected from special property short stature, extremely low birth weight, traumatic brain injury, metabolism syndrome and Noonan syndrome composition.
Another embodiment of the invention relates to pharmaceutical composition, and it comprises the hGH of poly-(ethylene glycol) of the present invention-modification, make up separately or with another kind of therapeutic agent, and at least a pharmaceutically acceptable excipient or carrier.The medicament that the hGH of poly-(ethylene glycol) of the present invention-modify can be mixed with the reagent that also contains pharmaceutically acceptable diluent, be used to prepare isosmotic solution, pH-regulator etc. then is so that to patient's administration.
Can be according to the difference of therapeutic purposes by subcutaneous, intramuscular, intravenous, pulmonary, intradermal or orally give said medicine.Dosage can also be based on the kind and the state of an illness of treatment patient's disease, for the adult, generally is 0.1mg-5mg by injection, and oral administration is 0.1mg-50mg.
The present invention used herein gather the hGH of (ethylene glycol)-modify or its agonist variant can with another kind of therapeutic agent coupling.Term " co-administered ", " using jointly " and " combination/coupling " that relates to compd A and one or more other therapeutic agents used herein means and really refers to and comprise following implication:
о gives the combination of this class A and therapeutic agent simultaneously to the patient who has treatment to need, and jointly be mixed with single dosage form with this constituents this moment, and this dosage form discharges described composition in simultaneously mode basically to described patient;
о gives the combination of this class A and therapeutic agent basically simultaneously to the patient who has treatment to need, be separated from each other this constituents and be mixed with independent dosage form this moment, described dosage form simultaneously by described patient's picked-up, is given described patient so described composition discharges basically simultaneously basically;
о gives the combination of this class A and therapeutic agent successively to the patient who has treatment to need, be separated from each other this constituents and be mixed with independent dosage form this moment, these dosage forms are absorbed in continuous time by described patient, there is significant interval between each administration, so described composition discharged to described patient with the different basically time; With
о gives the combination of this class A and therapeutic agent successively to the patient who has treatment to need, jointly be mixed with single dosage form with this constituents this moment, this dosage form discharges described composition in a controlled manner, then they at the same time and/or during different time to described patient simultaneously, continuous and/or overlapping staggered giving.
Can comprise with the suitable example of other therapeutic agent of A, its pharmaceutically acceptable salt and/or its derivative form coupling, but be not limited to: aromatase inhibitor, such as exemestane, formestane, atamestane, fadrozole, letrozole, vorozole and Anastrozole; The free-fat acid regulator comprises fibric acid derivant (such as fenofibrate, clofibrate, gemfibrozil, bezafibrate and ciprofibrate) and nicotinic acid derivates, such as acipimox; Insulin sensitizer includes but not limited to biguanides, such as metformin; PPAR γ insulin sensitizer and thiazolidinediones (thiazolodeniones), such as troglitazone and rosiglitazone: troglitazone, 5-[[4-[3,4-dihydro-6-hydroxyl-2,5,7,8-tetramethyl-2H-]-.alpha.-5:6-benzopyran-2-yl] methoxyl group] phenyl] methyl 3-2,4-thiazolidinedione V411 (DIABII, Glaucanin); Pioglitazone (ACTOS, AD 4833, and U 72107, U 72107A, U 72107E, ZACTOS), and chemical name: 2,4-thiazolidinedione, 5-[[4-[2-(5-ethyl-2-pyridine radicals) ethyoxyl] phenyl] methyl]-, a hydrochlorate, (a/-); Rosiglitazone (Avandia, BRL 49653, BRL49653C) chemical name: 2,4-thiazolidinedione, 5-[[4-[2-(methyl-2-pyridinylamino) ethyoxyl] phenyl] methyl]; 25 bexarotenes-oral (LGD 1069 oral, Targretin is oral, Targretin, Targretyn are oral, Targrexin is oral) chemical name: 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydrochysene-2-naphthyl) vinyl] benzoic acid; ZD2079, (ICI D 2079) (chemical name: R)-and N-[2-4-(carboxymethyl) 30 phenoxy groups] ethyl)-N-(2-hydroxyl-2-phenethyl) ammonium chloride: Netoglitazone, (Isaglitazone, MCC 555, RWJ 241947) (chemical name: 5-[6 (2-fluorine benzyloxy) naphthalene-2-ylmethyl] thiazolidine-2, the 4-diketone); INS (D-chirality-inositol) (chemical name: D-1,2,3,4,5,6-Inositol), ON 2344 (DRF 2593); Dexlipotam, chemical name: 5 (R)-(1,2-dithiolane-3-yl) pentaloic 35 acid; HQL 975, chemical name: 3-[4-[2-(5-methyl-2-phenyl azoles-4-yl) ethyoxyl] phenyl]-2 (S)-(third amino) propanoic acid; YM 268, chemical name: 5,5 '-methylene-two (1, the 4-phenylene) dimethylene two (thiazolidine-2,4-diketone).I PPAR agonist in research and development comprises: (JTT 501 for Reglitazar, PNU182716, PNU 716) (chemical name: different azoles alkane diene-3,5-diketone, i 4-[[4-(2-phenyl-5-methyl)-1,3- azoles base] ethoxyl phenenyl-4] methyl-, (4RS)); (RP 297, chemical name: 10 5-(2,4-dioxo Thiazolidine Thiazolidine-5-ylmethyl)-2-methoxyl group-N-[4-(trifluoromethyl) benzyl Benzoylamide for I; R 119702 (C1 1037, and CS 011) chemical name: (/-)-5-[4-(5-methoxyl group-1H benzimidazolyl-2 radicals-ylmethoxy) benzyl] thiazolidine-2, the 4-dione hydrochloride; 15 DRF 2189, chemical name: 5-[[4-[2-(1-indyl) ethyoxyl] phenyl] methyl] thiazolidine-2, the 4-diketone; The cortisone synthetic inhibitor is such as ketoconazole, econazole or miconazole; Growth hormone is such as growth hormone (somatropin) or growth hormone (somatonorm) and derivant thereof, as human growth hormone's fusion rotein, such as ALBUTROPIN; The Polyethylene Glycol growth hormone is such as growth hormone, the BT005 (Bolder BioTechnology Inc.) of cysteine-Pegylation; Growth hormone cinogenic agent, such as, for example SM 130686 (Sumitomo) capromorelin (Pfizer), mecasermin (Fujisawa), Sermorelin (Salk Institute, Bio-Technology General), somatrem, somatomedin (C Llorente; Pharmacia Corporation) Examorelin, tabimorelin; CP 464709 (Pfizer), LY 426410 and LY 444711 (Lilly); As 8-(aminoalkoxy the imino group)-8H-dibenzo [a that discloses among the WO2002057241, e] triazol [4,5-c] [7] cycloheptene class, dibenzo [a that 2-described in WO2002056873 replaces, e] 1,2,3-triazol [4,5-c] [7] annulene-8-ketone, as U.S. Pat 4,411,890 and open source literature WO 89/07110, growth hormone-releasing peptide class GHRP-6 and GHRP-1 described in the WO 89/07111, B-HT920 described in WO 93/04081, hexarelin and GHRP-2 or growth hormone releasing hormone (GHRH, also called after GRF) and analog thereof; Somatomedin comprises IGF-1 and IGF-2 and derivant thereof, such as the reorganization fusant of a Somatokine-insulin-like growth factor-i BP-3 conjugated protein with it; α-2-2-adrenergic agonist components is such as clonidine, xylazine, detomidine and medetomidine; Or 5-hydroxy tryptamine 5HTID agonist, such as surnitriptan or suppress somatostatin or the activating agent of its release, such as physostigmine and pyridostigmine, ThGRF1-44 (Theratechnologies); L 165166 (Merck﹠amp; Company); Dipeptidase derivant described in WO9858947; Inhibitors of dipeptidyl IV, the amino described in US6521644, WO95/15309 and WO98/19998-acyl-pyrrolidine nitrile; Beta-amino heterocycle depeptidyl peptidase inhibitors, those inhibitor described in US20030100563 and WO2003082817; Growth hormone releasing compounds described in following document: US20030055261, US20030040483, EP18072, EP83864, WO89/07110, WO89/01711, WO89/10933, WO88/9780, WO83/02272, WO91/18016, WO92/01711, WO93/04081, WO9514666, EP0923539, U.S. Pat 5,206,235,5,283,241,5,284,841,5,310,737,5,317,017,5,374,721,5,430,144,5,434,261,5,438,136,5,494,919,5,494,920,5,492,916,5,536,716 and 5,578,593, WO94/13696, WO94/19367, WO95/03289, WO95/03290, WO95/09633, WO95/11029, WO95/12598, WO95/13069, WO95/14666, WO95/16675, WO95/16692, WO95/17422, WO95/17423, WO95/34311 and WO96/02530; Piperidines described in US5804578, US5783582, WO2004007468, pyrrolidines and hexahydro-1 H-azepines class; Acylamino-spiroperidol class, the chemical compound described in those WO0104119; 2-amino-5-pyrimidine acetic acid compound, comprise the 2-[(5 described in US6329383,6-dimethyl-2-benzimidazolyl) amino]-4-hydroxyl-6-methyl-5-pyrimidine acetic acid (2) and 2-[(5,6-dimethyl-2-benzimidazolyl) amino]-4-hydroxyl-6-methyl-5-pyrimidine ethyl acetate; Benzimidazole described in EP1155014; The peptide class of similar Peptidyl compounds relevant and U.S. Pat 4,411,890 with GRF; Antagonists of gonadotropin-releasing hormone is described in chemical compound among WO0170228, WO0170227, WO0170228, WO0069433, WO0004013, W0995156, WO9951595, WO9951231-4, WO9941251-2, WO9921557, the WO9921553 and the 6-azaindole chemical compound described in WO0053602, WO0053185, WO0053181, WO0053180, WO0053179, WO0053178, US6288078 such as those; The IGF-1 sercretogogue; Insulin like growth factor-2 (IGF-2 or SM-A) and IGF-2 sercretogogue; Tubocurarine antagonist and the chemical compound that is suppressed to bfgf receptor-3 (FGFR-3) tyrosine kinase.
The polymer material that comprises is also preferably at room temperature for water miscible.The non-limiting tabulation of this base polymer comprises poly-(epoxyalkane) homopolymer such as poly-(ethylene glycol) or poly-(propylene glycol), poly-(oxyethylation polyhydric alcohol), its copolymer and block copolymer thereof, and condition is that the water solublity of block copolymer is maintained.
Alternative as based on the polymer of PEG can be used nonantigenic effectively material, such as glucosan, poly-(vinylpyrrolidone), poly-(acrylamide), poly-(vinyl alcohol), based on the polymer of carbohydrate etc.In fact, can according to be used for transforming poly-(epoxyalkane) similarly mode carry out the α of these polymer materials-and activation of ε-end group and apparent to those skilled in the art thus.Those skilled in the art will recognize that above-mentioned tabulation only is all polymer materials indicative and that concern has character described herein.With regard to purpose of the present invention, " nonantigenic effectively " means and is interpreted as the mammal avirulence in this area and can not causes all substances of significant immunogenic response.
Definition
Be to exchange the abbreviation of use and the tabulation of corresponding meaning in this article below:
-g gram
-mg milligram
-ml or mL milliliter
-RT room temperature
-PEG gathers (ethylene glycol)
-full content of all open source literatures, patent and the patent application of quoting from this description is incorporated herein by reference, just as each open source literature, patent or patent application are incorporated herein by reference especially and respectively separately.
Although purpose is clear to understand by explaining and example is described above-mentioned invention in detail, can under the situation that does not break away from essence of the present invention and scope, change according to instruction of the present invention it will be apparent to those skilled in the art with modification.Provide the following example only being illustrated as purpose, but be not intended to limit above with the described scope of the present invention of wide region term.
In the following example, hGH is the hGH of SEQ ID NO:1.Should understand other hGH polypeptide class also can be as the illustrative similar fashion of embodiment subsequently by Pegylation.
Embodiment
Glycerol branched 43K PEG aldehyde hGH
Wherein:
(CH
2CH
2O)
nMean molecule quantity and each (CH with about 3Kd
2CH
2O)
mMean molecule quantity with about 20Kd.
(GL3-400AL2)
Present embodiment has shown the terminal Pegylation hGH by reductive alkylation generation N-.Pass through reductive alkylation, relative pKa value by utilizing primary amine on the N-end is poor with respect to the pKa value of the locational primary amine class of the epsilon-amino of lysine residue, make about 43, the glycerol branched PEG aldehyde reagent of 000MW (GL3-400AL2 NOF company) and the terminal coupling of the N-of hGH.Make with 4,7 or 10mg/mL be dissolved in 25mM MES (the Sigma Chemical of pH5.8, St.Louis, MO) or the hGH protein of the 20mM HEPES of pH7.0 and glycerol branched 43K PEG aldehyde to obtain relative PEG:hGH mol ratio be to react in 1.5: 1,2: 1,3.4: 1,4: 1 or 5: 1 by adding reagent.By add the pyridine borine (Sigma Chemical, St.Louis, MO) to final concentration be the 10mM catalytic reaction.Make be reflected at 4 ℃ down and the dark place carried out 16-87 hour.Go into suitable buffer cessation reaction so that carry out purification by dilution.
Table 1 shows the percentage ratio of the kind of many-Pegylation, the conjugate of one-Pegylation, unreacted hGH and at pH5.8 with mol ratio under react the final purification productive rate of 63 hours glycerol branched 43K PEG aldehyde hGH at 1.5: 1: 1.
Synthetic and the purification process of 43K PEG aldehyde hGH conjugate that table 1 is glycerol branched
Synthetic and the purification productive rate of glycerol branched 43K PEG aldehyde-hGH | |
Kind in the reactant mixture: | |
Glycerol branched 43K PEG aldehyde-hGH | |
Many-the |
4% |
One-PEG product | 68% |
Unreacted hGH | 28% |
The purification |
35% |
The purification of Pegylation hGH
Use single ionic exchange chromatography step from reactant mixture purification Pegylation hGH kind to>95% (SEC analyzes, accompanying drawing 1).
Anion-exchange chromatography
Use single ionic exchange chromatography step from reactant mixture purification PEG hGH kind to>95% (SEC analyzes, accompanying drawing 1).Use hGH and the many-Pegylation hGH kind apoplexy due to endogenous wind purification one-Pegylation hGH of anion-exchange chromatography from unmodified.Using 25mM HEPES, pH7.3 (buffer A) equilibrated Q-Sepharose Hitrap post (5mL) (AmershamPharmacia Biotech, Piscataway, NJ) or Q-Sepharose post (26/20, the 70mL bed volume) (Amersham Pharmacia Biotech, Piscataway NJ) goes up the aforesaid typical glycerol branched 43K PEG aldehyde hGH reactant mixture of purification (80 or 1500mg protein).With buffer A with this reactant mixture dilution 7X and with 2.5mL/ minute flow velocity upper prop.Buffer A column scrubber with 3-10 column volume.The LINEAR N aCl gradient of using the buffer A of 20 column volumes and 0-100mM subsequently different hGH kind of eluting from the post.According to absorbance (A at 280nm
280) monitor eluent and collect the fraction of suitable size.Fraction is compiled according to the degree of Pegylation, for example one-, two-, three-etc. (as estimating among the embodiment 3).Then with gleanings Centriprep YM10 concentrator (Amicon, Technology Corporation, Northborough, MA) in or be concentrated into 0.5-5mg/mL by diafiltration.Pass through A
280, the extinction coefficient of use 0.78 are measured the protein concentration of gleanings.
The biochemical sign
Characterize the Pegylation hGH gleanings of purification by non-reduced SDS-PAGE, non-degeneration size exclusion chromatography and peptide mapping.
Size exclusion high performance liquid chromatography (SEC-HPLC)
Use non-degeneration SEC-HPLC to estimate the product of reactant mixture, anion exchange purification gleanings and the final purification of glycerol branched 43K PEG aldehyde and hGH.Post TSK G4000PWXL (Tosohaas) or the ShodexKW-804 (Waters Corp) of use in 20mM phosphate pH7.2,150mM NaCl, with 0.5mL/ minute flow velocity (Superdex 2007.8mm * 30cm randomly, Amersham Bioscience, Piscataway, NJ), carry out the non-degeneration SEC-HPLC of analytical type.Pegylation has increased proteinic hydrodynamics volume greatly, causes migrating to retention time early.In PEG aldehyde hGH reactant mixture, observed the hGH of new kind together with unmodified.Use the Q-Sepharose chromatography to separate these Pegylations and kind non--Pegylation, and a PEG-aldehyde hGH kind that confirms the gained purification subsequently when non-degeneration SEC as unimodal eluting (>95% purity, accompanying drawing 1).The Q-Sepharose chromatographic step has effectively been removed free PEG, hGH and poly ethylene glycol hGH kind from one-Pegylation hGH.
SDS-PAGE
SDS-PAGE is used to estimate the reaction of glycerol branched 43K PEG aldehyde and hGH and the end-product of purification.SDS-PAGE is at 1mm thickness 10-NuPAGE gel (Invitrogen, Carlsbad, CA) go up and under reduction and non-reduced condition, to carry out and use Novex ColloidalCoomassie (TM) G-250 staining kit (Invitrogen, Carlsbad CA) dye.
The N-end sequence
Automatization's edman degradation chemistry is used to measure NH
2-terminal protein matter sequence.(Perkin Elmer, Wellesley MA) are used for degraded to AppliedBiosystems Model 494 Procise sequenators.Analyze by RP-HPLC, identify corresponding PTH-AA derivant according to the on-line operation mode of the Applied Biosystems Model140C PTH analyser of PerkinElmer/Brownlee 2.1mm i.d.PTH-C18 post being installed with use.
Peptide mapping
Carry out trypsinization and general each digestion use 25ug material with the concentration of 1mg/mL.Add trypsin so that the ratio of trypsin and PEG-hGH is 1: 30 (w/w).The Tris buffer is with 30mM, and pH7.5 exists.At room temperature with sample incubation 16 ± 0.5 hours.Make the reaction quencher by adding 50uL 1N HCl/mL digestion solution.Before sample is put into automatic sampler with diluted sample to final concentration at 6.25% acetonitrile 0.25mg/ml.At first add acetonitrile (to 19.8% acetonitrile), mix gently and add entry then to final volume (4 times to initial volume).Can remove extra digestion solution and under-20 ℃, store and reach for 1 week.
Waters Alliance 2695 HPLC systems are used for analyzing, and but, other system should produce similar result.Use has the particulate AstecC-4 polymer 25cm of 5um * 4.6mm post.Experiment is carried out with the typical applied sample amount of 50ug protein/sample at ambient temperature.Buffer A is 0.1% trifluoroacetic acid in water; Buffer B is 0.085% trifluoroacetic acid in acetonitrile.Linear gradient elution sample with 0-45%B in 90 minutes.
The Waters 996 PDA detector detected peaks of use image data between 210-300nm.The chromatogram of the extraction at 214nm place is used for sample analysis.
(PEG: hGH) Fan Ying glycerol branched 43K PEG aldehyde carries out trypsin mapping (accompanying drawing 2) to hGH with 2: 1 mol ratio.The terminal trypsin fragment of N-is called T-1.The T-1 percentage ratio prompting that exists of comparing with Pegylation hGH is not modified greater than 99% PEG and is positioned at the N-end, and remainder obviously is connected with one of several possible lysine residues.
Table 2
T-1 content relatively
The T-1% that exists | Compare the T-1% of existence with Pegylation hGH not | |
hGH | 28.0% | 100% |
Glycerol branched 43K PEG aldehyde/hGH | Be lower than 1% | Be lower than 1% |
Extracorporeal biology
Be designed for the ability of using the glycerol branched 43KPEG aldehyde hGH identification people receptor of Biacore 3000 instrument tests between assessment conjugate and the growth hormone receptor (hGHR) (28kDa extracellular domain) in the interactional test.From surface plasma resonance (SPR) result of experiment as shown in following table 3.
Table 3.Biacore result of the test
Sample | Avg.k a×10 5(M -1s -1±stdev) a | Avg.k d (s -1±stdev) a | K D=k d/k a,mM | K with respect to hGH D |
hGH(n=9) | 3.07±8.20 | 38.8±3.6 | 0.13±0.03 | 1.000 |
43K PEG-hGH (n=3) | 0.28±0.03 | 90.0±31.0 | 3.22±0.10 | 0.041 |
aAt 37 ℃ of following and HEP-BES buffer (0.01 M Hepes, pH7.4, + 0.15MNaCl, 3mM EDTA and 0.005% surfactant P20) in, use the human growth hormone's conjugated protein (28kDa extracellular domain) who passes through with amine coupling chemistry labelling on the CM5 chip of Δ RU ≈ 3000-5000, with 50uL/ minute measurement of rate of flow k
a(on-rate) and k
d(off-rate).Be expressed as the ka of every M per second, be expressed as the kd of per second, be the meansigma methods ± standard deviation of at least 3 mensuration on 1 chip.Data hypothesis measuring h GH with 1: 1 ratio in conjunction with the high-affinity site 1 on the GHBP.
Pharmacodynamic study
Effect in the body in the rat test in effectiveness-11-days (weight increase, tibia growth, serum BUN prevent)
Rat body weight increases
Will be the HPX female Sprague Dawley rat prescreen rate of growth of Harlan laboratory 4-10 days.The group that rat is divided into 6 animals.Since the 0th day, control rats was accepted 1 subcutaneous injection 0.3mg/kg hGH or continuous 11 days of carrier every day.Test group was accepted the glycerol branched 43KPEG aldehyde hGH of 1.8mg/kg single (once/weekly) dosage at the 0th and 6 day.Weigh to animal every day.Accompanying drawing 4 is presented at hGH in the representational research and the glycerol branched 43K PEG aldehyde hGH effect to weight increase.
With the data of research representative shown in the accompanying drawing 4 with add that from growth researchs in historical 11-days the data of the extra increment study that uses the glycerol branched PEG aldehyde hGH conjugate of 43K merge to the rat of hGH (reference substance) treatment, the weight increase that on average increases progressively that uses the 1.8mg/kg conjugate to treat 1 time animal weekly is 109% of the value that obtains according to hGH administration every day (accumulation 3.3mg/kg).
Rat tibia length
When 11-days body weight increase animal in the research at the 11st day, put to death, take out the left side tibia and clap X-ray photograph and use caliper to measure bone length.Accompanying drawing 5 shows the tibia length measurements of glycerol branched 43KPEG aldehyde hGH treatment animal.
Rat BUN level
As the metabolic biomarker in hGH-treatment back, measure the blood urea nitrogen level by the 11st day blood sample.Accompanying drawing 6 demonstration hGH every day and 1 time/glycerol branched weekly 43K PEG aldehyde hGH treatment all cause blood urea nitrogen significantly to reduce.
Rat body weight increases 6-days dose escalation study
Glycerol branched 43K PEG aldehyde hGH with different single doses treats HPX rat, or uses the hGH treatment every day, and monitors weight increase 6 days.Accompanying drawing 7 shows the weight increase that difference treatment group is obtained.Blood sampling and measure serum I GF-1 level at the appointed time by ELISA.Meansigma methods ± SEM is drawn.To organize (n=6) meansigma methods is used to use one way analysis of variance that the value of measuring is calculated the IGF-1 response, and to 0.067,0.2,0.6 and 1.8mg/kg administration group measured 20,040,22,958,28 respectively, 129 and 37,839 AUCd0-6 (ng-hr/mL) value.
Rat body weight increases 11-days dose escalation study
In second research, the 0th day and once more the 6th day with 1.8mg/kg or more high dose be the glycerol branched PEG aldehyde hGH conjugate of 5.1mg/kg43K treatment animal.Table 4 demonstration gives carrier with every day (QD11) or gives hGH (with the 0.3mg/kg/ days) value that the back obtains every day to compare weight increase relevant with dosage in the time of the 6th and 11 day.
Table 4. is at the 6th weight increase relevant with dosage during with 11 days
The 0th day (g) | The 6th day (g) | The 11st day (g) | |
Carrier (QD11) | 106.2±2.3 | 106.2±2.8 (0.07±0.7) | 106.7±2.8 (0.5 1±0.6) |
hGH(0.3mg/kg)(QD11) | 109.2±1.3 | 123.1±1.4 * (13.9±0.5) | 136.7±1.6 (27.5±0.7) * |
Glycerol branched 43K PEG aldehyde hGH (1.8mg/kg) (D0,6) | 106.8±1.3 | 120.6±1.1 * (13.8±0.7) | 133.6±1.0 (26.7±0.8) * |
Glycerol branched 43K PEG aldehyde hGH (5.1mg/kg) (D0,6) | 106.1±1.4 | 127.1±1.3 * (21.0±1.0) | 142.5±1.4 (36.4±1.2) * |
* compare p<0.05 with carrier, compares p<0.05 with hGH; Increase increment value (g) as the average weight of measuring from the 0th day
BW, body weight; HGH, the human growth hormone.
Value is represented meansigma methods ± SEM (standard error of meansigma methods).
Value representative in the round parentheses is from the meansigma methods ± SEM of the 0th day change.
IGF-1 research
Use increases the animal of studying from 6-days body weight.Show the serum I GF-1 level of measuring by ELISA during different time in this research process in blood sampling and the accompanying drawing 8.With immunoassay kit (Diagnostic System Laboratories) monitoring rat IGF-1 level.
Pharmacokinetic
In the Sprague-Dawley male rat of normal cannulate, carry out pharmacokinetic.Use every group of 6 rats to carry out single subcutaneous the injecting fast of the intravenous single dose injection of 1.0mg/kg or 1.8mg/kg hGH or glycerol branched 43K PEG aldehyde hGH.Blood sampling is used for the evaluation of relevant PK parameter in 1-5 days aptly.When each sampling, use immunoassay monitoring hGH and glycerol branched 43K PEG aldehyde hGH blood levels.Table 4 shows the P of Rats K parameter of glycerol branched 43K PEG aldehyde hGH.Pegylation to act on observed elimination half-life aspect apparent, because this parameter has surpassed 6 hours for conjugate, and be 1.35 ± 0.2 (Clark according to reports from the data to hGH report of similar research, document is the same), 0.77-1.7 (Jorgensen etc. " Polyethyleneglycol-conjugated proteins ", PSTT 1 (8), in November, 1998) or 1 hour (Genotropin (PNU-180307) Investigator Brochure).
The hGH immunoassay
HGH in use hGH AutoDELFIA test kit fluorescence immunoassay (Perkin-Elmer) the mensuration rat plasma and glycerol branched 43K PEG aldehyde hGH protein concentration level.
Table 5.
Glycerol branched 43K PEG aldehyde hGH | |||
Approach | iv/sc | ||
Dosage | mg/kg | 1.0/1.8 | |
T1/2 | h | 6.44±2.38 | |
AUCO-∞,,iv | μg·h/mL | 489±16 | |
AUC0-∞,,sc | μg·h/mL | 97.7±2.1 | |
CL amounts to | mL/hr/kg | 2.05±0.07 | |
Vss | mL/kg | 27.2±2.8 | |
| h | 12±0 | |
Cmax | μg/ml | 1.96±0.05 | |
F | % | 11.1±0.2 | |
*AUCO-∞,, sc proofreaies and correct 1.8mpk |
In addition, directly measure the dependency of plasma drug level between reacting with IGF-1 in the comprehensive study design behind the subcutaneous glycerol branched 43K PEG aldehyde hGH (1.8mg/kg) that gives single dose to HPX female rodent.
Claims (12)
2. the described PEG-hGH conjugate of claim 1, wherein said (CH
2CH
2O)
nPart has mean molecule quantity and the each (CH of about 3Kd
2CH
2O)
mPart has the mean molecule quantity of about 20Kd.
3. claim 1 or 2 described PEG-hGH conjugates, wherein said human growth hormone comprises the aminoacid sequence of SEQ ID NO:1.
4. the described PEG-hGH conjugate of claim 3, wherein the terminal phenylalanine of the N-of PEG and SEQ ID NO:1 is puted together.
5. the described PEG-hGH conjugate of claim 4, wherein said conjugate are one-Pegylation.
6. the described PEG-hGH conjugate of claim 4, wherein at least 80% among the PEG puts together with the α amino of the terminal phenylalanine of N-of SEQ ID NO:1.
7. the described PEG-hGH conjugate of claim 5, wherein at least 90% among the PEG puts together with the α amino of the terminal phenylalanine of N-of SEQ ID NO:1.
8. the described PEG-hGH conjugate of claim 5, wherein at least 95% among the PEG puts together with the α amino of the terminal phenylalanine of N-of SEQ ID NO:1.
9. the described PEG-hGH conjugate of claim 5, wherein at least 98% among the PEG puts together with the terminal phenylalanine of the N-of SEQ ID NO:1.
10. treatment has growth or stunted patient's method, comprises claim 1,2,3,4,5,6,7, the 8 or 9 described human growth hormone-PEG conjugates of described patient being treated effective dose.
11. the group that the described method of claim 10, wherein said growth or dysplasia are selected from growth hormone deficiency (GHD), Turner syndrome, chronic renal insufficiency and form less than gestational age (SGA).
12. the described method of claim 11, wherein said growth or dysplasia are selected from erectile dysfunction, the HIV lipodystrophy, fibromyalgia, osteoporosis, dysmnesia, depression, Crohn disease, skeleton development is bad, traumatic brain injury, subarachnoid hemorrhage, Noonan syndrome, mongolism, the special property sent out short stature (ISS), end stagerenaldisease (ESRD), extremely low birth weight (VLBW), the bone marrow stem cell rescue, metabolism syndrome, the glucocorticoid myopathy, short stature that causes because of the glucocorticoid among child treatment and short and small premature infant's growth lag behind.
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Cited By (4)
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CN101831067A (en) * | 2010-05-31 | 2010-09-15 | 王二新 | Polyethylene glycol ester conjugate and application thereof in medicine preparation |
CN102367290A (en) * | 2011-04-26 | 2012-03-07 | 刘超 | Chain-functionalized multi-level branched polyethylene glycol and synthesis method thereof |
CN102367290B (en) * | 2011-04-26 | 2013-05-08 | 厦门赛诺邦格生物科技有限公司 | Chain-functionalized multi-level branched polyethylene glycol and synthesis method thereof |
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