CN102690804A - Polyethylene-glycol-modified recombinant homosapiensarginase I, preparation method thereof and application thereof - Google Patents

Abstract

The invention belongs to the biotechnology field, providing a polyethylene-glycol-modified recombinant homosapiensarginase I. Newly prepared artificial molecules in the invention are characterized by specific activity in vivo, a prolonged half-life period, low immunogenicity, a uniform and stable structure, as well as easiness in process and quality control. The invention also provides a preparation method for the target molecules. The polyethylene-glycol-modified recombinant homosapiensarginase I has good application prospect in human tumor treatment.

Description

A kind of polyethyleneglycol modified recombinant human arginase I its preparation method and application

Technical field

The invention belongs to biomedicine field, relate to the chemical modification technology of recombinant protein.

Background of invention

Arginase (Arginase) is a kind of enzyme in the ornithine cycle, and the hydrolysis of catalysis l-arginine produces ornithine and urea, is playing an important role aspect the body nitrogen metabolism.Arginase has two kinds (the I type is claimed extractum hepatis propylhomoserin enzyme, and the II type claims liver outer arginase) in the human body, and I type arginase expression of gene receives substrate l-arginine and coenzyme M n ²⁺Induce (Brock, A.A et al, Dietary manganese deficiency decreases rat hepatic arginase activity.J.Nutr.1994; 124:340-344), being present in the outer II type arginase of liver has tissue distribution widely, in different tissues and organ, brings into play different effects; Comprise and participate in (Russel, D.H, et al such as polyamines metabolism, NO be synthetic; Polyamine biogenesis in the rat mammary gland during pregnancy and lactation.Bilchem.1972; 130:71-76), compare with I type arginase, its vigor is lower.

Arginase carries out nutrition through the decomposition l-arginine to tumour cell to be deprived, less to normal tissue influence, becomes the another selection of antineoplaston.Traditional chemicotherapy method has been difficult to satisfy the requirement of patient to quality of life at present, and the method that amino acid is deprived is injected new vitality to cancer therapy.Though it can cause some imbalance of physiology and metabolism aspect, in body, introduces toxic substance unlike traditional modes such as chemicotherapy, make originally to make the matter worse with regard to the sick body that cannot withstand a single blow.

That find the earliest that arginase has function of tumor inhibition is Bach and Lansitksi; They handled rat Walker256 sarcoma four days with the beef liver arginase of purifying; Find tumor growth 31%-71% (the Bach SJ that slowed down; Lasnitzki I.Some aspects of the role of arginine and arginase in mouse carcinoma.Enzymologia.1947,12:198-205).The arginase anti tumor activity in vitro is from nineteen sixty existing report (Bach SJ, HawkinsRA, Swaine D.A short method forthe purification of arginase from ox liver.Biochem J1963; 89:263-5), report such as Curtie arginase discharges from LPS and the post-stimulatory scavenger cell of zymosan, can cause the apoptosis of Chinese Rat Pulmonary V79, lymphoma cell L5178Y, HSN rat sarcoma cell.Storr and Burto report are handled with the arginase in mouse and cattle liver source, and the growth of lymphosarcoma cell is suppressed under the situation that arginine-level is brought down below 8umol/L and continues 24 hours.The anti tumor activity in vitro that Scott and Wheatley etc. exhaust l-arginine has carried out comparatively systematic research (Scott etc.; Single amino aacid (arginine) deprivation:rapid and selective death of cultured transformed and malignant cells.Br.J.Cancer 83; 800-810); In 24 kinds of tumour cells of test; The all cells strain is dead in 5 days after the arginase effect, and the subject cell strain comprises cells such as mammary cancer, carcinoma of the colon and rectum, lung cancer, prostate cancer and ovarian cancer.

People's liver cancer (Human hepatocellular carcinoma; HCC) cell and human melanoma cell belong to l-arginine auxotrophy cell; Its argininosuccinate synthetase (argininosuccinate synthetase; ASS) express defective, possibly methylated thereby be suppressed, on transcriptional level, reduce relevant (Dillon BJ et al.Incidence and distribution of argininosuccinate synthetase deficiency in human cancers:a method for identifying cancers sensitive to arginine deprivation.Cancer2004 with the promotor of ASS gene; 100:826-33).L-arginine changes imido enzyme (arginine deiminase; ADI) aspect the treatment ASS defective type tumour clear and definite curative effect is being arranged; But many non-ASS defective type HCC cells have opposing (Shen LJ et al.Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culturecorrelates with the endogenous enzyme, argininosuccinate synthetase.Cancer Lett 2003 to ADI; 191:165-70).Foreign literature report recombinant human arginase I (recombinant human arginase I; RhArg I) non-ASS defective type HCC cell had clear and definite restraining effect; All five strain HCC cells are all responsive to rhArg I; But ADI is to these cell unrestraint effects, and these cells are all expressed ASS, but does not express ornithine transcarbamylase (ornithine transcarbamylase; OTC); RhArg I degraded l-arginine produces ornithine, and OTC is converted into N.delta.-carbamylornithine with ornithine, and N.delta.-carbamylornithine is again through ASS and argininosuccinate lyase (argininosuccinate lyase; ASL) change into l-arginine; With OTC transfection HCC cell can make this cell to rhArg produce opposing (Paul Ning-Man Cheng et, Pegylated Recombinant Human Arginase (rhArg-peg5,000mw) Inhibits the In vitro and In vivo Proliferation of Human Hepatocellular Careinoma through Arginine Depletion.Cancer Res 2007; 67: (1) 309-317) (ornithine cycle and relevant enzyme effect node synoptic diagram are seen Figure of description 1).

Ornithine cycle is incomplete (mechanism is unclear) in some cells; The meta-bolites ornithine can not get into circulation again and produce l-arginine (Paul Ning-Man Cheng et al; Pegylated Recombinant Human Arginase (rhArg-peg5,000mw) Inhibits the In vitro and In vivo Proliferation of Human Hepatocellular Carcinoma through Arginine Depletion.Cancer Res 2007; 67: (1) 309-317), thereby change the imines enzyme with l-arginine and compare, arginase is an arginic more strong enzyme in the degraded circulation of blood; See that from clinical therapeutic efficacy the value that Arg is developed to antitumor drug is bigger than ADI.

Recombinant human arginase I success obtains to express (Masaki IKEMOTO et al in intestinal bacteria; Expression of human liver arginase in Escherichia coli.Biochem; J.1990; (270): 697-703), but its short body-internal-circulation transformation period (being shorter than 30 minutes) make and under the situation that can not carry out frequent heavy dose of administration, use it for tumour patient to reduce in the blood very difficulty of arginine-level.The arginase in another kind of beef liver source is not expected as the drug candidate of treatment human tumor always, and reason is that itself and the arginic avidity of substrate are lower under physiological condition, ph optimum 9.6, and the transformation period has only several minutes.Since the 1980s, these all are considered to the major defect of beef liver arginase.The research of Savoca shows that the ox arginase does not have anti-tumor activity (Savoca KV et al to the mouse of transplanting Taper liver cancer; Cancer therapywith chemically modified enzymes.II.The therapeutic ettectiveness of arginase; And arginase modified by the covalent attachment of polyethylene glycol; On the taper liver tumor and the L5178Y murine leukemia.Cancer Biochem Biophys1984; 7:261-268), some other investigator thinks that also the ox arginase does not have anti-tumor activity.

Though the external tumor-inhibiting action of arginase is clear and definite; Attempt in the body to exhaust the multinomial research of the treatment cancer (Br.J.Cancer such as Storr that do not achieve the desired result but use it for through l-arginine; 1974; V30:50-59); This is because have vivo acid homeostatic mechanism (amino acid homeostatic mechanism) during body reply amino acid starvation, decompose approach through activator matter when amino acid is deficient and l-arginine is discharged into participates in circulation in the blood, has replenished the l-arginine that the arginase metabolism causes and has exhausted.For overcoming this homeostatic mechanism of body, realize having the l-arginine of treatment meaning to exhaust the continuous action that just needs the l-arginine degrading enzyme, thereby will carry out modification, to reach long-acting to it.

Tepic is at USP UP 6; 261; Describe in 557 l-arginine degrading enzyme and the combination of protein decomposing inhibitor Regular Insulin are used; Replenish this approach of l-arginine with blocking-up through degraded body muscle, but the application of Regular Insulin receives the restriction of patient's glucose level, will cause danger if patient's glucose level can not remain in the limited normal range.

Savaca in 1979 etc. use 2,4, and 6-three chloro-S-triazines (cyanuryl chloride) are as coupling agent; With the arginase in cattle liver source and the covalently bound (Savoca of mono methoxy polyethylene glycol of 5KD; K.V. etc., 1979, Biochimied et Biophysica Acta 578; 47-53); Though polyethyleneglycol modifiedly can reduce proteic immunogenicity to a certain extent, the ox arginase is a heterologous protein as far as the people after all by extending to 12 hours less than one hour transformation period in the body of mouse with arginase; And the molecule after modifying only kept that protoenzyme lives 65%, live loss of enzyme is bigger.

The PCT patent (PCT/CN2002/0006352002.9.9) of Hong Kong Condar medicine ltd; Adopt subtilis to express with 6 histidine-tagged human arginase I; As modifier, decorating site is the amino of Methionin or N-terminal with the mPEG-SPA of molecular weight 5KD, and the PEG and the Arg mol ratio of modifying the after product molecule are 10-12; Be a kind of structure mixture, the transformation period extends to 3 days in the body.This modification mode makes the PEG molecule that number of connection does not wait on the Arg molecule, product conformation heterogeneity, and the production quality control difficulty, preparation process is bigger to the quality influence of product between criticizing, the technology controlling and process difficulty.

The arginic enzyme of another kind of degraded is that l-arginine changes imines enzyme (ADI), and this is a kind of enzyme that is derived from mycoplasma.Its long-acting molecule is U.S. Phoenix Pharmacologics; The PEG modifier of Inc development; Got at present II phase clinical study (Pegylated Arginine Deiminase Treatment of Patients With Unresectable Hepatocellular Carcinoma:Results From Phase I/II Studies.Journal of Clinical Oncelogy 2004 (22): 10 such as Francesco Izzo, 1815-1822).Result of study shows that it has clear and definite restraining effect to some HCC cell and MC; These tumour cells rely on exogenous l-arginine and keep its growth; The auxotrophic mechanism of its l-arginine is complicated, and basic reason is that the promotor of ASS gene is methylated thereby is suppressed.The shortcoming of this molecule of pharmaceutical is that it is a mycoplasma species enzyme, is still a problem though carried out its immunogenicity of PEG modification.Existing report autoantibody can detect in preceding 5 weeks in its II phase clinical study, and its titre continues to rise afterwards, and this will cause the invalid of successive treatment; Secondly ADI changes l-arginine into N.delta.-carbamylornithine and free amine group, and it is compensatory when blood ammonia levels is high, can to form the mistake of liver cirrhosis and liver function, hepatogenic encephalopathy can occur some patients.

Summary of the invention

The objective of the invention is to develop a kind of molecule of pharmaceutical that is superior to above-mentioned several kinds of schemes, make it have height ratio is lived, prolonged in the body transformation period, reduced immunogenicity, structure stable homogeneous, and be easy to carry out the control of technology and quality; Another object of the present invention provides the application of this decorating molecule in preparation human liver cancer and melanoma medicine.

The object of the invention is realized through following technology:

According to prior art; Can prepare human arginase I (Masaki IKEMOTO through recombinant gene; Masayoshi TABATA, Toshio MIYAKE, et al.Expression of human liver arginase in Escherichia coli.Biochem; J.1990, (270): 697-703); And activated polyglycol can obtain through multiple commercial sources.

The present invention adopts the mono methoxy polyethylene glycol propionic aldehyde as modifier; The control reaction conditions; Realizing that peg molecule and recombinant human arginase I molecule N-terminal are amino is connected, the title product structure homogeneous that after separation and purification, obtains, and controllability is strong between the criticizing of preparation technology; More less before and after the modification than fall alive, reach the purpose that increases pharmaceutical protein stability, interior transformation period of extension body, reduces drug effect in immune prototype, the enhancing body.

The present invention adopts recombinant gene from people's liver, to obtain the mRNA of human arginase I; Again coli expression carrier is arrived in human arginase I gene clone; Transfection Escherichia coli, screening obtains the high expression level bacterial strain, and acquisition purity is greater than 95% recombinant human arginase I behind fermentation, purifying; Select the mono methoxy polyethylene glycol propionic aldehyde as modifier; The control reaction conditions; N-terminal amino to recombinant human arginase I carries out pointed decoration; Modifier obtains the target molecule of structure homogeneous behind gel exclusion chromatography and ion exchange chromatography, it is 20-30% that external l-arginine edman degradation Edman detects enzyme work fall after modification.

Polyethyleneglycol modified is the protein modified technology that grows up in the later stage seventies 20th century.With protein molecule coupling on the activated polyglycol; Through influencing the protein molecule space structure, the various character of protein are changed: chemicalstability increases, the ability of opposing protease hydrolysis improves, stronger, the immunogenicity of drug effect and toxicity reduces, plasma clearance reduces, the transformation period prolongs etc. in the body in the body.

Constitute in the proteinic common amino acid at 20 kinds, the side-chain radical that only has the polar amino-acid residue can carry out chemically modified.Reaction amino acid commonly used comprises Methionin, halfcystine, Histidine, l-arginine, aspartic acid, L-glutamic acid, Serine, Threonine, tyrosine, the amino and C-end carboxyl of N-end.Reactive group on these amino-acid residues is nucleophilicity more, and its nucleophilic activity is successively decreased by following order successively: sulfydryl＞α amino＞ε amino＞carboxyl＞hydroxyl.Different according to reaction property between chemical modifier and the protein, modification reaction mainly is divided into types such as acylation reaction, alkylated reaction, redox reaction, aromatic nucleus substitution reaction.Sulfydryl is present on proteinic disulfide linkage and the avtive spot usually, and if carboxyl not with protein on amino intermolecular or intramolecularly neutralization reaction takes place, be difficult to activation.Therefore, the site that protein or peptide molecule react with modifier the most easily is the amino that is exposed on the amino-acid residue of molecular surface, comprises α amino or ε amino.

It is active that the free amine group on protein molecule surface has higher nucleophilic reaction, generally is not in the position, active site, thereby become in the chemically modified the most frequently used by modification group.The ε that main adorned amino is Methionin is amino, α is amino and terminal amino group.Free amine group content in protein molecule is higher; When polyoxyethylene glycol and the reaction of amino acid whose free amine group, be mostly to modify at random, often have several amino to be modified; Cause the generation of polymorphic mixture; Even only combine a polyoxyethylene glycol, also possibly be combined on the different amino sites, produce the site isomer.Amido modified the most frequently used modifier comprises SS-PEG, SC-PEG, SPA-PEG, NHS-PEG and ALD-PEG etc.

Because protein has only a terminal amino group, therefore can avoid the generation of polymorphic mixture and site isomer to its modification.The derivatization that generates a kind of single polyoxyethylene glycol product through reductive alkylation has utilized the amino reactivity worth amino different with the protein molecular N-terminal of the ε on the Methionin; At control reaction pH; Have under the condition of specific reductant existence, can realize the pointed decoration of protein molecular.

The modifier one of which end that the present invention selects is sealed by mono methoxy, and the other end is the activation aldehyde radical, molecular weight ranges 5KD-40KD, and most preferably molecular weight is 20KD.The existing at present commercially available prod of this kind activated PEG also can prepare by reference, modifies through each index of quality examination up to standard promptly can be used for.

The reaction pH condition that the present invention selects is 4-7; This pH value can provide the difference of pK value between the amino and N-terminal α amino of ε on the lysine residue; The keying action of PEG and Arg mainly occurs in the N-terminal of Arg, and tangible modifying function does not take place other group such as lysine side-chain amino with reactive behavior.The pH value also influences the feed ratio of albumen and modifier in the reaction system, if the pH value is low excessively, the amino reactive behavior of N-terminal α is relatively poor, needs more PEG to realize higher reactivity.The reaction pH that the present invention selects is 4-7, preferred 4.5-6.0.Usually the mol ratio of PEG and protein molecular is low more in the reaction system, and the PEG molecule number that can be connected on the protein molecular is few more, and the mol ratio of choice reaction time Arg of the present invention and PEG is 1: 1-1: 10, and preferred 1: 3 to 1: 6.As far as reductive alkylation; Preferred reductive agent can only reduce in the initial stage of reductive alkylation step; The available reductive agent comprises sodium hydride, SODIUM CYANO BOROHYDRIDE, n n dimetylaniline borane, Trimethylamine 99 borane, pyridine borane, and preferred reductive agent is a SODIUM CYANO BOROHYDRIDE.

What the present invention was used is recombinant human arginase I by decorating molecule; Form theoretical molecular 34734.94 dalton, iso-electric point 7.09 by 322 amino acid; 3 halfcystines are arranged in the peptide chain, prepare purity and promptly can be used for modification reaction greater than 95% recombinant human arginase I.

The protein concentration of Arg is controlled at 2-5mg/ml; Regulate the pH value to 4-6mg/ml, adding SODIUM CYANO BOROHYDRIDE to final concentration is 20mM, and the add-on of PEG is 3-6 a times of Arg mole number; Whole system at room temperature stirring reaction 10-15 hour adds the glycocoll termination reaction.

Its molecular weight difference of mixture after PEG modifies is more obvious; Suit to separate with the gel chromatography medium; Select for use Sephacryl S200 chromatography as the isolating the first step of modified mixture, use the Arg enzyme is lived glycocoll-sodium hydrate buffer solution of favourable pH9.8 as balance and elution buffer, can remove unreacted Arg in the reaction mixture through this step operation; Make PEG-Arg be able to preliminary purification, purity can reach about 90%.Another effect of this step is that the PEG and the reductive agent that have neither part nor lot in reaction in the reaction mixture are effectively removed.Used PEG is that molecular weight is the neutral molecule of 20KD, and reductive agent is the inorganic molecules material, and they do not produce adsorption with filler; And the molecular weight of target modifier is 55KD, and it is bigger in the wash-out behavior of S200 column chromatography and PEG, reductive agent difference, can reach effective removal of PEG and reductive agent a step.

There is some difference on surface charge effect for the many modifiers of PEG and mono-modified thing, utilizes this point to reach making with extra care sample with can not effectively being removed by complete isolating many modifiers of PEG composition in the S200 sample.According to Arg more stable characteristics under weak basic condition; Selection is applicable to that the DEAE of weak base condition is as the proteic solid-phase media of binding purposes; After the PB balance with pH8.0; Modify component with the 0.O5MNaCl wash-out, 0.2M NaCl wash-out molecules of interest can reach more than 95% through this step sample purity more.

After above each step obtains to meet the modified protein stoste of pharmacy quality standard, promptly can be used for half-finished preparation.For therepic use, title product of the present invention can be formulated in the aseptic biocompatible pharmaceutical carrier and use, and we have developed injection liquid and two kinds of formulations of freeze-dried powder.

Liquid dosage form consists of (1ml/ props up): PEG-Arg5mg, 20mM phosphate buffered saline buffer, 0.01% tween 80,0.1M sodium-chlor, pH8.0.

Freeze-dried formulation consists of (1ml/ props up): PEG-Arg5mg, 20mM phosphate buffered saline buffer, 0.1M sodium-chlor, 5% N.F,USP MANNITOL, pH8.0.Process freeze-dried powder through freeze-drying after the dosing, add injection water dissolving back before the use and use.

Title product of the present invention can be used for people's liver cancer and melanomatous treatment, administration frequency be per 7 days once.The consumption of the PEG-Arg that plays effectiveness in the treatment is confirmed through clinical trial, at first should in the animal model that is suitable for, measure curative effect and confirm safe and effective dosage, and administering mode is intramuscular injection.

Description of drawings:

Fig. 1 ornithine cycle and relevant enzyme effect node synoptic diagram:

Groups of people's liver cancer cell is expressed argininosuccinate synthetase (ASS) and the argininosuccinate lyase (ASL) in the ornithine cycle, but does not express ornithine transcarbamylase (OTC).The ornithine that PEG-Arg degraded l-arginine produces in the blood gets into cell once more, but can not utilize through ornithine cycle owing to lack ornithine transcarbamylase (OTC) again; L-arginine changes imines enzyme (ADI) degraded l-arginine and produces N.delta.-carbamylornithine; N.delta.-carbamylornithine gets into cell; Be transformed into l-arginine again by the cell utilization through argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), thereby the part patient resists to the treatment generation of ADI in the generation clinical study.By contrast, arginase (Arg) is wider to the treatment spectrum of human liver cancer cell than l-arginine commentaries on classics imines enzyme (ADI).

Fig. 2 arginase is to arginic Degradation in the rat serum:

Rats by intraperitoneal injection (500,1,000; 2000; 4000IU/ is only) behind the recombinant human arginase (Arg), detect the variation of arginine-level in the blood, the result shows that Arg has only maximum dose level group (4000IU/ is only) can make in the blood arginine-level reduce to 20uM in back 5 hours in injection; Arginine-level gos up gradually in the blood subsequently, returns to normal level to injecting after 2 days.

Fig. 3 polyoxyethylene glycol arginase is to arginic Degradation in the rat serum:

Rats by intraperitoneal injection (500; 1; 000,2000,4000IU/ is only) behind the BCT-100 (PEG-Arg); Detect the variation of arginine-level in the blood, the result show PEG-Arg than low dose group (1000IU/ only) can make arginine-level in the blood reduce to detection less than level and sustainable 6 days.Compare with Fig. 2 result, explain that recombinant human arginase strengthens drug effect in the body, the purpose of interior transformation period of extension body through polyethyleneglycol modified having reached.

Embodiment

According to the present invention, the preparation of PEG-Arg and its active checking are presented at hereinafter, and embodiment has more at large described the present invention, but does not limit the present invention, and the Arg among the embodiment is the recombinant human arginase I of escherichia coli expression.

The preparation of embodiment 1. recombinant human arginase I

Pcr amplification people I type arginase gene (hAI); Insert among the coli expression carrier pET30a; Make up recombinant expression plasmid pET30a-hAI, transformed into escherichia coli host bacterium BL21 (DE3) makes up bacillus coli gene engineering bacteria BL21 (DE3) (pET30a-hAI); IPTG induces the back to express the .SDS-PAGE analysis revealed, and the BL21 after inducing (DE3) (pET30a-hAI) has tangible protein expression at about 35kD place.By fermentation, final purity is more than 95% behind the purifying.The detailed step reference literature (Masaki IKEMOTO, Masayoshi TABATA, Toshio MIYAKE, et al.Expression of human liver arginase in Escherichia coli.Biochem, J.1990, (270): 697-703)

Embodiment 2. decorating sites are in the preparation of PEG (the 20KD)-Arg at the amino place of Arg N-terminal α

The protein concentration of regulating Arg is to 4.0mg/ml; The pH value of adjusting protein solution is 5.2, and it is 20mM that the adding SODIUM CYANO BOROHYDRIDE makes its final concentration, and abundant dissolving back adding is equivalent to the Arg mole number and measures mPEG-ALD (20KD) for 4 times; Stirring reaction is 12 hours under the room temperature, adds the glycocoll termination reaction.Modify efficient in the reaction and detect with the SDS-PAGE method, reacted 12 as a child, SDS-PAGE result shows that 70% Arg is modified, mono-modified ingredients constitute 80-90% in the adorned component, and the time further prolongs, and the modification rate no longer improves.The centrifugal deposition of abandoning behind the reaction terminating, the supernatant part directly goes up Sephacryl-S ₂₀₀Post adds 0.15M sodium-chlor as elutriant with 50mM glycocoll-sodium hydroxide (pH9.8) damping fluid, can not adorned Arg molecule be removed through this step.Merge through Sephacryl-S according to the electrophoresis purity situation ₂₀₀Fraction collection appearance behind the post; With 50mM glycocoll-sodium hydroxide (pH9.8) damping fluid after Sephadex G25 desalination; Go up the DEAE Sepharose FF post of having crossed with the PB balance of pH8.0 again; Modify component with the 0.05MNaCl wash-out, 0.2M NaCl wash-out molecules of interest can reach more than 95% through this step sample purity more.

The preparation of embodiment 3.PEG-Arg injection liquid

After the PEG-Arg stoste that obtains via EXAMPLE l is measured protein concentration with the Lowry method, the 5000mgPEG-Arg adding is contained in the liquid of following material: Sodium phosphate, dibasic 6.78g, SODIUM PHOSPHATE, MONOBASIC 0.17g, 0.1ml tween 80, sodium-chlor 5.844g.Divide in the cillin bottle of the 2ml that packs into every can amount lml after the sterile filtration.

The preparation of embodiment 4. injection PEG-Arg freeze-dried powders

After the PEG-Arg stoste that obtains via embodiment 1 is measured protein concentration with the Lowry method, the 5000mgPEG-Arg adding is contained in the liquid of following material: Sodium phosphate, dibasic 6.78g, SODIUM PHOSPHATE, MONOBASIC 0.17g, sodium-chlor 5.844g, N.F,USP MANNITOL 50g.Divide in the cillin bottle of the 2ml that packs into after the sterile filtration, after lyophilize, promptly get.

Embodiment 5.PEG-Arg is to the inhibition test of the tumour cell of vitro culture

Select rat liver cancer cell MH134, mouse fibroma cell Meth A, human liver cancer cell 7402,7721, HepG2 for use, human melanoma cell SK-MEL-28 carries out the in-vitro cell growth inhibition test.Cell concn is 1.0 * 10 ⁴/ hole, substratum are the RPM1640 substratum that contains 10% foetal calf serum, separating tests group and control group; The test group culture plate adds respectively that final concentration is 0.2,0.4,0.6,0.8,1.0, the given the test agent (ADI, Arg, PEG-Arg) of 1.2IU/ml; Under 37 ℃, 5% carbon dioxide conditions, hatched 3-7 days, every afterwards hole adds 0.02mlMTS reagent, hatches 4 hours again; Detect absorbance value in 490nm, kill the required sample of 50% cell and be defined as IC than unit alive ₅₀

Embodiment 6.PEG-Arg is to arginic Degradation in the rat body

The male and female rat, mean body weight 250g, stable back random packet, abdominal injection various dose (500; 1,000,2000,4000IU/ is only) given the test agent (Arg, PEG-Arg); The tail vein is got blood as basic value before the injection, gets blood sample one time in 1 to 6 day per two days after the administration, and it is with fixed attention anti-to add EDTA when getting blood; Educate 30 minutes deposition foreign proteins with 50% Tricholroacetic Acid mixing ice, centrifugal 15 minutes of 13000g, supernatant part arginine-level adopt high-speed amino acid analyzer to analyze.(l-arginine concentration changes with time curve is seen Figure of description 2 and accompanying drawing 3 in the blood).

Claims (6)

1. a polyethyleneglycol modified recombinant human arginase I is primarily characterized in that each recombinant human arginase I molecule and single peg molecule coupling, and connection site is the N-terminal α amino of arginase I molecule.

2. polyethyleneglycol modified recombinant human arginase I as claimed in claim 1, its another characteristic is: said polyoxyethylene glycol one of which end seals with mono methoxy, and it is 10KD-40KD that the other end has an aldehyde radical and a molecular weight with reactive behavior.

3. polyethyleneglycol modified recombinant human arginase I as claimed in claim 1, its another characteristic is: said recombinant human arginase I is through the human arginase I of recombinant gene preparation or has identical active two mutants.

4. method for preparing polyethyleneglycol modified according to claim 1 recombinant human arginase I, its key step is:

(1) alkylated reaction:

At room temperature, pH4-6, have under the condition that reductive agent exists; Recombinant human arginase I is mixed with activated polyglycol aldehyde; The feed ratio of recombinant human arginase I and polyoxyethylene glycol (mol ratio) is 1: 1-1: 10; The molecular weight of polyoxyethylene glycol is 10KD-40KD, reacts 10-15 hour, adds the glycocoll termination reaction

(2) isolation and purification of reaction mixture:

Through the recombinant human arginase I molecule and the unreacted peg molecule of gel exclusion chromatography removal unmodified,, ion exchange chromatography can obtain polyethyleneglycol modified recombinant human arginase I title product after removing many modification components again.

5. a pharmaceutical composition is characterized in that said composition is a main active substances with the described molecule of claim 1.

6. described polyethyleneglycol modified recombinant human arginase I of claim 1 and the described pharmaceutical composition of claim 5 application that is used for treating human liver cancer or melanomatous medicine in preparation.

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