CN1536079A - Modified arginine deiminase - Google Patents
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- CN1536079A CN1536079A CNA031162622A CN03116262A CN1536079A CN 1536079 A CN1536079 A CN 1536079A CN A031162622 A CNA031162622 A CN A031162622A CN 03116262 A CN03116262 A CN 03116262A CN 1536079 A CN1536079 A CN 1536079A
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Abstract
The present invention relates to a modified arginine deiminase having no connecting gene, it is a compound with formula (1) structure, said formula (1) is ADI-(PEG)n (I): in which PEG represents polyethylene glycol whose average molecular weight is 1000-20000 Da, ADI represents arginine deiminase, and 'represents covalent bond between PEG and ADI, and n is integral number of 2-30. Said invention also provides preparation method of modified arginine deiminase and its correspondent medicine composition. Said invented compound has obvious effect for resisting tumor, more stable chemical property and lower immunogenicity.
Description
Technical field
The present invention relates to polyethyleneglycol modified arginine deiminase, and the application aspect the treatment tumour.
Background technology
China is one of primary hepatocarcinoma state occurred frequently, and annual morbidity 5-10/10 ten thousand populations have 130,000 people to die from primary hepatocarcinoma every year approximately, account for the 3rd of whole malignant tumours.Other countries and area in the Asia, for example Japan and Taiwan, liver cancer also is one of main cancer form.The medicine of development treatment liver cancer is badly in need of very much, and very important meaning is arranged.
Tumour cell has different nutritional needss with normal cell, some non-essential amino acid, and normal cell can independently synthesize, and tumour cell has been lost the ability of synthetic this class of amino acid.Can the degrade enzyme of this class of amino acid of utilization, optionally " hunger " tumour cell is a kind of highly selective, hypotoxic tumor therapeuticing method in theory.A famous example is an asparaginase, is used for the treatment of acute lymphoblastic leukemia.Some human body tumour cell can not synthesize arginine, thereby the arginine degrading enzyme might be developed to anti-tumor drug theoretically.
Normocellular growth does not need arginine, because they can be by synthesizing arginine by argininosuccinate synthetase and the catalytic two-step reaction of arginosuccinate lyase from citrulline.Yet hepatoma, melanoma and some other sarcoma are not expressed argininosuccinate synthetase, thereby they are arginic auxotroph.Difference in this metabolism can be used to develop to treating the safe and effective therapeutical agent of these diseases.Arginine deiminase energy catalysis arginine can be used to remove arginine to the conversion of citrulline.Therefore, arginine deiminase can be used to treat hepatoma, melanoma and some other sarcomas.
The natural arginine deiminase can find in multiple microorganism.Takaku etc. are at Int.J.Cancer, 51:244-249 (1992) and United States Patent (USP) NO.5474928 have described the arginine deiminase from mycoplasma arginini, in vivo, external restraining effect to various tumor cell strains, wherein the restraining effect to the tumor cell of liver strain is especially remarkable.
But arginine deiminase is used the very strong immunogenicity of existence, and is disposed fast in patient's blood circulation as a kind of heterologous protein on human body.
Protein PEGization is to overcome the immunogenic a kind of effective ways of heterologous protein.By the polyoxyethylene glycol covalent modification, can reduce the immunogenicity of foreign protein, prolong half-life.Compare with the natural arginine deiminase, polyethyleneglycol modified arginine deiminase is more effective when the treatment tumour.The adenosine desaminase (PEG-ADA) of PEGization, asparaginase (PEG-ASP) are in clinical application for many years.
Polyoxyethylene glycol (PEG) molecule must be by the activation of activating group, could with reactive group reactions such as the amino of protein surface, sulfydryl, carboxyl, imidazolyl, be connected on the protein molecule with the covalent linkage form.Common activating group comprises a linking group, comprises or does not comprise a leavings group, and leavings group comes off in the reaction process, and PEG is covalently bound by linking group and protein.(comprise such as maleimide groups (comprising succsinic acid succinimide ester (SS), propionic acid succinimide ester (SPA), carboxylic formic acid succinimide ester (SCM), succsinic acid imino-succinic diamide (SSA) or N-hydroxy-succinamide (NHS)), epoxy group(ing), oxygen base carbonylic imidazole base, for example, carbonyl dimidazoles base (CDI)), nitrophenyl (comprises, for example, carbonic acid nitro phenyl ester (NPC) or carbonic acid trichlorine phenyl ester (TPC)), isocyanate group, ethene sulfuryl, tyrosine-based, halfcystine base, Histidine base or primary amine.
Takaku etc. have described among 84:1195-1200 (1993) and the flat 4-121187 of Japanese Patent NO. with polyoxyethylene glycol by the chemically modified of cyanuryl chloride linking group to arginine deiminase at Jpn.J.Cancer Res..Clark etc. have described in United States Patent (USP) NO.6183738 with polyoxyethylene glycol and by succinyl-succinimide (SS-PEG) linking group arginine deiminase have been carried out chemically modified.There is a residual linking group in this class modifying method between protein and PEG molecule.The existence of this linking group is not essential, and the existing of linking group (1) target site of enzymolysis or hydrolysis may be provided, cause the instability of connector, for example the ester bond among the SS-PEG; (2) linking group may have immunogenicity or potential toxicity, for example cyanuryl chloride.
Therefore, this area presses for the arginine deiminase of the new modification of exploitation, and described arginine deiminase not only has outstanding antitumous effect, and more stable, immunogenicity is low.
Summary of the invention
Purpose of the present invention just provides a kind ofly has outstanding antitumous effect, and the arginine deiminase of the modification more stable, that immunogenicity is low.
In a first aspect of the present invention, a kind of ADI-PEG compound that does not have linking group is provided, it has formula (I) structure:
ADI-(PEG)n (I)
Wherein PEG represents that molecular-weight average is the polyoxyethylene glycol of 1000-20000Da, and ADI represents arginine deiminase, and the covalent linkage between "-" expression PEG and the ADI, n is the integer of 2-30.
In another preference, wherein said arginine deiminase is the arginine deiminase of Mycoplasma microorganism.
In another preference, wherein said arginine deiminase is the arginine deiminase of mycoplasma arginini, mycoplasma hominis or mycoplasma arthritidis.More preferably, wherein said arginine deiminase is the arginine deiminase of mycoplasma arginini.
In another preference, the molecular weight of described PEG is 4000-6000Da, and arginine deiminase has the aminoacid sequence shown in the SEQ ID NO:2.
In another preference, the n of described compound is 5-17, more preferably is 7-15, is 9-12 best.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it contains formula of the present invention (I) compound and pharmaceutically acceptable carrier.
In a third aspect of the present invention, the purposes of formula of the present invention (I) compound is provided, it is used to prepare medicine, and this medicine is used for the treatment of hepatoma.
In a fourth aspect of the present invention, the method for a kind of preparation formula (I) compound is provided,
ADI-(PEG)n (I)
Wherein PEG represents that molecular-weight average is the polyoxyethylene glycol of 1000-20000Da, and ADI represents arginine deiminase, and the covalent linkage between "-" expression PEG and the ADI, n is the integer of 2-30,
The method comprising the steps of:
(a) provide the activated PEG precursor of formula (II):
mPEG-X
MPEG is a mono methoxy polyethylene glycol in the formula, and X is a leavings group,
(b) activatory PEG precursor is mixed with arginine deiminase, at 0-30 ℃, reaction is 0.5-2 hour in the damping fluid of PH6-8, forms formula (I) compound;
(c) separation and purification formula (I) compound.
Description of drawings
Fig. 1 has shown the arginine deiminase nucleotide sequence, and the codon TGA of 5 coding colors propylhomoserins is nonsense codon in intestinal bacteria, changes TGG into.(promptly 606,792,819,885 and 1230 A becomes G).
Fig. 2 has shown the aminoacid sequence of arginine deiminase.
Fig. 3 has shown natural and the HPLC collection of illustrative plates PEGization arginine deiminase.
Fig. 4 A has shown the blood plasma arginine concentration of using behind natural and the PEGization arginine deiminase.
Fig. 4 B has shown the blood plasma citrulline concentration of using behind natural and the PEGization arginine deiminase.
Fig. 5 has shown natural and the immunogenicity PEGization arginine deiminase.
Fig. 6 has shown arginine deiminase anti-tumor in vivo activity.
Embodiment
The inventor is through extensive and deep discovering, with molecular weight is that the PEG of 1000-20000Da directly is connected formed modification type ADI with arginine deiminase (ADI), compare with the modification type ADI of existing band linking group, has more excellent comprehensive characteristic, be outstanding antitumous effect, chemical property is more stable, immunogenicity is lower.
In the present invention, use following abbreviation:
PEG, polyoxyethylene glycol;
MPEG, mono methoxy polyethylene glycol;
TMPEG/mPEG-tresyl, trifluoroethyl alkylsulfonyl mono methoxy polyethylene glycol;
MPEG-Cl: chlorination mono methoxy polyethylene glycol;
ADI, arginine deiminase.
As used herein, " polyoxyethylene glycol " or " PEG " is meant the mixture of the straight or branched form polycondensate of oxyethane and water, by general molecular formula H (OCH
2CH
2)
nOH represents, and is at least 4 at this n.About molecular-weight average of together representing it with " polyoxyethylene glycol " or " PEG " together with the numeric suffix of back.For example, PEG5000 is meant that molecular-weight average is approximately 5000 polyoxyethylene glycol.
" hepatoma " can be a kind of pernicious or innocent tumour of liver, comprises for example hepatocellular carcinoma.
" patient " is meant animal, preferably is meant Mammals, more preferably refers to the people.
As used herein, term " arginine deiminase that PEG modifies " or " PEG-ADI " refer to the compound of formula (I) structure:
ADI-(PEG)n (I)
Wherein PEG represents that molecular-weight average is the polyoxyethylene glycol of 1000-20000Da, and ADI represents arginine deiminase, and the covalent linkage between "-" expression PEG and the ADI.
Among the present invention, arginine deiminase and gene thereof can be to obtain from any source, comprise that reorganization produces or synthetic.For example the arginine deiminase gene can perhaps adopt chemical process complete synthesis from the mycoplasma genomic clone; The arginine deiminase gene can adopt native sequences, the majorizing sequence that perhaps adopts the gene engineering method sudden change to produce; Can adopt the sequence of a mycoplasma species sequence or multiple mycoplasma optimum combination.Preferably, arginine deiminase adopts the sequence in mycoplasma arginini, humanoid mycoplasma, mycoplasma arthritidis source, and preferred, arginine deiminase adopts the sequence in mycoplasma arginini source.A kind of particularly preferred ADI sequence is listed in Fig. 2.
In of the present invention, the molecular-weight average of polyoxyethylene glycol is from 1000 to 20000; Preferably be 2000-12000; More preferably be 3000-8000, more preferably from 4000-6000; Be about 5000Da best.
In the present invention, by activating group PEG is linked to each other with ADI.Used activating group itself is a leavings group, and activating group comes off in reaction, and PEG directly links to each other with protein.Such as organic alkylsulfonyl (comprising tosyl group, trifluoromethyl sulfonyl, trifluoroethyl alkylsulfonyl), halogen (F, Cl, I).An exception is acetaldehyde activatory PEG, and acetaldehyde does not come off in reaction as activating group, but oxyethyl group and PEG that the reaction back forms can not distinguish.The preferred activating group of the present invention is trifluoroethyl alkylsulfonyl (tresyl) base or Cl.
(linkerless) polyethyleneglycol modified technology of no linking group of the present invention is a kind of method that PEG is directly linked to each other with albumen.In this reaction, the PEG molecule is by a leavings group activation, and activating group is left away in ligation, thereby makes PEG and albumen directly covalently bound.People such as Malik F have described with trifluoroethyl SULPHURYL CHLORIDE activatory PEG (mPEG-tresyl) modified granulocyte-huge method of having a liking for colony-stimulating factor at Exp.Hematol.20:1028-1035 (1992) and Bri.J.Hematol.82:654-663 (1992).Francis G.E. etc. has described the method with halogenated PEG modified protein in WO9832466.The disclosure of these documents all is incorporated herein by reference.
The present invention also provides the method for the arginine deiminase of preparation modification, and it generally includes following steps:
(a) (a) provide the activated PEG precursor of formula (II):
mPEG-X
MPEG is a mono methoxy polyethylene glycol in the formula, and X is a leavings group,
In the present invention, preferred X is organic SULPHURYL CHLORIDE (comprising trifluoromethyl SULPHURYL CHLORIDE, trifluoroethyl SULPHURYL CHLORIDE, Phenylmethylsulfonyl chloride) or halogen (comprising F, Cl, I).Particularly preferred leavings group is trifluoroethyl SULPHURYL CHLORIDE (tresyl) and chlorine (Cl).
(b) activatory PEG precursor is mixed with arginine deiminase, at 0-30 ℃, PH6-8, reaction is 0.5-2 hour in suitable buffered soln such as the phosphate buffer solution, forms formula (I) compound;
(c) separation and purification formula (I) compound.Separating step can adopt ordinary methods such as column chromatography or affinity chromatography.A kind of preferable methods is that unreacted PEG is removed in dialysis earlier, obtains formula (I) compound by column chromatography or affinity chromatography then.
The degree of modification that increases protein or enzyme can increase the circulating half-life of modifier, but can reduce the specific activity of albumen or enzyme yet increase degree of modification, thereby need reach balance between the two.This point is conspicuous for those skilled in the art.Among the present invention, by the mol ratio of adjusting PEG and ADI, and controlling reaction time, can control degree of modification.Primary amino 30%-70% on the preferred arginine deiminase of the present invention is modified by PEG, and the primary amino that is more preferably 40%-60% is modified by PEG.
The albumen that PEG modifies is normally inhomogenous, but the PEG molecule that the protein molecule number of connection does not wait needs separation and purification to go out the more part of homogeneous sometimes.Among the present invention, a kind of preferred ingredients is both to be the mixture that each ADI subunit on average connects about 9-12 PEG molecule.For example modify the ADI product with mPEG-tresyl, mPEG-Cl, use column chromatography method subsequently, the isolated molecule amount is 200, and the component about 000Da gets final product the mixture that each ADI subunit on average connects about 9-12 PEG molecule.
The treatment effective dose of compound of the present invention is effectively to suppress the dosage of tumor growth, and is general, and treatment begins with low dose, so that constantly increase dosage the suitableeest under this condition.Before injection, PEG-ADI can or be that solution that any other is fit to known to those skilled in the art mixes with phosphate buffered saline buffer.The PEG-ADI preparation can be with solid (lyophilize) or liquid preparation form administration as demand.
The method of using compound of the present invention is determined on a case-by-case basis.Usually, using Compound P EG-ADI of the present invention can finish by following approach: in the oral cavity, in the nasal cavity, intraperitoneal, parenteral, intravenously, intralymphatic, tumour, in the intramuscular, a matter, in the intra-arterial, subcutaneous, intraocular, synovial membrane chamber, through epithelium, applied dermally.
In another aspect of this invention, also passed through a kind of pharmaceutical composition, it contains PEG-ADI of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount usually.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, PEG-ADI of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that PEG-ADI with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Major advantage of the present invention is:
With the PEG-ADI modifier that no linking group (linkerless) PEG modification technique obtains, PEG directly links to each other with ADI, and the C-N key of formation is very stable.Compare with the modification type ADI of existing band linking group, have more excellent comprehensive characteristic, promptly outstanding antitumous effect, chemical property is more stable, immunogenicity is lower.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The production of reorganization ADI
The gene of arginine deiminase according to T.Ohno etc. at Infect.Immun., the sequence of the mycoplasma arginini of announcing among the 58:3788-3795 (1990) is synthetic, open reading frame comprises 1230 base pairs (Fig. 1 and SEQ ID NO:1), wherein the codon TGA of 5 coding colors propylhomoserins is colibacillary nonsense codons, changes TGG into.SEQ ID NO:1 410 the amino acid whose arginine deiminases (as SEQ ID NO:2 and shown in Figure 2) of encoding.
Carry out expression and renaturation in the intestinal bacteria by the following method: specifically, synthetic ADI gene inserts pET32 carrier (Novagen company) SpeI, BamHI site, and member is built expression plasmid pET32-ADI.PET32-ADI transforms conventional coli strain BL21, and transformed bacteria is cultivated in 500ml LB substratum, the IPTG abduction delivering of usefulness 1.0mM 4 hours.Bacterium is used ultrasonication, centrifugal collection inclusion body.Inclusion body sex change damping fluid (50mM Tris-Cl, pH8.5,6M Guanidinium hydrochloride 10mM dithiothreitol (DTT)) 37 ℃, 1 hour.Dissolving supernatant liquor renaturation in the phosphoric acid buffer of pH7.5, the 10mM of 10 times of volumes was hatched 90 hours for 15 ℃.Adopt DEAE-Sepharose and arginine affinitive layer purification.
The product of purifying is 45KD in sex change SDS-PAGE, is 90KD in non-sex change electrophoresis, illustrates that arginine deiminase exists with dimeric forms.The N-terminal order-checking shows that the methionine(Met) of translation initiation codon coding is by the place to go.ADI is about 0.3mM in conjunction with arginic Km value, and the suitableeest enzyme reaction condition is 41 ℃, pH6.4.Stability test is presented under the physiological condition and placed 5 days, and enzymic activity keeps more than 50%.
ADI and TMPEG are crosslinked
The ADI and the molecular-weight average of preparation are the TMPEG of 5000Da among the embodiment 1 that present embodiment adopts.
The crosslinking reaction of PEG and ADI is at pH7.5, and the phosphoric acid buffer of 50mM that adds the sodium-chlor of 0.125M carries out the mass ratio of PEG and ADI 30: 1, stirring at room 2 hours.After reaction finished, unconjugated PEG removed with the ultra-filtration membrane dialysis in the mixture, and binding substances carries out degree of modification and enzyme assay.
Be used for measuring the degree of modification of arginine deiminase with following experiment:
At J.Biol.Chem., the method that 252.3582-(1977) describes is carried out, and promptly uses trinitro-benzene-sulfonic acid (TNBS) method titration free amine group with reference to A.Abuchowski etc., changes by free amine group before and after relatively PEG modifies, and determines degree of modification.
Be used for determining modification back arginine deiminase activity with following experiment:
At Meth.Enzymol., the method for describing among the 3:639-642 is carried out with reference to Oginsky etc.At 0.1MNa
2PO
4, pH7.0 (BUN measures damping fluid) 10ul sample places 96 hole titer plate, adds 40ul and measures 0.5mM arginine in the damping fluid at BUN, covers the culture plate lid, and incubation is 15 minutes under 37C.Add the complete BUN reagent of 20ul (Sigma Diagnostics), culture plate incubation 10 minutes under 100 C.Then culture plate is cooled to 22, and under 490nm, analyzes with microplate reader (Biored).Per minute can be converted into 1umol L-arginine the enzyme amount location 1.0IU of L-citrulline.Measure the enzymic activity of modifying front and back with this method.Measure protein content with the Xylene Brilliant Cyanine G method, calculate the ratio of modifying the front and back zymin and live.
In the present embodiment, remove unconjugated PEG after, the total degree of modification of binding substances is 51%, modify that back residual enzyme activity accounts for that initial enzyme lives 57%.
Binding substances is further used Sephacryl S-300HR (Pharmacia) gel chromatography, and it is about 200 to collect molecular weight, and the component about 000Da (annotate: after measured, in this mixture, each ADI on average is connected with 9-12 PEG molecule) is used for experimentation on animals.
Embodiment 3
ADI and mPEG-C1's is crosslinked
The ADI and the molecular-weight average of preparation are the mPEG-Cl of 5000Da among the embodiment 1 that present embodiment adopts.
The crosslinking reaction of mPEG-Cl and ADI is carried out at the phosphoric acid buffer of pH7.0,20mM, and the mol ratio of PEG and ADI is about 40: 1, stirring at room 2 hours.After reaction finished, unconjugated PEG removed with the ultra-filtration membrane dialysis in the mixture, and binding substances carries out degree of modification and enzyme assay.With Sephacryl S-300HR (Pharmacia) gel chromatography, collect molecular weight 200, the component about 000Da.In the present embodiment, remove unconjugated PEG after, the total degree of modification of binding substances is 53%, modify that back residual enzyme activity accounts for that initial enzyme lives 55%.
Binding substances is further used Sephacryl S-300HR (Pharmacia) gel chromatography, collects molecular weight 200, and the component about 000Da is used for experimentation on animals.
Circulating half-life
The molecular weight 200 of embodiment 2 preparations, 000Da PEG-ADI binding substances is used for circulating half-life and measures.
Circulating half-life with single-dose after, the time that the rising of the reduction of arginine concentration and citrulline concentration continues in the serum is weighed.With reference to the method that Takaku etc. uses at Jpn.J.Cancer Res., male BDF1 rat, the intravenously single-dose, dosage be 5IU/ only, different time after the administration, tail vein are got blood, ordinary method prepares serum ,-70 ℃ of preservations.Handle sample with the NDB-F fluorescent reagent during mensuration, use u-BondapackC
18The content of post assay determination arginine, citrulline on HPLC.
The content of arginine, citrulline such as Fig. 4 A and 4B, and shown in the Table I.
Table I blood plasma arginine, citrulline concentration (uM)
Time natural A DI PEG-ADI
Arginine citrulline arginine citrulline
Contrast 175.0 ± 21.3 69.4 ± 24.9 204.0 ± 31.2 74.6 ± 2.0
1 day＜5.0 243.1 ± 31.0＜5.0 277.0 ± 2.0
3 days＜5.0 286.6 ± 27.5＜5.0 245.9 ± 18.9
5 days 127.3 ± 17.8 63.4 ± 19.9＜5.0 304 ± 79.2
8 days 145.6 ± 21.3 60.4 ± 20.8＜5.0 237.5 ± 45
15 days 91.4 ± 5.4 75.5 ± 2.7 131.1 ± 40.3 80.9 ± 13.3
The result shows that natural A DI is eliminated very soon, and about 7-8 of ADI-PEG5000 transformation period days.
Immunogenicity
The molecular weight 200 of natural A DI, embodiment 2 preparations, the immunogenicity determining of 000Da PEG-ADI binding substances carries out by the following method: by 1 time weekly, the ADI of the natural or modification of intravenous injection 0.5IU, continuous 12 weeks, immune BalbC mouse.When 4,8,12 weeks of testing after beginning, the animal eye socket is got blood, and separation of serum is stored in-70C, measures the antibody of anti-ADI with the ELISA method.Antibody titers is defined as being worth than background absorption OD the high dilution of high 2 times serum.
The antibody titers of Table II ADI, PEG-ADI immunized mice
Detect antigen injecting pathway immunizing antigen antibody titers
ADI IV ADI 625000
ADI IV PEG-ADI 450
PEG-ADI IV ADI 3250
PEG-ADI IV PEG-ADI 150
The result as shown in Figure 5.Natural ADI injects 4 times and promptly produces above 10
5Titer antibody, the PEG-ADI injection does not produce detectable antibody 4 times.8 generations of PEG-ADI injection only produce about 10
2Titer antibody has significantly reduced immunogenicity.
Anti-tumour effect
Detect the ability that PEG5000-ADI suppresses the hepatoma growth in vivo.10 every group of nude mices are injected 10
6SK-Hep1 human liver cell oncocyte.Tumor growth is after 2 weeks, with the PEG5000-ADI of 5.0IU, 1 time weekly, intravenous injection.Add up the mortality ratio of control group, treatment group weekly.
The result as shown in Figure 6.Untreated animal all die in 3 weeks, and the animal that PEG5000-ADI handles has the long life-span.Survival rate is about 70% after 12 weeks.The animal of all survivals euthanasia after 3 months, tumour is not found in postmortem.
Table III ADI on the BDF1 mouse to the anti-tumour effect of SK-Hep 1 human liver cell knurl strain
Handle survival fate (number of animals)
Control group 8 (3) 13 (4) 19 (3)
2?IU PEG-ADI?41(2)80(1)180(7)
5?IU PEG-ADI?41(0)80(3)180(7)
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Fudan Zhangjiang biomedical Co., Ltd
＜120〉arginine deiminase of Xiu Shiing
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Leu?Lys?Ala?Asn?Asp?Ile?Asn?Val?Val?Glu?Leu?Ile?Asp?Leu?Val?Ala
65 70 75 80
Glu?Thr?Tyr?Asp?Leu?Ala?Ser?Gln?Glu?Ala?Lys?Asp?Lys?Leu?Ile?Glu
85 90 95
Glu?Phe?Leu?Glu?Asp?Ser?Glu?Pro?Val?Leu?Ser?Glu?Glu?His?Lys?Val
100 105 110
Val?Val?Arg?Asn?Phe?Leu?Lys?Ala?Lys?Lys?Thr?Ser?Arg?Lys?Leu?Val
115 120 125
Glu?Ile?Met?Met?Ala?Gly?Ile?Thr?Lys?Tyr?Asp?Leu?Gly?Ile?Glu?Ala
130 135 140
Asp?His?Glu?Leu?Ile?Val?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe?Thr?Arg
145 150 155 160
Asp?Pro?Phe?Ala?Ser?Val?Gly?Asn?Gly?Val?Thr?Ile?His?Tyr?Met?Arg
165 170 175
Tyr?Lys?Val?Arg?Gln?Arg?Glu?Thr?Leu?Phe?Ser?Arg?Phe?Val?Phe?Ser
180 185 190
Asn?His?Pro?Lys?Leu?Ile?Asn?Thr?Pro?Trp?Tyr?Tyr?Asp?Pro?Ser?Leu
195 200 205
Lys?Leu?Ser?Ile?Glu?Gly?Gly?Asp?Val?Phe?Ile?Tyr?Asn?Asn?Asp?Thr
210 215 220
Leu?Val?Val?Gly?Val?Ser?Glu?Arg?Thr?Asp?Leu?Gln?Thr?Val?Thr?Leu
225 230 235 240
Leu?Ala?Lys?Asn?Ile?Val?Ala?Asn?Lys?Glu?Cys?Glu?Phe?Lys?Arg?Ile
245 250 255
Val?Ala?Ile?Asn?Val?Pro?Lys?Trp?Thr?Asn?Leu?Met?His?Leu?Asp?Thr
260 265 270
Trp?Leu?Thr?Met?Leu?Asp?Lys?Asp?Lys?Phe?Leu?Tyr?Ser?Pro?Ile?Ala
275 280 285
Asn?Asp?Val?Phe?Lys?Phe?Trp?Asp?Tyr?Asp?Leu?Val?Asn?Gly?Gly?Ala
290 295 300
Glu?Pro?Gln?Pro?Val?Glu?Asn?Gly?Leu?Pro?Leu?Glu?Gly?Leu?Leu?Gln
305 310 315 320
Ser?Ile?Ile?Asn?Lys?Lys?Pro?Val?Leu?Ile?Pro?Ile?Ala?Gly?Glu?Gly
325 330 335
Ala?Ser?Gln?Met?Glu?Ile?Glu?Arg?Glu?Thr?His?Phe?Asp?Gly?Thr?Asn
340 345 350
Tyr?Leu?Ala?Ile?Arg?Pro?Gly?Val?Val?Ile?Gly?Tyr?Ser?Arg?Asn?Glu
355 360 365
Lys?Thr?Asn?Ala?Ala?Leu?Glu?Ala?Ala?Gly?Ile?Lys?Val?Leu?Pro?Phe
370 375 380
His?Gly?Asn?Gln?Leu?Ser?Leu?Gly?Met?Gly?Asn?Ala?Arg?Cys?Met?Ser
385 390 395 400
Met?Pro?Leu?Ser?Arg?Lys?Asp?Val?Lys?Trp
405 410
Claims (10)
1. an ADI-PEG compound that does not have linking group is characterized in that, has formula (I) structure
ADI-(PEG)n (I)
Wherein PEG represents that molecular-weight average is the polyoxyethylene glycol of 1000-20000Da, and ADI represents arginine deiminase, and the covalent linkage between "-" expression PEG and the ADI, n is the integer of 2-30.
2. compound as claimed in claim 1, wherein said arginine deiminase are the arginine deiminases of Mycoplasma microorganism.
3. compound as claimed in claim 2, wherein said arginine deiminase are the arginine deiminases of mycoplasma arginini, mycoplasma hominis or mycoplasma arthritidis.
4. compound as claimed in claim 3, wherein said arginine deiminase are the arginine deiminases of mycoplasma arginini.
5. compound as claimed in claim 4 is characterized in that, the molecular weight of described PEG is 4000-6000Da, and arginine deiminase has the aminoacid sequence shown in the SEQ ID NO:2.
6. the described compound of claim 1 is characterized in that, n is 5-17.
7. the described compound of claim 1 is characterized in that, n is 7-15.
8. a pharmaceutical composition is characterized in that, it contains described compound of claim 1 and pharmaceutically acceptable carrier.
9. the purposes of compound according to claim 1 is characterized in that be used to prepare medicine, this medicine is used for the treatment of hepatoma.
10. the method for a preparation formula (I) compound,
ADI-(PEG)n (I)
Wherein PEG represents that molecular-weight average is the polyoxyethylene glycol of 1000-20000Da, and ADI represents arginine deiminase, and the covalent linkage between "-" expression PEG and the ADI, n is the integer of 2-30,
It is characterized in that the method comprising the steps of:
(a) provide the activated PEG precursor of formula (II):
mPEG-X
MPEG is a mono methoxy polyethylene glycol in the formula, and X is a leavings group,
(b) activatory PEG precursor is mixed with arginine deiminase, at 0-30 ℃, reaction is 0.5-2 hour in the damping fluid of pH6-8, forms formula (I) compound;
(c) separation and purification formula (I) compound.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031162622A CN1536079A (en) | 2003-04-09 | 2003-04-09 | Modified arginine deiminase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031162622A CN1536079A (en) | 2003-04-09 | 2003-04-09 | Modified arginine deiminase |
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| Publication Number | Publication Date |
|---|---|
| CN1536079A true CN1536079A (en) | 2004-10-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031162622A Pending CN1536079A (en) | 2003-04-09 | 2003-04-09 | Modified arginine deiminase |
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| CN (1) | CN1536079A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101002945A (en) * | 2006-01-20 | 2007-07-25 | 清华大学 | Novel complexus used for treating tumor |
| CN101591649B (en) * | 2009-07-10 | 2013-02-13 | 江苏泰康生物医药有限公司 | Methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof |
| CN107073085A (en) * | 2014-09-16 | 2017-08-18 | 波拉里集团 | Arginine deiminase for the cross reactivity to ADI PEG20 antibody with reduction for the treatment of of cancer |
| CN108324951A (en) * | 2018-04-16 | 2018-07-27 | 薛剑峰 | A kind of arginine deiminase injection and preparation method thereof |
| CN110121340A (en) * | 2016-11-02 | 2019-08-13 | 波拉里集团 | The composite of PEGylated arginine deiminase |
-
2003
- 2003-04-09 CN CNA031162622A patent/CN1536079A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101002945A (en) * | 2006-01-20 | 2007-07-25 | 清华大学 | Novel complexus used for treating tumor |
| CN101002945B (en) * | 2006-01-20 | 2012-09-05 | 清华大学 | Novel complex used for treating tumor |
| CN101591649B (en) * | 2009-07-10 | 2013-02-13 | 江苏泰康生物医药有限公司 | Methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof |
| CN107073085A (en) * | 2014-09-16 | 2017-08-18 | 波拉里集团 | Arginine deiminase for the cross reactivity to ADI PEG20 antibody with reduction for the treatment of of cancer |
| CN110121340A (en) * | 2016-11-02 | 2019-08-13 | 波拉里集团 | The composite of PEGylated arginine deiminase |
| CN108324951A (en) * | 2018-04-16 | 2018-07-27 | 薛剑峰 | A kind of arginine deiminase injection and preparation method thereof |
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