CN103554187B

CN103554187B - Platinum complexes, protein complex of being obtained by this platinum compound and preparation method thereof

Info

Publication number: CN103554187B
Application number: CN201310428915.8A
Authority: CN
Inventors: 王震; 刘扬中; 师红东; 程芹芹; 张文星
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2013-09-18
Filing date: 2013-09-18
Publication date: 2016-03-30
Anticipated expiration: 2033-09-18
Also published as: CN103554187A

Abstract

The invention discloses a class contain the tetravalence platinum complex of imidazole group and be combined protein complex formed and preparation method thereof with azurin mutant.Be specifically related to azurin and a class tetravalence platinum complexes, described azurin has a Histidine to be suddenlyd change on its copper binding site, and platinum complex used contains an imidazole group, and the two is combined by coordination and is configured to albumen composition.To be tetravalence platinum complexes to replace in azurin by the imidazole group of Histidine that suddenlys change and copper atom coordination by imidazole group for the combination of tetravalence platinum complexes and azurin mutant, the coordination of tetravalence platinum complexes to be attached in the copper atom of azurin mutant in the heart.Growth of tumour cell Inhibition test result shows, the azurin containing tetravalence platinum complex has good growth of tumour cell restraining effect, has the application prospect as antitumor drug.

Description

Platinum complexes, protein complex of being obtained by this platinum compound and preparation method thereof

Technical field

The invention belongs to protein drug formulation art, be specifically related to protein drug and preparation method thereof, be specifically related to azurin and a class tetravalence platinum complexes, described azurin has a Histidine to be suddenlyd change on its copper binding site, platinum complex used contains an imidazole group, and the two is combined by coordination and is configured to albumen composition.

Background technology

Cancer is always the synonym of incurable disease.Since 21 century, along with industrialized development, the living environment of the mankind is in continuous deterioration, and the quantity of cancer patients also cumulative year after year.The data that International Union Against Cancer in 2010 issues in the 21st the anticancer conference in the world show, 2008, there were 1,270 ten thousand people's cancer strickens in the whole world, and death toll is up to 7,600,000, accounts for deaths worldwide's then 13%.In China, about newly-increased 2,200,000 cancer patientss every year, and have 1,300,000 people to die from cancer.Cancer is with replace cardiovascular disorder to become the first killer that people's life is captured by China.

Platinum compound is the key agents of current clinical anticancer, and what be mainly used in clinical treatment at present is divalence platinum complexes, and as cis-platinum, carboplatin, oxaliplatin etc., but it still has to be solved to problems such as the toxic side effect of body and resistances.And tetravalence platinum compound is also because to little, the fat-soluble increase of the toxic side effect of body by extensive concern.Cis-two chloro-trans-diacetyl-ammonia-methylamine closes platinum (IV) (JM216); and JP-A61-33192; JP-A-61-7283; the tetravalence platinum mentioned in JP-A-1-294684 all has outstanding water-soluble and low renal toxicity; but its antitumous effect is not very satisfactory, so need to find new approach.

Bacterioprotein is as the nowadays also heating gradually of potential cancer therapy drug, and azurin is one of focus wherein.Continue 2002, Illinois, US university Yamada etc. have caused the extensive concern of scientific circles since finding its suppression for growth of tumour cell.Have been found that it has cytostatic effect for kinds of tumor cells (MCF-7, J774, U2OScells etc.) at present, and similar effect has not been had for corresponding normal cell.And this patent describes is exactly that a mutant of bacterioprotein azurin is combined with the tetravalence platinum complexes that a class aglucon contains imidazole ring the class medicine formed by coordination.

Summary of the invention

In order to solve the problem, the object of the invention is to be combined with the tetravalence platinum complexes that a class aglucon contains imidazole ring the class mixture formed by a class mutant of bacterioprotein azurin by coordination, make it that there is the two common advantage, wish that this mixture is adding the tumor cell killing potential of azurin simultaneously, reduce again tetravalence platinum complexes to Normocellular toxic side effect.

In order to realize object of the present invention, first, the invention provides a kind of platinum complexes, its structural formula is as follows:

Wherein, two R ₁part can be the same or different, and it is by nitrogen-atoms and Pt Atomic coordinate, described R ₁for ammonia (NH ₃) or replace amine; Two L parts can be the same or different, and it is selected from halogen, carboxylate radical or substituted carboxylic acid root; Described R ₂for the substituting group containing imidazole group.

Wherein, described replacement amine is an ethylamine, diethylamide, a n-propyl amine, di-n-propyl amine, an isopropylamine, diisopropylamine, a n-butylamine, di-n-butyl amine, an isobutylamine, diisobutyl amine, a n-pentyl amine, two n-pentyl amine, an isoamylamine, diisoamyl amine or cyclohexanediamine; Described halogen is selected from Cl, Br or I; Described carboxylate radical is acetate moiety, propionate, butanic acid root, succinic, benzoate anion, toluylic acid root or p-methylbenzoic acid root; Described substituted carboxylic acid root is Monochloro Acetic Acid root, dichloro acetic acid root or trichoroacetic acid(TCA) root; Described substituting group comprises C (O) NHCH (COOH) CH ₂-Im or (CH ₂) _n-Im, wherein n=1-6, Im are imidazole group.

Further, the invention provides a kind of albumen composition, the part of the platinum complexes containing imidazole ring as pharmaceutical carrier with azurin mutant, is combined with this azurin mutant coordination and obtains by it; Wherein, described azurin mutant has the aminoacid sequence as shown in SEQIDNO:1, or it has the active fragment of equivalent biological or its examples of conservative variations or derivative.

Further, present invention also offers a kind of azurin mutant, it has the aminoacid sequence as shown in SEQIDNO:1, or it has the active fragment of equivalent biological or its examples of conservative variations or derivative.

Described azurin mutant is glycine or L-Ala by its copper binding site Histidine mutagenesis.Term used herein " has the fragment that equivalent biological is active " or its " examples of conservative variations " refers to that described fragment is different from the aminoacid sequence described in SEQIDNO:1 respectively, but remain described amino acid whose fundamental characteristics, as basic biological nature, structural performance, control characteristic or biochemical characteristic.Usually, the difference on described amino acid levels is limited, with make the sequence of reference polypeptide and its examples of conservative variations closely similar generally, and be identical in many regions.Difference Ke Yi Shi ー on examples of conservative variations and reference polypeptide aminoacid sequence or the same or analogous amino acid whose conservative property of multiple character are replaced, insert and are deleted.Replace or insert amino-acid residue can be encoded by genetic code, also can can't help genetic code coding.The variant of polynucleotide or polypeptide can produce (as allele variant) naturally, or it can the spontaneous variant of right and wrong.The variant that the non-natural of polynucleotide and polypeptide produces produces by induced-mutation technique or direct synthesis.

In conjunction with content disclosed by the invention, and conservative amino acid substitutions well known in the art, those of ordinary skill in the art know, azurin mutant of the present invention is not limited to the aminoacid sequence described in SEQIDNO:1, also comprise the fragment with the equivalent biological activity of substantially the same biological function obtained after above-mentioned conservative property is replaced, or its examples of conservative variations.

Further, present invention also offers a kind of nucleotide sequence of described azurin mutant of encoding, it comprises the nucleotide sequence shown in SEQIDNO:2, or its degeneracy variant." degeneracy variant " of the present invention refers to the degeneracy due to genetic code, the nucleotide sequence of described azurin mutant of encoding may have multiple based composition, its polynucleotide can be different from the polynucleotide of reference, but remain the basic of code for said proteins.The change of degeneracy Variant nucleotide sequences does not change the aminoacid sequence of described protein.Corresponding nucleotide sequence variants refers to the nucleotide sequence of the fragment that the described azurin mutant equivalent biological of coding is active or its examples of conservative variations or derivative.

Further, the invention provides the method preparing described albumen composition, the part of the platinum complexes containing imidazole ring as pharmaceutical carrier with azurin mutant, is combined with this azurin mutant coordination and obtains by it.

More specifically, the described method preparing albumen composition, specifically comprises the steps:

The first step: be connected on prokaryotic expression carrier by the cDNA of azurin, obtains the expression plasmid of an azurin;

Second step: the codon mutation of the Histidine on the coding azurin copper binding site in the expression plasmid obtained in the first step is the codon of glycine or L-Ala by utilization site-directed mutagenesis technique, obtains the expression plasmid for expressing azurin mutant;

3rd step: be transformed in prokaryotic expression system by above-mentioned expression plasmid, expresses and obtains azurin mutant, and carry out purifies and separates;

4th step: the described tetravalence platinum complex containing imidazole group of preparation;

5th step: by under cupric ion existent condition, adds the tetravalence platinum complex containing imidazole group, can obtain albumen composition after mixing in azurin mutant.

Preferably, in the 5th step: the concentration of described cupric ion or be 1:1 – 5:1 with the pass of azurin mutant amount; The condition of described reaction is normal temperature; Described azurin mutant is 1:1 – 10:1 with the amount ratio of tetravalence platinum complex.

Further, the invention provides a kind of pharmaceutical composition, it contains the described albumen composition for the treatment of effective dose, also containing other pharmaceutically acceptable auxiliary materials.

Further, the invention provides a kind of tumor therapeutic agent, it contains the described albumen composition for the treatment of effective dose, also containing other pharmaceutically acceptable auxiliary materials; Also optional other known antitumor drug comprising one or more effective doses.

" treatment effective dose " of the present invention refers to the amount that can produce positively effect for tumour to be treated.Concrete, according to the difference of the disease stage of tumor type to be treated, patient, general status (comprising age, body weight) and the factor such as administering mode, route of administration, clinician can adopt concrete " treatment effective dose " according to its micro-judgment.Generally speaking, described treatment effective dose is in the scope of 0.01mg-10mg platinum/every kg body weight.

In composition of the present invention, auxiliary material is that formulation arts personnel know, and includes but not limited to that vehicle, disintegrating agent, tackiness agent, tinting material etc. all can use, as long as described assistant agent does not affect the effect of albumen composition of the present invention.

Of the present invention for the therapeutical agent of oncotherapy in can also contain other known antitumor drug extraly, thus together with albumen composition of the present invention, produce synergy, treatment tumour.

Beneficial effect of the present invention is as follows:

The present invention is combined with the tetravalence platinum complexes that a class aglucon contains imidazole ring the class mixture formed by a class mutant of bacterioprotein azurin by coordination, make it that there is the two common advantage, add the tumor cell killing potential of azurin simultaneously, reduce again tetravalence platinum complexes to Normocellular toxic side effect.

To be tetravalence platinum complexes to replace in azurin by the imidazole group of Histidine that suddenlys change and copper atom coordination by imidazole group for the combination of tetravalence platinum complexes provided by the invention and azurin mutant, the coordination of tetravalence platinum complexes to be attached in the copper atom of azurin mutant in the heart.Growth of tumour cell Inhibition test result shows, the azurin containing tetravalence platinum complex has good growth of tumour cell restraining effect, has the application prospect as antitumor drug.

Accompanying drawing explanation

The collection of illustrative plates of prokaryotic expression carrier pST-GB1 used in Fig. 1 embodiment 1;

The Tricine-SDS-PAGE electrophorogram of azurin mutant in Fig. 2 embodiment 3;

Fig. 3 embodiment 4 tetravalence platinum complex one dimension nuclear magnetic spectrogram;

The growth of tumour cell Inhibition test result of tetravalence platinum complex and corresponding protein drug mixture in Fig. 4 embodiment 9.

Embodiment

Below in conjunction with the drawings and specific embodiments, concrete technical scheme of the present invention is specifically described.

Enzyme described in the present invention, test kit, carrier, Host Strains etc. can be obtained by commercial sources, if the bacterial strain of intestinal bacteria (Escherichiacoli) BL21 and Top10 is purchased from NEB company, Pseudomonas aeruginosa Pseudomonasaeruginosa (Schroeter) Migula ( 15692 ^tM) bacterial strain purchased from American Type culture collection warehousing (ATCC), the enzymes such as SspI and DpnI are purchased from Takara company, PCR primer, bacterial genomes DNA extraction agent box and pcr amplification test kit are purchased from Shanghai Sheng Gong bio-engineering corporation etc., and described pharmaceutical chemicals and reagent are if imidazoles, sodium-chlor, cupric chloride etc. are purchased from Chemical Reagent Co., Ltd., Sinopharm Group.Unreceipted actual conditions person in embodiment, routinely or manufacturers suggestion condition carry out.

Embodiment 1: the expression vector building azurin

Cut containing a N end containing (His) with SspI enzyme (purchased from Takara company) enzyme ₆accompanying drawing 1 is shown in by the prokaryotic expression carrier pST-GB1(collection of illustrative plates of GB1 label of-label, C end containing TEV restriction enzyme site), enzyme is cut 4 hours (actual conditions see the following form 1-1).Agarose gel electrophoresis, glue reclaim obtain SspI enzyme cut after pST-GB1 carrier.Wherein, pST-GB1 carrier is the plasmid that this laboratory is transformed on the basis of commercial pET21a, by the sequence excision on pET21a between NdeI and BamHI restriction enzyme site, and connects containing (His) ₆-label, GB1 hydrotropy label and one section of one section of sequence containing the LIC sequence of SspI enzyme restriction enzyme site.Using SEQIDNO:3 as forward primer, SEQIDNO:4 is as reverse primer (SEQIDNO:3 and SEQIDNO:4 is all purchased from Shanghai Sheng Gong bio-engineering corporation), be that (genomic dna is extracted from pseudomonas aeruginosa strains by the bacterial genomes DNA extraction agent box of Shanghai Sheng Gong bio-engineering corporation template with the genomic dna of the Pseudomonas aeruginosa (PseudomonasAeruginosa) containing azurin gene, bacterial strain is purchased from ATCC), to be seen the following form 1-2 by the cDNA(actual conditions of the pcr amplification test kit amplification azurin of raw work, 1-3).After agarose gel electrophoresis, glue reclaims the cDNA obtaining azurin.

The azurin cDNA(that pST-GB1 carrier after cutting with T4DNA polysaccharase (purchased from Takara company) 37 DEG C for the treatment of S spI enzyme enzymes and pcr amplification obtain is in Table 1-4).And by both 1:5 mixing in molar ratio, 30 DEG C of water-baths connect 30 minutes, obtain the plasmid of expressing azurin albumen.

Plasmid is proceeded in intestinal bacteria Top10 competence bacterial strain, coated plate, 37 DEG C hatch 24 hours after, obtain mono-clonal transformant.By this clone's enlarged culturing, extracting plasmid obtains a large amount of plasmid, for embodiment 2.

Table 1-1:SspI enzyme enzyme cuts pST-GB1 plasmid

pST-GB1	16μL
		ddH ₂O	10μL
10xSspI damping fluid	3μL
		SspI enzyme	1μL

Table 1-2: the PCR reaction system of the cDNA of amplification azurin

ddH ₂O	30.5μL
		10xPCR damping fluid	5μL
2mM dNTP(mix)	5μL
		25mM MgCl ₂	3μL
10mM forward primer	2.5μL
		10mM reverse primer	2.5μL
The genomic dna of Pseudomonas aeruginosa	1μL
		Taq archaeal dna polymerase	0.5μL

Table 1-3: the PCR thermal circulation parameters of the cDNA of amplification azurin

Table 1-4:T4DNA polysaccharase treatment S spI enzyme enzyme cut after pST-GB1 plasmid and the azurin cDNA that obtains of pcr amplification

The pST-GB1 plasmid that SspI enzyme is cut	15μL	Azurin cDNA	15μL
				0.1%BSA	2μL	0.1%BSA	2μL
10mM dCTP	1μL	10mM dGTP	1μL
				10xT4DNA polymerase buffer	2μL	10xT4DNA polymerase buffer	2μL
T4DNA polysaccharase	0.2μL	T4DNA polysaccharase	0.2μL

Embodiment 2: fixed-point mutation method builds the molecular cloning vector of azurin mutant protein.

Use site-directed mutagenesis technique to be hydrophobic amino acid glycine by the Histidine mutagenesis on the copper binding site of azurin to the plasmid that obtains in embodiment 1, obtain the plasmid of azurin mutant, concrete steps are as follows:

Be forward primer with SEQIDNO:5, take SEQIDNO:6 as reverse primer (SEQIDNO:5 and SEQIDNO:6 is all purchased from Shanghai Sheng Gong bio-engineering corporation), with the plasmid obtained in embodiment 1 for template, use TakaraPrimerSTAR ^tMhSDNAPolymerasewithGCBuffer test kit pcr amplification (actual conditions is in Table 2-1 and table 2-2); After agarose gel electrophoresis, glue reclaims PCR primer; By DpnI enzyme (purchased from Takara company) 37 DEG C process PCR primer 1 hour (see table 2-3); PCR primer after being cut by enzyme is transformed in intestinal bacteria Top10 competence bacterial strain, coated plate, obtains the mono-clonal transformant proceeding to this cloning vector.By this clone's enlarged culturing, extracting plasmid obtains a large amount of plasmid, i.e. the molecular cloning vector of azurin mutant.The plasmid of order-checking azurin mutant, the aminoacid sequence of gained azurin mutant is as shown in SEQIDNO1.

Table 2-1: the PCR reaction system of rite-directed mutagenesis azurin

ddH ₂O	31μL
		5xPrime STAR buffer	10μL
2mM dNTP	5μL
		10mM forward primer	1μL

10mM reverse primer	1μL
		The plasmid that embodiment 1 obtains	1μL
Primer STAR	1μL

Table 2-2: the PCR thermal circulation parameters of rite-directed mutagenesis azurin

Table 2-3:DpnI enzyme enzyme cuts PCR primer

PCR primer	17μL
		10xtango Buffer	2μL
DpnI enzyme	1μL

Embodiment 3: the expression and purification of azurin mutant

The plasmid obtained in embodiment 2 is proceeded in BL21 (DE3) competence bacterium, spread plate, picking mono-clonal transformant, cultivate in the LB substratum (often liter of substratum is containing 0.1 milligram of penbritin) of enlarged culturing to 1 liter; To OD ₆₀₀when=0.8, add in cupric chloride (final concentration is 20 μm of ol/L) and isopropyl-beta D-thio galactopyranoside (IPTG, final concentration is 0.2mmol/L), induce 10 hours for 25 DEG C.

25 DEG C of conditions, receive bacterium 20 minutes with 4000 rpms of rotating speeds; With the resuspended bacterium of the resuspended damping fluid of Ni-NTA (30 milliliters of damping fluids often rise bacterium); Ultrasonication bacterium, the broken liquid of 16000 rpms of eccentric cells 30 minutes under 4 DEG C of conditions, suction filtration supernatant, obtains the supernatant of cytoclasis liquid.

Poured into by supernatant in Ni-NTA affinity chromatography resin column (Shanghai Sheng Gong bio-engineering corporation), under 4 DEG C of conditions, rotary shaker shakes 30 minutes, makes (His) ₆-GB1-azurin mutant fusion protein is attached on Ni-NTA affinity chromatography resin column; Put and penetrate liquid, wash assorted damping fluid with 10 milliliters and wash away foreign protein; With 20 milliliters of elution buffer wash-out fusion roteins.Collect elution buffer, cut 12 hours with tobacco etch virus enzyme 16 DEG C of enzymes.

By dialysis or ultrafiltration, the imidazoles in removing damping fluid.Enzyme after being cut by enzyme is cut liquid and is poured in Ni-NTA affinity chromatography resin column, rotates 30 minutes, make (His) in rotary shaker under 4 DEG C of conditions ₆-GB1 label, (His) ₆-GB1-azurin mutant fusion protein and tobacco etch virus enzyme are attached on Ni-NTA affinity chromatography resin column.Collection penetrates liquid, washes assorted damping fluid and washes away azurin mutant on Ni-NTA affinity chromatography resin column, collect and wash assorted damping fluid with 10 milliliters.

By collect penetrate liquid and wash in assorted damping fluid add excessive dithiothreitol (DTT) and ethylenediamine tetraacetic acid (EDTA), ultrafiltration and concentration, with Hiload16/60Superdex75(purchased from GEHealth) molecular sieve chromatography post is further purified, and obtains azurin mutant.

The Tricine-SDS-PAGE electrophorogram of the azurin mutant obtained is shown in accompanying drawing 2, left lane is that protein standard Marker(is purchased from ThermoScience), molecular weight is respectively 66.2kDa, 45.0kDa, 35.0kDa, 25.0kd, 18.4kDa and 14.4kDa from top to bottom, and the right swimming lane is azurin mutant.The resuspended damping fluid of note: 1.Ni-NTA: 50mmol/L phosphoric acid buffer pH8.0(formula sees the following form 3-1), 200mmol/L sodium-chlor

2. wash assorted damping fluid: 50mmol/L phosphoric acid buffer pH8.0,200mmol/L sodium-chlor, 20mmol/L imidazoles

3. elution buffer: 50mmol/L phosphoric acid buffer pH8.0,200mmol/L sodium-chlor, 250mmol/L imidazoles

Table 3-1:0.1mol/L phosphoric acid buffer pH8.0 formula

Embodiment 4:c, c, t-[Pt (NH ₃) ₂cl ₂(succinicacid-histidine) (OH)] preparation

The cis-platinum of 1mmol is dissolved in 5 ml waters, with the hydrogen peroxide of 10mmol 50 DEG C of stirring reactions 2 hours, stirred overnight at room temperature 10 hours, rotary evaporation removes desolventizing and obtains solid, through cold water, each repetitive scrubbing of ether 3 times, dry under vacuum environment, obtain yellow solid.

By the dimethyl formamide of dissolution of solid obtained above for 1mmol at 5 milliliters, with the Succinic anhydried of 1mmol 30 DEG C of stirring reactions 12 hours, rotary evaporation obtains solid except desolventizing, respectively washs 3 times with acetone, ether, dry in vacuum environment, obtain light yellow solid.

Be dissolved in by every 2mmol Histidine in the dimethyl formamide (containing 1-5%N, N-diisopropylethylamine) of 10 milliliters and react 5 hours with excessive fluorenylmethyloxycarbonyl and trityl at 30 DEG C, the amino of its main chain and the imidazolyl of side chain are protected.

The resin reaction after dimethyl formamide activates of the Histidine mixed solution of above-mentioned fluorenylmethyloxycarbonyl and trityl as protecting group and 1mmol 3 hours; add 20% piperidines and cut fluorenylmethyloxycarbonyl and trityl-protecting group on Histidine, wash away the fluorenylmethyloxycarbonyl, trityl and the Histidine that is not connected on resin that cut with dimethyl formamide.Now obtain the Histidine be combined on resin.

By the tetravalence platinum complexes of monosubstituted for the axis of 2mmol succinic acid and 1mmol1-hydroxybenzotriazole, 1mmol benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate and 1mmolN, N-diisopropylethylamine reacts 12 hours with the above-mentioned Histidine 30 DEG C be combined on resin in dimethyl formamide environment, TFA with 1% cuts the material be combined on resin, with dimethyl formamide, Virahol, normal hexane respectively washs 3 times and is separated from resin by this material, rotary evaporation removes desolventizing and obtains solid, with acetone, ether respectively washs 3 times, dry in vacuum environment, obtain light yellow solid, structural formula is as shown below:

Obtained light yellow solid one dimension nuclear magnetic spectrogram is shown in accompanying drawing 3, can prove that product is c, c, t-[Pt (NH ₃) ₂cl ₂(succinicacid-histidine) (OH)].Wherein chemical shift is δ (ppm)=4.69 corresponding D ₂o solvent peak; H on C2 on δ (ppm)=8.56 corresponding imidazole ring; H on δ (ppm)=7.22 corresponding imidazole ring on C4; H on main chain on δ (ppm)=4.44 corresponding histidine residues on C7; Two H on side chain on δ (ppm)=3.18 and δ (ppm)=3.06 corresponding histidine residues on C6; Four H on the Succinic anhydried of δ (ppm)=2.46 and δ (ppm)=2.58 difference corresponding tetravalence platinum coordination on C13 and C14.

Embodiment 5:c, c, t-{Pt [(1R, 2R)-cyclohexane-1,2-diamine] (ethanedioato-O, O ') (succinicacid-histidine) (OH)] preparation

Replace the raw material cis-platinum in embodiment 4 with oxaliplatin, adopt identical condition, method and step, finally can prepare

C, c, t-{Pt [(1R, 2R)-cyclohexane-1,2-diamine] (ethanedioato-O, O ') (succinicacid-histidine) (OH) }, structural formula is as follows:

Embodiment 6:c, c, t-[Pt (NH ₃) ₂cl ₂(succinicacid-histidine) (OH)] preparation of protein drug mixture of coordination

By the 1mmol azurin mutant protein of expression in embodiment 3 and the CuCl of 1mmol ₂after mixing, obtain in conjunction with cupric azurin mutant, add the c that 0.2mmol embodiment 4 is synthesized, c, t-[Pt (NH ₃) ₂cl ₂(succinicacid-histidine) (OH)], in aqueous, mix under 25 DEG C of conditions, react 1 hour, can c be obtained, c, t-[Pt (NH ₃) ₂cl ₂(succinicacid-histidine) (OH)] mixture of azurin mutant of combination of coordination.

Embodiment 7:c, c, t-{Pt [(1R, 2R)-cyclohexane-1,2-diamine] (ethanedioato-O, O ') (succinicacid-histidine) (OH) } preparation of protein drug mixture of coordination

By the 1mmol azurin mutant protein of expression in embodiment 3 and the CuCl of 1mmol ₂after mixing, obtain in conjunction with cupric azurin mutant, add 0.1mmolc, c, t-{Pt [(1R, 2R)-cyclohexane-1, 2-diamine] (ethanedioato-O, O ') (succinicacid-histidine) (OH) }, in aqueous, mix under 25 DEG C of conditions, react 1 hour, c can be obtained, c, t-{Pt [(1R, 2R)-cyclohexane-1, 2-diamine] (ethanedioato-O, O ') (succinicacid-histidine) (OH) } mixture of azurin mutant of coordination.

Embodiment 8:c, c, t-{Pt (NH ₃) ₂(dichloroaceticacid) [OC (O) (CH ₂) ₃-imidazole] (OH) } preparation of protein drug mixture of coordination

By the 1mmol azurin mutant protein of expression in embodiment 3 and the CuCl of 1mmol ₂after mixing, add 0.5mmolc, c, t-{Pt (NH ₃) ₂(dichloroaceticacid) [OC (O) (CH ₂) ₃-imidazole] (OH) }, in aqueous, mix under 25 DEG C of conditions, react 1 hour, can c be obtained, c, t-[Pt (NH ₃) ₂(dichloroaceticacid) (OCH ₂(O) (CH ₂) ₃-imidazole) (OH)] mixture in conjunction with azurin mutant of coordination.

Embodiment 9: the medicinal effect test of platinum complexes prepared by the present invention

One, cell cultures

By MCF-7 breast cancer cell and MCF-10A normal cell in containing rpm I-1640 substratum of 10% calf serum in 37 DEG C, 5%CO ₂cultivate in incubator.Logarithmic phase cell is selected in experiment.

Two, mtt assay measures cytoactive

Cytotoxicity experiment selects 20000 every milliliter, logarithmic phase cell to be seeded in 96 orifice plates, every hole 100 microlitre, cultivate and change fresh substratum after 24 hours, experimental group, control group and blank group are set, experimental group adds the medicine of different concns gradient, control group does not add medicine, and blank group does not have inoculating cell and do not add medicine; Continuation cultivation is tetrazolium bromide (MTT) method mensuration cytoactive after 72 hours, calculates the cell survival rate of experimental group, thus obtains the cytotoxicity of medicine.

1, collect logarithmic phase cell, adjustment concentration of cell suspension, every hole adds 100 microlitres, and bed board makes cell density to be measured be 2000 every holes, (marginal pore sterile PBS buffer is filled).

2,5%CO ₂, hatch for 37 DEG C, be paved with (96 hole flat underside) at the bottom of hole, add the medicine of concentration gradient to cell monolayer, each concentration establishes 3 multiple holes.

3,5%CO ₂, hatch 72 hours for 37 DEG C, observe under inverted microscope.

4, discard old substratum and add fresh culture 100 microlitre, then every hole adds 25 pi of MTT solution (5mg/mL, i.e. 0.5%MTT), continues cultivation 2 hours.

6, every hole adds 100 microlitres of cells lysates, 5%CO ₂, 37 DEG C of overnight incubation, make crystallisate fully dissolve.The absorbance A value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.

Survival rate (%)=[A value-blank (B) the A value of experimental group (S)]/[A value of the A value-blank (B) of control group (C)]

Experimental result shows: the tetravalence platinum in above-described embodiment and tetravalence platinum albumen composition all have good cytotoxicity for MCF-7 cell, and tetravalence platinum albumen composition for normal MCF-10A cytotoxicity and azurin similar, effect is weak compared with tetravalence platinum.

Growth of tumour cell Inhibition test the results are shown in Figure 4, and the Azurin wherein in figure is the azurin mutant of preparation in embodiment 3; His-Pt in figure is the c of preparation in embodiment 4, c, t-[Pt (NH ₃) ₂cl ₂(succinicacid-histidine) (OH)]; His-Pt2 in figure is the c of preparation in embodiment 5, c, t-{Pt [(1R, 2R)-cyclohexane-1,2-diamine] (ethanedioato-O, O ') (succinicacid-histidine) (OH) }; Im-Pt in figure is the tetravalence platinum complexes c in embodiment 8, c, t-{Pt (NH ₃) ₂(dichloroaceticacid) [OC (O) (CH ₂) ₃-imidazole] (OH) }; Azurin-His-Pt1 in figure is the protein drug mixture obtained in embodiment 6; Azurin-His-Pt2 in figure is the protein drug mixture of preparation in embodiment 7; Azurin-Im-Pt in figure is the protein drug mixture obtained in embodiment 8.(note: the albumen of above-mentioned protein drug mixture is all identical with the blending ratio of platinum medicine, for albumen and tetravalence platinum medicine mol ratio are 5:1, albumen composition concentration refers to the concentration of platinum in mixture)

The above is only the preferred embodiment of the present invention, it is to be noted, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention, and, after having read content of the present invention, relevant technical staff in the field can make various change or amendment to the present invention, and these equivalent form of values fall into the application's appended claims limited range equally.

Claims

1. a platinum complexes, its structural formula is as follows:

Wherein, two R ₁part can be the same or different, this part by nitrogen-atoms and Pt Atomic coordinate, described R ₁for ammonia (NH ₃) or replace amine; Two L parts can be the same or different, and comprise halogen, carboxylate radical or substituted carboxylic acid root; Described R ₂for the substituting group containing imidazole group; The described substituting group containing imidazole group is C (O) NHCH (COOH) CH ₂-Im or (CH ₂) _n-Im, wherein n=1-6, Im are imidazole group.

2. platinum complexes according to claim 1, it is characterized in that, described replacement amine is an ethylamine, diethylamide, a n-propyl amine, di-n-propyl amine, an isopropylamine, diisopropylamine, a n-butylamine, di-n-butyl amine, an isobutylamine, diisobutyl amine, a n-pentyl amine, two n-pentyl amine, an isoamylamine, diisoamyl amine or cyclohexanediamine.

3. platinum complexes according to claim 1, is characterized in that, described halogen is Cl, Br or I; Described carboxylate radical is acetate moiety, propionate, butanic acid root, succinic, benzoate anion, toluylic acid root or p-methylbenzoic acid root; Described substituted carboxylic acid root is Monochloro Acetic Acid root, dichloro acetic acid root or trichoroacetic acid(TCA) root.

4. an albumen composition, is characterized in that, the platinum complexes containing imidazole group described in claims 1 to 3 any one as pharmaceutical carrier with azurin mutant, is combined with this azurin mutant coordination and obtains by it; Wherein, described azurin mutant has the aminoacid sequence as shown in SEQIDNO:1.

5. albumen composition according to claim 4, it is characterized in that, its preparation method is, uses azurin mutant described in claim 4 as pharmaceutical carrier, to be combined by the cupric coordination of the part of the platinum complexes containing imidazole group in this azurin mutant and to obtain.

6. albumen composition according to claim 4, is characterized in that, it specifically comprises the steps:

4th step: the tetravalence platinum complex of preparation containing imidazole group;

7. a pharmaceutical composition, is characterized in that, albumen composition according to any one of its claim 4 or 6 containing treatment effective dose, also containing other pharmaceutically acceptable auxiliary materials.

8. a tumor therapeutic agent, is characterized in that, albumen composition according to any one of its claim 4 or 6 containing treatment effective dose, also containing other pharmaceutically acceptable auxiliary materials.