CN102617852B - Maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, preparation methods for maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, and application of maleimide-polyglutamic acid-aspartic acid polymer and composite thereof - Google Patents
Maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, preparation methods for maleimide-polyglutamic acid-aspartic acid polymer and composite thereof, and application of maleimide-polyglutamic acid-aspartic acid polymer and composite thereof Download PDFInfo
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Abstract
The invention discloses a maleimide-polyglutamic acid-aspartic acid polymer. The maleimide-polyglutamic acid-aspartic acid polymer is formed by the covalent bonding of a gamma-polyglutamic acid-aspartic acid polymer and amine compounds containing maleimide groups, wherein the bonding is realized by forming an amido bond between a free carboxyl group of the gamma-polyglutamic acid-aspartic acid polymer and amino groups on the amine compounds containing the maleimide groups under the coaction of a condensing agent and a catalyst. The invention also discloses a maleimide-polyglutamic acid-aspartic acid composite and a preparation method and application thereof. By coupling biological active molecules with a sulfydryl group, the stability and in-vivo activity of the maleimide-polyglutamic acid-aspartic acid polymer can be improved, and the in-vivo half life of the maleimide-polyglutamic acid-aspartic acid polymer can be prolonged; and meanwhile, the polymer which is coupled with targeting molecules with the sulfydryl group and is combined with an anti-tumor medicine can become a polymer medicine with active targeting.
Description
Technical field
The present invention relates to maleimide-polyglutamic acid-aspartic acid polymkeric substance and its mixture, and preparation method thereof and purposes, belong to the biological medicine technology field.
Background technology
Gamma-polyglutamic acid-is a kind of aminoacid polymers that is produced by microbial fermentation, is formed by alpha-amino group and the γ-carboxyl condensation of L-glutamic acid.Gamma-polyglutamic acid-has non-immunogenicity, biocompatibility and degradability, is widely used in every field.Gamma-polyglutamic acid-is as pharmaceutical carrier, can improve the water-soluble of medicine, the time of releasing of prolong drug, the toxic side effect of reduction medicine, can be used for the biological medicine technology field, disclose the scale operation of the clinical development of polyglutamic acid-therapeutic agent conjugates as patent 00817029.0; Patent 200610024353.0 discloses gamma-polyglutaic acid-CDDP complex, preparation and purposes; 200910045189.5 gamma-polyglutamic acid-asparaginic acid complex and preparation method and purposes are disclosed and for example, form gamma-polyglutamic acid-asparaginic acid complex by gamma-polyglutamic acid-and the reaction of aspartic acid generation acid amides, gamma-polyglutamic acid-is after aspartic acid is modified, as the antitumor drug carrier, can improve the drug loading of cis-platinum.But above-mentioned gamma-polyglutamic acid-and gamma-polyglutamic acid-asparaginic acid carrier be the coupling part directly, does not also possess initiatively target function.
Summary of the invention
In order to solve above-mentioned shortcoming, the invention provides a kind of maleimide-polyglutamic acid-aspartic acid polymkeric substance; Be the transformation to natural polymer, obtain the novel medicament carrier, can with the site-specific coupling of band sulfydryl part, expanded the range of application of polyglutamic acid pharmaceutical carrier.The present invention adopts the amine that contains maleimide base group that the gamma-polyglutamic acid-asparaginic acid polymkeric substance is carried out chemically modified, obtains maleimide-polyglutamic acid-aspartic acid polymkeric substance.Maleimide-polyglutamic acid-aspartic acid polymkeric substance can with the coupling of band sulfydryl part locus specificity, this is because the two keys in the maleimide base group and sulfydryl generation Michael addition reaction cause; Part comprises biologically active molecules and targeted molecular.Maleimide-polyglutamic acid-aspartic acid polymkeric substance and the coupling of band sulfydryl biologically active molecules can increase its stability, prolong the transformation period in its body, carry its high in-vivo activity.Maleimide-polyglutamic acid-aspartic acid polymkeric substance and the coupling of band sulfydryl targeted molecular again further combined with antitumor drug, become the initiatively polymer drug of target of tool.
Maleimide-polyglutamic acid provided by the invention-aspartic acid polymkeric substance is the polymkeric substance that is formed by the aminated compounds that contains maleimide base group and gamma-polyglutamic acid-asparaginic acid polymkeric substance covalent attachment, bonding wherein is by the carboxyl of polyglutamic acid-aspartic acid and contains and form amido linkage between the amino of aminated compounds of maleimide base group under condensing agent and catalyzer acting in conjunction and realize that its general formula is:
The positive integer of n=30～1000;
The positive integer of m=1/10 n～n;
The positive integer of z=1/50 n～1/2 n;
γ-PGA is gamma-polyglutamic acid-;
Asp is aspartic acid;
Mal is the aminated compounds that contains maleimide base group, the one end is maleimide base group, the other end is amino, include but not limited to N-(2-amino-ethyl)-6-maleimide hexanamide, N-(2-amino-ethyl) maleimide, N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamide; Its general formula is:
R is the carbochain of dissimilar different lengthss, wherein including but not limited to hydrophilic radicals such as acid amides and ehter bonds.
In maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance, carboxylic group is 50:1～2:1 with the molecule number ratio of maleimide base group, is preferably 20:1～4:1, more preferably 10:1.
The molecular weight of maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance is 2000Da～100KDa, is preferably 4000Da～80KDa, more preferably 5000Da～40KDa.
Wherein, condensing agent and catalyzer play activated carboxyl respectively and reduce the effect (reference [Christian A. G. N. Montalbetti et al. Tetrahedron 61 10827 – 10852]) that side reaction takes place.
Another object of the present invention is to provide a kind of maleimide-polyglutamic acid-asparaginic acid complex.Maleimide-polyglutamic acid provided by the invention-asparaginic acid complex refers to the mixture that the part of the maleimide base group locus specificity coupling band sulfydryl of maleimide-polyglutamic acid-aspartic acid polymkeric substance forms, and bonding wherein is the sulfydryl generation Michael addition reaction generation by the two keys in the maleimide base group and part.
Among the present invention, the part of band sulfydryl comprises biologically active molecules and targeted molecular.Biologically active molecules is albumen or the polypeptide with pharmaceutical value, includes but not limited to bioactive peptide, proteolytic enzyme, cytokine.Targeted molecular is that the mediation mixture is combined with tumor cell specific and is improved the part of result for the treatment of, includes but not limited to the cancer target peptide, antibody and antibody fragment.
Maleimide-polyglutamic acid of the present invention-asparaginic acid complex, its general formula is:
The positive integer of n=30～1000;
The positive integer of m=1/10 n～n;
The positive integer of z=1/50 n～1/2 n;
Mal is the amine that contains maleimide base group, it is characterized in that an end is maleimide base group; The other end is amino; Include but not limited to N-(2-amino-ethyl)-6-maleimide hexanamide, N-(2-amino-ethyl) maleimide, N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamide; Its general formula is:
R is the carbochain of dissimilar different lengthss, wherein including but not limited to hydrophilic radicals such as acid amides and ehter bonds.
γ-PGA is gamma-polyglutamic acid-.
Asp is aspartic acid.
Y is bioactive molecules for the part of band sulfydryl.Y is including, but not limited to bioactive peptide, proteolytic enzyme, cytokine.Maleimide-polyglutamic acid of the present invention-asparaginic acid complex has the biologic activity that part brings, and becomes the polymer drug with good pharmacodynamic properties.
Another object of the present invention is to provide maleimide-polyglutamic acid-aspartic acid-medicinal composition, maleimide-polyglutamic acid-aspartic acid polymkeric substance combines with the part that has sulfydryl, combining with antitumor drug forms again, and its general structure is:
The positive integer of n=30～1000;
The positive integer of m=1/10 n～n;
The positive integer of z=1/50 n～1/2 n;
Mal is the amine that contains maleimide base group, it is characterized in that an end is maleimide base group; The other end is amino; Include but not limited to N-(2-amino-ethyl)-6-maleimide hexanamide, N-(2-amino-ethyl) maleimide, N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamide; Its general formula is:
R is the carbochain of dissimilar different lengthss, wherein including but not limited to hydrophilic radicals such as acid amides and ehter bonds.
γ-PGA is gamma-polyglutamic acid-.
Asp is aspartic acid.
X is antitumor drug.Y is including, but not limited to cis-platinum, camptothecine, taxol.
Y is targeted molecular for the part of band sulfydryl.Y is including, but not limited to the target peptide, antibody and antibody fragment.The Y targeted molecular only has targeting, needs bound drug such as antitumor drug.Maleimide-polyglutamic acid of the present invention-aspartic acid-medicinal composition has the active targeting that part brings, and has the anti-tumor activity that medicine brings simultaneously, thereby becomes targeted drug.
Another object of the present invention is to provide the preparation method of maleimide-polyglutamic acid-asparaginic acid complex, carry out the acid amides reaction with water-soluble poly L-glutamic acid-aspartic acid polymkeric substance and the amine that contains maleimide base group at the organic phase mild conditions, prepare maleimide-polyglutamic acid-aspartic acid polymkeric substance, react with the part that has sulfydryl further and obtain described maleimide-polyglutamic acid-asparaginic acid complex.
Preparation method of the present invention specifically may further comprise the steps:
(a) obtain the gamma-polyglutamic acid-polymer by microbial fermentation and purge process, adopt the high temperature acidolysis legal system to be equipped with lower molecular weight gamma-polyglutamic acid-sodium salt;
(b) the protected aspartic acid of lower molecular weight gamma-polyglutamic acid-sodium salt and carboxyl is dissolved in distilled water, and under the condensing agent effect, the amido linkage of formation obtains the gamma-polyglutamic acid-asparaginic acid sodium salt;
(c) with souring method lower molecular weight gamma-polyglutamic acid-asparaginic acid sodium salt is converted into Hydrogen, obtains the acid type gamma-polyglutamic acid-asparaginic acid;
(d) molecular weight acid type gamma-polyglutamic acid-asparaginic acid is dissolved in inert reaction solvent with the amine that contains maleimide base group, under the existence effect of condensing agent and catalyzer, the amido linkage that forms between amino by the described amine that contains maleimide base group and the carboxyl of Hydrogen gamma-polyglutamic acid-asparaginic acid is attached to maleimide base group on the gamma-polyglutamic acid-asparaginic acid;
(e) use the ether termination reaction, and the extractive reaction mixture, phase liquid down collected;
(f) impurity and unreacted small-molecule substance are removed in dialysis, and obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance by cryodesiccated mode, are white floss;
(g) with the part of maleimide-polyglutamic acid-aspartic acid polymkeric substance and band sulfydryl, i.e. biologically active molecules, as bioactive peptide, proteolytic enzyme, cytokine is dissolved in the deionized water, reaction 12h; Obtain maleimide-polyglutamic acid-asparaginic acid complex through the exclusion chromatography separation and purification, i.e. biologically active molecules-maleimide-polyglutamic acid-asparaginic acid complex.
Another object of the present invention is to provide the preparation method of maleimide-polyglutamic acid-asparaginic acid complex-medicine, may further comprise the steps:
(a) obtain the gamma-polyglutamic acid-polymer by microbial fermentation and purge process, adopt the high temperature acidolysis legal system to be equipped with lower molecular weight gamma-polyglutamic acid-sodium salt;
(b) the protected aspartic acid of lower molecular weight gamma-polyglutamic acid-sodium salt and carboxyl is dissolved in distilled water, and under the condensing agent effect, the amido linkage of formation obtains the gamma-polyglutamic acid-asparaginic acid sodium salt;
(c) with souring method lower molecular weight gamma-polyglutamic acid-asparaginic acid sodium salt is converted into Hydrogen, obtains the acid type gamma-polyglutamic acid-asparaginic acid;
(d) molecular weight acid type gamma-polyglutamic acid-asparaginic acid is dissolved in inert reaction solvent with the amine that contains maleimide base group, under the existence effect of condensing agent and catalyzer, the amido linkage that forms between amino by the described amine that contains maleimide base group and the carboxyl of Hydrogen gamma-polyglutamic acid-asparaginic acid is attached to maleimide base group on the gamma-polyglutamic acid-asparaginic acid;
(e) use the ether termination reaction, and the extractive reaction mixture, phase liquid down collected;
(f) impurity and unreacted small-molecule substance are removed in dialysis, and obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance by cryodesiccated mode, are white floss;
(h) with the part (targeted molecular of maleimide-polyglutamic acid-aspartic acid polymkeric substance with the band sulfydryl, as target peptide, antibody and antibody fragment) be dissolved in the deionized water, 4 ℃ were reacted 12 hours, add antitumor drug, in 37 ℃ of lucifuge stirring reactions 48 hours, dialysed 48 hours in regulator solution pH to 7.0～8.0, with 0.22 mm membrane filtration, obtain targeted molecular-maleimide-polyglutamic acid-aspartic acid-antitumour drug complex.
Among the present invention, gamma-polyglutamic acid-asparaginic acid, its molecular weight is 1000Da～100KDa, is preferably 4000Da～80KDa, more preferably 4000Da～30KDa.
Among the present invention, the acid type gamma-polyglutamic acid-asparaginic acid is transformed by the gamma-polyglutamic acid-asparaginic acid sodium-salt form, and the gamma-polyglutamic acid-asparaginic acid sodium salt was dialysed 48 hours under sour condition, obtained through lyophilize.
Among the present invention, Mal refers to contain the aminated compounds of maleimide base group, and molecule one end is maleimide base group, and the other end is amino; Include but not limited to N-(2-amino-ethyl)-6-maleimide hexanamide, N-(2-amino-ethyl) maleimide, N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamides etc. are preferably N-(2-amino-ethyl)-6-maleimide hexanamide.
Among the present invention, maleimide-polyglutamic acid-aspartic acid polymkeric substance, its molecular weight is 2000Da～100KDa, is preferably 4000Da～80KDa, more preferably 5000Da～40KDa.
The preparation method of maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance, the carboxyl of the described polyglutamic acid-aspartic acid of step (d) is 25:1～1:5 with the mol ratio that contains the amine of maleimide base group, be preferably 10:1～1:2, more preferably 5:2; Temperature of reaction is-20～150 ℃, is preferably 0～70 ℃, and more preferably 0～30 ℃, the reaction times is 1～150h, is preferably 24～90h, more preferably 72h.
The preparation method of maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance, described solvent can be distilled water or inert organic solvents, wherein said inert organic solvents includes but not limited to dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), acetonitriles etc. are preferably dimethyl sulfoxide (DMSO) (DMSO).
Maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance is at condensing agent and catalyzer and leaves and react, and forms amido linkage.The condensing agent that is fit to is to know in the organic chemical synthesis, include but not limited to N, N-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), N, N-DIC (DIC), I-hydroxybenzotriazole (HOBt), N-hydroxy thiosuccinimide (sulfo-NHS), N, the N'-carbonyl dimidazoles, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus etc. are preferably 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and I-hydroxybenzotriazole (HOBt).The catalyzer that is fit to comprises organic bases, includes but not limited to the 4-Dimethylamino pyridine, and 4-tetramethyleneimine pyridine and triethylamine etc. are preferably triethylamine.
Another object of the present invention is to provide the application of maleimide-polyglutamic acid-aspartic acid polymkeric substance as pharmaceutical carrier.The present invention also provides maleimide-polyglutamic acid-asparaginic acid complex or the application of maleimide-polyglutamic acid-aspartic acid-medicinal composition in preparation prevention or treatment disease medicament.Maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance can combined belt sulfydryl biologically active molecules, increase its stability, prolong the transformation period in its body, improve its activity in vivo, the carrier that serves as high biologically active molecules can be used for prophylactic treatment immunity, viral and metabolic disease.Maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance can combined belt sulfydryl targeted molecular, again further combined with antitumor drug, has target function, serve as the targeting vector of antitumor drug, tumour includes but not limited to liver cancer, mammary cancer, intestinal cancer, the esophageal carcinoma, prostate cancer etc.
The present invention can with the band sulfydryl biologically active molecules of maleimide-polyglutamic acid-aspartic acid polymkeric substance coupling, refer to have albumen or the polypeptide of pharmaceutical value, including, but not limited to bioactive peptide, cytokine, somatomedin and hormone etc., for example Regular Insulin, human glucagon-like-peptide 1 and Interferon, rabbit etc.
The present invention can with the targeted molecular of the band sulfydryl of maleimide-polyglutamic acid-aspartic acid polymkeric substance coupling, including, but not limited to the target peptide, antibody and antibody fragment, for example, Cetuximab, Herceptin and shellfish are cut down pearl monoclonal antibody etc.
The present invention can with the medicine that maleimide-polyglutamic acid-the aspartic acid polymkeric substance is combined, refer to cancer therapy drug direct or indirect and that maleimide-polyglutamic acid-aspartic acid polymkeric substance carboxyl is combined, including, but not limited to cis-platinum, taxol, camptothecine, morellic acid, Zorubicin and alkane promise ketone etc.
The present invention innovates part and is to transform in the gamma-polyglutamic acid-asparaginic acid polymer base, prepare maleimide-polyglutamic acid-aspartic acid polymkeric substance, this polymkeric substance is as pharmaceutical carrier, can with the band sulfydryl the biologically active molecules combination, the carrier that serves as high biologically active molecules, increase the stability of biologically active molecules, the transformation period in the extension body, improve activity in vivo; Simultaneously, the targeted molecular that this polymkeric substance also can the combined belt sulfydryl, and further combined with antitumor drug, the targeting vector that serves as medicine, improve curative effect of medication, reduce the toxic side effect of medicine, further widened gamma-polyglutamic acid-and gamma-polyglutamic acid-asparaginic acid as the application of pharmaceutical carrier.
Description of drawings
Maleimide-polyglutamic acid that Fig. 1 obtains for the present invention-aspartic acid polymkeric substance utilizes AVANCE 500MHZ nmr determination
1The H nuclear magnetic resonance map.
Fig. 2 is the lab diagram that maleimide-polyglutamic acid-aspartic acid polymkeric substance is connected with target peptide TP13.1 is albumen marker, and 2 is target peptide TP13, and 3 is target peptide TP13-maleimide-polyglutamic acid-asparaginic acid complex, and 4 is maleimide-polyglutamic acid-aspartic acid polymkeric substance, and 5 is polyglutamic acid-aspartic acid and target peptide TP13 mixture.
Fig. 3 is the release in vitro curve (physiological saline) of cis-platinum in target peptide TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex.
Fig. 4 is the lab diagram of the specificity combination of target peptide TP13-maleimide-polyglutamic acid-asparaginic acid complex and liver cancer cell SMMC-7721.
Fig. 5 is the lab diagram of the specificity combination of target peptide TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex and liver cancer cell SMMC-7721.
Fig. 6 is target peptide TP13-maleimide-polyglutamic acid-aspartic acid-cytotoxicity experiment figure of 48 hours of CDDP complex effect liver cancer cell SMMC-7721.
Fig. 7 investigates target peptide TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex in normal male kunming mice toxicity in vivo lab diagram from the survival rate angle.
Fig. 8 investigates target peptide TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex in normal male kunming mice toxicity in vivo lab diagram from the body weight change angle.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content of mentioning specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
The preparation of embodiment 1 acid type gamma-polyglutamic acid-asparaginic acid
Genus bacillus (Bacillus licheniformis ATCC 9945a) fermentative preparation obtains gamma-polyglutamic acid-, and reference literature [Haifeng Ye et al. Biomaterial 27 5958-5965] is slightly changed.Be inoculated in fermention medium (maltose 50 gl with 4% inoculum size
-1, yeast extract paste 10 gl
-1, Sodium Glutamate 30 gl
-1, NaCl 10gl
-1, KH
2PO
45gl
-1, MgSO
47H
2O 0.5gl
-1), 37
0C, 220 rmin
-1Shake-flask culture 72 hours.After the fermentation ends, 4 times of fermented liquid adding distil water dilutions are transferred fermented liquid pH to 2.0～3.0,12000 rmin with hydrochloric acid
-1, the centrifugal removal bacterial sediment of 30 min, to regulate supernatant liquor pH to 7.0～8.0 then, add 3 times of dehydrated alcohols that volume is ice-cold again, stirring obtains the gamma-polyglutamic acid-precipitation, to precipitate and be dissolved in again in the distilled water, and remove insolubles, impurity and small-molecule substance are removed in dialysis (molecular weight cut-off MW:10000), lyophilize obtains the white powder material and is the gamma-polyglutamic acid-macromole, and output can reach 30 gl
-1
2% macromole gamma-polyglutamic acid-solution 200 ml, transfer pH to 2.0, effect 25 min under 121 ℃, 0.1MPa condition obtain the lower molecular weight gamma-polyglutamic acid-through the dialysis lyophilize, identify the molecular weight of degraded product by 1% agarose gel electrophoresis and gel permeation chromatography.By gel permeation chromatography lower molecular weight gamma-polyglutamic acid-of the present invention molecular weight at 2500Da～30KDa.
The preparation reference literature of gamma-polyglutamic acid-asparaginic acid [Zhen Feng et al. Journal of Biomaterials Science, Polymer Edition 22 2023-2040] is slightly changed.Lower molecular weight gamma-polyglutamic acid-(γ-PGA; 8.5 g); be dissolved in the distilled water (50 ml); add the aspartic acid (H-Asp (OtBu)-OtBu that carboxyl is protected by Otbu; 17 g) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC, 12.75 g), through deprotection and separation and purification; lyophilize obtains gamma-polyglutamic acid-asparaginic acid.
Gamma-polyglutamic acid-asparaginic acid (10 g), be dissolved in the 100 ml deionized waters after, add hydrochloric acid and regulate and transfer pH to 2.0, the dialysis purifying, lyophilize gets the acid type gamma-polyglutamic acid-asparaginic acid.Measure gamma-polyglutamic acid-asparaginic acid sodium salt of the present invention by multi-angle laser light scattering instrument and size exclusion chromatography, coupling, its weight-average molecular weight, the weight average molecular radius, polydispersity coefficient is respectively 1,14 * 10
4Da, 31.1nm, 1.792 ± 0.021.Nmr determination (Bruker Avance 500 spectrometer) is measured, and by the gamma-polyglutamic acid-asparaginic acid that this scheme obtains, wherein glutaminic acid residue is about 1:0.82 than asparagicacid residue number ratio.
The preparation method of embodiment 2 maleimides-polyglutamic acid-aspartic acid polymkeric substance
Reaction scheme 1:
Get the acid type gamma-polyglutamic acid-asparaginic acid (PGA-Asp that above embodiment 1 prepares; 37mg); be dissolved in dimethyl sulphoxide solution (DMSO; 10 ml) in, (HOBt is 80mg) with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC to add I-hydroxybenzotriazole; 120mg); drip the triethylamine of 150 μ L and the solution (400mg is dissolved in 1mlDMFO) of N-(2-amino-ethyl)-6-maleimide hexanamide, 37 ℃ of nitrogen protections were stirred 72 hours.The ether termination reaction that in reaction system solution, adds 15 times of volumes, fierce concussion, mixed solution is divided into two-layer up and down, and lower floor's liquid is light yellow.With lower floor's liquid collecting, (pH 7.0, and 0.2M), liquid dialysis 48 hours, lyophilize obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance white floss to add isopyknic phosphoric acid buffer.Weight-average molecular weight by gel permeation chromatography maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance is 1,63 * 10
4Da.Nmr determination (Bruker Avance 500 spectrometer) is measured the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp1 that obtains by this scheme, see Fig. 1), wherein maleimide base group is about 0.43:1 than glutaminic acid residue number ratio, and general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=21(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid.
Reaction scheme 2:
Get the acid type gamma-polyglutamic acid-asparaginic acid (PGA-Asp of above embodiment 1 preparation; 37mg); be dissolved in dimethyl sulphoxide solution (DMSO; 10 ml) in, (HOBt is 80mg) with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC to add I-hydroxybenzotriazole; 120mg); drip the triethylamine of 75 μ L and the solution (200mg is dissolved in 1mlDMFO) of N-(2-amino-ethyl)-6-maleimide hexanamide, 37 ℃ of nitrogen protections were stirred 72 hours.The ether termination reaction that in reaction system solution, adds 15 times of volumes, fierce concussion, mixed solution is divided into two-layer up and down, and lower floor's liquid is light yellow.With lower floor's liquid collecting, (pH 7.0, and 0.2M), liquid dialysis 48 hours, lyophilize obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance white floss to add isopyknic phosphoric acid buffer.Weight-average molecular weight by gel permeation chromatography maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance is 1,54 * 10
4Da.Nmr determination (Bruker Avance 500 spectrometer) is measured the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp2) that obtains by this scheme, wherein maleimide base group is about 0.33:1 than glutaminic acid residue number ratio, and general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=17(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid.
Reaction scheme 3:
Get the acid type gamma-polyglutamic acid-asparaginic acid (PGA-Asp of above embodiment 1 preparation; 37mg); be dissolved in dimethyl sulphoxide solution (DMSO; 10 ml) in, (HOBt is 80mg) with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC to add I-hydroxybenzotriazole; 120mg); drip the triethylamine of 30 μ L and the solution (40mg is dissolved in 1mlDMFO) of N-(2-amino-ethyl)-6-maleimide hexanamide, 37 ℃ of nitrogen protections were stirred 72 hours.The ether termination reaction that in reaction system solution, adds 15 times of volumes, fierce concussion, mixed solution is divided into two-layer up and down, and lower floor's liquid is light yellow.With lower floor's liquid collecting, (pH 7.0, and 0.2M), liquid dialysis 48 hours, lyophilize obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance white floss to add isopyknic phosphoric acid buffer.Weight-average molecular weight by gel permeation chromatography maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance is 1,42 * 10
4Da.Nmr determination (Bruker Avance 500 spectrometer) is measured the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp3) that obtains by this scheme, wherein maleimide base group is about 0.24:1 than glutaminic acid residue number ratio, and general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=12(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid.
Reaction scheme 4:
Get the acid type gamma-polyglutamic acid-asparaginic acid (PGA-Asp of above embodiment 1 preparation; 37mg); be dissolved in dimethyl sulphoxide solution (DMSO; 10 ml) in, (HOBt is 80mg) with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC to add I-hydroxybenzotriazole; 120mg); drip the triethylamine of 21 μ L and the solution (24mg is dissolved in 1mlDMFO) of N-(2-amino-ethyl)-6-maleimide hexanamide, 37 ℃ of nitrogen protections were stirred 72 hours.The ether termination reaction that in reaction system solution, adds 15 times of volumes, fierce concussion, mixed solution is divided into two-layer up and down, and lower floor's liquid is light yellow.With lower floor's liquid collecting, (pH 7.0, and 0.2M), liquid dialysis 48 hours, lyophilize obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance white floss to add isopyknic phosphoric acid buffer.Weight-average molecular weight by gel permeation chromatography maleimide-polyglutamic acid of the present invention-aspartic acid polymkeric substance is 1,23 * 10
4Da.Nmr determination (Bruker Avance 500 spectrometer) is measured the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp4) that obtains by this scheme, wherein maleimide base group is about 0.07:1 than glutaminic acid residue number ratio, and general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=4(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid.
Reaction scheme 5:
With reference to identical ratio in preceding four kinds of reaction scheme, with N, dinethylformamide with identical experimental program, can obtain similar experimental result as solvent respectively.
Reaction scheme 6:
With reference to identical ratio in preceding four kinds of reaction scheme, with N, N-dicyclohexylcarbodiimide (DCC) with identical experimental program, can obtain similar experimental result as condensing agent respectively.
Reaction scheme 7:
With reference to identical ratio in preceding four kinds of reaction scheme, with N, N-DIC (DIC) with identical experimental program, can obtain similar experimental result as condensing agent respectively.
Reaction scheme 8:
With reference to identical ratio in preceding four kinds of reaction scheme, as catalyzer, with identical experimental program, can obtain similar experimental result respectively with the 4-Dimethylamino pyridine.
Reaction scheme 9:
With reference to identical ratio in preceding four kinds of reaction scheme, as reactant, with identical experimental program, can obtain similar experimental result respectively with N-(2-amino-ethyl) maleimide, general structure is:
Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid.
Reaction scheme 10:
With reference to identical ratio in preceding four kinds of reaction scheme, as reactant, with identical experimental program, can obtain similar experimental result respectively with N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamide, general structure is:
Mal is N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid.
Maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp1 with 1 preparation of embodiment 2 reaction scheme, 48mg) be dissolved in the deionized water (10 ml), add recombinanthumaninterferon (IFN α-2b, 10mg), 4 ℃ were reacted 12 hours, this preparation condition is conducive to keep recombinanthumaninterferon's activity, adopt dialysis, method such as ultrafiltration and Size Exclusion Chromatograph SEC is carried out separation and purification, IFN-maleimide-polyglutamic acid-the asparaginic acid complex that obtains, general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=21(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid; IFN is recombinanthumaninterferon (IFN α-2b).
The determination of activity of embodiment 4 IFN maleimide-polyglutamic acid-asparaginic acid complexes
With IFN-maleimide-polyglutamic acid-asparaginic acid complex and the recombinanthumaninterferon of embodiment 3 preparations, measure its biologic activity according to " the Chinese biological goods rules " P372-P375 described " Interferon, rabbit titration (cytopathic-effect inhibition assay) " that is published (version in 2000) by Chemical Industry Press.The results are shown in table 1.The result shows that IFN-maleimide-polyglutamic acid-asparaginic acid complex has kept its biologic activity.
The preparation of embodiment 5 C225-maleimide-polyglutamic acid-asparaginic acid complexes
Get the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp1 of above embodiment 2 reaction scheme 1 described preparation, 48mg), in the dissolving deionized water (10 ml), add Cetuximab (C225,8mg), 4 ℃ were reacted 12 hours, this preparation condition is conducive to keep the activity of the monoclonal antibody of anti-EGFR, adopt dialysis, method such as ultrafiltration and Size Exclusion Chromatograph SEC is carried out separation and purification, obtain C225-maleimide-polyglutamic acid-asparaginic acid complex that this scheme obtains, general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=21(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid; C225 be Cetuximab (monoclonal antibody of anti-EGFR, cetuximab, C225).
The determination of activity of embodiment 6 C225-maleimide-polyglutamic acid-asparaginic acid complexes is to the cell in vitro inhibiting rate of MCF-7 MCF27
Will
MCF27The cell kind is in 96 orifice plates (l * 10
4Individual cells/well) cultivate 12h in, (the drug level scope is at 0.25～512mg.mL to change the DMEM-F12 perfect medium of McAb-maleimide-polyglutamic acid-asparaginic acid complex that the monoclonal antibody (McAb) that contains anti-EGFR and embodiment 5 prepare behind the cell attachment respectively
-1), each concentration is done 3 multiple holes, organizes in contrast with not dosing group, changes the substratum that once contains medicine, cultivates 12 days in per 3 days.It is the MTT damping fluid of 5 mg/mL that every hole adds 20mL concentration, continues to cultivate 2 hours, careful sucking-off nutrient solution, and every hole adds 200 μ L dimethyl sulfoxide (DMSO), jolts several seconds gently, measures its absorbance value at 570 nm places on microplate reader.C225 concentration is 58mg.mL in McAb-maleimide-polyglutamic acid-asparaginic acid complex
-1The time, the MCF27 cell proliferation inhibition rate is about 50%; This shows that C225-maleimide-polyglutamic acid-asparaginic acid complex has kept its biologic activity.
The preparation of embodiment 7 TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex
Get scheme 3 preparation-obtained maleimide-polyglutamic acids-asparaginic acid complex (Mal-PGA-Asp3 of embodiment 2,48mg), in the dissolving deionized water (10 ml), add target peptide (TP13,6mg), 4 ℃ of reactions 12 hours (SDS-PAGE detects Mal-PGA-Asp coupling TP13 and the results are shown in Figure 1) add cis-platinum 18 mg, transfer pH value of solution to 7.0～8.0, in 37
0C lucifuge stirring reaction 48 hours was dialysed 48 hours, with 0.22 mm membrane filtration, obtained TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex (TP13-Mal-PGA-Asp3-Pt) solution that this scheme obtains, and general structure is:
Wherein, n=51(mean value); M=42(mean value); Z=12(mean value); Mal is N-(2-amino-ethyl)-6-maleimide hexanamide; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid; TP13 is target peptide (aminoacid sequence: CYHWYGYTPQNVI); CDDP is that cis dichloro diamines closes platinum.
The preparation of embodiment 8 TP13-maleimide-polyglutamic acids-aspartic acid-CDDP complex freeze-dried powder
Get the TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex (being equivalent to cis-platinum 100mg) of embodiment 7 preparations, add an amount of water for injection dissolving, add N.F,USP MANNITOL 0.8 g and stir, regulate pH 5.0 with hydrochloric acid, add 0.5% needle-use activated carbon, 60
o C insulation 20 minutes, take off charcoal after, filtrate adds sterilized water for injection to 40ml, under the aseptic condition, with 0.22 mm filtering with microporous membrane, be sub-packed in the 10ml cillin bottle, every bottle of 1ml lyophilize, outlet rolls lid and gets final product.
The release in vitro curve of cis-platinum in embodiment 9 TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex
TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex solution with embodiment 7 described preparations changes the dialysis band over to, dialyse as extracellular fluid dialysis with physiological saline, take a sample in predetermined time, measure the content of cis-platinum, be depicted as the release in vitro curve (Fig. 3) of cis-platinum in TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex (TP13-Mal-PGA-Asp3-Pt).The result shows: in physiological saline, cis-platinum in TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex is discharged gradually, it is characterized by: discharge in preceding 15 h soon, the accumulative total burst size reaches 50%, slowly slowing down subsequently, is 76% at 72h accumulative total release rate.
Get the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp of above embodiment 2 reaction scheme 3 described preparations, 4.8mg) and with the target peptide (FITC-TP13 of FITC mark, 0.6mg) be dissolved in the deionized water of 1ml, 4 ℃ were reacted 12 hours, use the hyperfiltration process purifying, obtain this scheme and obtain pure FITC-TP13-maleimide-polyglutamic acid-asparaginic acid complex (FITC-TP13-PGA-Asp).Liver cancer cell SMMC-7721 is through trysinization, collects resuspendedly, cell suspension is divided into 4 manages.First pipe is blank cell, second tube cell and FITC-TP13 (40mg/mL), the FITC-TP13-PGA-Asp of the 3rd tube cell and (being equivalent to 40 mg/mL FITC-TP13), the 4th tube cell and (40 mg/mL, be the irrelevant peptide of FITC mark) FITC-F13 fostered 2 hours at 37 ℃, use D-Hank ' s solution to give a baby a bath on the third day after its birth time, be used for flow cytometer and detect, each 20,000 cells of analyzing and testing.The result shows that (Fig. 4) TP13-maleimide-polyglutamic acid-asparaginic acid complex and liver cancer cell SMMC-7721 have the specificity combination.
Embodiment 11 TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex and the combination of liver cancer cell SMMC-7721 specificity
Get the maleimide-polyglutamic acid-aspartic acid polymkeric substance (Mal-PGA-Asp of above embodiment 2 reaction scheme 3 described preparations, 4.8mg) and with the target peptide (FITC-TP13 of FITC mark, 0.6mg) be dissolved in the deionized water of 1ml, 4 ℃ were reacted 12 hours, add cis-platinum (1.8mg) in 37 ℃ of lucifuge reactions 48 hours, use the hyperfiltration process purifying, obtain this scheme and obtain pure FITC-TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex (FITC-TP13-PGA-Asp-Pt).With SMMC-7721 cell suspension kind in having put 6 orifice plates of cover glass in advance, incubated overnight, make cell attachment, hatched 2 hours with the FITC-TP13-Mal-Aps-PGA of (being equivalent to 0.1mg/ml FITC-TP13), wash 3 times with D-Hanks, add Paraformaldehyde 96 then and fix 30 minutes, carefully will fixedly there be the cover glass of cell to take out, drip 80% glycerine (5 * PBS of 800 mL glycerine+200 mL mixes) at slide glass, cover glass is placed on the slide glass, observes with laser confocal microscope.The result shows that (Fig. 5) TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex and liver cancer cell SMMC-7721 have the specificity combination.
With SMMC-7721 cell kind in 96 orifice plates (l * 10
4Individual cells/well) cultivates 12h in, change respectively behind the cell attachment and contain free cis-platinum, (the drug level scope is at 5～200mg.mL for the DMEM perfect medium of gamma-polyglutamic acid-asparaginic acid-CDDP complex and TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex (TP13-Mal-PGA-Asp3-Pt)
-1), each concentration is done 3 multiple holes, organizes in contrast with not dosing group, cultivates 24 and 48 h.It is the MTT damping fluid of 5 mg/mL that every hole adds 20mL concentration, continues to cultivate 2 hours, careful sucking-off nutrient solution, and every hole adds 200 μ L dimethyl sulfoxide (DMSO), jolts several seconds gently, measures its absorbance value at 570 nm places on microplate reader.
Gamma-polyglutamic acid-asparaginic acid-CDDP complex and TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex have all kept anti-tumor biological and have learned active, under the Isodose condition, TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex is than the vitro cytotoxicity height (Fig. 6) of gamma-polyglutamic acid-asparaginic acid-CDDP complex, and this is because TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex has targeting.
The toxicity in vivo of embodiment 13 TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex
Totally 30 of male mouse of kunming, body weight 14～17 g are divided into three groups at random, 10 every group, inject salt solution in the physiology respectively; Free cis-platinum 4 mg/kg, TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex (TP13-Mal-PGA-Asp3-Pt) 4 mg/kg, medication is intraperitoneal administration, be administered three times altogether, respectively at administration in the 0th, 2,4 day, record survival condition and the body weight change situation of mouse every day, the results are shown in Figure 6 and Fig. 7.
Be the toxicity in vivo of investigating medicine from the angle of normal mouse survival rate from Fig. 7, the result shows: the toxicity of the free normal mouse of CDDP is very big, mouse death rate 55% after 7 days; And TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex administration group mouse does not have death.
Fig. 8 is the toxicity in vivo of investigating medicine from the angle of normal mouse body weight change, and the result shows: CDDP administration group and control group mice body weight have extremely significant difference; And TP13-maleimide-polyglutamic acid-aspartic acid-CDDP complex administration group and control group mice body weight indifference show that its toxicity significantly reduces.
Protection content of the present invention is not limited to above embodiment.Under the spirit and scope that do not deviate from inventive concept, variation and advantage that those skilled in the art can expect all are included in the present invention, and are protection domain with the appending claims.
Claims (15)
1. maleimide-polyglutamic acid-aspartic acid polymkeric substance, it is characterized in that, described polymkeric substance is the polymkeric substance that is formed by gamma-polyglutamic acid-asparaginic acid polymkeric substance and the aminated compounds covalent attachment that contains maleimide base group, and bonding wherein is by the free carboxy of gamma-polyglutamic acid-asparaginic acid polymkeric substance and contains between the amino on the aminated compounds of maleimide base group under condensing agent and catalyzer acting in conjunction the formation amido linkage and realize; Its structural formula is:
Wherein, the positive integer of n=30～1000; The positive integer of m=1/10n～n; The positive integer of z=1/50n～1/2n; γ-PGA is gamma-polyglutamic acid-; Asp is aspartic acid; Mal is the aminated compounds that contains maleimide base group, and its structural formula is:
Wherein, R is carbochain.
2. maleimide-polyglutamic acid as claimed in claim 1-aspartic acid polymkeric substance, it is characterized in that, Mal is N-(2-amino-ethyl)-6-maleimide hexanamide, N-(2-amino-ethyl) maleimide, N-(2-(2-(2-amino ethoxy) oxyethyl group) ethyl)-2-maleimide ethanamide.
3. maleimide-polyglutamic acid as claimed in claim 1-aspartic acid polymkeric substance is characterized in that, described polymericular weight is 2000Da～100KDa.
4. maleimide-polyglutamic acid-asparaginic acid complex, it is characterized in that, described mixture is the formation that combines with the part that has sulfydryl as each described maleimide-polyglutamic acid-aspartic acid polymkeric substance of claim 1-3, bonding wherein is the sulfydryl generation Michael addition reaction generation by the two keys in the maleimide base group and described part, and its structural formula is:
Wherein, the positive integer of n=30～1000; The positive integer of m=1/10n～n; The positive integer of z=1/50n～1/2n; Mal is the aminated compounds that contains maleimide base group; Y is the part that has sulfydryl.
5. maleimide-polyglutamic acid as claimed in claim 4-asparaginic acid complex is characterized in that, Y is bioactive molecules, and described bioactive molecules is selected from bioactive peptide, proteolytic enzyme and cytokine.
6. maleimide-polyglutamic acid-aspartic acid-medicinal composition, it is characterized in that, described mixture combines with the part that has sulfydryl for each the described maleimide-polyglutamic acid-aspartic acid polymkeric substance as claim 1-3, combining with antitumor drug forms again, and its structural formula is:
Wherein, the positive integer of n=30～1000; The positive integer of m=1/10n～n; The positive integer of z=1/50n～1/2n; Mal is the aminated compounds that contains maleimide base group; Y is the part that has sulfydryl; X is antitumor drug, and described antitumor drug is selected from cis-platinum, camptothecine, taxol.
7. maleimide-polyglutamic acid as claimed in claim 6-aspartic acid-medicinal composition is characterized in that, Y is targeted molecular, and described targeted molecular is selected from the target peptide, antibody and antibody fragment.
8. the preparation method of maleimide-polyglutamic acid-asparaginic acid complex, it is characterized in that, described method is carried out the acid amides reaction with water-soluble poly L-glutamic acid-aspartic acid polymkeric substance and the amine that contains maleimide base group at the organic phase mild conditions, prepare maleimide-polyglutamic acid-aspartic acid polymkeric substance, further obtain maleimide-polyglutamic acid as claimed in claim 4-asparaginic acid complex with the part reaction that has sulfydryl.
9. preparation method as claimed in claim 8 is characterized in that, may further comprise the steps:
(a) obtain the gamma-polyglutamic acid-polymer by microbial fermentation and purge process, adopt the high temperature acidolysis legal system to be equipped with lower molecular weight gamma-polyglutamic acid-sodium salt;
(b) the protected aspartic acid of lower molecular weight gamma-polyglutamic acid-sodium salt and carboxyl is dissolved in distilled water, under the condensing agent effect, forms amido linkage, obtains the gamma-polyglutamic acid-asparaginic acid sodium salt;
(c) with souring method lower molecular weight gamma-polyglutamic acid-asparaginic acid sodium salt is converted into Hydrogen, obtains the acid type gamma-polyglutamic acid-asparaginic acid;
(d) the acid type gamma-polyglutamic acid-asparaginic acid is dissolved in inert organic solvents with the amine that contains maleimide base group, under the existence effect of condensing agent and catalyzer, the amido linkage that forms between amino by the described amine that contains maleimide base group and the carboxyl of Hydrogen gamma-polyglutamic acid-asparaginic acid is attached to maleimide base group on the gamma-polyglutamic acid-asparaginic acid;
(e) use the ether termination reaction, and the extractive reaction mixture, phase liquid down collected;
(f) impurity and unreacted small-molecule substance are removed in dialysis, and obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance by cryodesiccated mode, are white floss;
(g) maleimide-polyglutamic acid-aspartic acid polymkeric substance and the part that has sulfydryl are dissolved in the deionized water reaction 12h; Obtain maleimide-polyglutamic acid as claimed in claim 4-asparaginic acid complex through the exclusion chromatography separation and purification.
10. the preparation method of maleimide-polyglutamic acid-aspartic acid-medicinal composition is characterized in that, may further comprise the steps:
(a) obtain the gamma-polyglutamic acid-polymer by microbial fermentation and purge process, adopt the high temperature acidolysis legal system to be equipped with lower molecular weight gamma-polyglutamic acid-sodium salt;
(b) the protected aspartic acid of lower molecular weight gamma-polyglutamic acid-sodium salt and carboxyl is dissolved in distilled water, under the condensing agent effect, forms amido linkage, obtains the gamma-polyglutamic acid-asparaginic acid sodium salt;
(c) with souring method lower molecular weight gamma-polyglutamic acid-asparaginic acid sodium salt is converted into Hydrogen, obtains the acid type gamma-polyglutamic acid-asparaginic acid;
(d) the acid type gamma-polyglutamic acid-asparaginic acid is dissolved in inert organic solvents with the amine that contains maleimide base group, under the existence effect of condensing agent and catalyzer, the amido linkage that forms between amino by the described amine that contains maleimide base group and the carboxyl of Hydrogen gamma-polyglutamic acid-asparaginic acid is attached to maleimide base group on the gamma-polyglutamic acid-asparaginic acid;
(e) use the ether termination reaction, and the extractive reaction mixture, phase liquid down collected;
(f) impurity and unreacted small-molecule substance are removed in dialysis, and obtain maleimide-polyglutamic acid-aspartic acid polymkeric substance by cryodesiccated mode, are white floss;
(h) maleimide-polyglutamic acid-aspartic acid polymkeric substance and the part that has sulfydryl are dissolved in the deionized water, 4 ℃ were reacted 12 hours, add antitumor drug, regulator solution pH to 7.0～8.0, in 37 ℃ of lucifuge stirring reactions 48 hours, dialysed 48 hours, and with 0.22 μ m membrane filtration, obtained maleimide-polyglutamic acid as claimed in claim 6-aspartic acid-medicinal composition.
11., it is characterized in that the carboxyl of polyglutamic acid-aspartic acid is 25: 1～1: 5 with the mol ratio that contains the amine of maleimide base group in the described step (d) as claim 9 or 10 described preparation methods; Temperature of reaction is-20～150 ℃; Reaction times is 1～150h.
12. as claim 9 or 10 described preparation methods, it is characterized in that described inert organic solvents is dimethyl sulfoxide (DMSO), N, dinethylformamide, acetonitrile.
13. as claim 9 or 10 described preparation methods, it is characterized in that described condensing agent is N, the N-dicyclohexylcarbodiimide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, N, N-DIC, I-hydroxybenzotriazole, the N-hydroxy thiosuccinimide, N, N '-carbonyl dimidazoles, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus; Described catalyzer is the 4-Dimethylamino pyridine, 4-tetramethyleneimine pyridine and triethylamine.
14. maleimide-polyglutamic acid as claimed in claim 1-aspartic acid polymkeric substance or the described maleimide-polyglutamic acid of claim 4-asparaginic acid complex or maleimide-polyglutamic acid as claimed in claim 6-aspartic acid-medicinal composition are as the application of pharmaceutical carrier.
15. maleimide-polyglutamic acid as claimed in claim 1-aspartic acid polymkeric substance or the described maleimide-polyglutamic acid of claim 4-asparaginic acid complex or the maleimide-polyglutamic acid as claimed in claim 6-aspartic acid-medicinal composition application in preparation prevention or treatment disease medicament.
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