CN105797169A - Anti-tumor macromolecular prodrug compound, and preparation method and application thereof - Google Patents
Anti-tumor macromolecular prodrug compound, and preparation method and application thereof Download PDFInfo
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- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
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Abstract
The invention discloses an anti-tumor macromolecular prodrug compound, and a preparation method and application of the anti-tumor macromolecular prodrug compound. The anti-tumor macromolecular prodrug compound is prepared from heparin or a derivative-a doxorubicin type macromolecular prodrug of the heparin and a hyaluronic acid-natural active component type macromolecular prodrug in a compounding way. The invention also provides the preparation method of the anti-tumor macromolecular prodrug compound and the application of the anti-tumor macromolecular prodrug compound in preparing anti-tumor drugs. Compared with the prior art, by compounding two kinds of macromolecular prodrugs, not only are multiple tumor targeting properties obtained, but also multi-drug resistance of tumor can be effectively reversed; the two kinds of macromolecular prodrugs work together and are in mutual synergy, so that the treating effect is obviously enhanced.
Description
Technical field
The present invention relates to antitumor macromolecular prodrug complex and its preparation method and application, belong to macromolecular material and
Field of pharmaceutical preparations.
Background technology
Amycin (doxorubicin, DOX) has another name called doxorubicin, Dox, is a kind of common anthracycline
Antitumor antibiotics, its has a broad antifungal spectrum, clinic is mainly used in treatment breast carcinoma, ovarian cancer, lung bronchogenic carcinoma, malignant lymphoma
Etc. malignant tumor, oncotherapy occupies critical role.Similar to most of antitumor drug, during life-time service amycin,
Normal tissue organ, has obvious toxic and side effects such as heart, kidney, bone marrow etc..Amycin clinical adverse is many, the most sternly
Weight for dose dependent cardiac toxicity.It is reported, the accumulated dose of amycin Clinical practice should control 450～600mg/
m2, to alleviate cardiac toxicity, it is to avoid myocardial damage and the generation of chronic heart failure.Additionally, tumor multi-medicine drug-resistant
(multidrug resistance, MDR) is the main cause that adriamycin chemotherapy is failed.Tumor multi-medicine drug-resistant refers to that tumor cell exists
A kind of cancer therapy drug produces toleration to multi-medicament under stimulating, and Forming Mechanism specifically includes that multidrug resistance gene (MDR1 base
Cause) and the overexpression of P-glycoprotein (P-glycoprotein, P-gp) of coding；The activity increasing of glutathion and relevant enzyme
High；The change of topoisomerase II (Topo II)；DNA damage repair ability strengthens；Multidrug-associated protein (multidrug
Resistance-associated protein, MRP) express increase etc..Therefore, overcome tumor multi-medicine drug-resistant to become to capture
The prerequisite of cancer.At present, the clinical practice stage is furtherd investigate or entered to existing multiple therapy methods and means, such as combination
Chemotherapeutic sensitizer (the patent of Publication No. CN103372210A: berberine combined chemotherapy medicine answering in antineoplaston
With), improve the form of administration (patent of Publication No. CN104056275A: multi-functional active targeting hyaluronic acid-poly lactic acid carrier
Synthesis and antitumor drug micelle preparation method thereof；The patent of Publication No. CN102357074A: artitumor multi-medicine-resistant targeting
Liposome), exploitation new type antineoplastic medicine (epirubicin, Perarubicin, mitoxantrone, aklavine, de-methoxy are soft
Erythromycin) etc..Although but these technology improve the deficiency of amycin clinical practice to a certain extent, but still suffering from each
Defect: precipitation and flocculation easily occur when doxorubicin hydrochloride and other drug or chemotherapeutic sensitizer combination, flocky precipitate enters
Blood vessel may result in arteriolar embolism；Although Liposomal formulation can be reduced the toxicity of amycin, but there is also envelop rate
Leakage low, easy, stability, bone marrow toxicity increase, and produce the problems such as new dermal toxicity；Common micellar preparation rate of releasing drug is not
Controlled, and in terms of reverse multiple drug resistance of tumor, there is limitation；It is secondary that life-time service new type antineoplastic medicine the most still faces poison
The problems such as effect and multidrug resistance.These defects hinder the development of above-mentioned technological industrialization to some extent and clinic should
With.
As it has been described above, in chemotherapy of tumors, combination chemotherapeutics and multidrug-resistance reversal agent can carry to a certain extent
High curative effect.But, the traditional multidrug-resistance reversal agent (such as verapamil, ciclosporin A etc.) with P-gp inhibitor as representative is because of poison
The factors such as side effect is big, crossover pharmacokinetic reaction, not can apply to clinic so far.Comparatively speaking, Chinese medicine is many as tumor
Medicine reversal agent of drug resistance has following superiority: action target spot is many, and self untoward reaction is few, can hold concurrently while reverse multidrug drug resistance
Have killing tumor cell, regulate and improve the effects such as body's immunity.If curcumin (curcumin, CUR) is the master of Rhizoma Curcumae Longae
Want effective ingredient.In recent years, the adjuvant therapy of tumors effect of curcumin has caused the attention of people, including its antitumor
Activity, attenuation synergistic effect and reverse multidrug resistant characterization.Use as collaborative in curcumin and paclitaxel (paclitaxel, PTX)
Proliferation of Human Ovarian Cell OC can be suppressed3Propagation, and the two combined chemotherapy has synergism and attenuation (Liu Guanyuan, beam Hua Mao, Zeng Xin
Red, etc. paclitaxel and the curcumin combination inhibitory action [J] to abortion syndrome OC3. journal of Shandong university: medicine,
2004,42 (3): 325-327.).Prior art have studied the curcumin auxiliary therapeutic action to amycin, research display, with Ah
Mycin group is compared, and combination curcumin and amycin are obviously enhanced the cytotoxicity of amycin, and alleviate amycin bad instead
Should.Comprehensive literature is reported, curcumin reverse multiple drug resistance of tumor mechanism is as follows: (1) reverses protein called membrane transporters P-gp mediation
Tumor multi-medicine drug-resistant；(2) tumor multi-medicine drug-resistant of Revertase System-mediated；(3) tumor of apoptosis-related genes mediation is reversed
Multidrug resistance；(4) curcumin reverses the tumor multi-medicine drug-resistant of DNA repair mechanism mediation.Additionally, Quercetin, rheum emodin, white hellebore
The natural activity components such as alcohol, chrysin, procyanidin, silymarin not only have adjunct antineoplastic activity, it is also possible to by many
Kind of mechanism carrys out reverse multiple drug resistance of tumor, be a great Prospect of R & D of class chemotherapeutic sensitizer (Li Xuetao, Tan Jingjing, explain Rong Ping,
Deng. Chinese traditional medicine reverse antitumor drug Research Progress of Multidrug Resistance [J]. Liaoning University of TCM's journal, 2013 (1): 155-
158.).But, mostly there is the shortcomings such as low, the poor permeability of dissolubility due to these natural active components, seriously limit it clinical
Application, affects its probability becoming natural multidrug-resistance reversal agent further.
In recent years, as a kind of novel reverse multidrug resistance strategy, nanometer system is applied to the targeting of antitumor drug and passs
Send and extensively proved and approve.Wherein, hydrophilic high molecular material and hydrophobic drug two formed by chemical coupling
Parent's property macromolecular prodrug because its particle diameter is little, lyotropy is good, good stability, slow controlled release, passive target (EPR effect), can avoid resistance to
The advantages such as the identification of medicine mechanism associated protein, are paid high attention to by researchers.But, it is based solely on the mediation of EPR effect and receives
The cancer target of rice corpuscles delivers and still suffers from certain limitation (such as targeting limited efficacy, being not suitable for all types tumor),
Therefore, modify strategy further combined with active targeting, be beneficial to strengthen the tumor cell ingestion efficiency to medicine, thus enter one
Step reverse multiple drug resistance of tumor.Additionally, macromolecular prodrug release is restricted by many factors, if medicine is before arriving tumor locus
Release in advance, will cause toxic and side effects increase, target site concentration reduce, medicine discharged at tumor locus and then can cause slowly
Drug resistance increases, and drug effect reduces.Therefore, it is achieved the controllable release of macromolecular prodrug Chinese medicine is the most challenging.
The selection of macromolecular skeleton material is the key link preparing macromolecular prodrug.Polysaccharide molecule has good biology
The compatibility, biodegradability, wide material sources, cheap and easy to get, be prone to carry out the advantages such as structure of modification, be build amphipathic big point
The excellent component of sub-prodrug.Heparin and derivant thereof are by glucamine, L-idose aldehyde glycosides, N-Acetyl-D-glucosamine and D-Portugal
The acid mucopolysaccharide sulfuric ester of grape alduronic acid alternately composition.Research shows, it is thin that heparin and derivant thereof have suppression malignant tumor
The effects such as born of the same parents' propagation, interference tumor angiogenesis, suppression malignant tumor infiltration and transfer.On its molecule rich in a large amount of carboxylics
Base is also the major site of structure of modification, and modified heparin or derivatives thereof cellular uptake increases, and anticoagulating active reduces, thus
The generation of toxic and side effects can be reduced further.Study display, heparin and derivant thereof further and can occur special with tumor cell
Property interact, and absorbed by Tumor Differentiation vascular endothelial, therefore there is the differentiation tumor epithelial cell targeting of sensitivity.Thoroughly
Bright matter acid (hyaluronic acid, HA) is a kind of natural line being made up of NAG and D-glucuronic acid monosaccharide
Property polysaccharide, has good biocompatibility, degradability, high viscoelasticity and non-immunogenic.Additionally, hyaluronic acid can be with
The CD44 receptor of tumor cell surface overexpression combines, and enters cell by endocytosis, thus has significant tumor target
Tropism.
In sum, in clinical cancer therapy, combination antitumor drug and chemotherapeutic sensitizer are (as tumor multi-medicine drug-resistant is inverse
Turn agent, anti-tumor active substance etc.) synergism and multiple-effect can be produced, one of means being an up drug effect.But, anti-
Tumour medicine and chemotherapeutic sensitizer itself are respectively provided with the shortcomings such as bioavailability is low, tumor-selective is poor, it is impossible to obtain preferably
Therapeutic effect, meanwhile, medicine or chemotherapeutic sensitizer are likely to cause toxic and side effects to increase in the accumulation of normal structure.Application nanometer
Technology delivers the focus that antitumor drug is delivery system research in recent years, but common nanometer system still suffers from targeting effect
The defects such as rate is low, cannot efficiently overcome tumor multi-medicine drug-resistant, rate of releasing drug uncontrollable.
Summary of the invention
Goal of the invention:
Present invention aims to above-mentioned application problem, the invention provides a kind of antitumor macromolecular prodrug and be combined
Thing, this macromolecular prodrug complex compensate for the defect that natural activity component dissolubility is low, and functional material (heparin or its spread out
Biology, hyaluronic acid, natural activity component) introducing overcome macromolecular prodrug deliver amycin enter tumor cell time,
Deficiency at aspects such as rate of releasing drug, targeting and reverse multidrug resistance.Macromolecular prodrug complex is passive via EPR effect
After being targeted to tumor tissues, enter tumor cell, subsequently tumor cell inner acidic pH by CD44 receptor mediated endocytosis
Environment causes hydrazone bond fission in macromolecular prodrug complex to discharge amycin, and at the relevant reverse mechanism of natural activity component
Under effect, push amycin and enter nucleus performance tumor cytotoxicity effect.I.e. by multiple targeting, (hydrazone key triggers
PH response release, the passive target of EPR effect, the CD44 receptor active targeting of hyaluronic acid mediated, heparin or derivatives thereof are situated between
The differentiation tumor epithelial cell targeting led), and reverse multiple drug resistance of tumor effect (endocytic mechanism of macromolecular prodrug complex and
The reverse multiple drug resistance of tumor activity of natural activity component), amycin, natural activity component and heparin or derivatives thereof in addition
Synergistic antitumor effect, dramatically increases amycin in the distribution of tumor locus and accumulation, improves therapeutic effect and also reduce amycin
Potential toxic and side effects.Therefore, the present invention creatively constructs a kind of macromolecular prodrug complex systems, not only can significantly carry
Its tumor-targeting high, the most all right effectively reverse multiple drug resistance of tumor, and also two kinds of macromolecular prodrug components are worked in coordination with and are made
With, it is mutually improved, significantly improves oncotherapy effect.
It is a further object to provide the preparation method of above-mentioned macromolecular prodrug complex.
Last purpose of the present invention is to provide the application in preparing antitumor drug of the above-mentioned macromolecular prodrug complex.
Technical scheme:
For realizing the purpose of foregoing invention, the technical scheme that the present invention takes is as follows:
A kind of antitumor macromolecular prodrug complex, its be by heparin or derivatives thereof-amycin type macromolecular prodrug with
Hyaluronic acid-natural activity component type macromolecular prodrug is composited.
As preferably, described heparin or derivatives thereof-amycin type macromolecular prodrug, is with tert-butyl carbazate (Boc
Hydrazine) it is linking arm, after first reacting with heparin or derivatives thereof, then remove tertbutyloxycarbonyl (Boc group), continue with amycin
Reaction, it is achieved heparin or derivatives thereof and the connection of amycin.
Preferred as another kind, described heparin or derivatives thereof goes sulphation liver selected from heparin, low molecular weight heparin, N-O-
Element or N-remove sulfated heparin.
Preferred as another kind, described hyaluronic acid-natural activity component type macromolecular prodrug, is to utilize hyaluronic acid master
Containing substantial amounts of carboxyl on chain, by the method for chemical coupling, introduce the natural activity with reverse multiple drug resistance of tumor function
Component is formed.
Preferred as another kind, described natural activity component is selected from curcumin, Quercetin, rheum emodin, resveratrol, Cortex Populi dividianae
Element, procyanidin or silymarin.
Specifically, with diazanyl as linking arm, connect heparin by amide reaction and schiff base reaction respectively or it derives
Thing and amycin, synthesized the heparin or derivatives thereof-amycin type macromolecular prodrug with pH response drug release feature, in preparation
During with Boc hydrazine replace hydrazine hydrate, after first reacting with heparin or derivatives thereof, then remove Boc blocking group, with amycin
Continue reaction, thus reduce the occurrence probability of side reaction, it is achieved heparin or derivatives thereof is efficiently connected with amycin；Utilize thoroughly
Containing substantial amounts of carboxyl on bright matter acid main chain, by the method for chemical coupling, introduce and there is reverse multiple drug resistance of tumor function
Natural activity component, the hyaluronic acid of formation-natural activity component type macromolecular prodrug improves natural activity component itself
Stability and dissolubility, and have amphipathic, play enhancing system stability in prodrug complex and prevent that amycin is prominent to be released
Effect.
The preparation method of described macromolecular prodrug complex, comprises the following steps:
One, the preparation of heparin or derivatives thereof-amycin type macromolecular prodrug
Being dissolved in suitable organic solvent by above-mentioned heparin or derivatives thereof, using Boc hydrazine is linking arm, 1-ethyl-(3-bis-
Dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS) be that activator carries out condensation reaction, obtains Boc hydrazine
The reactive intermediate of the heparin or derivatives thereof modified；Trifluoroacetic acid (TFA) method removing Boc group is used to obtain with hydrazide group
The reactive intermediate of heparin or derivatives thereof；Doxorubicin hydrochloride is scattered in dichloromethane, adds appropriate triethylamine (TEA)
Reaction, obtains desalination amycin；Finally by the reactive intermediate of desalination amycin with the heparin or derivatives thereof with hydrazide group
It is scattered in appropriate reaction solvent, prepares heparin or derivatives thereof-amycin type macromolecular prodrug by schiff base reaction.
Two, the preparation of hyaluronic acid-natural activity component type macromolecular prodrug
Hyaluronic acid is dissolved in suitable organic solvent, with DMAP (DMAP) as catalyst, 1-ethyl-
(3-dimethylaminopropyl) carbodiimide (EDC) is dehydrant, first by after the activated carboxylic in hyaluronic acid backbone, then by sky
Add after so active component is dissolved in suitable organic solvent, by esterification, prepare hyaluronic acid-natural activity component type and divide greatly
Sub-prodrug.
Three, the preparation of antitumor macromolecular prodrug complex
Described preparation process one, two, wherein suitably organic solvent selected from Methanamide or Methanamide and oxolane or
Methanamide and the mixed solvent of DMF.
Described preparation process one, wherein appropriate reaction solvent selected from PBS (pH=6.5), dimethyl sulfoxide or
PBS (pH=6.5) and the mixed solvent of dimethyl sulfoxide.
Described preparation process three, wherein said certain proportion is: amycin: the mass ratio of natural activity component is 1:
(1～20).
Concrete preparation method is as follows:
One, the preparation of heparin or derivatives thereof-amycin type macromolecular prodrug
(1) heparin or derivatives thereof is dissolved in suitable organic solvent, under inert gas shielding, 1-ethyl-(3-diformazan
Base aminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS) be activator, by heparin or derivatives thereof main chain
Activated carboxylic；After Boc hydrazine is dissolved with suitable organic solvent equally, it is slowly dropped in the solution of heparin or derivatives thereof, control
System reaction to completely, precipitates with appropriate solvent, filters to obtain precipitate, vacuum drying, i.e. obtains heparin that Boc hydrazine modifies or it spreads out
Biological reactive intermediate.
Synthetic route formula is as follows:
(R1-COOH is that heparin, low molecular weight heparin, N-go sulfated heparin or N-O-to remove sulfated heparin)
(2) reactive intermediate of the heparin or derivatives thereof modified by Boc hydrazine is scattered in dichloromethane and trifluoroacetic acid
(TFA) in solution, suspendible is ultrasonic, controls reaction to the removing of Boc group completely, and sucking filtration obtains precipitate, redissolves, dialysis, freezing dry
Dry, i.e. obtain the reactive intermediate of the heparin or derivatives thereof with hydrazide group.
Synthetic route formula is as follows:
(3) under the conditions of lucifuge, being scattered in dichloromethane by doxorubicin hydrochloride, add appropriate triethylamine (TEA), water-bath surpasses
Sound, controls to react to completely, is extracted to water layer with water colourless, collects dichloromethane layer rotation steaming and volatilizes, obtains desalination amycin.
Synthetic route formula is as follows:
(4), under the conditions of lucifuge, the reactive intermediate of desalination amycin with the heparin or derivatives thereof with hydrazide group is divided
Dissipate in appropriate reaction solvent, control to react to completely, reactant liquor is added dilute, dialysis, it is filtered to remove insoluble matter, freezing
It is dried, i.e. obtains heparin or derivatives thereof-amycin type macromolecular prodrug.
Synthetic route formula is as follows:
DOX+R1-CONHNH2→R1-CONHN=DOX
Suitable organic solvent described in step (1) be Methanamide or Methanamide with oxolane or Methanamide with
The mixed solvent of DMF；Described heparin or derivatives thereof, the mol ratio of EDC, NHS, Boc hydrazine are 1:
(3～20: (3～20): (3～60)；Described control is reacted for first to add in heparin or derivatives thereof solution by above-mentioned mol ratio
Entering activator EDC and NHS, after reaction 0.2～2h, add Boc hydrazine, control reaction droplet speed is every and is spaced 2～30 seconds, drips
After room temperature reaction 6～72h again；Described suitable precipitation solvent is ice acetone, ice ether, ice ethanol or glacial acetic acid ethyl ester；Described
The vacuum drying time be 0.5～1d.
The mol ratio of the dichloromethane described in step (2) and trifluoroacetic acid is (1～3): 1；Described control reaction is
By above-mentioned mol ratio, reactive intermediate is added in dichloromethane and trifluoroacetic acid, ultrasonic reaction 0.5～6h；During described dialysis
Between be 0.5～3d.
The mol ratio of the doxorubicin hydrochloride described in step (3) and triethylamine is 1: (2～5)；Described control reaction is
By above-mentioned mol ratio, doxorubicin hydrochloride and triethylamine are added in dichloromethane, water bath sonicator 0.2～1h.
Appropriate reaction solvent described in step (4) is PBS (pH=6.5), dimethyl sulfoxide or PBS buffering
Liquid (pH=6.5) and the mixed solvent of dimethyl sulfoxide.Described desalination amycin and heparin or its with hydrazide group derive
The mol ratio of the reactive intermediate of thing be mol ratio be (1～5): 1.Described control reaction is room temperature reaction 6～72h；Described
The dilution volume ratio of reactant liquor and water be 1: (1～3)；Described dialysis time is 0.5～3d.
Two, the preparation of hyaluronic acid-natural activity component type macromolecular prodrug
Hyaluronic acid is dissolved in suitable organic solvent, under inert gas shielding, with DMAP (DMAP)
For catalyst, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) is dehydrant, by the carboxylic in hyaluronic acid backbone
Base activates；After natural activity component is dissolved with appropriate reaction solvent equally, it is slowly dropped in hyaluronic acid solution, controls reaction
To completely, precipitate with appropriate solvent, filter to obtain precipitate, redissolve, ultrasonic, dialysis, lyophilization, i.e. obtain hyaluronic acid-sky
So active component type macromolecular prodrug.According to the natural activity component toleration to light, choose whether that lucifuge is reacted.
Synthetic route formula is as follows:
(R2-COOH is hyaluronic acid；R3-OH is curcumin, Quercetin, rheum emodin, resveratrol, chrysin, former cyanine
Element, silymarin)
Described suitable organic solvent is Methanamide or Methanamide and oxolane or Methanamide and N, N-diformazan
The mixed solvent of base Methanamide；Described hyaluronic acid, catalyst, dehydrant, the mol ratio of natural activity component are 1:
(0.2～0.5): (3～20): (1～5).
Described control reaction, for adding catalyst and activator by above-mentioned mol ratio, after reaction 0.2～2h, adds natural
Active component, controls reaction and drips speed and be every interval 2～30 seconds, room temperature reaction 6～72h again after dripping；Described is the most heavy
Shallow lake solvent is ice acetone, ice ether, ice ethanol or glacial acetic acid ethyl ester；Described is ultrasonic for Probe Ultrasonic Searching 10～60min；Described
Dialysis time is 0.5～3d.
Three, the preparation of antitumor macromolecular prodrug complex
By step one, in two the dried frozen aquatic products of two kinds of macromolecular prodrugs of preparation by molten in every 1mL water or other polar solvents
After solving the ratio dissolving of 3～50mg, it is mixed to get antitumor macromolecular prodrug complex by a certain percentage.
Described certain proportion is: amycin: the mass ratio of natural activity component is 1: (1～20).
Finally, present invention also offers the application in preparing antitumor drug of the described macromolecular prodrug complex.Described
Macromolecular prodrug complex processes through ultrasonic or high pressure homogenize, makes solution type preparation or lyophilizing is prepared as lyophilized formulations, it is possible to
So that the absorption of this solution is prepared as solid preparation on solid adjuvant material surface.
Described through ultrasonic or high pressure homogenize process, make the macromolecular prodrug complex solution that particle diameter is 10～1000nm.
Technique effect: the present invention has a following benefit relative to prior art:
1. heparin or derivatives thereof-amycin type macromolecular prodrug that the present invention provides, has good water solubility, normal raw
Stablize the advantages such as good under reason environment, efficiently solve in Clinical practice, heparin or derivatives thereof and doxorubicin hydrochloride physical mixed
Time easily occur precipitation problem.Additionally, this macromolecular prodrug can under tumor tissues sour environment fast degradation and discharge Ah
Mycin.Preparation method is simple and easy to do, it is simple to operation is promoted.
2. hyaluronic acid-natural activity component type macromolecular prodrug that the present invention provides, effectively overcomes natural activity group
Dividing the defects such as low, the poor bioavailability of dissolubility own, this macromolecular prodrug has tumor-targeting concurrently and multi-medicine tolerant reversal is special
Property, it is expected to improve the shortcomings such as low, the tumor multi-medicine drug-resistant of antitumor drug tumor-selective, is to build macromolecular prodrug complex
Excellent component, and the synthesis condition of this material is gentle, reaction is simple, and cost of material is low, it is easy to industrialized production.
3. the antitumor macromolecular prodrug complex that the present invention provides has the advantage that (1) significantly tumor-targeting.
Macromolecular prodrug complex particle diameter is Nano grade, can realize passive cancer target by EPR effect；Utilize heparin or its derive
The differentiation tumor epithelial cell affinity of thing and hyaluronic acid-CD44 receptor-mediated active tumor-targeting, it is achieved higher
Cancer target efficiency, thus improve the tumor cell ingestion efficiency to macromolecular prodrug；The pH sensitivity utilizing hydrazone key realizes swollen
The quick release in location in oncocyte, overcomes general macromolecular prodrug and enters rate of releasing drug defect slowly after born of the same parents.Therefore, this sets
Meter achieves from blood circulation to tumor tissues again to targeting layer by layer and the tumor inner position release of tumor cell, is effectively improved Ah mould
Element is in the accumulation of tumor locus, and reduces its heart nephrotoxicity.(2) reverse multiple drug resistance of tumor characteristic.Macromolecular prodrug complex
It is effectively increased stability and the reverse multiple drug resistance of tumor activity of natural activity component；Meanwhile, in conjunction with the endocytosis of nano-micelle
Mechanism and the multi-medicine tolerant reversal function of natural activity component mediation, macromolecular prodrug complex is expected to efficiently, overcomes in an orderly manner
Heavy drug resistance obstacle in amycin delivery.(3) collaborative tumor inhibition effect.In the suppression giving full play to heparin or derivatives thereof
On the basis of the antitumor auxiliary therapeutic actions such as Tumor angiogenesis and suppression tumor growth effect, natural activity component presses down in addition
Tumor cell proliferation processed the effect of inducing apoptosis of tumour cell, in conjunction with amycin wide spectrum active anticancer and to various entities
The good lethal effect of tumor, has significant synergistic antitumor therapeutic effect.(4) controlled drug release feature.By regulation macromole
The different ratio of two kinds of components in prodrug complex, is possible not only to dose ratio amycin being adjusted flexibly with natural activity component,
Also the rate of releasing drug of amycin in scalable macromolecular prodrug complex.The method carries the nano-carrier phase of medicine type with physics bag
Ratio, preparation technology is simple, and easy and simple to handle, controllability is high.Additionally, do not have the amphipathic hyaluronic acid-sky of acid-sensitive degraded feature
So active component type macromolecular prodrug can strengthen the stability of macromolecular prodrug complex systems, prevents amycin in complexity
Physiological environment occurs phenomenon of burst release.(5) safety of height.Heparin or derivatives thereof and hyaluronic acid that the present invention uses are equal
For high security, asepsis environment-protecting, biodegradable pharmaceutic adjuvant, natural activity component is the efficient natural multidrug resistance of low toxicity
Inversion agent.
Accompanying drawing illustrates:
Fig. 1: low molecular weight heparin-amycin/hyaluronic acid-curcumin (amycin: curcumin=1: 2, w: w) in difference
Release profiles (n=3) in the phosphate buffer of pH.
Detailed description of the invention
Below by embodiment to present invention further instruction in addition, but following embodiment is not limiting as the power of this patent
Profit scope.
Embodiment 1: the synthesis of low molecular weight heparin-amycin macromolecular prodrug
Weigh 1mmol low molecular weight heparin (LMWH) to add in 10mL Methanamide, magnetic agitation 1h to being completely dissolved, nitrogen
1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide is added under protection and condition of ice bath
(NHS), after reaction 0.5h, be slowly added dropwise Boc hydrazine formamide solution (mol ratio of LWMH, EDC, NHS, Boc hydrazine is followed successively by 1:
5: 5: 20), drip speed and be every interval 3 seconds, room temperature reaction 24h again after dropping, uses ice acetone precipitation, cyclic washing precipitation
Thing, is vacuum dried 12h, obtains the LMWH reactive intermediate that Boc hydrazine is modified；The LMWH reactive intermediate that 20mg Boc hydrazine is modified
Being scattered in 6mL dichloromethane, add 4mL trifluoroacetic acid, ultrasonic reaction 2h, sucking filtration obtains precipitate, after redissolving with water, and dialysis
2d, lyophilization must be with the LMWH reactive intermediate of hydrazide group；Under the conditions of lucifuge, 2.5mg doxorubicin hydrochloride is scattered in 1mL
In dichloromethane, dropping 100uL triethylamine (TEA), water bath sonicator 30min, be extracted to water layer with water colourless the most by several times, collects
Dichloromethane layer rotation steaming volatilizes, and obtains desalination amycin (DOX)；Under the conditions of lucifuge, by 0.3mmolDOX and 0.1mmol with acyl
The LMWH reactive intermediate of diazanyl is dissolved in the mixed solvent (v: v=1 of 10mLPBS buffer (pH=6.5) and dimethyl sulfoxide
: 1) in, room temperature reaction 12h, reactant liquor adds the water dilution of 2 times amount by reaction after terminating, transposition is dialysed 3d in bag filter, mistake
Filtering insoluble matter, lyophilization obtains end-product low molecular weight heparin-amycin macromolecular prodrug (LMWH-DOX).
Embodiment 2: the synthesis of heparin-amycin macromolecular prodrug
Weighing 1mmol amount heparin (heparin) and add in 10mL Methanamide, magnetic agitation 1h is to being completely dissolved, and nitrogen is protected
Protect and add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide under condition of ice bath
(NHS), after reaction 2h, (mol ratio of heparin, EDC, NHS, Boc hydrazine is followed successively by 1 to be slowly added dropwise the formamide solution of Boc hydrazine
: 10: 15: 60), drip speed and be every interval 5 seconds, room temperature reaction 24h again after dropping, use ice acetone precipitation, cyclic washing is heavy
Shallow lake thing, is vacuum dried 12h, obtains the heparin activity intermediate that Boc hydrazine is modified；In the middle of the heparin activity that 20mg Boc hydrazine is modified
Body is scattered in 6mL dichloromethane, adds 4mL trifluoroacetic acid, ultrasonic reaction 2h, and sucking filtration obtains precipitate, after redissolving with water, and dialysis
2d, lyophilization must be with the heparin activity intermediate of hydrazide group；Under the conditions of lucifuge, 2.5mg doxorubicin hydrochloride is scattered in 1mL
In dichloromethane, dropping 100uL triethylamine (TEA), water bath sonicator 30min, be extracted to water layer with water colourless the most by several times, collects
Dichloromethane layer rotation steaming volatilizes, and obtains desalination amycin (DOX)；Under the conditions of lucifuge, by 0.5mmolDOX and 0.1mmol with acyl
The heparin activity intermediate of diazanyl is dissolved in the mixed solvent (v: v=1 of 10mLPBS buffer (pH=6.5) and dimethyl sulfoxide
: 1) in, room temperature reaction 12h, reactant liquor adds the water dilution of 3 times amount by reaction after terminating, transposition is dialysed 2d in bag filter, mistake
Filtering insoluble matter, lyophilization obtains end-product heparin-amycin macromolecular prodrug.
Embodiment 3: the synthesis of hyaluronic acid-curcumin macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 3mmol curcumin (CUR) are dissolved in Methanamide and DMF
Mixed solvent (v: v=1: 1) in.Under nitrogen protection, by 5mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide
(EDC) add in HA solution with 0.5mmol DMAP (DMAP), after priming reaction 1h, be slowly added dropwise CUR solution,
Dripping speed be every and be spaced 2 seconds, room temperature reaction 48h again after dripping, reaction is heavy by glacial acetic acid ethyl ester precipitation, cyclic washing after terminating
Forming sediment, after redissolving with water, Probe Ultrasonic Searching 30min, dialyse 1d, and lyophilization i.e. obtains hyaluronic acid-curcumin macromolecular prodrug
(HA-CUR)。
Embodiment 4: the synthesis of hyaluronic acid-Quercetin macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 2mmol Quercetin (QU) are dissolved in Methanamide.Under nitrogen protection, will
3mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.2mmol DMAP (DMAP) add
In HA solution, after priming reaction 0.5h, it is slowly added dropwise QU solution, drips speed and be every interval 5 seconds, room temperature reaction again after dripping
24h, reacts and uses ice ether sedimentation, cyclic washing to precipitate after terminating, and after redissolving with water, Probe Ultrasonic Searching 30min, dialyse 2d, freezing
It is dried, i.e. obtains hyaluronic acid-Quercetin macromolecular prodrug (HA-QU).
Embodiment 5: the synthesis of hyaluronic acid-rheum emodin macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 5mmol rheum emodin are dissolved in Methanamide.Under nitrogen protection, will
10mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.5mmol DMAP (DMAP) add
Enter in hyaluronic acid solution, after priming reaction 0.5h, be slowly added dropwise solution, drip speed and be every interval 5 seconds, room temperature again after dripping
Reaction 24h, reaction precipitates with glacial acetic acid ethyl ester after terminating, and cyclic washing precipitates, after redissolving with water, Probe Ultrasonic Searching 30min, dialysis
2d, lyophilization, i.e. obtain hyaluronic acid-rheum emodin macromolecular prodrug.
Embodiment 6: the synthesis of hyaluronic acid-resveratrol macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 3mmol resveratrol are dissolved in the mixed solvent of Methanamide and oxolane
In (v: v=1: 1).Under nitrogen protection, by 15mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and
0.5mmol DMAP (DMAP) adds in hyaluronic acid solution, after priming reaction 1h, is slowly added dropwise solution, drips speed
Being spaced 15 seconds for every, room temperature reaction 36h again after dripping, ice acetone precipitation, cyclic washing precipitation use in reaction after terminating, use water
After redissolution, Probe Ultrasonic Searching 30min, dialyse 2d, and lyophilization i.e. obtains hyaluronic acid-resveratrol macromolecular prodrug.
Embodiment 7: the synthesis of hyaluronic acid-chrysin macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 5mmol chrysin are dissolved in Methanamide.Under nitrogen protection, will
20mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.5mmol DMAP (DMAP) add
Enter in hyaluronic acid solution, after priming reaction 2h, be slowly added dropwise solution, drip speed and be every interval 10 seconds, room temperature again after dripping
Reaction 48h, reacts and uses ice acetone precipitation, cyclic washing to precipitate after terminating, after redissolving with water, and Probe Ultrasonic Searching 30min, dialyse 2d,
Lyophilization, i.e. obtains hyaluronic acid-chrysin macromolecular prodrug.
Embodiment 8: the synthesis of hyaluronic acid-procyanidin macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 2mmol procyanidin are dissolved in Methanamide and DMF
In mixed solvent (v: v=1: 1).Under nitrogen protection, by 5mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC)
Add in hyaluronic acid solution with 0.2mmol DMAP (DMAP), after priming reaction 1h, be slowly added dropwise solution, drip
Speed is every and is spaced 2 seconds, room temperature reaction 24h again after dripping, and ice acetone precipitation use in reaction after terminating, cyclic washing precipitates, use
After water redissolves, Probe Ultrasonic Searching 60min, dialyse 3d, and lyophilization i.e. obtains hyaluronic acid-procyanidin macromolecular prodrug.
Embodiment 9: the synthesis of hyaluronic acid-silymarin macromolecular prodrug
Respectively 1mmol hyaluronic acid (HA) and 3mmol silymarin are dissolved in Methanamide.Under nitrogen protection, will
10mmol1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.3mmol DMAP (DMAP) add
Entering in hyaluronic acid solution, after priming reaction 2h, be slowly added dropwise solution, drip speed and be every interval 5 seconds, after dripping, room temperature is anti-again
Answering 36h, reaction to use after terminating or glacial acetic acid ethyl ester precipitation, cyclic washing precipitates, after redissolving with water, and Probe Ultrasonic Searching 20min, dialysis
2d, lyophilization, i.e. obtain hyaluronic acid-silymarin macromolecular prodrug.
Embodiment 10: the preparation of antitumor macromolecular prodrug complex and particle diameter and the mensuration of critical aggregation concentration (CAC)
1. the preparation of antitumor macromolecular prodrug complex: according to the character of natural activity component, it is considered to the need of keeping away
Under conditions of light, taking heparin or derivatives thereof-amycin type macromolecular prodrug 18mg and the hyaluronic acid-sky of different proportion respectively
So active component type macromolecular prodrug is dissolved in 3mL water, and wherein the mass ratio of amycin and natural activity component is 1: (1～
20), 30min is stirred at room temperature, then ultrasonic 30min under ice bath, 0.8 μm membrane filtration, obtain antitumor macromolecular prodrug multiple
Polymer solution, further lyophilizing obtains lyophilized formulations.
2. particle size determination: prepare antitumor macromolecular prodrug complex solution by 1, take 1mL and be diluted with water to 3mL,
It is measured with particle size determination instrument (Malvem Instruments, Malvern, UK), the results are shown in Table 1.As seen from table, it is prepared into
The antitumor macromolecular prodrug complex arrived, micelle particle diameter reaches nanoscale, and even particle size distribution.
3. critical aggregation concentration (CAC) measures: use fluorescent spectrometry, surveys as fluorescence pointer with pyrene (Pyrene, Py)
The CAC value of the antitumor macromolecular prodrug complex of preparation in fixed 1.Pyrene is a kind of hydrophobic condensed-nuclei aromatics compounds,
Dissolubility in water is minimum, and about 1.0 × 10-7Mol/L, the change to environment polarity is the most sensitive.Concentration when amphipathic molecule
During less than CAC, will not assemble in the solution, the pyrene of trace is dissolved in the water of polarity；When the concentration of amphipathic molecule is higher than CAC
Time, amphipathic molecule assembles, and pyrene distributes to the hydrophobic part of amphipathic molecule kernel, and enters nonpolar environment, performance
For a series of changes of fluorescence spectrum, such as the increase of fluorescence intensity, emission spectrum vibrates fine structure (the
Vibrational fine structure of the emission spectra) change, (0,0) wave band in excitation spectrum
Red shift, moves to 338nm from 333nm.Therefore, by with I in the excitation spectrum of pyrene338/I333(excitation spectrum medium wavelength is respectively for ratio
Fluorescence intensity level for 338nm and 333nm) or emission spectrum in I372/I385Ratio (scan under fixing excitation wavelength,
I372、I385Represent respectively in emission spectrum first and the fluorescence intensity level at three strongest ones peak) log concentration of amphiphilic species is made
Figure, the point (knee of curve) that in figure, slope changes is the critical point forming micelle, and corresponding concentration is amphipathic thing
The CAC of matter.The results are shown in Table 1.As seen from table, compared with Small molecular surfactant, the CAC of antitumor macromolecular prodrug complex
The lowest so that it is there is good stability, the most nano level particle diameter in physiological conditions, it can be made to escape RE system
The emptying mechanism of system and kidney, extends plasma half-life.
The sign of table 1 antitumor macromolecular prodrug complex
Embodiment 11: doxorubicin in vitro release experiment
So that low molecular weight heparin-(amycin: curcumin=1: 2, w: as a example by w), adopts amycin/hyaluronic acid-curcumin
With amycin external drug release behavior under at condition of different pH in dialysis Effect of Anti tumor macromolecular prodrug complex.Accurate title
Take low molecular weight heparin-amycin/hyaluronic acid-curcumin (amycin: curcumin=1: 2, w: lyophilized formulations w), 5% Portugal
Grape sugar juice dissolves, and to be diluted to doxorubicin concentration be 0.5mg/mL.Preparation pH (the 7.4/5.8/ containing 0.1% Tween 80
4.5) phosphate buffer is as release medium.Take 1mL macromolecular prodrug complex solution and be placed in bag filter (MWCO 3500)
In, it is put in the dissolution beaker filling 50mL release medium, in 90～100 vibration velocitys, lucifuge, enters under 37 ± 0.5 DEG C of medium temperatures
Row doxorubicin in vitro release experiment.The release outside bag filter is updated by the blank dissolution medium setting time interval same volume
Liquid, HPLC measures release liquid doxorubicin content.HPLC chromatogram condition: chromatographic column is Lichrospher C18(4.6 × 250mm, 5 μ
M), flowing is methanol-5% acetic acid (70: 30) mutually, and flow velocity is 1mL/min, and column temperature is 40 DEG C, UV-detector, and detection wavelength is
480nm, sample size is 20 μ L.Result is shown in Fig. 1, at low molecular weight heparin-amycin/hyaluronic acid-curcumin (amycin: Rhizoma Zingiberis Recens
Flavin=1: 2, w: in w), the release of amycin has obvious slow-releasing and pH sensitivity equally, and amycin is pH's 7.4
Under physiological condition, 72h preparation is 12.7%, and under the conditions of pH 5.8 and 4.5, in 72h, cumulative release amount respectively reaches
62.6% and 84.9%.Release experiment result shows, antitumor macromolecular prodrug complex can be protected in blood circulation
Hold relatively stable and complete structure and less release amycin, pass through EPR effect passive target, hyaluronic acid and low molecule subsequently
The multiple targeting mechanism such as the active targeting of amount mediated by heparin, are gathered in tumor locus, and in tumor cell, the microenvironment of low pH is touched
Give and discharge rapidly amycin, and then play therapeutical effect.
Embodiment 12: Cytotoxic evaluation
With free amycin for comparison, employing mtt assay investigates the antitumor macromolecular prodrug complex in embodiment 10
Cytotoxicity in human breast carcinoma drug-resistant cell strain MCF-7/ADR.Take MCF-7/ADR cell with 5 × 103Individual/hole is inoculated in
In 96 orifice plates, after 37 DEG C hatch 72h, suck culture fluid, be separately added into the above-mentioned macromolecular prodrug complex 50 μ L of variable concentrations,
Continue to hatch certain time.Taking 25 μ L Methyl thiazoly tetrazolium assay (MTT, 5mg/mL) to add in each hole, after continuing to hatch 4h, suction is abandoned
Supernatant in hole, each hole adds 100 μ L DMSO, shaking, makes crystallization fully dissolve.Under 570nm, measure sample by microplate reader inhale
Light value (ODsample).And measure the light absorption value of matched group (n.s) in the same way, it is designated as ODcontrol.Calculate by formula (1)
Subject cell strain survival rate, and calculate the half suppression to MCF-7/ADR cell of the above-mentioned macromolecular prodrug complex according to result
Concentration IC50.The results are shown in Table 2.
ODsampleIt is the absorbance for prospect hole with material or vehicle treated, ODcontrolIt is only to process with blank culture fluid
The absorbance of control wells；Cell viability is cell survival rate.
As shown in Table 2, owing to the outer row of free amycin is acted on by MCF-7/ADR drug-resistant cell strain, free amycin pair
Mdr cell does not has obvious lethal effect, and each antitumor macromolecular prodrug complex group is all to having significant cytotoxicity
Effect, such as low molecular weight heparin-amycin/hyaluronic acid-curcumin (amycin: curcumin=1: 5, w: w) group and low molecule
Amount heparin-amycin/hyaluronic acid-silymarin (amycin: silymarin=1: 5, w: w) organize the IC after hatching 72h50Value
It is respectively 0.97 ± 0.06 and 0.87 ± 0.17 μ g/mL..Result shows that antitumor macromolecular prodrug complex has the most inverse
Turn tumor multi-medicine drug-resistant effect, and enhance the amycin lethal effect to mdr cell.
The table 2 antitumor macromolecular prodrug complex half-inhibition concentration IC to MCF-7/ADR cell50Value (n=5)
Embodiment 13: pharmacodynamic evaluation
Set up MCF-7/ADR model of nude mice bearing tumor, at random MCF-7/ADR tumor bearing nude mice is randomly divided into 5 groups: negative control
Group (normal saline), positive controls (doxorubicin hydrochloride), low molecular weight heparin-amycin/hyaluronic acid-curcumin (Ah mould
Element: curcumin=1: 5, w: w) group, heparin-amycin/hyaluronic acid-resveratrol (amycin: resveratrol=1: 5, w: w)
Group, low molecular weight heparin-amycin/hyaluronic acid-procyanidin (amycin: procyanidin=1: 5, w: w) group.Amycin is given
Pharmaceutical quantities is 5mg/kg, and natural activity component dosage is obtained by quality proportioning conversion corresponding in each group.Often group 5,
Respectively at 0, tail vein injection was administered on 2nd, 4,6,8,10,12, put to death nude mice in the 14th day, stripped separation tumor, claimed tumor weight, surveyed
Amount tumor volume, calculates tumour inhibiting rate, carries out the pharmacodynamic study of each antitumor macromolecular prodrug complex group.The results are shown in Table 3.Result
Show, low molecular weight heparin-amycin/hyaluronic acid-curcumin group, heparin-amycin/hyaluronic acid-resveratrol group and
The tumour inhibiting rate of low molecular weight heparin-amycin/hyaluronic acid-procyanidin group is respectively 65.47 ± 5.15,57.72 ± 6.27,
61.36 ± 4.79%, it is all remarkably higher than normal saline group (p ＜ 0.01) and doxorubicin hydrochloride group (p ＜ 0.05).Additionally, due to
Doxorubicin hydrochloride solution heart nephrotoxicity is relatively strong, all causes weight loss very fast after nude mice successive administration, and low molecular weight heparin-Ah
Mycin/hyaluronic acid-curcumin group, heparin-amycin/hyaluronic acid-resveratrol group and low molecular weight heparin-amycin/
The nude mouse weight average of hyaluronic acid-procyanidin group shows a rising trend, and shows that antitumor macromolecular prodrug complex is identical
There is under dosage higher tumor killing effect and less toxic and side effects.
The pharmacodynamic evaluation of the different antitumor macromolecular prodrug complex of table 3
Note: * p ＜ 0.05 compared with doxorubicin hydrochloride group；Δ p ＜ 0.01 compared with normal saline group.
Claims (10)
1. an antitumor macromolecular prodrug complex, it is characterised in that it is to be divided greatly by heparin or derivatives thereof-amycin type
Sub-prodrug is composited with hyaluronic acid-natural activity component type macromolecular prodrug.
Antitumor macromolecular prodrug complex the most according to claim 1, it is characterised in that described heparin or its derive
Thing-amycin type macromolecular prodrug, is with tert-butyl carbazate as linking arm, after first reacting with heparin or derivatives thereof,
Remove tertbutyloxycarbonyl again, continue reaction with amycin, it is achieved heparin or derivatives thereof and the connection of amycin.
Antitumor macromolecular prodrug complex the most according to claim 1, it is characterised in that described heparin or derivatives thereof
Sulfated heparin or N-is gone to remove sulfated heparin selected from heparin, low molecular weight heparin, N-O-.
Antitumor macromolecular prodrug complex the most according to claim 1, it is characterised in that described hyaluronic acid-natural
Active component type macromolecular prodrug, is to utilize in hyaluronic acid backbone containing substantial amounts of carboxyl, by the method for chemical coupling, draws
The natural activity component entering to have reverse multiple drug resistance of tumor function is formed.
Antitumor macromolecular prodrug complex the most according to claim 1, it is characterised in that described natural activity component is selected
From curcumin, Quercetin, rheum emodin, resveratrol, chrysin, procyanidin or silymarin.
6. the preparation method of antitumor macromolecular prodrug complex described in any one of claim 1-5, it is characterised in that include with
Lower step:
(1) preparation of heparin or derivatives thereof-amycin type macromolecular prodrug:
Being dissolved in suitable organic solvent by heparin or derivatives thereof, employing tert-butyl carbazate is linking arm, 1-ethyl-(3-
Dimethylaminopropyl) carbodiimide, N-Hydroxysuccinimide be that activator carries out condensation reaction, obtains tert-butyl carbazate
The reactive intermediate of the heparin or derivatives thereof modified；Trifluoroacetic acid method removing tertbutyloxycarbonyl is used to obtain with hydrazide group
The reactive intermediate of heparin or derivatives thereof；Doxorubicin hydrochloride is scattered in dichloromethane, adds the reaction of appropriate triethylamine,
To desalination amycin；Finally the reactive intermediate of desalination amycin with the heparin or derivatives thereof with hydrazide group is scattered in suitable
When, in reaction dissolvent, preparing heparin or derivatives thereof-amycin type macromolecular prodrug by schiff base reaction；
(2) preparation of hyaluronic acid-natural activity component type macromolecular prodrug:
Hyaluronic acid is dissolved in suitable organic solvent, with DMAP as catalyst, 1-ethyl-(3-dimethylamino
Base propyl group) carbodiimide is dehydrant, first by after the activated carboxylic in hyaluronic acid backbone, then is dissolved in suitable by natural activity component
When adding after organic solvent, by esterification, prepare hyaluronic acid-natural activity component type macromolecular prodrug；
(3) preparation of antitumor macromolecular prodrug complex:
By two kinds of macromolecular prodrugs lyophilization respectively of preparation in step (1), (2), obtain lyophilized formulations, use pharmacy the most respectively
After upper acceptable solvent dissolves, it is mixed to get antitumor macromolecular prodrug complex by a certain percentage.
The preparation method of antitumor macromolecular prodrug complex the most according to claim 6, it is characterised in that step (1) or
(2) suitable organic solvent described in is selected from Methanamide or Methanamide and oxolane or Methanamide and DMF
Mixed solvent.
The preparation method of antitumor macromolecular prodrug complex the most according to claim 6, it is characterised in that in step (1)
Described appropriate reaction solvent is selected from the PBS of pH=6.5, dimethyl sulfoxide or pH=6.5PBS buffer and dimethyl
The mixed solvent of sulfoxide.
The preparation method of antitumor macromolecular prodrug complex the most according to claim 6, it is characterised in that in step (3)
Described certain proportion is: amycin: the mass ratio of natural activity component is 1: (1～20).
10. the application of antitumor macromolecular prodrug complex described in any one of claim 1-5, it is characterised in that described big point
The application in preparing antitumor drug of the sub-prodrug complex.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107952082A (en) * | 2017-11-22 | 2018-04-24 | 中国药科大学 | A kind of multi-functional synergistic pharmaceutical combination and its construction method based on adriamycin |
| CN110339181A (en) * | 2019-07-02 | 2019-10-18 | 中国药科大学 | A kind of pH response type nano preparation and its preparation method and application based on click-reaction |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1393720A1 (en) * | 2002-08-27 | 2004-03-03 | Universiteit Utrecht | Vesicle-encapsulated corticosteroids for treatment of cancer |
| CN101745119A (en) * | 2010-01-25 | 2010-06-23 | 中国药科大学 | Polysaccharide conjugate of carboxylic acid drug, preparation method thereof and application thereof |
| CN102675385A (en) * | 2011-03-08 | 2012-09-19 | 斯涛利科技发展(天津)有限公司 | Preparation method for doxorubicin13-position hydrazone derivative |
| CN102988999A (en) * | 2012-05-09 | 2013-03-27 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| CN103705939A (en) * | 2013-12-30 | 2014-04-09 | 中国药科大学 | Preparation of amphiphilic ursolic acid-polysaccharide coupled substance and application thereof in treating tumors |
| CN103768080A (en) * | 2013-12-30 | 2014-05-07 | 浙江工业大学 | Targeting preparation for resisting drug-resistant tumor, as well as preparation method and application thereof |
-
2016
- 2016-04-22 CN CN201610268355.8A patent/CN105797169B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1393720A1 (en) * | 2002-08-27 | 2004-03-03 | Universiteit Utrecht | Vesicle-encapsulated corticosteroids for treatment of cancer |
| CN101745119A (en) * | 2010-01-25 | 2010-06-23 | 中国药科大学 | Polysaccharide conjugate of carboxylic acid drug, preparation method thereof and application thereof |
| CN102675385A (en) * | 2011-03-08 | 2012-09-19 | 斯涛利科技发展(天津)有限公司 | Preparation method for doxorubicin13-position hydrazone derivative |
| CN102988999A (en) * | 2012-05-09 | 2013-03-27 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| CN103705939A (en) * | 2013-12-30 | 2014-04-09 | 中国药科大学 | Preparation of amphiphilic ursolic acid-polysaccharide coupled substance and application thereof in treating tumors |
| CN103768080A (en) * | 2013-12-30 | 2014-05-07 | 浙江工业大学 | Targeting preparation for resisting drug-resistant tumor, as well as preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| S. MANJU等: "Conjugation of curcumin onto hyaluronic acid enhances its aqueous solubility and stability", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》 * |
| YUMIN ZHANG等: "Co-delivery of doxorubicin and curcumin by pH-sensitive prodrug nanoparticle for combination therapy of cancer", 《SCIENTIFIC REPORTS》 * |
| 刘文，等: "pH敏感型K5多糖-阿霉素前体药物的制备及其性能", 《食品与生物技术学报》 * |
| 宦梦蕾: "肿瘤靶向阿霉素前体药物的设计合成及其活性研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107952082A (en) * | 2017-11-22 | 2018-04-24 | 中国药科大学 | A kind of multi-functional synergistic pharmaceutical combination and its construction method based on adriamycin |
| CN107952082B (en) * | 2017-11-22 | 2021-07-13 | 中国药科大学 | Multifunctional synergistic pharmaceutical composition based on adriamycin and construction method thereof |
| CN110339181A (en) * | 2019-07-02 | 2019-10-18 | 中国药科大学 | A kind of pH response type nano preparation and its preparation method and application based on click-reaction |
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|---|---|
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