CN102125547B - Pharmaceutical composition containing gambogic acid medicament and preparation method thereof - Google Patents
Pharmaceutical composition containing gambogic acid medicament and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition containing a gambogic acid medicament and a preparation method thereof. The pharmaceutical composition containing the gambogic acid medicament is characterized by containing the gambogic acid medicament which forms micelle with an amphipathic high polymeric conjugate. In the invention, the gambogic acid medicament is coated in the amphipathic high polymeric conjugate by physical embedding and chemical conjugation, thus greatly improving the solubility of the gambogic acid medicament, notably improving the storage stability and antitumor activity and improving the safety of the medicament. Compared with gambogic acid medicaments, the pharmaceutical composition has improved curative effects and reduced irritation on treating liver cancer, cervical cancer and other cancers. The invention also provides preparation methods of the amphipathic high polymeric conjugate and the pharmaceutical composition containing the gambogic acid medicament. The preparation method is simple, has a mature process and high yield, and is suitable for industrialized production.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, relate to a kind of preparation method that contains pharmaceutical composition He this pharmaceutical composition of gambogic acid medicament.The feature of this pharmaceutical composition is that the poly-conjugate of amphipathic height wraps up gambogic acid medicament with micelle form.
Background technology
Cancer has become the multiple commonly encountered diseases of serious threat human life health in the world.The clinical treatments such as operation, chemotherapy are the effective means of removing tumor tumor body, but operation only can be excised macroscopic tumor body, to sightless subclinical focus with shifted or the tumor cell that infiltrates normal structure but can't be removed; And conventional chemotherapy due to existing anticarcinogen to tumor tissues and cell non-selectivity almost, the ubiquity clinical efficacy is low, toxicity large, metastasis is difficult to the problems such as control, patient's medication poor compliance, thereby causes the bad situation of some patients were refusal medication or medication failure.Antitumor drug treatment cancer is one of main method of present clinical treatment, but because existing antitumor drug is mostly poorly soluble, oral absorption is relatively poor, bioavailability is lower, and metabolism is very fast in vivo, keeps the blood drug level time shorter, often needs increased dosage amount or increases administration number of times in order to improve curative effect, normal cell and cancerous cell are lacked selectivity in addition, bring thus larger toxic and side effects.Therefore developing long-acting, slow release and targeting antineoplastic medicine thing preparation is the emphasis of current research.
That the outstanding advantage of antineoplastic target preparation is is with strong points, effect is remarkable, side effect is low; Antitumor drug is prepared into the drug-supplying system intensifier target tropisms such as liposome, microcapsule (ball), improves curative effect, become the effective way of cancer target administration, but liposome is as pharmaceutical carrier, to have still that envelop rate is low, targeting distributes undesirable, store in the shortcomings such as stability is not good enough; Microcapsule (ball) particle diameter is larger, can't see through mucosa or directly drug conveying be arrived target tissue through the body circulation, is not suitable for injecting drug use; And liposome, microcapsule (ball) effect aspect some solid tumor for the treatment of (as breast carcinoma, carcinoma of prostate, colon cancer etc.) is not satisfactory.High poly-conjugate is a kind of very effective means, it is with micromolecule hydrophobic drug access macromolecule carrier, formed a kind of new polymers, particularly the nano grade polymer particle is due to its small-size effect and surface and interface effect etc., not only can improve drug effect, alleviate untoward reaction, and facilitate the patient to use.Its outstanding advantage also is: 1. increase the dewatering medicament solubility; 2. passive target and slow-releasing and controlled-releasing action; 3. avoid being removed fast by kidney prolong drug plasma half-life etc.
Resina garciniae is to extract in the secreted dry resin of Garcinia maingayii, mainly comprises the compositions such as gamlogic acid, neogambogic acid, allogambogic acid.Gamlogic acid is the main active of Resina garciniae, and kinds of tumors is had obvious inhibitory action.Result of study shows, gamlogic acid has higher distribution and grows in tumor tissues persistent period.The result of study of the molecular level of the research gamlogic acid mechanism of action shows: gamlogic acid has efficiently, the characteristics of low toxicity, multiaction target spot, kill cancer cell and on the not impact of normal hemopoietic system and leukocyte, this provides application prospect for seeking new antitumor drug optionally.The gamlogic acid utmost point is insoluble in water, and at present the gamlogic acid injection of report mainly uses the boric acid solution preparation, or adds the solubilizing agents such as L-arginine, meglumine, tween and prepare the gamlogic acid lyophilized formulations.These solubilizing agent meetings of life-time service cause a series of untoward reaction, or need larger dose just can obtain to stablize effective solubilizing effect.
The inventor finds that in early-stage Study the poly-conjugate of amphipathic height has good solubilization, and is as good in solubilizing effects such as paclitaxel, amycin for some insoluble drug.The poly-conjugate of amphipathic height used herein is take natural polysaccharide as skeleton, at the carboxyl of polysaccharide or be linking arm by simple Alkylenediamine on the carboxyl that derivatization forms, introduce carboxylic hydrophobic drug, make conjugate not only have the hydrophobic drug part but also have hydrophilic polysaccharide part, greatly strengthened amphipathic, and have the feature of polymer micelle concurrently: 1) but self assembly forms nano-micelle in aqueous solution, avoided the use of organic solvent, surfactant, cross-linking agent or heating condition; 2) under the dual function of polysaccharide molecule chain and hydrophobic drug, significantly reduce the critical micelle concentration of micelle, obviously extend stabilization time, and improve drug loading and the envelop rate of medicine; 3) be to be combined with carrier in non-covalent mode due to solubilising at the antitumor drug of conjugate kernel, make the medicine of delivery comparatively to be easy to discharge, mutually auxiliary with the drug release behavior of chemical coupling, reach procedural release effect; The mechanism of action that 4) can pass through the antitumor drug of physics solubilising and chemical coupling replenishes mutually, strengthens anti-tumor activity, reaches the effect of therapeutic alliance.CN101791411A discloses these technology.
Adopt the poly-conjugate parcel of amphipathic height gambogic acid medicament, and use other carriers parcel gambogic acid medicaments to compare to have clear superiority.Such as with gamlogic acid, phospholipid, cholesterol three according to certain weight ratio, adopt the thin-film ultrasonic legal system to get the gamlogic acid liposome.But there is following defective in the gamlogic acid liposome: physics, poor chemical stability, the easy seepage of wrapped medicine after the storage certain hour; Envelop rate is lower, envelop rate the highest only 80% left and right; Gamlogic acid liposome particle diameter is larger, lacks vascular permeability, is difficult for the intercellular substance by liver blood vessel, easily by reticuloendothelial system phagocytic; The preparation technology of gamlogic acid liposome and quality control technological requirement are higher, more difficultly realize the large production of industrialization.
For above problem, this patent adopts the pharmaceutical composition of gambogic acid medicament and the poly-conjugate of amphipathic height, has built a kind of new type antineoplastic medicine transmission system.In this way, gamlogic acid is wrapped in high poly-conjugate in the mode of physically trapping and chemical coupling, and this pharmaceutical composition can strengthen pharmacological action greatly, improve drug safety, realize the therapeutic alliance of medicine, and drug effect strengthens significantly, zest obviously reduces, and reaches desirable release effect.
Summary of the invention
The objective of the invention is for above-mentioned technical problem, a kind of pharmaceutical composition that contains gambogic acid medicament is provided.This pharmaceutical composition utilizes the poly-conjugate parcel of amphipathic height gambogic acid medicament, and this pharmaceutical composition closes that safety is good, drug loading is high, physiologically active good, drug effect improves, toxic and side effects reduces, good stability.
Another object of the present invention is to provide and comprises the above-mentioned preparation method that contains the pharmaceutical composition of gambogic acid medicament.
Another object of the present invention is to provide the above-mentioned application of pharmaceutical composition in pharmacy that contains gambogic acid medicament.
In the present invention, the poly-conjugate of amphipathic height has solubilization to gambogic acid medicament, and solubilizing effect is better than the insoluble anti-tumor medicaments such as paclitaxel, amycin.But conjugate self assembly in aqueous medium is the nano-micelle with anti-tumor activity, wraps up gambogic acid medicament, has effectively solved the solubility problem of gamlogic acid.
Contain the medicinal composites that the gambogic acid medicament described in the pharmaceutical composition of gambogic acid medicament comprises that mainly total cambogic acid, gamlogic acid, neogambogic acid, allogambogic acid and said medicine and basic amino acid, alkali metal ion, nitrogenous organic base compound, glucosamine form.
Contain the poly-conjugate of the amphipathic height described in the pharmaceutical composition of gambogic acid medicament, the polysaccharide of wherein selecting comprises that the polysaccharide derivates that the polysaccharide that contains carboxyl originally comprises unfraction heparin, low molecular weight heparin, desulfated heparin, hyaluronic acid, chrondroitin, poly-sulfated chrondroitin, alginic acid and contains carboxyl comprises polysaccharide chitosan, carboxymethyl chitosan, succinyl-chitosan, glucosan, fungus polysaccharide, carboxymethyl lentinan.described hydrophobic group is carboxylic pharmaceutical active or pharmacologically active molecule, comprise gamlogic acid, neogambogic acid, allogambogic acid, all-trans-retinoic acid, 9-cis-retinoic acid, methotrexate, aminopterin, Raltitrexed, pemetrexed, chlorambucil, amino-laevulic acid, the ginsenoside, oleanolic acid, ursolic acid, aspirin, diclofenac, ketoprofen, ibuprofen, fenbufen, mefenamic acid, meclofenamic acid, indomethacin, sulindac, acemetacin, benzydamine, etodolac, naproxen, ibuprofen, norfloxacin, ofloxacin, ciprofloxacin, pipemidic acid, levofloxacin, enoxacin, lomefloxacin, pefloxacin, fleroxacin, tosufloxacin, sparfloxacin, tomefloxacin, balofloxacin, edatrexate and comprise gallbladder acid, ursodesoxycholic acid, lithocholic acid, chenodeoxycholic acid is at interior bile acids.
The preparation method of the poly-conjugate of described amphipathic height comprises the following steps:
Carboxylic hydrophobic drug is dissolved in suitable organic solvent, the employing Alkylenediamine is linking arm, dicyclohexyl carbodiimide (DCC), N-Hydroxysuccinimide (NHS) obtain the reactive intermediate of a free end amino for activator carries out condensation reaction; To contain carboxyl or be dissolved in reaction dissolvent through the derivative polysaccharide that changes into carboxyl, passing through 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC) with the reactive intermediate that obtains is activator, further carboxyl and amino condensation reaction.
Described preparation method, wherein suitable organic solvent is preferably from DMF, oxolane, dimethyl sulfoxide.
Described preparation method, wherein linking arm is the Alkylenediamine structure of carbon number 2～12.
Described preparation method, wherein reaction dissolvent is preferably from the mixed solvent of water or Methanamide or DMF and water or Methanamide and water or DMF and Methanamide.
The pharmaceutical composition that contains gambogic acid medicament in the present invention, its preferred preparation method is as follows: the poly-conjugate of amphipathic height and water are 3～50: 1000 ratio dissolving by weight, obtain the conjugate nano-micelle; With the indissoluble for the treatment of effective dose or the gambogic acid medicament that is slightly soluble in water with after acceptable solvent dissolving pharmaceutically, after described conjugate nano-micelle mixes, process through ultrasonic or high pressure homogenize, solution is removed organic solvent and micromolecule with dialysis or ultrafiltration or post partition method, makes solution type preparation.
One or more mixed solvents in described pharmacy acceptable solvent particular methanol, ethanol, dichloromethane, chloroform, dimethyl sulfoxide, DMF.The further preferred alcohol of pharmacy acceptable solvent.The micelle medicine carrying amount that makes with this method is high, and envelop rate is high, and the sustainable release of medicine extends the time in blood circulation.
Research and development find, take the pharmaceutical composition of gamlogic acid-hyaluronic acid conjugate and gamlogic acid as example, gamlogic acid-hyaluronic acid conjugate is different from the consumption of gamlogic acid, the drug loading difference.In the very few or too much situation of gamlogic acid amount, the medicine carrying effect is all undesirable.The inventor determines that through experiment is final the mass ratio of the poly-conjugate of amphipathic height and gamlogic acid is 1: 0.01-1: 1.Further the mass ratio of the high poly-conjugate of preferred amphiphilic and gamlogic acid is 1: 0.4-1: 0.6.The micelle that the gamlogic acid that the employing optimum process prepares and gamlogic acid-hyaluronic acid conjugate form, drug loading reaches as high as 38.7%, and envelop rate can reach 88.2%, and the micelle particle diameter that makes reaches Nano grade.Medicament contg in the present invention in gamlogic acid micelle unit volume is high, has stable drug loading and envelop rate.Pharmaceutical composition of the present invention is through the overstability contrast test, proves that it compares with gamlogic acid crude drug and gamlogic acid liposome and have better stability.The gamlogic acid crude drug is obviously descending through medicament contg after certain hour, and gamlogic acid liposome medicine after certain hour also has Seepage, and in same time, the micelle size of this pharmaceutical composition is substantially constant, and content of dispersion is stable.
Also the above-mentioned pharmaceutical composition lyophilizing that contains gambogic acid medicament can be made lyophilized formulations.Can add freeze drying protectant when making lyophilized formulations, freeze drying protectant can be selected from one or more in dextran, mannitol, glucose, lactose, sucrose, trehalose.The amount of freeze drying protectant is according to the micellar solution volume calculation, and every part of micellar solution preferably adds 0.01-0.1 part freeze drying protectant, filters postlyophilization.
The lyophilized formulations that contains gambogic acid medicament of said method preparation does not contain any organic solvent, and before clinical use, available water for injection, glucose injection or normal saline redissolve, and dissolves rapidly the solution clarification when lyophilized formulations of the present invention dissolves again.Gamlogic acid micelle freeze-drying powder pin can significantly improve bin stability, can reduce again the insecurity of clinical application.
Pharmaceutical combination formulation of the present invention can be applied to by the mode of oral or parenteral the patient who needs this treatment, be used for when oral, conventional solid preparation such as tablet, capsule, powder, granule etc. be can be made into, liquid preparation such as water or oil-suspending agent or other liquid preparations such as syrup, oral liquid etc. made; When being used for parenteral, can be made into injection solution, powder pin, water or oiliness suspending agent etc.Preferred form is injection powder pin, tablet, coated tablet.
Another object of the present invention is to provide the pharmaceutical composition of a kind of Hepatoma therapy, cervical cancer and other tumors, and its curative effect is better than gamlogic acid, and zest and toxicity all reduce.The present invention also provides the purposes of aforementioned pharmaceutical compositions aspect preparation Hepatoma therapy, cervical cancer and other tumour medicines.
The present invention finds, (family's rabbit ear vein instils the pharmaceutical composition that contains gamlogic acid, is equivalent to 1mg gamlogic acid/20ml/kg) without the blood vessel zest.Carry out hemolytic test of the present invention with 2% rabbit erythrocyte suspension, show that the following dosage of 0.5ml has no erythrocyte hemolysis and clustering phenomena in 2h.This pharmaceutical composition is when the blood vessel administration, and the local irritation side effect of generation is lower than alone gamlogic acid.As: rabbit auricular vein sterile working slowly instil respectively combination preparation, 0.9% sodium chloride injection of gamlogic acid sodium salt, the pharmaceutical composition that contains gamlogic acid such as all-trans-retinoic acid-hyaluronic acid and gamlogic acid, every other day once, continuous 3 times.Result shows, compares with matched group: contain the pharmaceutical composition of gamlogic acid to rabbit auricular vein nonirritant, tissue slice checks that rabbit auricular vein blood vessel structure is normal, without endothelial injury, forms and other pharmacological variation without thrombosis; Gamlogic acid is irritant to the rabbit auricular vein, and tissue slice checks that auricular vein slightly expands, and the part endotheliocyte is downright bad, come off, the visible inflammatory reaction of surrounding tissue.
The present invention also finds, the pharmaceutical composition that contains gambogic acid medicament has increased the dissolubility of gambogic acid medicament in water, and drug effect also significantly strengthens simultaneously.Adopt tetramethyl azo azoles salt colorimetry, find that all-trans-retinoic acid-hyaluronic acid and its drug effect to s and hepatoma Hep G 2 cells of pharmaceutical composition of gamlogic acid formation significantly improve than the gamlogic acid crude drug, be meet clinical demand both have good aqueous solubility and a high medicine of drug effect.
Beneficial effect of the present invention:
One, the poly-conjugate of gambogic acid medicament and amphipathic height is combined in the present invention, the nano-micelle parcel antitumor drug that adopts hydrophilic shell and hydrophobic core to form, can the extension body internal recycle, reduce engulfing of reticuloendothelial cell, increase cancer target, improve safety.
Two, in the pharmaceutical composition that contains gambogic acid medicament provided by the invention, the poly-conjugate of amphipathic height utilizes the polysaccharide graft hydrophobic group, can be in water spontaneous formation nano-micelle, the bag that not only can be used for antitumor drug carries, and the nano-micelle structure that forms due to hydrophilic shell and hydrophobic core, can the extension body internal recycle, reduce engulfing of reticuloendothelial cell, increase cancer target, therefore can by the mode of physically encapsulation and chemical coupling antitumor drug, reach the effect of therapeutic alliance cancer;
Three, the pharmaceutical composition that contains gambogic acid medicament provided by the invention can be used for injection, oral, external or mucosa delivery.Have tight security, particle diameter can be controlled in 10～1000nm, and dissolubility is good, good stability.
Four, the pharmaceutical composition that contains gambogic acid medicament provided by the invention has improved the defective of present gambogic acid preparation, and is economical and practical, has good antitumor action, and without the blood vessel zest, drug effect is high.
The specific embodiment
To the present invention's further instruction in addition, but following embodiment does not limit the interest field of this patent below by embodiment.
Embodiment 1: all-trans-retinoic acid-hyaluronic acid conjugate synthetic
Get 10mmol all-trans-retinoic acid, 12mmol dicyclohexyl carbodiimide (DCC), 15mmol N-Hydroxysuccinimide (NHS); be dissolved in the 30ml DMF, under lucifuge, nitrogen protection; then ice bath reaction 30min rises to room temperature reaction 24h.After reaction finishes, the elimination precipitation, and add a large amount of ethyl acetate washing precipitations.Filtrate is extracted, the combined ethyl acetate layer, the rotary evaporation desolventizing obtains the middle ester of all-trans-retinoic acid activation.Ester in the middle of the activation of 1mmol all-trans-retinoic acid is dissolved in the 10ml dichloromethane, under condition of ice bath, slowly splash in the dichloromethane solution of 3mmol/ml ethylenediamine, after thin layer chromatography (TLC method) monitoring reaction extremely fully, reactant liquor is extracted, merge organic layer, silica gel column chromatography separation and purification products therefrom, i.e. all-trans-retinoic acid reactive intermediate.Get 26mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide, add and contain in the hyaluronic formamide solution of 0.1mmol, room temperature reaction 15min, the N that will contain 26mmol all-trans-retinoic acid reactive intermediate, the solution of dinethylformamide slowly adds in above-mentioned reactant liquor, room temperature reaction 24h.Reaction adds the acetone precipitation product after finishing, and sucking filtration must precipitate.Add water redissolution precipitation, the 3d that dialyses in water, lyophilization namely gets end product all-trans-retinoic acid-hyaluronic acid conjugate.
Embodiment 2: gamlogic acid-hyaluronic acid conjugate synthetic
Get 10mmol gamlogic acid, 12mmol dicyclohexyl carbodiimide (DCC), 15mmol N-Hydroxysuccinimide (NHS); be dissolved in 30mlN, in dinethylformamide, under lucifuge, nitrogen protection; then ice bath reaction 30min rises to room temperature reaction 24h.After reaction finishes, the elimination precipitation, and add a large amount of ethyl acetate washing precipitations.Filtrate is extracted, the combined ethyl acetate layer, the rotary evaporation desolventizing obtains the middle ester of gamlogic acid activation.Ester in the middle of the activation of 1mmol gamlogic acid is dissolved in the 10ml dichloromethane, under condition of ice bath, slowly splash in the dichloromethane solution of 3mmol/ml ethylenediamine, after thin layer chromatography (TLC method) monitoring reaction extremely fully, reactant liquor is extracted, merge organic layer, silica gel column chromatography separation and purification products therefrom, i.e. gamlogic acid reactive intermediate.Get 26mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide, add and contain in the hyaluronic formamide solution of 0.1mmol, room temperature reaction 15min, the N that will contain 26mmol gamlogic acid reactive intermediate, the solution of dinethylformamide slowly adds in above-mentioned reactant liquor, room temperature reaction 24h.Reaction adds the acetone precipitation product after finishing, and sucking filtration must precipitate.Add water redissolution precipitation, the 3d that dialyses in water, lyophilization namely gets end product gamlogic acid-hyaluronic acid conjugate.
Embodiment 3: contain the preparation of the self-assembled nano micelle compositions of gamlogic acid and gamlogic acid-hyaluronic acid conjugate
(1) dialysis
Gamlogic acid-hyaluronic acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Gamlogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
(2) emulsion-solvent evaporation method
Gamlogic acid-hyaluronic acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Gamlogic acid 9mg is dissolved in dichloromethane.Then both mix, Probe Ultrasonic Searching 30min, the uncovered stirring of room temperature is spent the night, and makes the dichloromethane volatilization, and centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 4: contain the preparation of the self-assembled nano micelle compositions of gamlogic acid and gamlogic acid-heparin thing
Gamlogic acid-heparin thing 18mg is dissolved in 3ml water and stirs 1h.Gamlogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 5: contain the preparation of the self-assembled nano micelle compositions of gamlogic acid and all-trans-retinoic acid-hyaluronic acid conjugate
All-trans-retinoic acid-hyaluronic acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Gamlogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 6: contain the preparation of the self-assembled nano micelle compositions of gamlogic acid and methotrexate-chondroitin sulfate conjugate
Methotrexate-chondroitin sulfate conjugate 18mg is dissolved in 3ml water and stirs 1h.Gamlogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 7: contain the preparation of the self-assembled nano micelle compositions of neogambogic acid and gamlogic acid-hyaluronic acid conjugate
Gamlogic acid-hyaluronic acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Neogambogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 8: contain the preparation of the self-assembled nano micelle compositions of neogambogic acid and aspirin-alginic acid conjugate
Aspirin-alginic acid acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Neogambogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 9: contain the preparation of the self-assembled nano micelle compositions of neogambogic acid and ursodesoxycholic acid-hyaluronic acid conjugate
Ursodesoxycholic acid-hyaluronic acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Neogambogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 10: contain the preparation of the self-assembled nano micelle compositions of allogambogic acid and ursodesoxycholic acid-hyaluronic acid conjugate
Ursodesoxycholic acid-hyaluronic acid conjugate 18mg is dissolved in 3ml water and stirs 1h.Allogambogic acid 9mg is dissolved in ethanol (methanol).Then both mix, after Probe Ultrasonic Searching 30min, the redistilled water dialysed overnight, centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
Embodiment 11: the mensuration of gamlogic acid content in the pharmaceutical composition self-assembled nano micelle
Carry out assay with HPLC (LC-2010C, Shimadzu, Japan) method.Mobile phase is methanol: water=93: 7 (v/v), contain 0.1% glacial acetic acid, and chromatographic column is Lichrospher C
18(150 * 4.6 μ m), pillar particle diameter are 5 μ m.Flow velocity is 1.0mL/min, and the detection wavelength is 360nm (SPD-10A, UV detector, Shimadzu, Japan), and column temperature is 30 ℃, and the injected sample volume is 20 μ l.Drug loading with formula (1) calculation sample.The results are shown in Table 1.
Table 1 embodiment 3～6 is loaded with the poly-conjugate self-assembled nano micelle of amphipathic height of gamlogic acid
Amphipathic height gathers conjugate preparation technology drug loading (%)
Embodiment 3 dialysis 38.7
Embodiment 3 emulsion-solvent evaporation methods 29.5
Embodiment 4 dialysis 34.5
Embodiment 5 dialysis 31.2
Embodiment 6 dialysis 28.9
Embodiment 12: the mensuration of neogambogic acid content in the pharmaceutical composition self-assembled nano micelle
Carry out assay with HPLC (LC-2010C, Shimadzu, Japan) method.Mobile phase is methanol: 1.0mol/L phosphoric acid=90: 10 (v/v), chromatographic column are Lichrospher C
18(150 * 4.6 μ m), pillar particle diameter are 5 μ m.Flow velocity is 1.0mL/min, and the detection wavelength is 360nm (SPD-10A, UV detector, Shimadzu, Japan), and column temperature is 30 ℃, and the injected sample volume is 20 μ l.The results are shown in Table 2.
Table 2 embodiment 7 is loaded with the poly-conjugate self-assembled nano micelle of amphipathic height of neogambogic acid
Amphipathic height gathers conjugate preparation technology drug loading (%)
Embodiment 7 dialysis 34.8
Embodiment 7 emulsion-solvent evaporation methods 24.9
Embodiment 13: the study on the stability of the pharmaceutical composition self-assembled nano micelle of gamlogic acid and all-trans-retinoic acid-hyaluronic acid conjugate
Particle diameter, current potential and medicament contg after redissolving in the drug regimen lyophilized formulations water investigated embodiment 3～5 under aqueous environments as index, places the stability of 0～4 ℃; Result such as table 3.But result shows the pharmaceutical composition stable existence of the poly-conjugate of gambogic acid medicament and amphipathic height with this understanding.
Table 3 embodiment 3～5 contains the stability of the self-assembled nano micelle of gamlogic acid and the poly-conjugate of amphipathic height
Embodiment 14: the pharmaceutical composition medicine carrying situation that contains gambogic acid medicament
The self-assembled nano micelle compositions that contains gamlogic acid and all-trans-retinoic acid-hyaluronic acid conjugate is according to preparation under 5 of embodiment, and the gamlogic acid liposome is to utilize gamlogic acid, phospholipid, cholesterol three according to certain weight ratio, adopts the thin-film ultrasonic legal system standby.Result is as shown in table 4.
Table 4 contains the medicine carrying situation of the pharmaceutical composition of gambogic acid medicament
Result shows: the drug loading of the pharmaceutical composition micelle that the poly-conjugate parcel of amphipathic height gamlogic acid forms, envelop rate are all apparently higher than the gamlogic acid liposome, and the envelop rate maximum can reach 88.2%, and the envelop rate of gamlogic acid liposome is only up to 80.2%.The envelop rate of gamlogic acid liposome drops to 57.1% in the time of the 7th day, particle diameter also increases to rapidly 410.6nm by 232.1nm, and the envelop rate and the particle diameter that contain the polymer micelle of gamlogic acid remain unchanged substantially, illustrate that the stability of the polymer micelle that contains gamlogic acid will obviously be better than the gamlogic acid liposome.
Embodiment 15: laser confocal microscope (cLsM) is observed the cellular uptake experiment of the pharmaceutical composition that contains gambogic acid medicament
1, preparation technology
All-trans-retinoic acid-hyaluronic acid conjugate 9mg is dissolved in 3ml water and stirs 1h.Coumarin 1 .5ug is dissolved in dichloromethane.After both mix, Probe Ultrasonic Searching 30min, the uncovered stirring of room temperature is spent the night, and makes the dichloromethane volatilization, and centrifugal (3000rpm) 15min is with 0.45 μ m membrane filtration, lyophilization.
2, observational technique
The breast carcinoma B21 cell of hatching is in advance made cell suspension, respectively with 1 * 10
5/ ml cell concentration adds in 24 orifice plates, and every hole 300 μ l establish five multiple holes, put 37 ℃ of 5%CO
2Cultivate 24h in incubator.Remove cell suspension, every hole adds respectively 10ul to carry the poly-conjugate micelle of height of coumarin and the DMEM culture medium that 300ul does not contain serum, 37 ℃ of 5%CO
2After hatching 0.5,1,2,4h, remove supernatant liquid in incubator, wash three times with PBS, 4% paraformaldehyde is removed after solidifying 20min, then washes 3 times with PBS, after Hoechs dyeing 15min room temperature lucifuge, then washes 3 times with PBS, and each jolting 10min removes; After mounting, observe under the laser confocal microscopy and take pictures.
According to experimental result, the poly-conjugate micelle of height that carries coumarin has entered core when 1h, and the interior fluorescence intensity of core is apparently higher than endochylema, and in core, fluorescence intensity strengthens along with the time increases.Illustrate that carrier micelle has the core targeting and can successfully enter core, and this cellular uptake has time dependence.
Embodiment 16: the extracorporeal anti-tumor function that contains the pharmaceutical composition of gambogic acid medicament
Take the logarithm trophophase hepatoma Hep G 2 cells, with 0.25% trypsinization, washing, centrifugal after, it is prepared into cell suspension, get 180 μ l cell suspension (0.5 * 10
5Individual/as ml) to be inoculated in 96 orifice plates, cell plates are placed in 37 ℃ of incubators, at 5%CO
2Under hatch 24h, microscopically is observed the visible cell adherent growth.Add respectively 20 μ l variable concentrations (5,10,20,30,40, the polymer micelle solution that contains gamlogic acid of gamlogic acid solution 50,100 μ g/ml), corresponding same concentrations (embodiment 5 preparations), the poly-conjugate carrier solution (1000 μ g/ml) of amphipathic height, the blank group adds isopyknic culture fluid.After 24h, add the MTT solution 31.5 μ l of 5mg/ml in every hole, continue to cultivate 4h, suck original fluid, every hole adds 200 μ lDMSO dissolving crystallized, measures the OD value in every hole with microplate reader in the 570nm place, calculate the inhibitory rate of cell growth under variable concentrations, calculate IC
50Above-mentioned experiment, every group is repeated 5 times, and each concentration is established 3 multiple holes.Result is as shown in table 5.
Cell moves suppression ratio=(1-experiment OD value/blank group OD meansigma methods) * 100%
Table 5 is respectively organized medicine to the inhibitory action of hepatoma Hep G 2 cells
Medicine IC
50(μ g/mL)
The polymer micelle 1.65 that contains gamlogic acid
Gamlogic acid crude drug 3.57
Result shows, the polymer micelle and the gamlogic acid crude drug that contain gamlogic acid all have obvious growth inhibited effect to the human hepatoma HepG2 cell in 0.5-10.0 μ g/mL concentration range, and along with increasing of drug level, growth inhibited effect to the HepG2 cell also strengthens, and shows obvious dose dependent.Study the IC that also discovery contains the polymer micelle of gamlogic acid
50Little than gamlogic acid crude drug illustrates that the polymer micelle cytotoxicity that contains gamlogic acid will be higher than the gamlogic acid crude drug.Because the screening of antitumoral compounds is that cytotoxicity with compound embodies routinely, so the pharmaceutical composition that gamlogic acid is made the carrier micelle form has more excellent anti-tumor activity, drug effect significantly improves.
Embodiment 17:HE dyeing observation of cell form
The take the logarithm HepG2 cell of trophophase is used 0.25% trypsinization, and adjusting cell density is 1 * 10
5Individual/mL, be inoculated in 6 orifice plates on clean coverslip, be divided into following 4 groups: the blank micelle group of (1) blank group (2): the amphipathic height of 100 μ g/mL gathers conjugate (3) carrier micelle group: the gamlogic acid polymer micelle of 2 μ g/mL and 10 μ g/mL (embodiment 5 preparations) (4) crude drug group: the gamlogic acid of 2 μ g/mL.
After effect 48h, carry out HE dyeing, sop up culture fluid, PBS liquid washboard slide, fixedly more than 30min, PBS washes 2 * 1min to cell fixative room temperature, hematoxylin dye liquor dyeing 5～10min, tap water embathes, and the dilute hydrochloric acid alcoholic solution embathes the color separation several seconds, and tap water embathes, nucleus oil blackeite 3～5min in light ammonia, tap water embathes, Yin Hong dye liquor dyeing 5～10min, and tap water embathes, 70%, 80%, 95% gradient alcohol dehydration, dimethylbenzene is transparent, and neutral gum mounting, microscopically are observed and respectively organized cellular morphology.
Result shows: the growth of matched group HepG2 cell attachment is found in microscopic examination, and granule is less, and kernel, nuclear membrane profile are obvious, and the flanking cell growth is merged in flakes; The HepG2 cell of processing through the polymeric micelle medicine composition that contains gamlogic acid of gamlogic acid crude drug and 2 μ g/mL is found cell progressively by adherent and come off after 24h, cell quantity reduces, and the refractivity variation illustrates that the growing multiplication of cell obviously is suppressed; Through the HepG2 cell that the polymeric micelle medicine composition that contains gamlogic acid and the gamlogic acid crude drug of 10 μ g/mL were processed, this phenomenon is more obvious, and cell almost all comes off, and quantity significantly reduces, and cell growth inhibition gets very obvious.
Claims (6)
1. a pharmaceutical composition that contains gambogic acid medicament, is characterized in that containing the poly-conjugate of gambogic acid medicament and amphipathic height, and the poly-conjugate of amphipathic height wraps up gambogic acid medicament with micelle form; Described gambogic acid medicament is selected from the medicinal composition of total cambogic acid, gamlogic acid, neogambogic acid, allogambogic acid and said medicine and basic amino acid, alkali metal ion, nitrogenous organic base compound formation; Described amphipathic height gathers conjugate, it is characterized in that described polysaccharide for originally containing hyaluronic acid, heparin, chondroitin sulfate, the alginic acid of carboxyl, described hydrophobic group is carboxylic pharmaceutical active or pharmacologically active molecule gamlogic acid, all-trans-retinoic acid, methotrexate, aspirin, ursodesoxycholic acid; The weight ratio of the poly-conjugate of described amphipathic height and gambogic acid medicament is 1: 0.4-1: 0.6.
2. the preparation method of pharmaceutical composition according to claim 1, comprise the steps: the poly-conjugate of amphipathic height with water by weight being 3-50: 1000 ratio is dissolved, and obtains the conjugate nano-micelle; With the pharmaceutically acceptable solvent dissolving of the gambogic acid medicament for the treatment of effective dose, after described conjugate nano-micelle mixes, process through ultrasonic or high pressure homogenize, solution is removed organic solvent and micromolecule with evaporation or dialysis or ultrafiltration or post partition method, make solution type preparation, perhaps this solution absorbs is prepared into solid preparation on the solid adjuvant material surface or lyophilizing is prepared into lyophilized formulations.
3. the preparation method of the poly-conjugate of amphipathic height in pharmaceutical composition according to claim 2, it is characterized in that comprising the following steps: carboxylic hydrophobic drug is dissolved in suitable organic solvent, the employing Alkylenediamine is linking arm, dicyclohexyl carbodiimide, N-Hydroxysuccinimide are that activator carries out condensation reaction, obtain the reactive intermediate of a free end amino; To contain carboxyl or change into carboxylic polysaccharide and be dissolved in reaction dissolvent through derivative, with the reactive intermediate that obtains be activator by 1-ethyl-(3-dimethylaminopropyl) carbodiimide, further carboxyl and amino condensation reaction; The high poly-conjugate of gained namely has well amphipathic, can spontaneous formation nano-micelle in aqueous medium.
4. pharmaceutical composition according to claim 1, is characterized in that said composition can be used for injection, oral or topical administration.
5. the application of pharmaceutical composition according to claim 4 in pharmacy, it is characterized in that described drug administration by injection is selected from injection, freeze-dried powder, oral administration is selected from tablet, capsule, pill, syrup, granule, oral solution, and topical administration is selected from patch, liniment, lotion, gel, varnish, ointment.
6. pharmaceutical composition according to claim 1, the application in preparation Hepatoma therapy, cervical cancer and other tumour medicines.
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