CN101015698B - Medicament using rare earth transferring complex compound as vector - Google Patents
Medicament using rare earth transferring complex compound as vector Download PDFInfo
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- CN101015698B CN101015698B CN2007100641532A CN200710064153A CN101015698B CN 101015698 B CN101015698 B CN 101015698B CN 2007100641532 A CN2007100641532 A CN 2007100641532A CN 200710064153 A CN200710064153 A CN 200710064153A CN 101015698 B CN101015698 B CN 101015698B
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Abstract
This invention relates to a medicine with rare earth transferring complex compound as medicine carrier. The invention provides a medicine comprising effective composition and carrier, wherein carrier is rare earth transferring complex compound. The medicine with rare earth transferring complex compound as carrier can be used as anticancer drug with orient transportation, for oral administration, and absorbent through lung permeation.
Description
Technical field
The present invention relates to a kind of with the medicine of using rare earth transferring complex compound as pharmaceutical carrier.
Background technology
Along with a large amount of novel therapeutic medicines, particularly biochemical drug is widely used in treating multiple disease, many defectives of existing drug-supplying system are also obvious day by day: the low diffusibility (as enterocyte and blood brain barrier) of striding biological barrier, low stability in biological fluid, low affinity for the particular pathologies site, non-special toxicity, and other negative effect that causes because of high dose or the like.A strategy that addresses the above problem is that foundation is the transportation system of mechanism with the receptor mediated endocytosis
This medicament transport approach of transferrins/TfR has embodied great potential at cancer therapy drug, protein and therapeutic gene in the transportation of the tumor cell of elementary neoplasm.Can improve permeability, retentivity and the targeting specific of macromolecular drug based on the medicament transport system of transferrins/TfR, not only improve drug effect, improve selectivity and medicine-releasing performance, also reduce toxicity simultaneously.The reason that this medicament transport approach has above-mentioned advantage is in the tumor cell that the expression of TfR is than normal cell height; In addition, in blood brain barrier and digestive tract barrier, TfR also has higher expression, and with the transport pathway of TfR mediated endocytosis as therapeutant, the target of crossing over blood brain barrier or other physiologic barriers will be no longer remote.
The medicament transport system of transferrins/TfR is attempted being used for the transportation of following medicine:
The metal medicine: as the extensive use in radiation treatment
67Ga and
111In
3+
The location transportation of antitumor drug: comprise transhipment antineoplastic gene or medicine, as amycin
Chlorambucil (Chlorambucil) and CRM107 (a kind of diphtheria toxin, diphtherotoxin albumen of rite-directed mutagenesis) etc.
Liposome/nano-particle that transferrins is modified: the medicine of preparation oral administration and pulmonary route administration, as insulin-transferrin complex of protein, reduced the blood glucose of suffering from diabetes rat behind this complex oral administration.
But, directly as the method for pharmaceutical carrier important limitation is arranged with transferrins, because under physiological condition, the endogenous plasma transferrins have higher concentration, the transferrins carrier to relatively small amount forms serious competition like this; In addition, the higher melanocyte transferrins of Chang Yong efficient is easily by the very fast decomposition of liver.Can reduce the efficient of medicament transport so greatly.Present solution comprises: monoclonal antibody (as OX26) or its Fab fragment of using oligomerization transferrins and the various TfRs of application (TfR), improved the cost of transport of drug system so greatly, the latter and be subjected to the various limitations that monoclonal antibody self is used.
Transferrins is except with the form in conjunction with ferrum, and (Ni Jia inherits volume, rare earth biological inorganic chemistry, Science Press, 2002 can also to comprise rare earth ion in conjunction with multiple trivalent metal ion; Smith, C.A., et al., 1994.J.Am.Chem.Soc.116 7889-7890; Luk C.K.Biochemitry 1971 102838-284 3; Yang Binsheng etc., 2002. chemical journal 60:737-74 3), studies show that using rare earth transferring have the transmembrane transport ability same with transferrins (Du X, et al., Eur.J.Biochem.2002,269:6082-6090).Using rare earth transferring has following superior character, comprising: (1) using rare earth transferring complex compound is a kind of more stable protein complex, and the binding constant of rare earth ion and transferrins is 10
8-10
10(2) using rare earth transferring complex compound can with TfR TfR combination, and its affinity is all stronger than apotransferrin and transferrins, transferrins and its receptor TfR affinity are much higher than apotransferrin, and the affinity of using rare earth transferring (as terbium transferrins Tb-Tf) and TfR is high more about more than 6 times than transferrins.Therefore (3) using rare earth transferring can be absorbed by cell, and using rare earth transferring reaches 1 μ M to the toxicity of blood cell K562 cell strain.(4) on external blood brain barrier model, using rare earth transferring can carry binding molecule such as label fluorescein (FITC) sees through blood brain barrier, and the existence of transferrins is limited to the reduction of its blood brain barrier permeability.On the contrary, using rare earth transferring can reduce the blood brain barrier permeability of transferrins greatly.
Summary of the invention:
In order to solve the above-mentioned defective that existing medicine and pharmaceutical carrier exist, the present invention is pharmaceutical carrier with the using rare earth transferring complex compound, to solve the problems referred to above that prior art exists.
Technical scheme of the present invention is as follows:
The invention provides a kind of medicine, comprise active ingredient and pharmaceutical carrier, pharmaceutical carrier wherein is a using rare earth transferring complex compound.
In the said medicine, the combination of active ingredient and pharmaceutical carrier can be selected from using rare earth transferring complex compound and active ingredient is modified drug-loaded liposome method, cyclodextrin inclusion compound method and using rare earth transferring modification medicament-carried nano granule method by chemical bond coupling method, using rare earth transferring complex compound and active ingredient by biotin/antibiotin method coupling method, using rare earth transferring.
Above-mentioned using rare earth transferring complex compound and active ingredient are as follows by chemical bond coupling method step:
(1) using rare earth transferring solution is dissolved into the solution of 5-10mg/ml, with protein solution by in advance with 0.1mol/L pH8.0 NaHCO
3The PD-10 desalting column that balance is good is collected protein component;
(2) according to the structure choice commodity coupling reagent of medicine such as SPDP, maleimide, carbodiimide etc., according to the method pharmacological activation molecule in the reagent handbook;
(3), select corresponding coupling reagent such as SPDP, maleimide, carbodiimide etc. according to the reagent handbook, and press the method activation using rare earth transferring in the handbook according to the commodity coupling reagent kind of said medicine Molecular Selection;
(4) using rare earth transferring after will activating and drug molecule are according to 1: the mixed of 1-5,4 ℃ incubation 2-6 hour or spend the night;
(5) with above-mentioned reaction mixture in sample: the ratio of gel volume=1: 10, by Sephadex G-25 gel column, collect protein component, promptly obtain using rare earth transferring-drug molecule conjugate, the product lyophilizing is standby.
Above-mentioned using rare earth transferring complex compound and active ingredient are as follows by biotin/antibiotin method coupling method step:
(1) using rare earth transferring solution is dissolved into the solution of 5-10mg/ml, with protein solution by in advance with 0.1mol/L pH8.0 NaHCO
3The PD-10 desalting column that balance is good is collected protein component;
(2) the active ester BNHS of commodity biotin succinimide is dissolved in the solution that DMSO becomes 20mg/ml;
(3) using rare earth transferring solution and BNHS solution are pressed protein: the mixed of biotin=1: 2-5,4 ℃ incubation 2-6 hour or spend the night;
(4) with above-mentioned reaction mixture in sample: the ratio of gel volume=1: 10, by Sephadex G-25 gel column, collect protein component, promptly obtain protein: the biotinylation using rare earth transferring of biotin=1: 1-5;
(5) medicine is dissolved in suitable solvent, adds 1: 1 BNHS reaction, obtain the biotinylation drug molecule;
(6) with the biotinylation using rare earth transferring: the affinity element: the mixed of biotinylation drug molecule=1: 1: 3-5,4 ℃ incubation 2-6 hour.Yet with above-mentioned reaction mixture in sample: the ratio of gel volume=1: 10, by Sephadex G-25 gel column, collect protein component, promptly obtain using rare earth transferring-drug molecule BAB conjugate, the product lyophilizing is standby;
(7) the same manner can prepare using rare earth transferring-drug molecule BA conjugate.Concrete grammar is referring to (Xu Yiwei writes, " immunoassay technology " second edition, Science Press, Beijing in 1991, chapter 10 biotin avidin system and application thereof).
Above-mentioned using rare earth transferring modification drug-loaded liposome method step is as follows:
(1) using rare earth transferring solution is dissolved into the solution of 5-10mg/ml, with protein solution by in advance with 0.1mol/L pH8.0 NaHCO
3The PD-10 desalting column that balance is good is collected protein component;
(2) drug-loaded liposome of changing with conventional method (seeing the Lu Bin chief editor for details, " novel pharmaceutical formulation and new technique ", People's Health Publisher,, Beijing, chapter 4, liposome preparation technology in 2000) preparation PEG (2000-3400);
(3) liposome solutions adds the sodium periodate of 0.03mol/L, and room temperature was placed 30 minutes.The PEG aqueous solution that adds 0.10mol/L then, room temperature was placed 30 minutes;
(4) add using rare earth transferring solution (optimum protein/liposome ratio is determined by preliminary experiment), 4 ℃ incubation 2-6 hour;
(5) centrifugal 5000-10000rpm obtains the drug-loaded liposome that using rare earth transferring is modified.
Above-mentioned cyclodextrin inclusion compound method step is as follows:
(1) the 2g biotin is dissolved in chloroacetic chloride, room temperature reaction 2 hours, distilling under reduced pressure obtains the biotin acyl chlorides;
(2) with cyclodextrin and biotin acyl chlorides by 1: 2-5 is dissolved in the pyridine solution, 0 ℃ of reaction 2 hours, room temperature was placed 2 hours, distilling under reduced pressure obtains crude product.Crude product is used DMF: H on Sephadex LH-20 post
2O=1: 1 eluting is purified, and after concentrating after cyclodextrin is partly regained, pours in the acetone soln and precipitates.Obtain the biotinylation cyclodextrin;
(3) medicine is dissolved in appropriate solvent, dropwise adds biotinylation cyclodextrin saturated aqueous solution, stir under the uniform temperature, place 12 hours drug loading.Remove and desolvate, reclaim cyclodextrin.(concrete points for attention see Lu Bin chief editor, " novel pharmaceutical formulation and new technique ", People's Health Publisher,, Beijing, chapter 2, inclusion technique in 2000 for details);
(4) the biotinylation rare earth is pressed transferrins: the affinity element: biotinylation medicine carrying cyclodextrin=1: 1: the mixed of 1-3,4 ℃ incubation 2-6 hour, promptly obtain using rare earth transferring-drug molecule BAB conjugate.The product lyophilizing is standby.
Above-mentioned using rare earth transferring modification medicament-carried nano granule method step is as follows:
(1) using rare earth transferring solution is dissolved into the solution of 5-10mg/ml, with protein solution by in advance with 0.1mol/L pH8.0 NaHCO
3The PD-10 desalting column that balance is good is collected protein component;
(2) prepare various medicament-carried nano granules (seeing the Lu Bin chief editor for details, " novel pharmaceutical formulation and new technique ", People's Health Publisher,, Beijing, chapter 5, microencapsulation and miniature balling-up technology in 2000) with conventional method;
(3) according to the structure choice commodity coupling reagent of nanometer microencapsulation such as SPDP, maleimide, carbodiimide etc., according to the method pharmacological activation molecule in the reagent handbook;
(4), select corresponding coupling reagent such as SPDP, maleimide, carbodiimide etc. according to the reagent handbook, and press the method activation using rare earth transferring in the handbook according to the commodity coupling reagent kind of said medicine Molecular Selection;
(5) using rare earth transferring after will activating and nanometer microencapsulation mix (optimum protein/alloy granular solids ratio is determined by preliminary experiment), 4 ℃ incubation 2-6 hour;
(6) centrifugal 5000
-10000rpm obtains the medicament-carried nano granule that using rare earth transferring is modified.
Described using rare earth transferring complex compound prepares with the following method:
(1) transferrins Tf (2-20mg/ml) is dissolved in the hac buffer that contains EDTA (1-10mM) and Vc (10-50mM) (0.1M, pH=5.5) in, 4 ℃ incubation 2-6 hour or spend the night;
(2) Sephadex G-25 gel column is used 0.1M in advance, the Tris-HCl of pH=7.4 or other non-phosphate buffer solution balance);
(3) in sample: the ratio of gel volume=1: 10, above-mentioned Tf solution by the SephadexG-25 gel column, is collected protein component, obtain apotransferrin Apo-Tf;
(4) will add 1 in the above-mentioned Apo-Tf solution: the rare earth trivalent ion of 1-5 (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y), 4 ℃ incubation 2-6 hour or room temperature 0.5-1 hour;
(5) with above-mentioned reaction solution in sample: the ratio of gel volume=1: 10, by the SephadexG-25 gel column, collect protein component, promptly obtain using rare earth transferring complex compound.
The technique effect that the present invention realizes is as follows:
Said medicine adopts using rare earth transferring complex compound as pharmaceutical carrier, the orientation of useful as anti-cancer agents thing transportation, oral and see through that pulmonary absorbs the drug and cerebral nervous system (seeing through blood brain barrier) medicine.
Carrier-medicine macromole the conjugate and the medicament transport system that obtain with said method can be used for following application:
Directed cancer therapy drug transportation: since cancerous cell particularly the blood cancerous cell usually its TfR level be much higher than normal cell, therefore will more absorb the medicine of transferrins and load.The medicament transport system of making carrier with using rare earth transferring can improve the drug absorption rate of tumor, improves the cell selective of medicine, reduces the toxicity of medicine simultaneously.
Nervus centralis medicine:, therefore can realize the transport capacity of medicine to the central nervous system because using rare earth transferring can carry medicine by blood brain barrier.
Protein/polypeptide/nucleic acid drug oral or that absorb through lung: is the big obstacle of one for protein/polypeptide/nucleic acid drug by the digestive tract barrier.Because transferrins can see through barriers such as digestive tract and lung mucosa by its receptor-mediated cytophagy, so the medicament transport system that using rare earth transferring is made carrier can realize the oral of said medicine or improve its absorbability through the lung approach.
Compare with transferrins, oligomerization transferrins and TfR antibody, have following characteristic and advantage based on the medicament transport system of using rare earth transferring:
(1) high efficiency.Because using rare earth transferring is higher 6 times to the affinity of receptor than transferrins, promptly be equivalent to the oligomer of 6 transferrinss, but molecular weight is far smaller than oligomer.Therefore using rare earth transferring medicament transport system has higher medicament transport efficient, and is subjected to the competitive influence of endogenous transferrins in the blood little.
(2) preparation method is simple.The transferrins source is abundant, and the preparation using rare earth transferring only needs a step deferrization and a step rare earth incubation integrating step, than preparation of albumen oligomer and Monoclonal Antibody, method and step are all simple many, and preparation process does not influence the biological activity of transferrins.
(3) low price.Because raw material sources are abundant and preparation is simple, therefore greatly reduce production cost, so also and then the cost and price of reduction medicine.
The specific embodiment
Embodiment 1:
Amycin-using rare earth transferring complex targeted anticancer medicine preparation
Amycin is present a kind of common antibiotic cancer therapy drug.For improving its targeting type, can be prepared into amycin-using rare earth transferring complex medicine, method is summarized as follows:
The preparation using rare earth transferring: with transferrins Tf (Sigma Inc product) (2-20mg/ml) be dissolved in the hac buffer that contains EDTA (1-10mM) and Vc (10-50mM) (0.1M, pH=5.5) in, 4 ℃ are incubated overnight.Then with reaction solution by Sephadex G-25 gel column (using 0.1M in advance, the Tris-HCl buffer solution balance of pH=7.4), collect protein component, obtain apotransferrin Apo-Tf.To add 1 in the above-mentioned Apo-Tf solution: rare earth trivalent ion (La, Ce, Pr, Nd, Sm, Eu, the Gd of 2-5, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y), reaction in room temperature 0.5-1 hour afterwards by the SephadexG-25 gel column, is collected protein component, promptly obtains using rare earth transferring complex compound.
With amycin and using rare earth transferring with the glutaraldehyde method coupling (Singh, et al, AnticancerRes, 1998,18:1423-1427).Method is summarized as follows: using rare earth transferring is dissolved in the HEPES buffer solution of pH6.8, makes 10mg/ml solution.The glutaraldehyde of adding 1.25%, room temperature are placed and are spent the night.Reaction solution is collected protein portion by Sephadex G-25 gel desalting column.
Press amycin: transferrins=20
-100: 1 molar ratio joins using rare earth transferring solution after the activation with amycin.Mix homogeneously was placed 24 hours for 4 ℃.
Above-mentioned product is crossed Sephadex G-25 gel desalting column (removing not link coupled amycin), collect protein portion, promptly obtain amycin-using rare earth transferring complex.Lyophilizing is standby.
Amycin-using rare earth transferring complex can be made oral (enteric) or intravenous formulations.
Embodiment 2:
Using rare earth transferring decorated nanometer Realgar medicine
Arsenic compound can effectively be treated leukemia, and nanometer-size realgar is to substitute As
2O
3The alternative medicine that is expected.But the absorption of Realgar is bad, targeting ability in blood.Can modify to increase its absorbability and targeting with using rare earth transferring, method is summarized as follows:
Prepare using rare earth transferring with preceding method.
Using rare earth transferring be dissolved in PBS (0.1mol/L, pH6.3) in, concentration 2mg/mL.Add S-acetyl mercapto succinaldehyde by 1: 5 molar ratio.Room temperature reaction 1 hour.The azanol that adds 0.05mol/L then, 30 ℃ of reactions 30 minutes (deacetylated) obtain the activatory using rare earth transferring of sulfydryl.Be diluted to the 0.5mg/ml adapted.
In the 1g Realgar: the ratio of 100ml water adds Realgar powder in the ball mill, and rotating speed 500r/min ground 6 hours.Then with the Realgar suspension ultra-sonic dispersion that grinds 1 minute, min, the centrifugal bulky grain composition of removing of 4000rpm is crossed 0.2 micro-filtrate membrane filtration and is obtained the Realgar nano-particle.
Nanometer-size realgar and horse activation using rare earth transferring equal-volume are mixed room temperature incubation 0.5
-1 hour.The centrifugal precipitation that obtains of 15000rpm, and, obtain using rare earth transferring decorated nanometer Realgar medicine with a small amount of buffer solution washing.
Using rare earth transferring decorated nanometer Realgar medicine can suspend again, further makes oral drugs or intravenous injection.
Embodiment 3:
Using rare earth transferring is modified and is carried cisplatin albumin nanometer microsphere
Cisplatin is one of at present main cancer therapy drug.Cisplatin has bigger toxicity and can't orally utilize simultaneously.Can increase its targeting type and improve oral utilization rate as carrier with using rare earth transferring, utilize the Nano microsphere encapsulation can improve its sustained release efficient.Method is summarized as follows:
With the low biotinylated using rare earth transferring of ratio of preceding method preparation.Dilution is 0.5mg/ml solution.
Human serum albumin (200g/L) adds the active ester BNHS of biotin succinimide (20mg/ml, DMSO solution) by 1: 2 mol ratio, and room temperature reaction 1 hour obtains the biotinylation human serum albumin.
Carry cisplatin albumin nanometer microsphere [Zhang Ming etc. with the preparation of emulsifying polycondensation method, Acta Pharmaceutica Sinica, 1994,29:380], be summarized as follows: cisplatin is dissolved in human serum albumin (200g/L, contain 10% biotinylation human serum albumin, with the pH9.8 carbonate buffer solution is solvent) solution in, the organic facies that contains 5% polysorbate trioleate (chloroform: cyclohexane extraction=1: 4) mix with 5 times of volumes, with supersound process 10 minutes, make w/o type emulsion, add isopyknic o-phthaloyl chloride (25g/L is dissolved in identical organic solvent), carried out polycondensation reaction 5 minutes continuous the stirring.
Centrifugal 5000-10000rpm obtains medicine carrying microballoons, washes several times with petroleum ether, and vacuum drying is removed residual petroleum ether.
With the molar ratio adding affinity cellulose solution of biotinylated using rare earth transferring by 1: 1, room temperature was placed 1 hour.The above-mentioned medicine carrying microballoons of making is immersed this solution, and excess liq is removed in centrifugal filtration, and room temperature was placed 1 hour.Repeat this and immerse processing 2 times.Promptly obtain using rare earth transferring and modify the microsphere medicine bag, standby after the lyophilizing.
Using rare earth transferring is modified cisplatin microsphere medicine bag can oral drugs or intravenous injection.
Embodiment 4:
Using rare earth transferring is modified and is carried As
2O
3Liposome
As
2O
3Injection successfully is used for treating leukemia.But toxicity is bigger, and the treatment window is little.Can increase its targeting type with the using rare earth transferring carrier, reduce drug toxicity.Method is summarized as follows:
Prepare using rare earth transferring with preceding method.
Get dipalmitoyl phosphatidyl choline, two palmityl PEG and cholesterol and be dissolved in chloroform and the isopropyl alcohol mixed liquor, will contain As by 1: 0.1: 1 mol ratio
2O
3Buffer solution add in the above-mentioned mixed liquor, in Ultrasound Instrument, carry out supersound process 5 minutes (45 ℃), reduce pressure down in 45 ℃ then and remove organic solvent, obtain a kind of thick liquid, add an amount of buffer solution, continue evaporation again and removed micro-organic solvent in 15 minutes, place after 30 minutes, the gained liposome turbid liquor by Sephadex G-25 gel column, is separated and removes the As that does not wrap into
2O
3Collect and carry As
2O
3PEG liposome part.
Liposome solutions adds the sodium periodate of 0.03mol/L, and room temperature was placed 30 minutes.The PEG aqueous solution that adds 0.10mol/L then, room temperature was placed 30 minutes.
Add 0.1mg/ml using rare earth transferring solution, 4 ℃ incubation 2-6 hour.
Centrifugal 5000-10000rpm obtains the drug-loaded liposome that using rare earth transferring is modified.
Using rare earth transferring is modified and is carried As
2O
3Liposome can further be made oral drugs or intravenous injection.
Embodiment 5:
Neurologic agent: using rare earth transferring is modified neurotrophic factor (BDNF) the PEG liposome of brain source property.
For neurologic agent, blood brain barrier must can be striden across.Neurotrophic factor (BDNF) can be used as neuroprotective, treats a series of neurological sexual disorder.But, can not pass through blood brain barrier as pharmaceutical grade protein.Can address this problem with the using rare earth transferring carrier, and can realize oralization of medicine.Method is summarized as follows:
Prepare using rare earth transferring with preceding method.
Getting dipalmitoyl phosphatidyl choline, two palmityl PEG and cholesterol is dissolved in chloroform and the isopropyl alcohol mixed liquor by 1: 0.1: 1 mol ratio, the buffer solution that will contain neurotrophic factor (BDNF) adds in the above-mentioned mixed liquor, in Ultrasound Instrument, carry out supersound process 5 minutes (45 ℃), reduce pressure down in 45 ℃ then and remove organic solvent, obtain a kind of thick liquid, add an amount of buffer solution, continue evaporation again and removed micro-organic solvent in 15 minutes, place after 30 minutes, the gained liposome turbid liquor by Sephadex G-25 gel column, is separated and removes the BDNF that does not wrap into.Collect the PEG liposome part of carrying BDNF.
Liposome solutions adds the sodium periodate of 0.03mol/L, and room temperature was placed 30 minutes.The PEG aqueous solution that adds 0.10mol/L then, room temperature was placed 30 minutes.
Add 0.1mg/ml using rare earth transferring solution, 4 ℃ incubation 2-6 hour.
Centrifugal 5000-10000rpm obtains the drug-loaded liposome that using rare earth transferring is modified.
Using rare earth transferring is modified a year BDNF liposome can further make oral drugs or intravenous injection.
Embodiment 6:
Using rare earth transferring is modified insulin PEG liposome
The non-injection administration insulin is the emphasis problem that people study always.And the ability that can improve oral insulin and absorb through pulmonary by the using rare earth transferring carrier.Method is summarized as follows:
Prepare using rare earth transferring with preceding method.
Getting dipalmitoyl phosphatidyl choline, two palmityl PEG and cholesterol is dissolved in chloroform and the isopropyl alcohol mixed liquor by 1: 0.1: 1 mol ratio, the buffer solution that will contain insulin adds in the above-mentioned mixed liquor, in Ultrasound Instrument, carry out supersound process 5 minutes (45 ℃), reduce pressure down in 45 ℃ then and remove organic solvent, obtain a kind of thick liquid, add an amount of buffer solution, continue evaporation again and removed micro-organic solvent in 15 minutes, place after 30 minutes, the gained liposome turbid liquor by Sephadex G-25 gel column, is separated and removes the insulin that does not wrap into.Collect and carry insulin PEG liposome part.
Liposome solutions adds the sodium periodate of 0.03mol/L, and room temperature was placed 30 minutes.The PEG aqueous solution that adds 0.10mol/L then, room temperature was placed 30 minutes.
Add 0.1mg/ml using rare earth transferring solution, 4 ℃ incubation 2-6 hour.
Centrifugal 5000-10000rpm obtains the drug-loaded liposome that using rare earth transferring is modified.
Using rare earth transferring is modified a year insulin liposome can further be used for making oral drugs or respiratory tract spray delivery dosage form.
Embodiment 7:
Using rare earth transferring-cyclodextrin inclusion compound taxol drug
Paclitaxel is the cancer therapy drug of using always, but dissolubility is low and exist the medicine through Pgp to efflux mechanism in digestive tract, is difficult for doing oral.And can improve the ability of dissolubility and oral absorption by using rare earth transferring-cyclodextrin carrier.Method is summarized as follows:
Prepare the biotinylation using rare earth transferring as stated above
The 2g biotin is dissolved in chloroacetic chloride, room temperature reaction 2 hours, distilling under reduced pressure obtains the biotin acyl chlorides
With cyclodextrin and biotin acyl chlorides by 1: 2-5 is dissolved in the pyridine solution, 0 ℃ of reaction 2 hours, room temperature was placed 2 hours, distilling under reduced pressure obtains crude product.Crude product is used DMF: H on Sephadex LH-20 post
2O=1: 1 eluting is purified, and after concentrating after cyclodextrin is partly regained, pours in the acetone soln and precipitates.Obtain the biotinylation cyclodextrin.
Paclitaxel is dissolved in the ethanol of heating (75 ℃), makes saturated solution.Keep temperature to the b-cyclodextrin saturated aqueous solution (31.8g/L) that wherein drips 200 times of volumes, stirs and stopped heating in 30 minutes, continue the stirring 5 hours static placement of room temperature 12 hours then.Filter, precipitate 60 ℃ of dryings, obtain the cyclodextrin inclusion compound taxol drug.
With the biotinylation using rare earth transferring: the affinity element: biotinylation medicine carrying cyclodextrin is by=1: 1: the mixed of 1-3,4 ℃ incubation 2-6 hour, promptly obtain using rare earth transferring-cyclodextrin inclusion compound drug molecule BAB conjugate.The product lyophilizing is standby.
Using rare earth transferring-cyclodextrin inclusion compound drug conjugates can further be made oral cancer therapy drug.
Using rare earth transferring (TbTf) competition suppresses the absorption of K562 cell to transferrins Tf.503nhibiting concentration is TbTf/Tf=0.16, and the adhesion that TbTf and cell surface TfR TfR are described is about 6 times of Tf, and is 6 times of Tf as the efficient of pharmaceutical carrier with TbTf.TbTf-FITC is by the absorption of K562 cell.Left figure is the fluorescence microscope photo of the common incubation of TbTf-FITC and K562 cell after 40 minutes, TbTf-FITC green fluorescence in the outer and cell of visible cell.Right figure is the process that variable concentrations TbTf-FITC is absorbed by cell.The using rare earth transferring amount of fluorescence intensity representative picked-up in the cell.Illustrate that using rare earth transferring can carry covalently bound molecule and initiatively be absorbed by cell.
Using rare earth transferring fluorescent composition TbTf-FITC is subjected to the influence of transferrins Tf concentration by external blood brain barrier.When Tf/TbTf=1, only reduce blood brain barrier to about 20% of the turn-over capacity of TbTf-FITC.External blood brain barrier is mouse brain capillary endothelial cell/star spongiocyte contact model, prepares on 12 hole Transwell.
Transferrins fluorescent composition Tf-FITC is subjected to the influence of using rare earth transferring TbTf concentration by external blood brain barrier.When TbTf/Tf=1, Tf-FITC reduces about 82% in the turn-over capacity of blood brain barrier.External blood brain barrier is mouse brain capillary endothelial cell/star spongiocyte contact model, prepares on 12 hole Transwell.
Claims (4)
1. a medicine comprises active ingredient and pharmaceutical carrier, it is characterized in that, described pharmaceutical carrier is a using rare earth transferring complex compound.
2. medicine according to claim 1 is characterized in that described rare earth is meant La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, the combination of one or more among the Y.
3. medicine according to claim 1, it is characterized in that the combination of active ingredient and pharmaceutical carrier is selected from using rare earth transferring complex compound and active ingredient and modifies drug-loaded liposome method, cyclodextrin inclusion compound method and using rare earth transferring by chemical bond coupling method, using rare earth transferring complex compound and active ingredient by biotin/antibiotin method coupling method, using rare earth transferring and modify medicament-carried nano granule method in the described medicine.
4. according to any described medicine of claim 1-3, it is characterized in that the method for preparing described using rare earth transferring complex compound comprises the steps:
(1) transferrins is dissolved in contains 1-10mM EDTA and 10-50mM Vc, in the 0.1M hac buffer of pH=5.5, the preparation apotransferrin;
(2) will add 1 in the above-mentioned apotransferrin: the rare earth trivalent ion of 1-5, preparation using rare earth transferring complex compound.
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