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CN1709517A - Magnetic resonance tumour target contrast media and preparing method thereof - Google Patents

Magnetic resonance tumour target contrast media and preparing method thereof Download PDF

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Publication number
CN1709517A
CN1709517A CN 200510026505 CN200510026505A CN1709517A CN 1709517 A CN1709517 A CN 1709517A CN 200510026505 CN200510026505 CN 200510026505 CN 200510026505 A CN200510026505 A CN 200510026505A CN 1709517 A CN1709517 A CN 1709517A
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dtpa
poly
lysine
folic acid
magnetic resonance
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CN1317037C (en
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刘士远
袁正
钟高仁
姜庆军
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Second Military Medical University SMMU
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Second Military Medical University SMMU
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Abstract

The present invention relates to a contrast medium capable of specifically making tumor be developed in magnetic resonance image formation and its preparation method. Said invention uses DTPA-Poly-L-Lysine as carrier of magnetic resonance paramagnetic gadolinium contrast medium, it is coupled with folic acid to form macromolecular polymer, and its molecular weight is 30000-70000. Said macromolecular polymer has the characteristics of high carrier Gd3+ concentration, stable chemical property, high purity and high affinity with folic acid receptor, etc.

Description

A kind of magnetic resonance tumor targeted contrast medium and preparation method thereof
Technical field
The present invention relates to medical detectable technical field, relate in particular to and a kind ofly can specificity in nuclear magnetic resonance make contrast medium of tumor imaging and preparation method thereof.
Background technology
Clinical magnetic resonance imaging (Magnetic Resonance Imag-ing, MRI) use common contrast medium in and dynamically strengthen the accuracy that can improve pulmonary cancer diagnosis, but it is based on vessel density in tuberosity or the lump, reflection be the blood of pathological changes for characteristics, be not distinctive; Monoclonal antibody type target contrast agent is applied to the people and knows from experience the reaction of generation human antimouse antibody, adds that the mesenchyma stroma of tumors internal pressure raises, and the reason of link coupled macromole permeability difference makes that the targeting effect of monoclonal antibody type contrast medium is also desired not as people.Unger etc. utilize anti-CEA antibody to become anti-CEA antibody-Gd-DTPA to grip the MR imaging research that chemical compound is used for adenocarcinoma of colon altogether with two ethylenediamines, five gadolinium acetates (Gd-DTPA) coupling, found that to exist the antibody consumption big, shortcomings such as reinforced effects difference, and immunoreation takes place in human body, produce an anti-antibody and limited repeated use (list of references: E.C.Unger, W.G.Totty, D.M.Neufeld et al., Magneticresonance imaging using gadolinium labeled monoclonal antibody.Invest.Radiol.20 (1985), 693-700).
Summary of the invention
The objective of the invention is to utilize the characteristic of folic acid and folacin receptor high-affinity to realize targeted developing, a species specificity, selectivity, reinforced effects is good, toxic and side effects is low magnetic resonance tumor targeted contrast medium and preparation method thereof are provided.
The invention provides a kind of magnetic resonance tumor targeted contrast medium, it is to be the carrier of magnetic resonance paramagnetism gadolinium contrast medium with Diethylenetriamine five sour polylysine DTPA-Poly-L-Lysine, becomes the macromole polymer with folacin coupled, and molecular weight is 30,000~70,000 dalton.
A kind of magnetic resonance tumor targeted contrast medium, it is folic acid and polylysine, Magnevist Solution (Gadolinium diethylenetriamine pentacetic acid, union body GD-DTPA), i.e. folic acid-PL-Gd-DTPA.
The present invention also provides the preparation method of above-mentioned magnetic resonance tumor targeted contrast medium, may further comprise the steps:
1. DTPA bisgallic acid acid anhydride (CaDTPA) is synthetic
10g Diethylenetriamine five acid (DTPA) are suspended in the mixed liquor of 30ml acetic anhydride and 15ml pyridine, and in 60 ℃ of stirring reaction 6h, add an amount of ethanol after the cooling and filter, the ether washing, drying gets white solid DTPA bisgallic acid acid anhydride (CaDTPA) crude product.
2. DTPA-Poly-L-Lysine preparation
Get polylysine (Poly-L-Lysine) 10mg, be dissolved in 3ml 0.2mol/LNaH 2CO 3/ Na 2HCO 3In the buffer (pH=9.6), under the ice bath stirring, add DTPA bisgallic acid acid anhydride (CaDTPA) solution, the restir reaction is after 16 hours under the room temperature, take out reaction mixture, remove free CaDTPA with equilibrium dialysis, and be transformed into 0.2M NaAc/HAc (acetate/acetic) buffer (pH=5).Take out solution in the bag filter, put 4 ℃ of preservations behind the concentrating under reduced pressure, be used for the next step.
3. Gd-DTPA-Poly-L-Lysine preparation
Gd 2O 3Join among the 0.1mol/L HCl, be heated to 60 ℃, sustained response 10min, after the dissolving, the HCl of evaporating surplus promptly obtains GdCl fully 3Solution.GdCl with preparation 3Solution joins through in the DTPA-Poly-L-Lysine solution of concentrating under reduced pressure, and restir reaction was at room temperature removed free Gd with equilibrium dialysis after 24~30 hours then 3+Make Gd-DTPA-Poly-L-Lysine.Adopt the high-frequency inductor Rhizoma Nelumbinis to close plasma emission spectroscopy method (ICP-AES) measuring samples Gd 3+Content.
4. the coupling of folic acid and Gd-DTPA-Poly-L-Lysine
Folic acid is dissolved in 0.1mol/L NaH 2CO 3/ Na 2HCO 3Buffer (pH=9.6), add ECD (ethyl cysteinate dimer) and Gd-DTPA-Poly-L-Lysine then, restir reaction at room temperature is after 24~30 hours, remove the folic acid that on not connecting with Sephadex CL-4B chromatographic column, collect the union body of folic acid and Gd-DTPA-Poly-L-Lysine, freeze concentration promptly gets folic acid-PL-Gd-DTPA.
Folic acid-PL-Gd-DTPA vitro characterization:
1) Gd 3+Assay: adopt the high-frequency inductor Rhizoma Nelumbinis to close plasma emission spectroscopy method (ICP-AES) and measure.The Gd that is used for zooperal per molecule folic acid and Gd-DTPA-Poly-L-Lysine union body 3+Content is 56.
2) macromolecular purity testing: use TSK3000 (Bio-Rad) molecular sieve gel analytical column, use 0.01mol/l, the high pressure liquid chromatography (HPLC) of the PB liquid eluting of pH=7.4 is analyzed, and a big peak (>95%) occurs, less molecular weight peaks<2%.Cellulose acetate membrane electrophoresis and the analysis of molecular sieve column chromatography Sepharose CL-4B column chromatography are not found a large amount of than small molecular weight material yet, see Fig. 1.
3) determination of activity: use cell folacin receptor competition combined techniques proof folic acid (folate)-PL-Gd-DTPA activity, the result obtains and the similar competition curve of folic acid, illustrate that target contrast agent folate-PL-Gd-DTPA has kept folic acid and the bonded characteristic of folacin receptor high-affinity (KD~10-10M), see Fig. 2.
Folic acid-PL-Gd-DTPA tumor animal experiment:
1) preparation of animal model for tumour
Under aseptic condition, be cut into the tumor piece of about 2mm size in the subcutaneous human lung adenocarcinoma tumor body (H460 human lung carcinoma cell line) of nude mice with going down to posterity, transplant in nude mice (BALB/C mice, 6-7 week age) inclined to one side dorsal part infrascapular region, right side subcutaneously, about 30 days posterior tuberosities directly reach 1.0-1.5cm and are used for the thing imaging experiment;
2) laboratory animal MR imaging
Lotus people pulmonary carcinoma nude mice model (n=3) is used for the MRI experiment.Test the previous day, stop the rodent normal diet, do not have the folic acid diet.During experiment, with 1.5T superconduction High-Field MR scanner, row is unenhanced earlier.Finish unenhanced after, nude mice is fixed in the injection device, inject the target contrast agent folic acid-PL-Gd-DTPA (0.2mmol/Kg body weight) of preparation through caudal vein with the 1.0ml syringe; After injecting successfully, once more nude mice is fixed on the fixing head each time point scanning of row.The injection reagent after sweep time point selection: 30 ', 3h, 6h, 14h, 24h, 48h, 72h.
3) MR signal value measuring method
Get 5 circular region of interest on the identical aspect tumor body maximum diameter, area is 2mm, surveys its ROI value, calculates its meansigma methods then as far as possible.Measure liver, leg muscle ROI value simultaneously, method is the same.
4) date processing
Adopt SAS 6.12 version statistical softwares, data handled with the variance analysis method of randomized block design data, carry out in twos with the SNK-q check afterwards between comparison.
Found that tumor body signal value is obvious strengthening effect behind injection folic acid-PL-Gd-DTPA, and along with the prolongation of observing time, the local accumulative trend of the oriented tumor body of reagent folic acid-PL-Gd-DTPA, peak value appears at and strengthens back 48 hours approximately, maximum reinforcement rate 126.0%; And in liver, also become obvious strengthening effect, peak value appears at and strengthened approximately back 6 hours, afterwards downward trend, see Fig. 3 to Fig. 6, table 1.
Table 1 nude mice is respectively organized peak value reinforcement rate
The ROI value Peak value reinforcement rate
Before the injection Injection back (peak value)
The tumor body ??192.5±15.2 ??521.4±61.9 ??170.8%
Liver ??253.0±23.8???? ??476.2±44.2 ???? ??88.2% ????
Muscle ??185.3±19.0?? ??262.8±38.8?? ??41.8%??
The present invention utilizes the characteristic of folic acid and folacin receptor high-affinity to realize targeted developing, is the ligand-receptor guidance system, and it is in conjunction with characteristics such as have specificity, selectivity, saturability, affinity is strong and biological effect is obvious.Utilize the carrier of part,, increase medicine, reduce toxic and side effects, reach target tumor development purpose in the partial concentration of focus, raising curative effect by receptor-mediated effect for Gd-DTPA.
Description of drawings
Fig. 1 is high pressure liquid chromatography figure behind the reagent purification: a big peak (>95%) occurs, less molecular weight peaks<2%
Fig. 2 is to use cell folacin receptor competition combined techniques proof folate-PL-Gd-DTPA activity, and the result obtains and the similar competition curve of folic acid
Fig. 3 is the cross-section bit image of unenhanced time point nude mice
Fig. 4 is the cross-section bit image of 30 ' time point nude mice
Fig. 5 is the cross-section bit image of 48h time point nude mice
Fig. 6 is the cross-section bit image of 72h time point nude mice
The specific embodiment
Embodiment 1: a kind of magnetic resonance tumor targeted contrast medium, and it is the union body of folic acid and polylysine, Magnevist Solution GD-DTPA, i.e. folic acid-PL-Gd-DTPA, molecular weight is 30,000~70,000 dalton.
Embodiment 2: a kind of method for preparing embodiment 1 described magnetic resonance tumor targeted contrast medium, and it may further comprise the steps:
1. DTPA bisgallic acid acid anhydride (CaDTPA) is synthetic
10g Diethylenetriamine five acid (DTPA) are suspended in the mixed liquor of 30ml acetic anhydride and 15ml pyridine, and in 60 ℃ of stirring reaction 6h, add an amount of ethanol after the cooling and filter, the ether washing, drying gets white solid DTPA bisgallic acid acid anhydride (CaDTPA) crude product;
2. DTPA-Poly-L-Lysine preparation
Get polylysine (Poly-L-Lysine) 10mg, be dissolved in 3ml 0.2mol/LNaH 2CO 3/ Na 2HCO 3In the buffer (pH=9.6), under the ice bath stirring, add DTPA bisgallic acid acid anhydride (CaDTPA) solution, the restir reaction is after 16 hours under the room temperature, take out reaction mixture, remove free CaDTPA with equilibrium dialysis, and be transformed into 0.2M NaAc/HAc (acetate/acetic) buffer (pH=5).Take out solution in the bag filter, put 4 ℃ of preservations behind the concentrating under reduced pressure, be used for the next step;
3. Gd-DTPA-Poly-L-Lysine preparation
Gd 2O 3Join among the 0.1mol/L HCl, be heated to 60 ℃, sustained response 10min, after the dissolving, the HCl of evaporating surplus promptly obtains GdCl fully 3Solution.GdCl with preparation 3Solution joins through in the DTPA-Poly-L-Lysine solution of concentrating under reduced pressure, and restir reaction was at room temperature removed free Gd with equilibrium dialysis after 24~30 hours then 3+Make Gd-DTPA-Poly-L-Lysine.Adopt the high-frequency inductor Rhizoma Nelumbinis to close plasma emission spectroscopy method (ICP-AES) measuring samples Gd 3+Content;
4. the coupling of folic acid and Gd-DTPA-Poly-L-Lysine
Folic acid is dissolved in 0.1mol/L NaH 2CO 3/ Na 2HCO 3Buffer (pH=9.6), add ECD (ethyl cysteinate dimer) and Gd-DTPA-Poly-L-Lysine then, restir reaction at room temperature is after 24~30 hours, remove the folic acid that on not connecting with Sephadex CL-4B chromatographic column, collect the union body of folic acid and Gd-DTPA-Poly-L-Lysine, freeze concentration promptly gets folic acid-PL-Gd-DTPA.

Claims (3)

1, a kind of magnetic resonance tumor targeted contrast medium, it is characterized in that it is is the carrier of magnetic resonance paramagnetism gadolinium contrast medium with Diethylenetriamine five sour polylysine DTPA-Poly-L-Lysine, become the macromole polymer with folacin coupled, molecular weight is 30,000~70,000 dalton.
2, a kind of magnetic resonance tumor targeted contrast medium according to claim 1, it is the union body of folic acid and polylysine, Magnevist Solution GD-DTPA, i.e. folic acid-PL-Gd-DTPA.
3, a kind of method for preparing claim 1 or 2 described magnetic resonance tumor targeted contrast medium is characterized in that it may further comprise the steps:
1. DTPA bisgallic acid acid anhydride CaDTPA's is synthetic
10g Diethylenetriamine five sour DTPA are suspended in the mixed liquor of 30ml acetic anhydride and 15ml pyridine, in 60 ℃ of stirring reactions 6 hours, add an amount of ethanol after the cooling and filter, the ether washing, drying, white solid DTPA bisgallic acid acid anhydride CaDTPA crude product;
2. DTPA-Poly-L-Lysine preparation
Get polylysine Poly-L-Lysine10mg, be dissolved in the 3ml 0.2mol/LNaH of pH=9.6 2CO 3/ Na 2HCO 3In the buffer, under the ice bath stirring, add CaDTPA solution, the restir reaction is after 16 hours under the room temperature, take out reaction mixture, remove free CaDTPA with equilibrium dialysis, and be transformed into 0.2M NaAc/HAc buffer, pH=5, take out solution in the bag filter, put 4 ℃ of preservations behind the concentrating under reduced pressure;
3. Gd-DTPA-Poly-L-Lysine preparation
Gd 2O 3Join among the 0.1mol/L HCl, be heated to 60 ℃, sustained response 10min, after the dissolving, the HCl of evaporating surplus promptly obtains GdCl fully 3Solution, with the preparation GdCl 3Solution joins through in the DTPA-Poly-L-Lysine solution of concentrating under reduced pressure, and restir reaction was at room temperature removed free Gd with equilibrium dialysis after 30 hours then 3+, make Gd-DTPA-Poly-L-Lysine, adopt the high-frequency inductor Rhizoma Nelumbinis to close plasma emission spectroscopy method measuring samples Gd 3+Content;
4. the coupling of folic acid and Gd-DTPA-Poly-L-Lysine
Folic acid is dissolved in the 0.1mol/L NaH of pH=9.6 2CO 3/ Na 2HCO 3Buffer, add ethyl cysteinate dimer ECD and Gd-DTPA-Poly-L-Lysine then, restir reaction at room temperature is after 30 hours, remove the folic acid that on not connecting with Sephadex CL-4B chromatographic column, collect the union body of folic acid and Gd-DTPA-Poly-L-Lysine, freeze concentration promptly gets folic acid-PL-Gd-DTPA.
CNB2005100265056A 2005-06-06 2005-06-06 Magnetic resonance tumour target contrast media and preparing method thereof Expired - Fee Related CN1317037C (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101757642A (en) * 2010-03-03 2010-06-30 天津科技大学 Method for preparing gadolinium-containing nano particles
CN101564540B (en) * 2008-04-22 2011-05-11 上海交通大学医学院附属瑞金医院 Preparation method of Gd-DTPA-Polylysine-McAb linker
CN102803270A (en) * 2009-05-07 2012-11-28 加图立大学校产学协力团 Gadolinium complex, method for preparing same, and MRI contrast agent comprising same
CN102989015A (en) * 2012-12-10 2013-03-27 西安交通大学 Preparation method of CT imaging molecular probe of folate receptor targeted tumor
CN107349434A (en) * 2016-05-09 2017-11-17 中国科学院苏州纳米技术与纳米仿生研究所 A kind of dissaving polymer and its preparation method and application
CN108430520A (en) * 2015-11-06 2018-08-21 威斯康星校友研究基金会 Long-acting gadolinium base cancer target imaging and therapeutic agent
CN113514588A (en) * 2021-06-02 2021-10-19 原子高科股份有限公司 High performance liquid chromatography analysis method of relevant substances of cysteamine for injection

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101564540B (en) * 2008-04-22 2011-05-11 上海交通大学医学院附属瑞金医院 Preparation method of Gd-DTPA-Polylysine-McAb linker
CN102803270A (en) * 2009-05-07 2012-11-28 加图立大学校产学协力团 Gadolinium complex, method for preparing same, and MRI contrast agent comprising same
CN101757642A (en) * 2010-03-03 2010-06-30 天津科技大学 Method for preparing gadolinium-containing nano particles
CN102989015A (en) * 2012-12-10 2013-03-27 西安交通大学 Preparation method of CT imaging molecular probe of folate receptor targeted tumor
CN102989015B (en) * 2012-12-10 2014-12-10 西安交通大学 Preparation method of CT imaging molecular probe of folate receptor targeted tumor
CN108430520A (en) * 2015-11-06 2018-08-21 威斯康星校友研究基金会 Long-acting gadolinium base cancer target imaging and therapeutic agent
US11623007B2 (en) 2015-11-06 2023-04-11 Wisconsin Alumni Research Foundation Long-lived gadolinium based tumor targeted imaging and therapy agents
CN107349434A (en) * 2016-05-09 2017-11-17 中国科学院苏州纳米技术与纳米仿生研究所 A kind of dissaving polymer and its preparation method and application
CN113514588A (en) * 2021-06-02 2021-10-19 原子高科股份有限公司 High performance liquid chromatography analysis method of relevant substances of cysteamine for injection

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