CN101838345A - Fluorine-18-labelled galactosylated chitosan compound and preparation method thereof - Google Patents
Fluorine-18-labelled galactosylated chitosan compound and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a fluorine-18-labelled galactosylated chitosan compound and a preparation method thereof. The chemical expression of the compound is [18F] FB-GC. The radioactive fluorine-18-labelled galactosylated chitosan compound is prepared by reacting radioactive N- succinimide 4-[18F] fluorine benzoate ([18F] SFB) with galactosylated chitosan under an alkaline condition. The compound has the advantages of good chemical stability and biological performance, high chemical purity, simple preparation and low using cost and is applied to the technical fields of radiopharmaceutical chemistry and clinical nuclear medicine as a novel liver receptor PET developer.
Description
Affiliated technical field
The present invention relates to the radiopharmaceutical chemistry and the clinical nuclear medicine technical field of fluoro-18 marks, particularly relate to galactosylated chitosan compound of a kind of fluoro-18 marks and preparation method thereof.
Background technology
ASGP acceptor (Asialoglycoprotein receptor, ASGP-R) be writing a Chinese character in simplified form of asialoglycoprotein sugar albumin acceptor, being present on the hepatocyte of mammal film, is asialoglycoprotein glycoprotein (Asialoglycoprotein, single-minded site ASGP) in the mediation hepatic clearance blood.Nearly all plasma proteins all is a glycoprotein except albumin, and when being synthesized by liver and being discharged into blood, its sugar-chain end is a sialic acid.Sialic acid forms more weak being connected with other glycosyl, and is also unstable in blood, very easy being eliminated.Sialic acid partly is eliminated, and indicates the end in this glycoprotein life-span.The semi-lactosi structure all is positioned on terminal second in most of plasma proteins, therefore after terminal sugar is by enzymolysis, just can be used as the effect substrate of ASGP acceptor with the glycoprotein of semi-lactosi ending.Asialoglycoprotein glycoprotein is in case in cell surface and ASGP receptors bind, the rapid internalization of formed ligand-receptor mixture, enter about 5 minutes behind the prelysosome vesica owing to the effect of pH is dissociated, acceptor is recycled to plasma membrane, most of part is through the effect of lysosomal enzyme and the metabolism that is decomposed, the small portion part is then walked around lysosomal degraded and is entered bile, or passes cytolemma and return cell surface and still combine with acceptor.This process can be kept the running balance of plasma glycoprotein.The ASGP acceptor can reflect effective hepatocyte function to a certain extent, and when liver injury diseases such as hepatitis, liver cirrhosis or liver cancer took place, its quantity and activity all suffered damage.Hepatitis disease worldwide all is a kind of disease occurred frequently, and particularly liver cirrhosis secondary liver cancer patient is numerous, has a strong impact on human health and quality of life, and its importance has caused that now extensive concern appears suddenly.Vera in 1984 etc. by chemical synthesis process with semi-lactosi structure and human serum albumin coupling obtain new lactose albumin (galactosyl-neoglycoalbumin, NGA), and warp
99mCarry out liver imaging research behind the Tc mark.Kubota in 1986 etc. have developed a kind of
99mTc mark GSA (
99mTc-diethylenetriamine pentaaceticacid-galactosyl-human serum albumin),, simplified flag condition, reduced non-specific binding by increasing bifunctional linking reagent DTPA (diethylene triamine pentacetic acid (DTPA)) structure.Japan has utilized the ASGPR developer to set up three-dimensional hepatic model for nuclear medicine liver SPECT video picture, this imaging technique can truly, digitizing reflect liver function, so both can carry out correct assessment to liver function before the liver cirrhosis patient art, aid forecasting operation risk, formulation treatment plan, the peri-operation period mortality ratio of reduction operation on liver.
Positron emission computerized tomograph (PET) is the field of nuclear medicine advanced person's a clinical examination image technology.Compare with SPECT and to have higher resolving power and sensitivity, particularly PET and CT combines, improved localized accuracy.Vera in 1985 etc. have reported usefulness
68The NGA of Ga mark can be used for the PET video picture, its bio distribution in mouse with
99mTc-GSA is similar.Fluoro-18 is the most frequently used PET nucleic of nuclear medicine, and by accelerator production, it has good physicals and suitable transformation period (110min).The ASGP receptor developer of positron mark particularly has important meaning for setting up three-dimensional liver digital model for the liver video picture.Further investigation exploitation at present is used for the PET developer of liver rii receptor, and particularly the ASGP receptor developer of fluoro-18 marks is important topics that the present technique field need solve.
Summary of the invention
The purpose of this invention is to provide a kind ofly have that chemical stability is strong, biological property good, chemical purity is high, preparation is simple and use cost is low, is applied in the galactosylated chitosan compound of fluoro-18 marks in liver receptor positron emission computerized tomograph field.
Another object of the present invention provides the preparation method of the galactosylated chitosan compound of described fluoro-18 marks.
In order to achieve the above object, the present invention by the following technical solutions: a kind of galactosylated chitosan compound of fluoro-18 marks, by parafluorobenzoic acid ([
18F] FB) be connected the galactosylated chitosan compound that obtains fluoro-18 marks with the amino of galactose chitosan molecular chain, its developed by molecule formula be [
18F] FB-GC, its general structure is:
The preparation method of the galactose chitosan of fluoro-18 marks is as follows:
With radioactivity N-succinimide 4-[
18F] the fluorobenzoic acid ester ([
18F] SFB) obtain the galactosylated chitosan compound of described radioactive fluorine-18 mark with galactose chitosan (GC) prepared in reaction under alkaline condition, its preparation process is:
A. at K
2CO
3, K
2.2.2.[
18F] F
-After 20 minutes, after the NaOH hydrolysis, add HCl and obtain 4-[in reaction under the heating condition with 4-trimethylamine groups ethyl benzoate trifluoromethyl sulfonic acid
18F] fluorobenzoic acid;
B.4-[
18F] fluorobenzoic acid and O-(N-succinimide)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester reacted 5 minutes under heating condition, and with obtaining N-succinimide 4-[behind the acidifying with acetic acid
18F] the fluorobenzoic acid ester ([
18F] SFB);
C. galactose chitosan GC is dissolved in the N-succinimide 4-[that adds step b preparation in the damping fluid
18F] in the fluorobenzoic acid ester, under 10 ℃～40 ℃ conditions, react 10～60min behind the mixing;
D. step c gained solution is carried out purifying by gel chromatography.
Temperature of reaction described in the above-mentioned steps a is 90～140 ℃.
Temperature of reaction described in the above-mentioned steps b is 80～100 ℃.
Damping fluid described in the above-mentioned steps c is: 0.1mol/L pH8.6 borate buffer, consumption are 0.1mL～1mL;
It is the gel column (HiTrap Desalting gel column) of sephadex G 25 (Sephadex G25) that gel chromatographic columns described in the above-mentioned steps d is selected filler for use;
The consumption of galactose chitosan described in the above-mentioned preparation method is 1mg～5mg.
The present invention is with radioactivity N-succinimide 4-[
18F] the fluorobenzoic acid ester ([
18F] SFB) with galactose chitosan GC prepared in reaction under alkaline condition obtain described radioactive fluorine-18 mark galactosylated chitosan compound ([
18F] FB-GC), be a kind of novel liver cell asialoglycoprotein receptor (ASGPR) developer, introduce radioactive fluoro-18 on the amino in the galactose chitosan molecule, can be used for positron emission computerized tomograph (PET).[
18F] FB-GC chemical stability and biological property is good, chemical purity is high.With [
18F] FB-NGA compares, and it has better liver to be detained and higher spatial resolution, and can be used to prepare becomes novel liver receptor developer.
Detection shows: [
18F] FB-GC identifies by thin-layer chromatography and HPLC, its radiochemical purity is greater than 99%, and at room temperature places radiochemicsl purity no change after 4 hours, and stability is preferably arranged.Putting productive rate behind the decay correction is 4～8%.
Experiment shows, [
18F] the basic performance of FB-GC is as follows:
1.[
18F] FB-GC bio distribution in the normal mouse body
Get 15 normal Kunming small white mouses, in tail vein injection 0.1mL[
18F] FB-GC (about 0.037MBq contains 20 μ g GC).Sacrificed by decapitation behind injection back 10,60 and 120min.Take out tissues such as the heart, liver, lung, kidney, muscle, bone, blood, weighing is also surveyed its radiocounting in gamma counter.
[
18F] bio distribution of FB-GC in normal mouse see Table 1;
Table 1.[
18F] F B-GC in the normal mouse body bio distribution data (%ID/g ± sd, n=5) and with [
18F] F B-NGA (%ID/g ± sd, n=5) contrast of biological property
Bio distribution result's demonstration in normal mouse, [
18F] FB-GC has certain initial picked-up at liver, behind the injection 5min, the picked-up value of (11.13 ± 1.63) %ID/g arranged in liver.Radioactivity at internal organs such as the heart, lung, spleens concentrates lower.[
18F] FB-GC has preferably at liver and is detained, and the liver picked-up still has the picked-up value of (10.77 ± 0.95) %ID/g behind the injection 120min.With [
18F] FB-NGA compares, have better liver to be detained and faster blood remove.[
18F] FB-GC has higher picked-up in kidney, and this is that the character of water-soluble chitosan itself causes.Based on above bio distribution experimental result, add PET imaging technique self spatial resolution advantage, in the video picture in future, can obtain more distinct image.
2.[
18F] FB-GC in the mouse body, suppress the experiment
Get with batch 15 normal Kunming small white mouses, in tail vein preform injection 0.1mL inhibitor (NGA is dissolved in physiological saline, presses the dosage injection of 10mg/kg body weight), behind the 5min, in tail vein injection 0.1mL[
18F] FB-GC (about 0.037MBq contains 2 μ g NGA).Sacrificed by decapitation behind injection back 10,60, injection back and the 120min.Take out tissues such as the heart, liver, lung, kidney, muscle, bone, blood, weighing is also surveyed its radiocounting in the technetium analyser.
[
18F] FB-GC suppresses experimental result and sees Table 2 in the normal mouse body;
Table 2.[
18F] FB-GC in the normal mouse body, suppress experimental data (5min after the injection, %ID/g ± sd, n=5)
Suppress result of experiment and show, by preform injection NGA, can obvious suppression [
18F] picked-up (P＜0.001) of FB-GC in mouse liver, do not enter liver [
18F] FB-GC removes fast by kidney, and the radioactivity behind the injection 60min in blood only is the picked-up value of (0.31 ± 0.10) %ID/g.
Suppress the result of experiment explanation by preform injection NGA, can effectively suppress [
18F] picked-up of FB-GC in liver.Proof [
18F] FB-GC has affinity to the ASGP acceptor.
Above-mentioned every galactosylated chitosan compound that experiment showed, fluoro-18 marks of the present invention ([
18F] FB-GC) its radiochemical purity is greater than 99%, has good biological property, certain picked-up is arranged and have preferably to keep in liver, can satisfy condition, can be prepared as liver acceptor PET developer and be applicable clinically as liver acceptor PET developer.
Embodiment
Below by embodiment in detail the present invention is described in detail, a kind of galactosylated chitosan compound of fluoro-18 marks:
1. the preparation of galactose chitosan
0.005mmol the lactobionic acid of chitosan (molecular weight＜10000, deacetylation 80%) and 0.1mmol is dissolved in the MES damping fluid of 6mL (pH 6.0 for 2-(N-morpholino) ethanesulfonic acid, 0.1mol/L), is stirred to whole dissolvings.Add the EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) of 0.2mmol and the N-succinimide of 0.8mmol, room temperature reaction 48 hours.Gained solution was dialysed 3 days with deionized water, and lyophilize obtains lurid lyophilized powder.
2.4-[
18F] preparation of fluorobenzoic acid
Contain 6mg K at 1.5mL
2CO
3With 11mg K
2.2.2.[
18F] F
-Solution adds in the 10mL reaction flask, immerses in 120 ℃ of oil baths, feeds nitrogen (1mL/min) and dries up, and adding 0.5mL anhydrous acetonitrile is evaporated to dried, triplicate.Add the 4mg 4-trimethylamine groups ethyl benzoate trifluoromethyl sulfonic acid that is dissolved among the 1mL DMSO, confined reaction is 20 minutes under 120 ℃ of conditions.Add 0.5mL 0.2mol/L NaOH hydrolysis then.Reaction finishes postcooling to room temperature, behind the adding 0.5mL 1mol/L HCl, is diluted with water to 10mL, adds Sep-Pak C with the 10mL syringe behind the thorough mixing
18The Plus post is used the 10mL water wash, and elutant discards.After nitrogen dries up pillar, to another reaction flask, measure the product radioactive activity with 3mL acetonitrile (containing 0.1% trifluoroacetic acid) drip washing.
3.N-succinimide 4-[
18F] preparation of fluorobenzoic acid ester
To 4-[
18F] add the Bu of 10 μ L 40% in the fluorobenzoic acid
4The NOH aqueous solution feeds nitrogen (1mL/min) and dries up in 120 ℃ of oil baths, adding 0.5mL anhydrous acetonitrile is evaporated to dried, triplicate.To wherein adding 15mg O-(N-succinimide)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester (TSTU is dissolved in the 1mL anhydrous acetonitrile) is at 80～90 ℃ of confined reaction 5min then.Reaction finishes postcooling to room temperature, with 50% acidifying with acetic acid of 0.5mL, and is diluted with water to 10mL.Add Sep-PakC with the 10mL syringe behind the thorough mixing
18The Plus post is used the 10mL water wash, and elutant discards.After nitrogen dries up pillar, to another reaction flask, measure the product radioactive activity with the 2.5mL eluent methylene chloride.
4.[
18F] the fluorine galactose chitosan ([
18F] FB-GC) preparation
N-succinimide 4-[
18F] the fluorobenzoic acid ester feeds nitrogen (1mL/min) and dries up solvent in 50 ℃ of oil baths.Add 50 μ L DMSO with the dissolving of the resistates in the reaction flask, add 3mg galactose chitosan (GC is dissolved in 200 μ L 0.1mol/L pH8.6 borate buffers), room temperature reaction 30min.HiTrap desalination gel column (Sephadex G25) with 25mL leacheate (phosphoric acid buffer of 0.05mol/L pH 7.5) balance, is controlled flow velocity between 1～10mL/min.With the 0.25mL mark good [
18F] FB-GC sample on the 1mL syringe, with after the drip washing of 1.25mL leacheate, collect the 1mL leacheate earlier, the impurity that is removed, the rate of recovery＞95% [
18F] FB-GC solution.
Identify that by thin-layer chromatography and HPLC its HPLC retention time is 12.3min, radiochemical purity is greater than 99%.The HPLC condition is: high performance liquid chromatograph SHIMADZU (SCL-10Avp); KromaislC4 post 250 * 4.6mm, 5 μ m
A is water (containing 0.1%TFA) mutually, and B is acetonitrile (containing 0.1%TFA) mutually; The drip washing gradient is: 0～30min:30%～70%B phase; Flow velocity 1mL/min.
Claims (6)
1. the galactosylated chitosan compound of fluoro-18 marks, its developed by molecule formula be [
18F] FB-GC, its general structure is:
Wherein: [
18F] FB-GC is connected with the amino of galactose chitosan molecular chain by parafluorobenzoic acid to obtain.
2. the preparation method of the galactosylated chitosan compound of fluoro-18 marks is characterized in that: with radioactivity N-succinimide 4-[
18F] the fluorobenzoic acid ester ([
18F] SFB) obtain the galactosylated chitosan compound of described radioactive fluorine-18 mark with galactose chitosan (GC) prepared in reaction under alkaline condition, its preparation process is:
A. at K
2CO
3, K
2.2.2.[
18F] F-and 4-trimethylamine groups ethyl benzoate trifluoromethyl sulfonic acid react under heating condition after 20 minutes, after the NaOH hydrolysis, add HCl and obtain 4-[
18F] fluorobenzoic acid;
B.4-[
18F] fluorobenzoic acid and O-(N-succinimide)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester reacted 5 minutes under heating condition, and with obtaining N-succinimide 4-[behind the acidifying with acetic acid
18F] the fluorobenzoic acid ester ([
18F] SFB);
C. galactose chitosan GC is dissolved in the N-succinimide 4-[that adds step b preparation in the damping fluid
18F] in the fluorobenzoic acid ester, under 10 ℃～40 ℃ conditions, react 10～60min behind the mixing;
D. step c gained solution is carried out purifying by gel chromatography.
3. the preparation method of the galactose chitosan of fluoro-18 marks as claimed in claim 2 is characterized in that:
Reacting by heating temperature among the described step a is 90～140 ℃, and the reacting by heating temperature described in the step b is 80～100 ℃.
4. the preparation method of the galactose chitosan of fluoro-18 marks as claimed in claim 2 is characterized in that:
Damping fluid among the described step c is: 0.1mol/L pH8.6 borate buffer, consumption are 0.1mL～1mL.
5. the preparation method of the galactose chitosan of fluoro-18 marks as claimed in claim 2 is characterized in that:
It is the G25 gel column of dextrane gel that gel chromatographic columns in the described steps d is selected filler for use.
6. the preparation method of the galactose chitosan of fluoro-18 marks as claimed in claim 2 is characterized in that:
The consumption of described new lactose albumin is 1mg～5mg.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113354A (en) * | 2012-11-01 | 2013-05-22 | 张现忠 | PEGylated benzyltriazolyl pyridazinone compounds, and preparation method and application thereof |
| CN111840566A (en) * | 2019-04-30 | 2020-10-30 | 深圳市罗湖区人民医院 | Fluorinated chitosan used as bladder perfusion drug carrier and preparation method thereof |
| EP4095162A4 (en) * | 2019-04-30 | 2023-08-02 | Soochow University | Application of and preparation method for cationic polymer modified by fluorine-containing compound as drug carrier |
-
2010
- 2010-05-17 CN CN2010101748007A patent/CN101838345B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《Nuclear Medicine and Biology》 20061231 Eun-Mi Kim et.al Asialoglycoprotein-receptor-targeted hepatocyte imaging using 99m Tc galactosylated chitosan 529-534 1-6 第33卷, 第4期 2 * |
| 《The Journal of Nuclear Medicine》 20050131 Eun-Mi Kim,MS Hepatocyte-Targeted Nuclear Imaging Using 99m Tc-Galactosylated Chitosan:Conjugation,Targeting,and Biodistribution 141-145 1-6 第46卷, 第1期 2 * |
| 《武汉理工大学学报》 20071231 刘利等 半乳糖基化壳聚糖的合成与表征 29-31 1-6 第29卷, 第12期 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113354A (en) * | 2012-11-01 | 2013-05-22 | 张现忠 | PEGylated benzyltriazolyl pyridazinone compounds, and preparation method and application thereof |
| CN103113354B (en) * | 2012-11-01 | 2014-12-17 | 张现忠 | PEGylated benzyltriazolyl pyridazinone compounds, and preparation method and application thereof |
| CN111840566A (en) * | 2019-04-30 | 2020-10-30 | 深圳市罗湖区人民医院 | Fluorinated chitosan used as bladder perfusion drug carrier and preparation method thereof |
| EP4095162A4 (en) * | 2019-04-30 | 2023-08-02 | Soochow University | Application of and preparation method for cationic polymer modified by fluorine-containing compound as drug carrier |
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