CN104800865A - Targeted positron tracer for realizing PET (Positron Emission Tomography) imaging of positive tumors for HER2 (Human Epidermal Growth Factor Receptor 2) expression, and preparation method of targeted positron tracer - Google Patents

Targeted positron tracer for realizing PET (Positron Emission Tomography) imaging of positive tumors for HER2 (Human Epidermal Growth Factor Receptor 2) expression, and preparation method of targeted positron tracer Download PDF

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CN104800865A
CN104800865A CN201510218446.6A CN201510218446A CN104800865A CN 104800865 A CN104800865 A CN 104800865A CN 201510218446 A CN201510218446 A CN 201510218446A CN 104800865 A CN104800865 A CN 104800865A
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reagent
positron
tracer
ltvspwy
her2
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段小艺
李淼
朱媛
王丽
高俊刚
刘翔
尚进
王健生
郭佑民
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First Affiliated Hospital of Medical College of Xian Jiaotong University
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First Affiliated Hospital of Medical College of Xian Jiaotong University
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Abstract

The invention provides a targeted positron tracer for realizing PET (Positron Emission Tomography) imaging of positive tumors for HER2 (Human Epidermal Growth Factor Receptor 2) expression, and a preparation method of the targeted positron tracer. The positron tracer is a compound established on the basis of oligopeptide in the sequence of leucine-threonine-valine-serine-proline-tryptophan-tyrosine, 6-hydrazinonicotinoyl is connected to the amino terminal of the oligopeptide, the radionuclide [18F] fluorine is marked, and amino can be connected to the carboxyl terminal. The positron tracer disclosed by the invention is good in biodistribution in vivo, and verified by the cellular assay in vitro that the positron tracer has a better targeting property, so that the positron tracer can be used for the specificity imaging of positive tumors for HER2 expression, the selection and the curative-effect judgment of targeted therapeutic drugs. According to the invention, a complete, process-centered full-automatic synthesis method is provided, and the preparation process is simple and practicable, convenient and efficient, and the preparation method is favorable for batch and mass production and commercialized supply in the future.

Description

Targeting positron tracer of positive tumor PET imaging and preparation method thereof is expressed for HER2
Technical field
The present invention relates to the positron tracer for PET imaging, be specifically related to a kind ofly express positive malignancies targeting diagnosis, the positron tracer of medicament selection and therapeutic evaluation and synthetic method thereof for HER2.
Background technology
Human epidermal growth factor receptor 2 (Human Epidermal Growth Factor Receptor 2, HER2) in the generation, development of malignant tumor, there is important function, target therapeutic agent such as Iressa etc. for HER2 has been widely used in clinical, achieves certain effect.But due to individual variation and the Tumor Heterogeneity of tumor tissues HER2 gene expression, quite a few patient's targeted drug is failed to respond to any medical treatment or poor effect, causes unnecessary manpower and financial resources to expend, even delay the treatment best opportunity, cause disease progression.By Positron emission computed tomography (Positron Emission Tomography, PET) technology is conducive to choose reasonable target therapeutic agent at the expression of health check-up survey and dynamic monitoring specimens HER2 gene and change, evaluates therapeutic effect.
Positron radionuclide [ 18f] fluorine be optimal, be also PET tracer labelled nuclide the most frequently used at present, its physical half time is 109 minutes, and preparation process is simple, and labeling method is ripe, is suitable for clinical popularization and application.There is not yet the PET positron tracer research report for HER2 oligopeptide fragments at present both at home and abroad.
Based on oligopeptide molecule 18f labelling positron tracer just occurs in recent years.[ 18f] strategy of fluorine labelling peptide mainly comprises direct labelling method and Indirect Labelling.Direct labelling is minimum to the structure influence being labeled peptide, but severe reaction conditions, be unfavorable for that peptide keeps active, and poor selectivity, therefore be of little use; Indirect Labelling need to be labeled peptide modify to provide specific [ 18f] fluorine mark point, and protection may produce the group of side reaction in advance, will [ 18f] on fluorine labelling to specific reaction intermediate after, then coupled reaction intermediate and modified peptides obtain target product.The advantage of indirect method is that reaction condition is gentle, fast, selectivity is good, activity is high, shortcoming be be labeled peptide and [ 18f] introduce linking group between fluorine and can change the character being labeled peptide, therefore labeling method needs careful selection.Indirect Labelling mainly comprises: prothetic group method (activated carboxyl prothetic group method, aldehyde radical become hydrazone method, sulfydryl reducing process, click chemistry method), solid phase method, coordination compound method (AlF method), isotope exchange method (SiF method), aromatic ring method of substitution (electrophilic substitution method, nucleophilic displacement of fluorine method) etc.Wherein solid phase method mild condition, labelling time are short, but to the protection of peptide and curing operation complicated; Coordination compound method mild condition, productive rate is high, can realize by single step reaction, but in product body, defluorinate is serious; Isotope exchange method is easy to operate, but the labelling time is long, productive rate is low, product and raw material separation difficulty; The aromatic ring method of substitution response time is short, but has particular/special requirement to being labeled peptide structure, and condition is harsh, and product is complicated, difficult separation, and mark rate is low; Prothetic group method is modal method, and wherein the research of activated carboxyl prothetic group method is more, is the classical way reported the earliest, and major defect is that reactions steps is many, and complicated operation is large to yield impact; Sulfydryl reducing process and click chemistry method all need special intermediate and modified peptides, are unfavorable for application because being difficult to obtain; Relatively simple containing the operation of aldehyde radical prothetic group labelling method, reaction condition is gentle, and productive rate is higher, and the easy purification of product, body internal stability are good, just there are certain requirements reaction system design.
There is not yet up to now utilize positron radionuclide [ 18f] fluorine label L TVSPWY prepares any report of PET positron tracer.
Summary of the invention
The object of the present invention is to provide a kind of for HER2 express positive tumor PET imaging targeting positron tracer (referred to as [ 18f] HYNIC-LTVSPWY or [ 18f] HYNIC-LTVSPWY-NH 2) and preparation method thereof.
For achieving the above object, present invention employs following technical scheme:
Express a targeting positron tracer for positive tumor PET imaging for HER2, this positron tracer comprises the compound with structure as shown in Equation 1:
In formula 1, R 1for oligopeptide aminoterminal sloughs remaining part after a hydrogen atom, described oligopeptide has the aminoacid sequence as shown in SEQ.ID.NO.1, or R 1for:
R 2for 4-[ 18f] fluorobenzaldehyde (4-[ 18f] fluorobenzaldehyde, [ 18f] FBA) slough aldehyde radical oxygen atom after remaining part, or, R 2for 2-[ 18f] fluoro-2-deoxy-D-glucose (2-[ 18f] fluorine-2-deoxy-D-glucose, [ 18f] FDG) slough aldehyde radical oxygen atom after remaining part.
Described positron tracer also comprises solvent.
Described solvent is 20mmol/L sodium dihydrogen phosphate (NaH 2pO 4) mixture that is mixed to get according to the volume ratio of 80:20 of aqueous solution and ethanol (Ethanol).
The specific activity of described positron tracer is 370 ~ 740MBq/mL.
Express a preparation method for the targeting positron tracer of positive tumor PET imaging for HER2, comprise the following steps:
1) reagent is prepared:
By 2 ~ 3mg potassium carbonate (K 2cO 3) to be dissolved in 0.3 ~ 0.5mL water obtaining reagent 1-1, by 10 ~ 20mg amino-polyether (Kryptofix 2.2.2, K 2.2.2) to be dissolved in 1 ~ 2mL acetonitrile (Acetonitrile) obtaining reagent 1-2, reagent 1-1 and reagent 1-2 is mixed to get reagent 1; By 10 ~ 20mg trifluoromethanesulfonic acid-4-N, N, N-trimethyl ammonium benzaldehyde is dissolved in 0.5 ~ 1mL anhydrous dimethyl sulphoxide (dimethyl sulfoxide, DMSO) and obtains reagent 2; By sodium acetate (the sodium acetate of 0.1mol/L, NaAc) buffer solution (pH 3 ~ 4) and ethanol (Ethanol) by volume 9:1 mix, obtain reagent 3-1, by 1 ~ 2mg HYNIC-LTVSPWY or HYNIC-LTVSPWY-NH 2be dissolved in the reagent 3-1 of 1 ~ 2mL and obtain reagent 3, described HYNIC-LTVSPWY-NH 2for having the compound such as formula structure shown in 2-1:
Described HYNIC-LTVSPWY is for having the compound such as formula structure shown in 2-2:
By 20mmol/L NaH 2pO 4aqueous solution and ethanol are mixed to get reagent 4-1 according to the volume ratio of 80:20, get 1 ~ 2mL reagent 4-1 as reagent 4;
2) activity prepared by cyclotron be 200 ~ 400mCi [ 18f] fluorion ([ 18f] F -) pass into and be adsorbed on anion-exchange column, then by be adsorbed on anion-exchange column [ 18f] fluorion reagent 1 is eluted in the reaction bulb of synthesizer, then at nitrogen (N 2) or helium (He) protection under be warming up to 60 ~ 70 DEG C and keep 3 ~ 4min, and then evacuation keep 10 ~ 15min after being warming up to 85 ~ 95 DEG C, then stop evacuation and be down to room temperature;
3) through step 2) after, reagent 2 is added and is warming up to 105 ~ 110 DEG C after in described reaction bulb and keeps 10 ~ 15min to obtain mixture 1, thing 1 to be mixed is down to and is added 5 ~ 10mL water in the backward described reaction bulb of room temperature and obtain mixture 2, by mixture 2 successively by cation exchange column and reverse phase solid phase extraction post, 0.5 ~ 1.0mL dehydrated alcohol (anhydrous ethanol) is then used to be eluted in described reaction bulb by the adsorbate on reverse phase solid phase extraction post;
4) through step 3) after, reagent 3 is added in described reaction bulb, then be warming up to 100 ~ 105 DEG C and keep 10 ~ 15min, then room temperature is down to, then in described reaction bulb, add reagent 4 obtain mixture 3, with reagent 4-1 for mobile phase, be separated by the preparative liquid chromatography of mixture 3 through synthesizer, collecting retention time on γ chromatogram is the separated product of 13 ~ 18min.
Described synthesizer is GE Tracerlab FX FN automated synthesiser.
Described step 2) to step 4) in, temperature-rise period is heated by electric jacket or microwave heating completes.
Express a preparation method for the targeting positron tracer of positive tumor PET imaging for HER2, comprise the following steps:
1) reagent is prepared:
By 2 ~ 3mg K 2cO 3be dissolved in 0.3 ~ 0.5mL water and obtain reagent 1-1,10 ~ 20mg amino-polyether (Kryptofix 2.2.2, K 2.2.2) is dissolved in 1 ~ 2mL acetonitrile and obtains reagent 1-2; Reagent 1-1 and reagent 1-2 is mixed to get reagent 1; Tetra-acetylated for 10 ~ 20mg 1,3,4,6-trifyl mannose (1,3,4,6-tetraacetyl-mannose triflate) is dissolved in 1 ~ 2mL anhydrous acetonitrile and obtains reagent 2; By 0.1mol/L sodium acetate (sodium acetate, NaAc) buffer solution (pH 3 ~ 4) and ethanol by volume 9:1 mix, obtain reagent 3-1, by 1 ~ 2mg HYNIC-LTVSPWY or HYNIC-LTVSPWY-NH 2be dissolved in 1 ~ 2mL reagent 3-1 and obtain reagent 3, described HYNIC-LTVSPWY-NH 2for having the compound such as formula structure shown in 2-1:
Described HYNIC-LTVSPWY is for having the compound such as formula structure shown in 2-2:
By 20mmol/L NaH 2pO 4aqueous solution and ethanol are mixed to get reagent 4-1 according to the volume ratio of 80:20, get 1 ~ 2mL reagent 4-1 as reagent 4;
2) activity prepared by cyclotron be 200 ~ 400mCi [ 18f] fluorion ([ 18f] F -) pass into and be adsorbed on anion-exchange column, then by be adsorbed on anion-exchange column [ 18f] fluorion ([ 18f] F -) be eluted in the reaction bulb of synthesizer with reagent 1, then at nitrogen (N 2) or helium (He) protection under be warming up to 60 ~ 70 DEG C and keep 3 ~ 4min, and then evacuation keep 10 ~ 15min after being warming up to 85 ~ 95 DEG C, then stop evacuation and be down to room temperature;
3) through step 2) after, reagent 2 is added and is warming up to 80 ~ 90 DEG C after in described reaction bulb and keeps 4 ~ 5min, be then warming up to 105 ~ 110 DEG C and keep 4 ~ 5min;
4) through step 3) after, described reaction bulb is cooled to 35 DEG C, then 1 ~ 2mL 1mol/L sodium hydroxide (NaOH) aqueous solution to be added in described reaction bulb and to carry out hydrolysis 5min, hydrolysis terminates rear hydrochloric acid (HCl) adjust ph to 4 ~ 5, then leaves standstill 5 ~ 10min;
5) through step 4) after, reagent 3 is added in described reaction bulb, then be warming up to 100 ~ 105 DEG C and keep 10 ~ 15min, then room temperature is down to, then in described reaction bulb, add reagent 4 and obtain mixture 3 ', with reagent 4-1 for mobile phase, be separated by the preparative liquid chromatography of mixture 3 ' through synthesizer, collecting retention time on γ chromatogram is the separated product of 10 ~ 15min.
Described synthesizer is GE Tracerlab FX FN automated synthesiser.
Described step 2) to 3) and step 5) in, temperature-rise period is heated by electric jacket or microwave heating completes.
Beneficial effect of the present invention is embodied in:
Positron tracer of the present invention overcomes the shortcoming that conventional antibodies class positron indicator molecule amount is comparatively large, poor, in tumor the gathering of tissue permeability is restricted.First, this positron tracer is based upon on LTVSPWY oligopeptide structure (Leu-Thr-Val-Ser-Pro-Trp-Tyr, LTVSPWY) basis, has for HER2 targeting; Secondly, this positron tracer combined coefficient is high, calculating from end of bombardment, and preparation total time is 50 ~ 60min, effectively can reduce the waiting time of person under inspection; In addition, this positron tracer is mainly through renal excretion, and liver uptake ratio is low, and excretion is fast, thus can reduce person under inspection's Radioimmunoimaging.
Present invention employs [ 18f] FBA or [ 18f] FDG is connected with precursor (HYNIC modifies LVTSPWY), thus synthesis [ 18f] synthetic route of HYNIC-LTVSPWY, productive rate high (reaching 45%), precursor with positron radionuclide [ 18f] fluorine connects after (labelling), neither change the biology performance of precursor, can play imaging object again, preparation process is simple, be conducive to setting up for [ 18f] complete, procedure, the full-automatic synthetic method of HYNIC-LTVSPWY, accomplishing scale production uses with universalness, has higher practical value and social benefit.In order to suppress body endoproteinase to the hydrolysis of oligopeptide, extend biological half-life in its body, the present invention connects amino (-NH at its c-terminus on the basis of original aminoacid sequence (LTVSPWY) 2), experiment proves [ 18f] HYNIC-LTVSPWY-NH 2more be applicable to PET imaging.
Accompanying drawing explanation
Fig. 1 is HYNIC structural formula;
Fig. 2 is LTVSPWY structural formula;
Fig. 3 is HYNIC-LTVSPWY-NH 2structural formula;
Fig. 4 is the structural formula of the positron tracer that embodiment one is synthesized;
Fig. 5 is the structural formula of the positron tracer that embodiment two is synthesized;
Fig. 6 is synthetic route of the present invention;
Fig. 7 be different phase [ 18f] HYNIC-LTVSPWY-NH 2distribution in vivo;
Fig. 8 is cell in vitro binding tests, and wherein, (a) is MD-MA-231 cell, (b) SK-BR-3 cell.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is elaborated.
The invention provides a kind of for ErbB-2 (human epidermalgrowth factorreceptor-2, HER2) express positive tumor positron emission tomography (positron emissiontomography, PET) targeting positron tracer (referred to as [ 18f] HYNIC-LTVSPWY or [ 18f] HYNIC-LTVSPWY-NH 2) and preparation method thereof.
This positron tracer is based upon on oligopeptide LTVSPWY basis, on the aminoterminal of oligopeptide, connect 6-diazanyl nicotinoyl (6-hydrazinonicotinoyl, HYNIC), and c-terminus connects amino (-NH 2), labelling radionuclide [ 18f] fluorine ([ 18f] fluorine, [ 18f] F) obtain afterwards, structure is as shown in Figure 4, Figure 5; Or this positron tracer is based upon on oligopeptide LTVSPWY basis, connects 6-diazanyl nicotinoyl (6-hydrazinonicotinoyl, HYNIC) on the aminoterminal of oligopeptide, labelling radionuclide [ 18f] fluorine ([ 18f] fluorine, [ 18f] F) obtain afterwards.
Above-mentioned positron tracer (with [ 18f] HYNIC-LTVSPWY-NH 2for example) full-automatic synthetic method be described as follows:
Connect HYNIC (structure is as Fig. 1) at the aminoterminal of LTVSPWY oligopeptide (structure as Fig. 2, and SEQ.ID.NO.1), c-terminus connects amino-NH 2form precursor (i.e. HYNIC-LTVSPWY-NH 2, structure is as Fig. 3), using HYNIC as difunctional connector, and utilize positron radionuclide [ 18f] fluorine to precursor carry out labelling (HYNIC be used for [ 18f] FBA or [ 18f] FDG connect), adopt full-automatic synthetic technology synthesize [ 18f] HYNIC-LTVSPWY-NH 2.
Synthetic route (Fig. 6) is as follows:
1), after synthesizing LTVSPWY oligopeptide, HYNIC and-NH is adopted 2carry out modification and obtain precursor (biotechnology (Suzhou) company limited that shines by force synthesizes and provides).
2) adopt [ 18f] labelling trifluoromethanesulfonic acid-4-N, the synthesis of N, N-trimethyl ammonium benzaldehyde [ 18f] FBA, or prepare [ 18f] FDG (.4-such as [1] Li Gucai [ 18f] radiosynthesis of fluorobenzaldehyde. isotope, 2006,19 (2): 87-91; [2] Wang Mingwei etc. conventional 18the synthesis of F labelling intermediate and applied research thereof. nuclear technology, 2006,29 (1): 63-71).
3) based on the reaction mechanism generating hydrazone, will [ 18f] FBA or [ 18f] FDG is connected with precursor, obtain [ 18f] HYNIC-LTVSPWY-NH 2.
Embodiment one
By [ 18f] FBA synthesis [ 18f] HYNIC-LTVSPWY-NH 2(structure is as Fig. 4).Following steps 1) to step 8) completed by the full-automation of GE Tracerlab FX FN synthesizer.
1) 3mg K is taken 2cO 3be dissolved in 0.5mL water and obtain K 2cO 3solution; Take 15mg K 2.2.2 and to be dissolved in 1mL acetonitrile obtaining K 2.2.2 solution; By K 2cO 3solution and K 2.2.2 solution inject No. 1 storage pipe mixing of described synthesizer; By 15mg trifluoromethanesulfonic acid-4-N, N, N-trimethyl ammonium benzaldehyde injects No. 2 storage pipes of described synthesizer after being dissolved in the anhydrous DMSO of 500 μ L; 0.1mol/L NaAc buffer solution (pH3 ~ 4) and ethanol are mixed to get mixed solution with volume ratio 9:1, take 1mg HYNIC-LTVSPWY-NH 2, be dissolved in No. 3 storage pipes injecting described synthesizer after in mixed solution described in 1mL; By 20mmol/L NaH 2pO 4aqueous solution and ethanol are mixed with liquid chromatogram mobile phase (NaH 2pO 4aqueous solution: ethanol=80:20, v/v), get No. 4 storage pipes that described mobile phase 2mL injects described synthesizer, separately get described mobile phase 1000mL, after sonic oscillation 30min, inject the mobile phase bottle of described synthesizer; 0.5mL dehydrated alcohol is injected No. 5 storage pipes of described synthesizer; 10mL injection water (water for injection) is injected No. 6 storage pipes of described synthesizer;
2) with high-purity helium prepared by cyclotron [ 18f] F -(activity 200 ~ 400mCi) imports into and is adsorbed on QMA anion-exchange column.
3) with No. 1 storage pipe in mixed solution by be adsorbed on QMA anion-exchange column [ 18f] F -eluting enters reaction bulb, at N 2or under the protection of He, be warming up to 65 DEG C of maintenance 3.5min, be then warming up to 90 DEG C of evacuation, after keeping 10min, reaction bulb pressure is adjusted to atmospheric pressure, temperature is down to room temperature;
4) through step 3) after, the solution in No. 2 storage pipes is added reaction bulb, is warming up to 110 DEG C and keeps 10min; Room temperature is down to by question response bottle, injection water in No. 6 storage pipes is injected reaction bulb, then the liquid in reaction bulb is passed through cation exchange column (ion-exchange cartridge) LiChrolut SCX-cartridge (200mg successively, Merck company) and reverse phase solid phase extraction post (reverse phase-solid phase extractioncartridge) Sep-Pak C18light cartridge (Waters company), make product [ 18f] FBA is adsorbed on reverse phase solid phase extraction post, with the dehydrated alcohol in No. 5 storage pipes by 4-[ 18f] fluorobenzaldehyde is eluted to reaction bulb;
6) through step 5) after, No. 3 storage pipe solution are injected reaction bulb, is warming up to 100 DEG C and keeps 10 minutes;
7) through step 6) after, reaction bulb is down to room temperature, the mobile phase solution in No. 4 storage pipes is injected reaction bulb; Mixed solution in reaction bulb is injected preparative liquid chromatography be separated, flow velocity is 8mL/min, and UV-detector determined wavelength is 220nm, adopts γ detector to detect simultaneously, collects retention time on γ chromatogram and is about the separated product of 16min;
8) through step 7) after, by the separated product collected through germ tight filter (Merck MilliporeMILLEX-GS 0.22 μm of SLGSV255F) filter after obtain can for injection [ 18f] HYNIC-LTVSPWY-NH 2solution.
Through mass spectral analysis, the visible mass-to-charge ratio of separated product sample mass spectrum (m/z) is the quasi-molecular ions of 1104.5, and m/z value meets the compound molecular weight of expection, and the compound (Fig. 4) containing expection in separated product is described.
After testing, this programme synthetic yield can reach 45%, and radiochemical purity is greater than 98%.
Embodiment two
By [ 18f] FDG synthesis [ 18f] HYNIC-LTVSPWY-NH 2(Fig. 5).Step 1) to step 8) completed by the full-automation of GE Tracerlab FX FN synthesizer.
1) 3mg K is taken 2cO 3, be dissolved in 0.5mL water and obtain K 2cO 3solution; Take 15mg K 2.2.2, be dissolved in 1mL anhydrous acetonitrile and obtain K 2.2.2 solution; By K 2cO 3solution and K 2.2.2 solution inject No. 1 storage pipe mixing of described synthesizer; Take No. 2 storage pipes injecting described synthesizer after 20mg 1,3,4,6-tetra-acetylated trifyl mannose is dissolved in 1mL anhydrous acetonitrile; Preparation 1mol/L NaOH aqueous solution, gets No. 3 storage pipes that 1mL injects described synthesizer; By 0.1mol/L NaAc buffer solution (pH=3 ~ 4) and ethanol by volume 9:1 mix and be mixed to get mixed solution, take 1mg HYNIC-LTVSPWY-NH 2, be dissolved in mixed solution described in 1 ~ 2mL, inject No. 4 storage pipes of described synthesizer; With 20mmol/L NaH 2pO 4the mixed solution of aqueous solution and ethanol is preparative liquid chromatography mobile phase (NaH 2pO 4aqueous solution: ethanol=80:20, v/v), get No. 5 storage pipes that described mobile phase 2mL injects described synthesizer, by 0.5mL hydrochloric acid (1%, w/v) No. 6 storage pipes of described synthesizer are injected, separately get described mobile phase 1000mL, after sonic oscillation 30min, inject the mobile phase bottle of described synthesizer;
2) with high-purity helium prepared by cyclotron [ 18f] F -(activity 200 ~ 400mCi) imports into and is adsorbed on QMA anion-exchange column.
3) with No. 1 storage pipe in mixed solution by be adsorbed on QMA anion-exchange column [ 18f] F -eluting enters reaction bulb, at N 2or under He protection, be warming up to 65 DEG C of maintenance 3.5min, be then warming up to 90 DEG C, evacuation, keep 10min, then reaction bulb pressure is adjusted to atmospheric pressure, temperature is down to room temperature;
4) through step 3) after, the solution in No. 2 storage pipes is injected into reaction bulb, is warming up to 85 DEG C and keeps 5min; Be warming up to 105 DEG C after reaction terminates and keep 5min, evaporate to dryness acetonitrile;
5) through step 4) after, reaction bulb is cooled to 35 DEG C, the solution in No. 3 storage pipes is injected reaction bulb and carries out 5min hydrolysis; After reaction terminates, the hydrochloric acid of No. 6 storage pipes is injected reaction bulb, the pH value of solution in reaction bulb is adjusted to 4, place 5min;
6) through step 5) after, the solution in No. 4 storage pipes is injected reaction bulb, is warming up to 100 DEG C and keeps 10min;
7) through step 6) after, reaction bulb is down to room temperature, the solution in No. 5 storage pipes is injected reaction bulb; Mixed solution in reaction bulb is injected preparative liquid chromatography be separated, flow velocity is 8mL/min, and UV-detector determined wavelength is 220nm, adopts γ detector to detect simultaneously, collects retention time on γ chromatogram and is about the separated product of 13min;
8) through step 7) after, by the separated product collected through germ tight filter (Merck MilliporeMILLEX-GS 0.22 μm of SLGSV255F) filter after obtain can for injection [ 18f] HYNIC-LTVSPWY-NH 2solution.
Through mass spectral analysis, the visible mass-to-charge ratio of separated product sample mass spectrum (m/z) is the quasi-molecular ions of 1162.5, and m/z value meets the compound molecular weight of expection, and compound (Fig. 5) molecule containing expection in separated product is described.
After testing, this programme synthetic yield can reach 40%, and radiochemical purity is greater than 98%.
In above-mentioned embodiment one, two, if utilize 70W microwave to heat reaction bulb, then intensification required time is compared synthesizer and is carried common electric jacket and can shorten 1 ~ 2min, improves programming rate.
[ 18f] preparation and the above-mentioned embodiment one, two identical of HYNIC-LTVSPWY, be only that precursor is different, precursor is based upon on oligopeptide LTVSPWY basis, connects 6-diazanyl nicotinoyl on the aminoterminal of oligopeptide, and c-terminus does not carry out connecting amino modification.[ 18f] HYNIC-LTVSPWY structure is as follows:
or
The explanation of invention effect
(1) quality control:
1) differentiate: the radiochemical purity and the productive rate that adopt TLC and HPLC assay products.TLC condition: immobile phase silica gel G, thickness 0.25mm, developing solvent is acetonitrile: water=95:5 (v/v); HPLC condition: chromatographic column is octadecyl silane reversed phase chromatographic column (Inertsil ODS-SP, 4.6mm I.D. × 150mm, 5 μm, Shimadzu company), mobile phase is acetonitrile-trifluoroacetic acid aqueous solution (0.1%, v/v) mixed solution (acetonitrile: trifluoroacetic acid aqueous solution=75:25, volume ratio), isocratic elution, flow velocity 1mL/min.
2) check: pH value is 5.0 ~ 8.0 (Chinese Pharmacopoeia version in 2010, two, annex VI H).Detection of bacterial endotoxin: get appropriate this product (namely through germ tight filter filter after obtain can for injection [ 18f] HYNIC-LTVSPWY solution or [ 18f] HYNIC-LTVSPWY-NH 2solution), after baterial endotoxin test dilute with water 60 times, carry out detecting (Chinese Pharmacopoeia version in 2010, two, annex XI E) according to standard method, the endotoxin content of the every 1mL of this product is less than 15EU.Sterility test: get appropriate this product, carry out detecting (Chinese Pharmacopoeia version in 2010, two, annex XI H) according to standard method, this product meets the requirements.
3) specific activity: this product of accurate measuring certain volume, is placed in activity meter and measures activity, per sample volume and Activity Calculation specific activity thereof.This product specific activity scope is 370 ~ 740MBq/mL.
4) effect duration: calculating is greater than 6h from the demarcation moment.
(2) animal distribution in vivo
Select healthy new zealand white rabbit (body weight 3.0kg) as the object of observation, first use 3% pentobarbital solution (0.8mL/kg) practice processes to anaesthetize, then by auricular vein slowly inject [ 18f] HYNIC-LTVSPWY-NH 2solution (2.0mCi/kg), in 10,30,60,90, the capable PET/CT body scan of 120min, scanner is PHILIPS Co. GEMINI 64TF PET/CT imaging system, arranging CT tube voltage is 120V, tube current is 350mA, the every berth of PET gathers 3min, and the image post processing software adopting instrument to carry carries out date processing and image reconstruction, obtain [ 18f] HYNIC-LTVSPWY-NH 2scattergram in animal body.Different phase [ 18f] HYNIC-LTVSPWY-NH 2distribution situation in rabbit body see Fig. 7, figure in show this positron tracer ([ 18f] HYNIC-LTVSPWY-NH 2) mainly through renal excretion, liver and gastrointestinal tract uptake ratio low, meet in-vivo imaging requirement, [ 18f] HYNIC-LTVSPWY has similar result.
(3) cell in vitro binding tests
Select the human breast carcinoma SK-BR-3 cell of high expressed HER2 and HER2 to express negative MD-MA-231 cell as the object of observation, the latter is contrast.By cell culture to exponential phase, prepare cell climbing sheet, then instill FITC labelling [ 18f] HYNIC-LTVSPWY-NH 2, hatch 30min for 37 DEG C, in fluorescence microscopy Microscopic observation fluorescence distribution situation after PBS rinses.Result as shown in Figure 8, [ 18f] HYNIC-LTVSPWY-NH 2hatching rear surface of cell membrane with SK-BR-3 cell climbing sheet can clear display green fluorescence, sketch the contours of the profile (Fig. 8 b) of cell, and with MD-MA-231 cell incubation after visible fluorescence granule be dispersed in and be distributed in the visual field, cell outline display unclear (Fig. 8 a), point out thus positron tracer [ 18f] HYNIC-LTVSPWY-NH 2can with tumor cell surface HER2 specific binding, be applicable to high expressed HER2 tumor imaging.[ 18f] HYNIC-LTVSPWY has similar result.
(4) the present invention connects amino (-NH at c-terminus on the basis of original aminoacid sequence (LTVSPWY) 2), the oligopeptide fragments LTVSPWY-NH after modification 2in body, biodistribution is applicable to PET imaging more, with [ 18f] HYNIC-LTVSPWY compares, [ 18f] HYNIC-LTVSPWY-NH 2advantage is: add amino (-NH at the c-terminus of oligopeptide 2) modify, be conducive to the proteolytic cleavage site of closed oligopeptide, reduce its hydrolysis, increase oligopeptide stability in blood, extend the biological half-life of oligopeptide, improve tracer holdup time of pathological tissues and radioactivity dense poly-, amido modified rear tracer of this time synthesis ([ 18f] HYNIC-LTVSPWY-NH 2) demonstrating better distribution in vivo, blood clearance is high, and radioactive background is low, and picture quality is better.
The present invention establish for the preparation of [ 18f] HYNIC-LTVSPWY or [ 18f] HYNIC-LTVSPWY-NH 2complete set, procedure, fully automated synthesis and method of quality control.Experiment shows, [ 18f] HYNIC-LTVSPWY-NH 2there is suitable physics and chemistry and radiology character, ideal biological characteristics, can be used for the PET imaging of high expressed HER2 tumor, contribute to the specific diagnosis of this type of tumor, the selection of target therapeutic agent and therapeutic evaluation.

Claims (10)

1. express a targeting positron tracer for positive tumor PET imaging for HER2, it is characterized in that: this positron tracer comprises the compound with structure as shown in Equation 1:
In formula 1, R 1for oligopeptide aminoterminal sloughs remaining part after a hydrogen atom, described oligopeptide has the aminoacid sequence as shown in SEQ.ID.NO.1, or R 1for:
R 2for 4-[ 18f] fluorobenzaldehyde remaining part after sloughing aldehyde radical oxygen atom, or, R 2for 2-[ 18f] fluoro-2-deoxy-D-glucose remaining part after sloughing aldehyde radical oxygen atom.
2. a kind of targeting positron tracer of expressing positive tumor PET imaging for HER2 according to claim 1, is characterized in that: described positron tracer also comprises solvent.
3. a kind of targeting positron tracer of expressing positive tumor PET imaging for HER2 according to claim 2, is characterized in that: described solvent is the mixture that 20mmol/L biphosphate sodium water solution and ethanol are mixed to get according to the volume ratio of 80:20.
4. a kind of targeting positron tracer of expressing positive tumor PET imaging for HER2 according to claim 2, is characterized in that: the specific activity of described positron tracer is 370 ~ 740MBq/mL.
5. express a preparation method for the targeting positron tracer of positive tumor PET imaging for HER2, it is characterized in that: comprise the following steps:
1) reagent is prepared:
2 ~ 3mg potassium carbonate is dissolved in 0.3 ~ 0.5mL water and obtains reagent 1-1,10 ~ 20mg amino-polyether is dissolved in 1 ~ 2mL acetonitrile and obtains reagent 1-2, reagent 1-1 and reagent 1-2 is mixed to get reagent 1; By 10 ~ 20mg trifluoromethanesulfonic acid-4-N, N, N-trimethyl ammonium benzaldehyde is dissolved in 0.5 ~ 1mL anhydrous dimethyl sulphoxide and obtains reagent 2; By the sodium acetate buffer of 0.1mol/L and ethanol by volume 9:1 mix, obtain reagent 3-1, by 1 ~ 2mg HYNIC-LTVSPWY or HYNIC-LTVSPWY-NH 2be dissolved in the reagent 3-1 of 1 ~ 2mL and obtain reagent 3, described HYNIC-LTVSPWY-NH 2for having the compound such as formula structure shown in 2-1:
Described HYNIC-LTVSPWY is for having the compound such as formula structure shown in 2-2:
By 20mmol/L NaH 2pO 4aqueous solution and ethanol are mixed to get reagent 4-1 according to the volume ratio of 80:20, get 1 ~ 2mL reagent 4-1 as reagent 4;
2) activity prepared by cyclotron be 200 ~ 400mCi [ 18f] fluorion passes into and is adsorbed on anion-exchange column, then by be adsorbed on anion-exchange column [ 18f] fluorion reagent 1 is eluted in the reaction bulb of synthesizer, then under nitrogen or helium protection, be warming up to 60 ~ 70 DEG C and keep 3 ~ 4min, and then evacuation keep 10 ~ 15min after being warming up to 85 ~ 95 DEG C, then stop evacuation and be down to room temperature;
3) through step 2) after, reagent 2 is added and is warming up to 105 ~ 110 DEG C after in described reaction bulb and keeps 10 ~ 15min to obtain mixture 1, thing 1 to be mixed is down to and is added 5 ~ 10mL water in the backward described reaction bulb of room temperature and obtain mixture 2, by mixture 2 successively by cation exchange column and reverse phase solid phase extraction post, then with 0.5 ~ 1.0mL dehydrated alcohol, the adsorbate on reverse phase solid phase extraction post is eluted in described reaction bulb;
4) through step 3) after, reagent 3 is added in described reaction bulb, then be warming up to 100 ~ 105 DEG C and keep 10 ~ 15min, then room temperature is down to, then in described reaction bulb, add reagent 4 obtain mixture 3, with reagent 4-1 for mobile phase, be separated by the preparative liquid chromatography of mixture 3 through synthesizer, collecting retention time on γ chromatogram is the separated product of 13 ~ 18min.
6. a kind of preparation method expressing the targeting positron tracer of positive tumor PET imaging for HER2 according to claim 5, is characterized in that: described synthesizer is GE Tracerlab FX FN automated synthesiser.
7. a kind of preparation method expressing the targeting positron tracer of positive tumor PET imaging for HER2 according to claim 5, is characterized in that: described step 2) to step 4) in, temperature-rise period is heated by electric jacket or microwave heating completes.
8. express a preparation method for the targeting positron tracer of positive tumor PET imaging for HER2, it is characterized in that: comprise the following steps:
1) reagent is prepared:
By 2 ~ 3mg K 2cO 3be dissolved in 0.3 ~ 0.5mL water and obtain reagent 1-1,10 ~ 20mg amino-polyether is dissolved in 1 ~ 2mL acetonitrile and obtains reagent 1-2; Reagent 1-1 and reagent 1-2 is mixed to get reagent 1; Tetra-acetylated for 10 ~ 20mg 1,3,4,6-trifyl mannose is dissolved in 1 ~ 2mL anhydrous acetonitrile and obtains reagent 2; By 0.1mol/L sodium acetate buffer and ethanol by volume 9:1 mix, obtain reagent 3-1, by 1 ~ 2mgHYNIC-LTVSPWY or HYNIC-LTVSPWY-NH 2be dissolved in 1 ~ 2mL reagent 3-1 and obtain reagent 3, described HYNIC-LTVSPWY-NH 2for having the compound such as formula structure shown in 2-1:
Described HYNIC-LTVSPWY is for having the compound such as formula structure shown in 2-2:
By 20mmol/L NaH 2pO 4aqueous solution and ethanol are mixed to get reagent 4-1 according to the volume ratio of 80:20, get 1 ~ 2mL reagent 4-1 as reagent 4;
2) activity prepared by cyclotron be 200 ~ 400mCi [ 18f] fluorion passes into and is adsorbed on anion-exchange column, then by be adsorbed on anion-exchange column [ 18f] fluorion reagent 1 is eluted in the reaction bulb of synthesizer, then under nitrogen or helium protection, be warming up to 60 ~ 70 DEG C and keep 3 ~ 4min, and then evacuation keep 10 ~ 15min after being warming up to 85 ~ 95 DEG C, then stop evacuation and be down to room temperature;
3) through step 2) after, reagent 2 is added and is warming up to 80 ~ 90 DEG C after in described reaction bulb and keeps 4 ~ 5min, be then warming up to 105 ~ 110 DEG C and keep 4 ~ 5min;
4) through step 3) after, described reaction bulb is cooled to 35 DEG C, then being added by 1 ~ 2mL 1mol/L sodium hydrate aqueous solution in described reaction bulb and to carry out hydrolysis 5min, hydrolysis terminates rear salt acid for adjusting pH value to 4 ~ 5, then leaves standstill 5 ~ 10min;
5) through step 4) after, reagent 3 is added in described reaction bulb, then be warming up to 100 ~ 105 DEG C and keep 10 ~ 15min, then room temperature is down to, then in described reaction bulb, add reagent 4 and obtain mixture 3 ', with reagent 4-1 for mobile phase, be separated by the preparative liquid chromatography of mixture 3 ' through synthesizer, collecting retention time on γ chromatogram is the separated product of 10 ~ 15min.
9. a kind of preparation method expressing the targeting positron tracer of positive tumor PET imaging for HER2 according to claim 8, is characterized in that: described synthesizer is GE Tracerlab FX FN automated synthesiser.
10. a kind of preparation method expressing the targeting positron tracer of positive tumor PET imaging for HER2 according to claim 8, it is characterized in that: described step 2) to 3) and step 5) in, temperature-rise period is heated by electric jacket or microwave heating completes.
CN201510218446.6A 2015-04-30 2015-04-30 Targeted positron tracer for realizing PET (Positron Emission Tomography) imaging of positive tumors for HER2 (Human Epidermal Growth Factor Receptor 2) expression, and preparation method of targeted positron tracer Pending CN104800865A (en)

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