CN104815336B - A kind of folic acid dimer complex combined with folacin receptor targeting and its application - Google Patents
A kind of folic acid dimer complex combined with folacin receptor targeting and its application Download PDFInfo
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Abstract
The invention belongs to medical domain, discloses a kind of folic acid dimer complex combined with folacin receptor targeting and its application.The compound is X2Y Z A, wherein：X2The dimer of folic acid is connected for lysine or lysine connects the dimer of folic acid derivatives；Y is the conjugate with " (polyethylene glycol or polyethylene glycol analog derivative) m lysines " structure, m=1,2,3,4,5；Z is material and their derivatives such as DOTA, NOTA, CB TE2A, DTPA, NODAGA；A is the nano-particle of radionuclide, Magnetic Resonance Imaging agent, chemicals or load chemicals；It is connected between X, Y, Z and A each unit with covalent bond.The compound can be used for the medicine for preparing cancer target imaging and treatment, can effectively improve aggregation of the radionuclide in tumour and its transfer stove, improve diagnostic and therapeutic effect.
Description
Technical field
The invention belongs to medical domain, is related to a kind of folic acid dimer complex combined with folacin receptor targeting and its answers
With.
Background technology
Oophoroma is women fatal rate highest malignant tumour, and surgical operation joint platinum class, taxanes chemotherapy are current
Conventional means, and the multiple resistance of chemotherapy in ovarian cancer medicine is common, Postoperative recurrent rate and Lymph Node Metastasis rate are high, and nothing is effectively controlled
Treatment means, survival rate is less than 30% within 5 years[1], find new treatment means and improve oophoroma curative effect and survival rate as clinical at present
The problem faced.
Folacin receptor (folate receptor, FR) is a kind of glycosyl-phosphatidyl inositol coupling protein, mainly including α, β,
4 kinds of hypotypes of γ and δ.Wherein FR α acceptors are in the normal tissue as choroid plexus, placenta tissue and proximal tubular, urothelium are thin
Born of the same parents etc. also show low moderate expression, but are distributed mainly on cell tip surface, because without obvious blood supply and to folic acid and folate conjugate
With reference to few, and the tumor cell surface FR α acceptor quantities such as oophoroma, cervix cancer, breast cancer, liver cancer, nasopharyngeal carcinoma are high-level
Expression, it is seen that in 90% oophoroma[2], in normal cell, FR is optionally expressed on the surface of cell and in polarity point
Cloth, the medicine in blood circulation can not can not also enter normal cell close to this receptor, FR targeted drugs；But in malignant cell
FR distributions lose polarity, and the medicine in blood circulation can contact this receptor, and folic acid (folate acid, FA) acceptor and leaf
Acid and its derivative compatibility (Kd：10-9-10-10M) considerably beyond normal cell, it is often more important that, ovarian cancer tumor cell table
Face FR expression density increases and raised with tumour progression and grade of malignancy, therefore folacin receptor turns into the tumour such as oophoroma in recent years
The focus of radionuclide targeted imaging and Therapy study[3-7], this characteristic is also based on, can be by developer, medicine etc.
With folacin coupled, tumour cell is given in targeting, so as to which the diagnostic imaging such as nuclear medicine image, nuclear magnetic resonance applied to tumour shows
Picture, fluorescent imaging and oncotherapy such as chemotherapy, isotope therapy, immunization therapy, GEM 132 treatment and gene therapy.
FA γ-carboxyl is that FR targetings in recent years are swollen with after the coupling of other small molecules, remaining to keep the high-affinity with FR
The main reason for knurl imaging receives much concern, the small (molecular weight of folate molecule amount：441), without immune prototype, folate-targeted developer energy
Removed from blood rapidly, effectively reduce background tissue institute's raying and increase tumour and the contrast of background, be that it is used as targeting
The main advantage of medicine, imaging radionuclide is (such as99mTc,111In,66/67/68Ga and18F coupling agent DOTA) is passed through
(Tetraazacyclododecane tetra acetic acid), DTPA (diethylene triamine pentacetic acid (DTPA)) etc. mark folic acid,
For clinical (preceding) evaluation of oophoroma imaging[6-10].For folacin receptor active targeting images and treats, tumor cell surface
Folacin receptor density relatively lower slightly (1-3million/ cells) and it is easily saturated[11], folic acid, folacin monomer are in folic acid
Receptor negative tissue checkout time fast (half life：~10 minutes), it is most of to pass through by kidney excretion, and in proximal tubular
Cell surface FR mediations reabsorb and dense poly- in kidney and be detained[12], both turn into FR in recent years and target radiation treatment medicine
The bottleneck of research and development.Blood circulation time is short and tumor locus radioactive uptake is the main reason for limiting its curative effect less.Effectively increase
Radioisotope labeling folic acid and the like is added to be current folacin receptor targeting diagnosis in the intake and delay of oophoroma and control
Treat problem urgently to be resolved hurrily.
Folic acid, folacin, folic acid composite or modified with folic acid carrying medicament nano-particle by with cell membrane table
The FRa in face is combined, and is formed in depression and is swallowed born of the same parents, forms capsula interna, and in the presence of interior cyst membrane proton pump, intracapsular pH value is declined by 7
To 5, FR- ligand complex conformational changes, part is released into the cell, and FR returns to cell membrane, continues to play and part leaf
The specific binding of acid and its derivative and the effect of circulation transhipment medicine, and enter the folic acid separated after born of the same parents with acceptor and its derivative
Thing is then trapped in tumour cell, continues to be degraded.Therefore, after FR and folic acid (and its derivative) are combined and transported, remain to
Recycle and transport folic acid (and its derivative) again, exist for folic acid, folacin, folic acid composite and its isotope labeling thing
The tumor locus aggregation of the high expression of FR provides may.
In recent years, part polymer (such as integrin receptor αVβ3Target polypeptide RGD dimers, the RGD tetramers, six aggressiveness
Deng) and the preparation of nanoparticulate carriers and isotope labeling turn into that to improve neoplastic cell receptor targeting dense poly-[13-16].Wherein part leads to
The ligand concentration for crossing the polyvalency increase acceptor part of aglucon is its main mechanism, i.e., an aglucon and tumour in part polymer
The targeting of cell receptor combines, and will dramatically increase the ligand concentra around neighbouring acceptor, so as to accelerate and the combination adjacent to part
And slow down dissociation.Such as radioisotope labeling RGD2With integrin alphaVβ3Affinity and tumour Targeting distribution than RGD monomer
It is higher by an order of magnitude.Contemplate the dimer by synthesizing folic acid or folacin, folic acid composite in cell can be increased
Intake and tumor locus holdup time, so as to improve imaging quality or curative effect of medication.
In the market also without the radioisotope labeling folic acid dimer more than tumor locus radioactive uptake.
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expression in tumours with 68Ga-labelled mono-,di-and tetrameric RGD
peptides.Eur J Nucl Med,Mol Imaging.2011,38(1):128-37.
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RGD4 and 3PRGD2 for integrin targeted radionuclide therapy.Mol Pharm.2011,8
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The content of the invention
The purpose of the present invention is to provide a kind of folic acid dimer combined with folacin receptor targeting for above-mentioned technical problem
Compound.
It is a further object to provide the application of above-mentioned folic acid dimer complex.
The purpose of the present invention is realized by following technical proposal：
A kind of folic acid dimer complex for imaging and treating for cancer target, the compound have below general formula：
X2-Y-Z-A
Wherein：
X2The dimer of folic acid is connected for lysine or lysine connects the dimer of folic acid derivatives, i.e. (lysine-leaf
Acid)2Or (lysine-folic acid derivatives)2；
Y is the conjugate with " (polyethylene glycol or polyethylene glycol analog derivative) m- lysines " structure, m=1,2,3,4,
5；
Z is DOTA (Cyclen -1,4,7,10- tetraacethyls), NOTA (1,4,7-triaza
cyclononane-1,4,7-triacetic acid)、CB-TE2A
(Bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane)、DTPA
(diethylene triamine penta-acetic acid)、NODAGA(1,4,7-triazacyclononane,1-
glutaric acid-4,7-acetic acid)、Oxo-DO3A(1-oxa-4,7,10-triazacyclododecane-4,7,
10-triacetic acid)、PCTA(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-
Triene-3,6,9-triacetic acid), Hynic (hydrazino-nicotinamide) and their derivative；
A is the nano-particle of radionuclide, Magnetic Resonance Imaging agent (Gd), chemicals or load chemicals；
It is connected between X, Y, Z and A each unit with covalent bond.
Described folic acid dimer complex, wherein：
Described folic acid derivatives are that folinic acid, dihydrofoilic acid, tetrahydrofolic acid, 2- deaminize-hydroxyl folic acid, 1- denitrification leaves
Acid, 3- denitrifications folic acid or 8- denitrification folic acid；
The mean molecule quantity of described polyethylene glycol is 500~40000；
Described polyethylene glycol analog derivative has-O (CH2CH2O) n- structures, n=1,2,3,4,5, polyethylene glycols are spread out
Biological terminal reactive group is maleimide or n-hydroxysuccinimide, by forming stabilization with the carboxyl of lysine end
Covalent bond rise coupling and hydration；
The radionuclide for transmitting β-, the radionuclide of γ or β+ray；
The chemicals is taxol.
Described folic acid composite, wherein the imaging of transmitting β+ray is with radionuclide68Ga、64Cu、111In and99mTc, transmitting β-therapeutic nuclides be89Sr、90Y、177Lu。
X is connected by γ-carboxyl with the amino on Y with covalent bond, and Y is mainly used in Z and X coupling, and Z, which is used for combining, to be radiated
Property nucleic, chemicals, Magnetic Resonance Imaging agent, the nano-particle of load chemicals.
Application of the described folic acid dimer complex in the medicine of cancer target imaging and treatment is prepared.Described is swollen
The tumour that knurl is expressed for folacin receptor height, such as oophoroma, cervix cancer, breast cancer.
This research：1. by chemical synthesis FA dimers, the coupling agents such as DOTA, NOTA, NODAGA are connected by lysine,
And further combined with radionuclide, fluorescent material, chemicals, its general physicochemical property is investigated in the experiment of 2. inside and outsides；3.
Investigate its folacin receptor targeting binding specificity.4. experiment in vivo investigates its pharmacokinetics, toxicology, oophoroma is transplanted
The pharmacodynamics of knurl, Lymph Node Metastasis stove.
The external Solid phase synthesis folic acid dimer complex of this seminar first passage, its structure are (lysine-folic acid
)2- polyethylene glycol2- lysine [i.e. (K-folate)2-PEG2- K (hereinafter referred to as FA2), it can target and combine with folacin receptor, and with
Coupling agent (such as DOTA, DTPA, NOTA, NODAGA) connects, and the radionuclide of coupling agent mediation folic acid dimer is (such as68Ga
、64Cu、111In、99mTc、89Sr、90Y、177Lu mark).Wherein radionuclide can be resulting visualization radionuclide such as68Ga
、64Cu、111In and99mTc or Therapeutic radionuclides are such as89Sr、90Y、177Lu etc..It is external steady that experiment in vitro examines or check it
Qualitative, experiment in vivo investigates its internal pharmacokinetics and biodistribution, and passes through MicroPET/CT (or microSPECT/
CT) Imaging Evaluation its oophoroma tumor position radioactive uptake.
Beneficial effects of the present invention：
Folic acid dimer (FA prepared by the present invention2), and be coupled by chemical modification and developer or curative drug, energy
Combined with cell membrane surface folacin receptor targeting, reach the concentration of increase tumor by local developer or curative drug.Utilize
The folic acid dimer prepare radioisotope labeling thing, can be used as diagnose or treatment oophoroma target medicine, with compared with
Good effect.
Different from antibody, the part is without immunogenicity, and molecular weight is small, and blood removes fast, the rich reticuloendothelial system such as liver and spleen
Intake is less and removing is fast, and distribution is few in being organized in ovary, uterus etc., and in oophoroma, cervix cancer (and its transfer) focus portion
Position is dense poly- more, can be effectively as diagnosis and the carrier for the treatment of radionuclide.
With folic acid single phase ratio, folic acid dimer can be effectively increased its intake in the high expression region of folacin receptor, near
Amplify like rank character, aggregation of the radionuclide in tumour and its transfer stove can be effectively improved, improve diagnostic and treatment is imitated
Fruit.
The emphasis of the present invention is synthesis folic acid dimer and its label, it is intended to is taken the photograph by increasing tumor locus stability
Take, effectively improve imaging quality and treatment curative effect.The present invention for the high expression of folacin receptor tumour (such as oophoroma, cervix cancer,
Breast cancer etc.) and the targeting diagnosis of transfer stove and treatment new approaches and scientific basis are provided, and laid the foundation for it into clinic,
With preferable potential applicability in clinical practice.
Brief description of the drawings
Fig. 1 is DOTA-FA2HPLC figure.
Fig. 2 is DOTA-FA2Mass spectrogram.
Fig. 3 is68Ga-DOTA-FA2HPLC schemes.
Fig. 4 is68Ga-DOTA-FA2Oophoroma (SKOV3) mice with tumor microPET/CT fusion imagings.
Wherein：A, b, c are respectively Coronal 3D, cross-section tomographic image, sagittal plain 3D rendering.
Fig. 5 is lotus human ovarian cancer (SKOV3) and lung cancer (A549) nude mice tail vein injection177Lu-DOTA-FA2Different time
Tumor volume change curve.
Fig. 6 is lotus human ovarian cancer (SKOV3) nude mice tail vein injection177Lu-DOTA-FA21d (upper left) afterwards, 4d (upper right),
8d (lower-left), 16d (bottom right) tumor tissue pathology inspection result (HE：×10)
Embodiment
The invention will be further elaborated by the following examples.
Embodiment 1
First, DOTA-FA2Synthesis and its Quality Control：
The synthesis of folic acid-NHS 1. (succinimide) ester
(1) weigh 440mg folic acid to be dissolved in 40ml DMSO, add EDC.HCl (1- ethyls-(3- dimethylaminos third
Base) phosphinylidyne diimmonium salt hydrochlorate) 200mg ice baths reaction 1h；
(2) weigh 120mgNHS to add in reaction system, reacted at room temperature overnight after ice bath 1h；
(3) filter, boil off organic solvent, washed with distillation, centrifugation is lyophilized stand-by, labeled as A.
2. folic acid-Lys (DOTA) synthesis in solid state
(1) weighing 1.0g wang resins, (wang Resin Tianjin Nankais synthesize 1%DVB, 100-200mesh, 0.8-
1.0mmol/g) in the reaction tube of clean dried, appropriate DMF, swelling activation 30min or so are added, then weighs first ammonia
Base acid Fmoc-Lys (Dde)-OH (N- fluorenylmethyloxycarbonyls-N'- [1- (4,4- dimethyl -2,6- dioxo hexamethylenes subunit) ethyl] -
Lysine) 0.2mmol, DMAP (DMAP) 40mg, DIC 0.3ml is added in reaction tube, and DMF does solvent room temperature
React 3.5h.Reaction finishes is washed 4~6 times with DMF, adds appropriate pyridine and acetic anhydride, volume ratio 1:1, react 30min.Instead
It should finish and be washed 4~6 times with DMF.Then the Fmoc of amino acid is taken off with 20% piperidine solution, takes off common twice 15min, 10min+
5min.Washed 4 times with DMF again, methanol is washed 2 times, is taken out a small amount of resin ninhydrin detection reagent detection, is detected as blueness, you can
Carry out next step reaction.
(2) DOTA raw materials 0.3mmol, the HBTU (BTA-N, N, N', N'- tetra- of tBu (tert-butyl group) protections are weighed
MU hexafluorophosphoric acid ester) 0.3mmol, DMF make solvent, add 0.5ml DIEA (DIPEA) room temperature reactions
1h, reaction finishes is washed 4-6 times with DMF, and detection is colourless to be reacted in next step through row.
(3) DMF solution for adding 2% hydrazine hydrate takes off Dde (see Fmoc-Lys (Dde)-OH) 30min, 10min+10min+
10min, then to be washed 4 times with DMF, methanol is washed twice, is taken out a small amount of resin ninhydrin detection reagent detection, is detected as blueness,
Next step reaction can be carried out.
(4) FMOC-PEG2-COOH, 0.3mmol are weighed, HBTU 0.3mmol, DMF make solvent, add 0.5ml DIEA
1h is reacted at room temperature, reaction finishes is washed 4~6 times with DMF, and the Fmoc of amino acid is then taken off with 20% piperidine solution, is taken off twice altogether
15min, 10min+5min.Washed 4 times with DMF again, methanol is washed 2 times, takes out a small amount of resin ninhydrin detection reagent detection, detection
For blueness, you can carry out next step reaction.
(5) FMOC-Lys (Dde)-COOH, 0.3mmol, HBTU 0.3mmol, DMF are weighed and makees solvent, adds 0.5ml's
DIEA reacts at room temperature 1h, and reaction finishes is washed 4~6 times with DMF, and the Fmoc of amino acid is then taken off with 20% piperidine solution, takes off two
Secondary common 15min, 10min+5min.To be washed 4 times with DMF again, methanol is washed 2 times, takes out a small amount of resin ninhydrin detection reagent detection,
It is detected as blueness, you can carry out next step reaction.
(6) weigh A (folic acid-NHS esters) 0.3mmol, DMSO and make solvent, add DIEA 5 and drip, react at room temperature 1h；React
Finish and washed 4~6 times with DMF.
(7) DMF solution for adding 2% hydrazine hydrate takes off Dde 30min, 10min+10min+10min, then washes 4 with DMF
Secondary, methanol is washed twice, is taken out a small amount of resin ninhydrin detection reagent detection, is detected as blueness, you can carry out next step reaction.
(8) BOC { tertbutyloxycarbonyl }-Lys (Fmoc)-COOH, 0.3mmol, HBTU 0.3mmol, DMF are weighed and makees solvent,
0.5ml DIEA room temperature reaction 1h are added, reaction finishes is washed 4~6 times with DMF, then takes off amino acid with 20% piperidine solution
Fmoc { fluorenylmethyloxycarbonyl }, take off common twice 15min, 10min+5min.Washed 4 times with DMF again, methanol is washed 2 times, takes out a small amount of tree
Fat is detected with ninhydrin detection reagent, is detected as blueness, you can carries out next step reaction.
(9) weigh A (folic acid-NHS esters) 0.3mmol, DMSO and make solvent, add DIEA 5 and drip, react at room temperature 1h；React
Finish and washed 4~6 times with DMF；Methanol is washed twice, is drained.
(10) 2h finally is cut with 95% trifluoroacetic acid cutting liquid, reaction solution filters, and obtains the trifluoroacetic acid solution of folic acid, uses
Ether precipitates, and centrifugation, is then washed 3~5 times with ether again, obtains white solid, purified through HPLC, freezes, uses high-efficient liquid phase color
Molecular weight is carried out to target compound for spectrometer (HPLC) and mass spectrograph (MS) and chemical purity carries out quality analysis.
(11) the folic acid dimer of different PEG contents (PEGn) is synthesized, except in (4) step, with outside FMOC-PEGn-COOH,
Other building-up processes are identical with above-mentioned reaction.
3. quality analysis
Peptide sequence, molecular weight and chemistry are carried out to target compound using high performance liquid chromatograph (HPLC) and mass spectrograph (MS)
Purity carries out quality analysis.
HPLC is analyzed：Parameter：C18 posts, (4.6 × 250mm), mobile phase：Solvent orange 2 A be containing 0.1% trifluoroacetic acetonitrile,
Solvent B is as follows containing 0.1% trifluoroacetic ultra-pure water, eluent gradient：
Flow velocity：1.0ml/min, ultraviolet wavelength：220nm, injection volume are 10 μ l.
Mass spectral analysis condition：
Flow rate:0.2ml/min Run Time:1min
Buffer A:0.1%HCOOH in water Buffer B:0.1%HCOOH in Acetonitrile
Two, results
1.DOTA-FA2Molecular structural formula is shown in formula 1.
2. Quality Control image
(1)DOTA-FA2HPLC inspection results see Fig. 1.
DOTA-FA2Chemical purity is 98%, and in -4 DEG C or -20 DEG C storages, chemical property is relatively stable, HPLC image knots
Fruit shows, DOTA-FA2Appearance time is 9.524min.
(2)DOTA-FA2Mass spectrogram
DOTA-FA2Mass spectrogram result see Fig. 2.
Embodiment 2：68Ga-DOTA-FA2Preparation and inside and outside experimental study
First,68Ga-DOTA-FA2Preparation
Prepare DOTA-FA2Solution is 2mg/ml, and HEPES is 1M (pH：7), GaCl3For 1-10mCi/ml, 25 μ l 4- is taken
Hydroxyethyl piperazineethanesulfonic acid (HEPES) liquid (concentration 1M, collocation method：23.8gHEPES is dissolved in about 90ml water, uses NaOH
It is 6.8 to 8.2 to adjust pH, is then settled to 100ml with water), it is sequentially added into 0.7-5 μ L DOTA-FA2Solution (2mg/ml)
With 100 μ l's68GaCl3(radioactive concentration 1-10mCi/ml), it is placed in 95 DEG C of water-bath and heats, reaction 15-20min is produced
Arrive68Ga-DOTA-FA2.20 μ L rows HPLC are taken to check that parameter is the same, as a result sees Fig. 3.
Fig. 3 is shown68Ga-DOTA-FA2HPLC inspection results, radio-chemical purity 99.1%.
2nd,68Ga-DOTA-FA2 experiment in vitro
68After Ga-DOTA-FA2 synthesis, it is detected by radioactivity-HPLC, method and parameter are the same, as a result show
Show68The Ga-DOTA-FA2 ultraviolet peaks of HPLC and radioactivity peak occurs in same time, top coal drawing (97.2 ± 0.5%), and physiology
37 DEG C of incubations in salt solution, take 20ul to carry out HPLC inspections respectively after placing 2h, 4h, 6h, as a result have no68Ga dissociates peak, shows it
Vitro stability is good, and top coal drawing is (96.5 ± 0.4%) during 6h.
3rd,68Ga-DOTA-FA2Mice with tumor vivo biodistribution credit cloth
(1) choose lotus oophoroma (SKOV3, folate receptor-positive) nude mice 35 (female, 18 ± 2g of body weight), sampling with
Machine is divided into 7 groups, and (1-5 groups are68Ga-DOTA-FA2Experimental group, the 6th group is receptor blocking closed group, and the 7th group is68Ga-DOTA-FA
Control group), every group 5,1-5 groups are respectively in tail vein injection68Ga-DOTA-FA2(74kBq/0.1mL is 2.0 μ Ci/0.1mL)
10min, 30min, 60,120min afterwards, 4h, heart puncturing extracting blood 1.0ml, dissect and separate tumour, the heart, liver, spleen, lung, kidney,
The internal organs such as stomach, intestines, brain, muscle, bone, claim weight in wet base, γ calculating instruments (Perkin Elmer Wizard-1480, Shelton, CT)
Determine radiocounting, calculated after radiation decay correction down each time point main organs radioactivity account for injection gross activity it is every
The percentage (ID%) and/or the radioactivity of unit mass internal organs that minute counts account for the hundred of injection gross activity flicker number per minute
Divide rate (%ID/g).Stool and urine investigation radioactivity excretion is collected respectively and HPLC investigates its internal stability.Wherein, receptor blocking
Closed group：Every mouse is through vein preform injection DOTA-FA2(10mg/Kg body weight), then tail vein injection68Ga-DOTA-FA2Afterwards
120min is put to death, and binding specificity is investigated in the experiment of row biodistribution (experimentation is the same).68Ga-DOTA-FA control groups：Often
Mouse is respectively in tail vein injection68Ga-DOTA-FA (74kBq/0.1mL is 2.0 μ Ci/0.1mL) is put to death after 120min afterwards, row life
Thing distribution experiments, concrete operations are the same.
As a result：68Ga-DOTA-FA2After oophoroma lotus knurl tail vein injection, removed soon from blood, medicine mainly passes through
Liver and kidney excretion, the position radioactivity such as visible liver kidney spleen lung substantially lowers during 60min, and enteron aisle radioactive uptake slightly increases
Add, consideration may with it is a small amount of68Ga-DOTA-FA2, the normal structure such as ovary, uterus radiation relevant into enteron aisle is drained through liver and gall
Property intake be significantly lower than tumour, subcutaneous oophoroma/ovary tissue radioactive uptake ratio is (10.3 ± 0.9) during 120min, leaf
After acid acceptor blocks, oophoroma subcutaneous transplantation knurl radioactive uptake is substantially blocked during 120min, radioactive uptake (%ID/g)
For (2.19 ± 0.13), group (11.07 ± 1.83) is not blocked substantially less than.When radiopharmaceutical is injected intravenously 120min, ovary
Cancer pair68Ga-DOTA-FA2Intake be significantly higher than68Ga-DOTA-FA (7.36 ± 1.28), difference is statistically significant.As a result see
Table 1.
Table 1：Oophoroma mice with tumor is injected68Ga-DOTA-FA2Each internal organs radioactive uptake (%ID/g) of different time points afterwards
Note：Every group of mouse number is 5, and 60minB is receptor blocking closed group data, and 120minFA is68Ga-DOTA-FA pairs
According to a group data, data with_ Represent.
4th,68Ga-DOTA-FA2Oophoroma (SKOV3) mice with tumor microPET/CT is imaged
Oophoroma (SKOV3) lotus knurl tail vein injection 0.1mL (3.7MBq)68Ga-DOTA-FA260min afterwards, isoflurane
(Isoflurane) it is placed on MicroPET/CT equipment scanning beds and images after anaesthetizing.Micro PET/CT work stations are Inveon
Acquisition Workplace (IAW) 1.5.0.28, MicroCT acquisition parameter：80kV voltages, 500 μ A electric currents and 1100ms
Time for exposure, sweep time 10min, microPET time of developing 30min, data are dispersed through using IAW softwares, random counter,
Coincidence correction, coronal-plane, cross section, sagittal plane are rebuild by filtered back-projection (Filtered Back projection)
Faultage image is analyzed.Using Inveon ResearchWorkplace softwares, based on PET/CT fused images, tumour is delineated
Region of interest (Regions of Interest, ROI), calculates tumour radiotherapy intake and tumour/muscle (T/M) radioactivity is taken the photograph
Take ratio.
As a result see that Fig. 4, Fig. 4 are68Ga-DOTA-FA2Oophoroma (SKOV3) mice with tumor microPET/CT fusions imaging (a,
B, c are respectively Coronal 3D, cross-section tomographic image, sagittal plain 3D rendering), the visible obvious radioactivity of tumor locus is taken the photograph as seen from Figure 4
Take, tumour/muscle ratio is (9.7 ± 3.1), and subcutaneous oophoroma tumor position SUV is (9.84 ± 2.05), liver, spleen, lung
Obvious radioactive uptake, kidney and the visible obvious increased radioactivity of bladder are showed no Deng important organ, prompts folic acid dimer master
To pass through urinary system to drain, and drain comparatively fast, the visible a small amount of radioactive uptake distribution of enteron aisle, consider with some drugs via liver
Courage metabolism is relevant.
Embodiment 3：64Cu-DOTA-FA2Preparation and inside and outside experimental study
Except radionuclide is64CuCl2Outside, remaining is the same as embodiment 2.
As a result 1：64Cu-DOTA-FA2At once top coal drawing is (97.3 ± 2.56%) to drug labelling, and is placed in physiology salt in vitro
HPLC is carried out respectively after 37 DEG C of incubations 2h, 4h, 6h in water and checks that its top coal drawing investigates its vitro stability, is as a result had no64Cu is swum
From peak, show that its vitro stability is good, top coal drawing is (96.5 ± 1.62%) during 6h.
As a result 2：Oophoroma mice with tumor64Cu-DOTA-FA2After tail vein injection, vivo biodistribution credit cloth with68Ga-DOTA-
FA2Similar, it is that its vivo biodistribution characteristic distributions depends primarily on DOTA-FA to consider main cause2, show as mainly through uropoiesis
System (kidney and bladder) is drained, but is removed soon, and 120min may occur in which that kidney position radioactive uptake substantially lowers, and early stage liver can
See development, but it is dense it is poly- reduced rapidly with time lengthening, enteron aisle radioactive uptake slightly increases, brain position radioactive uptake continue compared with
Low, the normal structure such as ovary and uterus radioactive uptake is significantly lower than tumour, tumour/ovary tissue radioactive uptake during 120min
Ratio is (9.85 ± 0.74).After folacin receptor blocks, oophoroma subcutaneous transplantation knurl radioactive uptake is substantially hindered during 120min
Disconnected, radioactive uptake (%ID/g) is (1.41 ± 0.37), does not block group (9.23 ± 1.57) substantially less than.Radiopharmaceutical is quiet
120min after arteries and veins injection, subcutaneous oophoroma pair64Cu-DOTA-FA2Intake be significantly higher than68Ga-DOTA-FA (6.51 ± 1.32),
Difference is statistically significant.It the results are shown in Table 2.
Table 2：Oophoroma mice with tumor is injected64Cu-DOTA-FA2Each internal organs radioactive uptake (%ID/g) of different time points afterwards
Note：Every group of mouse number is 5, and 60minB is receptor blocking closed group data, and 120minFA is68Ga-DOTA-FA pairs
According to a group data, data withRepresent.
Embodiment 4：111In-DOTA-FA2Preparation and inside and outside experimental study
Except radionuclide is111Outside In, remaining is the same as embodiment 2.
As a result 1：111In-DOTA-FA2Top coal drawing (97.9 ± 1.9%), and 37 DEG C of incubations in physiological saline, placement 2h,
Take 20ul to carry out HPLC inspections after 4h, 6h respectively, as a result have no64Cu dissociates peak, shows that its vitro stability is good, top coal drawing during 6h
For (97.5 ± 0.39%).
As a result 2：Oophoroma lotus knurl tail vein injection111In-DOTA-FA2Afterwards, vivo biodistribution credit cloth with68Ga-DOTA-
FA2Similar, its blood background activity is removed fast, and the radiation of liver kidney position is shown as and mainly through liver and kidney excretion, during 60min
Property intake is obvious lowers, enteron aisle and bladder radiation intake increase, the internal organs radioactive uptake such as brain, lung continue relatively low, prostate
Normal structure radioactive uptake is significantly lower than tumour, during 120min tumour/ovary tissue radioactive uptake ratio for (9.91 ±
0.52).After folacin receptor blocks, oophoroma subcutaneous transplantation knurl radioactive uptake is substantially blocked during 120min, radioactive uptake
(%ID/g) is (1.47 ± 0.37), does not block group (9.83 ± 0.84) substantially less than.After radiopharmaceutical intravenous injection
120min, subcutaneous oophoroma pair64Cu-DOTA-FA2Intake (%ID/g) be significantly higher than68Ga-DOTA-FA (6.37 ± 0.47),
Difference is statistically significant.It the results are shown in Table 3.
Table 3：Oophoroma mice with tumor is injected111In-DOTA-FA2Each internal organs radioactive uptake (%ID/g) of different time points afterwards
Note：Every group of mouse number is 5, and 60minB is receptor blocking closed group data, and 120minFA is68Ga-DOTA-FA pairs
According to a group data, data withRepresent.
Embodiment 5：177Lu-DOTA-FA2Preparation and inside and outside experimental study
Except radionuclide is177Remaining is the same as embodiment 2 outside Lu.
Log P value experimental programs：Take isometric n-octyl alcohol and PBS solution (25.0mM, PH：7.4)(4ml：4ml) it is placed in true
Mixed in empty receiving flask, it is standby.Take preparation177Lu-DOTA-FA2With177Lu-DOTA-FA, isolated and purified through HPLC, under negative pressure
After organic solvent volatilization, the measure radiocounting of γ calculating instruments, the above-mentioned octanol/PBS mixed solutions prepared are then added, are held
Continuous concussion mixes 2h, and then high speed centrifugation (7000rpm, 15min), takes isometric organic phase and aqueous phase to distinguish by γ calculating instruments
Radiocounting is determined, calculates lipid P (organic phase counting/aqueous phase counts), experiment is repeated 3 times.
As a result 1：177Lu-DOTA-FA2Top coal drawing (98.7 ± 1.23), and 37 DEG C of incubations in physiological saline, placement 2h, 4h,
Take 20ul to carry out HPLC inspections after 6h respectively, as a result have no177Lu dissociates peak, shows that its vitro stability is good, and top coal drawing is during 6h
(96.1 ± 2.76%).177Lu-DOTA-FA2It is (1.97 ± 0.36) in n-octyl alcohol/PBS distribution coefficients, is higher than177Lu-DOTA-
FA (0.76 ± 0.11).
As a result 2：Oophoroma lotus knurl tail vein injection177Lu-DOTA-FA2Afterwards, vivo biodistribution credit cloth with68Ga-DOTA-
FA2It is similar,177Lu-DOTA-FA2Blood background activity is removed fast, is shown as and mainly through liver and kidney excretion, during 120min
Liver kidney position radioactive uptake substantially lowers, and enteron aisle and the internal organs radioactive uptake such as bladder radiation intake increase, brain, lung continue
Relatively low, ovary normal structure radioactive uptake is significantly lower than tumour, and tumour/ovary tissue radioactive uptake ratio is during 120min
(10.72±0.38).After folacin receptor blocks, oophoroma subcutaneous transplantation knurl radioactive uptake is substantially blocked during 120min, is put
Penetrating property intake (%ID/g) is (1.27 ± 0.36), does not block group (8.39 ± 1.1) substantially less than.Radiopharmaceutical is injected intravenously
120min afterwards, subcutaneous oophoroma pair177Lu-DOTA-FA2Intake (%ID/g) be significantly higher than177Lu-DOTA-FA(5.11±
0.73).It the results are shown in Table 4.
Table 4：Oophoroma mice with tumor is injected177Lu-DOTA-FA2Each internal organs radioactive uptake (%ID/g) of different time points afterwards
Note：Every group of mouse number is 5, and 60minB is receptor blocking closed group data, and 120minFA is68Ga-DOTA-FA pairs
According to a group data, data withRepresent.
Embodiment 6：177Lu-DOTA-FA2Pharmacodynamics test
Oophoroma (SKOV3：The high expression of FR) subcutaneous transplantation knurl animal model point 6 groups (every group 4), lotus lung cancer A549 (FR
Radiolucent table reaches) animal model be control group.Tail vein injection177Lu-DOTA-FA2(17.5MBq/0.1ml × 0.1ml), is controlled
Mouse body weight is every other day determined during treatment and determines tumour maximum major diameter and vertical minor axis with slide measure, passes through formula meter
Calculate gross tumor volume V, calculation formula V=ab2/ 2 (a is maximum major diameter, and b is vertical minor axis), while pay close attention to mice with tumor one
As situation such as diet, skin colour, the state of mind, active situation etc., dynamic monitoring medicine toxic side effect.In drug therapy
The previous day (i.e. -1d), treatment 1d, 4d, 8d, 16d, 32d, every group randomly chooses 3 execution respectively, takes tumour and important organ
Carry out pathological examination.
As a result：177Lu-DOTA-FA2Security is good, and (17.5MBq) mice with tumor body weight has no significant change after intravenous injection,
FR is high, and expression SKOV3 groups reach A549 group no significant differences, mice with tumor diet, skin colour, activity feelings with FR radiolucent tables
Condition is shown no obvious abnormalities.And mice with tumor subcutaneous transplantation knurl size variation is shown in that Fig. 5, Fig. 5 are shown in177Lu-DOTA-FA2Intravenous injection
Afterwards, SKOV3 oophoromas volume has no significant change, before gross tumor volume is slightly below treated during 16d, but no significant difference,
And the lung cancer A549 groups that FR expression is negative, gross tumor volume gradually increase,177Lu-DOTA-FA2The 11st day after intravenous injection, swell
Knurl volume (381.3 ± 114.9mm3) apparently higher than (51.3 ± 15.9mm before treatment3) and with time SKOV3 group (61.6 ±
26.1mm3).Pathological examination results are shown in that Fig. 6, Fig. 6 show that ovarian cancer tissue exists177Lu-DOTA-FA2 intravenous injection after 4 days it is visible
Stove shape necrosis, visible sheet is substantially downright bad during 8d, during which visible focal incomplete tumor, and gross tumor volume is not during 16d
See and be obviously reduced, but internal tumours cell is largely downright bad, exists without obvious tumour cell, and the main organs such as liver kidney have no obvious different
Often.
Embodiment 7.68Ga-DOTA-K-PEG4-(K-folate)2Synthesis and its inside and outside physicochemical property investigate
The specific experiment process of embodiment 7 with embodiment 1, wherein68Ga-(K-folate)2-PEG4-K-FA2Synthesis see
Embodiment 1 [one, 1.], except in (4) step, using FMOC-PEG4Outside-COOH, other building-up processes and the complete phase of above-mentioned reaction
Together.Prepare68Ga-DOTA-K-PEG4-(K-folate)2For mark rate up to 98.5%, vitro stability is good, position HPLC during 3h
Determine top coal drawing and be still up to 96.8%.Vivo biodistribution distribution results are shown：68Ga-DOTA-K-PEG4-(K-folate)2Lotus knurl
Remove fast after tail vein injection, in blood, medicine is mainly through kidney excretion, visible non-target organ increased radioactivity during 60min
It is obvious to lower, the increase of enteron aisle radioactive uptake, consider, the normal structure such as ovary, uterus relevant through liver and gall excretion with medicine
Radioactive uptake is significantly lower than tumour, during 120min subcutaneous oophoroma/ovary tissue radioactive uptake ratio for (11.7 ±
1.4) after, folacin receptor blocks, oophoroma subcutaneous transplantation knurl radioactive uptake is substantially blocked during 120min, radioactive uptake
(%ID/g) is (2.72 ± 0.27), does not block group (9.52 ± 0.97) substantially less than.
Claims (6)
1. a kind of folic acid dimer complex for imaging and treating for cancer target, it is characterised in that there is below general formula：
X2-Y-Z-A
Wherein：
X2The dimer of folic acid is connected for lysine or lysine connects the dimer of folic acid derivatives；
Y is the conjugate with " (polyethylene glycol or polyethylene glycol analog derivative) m- lysines " structure, m=1,2,3,4,5；
Z is DOTA, NOTA, CB-TE2A, DTPA, NODAGA, oxo-DO3A, PCTA, Hynic and their derivative；
A is the nano-particle of radionuclide, Magnetic Resonance Imaging agent, chemicals or load chemicals；
It is connected between X, Y, Z and A each unit with covalent bond；
The folic acid dimer complex uses external Solid phase synthesis.
2. folic acid dimer complex according to claim 1, it is characterised in that：
Described folic acid derivatives be folinic acid, dihydrofoilic acid, tetrahydrofolic acid, 2- deaminize-hydroxyl folic acid, 1- denitrifications folic acid,
3- denitrifications folic acid or 8- denitrification folic acid；
The mean molecule quantity of described polyethylene glycol is 500~40000；
Described polyethylene glycol analog derivative has-O (CH2CH2O) n- repetitive structures, n=1,2,3,4,5, polyethylene glycols are spread out
Biological terminal reactive group is maleimide or n-hydroxysuccinimide, by forming stabilization with the carboxyl of lysine end
Covalent bond rise coupling and hydration；
The radionuclide for transmitting β-, the radionuclide of γ or β+ray；
The chemicals is taxol.
3. folic acid composite according to claim 2, it is characterised in that transmitting β+ray imaging be with radionuclide68Ga、64Cu、111In and99mTc, transmitting β-radionuclide be89Sr、90Y、177Lu。
4. application of the folic acid dimer complex in the medicine of cancer target imaging and treatment is prepared described in claim 1.
5. application according to claim 4, it is characterised in that the tumour is the tumour of the high expression of folacin receptor.
6. application according to claim 5, it is characterised in that the tumour of the high expression of the folacin receptor is oophoroma, uterus
Neck cancer, breast cancer.
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