CN102617728A

CN102617728A - 18F labeled product of Exendin-4, preparation method and application

Info

Publication number: CN102617728A
Application number: CN2012101076757A
Authority: CN
Inventors: 张一帆; 梁胜; 乌海飞
Original assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Current assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Priority date: 2012-04-13
Filing date: 2012-04-13
Publication date: 2012-08-01
Anticipated expiration: 2032-04-13
Also published as: CN102617728B

Abstract

The invention discloses a [ ¹⁸ F]FB-Exendin-4 labeled product and its preparation method and application. The intermediate is prepared through a series of reactions of ¹⁸ ^F- and ethyl 4-trimethylamine benzoate trifluorosulfonate. N-succinimide 4-[ ¹⁸ F]fluorobenzoate ([ ¹⁸ F]SFB) is then combined with the polypeptide Exendin-4 to obtain the [ ¹⁸ F]FB-Exendin-4 labeled polypeptide. The radiochemical purity of the labeled product disclosed by the invention is greater than 98%, the labeling rate is 35%, and the stability in vitro is good.

Description

The 18F marked product of a kind of Exendin-4 and preparation method and application

Technical field

The present invention relates to the radiopharmaceuticals field.

Background technology

The method of the level diagnosis of insulinoma mainly contains functional trial and imaging examination etc.Functional trial comprises that stimulating hepatic vein to get blood through the arterial calcium agent surveys Regular Insulin (ASVS), gets blood through skin through the segmentation of hepatic portal arteries and veins and survey Regular Insulin (PTPC), put the pipe segmentation through the hilus lienis venipuncture and get the quick insulin assay of blood (PVS); Though and method susceptibility such as EUS, DSA are high; But be the inspection of wound property, clinically be difficult to widespread use.Conventional imaging localization method has CT, MRI etc., but sensitivity is not high.

Somatostatin receptor (SSTR) video picture is applied to the Position Research of insulinoma the earliest, uses maximum to be ¹¹¹In mark Octreotide, but be lower than 50% for the recall rate of insulinoma.This be because ¹¹¹Two hypotype SSTR of the main and SSTR of In-Octreotide ₂And SSTR ₅In conjunction with, and these two kinds of receptor subtypes distribute seldom in the insulinoma β cell, so susceptibility lowers.Because therefore insulinoma high expression level GLP-1 acceptor, can carry out the specificity video picture and the location of insulinoma through radioisotope labeling GLP-1 and analogue thereof.

The micromolecule polypeptide that GLP-1 is made up of 30 amino acid can mediate the secretion of insulin effect through the special GLP-1 acceptor in beta Cell of islet surface.But GLP-l can (transformation period only about 2min, therefore, have greatly been limited its application clinically in the body for dipeptidyl peptidase-IV, the quick down degraded of effect DPP-IV) at dipeptidyl peptidase-IV.The GLP-l analogue like Exendin-4 and Exendin-3, has the biological activity similar with GLP-1, can resist the degraded of dipeptidyl peptidase-IV (dipeptidyl peptidase-IV), long half time in the body.Therefore, carrying out the monitoring that video picture and the beta Cell of islet of islet cell tumor transplant through radioisotope labeling GLP-1 analogue is developed faster.

In early days, Gotthardt etc. passes through ¹²³The GLP-1 of I mark and analogue Exendin-3 thereof have carried out video picture research in the rat insulin knurl die body.Research shows, but the insulinoma specificity with ¹²³The Exendin-3 of I mark combines.But owing to lack tyrosine in the primary structure of Exendin-3, radioiodination is difficulty.Wild etc. passes through afterwards ¹¹¹In mark GLP-1 analogue Exendin-4, i.e. [Lys ⁴⁰(Ahx-DTPA- ¹¹¹In) NH ₂] exendin-4, carried out the insulinoma video picture of transgenic mouse Rip1Tag2 through pin hole (pinhole) SPECT/MRI and SPECT/CT, the demonstration tumour is obviously absorbed, and is higher than lung and pancreatic tissue.Consider ¹¹¹The Exendin-4 of In mark may cause higher relatively radiation absorbed dose to the patient, and Wild etc. passes through respectively afterwards ⁶⁸Ga with ^99mTc mark Exendin-4, SPECT and PET show the target/Ben Bigao of tumour, can show less insulinoma.But ⁶⁸Ga is the positron radionuclide of producer preparation, and spatial resolution is low, and clinical application is restricted. ^99mThough Tc is radionuclide commonly used, the sensitivity of clinical SPECT video picture is lower than the PET video picture.The PET video picture has spatial resolution and highly sensitive characteristics, is more suitable for the clinical diagnosis of insulinoma. ¹⁸F is the most frequently used positron radionuclide of PET/CT video picture, convenient sources, so we attempt passing through ¹⁸The FS that F mark Exendin-4 carries out the insulinoma video picture.

Because the molecular structure of polypeptide relative complex can not carry out directly that nucleophilic is fluoridized or close electricity is fluoridized, therefore, ¹⁸F labeling polypeptide class medicine must pass through midbody and could realize.At present, ¹⁸F mark midbody has been developed and widespread use fast, as carry out midbody through methods such as acylations, amidation, imidization, alkylation, photochemical connection, click chemistry, silication, boronation and solid phase synthesis ¹⁸The F mark.But the different markers method all has its advantage and limitation, wherein, through acylations approach mark synthetic N-succinimide 4-[ ¹⁸F] the fluorobenzoic acid ester ([ ¹⁸F] SFB),, be considered to optimum and be used as according to its synthetic body internal stability that reaches purification process, putting productive rate and label probe ¹⁸F mark midbody.And [ ¹⁸F] SFB has been widely used in the connection of various biomolecules such as monoclonal antibody, RGD (arginine-glycine-aspartic acid), Annexin-V.

(Eur J Nucl Med Mol Imaging 2012 such as Kiesewetter; 39:463 – 473) through the mark midbody [ ¹⁸F] FBEM carried out polypeptide Exendin-4 synthetic ([ ¹⁸F] FBEM-[Cys ⁴⁰]-exendin-4), and carried out the video picture of INS-1 insulinoma model mouse small animal position emission tomography (PET), the research demonstration, [ ¹⁸F] FBEM-[Cys ⁴⁰Putting of]-exendin-4 productive rate be 6.4% (with ¹⁸F ^-For initiator calculates), radiochemicsl purity>96%; The clear video picture of insulinoma.The Exendin-4 analogue of reported in literature is on the Exendin-4 basis, to carry out structural modification and get, and has just added Cys in Exendin-4 carboxyl section, and the polypeptide that contains Cys is oxidized easily, and stability will be worse than the polypeptide that does not contain Cys; Secondly, the marking method of reported in literature, comprehensively putting productivity ratio is lower, is merely 6.4%.

Summary of the invention

Technical problem to be solved by this invention is, provides a kind of vitro stability is good, putting productive rate is high Exendin-4's ¹⁸The F marked product.

Second technical problem to be solved by this invention is to provide a kind of Exendin-4's ¹⁸The preparation method of F marked product.

The 3rd technical problem to be solved by this invention be, provide [ ¹⁸F] application of FB-Exendin-4 in preparation insulinoma localization diagnosis medicine.

For realizing first purpose of the present invention, the present invention discloses a kind of Exendin-4's ¹⁸F marked product, said marked product be [ ¹⁸F] FB-Exendin-4.

Be to realize second purpose of the present invention, the present invention open [ ¹⁸F] preparation method of FB-Exendin-4, may further comprise the steps:

(1) ¹⁸F ^-With the reaction of 4-Trimethylamine 99 ethyl benzoate three fluoro sulfonates, preparation intermediate N succinimide 4-[ ¹⁸F] the fluorobenzoic acid ester [ ¹⁸F] SFB;

(2) [ ¹⁸F] SFB and Eexdin-4 coupling obtain [ ¹⁸F] FB-Exendin-4.

As a preferred version, step (1) comprising: through magnetic resonance acceleator PET trace preparation [ ¹⁸F] F ^-, to fill activation [ ¹⁸F] F ^-Reaction flask in add 4-Trimethylamine 99 ethyl benzoate three fluoro sulfonates, with NaOH, HCl reaction, reaction solution is through the drip washing of activatory Sep-Pak C18 post hydrochloric acid, N successively ₂Dry up, again with second eyeball wash-out obtain 4-[ ¹⁸F] fluorobenzoic acid; Add hydroxide tetrapropyl amine, azeotropic drying adds people O-(N-succinimide)-1,1,3, and acidifying with acetic acid is used in 3-tetramethyl-urea Tetrafluoroboric acid ester TSTU reaction then; Above-mentioned solution through activatory Sep-Pak C18 post, is used successively V: VSecond eyeball/water wash of=1/7 is used N ₂Dry up Sep-Pak C18 post, use the methylene dichloride wash-out; Chemical purity is penetrated in TLC, HPLC measuring, and activity instrumentation activity is calculated putting productive rate.

Step (2) comprising: will [ ¹⁸F] SFB is dissolved in the acetonitrile, adds Exendin-4, and it is 0.1M borax-borate buffer of 8.5 that Exendin-4 is dissolved in the pH value, reacts 30min under the room temperature; Separate through HPLC behind the reacting liquid filtering; Used chromatographic column is ZORBAX SB C18; 5 μ m, 9.4 mm * 150 mm, the HPLC system adopts the speed drip washing of following gradient with 3 mL/min: wherein A is the water that contains 0.1% trifluoroacetic acid for acetonitrile, the B that contains 0.1% trifluoroacetic acid mutually mutually; 0-20 min, A is raised to 90% from 16%; 20-30 min, A are 90%; Collect t _RThe peak of=18-19.5 min, products therefrom solution, the rotation evaporate to dryness, again with the dissolving of 0.9% NaCl injection liquid, must the title product compound [ ¹⁸F] FB-Exendin-4 is through Radio-TLC, and selecting developping agent for use is ETHYLE ACETATE and methylene dichloride, and both volume ratios are 3:1, and Radio-HPLC characterizes, and the Radio-HPLC analytical procedure is identical with the preparation method, but flow velocity is 1mL/min, chromatographic column is μ Bondapak ^TMC18,5 μ m, 300 mm * 3.9 mm.

The invention has the advantages that, the present invention successfully carried out [ ¹⁸F] SFB and polypeptide Exendin-4 ([ ¹⁸F] FB-Exendin-4) synthetic, the radiochemicsl purity of marked product is greater than 98%, mark rate be 35% (with ¹⁸F ^-For initiator calculates), vitro stability is good.

Description of drawings

Fig. 1 be [ ¹⁸F] synthetic route of SFB.

Fig. 2 be labeling polypeptide [ ¹⁸F] the radioactivity TLC collection of illustrative plates of FB-Exendin-4.

Fig. 3 be labeling polypeptide [ ¹⁸F] the radioactivity HPLC of FB-Exendin-4 analyzes collection of illustrative plates.

Fig. 4 is external competition binding curve, and the IC50 value that Graphpad Prism5 analyzes exendin-4 is 0.99nmol/L.

Fig. 5 is a saturation curve, ¹²⁵The equilibrium dissociation constant K of I-exendin-4 and GLP-1 acceptor _dValue is 56.64, maximum combined constant B _MaxBe 4.651 * 10 ⁷

Fig. 6 does ¹⁸F-FB-Exendin-4 insulinoma nude mice model 30min and 120min small animal position emission tomography (PET)/CT video picture.

Fig. 7 is the SUV value of each internal organs 30min and 120min.

The SUV value that Fig. 8 does not block and blocks for each internal organs 60min.

Fig. 9 is the %ID/g value of each internal organs of nude mice behind the 120min.

Embodiment

Below in conjunction with accompanying drawing to provided by the invention about [ ¹⁸F] preparation method of FB-Exendin-4 and the embodiment of application elaborate.

Embodiment 1, the preparation [ ¹⁸F] FB-Exendin-4

Material

Exendin-4 is by the Shanghai bio tech ltd (Shanghai, China) of shining by force, and is synthetic according to standard FMOC chemical process.4-fluorobenzoic acid (SB) is available from Switzerland Fluka company. ¹⁸F is provided by Ke Xing pharmaceutcal corporation, Ltd accelerator centre, through the proton irradiation oxygen enrichment ¹⁸O-H ₂0 obtains.High-performance liquid phase systems Dionex P680 summit HPLC analytical system (U.S. Dionex company) is furnished with Bioscan flow-count detector (U.S. Bioscan company); (Wisconsin USA) produces magnetic resonance acceleator for GE, Waukesha.

(1) N-succinimide 4-[ ¹⁸F] the fluorobenzoic acid ester ([ ¹⁸F] SFB) synthetic

[ ¹⁸F] (Wisconsin USA) produces fluorochemical for GE, Waukesha by magnetic resonance acceleator PET trace.Utilize 18O (p, n) 18F nuclear reaction, through proton irradiation contain in 1.5ml silver target (silver target) [ ¹⁸O] H ₂O, the incident proton can be 18MeV, electron beam current is 36 μ A.By magnetic resonance acceleator preparation [ ¹⁸F] F ^-After, be adsorbed onto on the Sep-Pak light QMA post (Sigma) and treat wash-out.[ ¹⁸F] F ^-Contain K through 1.5ml ₂CO ₃With Kryptofix 222 (K222; Sigma) mixing solutions of forming is eluted to (U.S. ALTECH company) in the V-type reaction flask, obtains anhydrous through the azeotropic evaporate to dryness ¹⁸F ^-

Labelled precursor [ ¹⁸F] synthesized reference literature method (the Kirchhoff TD of SFB; Merkesdal S; Frericks B; Et al. Intraarterial calcium stimulation (ASVS) for pancreatic insulinoma:comparison of preoperative localization procedures. Radiologe, 2003 (43): 301-305.; Suzuki K; Takahashi S; Aiura K; Et al. Evaluation of the usefulness of percutaneous transhepatic portal catheterization for preoperatively diagnosing the localization of insulinoma. Pancreas, 2002,24:96-102.; Wang GJ; Dou JT; Wang ZQ, et al. Comparison of endoscopic ultrasonography and digital subtraction angiography in localization of insulinomas. Chinese Journal of practical International Medicine, 2006; 26 (22): 1829-1830.), building-up process is referring to Fig. 1.

Simply be described as to filling activatory anhydrous ¹⁸F ^-Reaction flask in add 4-Trimethylamine 99 ethyl benzoate three fluoro sulfonates (5 mg, 16 μ mol are dissolved in 0.3 mL anhydrous acetonitrile), 90 ℃ of heating 10 min.After the reaction solution cooling, add NaOH 0.7 mL of people 1 mol/L, 100 ℃ of reaction 5 min, the cooling back adds HCl 0.9 mL of 1 mol/L, and reaction solution with the 0.01 mol/L hydrochloric acid drip washing of 2 mL, is used N earlier through an activatory Sep-Pak C18 post ₂Dry up Sep-Pak C18 post, again with 3 mL acetonitrile wash-outs obtain 4-[ ¹⁸F] fluorobenzoic acid.The aqueous solution of 20 μ L hydroxide tetrapropyl amine is added wherein, and 100 ℃ of azeotropic dryings add people 12 mg O-(N-succinimide)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester (TSTU) (being dissolved in the 0.25 mL acetonitrile), 90 ℃ of reaction 5 min.Use 5% acidifying with acetic acid of 3 mL then, dilute with 6 mL water again.With above-mentioned solution through an activatory Sep-Pak C18 post, use successively 10 mL acetonitrile/water ( V: V=1/7) N is used in drip washing ₂Dry up Sep-Pak C18 post, with 2 mL methylene dichloride wash-outs.Chemical purity is penetrated in TLC, HPLC measuring, and activity instrumentation activity is calculated putting productive rate.

(2) [ ¹⁸F] SFB and Eexdin-4 coupling

Will [ ¹⁸F] SFB is dissolved in the 50 μ L acetonitriles, adds 100 μ g Exendin-4 (be dissolved in 200 μ L pH values be 8.5 0.1M borax-borate buffer), reaction 30 min under the room temperature.Separate through HPLC behind the reacting liquid filtering; Used chromatographic column is ZORBAX SB C18; 5 μ m, 9.4 mm * 150 mm, the HPLC system adopts the speed drip washing of following gradient with 3 mL/min: wherein A is the water that contains 0.1% trifluoroacetic acid for acetonitrile, the B that contains 0.1% trifluoroacetic acid mutually mutually; 0-20 min, A is raised to 90% from 16%; 20-30 min, A are 90%.Collect t _RThe peak of=18-19.5 min, products therefrom solution, the rotation evaporate to dryness, again with the dissolving of 0.9% NaCl injection liquid, must the title product compound [ ¹⁸F] FB-Exendin-4 is through Radio-TLC, and selecting developping agent for use is ETHYLE ACETATE and methylene dichloride, and both volume ratios are 3:1, and Radio-HPLC characterizes, and the Radio-HPLC analytical procedure is identical with the preparation method, but flow velocity is 1mL/min, chromatographic column is μ Bondapak ^TMC18,5 μ m, 300 mm * 3.9 mm.

Embodiment 2, ¹⁸The vitro stability of F-FB-Exendin-4 is measured

2 kinds of affinity tags are placed saline water and foetal calf serum respectively, and place 37 ℃ of waters bath with thermostatic control, and respectively at 1,4, pure behind 8,12 h with the putting of HPLC method certification mark thing.

At first successfully synthesize labelled precursor ethyl-4-Trimethylamine 99 benzoic ether-three fluoro sulfonate, synthetic through three step one kettle ways, then through Sep-Pak C18 post separate obtain intermediate N succinimide-4-[ ¹⁸F] the fluorobenzoic acid ester ([ ¹⁸F] SFB).[ ¹⁸F] putting of SFB be pure greater than 95%.Exendin-4 with [ ¹⁸F] the SFB reaction, promptly obtain ¹⁸F-FB-Exendin-4.Radioactivity TLC has shown ¹⁸The R of F-FB-Exendin-4 _fValue is 0.0 (Fig. 2).The purifying of crude product HPLC shows a main radioactivity peak, ¹⁸The F-FB-Exendin-4 peak, RT t _R=17.2min (Fig. 3). ¹⁸The mark rate of F-FB-Exendin-4 is (35.6 ± 2.3) % (n=4), and putting is pure greater than 98%. ¹⁸F-FB-Exendin-4 all has preferably stability in foetal calf serum and saline water, put in 37 ℃ of waters bath with thermostatic control more than 12 h, and its putting is pure to be reached more than 90%.

Embodiment 3, cell in vitro competition combine experiment

The cultivation of RIN-m5f cell

Insulinoma clone RIN-m5f cell (Cancer Hospital of Chinese Academy of Medical Sciences is so kind as to give); RPMI1640 nutrient solution (Sigma company) with containing 15%FBS (Hyclone), 1% mycillin (GBICO company) is incubated in the petridish (Corning company) of diameter 10cm, places 37 ℃, 5%CO ₂Cell constant incubator (Heraeus company) in cultivate.

The saturation curve of acceptor and part

About about 3 days of RIN-m5f cell cultures is about cytogamy to 80%, with 0.25% trypsin Invitrogen company) digestion, centrifugal, after cell counting, reach in 24 orifice plates (Corning company), every porocyte number is about 5 * 10 ⁵Individual, place in 37 ℃ of constant incubators and cultivate, sucking-off cell culture fluid behind the 24h, 1 * PBS washes twice.Every hole adds different concns respectively ¹²⁵It is 500 μ L that I-exendin4, PBS damping fluid transfer to volume, makes it that final concentration is respectively 0,0.03,0.05,0.1,0.2,0.4,0.6,0.8,1.0nmol/L, and each concentration is all established 3 multiple holes.Be the deduction non-specific binding, respectively with 0,0.03,0.05,0.1,0.2,0.4,0.6,0.8,1.0nmol/L ¹²⁵All add the unlabelled exendin-4 of 5 μ g in the I-exendin4 hole, volume is 500 μ L, and each concentration is all established 3 multiple holes.Above-mentioned two groups all place 37 ℃ of constant incubators, hatch 40min.After reaction finishes; Wash 3 times to contain the cold PBS of 0.5%BSA; The sucking-off supernatant; It is the cold NaOH of 1mol/L 500 μ L that every hole adds concentration, behind the reaction 5min cell in every hole is laid and is moved to the γ immunity pipe from the petridish bottom blowing, and (Shanghai day ring company) surveys its per minute radiocounting (count per minute) through the γ calculating instrument.

The gained data combine the saturation curve fit procedure to handle through GraphPad Prism5 unit point, draw out ¹²⁵I-exendin4 receptors bind saturation curve.

Competition combines experiment

Cell is reached 24 orifice plates, and every porocyte number is about 5 * 10 ⁵Individual, place in 37 ℃ of constant incubators and cultivate, sucking-off cell culture fluid behind the 24h, 1 * PBS washes twice; Get ¹²⁵I-exendin-4 concentration is 0.4 nmol/L, adds concentration in every hole and is respectively the unlabelled exendin-4 of 0,0.1,1,10,100 nmol/L, puts in 37 ℃ of constant incubators and hatches 40min.Reaction is washed 3 times to contain the cold PBS of 0.5%BSA after finishing equally, the sucking-off supernatant, and it is the cold NaOH of 1mol/L 500 μ L that every hole adds concentration, behind the reaction 5min, cell is blown and beaten to get off to move to the γ immunity pipe from petridish, measures its cpm through the γ calculating instrument.

The gained data are handled through GraphPad Prism5 unit point competition binding curve fit procedure, try to achieve exendin-4 and suppress ¹²⁵The half inhibiting rate IC of I-exendin4 ₅₀Value.

The cell in vitro competition combines experiment

Because insulinoma cell strain RIN-m5f high expression level GLP-1 acceptor, competition combine experiment to show, exendin-4 suppresses ¹²⁵The half inhibiting rate IC of I-exendin4 ₅₀Value shows that for 0.35-2.81nmol/L (Fig. 4) unlabelled exendin-4 and GLP-1 acceptor have higher avidity.

Saturation curve

Through Graphpad Prism5 program, calculate ¹²⁵The equilibrium dissociation constant K of I-exendin-4 and GLP-1 acceptor _dValue is 56.64, maximum combined constant B _MaxBe 4.651 * 10 ⁷Show ¹²⁵I-SFB-exendin-4 can be special with the GLP-1 receptors bind, have saturability and reversibility, and have characteristics (Fig. 5) such as high-affinity.

Embodiment 4, nude mice model small animal position emission tomography (PET)/CT video picture

The foundation of insulinoma nude mice model

The female BALB/C nude mice (available from the institute of lab animals, Shanghai) in 4-5W age is raised by Medical College, Shanghai Communication Univ.'s Experimental Animal Center, and the raising condition is a SPF level aseptic condition.Experimentation on animals meets the ethic principle of protection of animal, raising and experiment through the approval of Ethics Committee of Medical College, Shanghai Communication Univ..

During the RIN-m5f cytogamy to 80% of cultivating, the sucking-off nutrient solution, 0.25% tryptic digestion is transferred to centrifuge tube, the centrifugal 5min of 1500 r/min, PBS is resuspended, centrifugal 2 times, uses the PBS of 150 μ l resuspended at last, through cell counting about 1 * 10 ⁷Totally 12 nude mices, every nude mice is in the RIN-m5f cell suspending liquid of right lower extremity dorsal part subcutaneous injection 150 μ l.After this, the conventional aseptic raising of nude mice, drinking-water is raised with 5% glucose injection (Shanghai drugmaker), and every 2-3 days blood glucose meter (the integrated blood glucose meter ACCU-CHEK Integra of Luo Shi) detects glucose level, and determination of blood glucose level is not less than 3mmol/L.Raise 2-3 about week, tumour is grown to 0.5-1.0cm, is used for small animal position emission tomography (PET)/CT video picture.

Nude mice model small animal position emission tomography (PET)/CT video picture

6 RIN-m5f model mouses respectively by the tail vein injection volume be 100ul contain 3.7MBq (100uCi) [ ¹⁸F] FB-exendin-4, injection every nude mice video picture in back is prepended in the aspiration box (induction chamber), and the isoflurane of feeding 5% is anaesthetized; Anesthesia back respectively at 30min and 120min with nude mice place small animal position emission tomography (PET)/CT (Inveon mPET/CT, Siemens Preclinical Solution, Knoxville, Tennessee, USA) scanning bed on, carry out the collection of 10min minute data.During video picture was gathered, nude mice was anaesthetized through the isoflurane that customization face shield (custom face mask) feeds 1-2%.

Nude mice model small animal position emission tomography (PET)/CT blocks video picture

3 insulinoma nude mice models contain the unlabelled Exendin-4 of 250 μ g by tail vein injection 100 μ l, and then by tail vein injection 100 μ l contain 3.7MBq [ ¹⁸F] FB-exendin-4.In addition 3 RIN-m5f model nude mices by tail vein injection 100 μ l contain 3.7MBq [ ¹⁸F] FB-exendin-4, as control group.Before the video picture nude mice is placed anesthesia in the aspiration box (induction chamber), injection [ ¹⁸F] 60min is positioned over animalcule with nude mice and fixingly in PET/CT is scanning bed behind the FB-exendin-4, carries out 10min minute IMAQ, and nude mice sucks the 1-2% isoflurane through breather mask and anaesthetizes during the video picture.

Small animal position emission tomography (PET)/CT picture processing

All small animal position emission tomography (PET) video pictures are rebuild through standard subclass expectation max methods (standard ordered-subset expectation maximization method).Reconstructed image is with horizontal shape position, and crown, sagittal plain shows; Show 512 continuous faultage images altogether, bed thickness 0.11mm, and carry out horizontal shape position through vision, crown position, the assessment of sagittal plain image.Image co-registration is carried out in CT video picture and PET video picture; Through major organs and tissues such as CT location liver, kidney, lungs, stomach, spleen and tumours; The region of interest of in the PET image, drawing (ROI) provides this tissue and the corresponding maximum SUV of organ (SUVmax) value automatically by computer program.

Outer each the internal organs increased radioactivity of nude mouse

Behind the nude mice dynamic acquisition 120min, dislocation is immediately put to death, at first heart extracting blood; Cut off peritonaeum then; Separate internal organs such as liver, spleen, stomach, heart, lung, pancreas, kidney, small intestine, colon, femur, muscle, weigh, place centrifuge tube; Measure the per minute radiocounting through the γ calculating instrument, the result representes with %ID/g (radioactivity of every gram tissue accounts for the percentage composition of ID).

Statistical study

All data are represented with

± SD; MV utilizes One-way ANOVA and Student ' s t test to handle, when there is significant difference P＜0.05.

The result

RIN-m5f model mouse small animal position emission tomography (PET)/CT video picture

The crown tomography (Fig. 6) of small animal position emission tomography (PET)/CT static image is visible behind RIN-m5f model mouse tail vein injection [18F] FB-Exendin-4; 30min tumor locus radioactivity is dense gathering obviously; Simultaneously visible lungs, the obvious development (Fig. 6 B) of organs such as liver, kidney and bladder; The 120min tumor imaging subtracts light, still visible lungs, and organs such as liver, kidney and bladder develop, but radioactivity reduces (Fig. 6 D).Fig. 6 A and 6C are respectively 30min and the crown faultage image of 120min nude mice CT, show that tumour is in nude mice right lower extremity dorsal part.

When static image 30min and 120min,, delineate each internal organs region of interest respectively by the PET image, and obtain corresponding SUVmax value (Fig. 7) through the CT location.It is thus clear that SUV is the highest for 30min kidney, liver, be lung, stomach, small intestine and tumour secondly; Tumour with the ratio of muscle tissue SUV is: 4.57.Kidney behind the 120min, liver SUV reduce, and wherein kidney reduces the most obviously, but kidney, liver still have higher SUV value; Lungs, stomach and small intestine SUV have reduction in various degree.SUV is all very low in the 120min endoskeleton muscle tissue.Because pancreas is difficult to clear demonstration in the CT image, therefore, does not carry out pancreas SUV pH-value determination pH.

Above result show [ ¹⁸F] the effective target tumor of FB-Exendin-4, the video picture with tumour cell GLP-1 receptors bind, simultaneously [ ¹⁸F] FB-Exendin-4 also with the mouse lung tissue bond, checking mouse lung tissue contains the GLP-1 acceptor, also the while further verified [ ¹⁸F] FB-Exendin-4 combines with the GLP-1 acceptor; [ ¹⁸F] FB-Exendin-4 mainly drains through kidney and liver, meets pharmacokinetic properties in the body of polypeptide.

Small animal position emission tomography (PET)/CT the video picture of model mouse Exendin-4 blocking-up

10min injection behind the model mouse tail vein injection 250 μ g Exendin-4 [ ¹⁸F] FB-Exendin-4, row small animal position emission tomography (PET)/CT video picture behind the 60min, the visible tumor locus of PET image is not seen tangible radioactive uptake, lungs are not seen development, liver develop subtract light, two kidneys obviously develop (Fig. 6 F).The crown faultage image of nude mice CT can clear positioning tumor position, arrow indication tumor locus (Fig. 6 E).

Carry out the SUVmax pH-value determination pH (Fig. 8) of tumour and each internal organs through the PET video picture of before the Exendin-4 blocking-up and blocking-up back 60min; It is thus clear that kidney, liver, stomach, small intestine, lung and tumour all have reduction in various degree; Wherein tumour reduces the most obvious; Lungs secondly, but show unmarked Exendin-4 specific inhibition [ ¹⁸F] FB-Exendin-4 combines with tumour cell, [ ¹⁸F] biological characteristics of FB-Exendin-4 is identical with Exendin-4.

The external test that each internal organs radiolabeled biotin of model mouse distributes

RIN-m5f model mouse tail vein injection 3.7MBq [ ¹⁸F] behind the FB-Exendin-4 120min, separate each internal organs, the result who records is expressed as %ID/g, and is as shown in Figure 9.After can clearly seeing 2h, in the kidney radiocounting the highest, be 0.27, secondly be that liver is 0.18; Tumour is 0.15, is higher than lung, pancreas, small intestine, large intestine, stomach and spleen respectively; %ID/g is minimum in the muscle tissue, is 0.037; Tumour is 7.1 with the muscle tissue ratio, and each internal organs 120min external beam radiotherapy property mensuration result of nude mice and video picture result are consistent.

We are through synthetic ¹⁸Intermediate N succinimide-4-that the F labeling polypeptide is commonly used [ ¹⁸F] the fluorobenzoic acid ester ([ ¹⁸F] SFB), the indirect labelling that has carried out.The research demonstration, [ ¹⁸F] SFB as the Exendin-4 of midbody mark ( ¹⁸F-SB-Exendin-4), its radiochemicsl purity is greater than 98%, mark rate 35%, and vitro stability is good.External combination experiment shows that the IC50 value is 0.35-2.81nmol/L; Equilibrium dissociation constant Kd value is 56.64; Maximum combined constant Bmax is 4.651 * 107, shows that 18F-SB-Exendin-4 and GLP-1 receptors bind have higher specificity.

We have set up RIN-m5F BALB/c nude mice insulinoma model, and through the visible clear video picture of tumour of small animal position emission tomography (PET)/CT video picture, and tumor imaging can be blocked by unlabelled Exendin-4, show [ ¹⁸F] FB-Exendin-4 can combine with the insulinoma cell-specific.Visible kidney of video picture simultaneously and liver obviously develop, and still visible liver of Exendin-4 blocking-up video picture and kidney develop, and be visible, [ ¹⁸F] FB-Exendin-4 is mainly through kidney and liver metabolism.From [ ¹⁸F] SUVmax of FB-Exendin-4 nude mice PET/CT video picture analyzes and can find out that except extraorgans such as liver kidneys, the SUVmax of organs such as lung, stomach and small intestine is higher, and the visible above-mentioned organ SUVmax of Exendin-4 blocking-up video picture obviously reduces.Gotthardt etc. show that through bio distribution and video picture research in the body of mouse and rat organs such as lung, stomach, pancreas all have the GLP-1 expression of receptor.But for the mankind; Except pancreas islet and duodenum (Brunner gland) have higher GLP-1 expression of receptor; In ileum, colon, kidney, lung and the pancreas organs such as (acinuses) a small amount of GLP-1 expression of receptor is only arranged, then do not see the GLP-1 expression of receptor in the tissues such as liver, spleen, Skelettmuskel and fat.Simultaneously, can avoid the eclipsed influence between the internal organs,, reduce the radiological effect of peripheral organs, have good potential applicability in clinical practice particularly through the accurate location of CT through the PET/CT video picture.

The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.

Claims

1. A ¹⁸ F labeled product of Exendin-4, characterized in that the labeled product is [ ¹⁸ F]FB-Exendin-4.

2. The preparation method of [ ¹⁸ F]FB-Exendin-4 described in claim 1, comprising the following steps:

(1) ¹⁸ F ^- reaction with ethyl 4-trimethylaminobenzoate trifluorosulfonate to prepare intermediate N-succinimide 4-[ ¹⁸ F]fluorobenzoate [ ¹⁸ F]SFB;

(2) [ ¹⁸ F]SFB is coupled with Eexdin-4 to obtain [ ¹⁸ F]FB-Exendin-4.

3. The preparation method of [ ¹⁸ F]FB- Exendin-4 according to claim 2, characterized in that, step (1) comprises: preparing [ ¹⁸ F]F ^- by cyclotron PET trace, and injecting activated [ ¹⁸ F ^] F- added 4-trimethylamine ethyl benzoate trifluorosulfonate in the reaction flask, reacted with NaOH and HCl in turn, the reaction solution was rinsed with activated Sep-Pak C18 column hydrochloric acid, and dried with _N2 , Then elute with acetonitrile to get 4-[ ¹⁸ F]fluorobenzoic acid; add tetrapropylamine hydroxide, azeotropically dry, add O-(N-succinimide)-1,1,3,3- Tetramethylurea tetrafluoroborate TSTU reacts, and then acidifies with acetic acid; Pass the above solution through the activated Sep-Pak C18 column, rinse with acetonitrile/water of V : V =1/7 in turn, blow with _N2 Dry the Sep-Pak C18 column and elute with dichloromethane; measure the radiochemical purity by TLC and HPLC, measure the activity with an activity meter, and calculate the radiochemical yield.

4. The preparation method of [ ¹⁸ F]FB-Exendin-4 according to claim 2, characterized in that step (2) comprises: dissolving [ ¹⁸ F]SFB in acetonitrile, adding Exendin-4, Exendin- 4 Dissolved in 0.1M borax-boric acid buffer solution with a pH value of 8.5, reacted at room temperature for 30 minutes; the reaction solution was filtered and separated by HPLC, the chromatographic column used was ZORBAX SB C18, 5 μm, 9.4 mm × 150 mm, and the HPLC system was as follows Gradient elution at a rate of 3 mL/min: phase A is acetonitrile containing 0.1% trifluoroacetic acid, phase B is water containing 0.1% trifluoroacetic acid; 0-20 min, A rises from 16% to 90%; 20-30 min, A is 90%; collect the peak at t _R =18-19.5 min, and the resulting product solution is spun to dryness, and re-dissolved with 0.9% NaCl injection to obtain the target product compound [ ¹⁸ F]FB-Exendin -4 is subjected to Radio-TLC, and the developing solvent is ethyl acetate and dichloromethane, the volume ratio of the two is 3:1, Radio-HPLC characterization, the Radio-HPLC analysis method is the same as the preparation method, but the flow rate is 1mL/min, The chromatographic column is μBondapak ^TM C18, 5 μm, 300 mm×3.9 mm.

5. The use of [ ¹⁸ F]FB-Exendin-4 according to claim 1 in the preparation of insulinoma imaging and diagnostic drugs.