CN103030689B - CART polypeptide compound, and preparation method and application of compound - Google Patents
CART polypeptide compound, and preparation method and application of compound Download PDFInfo
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- CN103030689B CN103030689B CN201210579256.3A CN201210579256A CN103030689B CN 103030689 B CN103030689 B CN 103030689B CN 201210579256 A CN201210579256 A CN 201210579256A CN 103030689 B CN103030689 B CN 103030689B
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Abstract
The invention relates to an <18>F labeled CART (***e and amphetamine-regulated transcript) polypeptide compound, namely <18>F-Al-NOTA-CART, and a preparation method and an application of the compound. The labeling method utilizes the characteristics that a dual-function chelating agent NOTA can be subjected to covalent binding with a non-active sequence end of polypeptide, and can also form a stable complex with metal ions, joins <18>F with CART peptide, and forms the polypeptide compound, namely <18>F-Al-NOTA-CART. <18>F-Al-NOTA-CART has the same biological property as CART peptide, and is good in stability; a labeled product is high in purity; the radiochemical purity is greater than 90%; in addition, the preparation method of <18>F-Al-NOTA-CART is simple; the whole reaction takes only 30min; the cost is low; and <18>F-Al-NOTA-CART can serve as an important tool for studying an action mechanism of CART peptide, and plays an important role in new drug development by taking CART peptide as a target.
Description
Technical field
The present invention relates to one
18the polypeptide compound of F mark
18f-Al-NOTA-CART and its preparation method and application, belongs to medicine and pharmacology technical field.
Background technology
Cocaine-and amphetamine-regulated transcript peptides (***e and amphetamine regulatedtranscript, CART peptide) is a kind of widely distributed neuropeptide material in vivo.Studies have shown that, CART peptide participate in habituation, appetite, stress, the physiological process such as anxiety and internal secretion.Especially CART peptide makes it become rapidly the study hotspot aspect neuropeptide with the effect research of the drug habit parties concerned.
First CART peptide is separated in the hypothalamic extract of sheep by the people such as Spiess (1981), thinks that at that time it is a kind of polypeptide that is similar to Somatostatin and so on, does not cause people's attention.Until people's discoveries such as nineteen ninety-five Douglass give after psychoactive drug Cocaine and amphetamine acute, the level of CART mRNA sharply increases, and some similar researchs are also in the news successively, and this has been a great concern CART peptide.Research subsequently finds that the cell nuclear colony of CART mRNA has the immunoreactivity of CART peptide, and WesternBlotting has also confirmed that CART peptide is widely distributed.In current known rat pituitary, enteron aisle, suprarenal gland, have 6 kinds of different CART peptides (from 4KD to 14KD) at least, comprise that preproCART, the proCART of macromolecule and small molecules measurer have physiologically active CART55-102 and 62-102.There are some researches show, CART peptide can suppress the feeding behavior of animal, reduces the body weight of obese rat, and is subject to the adjusting of leptin (leptin); The similar effect of rat ventral tegmental region injection CART peptide spiritedness stimulant, can change the behavior of rat; CART neurone and dopaminergic neuron are located altogether, and the metabolism of the interior Dopamine HCL of adjustable brain; CART peptide regulates the release of hypothalamic-pituitary hormone; CART peptide is relevant to pharmacological dependence etc.
Up to the present, rat, mouse, goldfish and the mankind's CART gene has been separated and order-checking completely, and they are all made up of 3 exons and 2 introns.The CART gene of large mouse forms two kinds of different products by alternative splicing, and that grows contains 129 amino acid, shorter contains 116 amino acid.The mankind's the about 2.5kb of CART gene, is positioned karyomit(e) 5q13-q14; CART gene forms the mRNA of two length by alternative splicing, thereby produces precursor (propeptide) proCART1-89 and the proCART1-102 of two different lengthss.What is interesting is, these two precursors all can find in rat, and only have proCART1-89 in the mankind.In precursor, comprise several cleavage sites, forming at least two kinds through translation post-treatment has bioactive CART peptide.ProCART1-102 can form CART55-102 and CART62-102; ProCART1-89 can form CART42-89 and CART49-89, and current most research all concentrates on CART55-102 and CART62-102.
CART peptide is also the peptide matters of current a few Tong Guo hemato encephalic barrier (BBB) of finding, if can be taking the structure of CART peptide as basis, antagonist or the blocker of synthetic CART peptide acceptor, the peptide matters of these and CART peptide structural similitude just can pass through BBB so, thus inhibition morphine; In addition, also studies have reported that CART peptide is a kind of endogenic neuroprotective, it can suppress the nerve injury due to A β (amyloid beta) neurotoxicity, improve cognitive function, to alzheimer's disease (Alzheimer ' s disease, AD) the different target spots in pathologic process all have impact, are expected to become the newtype drug for the treatment of AD; The people such as Elefteriou (Nature434:514-520 (2005)) think that CART peptide works in bone resorption, and the people such as DeokAh (Endocrinology147 (7): 3196-3202 (2006)) think that the conduction of CART signal is the reason that causes bone resorption to decline.
Therefore, illustrate the concrete mechanism of action of CART peptide, find CART acceptor and signal conductive process, be conducive to find and imitate or blocking-up CART effect, the agonist or the receptor antagonist that use as medical reagent, thus set for this platform for drug screening, significant.The acceptor of CART peptide is all attempted to find by receptors bind technology in many laboratories, but all ends in failure, and in brain or section, all there is no specific receptors bind.A series of laboratory evidence shows that CART peptide can activated G protein signal path in recent years, has greatly supported the existence of CART peptide g protein coupled receptor; But while verifying these results by receptors bind technology, exist the problems such as the low and easy variation of associativity, also need more to verify.
Main by using at present
125the method of I mark CART55-102 and CART62-102 is carried out receptors bind experiment (the Aleksandra Vicentic et al of CART peptide, CART(***e-and amphetamine-regulated transcript) peptide receptors:Specificbinding in AtT20 cells, European Journal of Pharmacology, 2005; Yiming Lin et al, CART peptide stimulation of G protein-mediatedsignaling in differentiated PC12 Cells:Identification of PACAP 6-38 asa CART antagonist, Neuropeptides, 2011).The ultimate principle of iodination reaction is as follows: by oxygenant make iodide (
125i
-) be oxidized to iodine molecule (
125i
2), iodine molecule again with polypeptide or protein molecule in tyrosine residues generation iodization.So no matter adopt which kind of iodine labeling method, must have the combinative group of iodine atom in the compound of mark, will contain tyrasamine base or histamine residue.The antigen such as protein or peptide class containing above-mentioned group in structure can directly carry out mark with radioiodine.As not containing above-mentioned group, radioiodine cannot mark, just can carry out iodine labeling must connect above-mentioned group in the structure of these compounds after.Therefore affect the factor of protein, polypeptide iodate efficiency, depend mainly on the quantity of tyrosine residues in protein, peptide molecule and the degree that they expose in molecular structure.Less in view of contained tyrasamine base or histamine residue in CART peptide, can cause the reduction of iodate efficiency; Directly the process of mark is difficult to control simultaneously, also may cause structure, stability, biological activity and the pharmacokinetics of CART peptide that unpredictable change occurs, therefore, in the urgent need to developing a kind of marking method that can highly efficient labeling CART peptide, and can improve the tracer agent of the sensitivity of PET video picture,, follow the trail of better the distribution situation of CART peptide at nervous center acceptor, and then the concrete mechanism of action of research CART peptide and its acceptor.
Summary of the invention
Technical problem to be solved by this invention is that the labeling effciency of CART peptide in prior art is low and then affect the problem of PET video picture, thereby provides a kind of labeling effciency high, and purity is high, and preparation is simple, good stability with
18cART polypeptide compound of F mark and preparation method thereof;
And further provide above-mentioned CART polypeptide compound as the application of PET tracer agent, with and the application in neural acceptor distributes at monitoring CART peptide.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of CART polypeptide compound, has structure as follows:
18f-Al-NOTA-CART, wherein, described CART peptide is the CART peptide that derives from people, rat, mouse or goldfish.
Described CART peptide comprises SEQ ID NO:1, SEQ ID NO:2, the aminoacid sequence shown in SEQ ID NO:3 or SEQ ID NO:4.
The aminoacid sequence of described CART peptide is as SEQ ID NO:1, and SEQ ID NO:2, shown in SEQ ID NO:3 or SEQ ID NO:4.
The preparation method of described CART polypeptide compound comprises the steps:
S01: pass through
18o(p, n)
18f reaction generates carrier free
18f
-, and be enriched on QMA post, use KHCO
3solution will
18f
-wash-out, adds acetonitrile azeotropic evaporate to dryness, regulates pH to 3.8-4.2 with Glacial acetic acid buffered soln, obtains required
18f
-reaction solution; Described
18o(p, n)
18f reaction can adopt mode well known to those skilled in the art and equipment to carry out, and selects magnetic resonance acceleator to react in various embodiments of the present invention, and described QMA post is first known and common type with those skilled in the art;
S02: get the acetate buffer solution that contains NOTA and selected CART peptide, add acetonitrile oil bath reaction at 40-50 DEG C, obtain NOTA-CART solution;
S03: to gained in described step S01
18f
-in reaction solution, add AlCl
3the NOTA-CART solution of preparing in solution and step S02, and with sodium acetate buffer solution regulate pH be 3.9-4.5, keep 95-105 DEG C of reaction;
S04: carry out purifying with reversed-phase HPLC post, obtain described polypeptide compound
18f-Al-NOTA-CART.
In described step S04, the condition of described HPLC is: the filler of selecting is octadecylsilane chemically bonded silica, and moving phase is acetonitrile-water, its ratio is 55: 45, the flow velocity of described moving phase is 0.8mL/min, and detection wavelength is 254nm, product when collection appearance time is 15min; In various embodiments of the present invention, select C18 post (Agilent, 5um, 4.6*250mm) to carry out purification process.
In described step S03, described NOTA-CART solution and described AlCl
3the mol ratio of solution is 1.5: 1～2: 1.
Further, described in contain in NOTA-CART solution, the mol ratio of described NOTA and described CART peptide is 1.5: 1～2: 1.
The concentration of the acetate buffer solution of the described NOTA of containing is 8～10mg/mL, the preferred 10mg/mL of the present invention, compound method is: the NOTA(1 of 0.25mg, 4,7-tri-azo-cycle nonane-Isosorbide-5-Nitraes, 7-nitrilotriacetic, No. CAS: 56491-86-2, is purchased from company of Hua Yi Esso Top (domestic)) be dissolved in the hac buffer that 25uL concentration is 0.1M; Or those skilled in the art can select other rational concentration also can realize the object of invention as required.
In described step S01, those skilled in the art can select according to the performance of selected equipment the elutriant of reasonable concentration, select KHCO in the present invention
3as elutriant, and described KHCO
3the concentration of solution is preferably 0.4mol/L.
The present invention also provides the CART preparing according to method described above polypeptide compound.
Further, the present invention also provides a kind of PET tracer agent, is described CART polypeptide compound.
The technology that the present invention also provides a kind of CART of detection peptide to distribute at nervous center acceptor, is used described PET tracer agent to carry out PET video picture to the distribution situation of CART peptide acceptor in nervous center.
Molecular imaging (Molecular Imaging) is image application method is carried out cell and molecular level quantitative and qualitative analysis research to the bioprocess under condition of living organism.Existing extensive application in clinical front medical research and clinical disease diagnosis, curative effect evaluation, prognosis judgement etc.Particularly the nuclear medicine molecular image based on computed tomography is the image technology of most active in current molecular image and tool prospect, it is the medical imaging technology using specific molecular in body as image contrast, can be in true, complete human or animal body, by physiology and the pathologic process of the direct showed cell of image or molecular level.It has erected interconnective bridge between clinical front molecular biology and clinical medicine, also becomes current conversion medical science (TranslationalMedicine) and studies one of important instrument.
PET full name is Positron Emission Computed Tomography (positron emissiontomography, PET), it is by extremely micro-positron radionuclide tracer agent is expelled in human body, then adopt special in-vitro measurements instrument (PET) to survey the distribution situation of these positron radionuclides at the each internal organs of Whole Body, show physiological metabolism and the structure of Whole Body major organs by the method for computerized tomograph.PET is that γ-camera is afterwards in radionuclide imaging major progress again, represent the last word of Nuclear MedicineImaging Technique, it has become endocrinopathy diagnosis and the diagnosis typing of research, the monitoring of medicine blood concentration, some tumour and transmissible disease and the important means of acceptor research, is widely used.
The sensitivity of PET technology and specificity depend on signal transmitter substance (tracer agent) and the distribution in vivo thereof of use significantly.The radionuclide using in PET scanning normally has short-decayed positron radiation isotropic substance, for example
11c (20min),
13n (10min),
15o (2min),
18f (110min),
131i (8 days) and
124i (4.2 days).These radionuclides are incorporated to such as in the conventional compound of the health of glucose (or glucalogue), water or ammonia, or are incorporated in the molecule of bind receptor or other drug site of action.
Technique scheme of the present invention has the following advantages compared to existing technology:
(1) of the present invention
18the polypeptide compound of F mark
18f-Al-NOTA-CART is to realize by bifunctional chelating agent NOTA
18the connection of F and CART peptide; Because NOTA can be connected by dehydration condensation with the N-terminal of CART peptide, then by chelatropic reaction by radioisotope labeling, chemical process is relatively simple and clear, and reaction product is single, is convenient to separation and purification; Need to be combined and cause label range limited with certain specific amino acids with respect to other marking methods, method of the present invention effectively avoided because in CART peptide tyrosine residues content number and degree of exposure in molecule problem that labeling effciency is exerted an influence, thereby improve the radio-labeling efficiency of CART peptide, set it as tracer agent carry out PET video picture process time, effectively improve the sensitivity of PET video picture, be convenient to CART peptide acceptor further to study;
(2) because fluorine is faint yellow gas at normal temperatures, chemical property is active, and nearly all material can be oxidized to fluorochemical by it; And after the hydrogen in compound molecule is replaced by fluorine, not biological active center if replace position, can not affect the biological activity of this compound; And due to
18the transformation period of F is relatively grown (110min), is conducive to some more complicated complex signs; In addition,
18the easy penetrate tissue of micromolecule polypeptide of F mark, and there is biospecificity; Therefore, of the present invention
18the CART polypeptide compound of F mark has the akin activity with CART, can effectively show the distribution situation of CART acceptor;
(3) due to
18f molecule is larger with respect to the volume of micromolecule polypeptide, usually can cause molecular structure distortion, and then affect its biological activity, thereby method of the present invention adopts
18mode and the Al metal ion of F mark bifunctional chelating agent form stable complex compound, have further stablized the biological activity of marked product, therefore of the present invention
18the polypeptide compound of F mark
18in F-Al-NOTA-CART,
18f is less on the polypeptide biological activity impact of mark; Therefore
18f-Al-NOTA-CART can obtain the biological property identical with CART peptide;
(4), by the use of bifunctional chelating agent, make of the present invention
18the polypeptide compound of F mark
18f-Al-NOTA-CART, its preparation process only needs 30min, simple for process, and labeling effciency is up to 45%, and radiochemicsl purity is high, and cost is low;
(5) utilizing CART polypeptide compound of the present invention is tracer agent, application PET imaging technique in vivo and in vitro each level is studied the physiological disposition of CART peptide, investigates CART peptide in neural acceptor distribution situation and for the mechanism of action of Psychoactive Substance Dependence target;
(6) experiment showed, of the present invention
18the polypeptide compound of F mark
18f-Al-NOTA-CART has good biological property, ICR Organs of Mice pair
18the SUV value of the radioactive uptake of F-Al-NOTA-CART no difference of science of statistics, illustrate that both have identical biological property; And
18f-Al-NOTA-CART target/non-target ratio has reached more than 4 times, can meet in CART peptide body and the research of interaction in vitro process.
Brief description of the drawings
For content of the present invention is more likely to be clearly understood, below in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein,
Fig. 1 is that embodiment 1 is prepared
18the radioactivity TLC figure of F-Al-NOTA-CART
Spectrum;
Fig. 2 is prepared to ICR injected in mice embodiment 1
18micro PET brain scans figure after F-Al-NOTA-CART30min;
Fig. 3 is prepared to ICR injected in mice embodiment 5
18micro PET brain scans figure after F-SFB-CART30min.
Embodiment
Embodiment 1
What the present embodiment was prepared has
18the CART polypeptide compound of F-Al-NOTA-CART structure is that the CART of aminoacid sequence shown in SEQ ID NO:1 is carried out
18f mark, described in there is the aminoacid sequence shown in SEQ ID NO:1 CART peptide derive from rat or mouse, its preparation process comprises the steps:
S01: adopt magnetic resonance acceleator, pass through
18o(p, n)
18f reaction generates carrier free
18f
-and be enriched on QMA post the KHCO that is 0.4mol/L by 200 μ L concentration
3solution will
18f
-wash-out enters reaction tubes, adds acetonitrile azeotropic evaporate to dryness twice, regulates pH to 4.0 with the Glacial acetic acid buffered soln of 0.1mol/L, obtains required
18f
-reaction solution, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 387mCi;
S02: get acetate buffer solution (NOTA concentration is 10mg/mL) the 25 μ L and the CART peptide 25 μ gs of aminoacid sequence as shown in SEQ ID NO:1 that contain NOTA, add the oil bath reaction at 45 DEG C of 25 μ L acetonitriles, obtain NOTA-CART solution, its concentration is 10mg/ml;
S03: to gained in described step S01
18f
-in reaction solution, add the AlCl that 3 μ L concentration are 2mg/mL
3the NOTA-CART solution 30 μ L that prepare in solution and step S02, and to regulate pH with the sodium acetate buffer solution of 0.1mol/L be 4.1,100 DEG C of reaction 15min;
S04: with filler be octadecylsilane chemically bonded silica, moving phase is acetonitrile: water=55: 45, detection wavelength is 254nm, the reversed-phase HPLC post that flow velocity is 0.8mL/min carries out purifying, collects the product of 15min, obtains described polypeptide compound
18f-Al-NOTA-CART, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 182mCi.
Embodiment 2
What the present embodiment was prepared has
18the CART polypeptide compound of F-Al-NOTA-CART structure is that the CART of aminoacid sequence shown in SEQ ID NO:2 is carried out
18f mark, described in there is the aminoacid sequence shown in SEQ ID NO:2 CART peptide derive from rat or mouse, its preparation process comprises the steps:
S01: adopt magnetic resonance acceleator, pass through
18o(p, n)
18f reaction generates carrier free
18f
-, and be enriched on QMA post the KHCO that is 0.4mol/L by 200 μ L concentration
3solution will
18f
-wash-out enters reaction tubes, adds acetonitrile azeotropic evaporate to dryness, regulates pH to 4.0 with the Glacial acetic acid buffered soln of 0.1mol/L, obtains required
18f
-reaction solution, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 379mCi;
S02: get acetate buffer solution (NOTA concentration is 10mg/mL) the 18 μ L and the CART peptide 25 μ gs of aminoacid sequence as shown in SEQ ID NO:2 that contain NOTA, add acetonitrile 25 μ L oil bath reaction at 45 DEG C, obtain NOTA-CART solution, its concentration is 10mg/ml;
S03: to gained in described step S01
18f
-in reaction solution, add the AlCl that 3 μ L concentration are 2mg/mL
3the NOTA-CART solution 22 μ L that prepare in solution and step S02, and to regulate pH with the sodium acetate buffer solution of 0.1mol/L be 4.1, keeps 100 DEG C to react 15min;
S04: with filler be octadecylsilane chemically bonded silica, moving phase is acetonitrile: water=55: 45, detection wavelength is 254nm, the reversed-phase HPLC post that flow velocity is 0.8mL/min carries out purifying, collects the product of 15min, obtains described polypeptide compound
18f-Al-NOTA-CART, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 171mCi.
Embodiment 3
What the present embodiment was prepared has
18the CART polypeptide compound of F-Al-NOTA-CART structure is that the CART of aminoacid sequence shown in SEQ ID NO:3 is carried out
18f mark, described in there is the aminoacid sequence shown in SEQ ID NO:3 CART peptide derive from people, its preparation process comprises the steps:
S01: adopt magnetic resonance acceleator, pass through
18o(p, n)
18f reaction generates carrier free
18f
-, and be enriched on QMA post the KHCO that is 0.4mol/L by 200 μ L concentration
3solution will
18f
-wash-out enters reaction tubes, adds acetonitrile azeotropic evaporate to dryness, regulates pH to 3.8 with the Glacial acetic acid buffered soln of 0.1mol/L, obtains required
18f
-reaction solution, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 400mCi;
S02: get acetate buffer solution (NOTA concentration is 10mg/mL) the 20 μ L and the CART peptide 25 μ gs of aminoacid sequence as shown in SEQ ID NO:3 that contain NOTA, add acetonitrile 25 μ L oil bath reaction at 40 DEG C, obtain NOTA-CART solution, its concentration is 10mg/ml;
S03: to gained in described step S01
18f
-in reaction solution, add the AlCl that 3 μ L concentration are 2mg/mL
3the NOTA-CART solution 25 μ L that prepare in solution and step S02, and to regulate pH with the sodium acetate buffer solution of 0.1mol/L be 4.5, keeps 95 DEG C to react 15min;
S04: with filler be octadecylsilane chemically bonded silica, moving phase is acetonitrile: water=55: 45, detection wavelength is 254nm, the reversed-phase HPLC post that flow velocity is 0.8mL/min carries out purifying, collects the product of 15min, obtains described polypeptide compound
18f-Al-NOTA-CART, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 172mCi.
Embodiment 4
What the present embodiment was prepared has
18the CART polypeptide compound of F-Al-NOTA-CART structure is that the CART of aminoacid sequence shown in SEQ ID NO:4 is carried out
18f mark, described in there is the aminoacid sequence shown in SEQ ID NO:4 CART peptide derive from rat and mouse, its preparation process comprises the steps:
S01: adopt magnetic resonance acceleator, pass through
18o(p, n)
18f reaction generates carrier free
18f
-, and be enriched on QMA post the KHCO that is 0.4mol/L by 200 μ L concentration
3solution will
18f
-wash-out enters reaction tubes, adds acetonitrile azeotropic evaporate to dryness, regulates pH to 4.2 with the Glacial acetic acid buffered soln of 0.1mol/L, obtains required
18f
-reaction solution, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 390mCi;
S02: get acetate buffer solution (NOTA concentration is 10mg/mL) the 22 μ L and the CART peptide 25 μ gs of aminoacid sequence as shown in SEQ ID NO:4 that contain NOTA, add the oil bath reaction at 50 DEG C of 25 μ L acetonitriles, obtain NOTA-CART solution, its concentration is 10mg/ml;
S03: to gained in described step S01
18f
-in reaction solution, add the AlCl that 3 μ L concentration are 2mg/mL
3the NOTA-CART solution 28 μ L that prepare in solution and step S02, and to regulate pH with the sodium acetate buffer solution of 0.1mol/L be 3.9, keeps 105 DEG C to react 15min;
S04: with filler be octadecylsilane chemically bonded silica, moving phase is acetonitrile: water=55: 45, detection wavelength is 254nm, the reversed-phase HPLC post that flow velocity is 0.8mL/min carries out purifying, collects the product of 15min, obtains described polypeptide compound
18f-Al-NOTA-CART, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 176mCi.
Embodiment 5
What the present embodiment was prepared has
18the CART polypeptide compound of F-SFB-CART structure is that the CART of aminoacid sequence shown in SEQ ID NO:1 is carried out
18f mark, described in there is the aminoacid sequence shown in SEQ ID NO:1 CART peptide derive from rat and mouse, its preparation process comprises the steps:
S11: adopt magnetic resonance acceleator, by
18o(p, n)
18the carrier free that F reaction generates
18f
-be enriched on QMA post, use K
2.2.2/ K
2cO
3solution will
18f
-wash-out enters reaction tubes, adds acetonitrile azeotropic evaporate to dryness twice, obtains
18f
-, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 390mCi;
S12: add 4-Trimethylamine 99 ethyl benzoate three fluoro sulfonate 12mg in the reaction solution of described step S11 gained, 90 DEG C of reaction 10min, cooling 10min; Add the NaOH0.5mL of 0.5M, 90 DEG C are hydrolyzed 5min, obtain the solution of clear; Cooling 10min, adds HCl7.5ml and the 1.5ml acetonitrile of 0.1M, then by the Sep-Pak C18 post of an activation, with 3ml acetonitrile wash-out, obtains 4-
18f-fluorobenzoic acid;
S13: add 12mg TSTU(O-(N-succinimide in the reaction solution of described step S11 gained)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester), confined reaction 5min at 90 DEG C, cooling 10min, add the HCl solution 5ml of 0.1M, by the Sep-PakC18 post of an activation, with 2ml acetonitrile wash-out, obtain again
18f-SFB(N-succinimide-4-
18f-fluorobenzoate);
S14: get 1mg CART and add the carbonate buffer solution 1ml of the 0.1M of pH=8.4 to put into reaction tubes, then fast fetching
18f-SFB0.05ml adds in reaction tubes, and 65 DEG C of oil bath reaction 30min, obtain
18f-SFB-CART, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 3.5mCi.
Embodiment 6
What the present embodiment was prepared has
125the CART polypeptide compound of I-CART structure is that the CART of aminoacid sequence shown in SEQ ID NO:1 is carried out to I
125mark, described in there is the aminoacid sequence shown in SEQ ID NO:1 CART peptide derive from rat and mouse, its preparation process comprises the steps:
S21: by 10 μ l CART peptides (10mg/ml) and 7 μ l NaI
125(8.0mCi), 20 μ l0.2mol/L PB(pH8.5), 10 μ l chloramine-Ts (5mg/ml), vortex concussion 30-60s at 15-60 DEG C;
S22: add 20 μ l Sodium Metabisulfites (10mg/ml), vortex vibration 2-5min termination reaction;
S23: with the C18 solid phase extraction column that balance is good, reaction solution is resolved to separation and purification, first with distilled water, the easy radioimpurity radioactive impurities such as free-iodine are washed away,
125i-CART is adsorbed on C18 solid phase extraction column, then with 10mM acidic alcohol will
125i-CART is wash-out from C18 post, obtains
125i-CART, adopting CRC-15R activity meter (CAPINTEC) to measure its radiation dose is 2.8mCi.
Labeling effciency and the putting of measuring respectively preparation-obtained CART polypeptide compound in embodiment 1-6 are pure.
The formula that calculates labeling effciency is:
18radiation dose (the mCi)/wash-out of labeling effciency=product of F mark CART enters in reaction tubes
18f
-radiation dose (mCi) * 100%;
125radiation dose (the mCi)/NaI of the labeling effciency=product of I mark CART peptide
125radiation dose (mCi) * 100%; Therefore,
In embodiment 1,
18labeling effciency=182/387*100%=47% of F-Al-NOTA-CART;
In embodiment 2,
18labeling effciency=171/379*100%=45% of F-Al-NOTA-CART;
In embodiment 3,
18labeling effciency=172/400*100%=43% of F-Al-NOTA-CART;
In embodiment 4,
18labeling effciency=176/390*100%=45% of F-Al-NOTA-CART;
In embodiment 5,
18labeling effciency=3.5*40/390*100%=36% of F-SFB-CART;
In embodiment 6,
125labeling effciency=2.8/8.0*100%*100%=35% of I-CART.
The pure detection of putting of product is to detect by thin-layer chromatography (TLC) method, analytical system is as follows: developping agent is V (acetonitrile)/V (water)=95:5, upholder is silica-gel plate, and the putting that obtains product by the detection of tlc analysis instrument is pure.
Only, taking embodiment 1 as example, embodiment 1 is prepared
18the radioactivity TLC collection of illustrative plates of F-Al-NOTA-CART as shown in Figure 1, according to peak area integration draw the putting of product pure be 98%.
The situations such as the reactivity worth of each marking method described in above-described embodiment 1-6 see the following form:
| Project | Marker site | Labeling effciency | The putting of product is pure | Total reaction is consuming time |
| Embodiment 1 | N-terminal | 47% | 98% | 30min |
| Embodiment 2 | N-terminal | 45% | 90% | 30min |
| Embodiment 3 | N-terminal | 43% | 93% | 30min |
| Embodiment 4 | N-terminal | 45% | 91% | 30min |
| Embodiment 5 | Tyrosine residues | 36% | 78% | 90min |
| Embodiment 6 | Tyrosine residues | 35% | 83% | 20min |
Embodiment 1-4 by adopt bifunctional chelating agent NOTA by CART peptide with
18f is connected, and whole preparation process only needs 30min, and technique is simple, and labeling effciency is high by approximately 45%, and cost is low, product radiochemicsl purity high (more than 90%, up to 98%); And connect by prothetic group SFB
18the whole process of F and CART peptide is loaded down with trivial details, need to repeatedly carry out chromatography and just can complete labeling process, and total about 90min consuming time, makes its application be subject to certain restriction; Although the direct mark CART of iodine peptide is simple to operate, consuming time less, the efficiency ratio of iodate mark is lower, is only 35% left and right; Directly mark may cause structure, stability, biological activity and the pharmacokinetics of CART peptide that unpredictable change occurs simultaneously, thereby affects every research of CART peptide, and its application also has certain limitation.
Embodiment 7
18the biology performance analysis of F-Al-NOTA-CART
Learn and measure check analysis by rat behavior
18the biology performance of F-Al-NOTA-CART.Used
18f-Al-NOTA-CART is prepared by embodiment 1-4 respectively, and contrast used is respectively has SEQ ID NO:1, SEQ ID NO:2, the CART peptide of the aminoacid sequence shown in SEQ ID NO:3 and SEQ ID NO:4.
Experiment 1
Experiment material: 27 of SD rats, body weight (200 ~ 220g)
Experimental technique: rat is divided into 9 groups at random: 12 of 3 of model group, CART groups,
1812 of F-Al-NOTA-CART groups; Wherein CART group is divided into again 4 groups, 3 every group, respectively organizes correspondence respectively and has SEQ ID NO:1, SEQ ID NO:2, the CART peptide of the aminoacid sequence shown in SEQ ID NO:3 and SEQ ID NO:4, is labeled as respectively CART-1, CART-2, CART-3, CART-4; Correspondingly,
18f-Al-NOTA-CART group is also divided into 4 groups, and 3 every group, prepared by each group respectively corresponding embodiment 1-4
18f-Al-NOTA-CART, is labeled as respectively
18f-Al-NOTA-CART-1,
18f-Al-NOTA-CART-2,
18f-Al-NOTA-CART-3,
18f-Al-NOTA-CART-4; Each rat sub-cage rearing (one, every cage).Not administration of described model group use in contrast, 24 10% chloral hydrate anesthesia of administration group, conventional tricorn heeling-in injection conduit, the rat fasting 24h after 7 days that performs the operation, freely drinks water.After fasting 24h administration group respectively intracerebroventricular injection contain CART,
18the artificial cerebrospinal fluid of F-Al-NOTA-CART 5 μ g/ ㎏.Feed behavior after the administration of observation rat, and after 24h, take leftover calculating food ration.Observe CART and
18the impact of F-Al-NOTA-CART on the rear feed behavior of rat hunger.
Experimental result: as shown in table 1
Table 1CART, the 18F-CART impact on hungry rat food ration
Note: respectively organize sample number n=3, and control group comparison:
*p<0.05,
*p<0.01
Experimental result shows, tail vein injection CART Toplink significantly suppresses the feed behavior of hungry mouse; Tail vein injection 18F-Al-NOTA-CART can suppress the feed behavior of mouse equally; This just illustrates that in each embodiment, the 18F-Al-NOTA-CART after mark has the biological property identical with CART peptide.
Embodiment 8
18f-Al-NOTA-CART and
18distribution and the mouse brain Micro PET video picture of F-SFB-CART in ICR Mice Body
For further investigating the biology performance of the CART of two kinds of marking method marks, we prepare embodiment 1
18f-Al-NOTA-CART and embodiment 5 prepare
18f-SFB-CART has carried out Micro PET scanning, divide 2 groups of ICR mouse, every group 6, scan method adopts animalcule anaesthetic device after being tail vein injection (50-100uCi/ only) 30min, passes into the mixed gas anesthetized mice of isoflurane and oxygen, then carry out Micro PET scanning 10min, image is rebuild through OSEM3D, delineates the area-of-interest of internal organs, and the picked-up SUV of computation organization value is as following table 2:
Table 2: 30min after injection
18f-Al-NOTA-CART and
18the distributed data (n=6, mean ± sd) of F-SFB-CART in ICR Mice Body
| ? | 18F-Al-NOTA-CART | 18F-SFB-CART |
| The heart | 5.21±0.68 | 4.89±0.33 |
| Liver | 8.72±0.94 | 9.54±0.29 |
| Kidney | 18.28±2.00 | 19.96±0.87 |
| Intestines | 4.41±0.39 | 4.23±0.38 |
| Brain | 3.67±0.76 | 3.17±0.43 |
| Flesh | 0.83±0.08 | 0.81±0.10 |
Micro PET scans I CR mouse brain figure as shown in Figures 2 and 3.
The demonstration of Micro PET scanning result,
18f-Al-NOTA-CART and
18f-SFB-CART in animal body, is mainly distributed in the histoorgans such as the heart, liver, kidney, intestines and brain, and mainly by renal excretion, after injection, the SUV value of 30min kidney picked-up reaches respectively 18.28 ± 2.00 and 19.96 ± 0.87, each internal organs to two kinds of markers (
18f-Al-NOTA-CART and
18f-SFB-CART) the SUV value no difference of science of statistics of radioactive uptake, shows that both have distribution and biological property in identical body.Particularly importantly, two kinds of SUV value result (3.67 ± 0.76 and 3.17 ± 0.43) demonstrations that markers absorb at brain, two markers can see through hemato encephalic barrier, and after injection, brain/muscle radioactivity ratio of 30min reaches 4 times more than.
Therefore, can carry out CART peptide animal or human swept-volume by Micro PET/PET, each level is studied the physiological disposition of CART peptide in vivo and in vitro, investigates CART peptide in neural acceptor distribution situation and for the mechanism of action of Psychoactive Substance Dependence target.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And the apparent variation of being extended out thus or variation are still among the protection domain in the invention.
Claims (3)
1. a method of preparing CART polypeptide compound, is characterized in that, described CART polypeptide compound has structure as follows:
18f-Al-NOTA-CART; Described method comprises the steps:
S01: pass through
18o(p, n)
18f reaction generates carrier free
18f
- ,and be enriched on QMA post, use KHCO
3solution will
18f
-wash-out, adds acetonitrile azeotropic evaporate to dryness, regulates pH to 3.8-4.2 with Glacial acetic acid buffered soln, obtains required
18f
-reaction solution;
S02: get the acetate buffer solution that contains NOTA and selected CART peptide mixes, add acetonitrile oil bath reaction at 40-50 DEG C, obtain NOTA-CART solution; The mol ratio of described NOTA and described CART peptide is 1.5:1～2:1, and described CART is the CART peptide that derives from people, rat, mouse or goldfish;
S03: to gained in described step S01
18f
-in reaction solution, add AlCl
3the NOTA-CART solution of preparing in solution and step S02, and with sodium acetate buffer solution regulate pH be 3.9-4.5, keep 95-105 DEG C of reaction; Described NOTA-CART solution and described AlCl
3the mol ratio of solution is 1.5:1～2:1;
S04: carry out purifying with reversed-phase HPLC post, obtain described polypeptide compound
18f-Al-NOTA-CART.
2. the method for preparing CART polypeptide compound according to claim 1, it is characterized in that, in described step S04, the condition of described HPLC is: the filler of selecting is octadecylsilane chemically bonded silica, moving phase is acetonitrile-water, and its ratio is 55:45, and the flow velocity of described moving phase is 0.8 mL/min, detection wavelength is 254nm, product when collection appearance time is 15min.
3. the method for preparing CART polypeptide compound according to claim 1 and 2, is characterized in that, the aminoacid sequence of described CART peptide is as SEQ ID NO:1, and SEQ ID NO:2, shown in SEQ ID NO:3 or SEQ ID NO:4.
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| CN103833829A (en) * | 2014-01-07 | 2014-06-04 | 江苏省原子医学研究所 | Radioactive <18>F-labeled imaging drug <18>F-AL-NOTA-IF7 targeting Anxa1 in tumor blood vessels and preparation method thereof |
| CN104059922A (en) * | 2014-04-03 | 2014-09-24 | 四川农业大学 | Cloning method of schizothorax prenati gene CART |
| CN106478820A (en) * | 2016-10-10 | 2017-03-08 | 米度（南京）生物技术有限公司 | A kind of liver cancer PET diagnosis tracer and preparation method thereof and purposes |
| CN108187078A (en) * | 2018-02-12 | 2018-06-22 | 中南大学湘雅医院 | Prostate cancer diagnosis imaging agent and preparation method thereof |
| CN108503695B (en) * | 2018-03-09 | 2021-06-08 | 南方医科大学南方医院 | Tracer based on GnRH polypeptide derivative and preparation method and application thereof |
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