CN102558291A - A dual mode molecule image probe - Google Patents
A dual mode molecule image probe Download PDFInfo
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- CN102558291A CN102558291A CN2010105926272A CN201010592627A CN102558291A CN 102558291 A CN102558291 A CN 102558291A CN 2010105926272 A CN2010105926272 A CN 2010105926272A CN 201010592627 A CN201010592627 A CN 201010592627A CN 102558291 A CN102558291 A CN 102558291A
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Abstract
The invention discloses a dual mode molecule image probe. The chelate of the present invention is a bonder. A chelating agent and polypeptide for specific object for measurement identification by a covalent connect to form a bonder and then the bonder and the radioactivity metal ion connects through a coordination bond to form the chelate. The dual mode probe of the present invention uses 153Gd3 + radio isotope as a single imaging core and connects the singularity molecule to the chelating agent, a center ion (153GD3 + radio isotope) and the chelating agent forms into the chelate and at the same time has MR imaging function and nuclear medicine imaging function. The dual mode probe of the present invention has high stability and enough signal intensity.
Description
Technical field
The present invention relates to a kind of bimodulus molecular image probe.
Background technology
Molecular imaging (Molecular Imaging) is the front subject that molecular biology, pharmaceutical chemistry and image technology intersect and merge; It is based on from cell and molecular level lesions showed, is species specific, brand-new live body (in vivo) developing method.Molecular imaging can make we understand on the whole disease generation, develop and lapse to, disclose biochemical change in position, reduce the inspection that wound is arranged, disease is carried out early detection, is differentiated qualitative and treatment is estimated.
The intension of molecular imaging has determined that it must be such as the early diagnosis of tumour with differentiate and bring into play irreplaceable effect aspect qualitative.The key of radical cure tumour is early diagnosis.The generation of tumour, development are accompanied by several genes and molecular changes, and what change at first is the molecular marker of tumour, like cell surface receptor, enzyme or signaling molecule etc.The task of molecular imaging is utilized molecular biological fundamental research achievement exactly; Center on associated molecule in the pathology incidence and development process; Make up the specific molecular probe, carry out the molecular level imaging with the image means, thereby reach early diagnosis and differentiate purpose qualitatively.
The core of molecular imaging is to make up target property molecular probe (molecular imaging probe).The general structure of molecular probe is: visualization portion (paramagnetic metals ion/MR imaging, radioactively labelled substance/nuclear medicine, fluorescent substance/optical imagery)+sequestrant/bifunctional linking reagent (being included as DTPA, DOTA, NOTA, MAG3, HYNIC etc.)+specific molecular (like polypeptide, antibody, other specific moleculars etc.).
Can be the key that the targeted molecular probe makes up with tumour target specificity bonded molecule, be the basis with the specific molecular, can make up the specific probe to tumour.Compare with cell with healthy tissues, some content receptor increases unusually under the neoplastic state, therefore can be used as the tumour-specific markers thing and carries out the specific molecular imaging.Common acceptor comprises integrates plain (α v β 3) acceptor, somatostatin receptor, folacin receptor, TfR, glucoreceptor, Her-2/neu acceptor, Dopamine Receptors, bombesin receptor etc.Wherein somatostatin receptor and integrin receptor α v β 3 use at most.
Somatostatin receptor
Somatostatin is a kind of active polypeptide that is present in the position endocrine cells such as human brain, hypothalamus, stomach and intestine, pancreas; Has physiological action widely; Can suppress secretions such as tethelin, intestinal hormone (gastrin, pancreatin, cholecystokinin, pancreatin secretin, glicentin, Regular Insulin and VIP etc.) and gastric acid secretion; Reduce the blood flow of internal organs; Mesenteric artery there is stronger contraction; Thereby reduce portal vein backflow blood volume, reduce portal venous pressure, so can be used to treat that endocrinopathy, intractable diarrhoea, acute and chronic pancreatitis, gastrointestinal fistula and pancreas fistula, digestive tract ulcer and erosive gastritis are also hemorrhage, various diseases such as tumour and esophageal varices bleeding.
The discovery of Somatostatin is in 1973, and Brazeau etc. separated and the peptide species of having purified from the sheep hypothalamus at that time, and structure is H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cy s-OH.Discover, 1 * 10
-9The M level; This material just can be in the secretion of vitro inhibition immunoreation rat or human growth hormone; Similar effect is also arranged in the rat body; Also has identical biological activity by this structure synthetic replica, Here it is Somatostatin-14 (SST-14) natural, that have 14 amino-acid residues.Later found to contain the another kind of spontaneous growth chalone-28 (SST-28) of 28 amino-acid residues again.SST-28 is the precursor of SST-14, and the precursor of Somatostatin-28 has 120 or more amino-acid residue, and it is former to be called precursor.
Long chalone is come to light the back just as medicine for treatment, progressively is applied to clinical.Yet this 14 natural peptide Somatostatins receive the effect of peptase, and the transformation period too short (1～3 minute), specificity is not high, and the medicine high price is expensive, can only the uninterrupted administration of vein, otherwise the high secreting phenomenon of knock-on property can appear.Above shortcoming has restricted the widespread use clinically of this medicine, for this reason nineteen eighty-two synthetic a kind of SSA SMS201-995, i.e. Sostatin (Octreotide), its structure such as Fig. 3.
Sostatin is a kind of polypeptide of being made up of 8 amino acid, has kept the major function group (solid black) of 14 peptides in the structure, and transform tryptophane wherein as the dextrorotation tryptophane; And the Threonine of the dexamphetamine propylhomoserin of N end and C end has the Degradation of resisting metabolic enzyme, thereby not only kept the pharmacological activity of spontaneous growth chalone, and specificity is higher; Act on byer force, stronger more than 30 times than spontaneous growth chalone (sst-14) effect, the transformation period rises to 90 minutes; Untoward reaction is few, use more convenient, but vein, subcutaneous or intramuscular injection; Even " knock-on " phenomenon does not take place after the life-time service drug withdrawal yet, popularize rapidly in clinical application.
In addition, Octroetide has blood vessel formation against function, can suppress the growth of nude mice people liver transplantation knurl.Octroetide all can obviously induce liver cancer cell (HepG2, SMMC-7721) apoptosis under (0.25,0.5,1.0,2.0and 4.0mg/L) concentration.Evidence suggests that Octreotide promotes human somatotropic hormone apoptosis of tumor cells through Activate SS TR2 acceptor.
Somatostatin is through (Somatostatin receptor SSTR) combines and the performance physiological function with somatostatin receptor in vivo.Somatostatin receptor is divided into different hypotype (subtype), has five kinds of hypotypes to be cloned and characterize (SSTR1-5) at present.The tumour of different sites and type is expressed the receptor subtype of different sorts and quantity.Most of tumours such as gi tract neuroendocrine tumor, one-tenth neuroblastoma, medulloblastoma, meningioma, gangliocytoma, mammary cancer, renal cell carcinoma, lymphoma, hepatocellular carcinoma, stomach and intestine pancreas tumor and the main high expression level SSTR2 of small cell lung cancer.Sarcoma and prostate cancer are expressed SSTR1, also express SSTR5.Some other tumour also possibly expressed SSTR1 like pheochromocytoma, cancer of the stomach and ependymoma, expresses SSTR2 or SSTR5 hypotype sometimes.
Spontaneous growth chalone 14 and 28 and these five kinds of hypotypes all with very high avidity is arranged.But the Somatostatin of synthetic is intended like thing, and like Octreotide, then have different combinations active with different hypotypes: Octreotide and SSTR2 and SSTR5 have very high avidity, and be lower with the avidity of SSTR3, and do not combine with SSTR1 and SSTR4.
Therefore, can utilize SSTR and OCT specificity bonded characteristics, can design the molecular probe that contains Octreotide, carry out the specificity video picture to the tumour of high expression level SSTR2/SSTR5 acceptor.
Integrin receptor
Integrating element is a receptoroid family that is positioned at cell surface, is the heterodimer transmembrane glycoprotein that is connected to form by non covalent bond by α, two chains of β, mediated cell and extracellular matrix (ECM) and intercellular adhesive attraction.Sticking with generation, development and infiltration, the transfer of tumour between the cell is closely related.Integrate plain RGD sequence through recognition ligand, combine with extracellular ligand, received signal stimulates, and participates in vasculogenesis.
The RGD peptide is a part of integrating plain (matrix metalloproteinase acceptor).Matrix metalloproteinase (MMP) is one type of proteolytic ferment that relies on divalent cations such as Zn2+, calcium 2+, can the degradation of cell epimatrix, and help tumor-infiltrated and shift.The RGD peptide is one type of small peptide that contains arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence, extensively is present in the organism, is to integrate recognition site plain and its ligand interaction.
Discover that integrating for 8 kinds plainly has expression on the vascular endothelial cell surface, comprise α 1 β 1, α 2 β 1, α 3 β 1, α 5 β 1, α 6 β 1, alpha 6 beta 4, α v β 3 and α v β 5.The plain molecule of multiple integration can combine with the RGD peptide, but mainly is α v β 3 and α v β 5, especially α v β 3, expresses tool selectivity.Research confirms; The integrin alpha v beta 3 acceptor not only has high expression level on the kinds of tumor cells surface; And strong expression is arranged at tumor tissues new vessel endothelial cell membrane; But in mature blood endothelial cell and most normal organ system, α v beta 3 receptor express lack or almost can not be visited and.Therefore, α v beta 3 receptor can be used as the good targets that the MR imaging shows vasculogenesis.Present stage, a large amount of research launched around integrating element.
TfR
HTrf's acceptor (human transferrin receptor, hTfR) as a molecular target once be used to directly as the treatment tumour with the carrier of transhipment medicine through hemato encephalic barrier.Moore etc. use it for the MR imaging.MR hTfR probe with the three-dimensional arrangement that contains iron can show hTfR expression of receptor and adjusting.This novel source of iron property acceptor probe can get into cell, and educate altogether in the process at test period through hTfR, can not produce immediately because the concentration of iron downward modulation that hTfR causes.This prompting hTfR can be used for live body and show genetic expression.Weissleder etc. use outward by MION of low molecular dextran (monocrystalline iron oxide nanoparticles) and TfR covalent coupling; Make up molecular probe; Specificity has shown ETR (Engineering Transferrin Receptor) positive tumor, starts the beginning of TfR rii gene.Thereafter Moore etc. continues to utilize the ETR acceptor through transforming, and carries out the MR video picture, has further affirmed TfR as reporter gene, carries out the feasibility of rii gene.Subsequently, the research of TfR rii gene increases gradually.
Folacin receptor
Under the physiological status; Vitamins B 9 (VB9) exists with the hydrophilic small molecules familiar form; As the one carbon unit shift reaction cofactor that comprises the Nucleotide de novo synthesis, it is synthetic at DNA and RNA, play a crucial role in the metabolism of apparent regulation process, cell increment and survival etc.The serum-concentration of circulation existence form-----5-methyl tetrahydrofolate (5-methyltetrahydrofolate) of VB9 is between 5-30nM.Folic acid deficiency to DNA duplicate and reparation, genetic expression have material impact, folic acid deficiency is to cause the immediate cause of fetal nerve systemic disease and cardiovascular disorder.
The wetting ability of folic acid and electronegativity make it under physiological status pH, can not pass through cytolemma with the mode of passive diffusion basically, must have carrier that it is transported in the cell.Folic acid has three kinds of carriers, and each works under different environment.Wherein a kind of carrier is called folacin receptor, and (folate receptor FR), has very high avidity with folic acid.FR is positioned cytolemma; Be a kind of glycosyl-phosphatidyl inositol (glycosyl phosphatidylinositol, the GPI) gp of grappling, but characteristic ground combines with the 5-methyl tetrahydrofolate; Dissociation constant relies on non-classical cell endocytic mode to transport folic acid in inferior nmole rank.
Folacin receptor is extended familys, is divided into four types, is called FRs-α respectively ,-β ,-γ and-δ.Research shows that FRs-α expresses very high like non-adenocarcinoma,mucoid, choriocarcinoma of testis, ventricles of the brain periosteum knurl, malignant pleural mesothelioma and the non-functional pituitary carcinoma etc. of ovary, uterus and uterine neck in most of malignant tumour; And other tumours, like mammary cancer, colorectal carcinoma and kidney, the FRs-alpha expression is seldom.Folacin receptor is conservative at the healthy tissues camber.Therefore, can be used for the tumour-specific video picture.
Konda etc., combine with the folacin receptor specificity as molecular probe with polyethyene diamine-folic acid dendendrimer (poly amidoamine-folate-dendrimer) PAMAM, show ovarian cancer (surpassing 80% ovarian cancer expression FR).External, friend's cell and the radiolabeled folic acid dendrimers combination rate of expressing FR are high by 2700% than control cells.And this combination can be suppressed by free folic acid, drops to the combination level that folacin receptor is expressed negative cells.In the experiment, oophoroma tumor is after injecting folic acid tree ridge portion in vivo, and signal strengthens 33%, obviously is different from non-specific, the ECS contrast medium Gd-HP-DO3A of same dose.In addition, this schedule of reinforcement does not occur in the saline water experimental group and folic acid is expressed negative tumour, and can be suppressed by free folic acid.The result shows, this have very high relaxation rate (1646mM (1) s (1)) macromole MR contrast medium, can be used to tumour cell and ovarian cancer that specificity shows the high expression level folacin receptor.After this, to folacin receptor, multiple imaging means is all tested, and comprises near infrared imaging, fluorescence imaging and nucleic fluorescence complex imaging etc., has expanded the application of folacin receptor imaging greatly.
It is the important step that makes up the molecular probe that can form images that ligands specific is connected (labeling process) with the imaging core.Early stage use direct labelling method is connected albumen or polypeptide with the imaging component, often condition of contact is relatively fiercer, and is therefore bigger to the damage of protein and polypeptide, and the two combination is insecure, comes off easily, thereby more uses at present is the indirect labelling method.The indirect labelling method be through bifunctional chelating agent (bifunctional chelating agents, BFCA) or bifunctional linking reagent (bifunctional linker, BFL) will form images component and specific molecular coupling.BFCA is just as a bridge block, and an end connects the target compound of wanting mark, and the other end complexing imaging component can be accomplished mark under common condition.Through the application of BFCA, do not change the characteristic of affinity tag, in conjunction with firm, and can avoid damage to affinity tag, reach the requirement of video picture.Therefore, select suitable bifunctional chelating agent extremely important to making up well behaved molecular probe.Bifunctional linking reagent is stablized chelated metal ions on the one hand; Alleviate its body physiological toxicity; Avoid metals ion directly to link to each other on the other hand, be unlikely to influence the polypeptide physiologically active, thereby guarantee the physiologically active and imaging usefulness of probe with specific proteins or peptide molecule.
Present stage, the imaging means of molecular imaging mainly contained PET (SPECT), MR and optical imagery.A little less than the optical imagery tissue penetration, be difficult to competent deep tissue video picture; The nuclear medicine image technology has the susceptibility height, and can quantitative advantage, but the nuclear medicine image spatial resolution is low, thereby is unfavorable for its further widespread use; MR is high because of spatial resolution, anatomical background clearly can be provided, and the advantage such as information such as function, metabolism of human body can be provided and receive increasing attention gradually.But compare with nuclear medicine, MR imaging sensitivity is poor slightly, and the more important thing is that MR is difficult to quantitatively, and this has greatly limited the development and the application of MR probe.
Multi-mode probe (multimodality probe) receives increasing attention because of advantage that can comprehensive different imaging modes.Having MR spatial resolution MR/SPECT (PET) bimodulus probe high and the highly sensitive advantage of nuclear medicine concurrently has become the main pattern of present this type of problem of solution.Yet multimode probe construction process adopts different separately imaging core (as adding nuclear medicine core such as 99mTc etc. with MR imaging core Gd, Fe, Mn etc. respectively) to come combined dual-mode or multimode probe in the current research; The probe molecule amount is very big; Cause stability of molecule to descend, in-vivo tissue distributes and the target-seeking ability drop; Because probe structure is complicated, and construction step is many, it is big to make up difficulty, is difficult for repetition in addition, and it is not little to enter into the clinical application difficulty.Therefore, how under the situation that guarantees probe specificity and bimodulus characteristic, make up that a kind of molecular weight is less, stable in properties, simple in structure, make up and be easy to very necessity of small molecules multimode/bimodulus probe.
Gadolinium (Gadolinium, Gd) general little than the SPIO class of the molecular weight of type probe.Gd is a kind of REE, and nuclear is outer to have a plurality of unpaired electrons, and spin magnetic moment is very big, is well suited for the MR imaging.The standard nucleidic mass of Gd is 157.25.Occurring in nature has 6 kinds of stable isotropic substances (being respectively 154Gd, 155Gd, 156Gd, 157Gd, 158Gd and 160Gd) and a kind of natural radioactive isotope 152Gd.The Gd isotropic substance one that adds synthetic has 29 kinds of ri.The natural radioactive isotope 152Gd transformation period is 1.08 * 10
14Year, be not suitable for imaging; Artificial radioisotope's transformation period, this wherein most of isotropic substance transformation period also was not suitable for being used for forming images less than 24.6 seconds all less than 74.7.
Immunofluorescence can be used to also show that receptors ligand combines situation.Immunofluorescence is special, sensitivity, and image is directly perceived, and can directly " see " part and corresponding receptor binding site, has very strong cogency.But compare with radioisotope method, shortcoming is exactly quantitatively difficult, can not calculate ligand receptor avidity, can't sets forth in detail binding kinetics details.Therefore, the gold standard of receptors bind experiment at present is
125The radioisotope method of I mark, this method is sensitive, reliable results, but quantitative Analysis receptor-ligand avidity, thus accurately reflect molecular probe video picture usefulness and BA.But
125Though the radioisotope method of I mark is a gold standard,
125The specific molecular surrogate of I mark can only partly reflect the usefulness of molecular probe, and under the practical situation, molecular probe is made as a whole have which kind of imaging modality and BA, and still difficulty is used fully
25The specific molecular surrogate of I mark is illustrated.Therefore, complete molecular probe itself have to
125I equally can just become the research focus by accurate quantification.
Fundamental research shows that most of elements of occurring in nature all have more than a kind of isotropic substance, and the element of often saying is actually the mixture that various isotropic substances constitute according to certain abundance.The maximum element of content of various molecules all has isotropic substance such as C, H, O, N, S, P etc. in constituting biological cell and organizing.C mainly contains 3 kinds of isotropic substances, and C12, C13 and C14 wherein are mainly the above two, and natural abundance (volumetric molar concentration) is respectively C120.9893 and C130.0107, C14＜10
-12, radioactivity is arranged, though abundance is extremely low, considerable using value is arranged but.Airborne C12 is become C14 by cosmic-ray bombardment, makes C12 and C14 in the atmosphere be maintained in a stable ratio.C14 in biological and the atmosphere keeps the same ratio.Just only decay is not additional in the dust for biology, and ratio has just become.Utilize this point can be to the carbonaceous thing division of history into periods.Hydrogen has 3 kinds of natural isotropic substances, is respectively protium, deuterium, tritium, and protium abundance on earth is 99.9885%, and deuterium abundance on earth has only 0.0115%, and the abundance of tritium is less than 0.000001%.Occurring in nature oxygen mainly exists with O16, O17, three kinds of isotopic forms of O18, and relative abundance is respectively 99.759%, 0.037%, 0.204%.N mainly contains two kinds of stable isotopes at nature, and 14N is 99.636%, and 15N is 0.364.The main coordination of S have S32, S33, S34 and S36, and abundance is respectively 94.93%, 0.76%, 4.29%, 0.02%.P mainly exists with P31, and abundance is near 100%, but is widely used in the certain biological study of radioactivity P32 and P33.
Summary of the invention
An object of the present invention is to provide a kind of inner complex.
Inner complex provided by the present invention is that the polypeptide of sequestrant and specific recognition test substance is connected to form binding substances through covalent linkage, the inner complex that binding substances is connected to form through co-ordination bond with radioactive metal ion again.
In the above-mentioned inner complex, said radioactive metal ion is 153Gd
3+Or other ri of Gd.
In the above-mentioned inner complex, said sequestrant is linear sequestrant or annular sequestrant.
In the above-mentioned inner complex, said linear sequestrant is the verivate of diethylene triamine pentacetic acid (DTPA) (DTPA) or diethylene triamine pentacetic acid (DTPA); NHS-MAG3 or hydrazino-nicotinamide (HYNIC);
In the above-mentioned inner complex, said annular sequestrant is 1,4,7 7-triazacyclononane nitrilotriacetics (NOTA), 1,4; 7,10-tetraazacyclododecanand-1,4,7,10-tetraacethyl (DOTA), 1; 4,7 7-triazacyclononane nitrilotriacetics (NOTA) and 1,4,7; 10-tetraazacyclododecanand-1,4,7, the verivate of the verivate of 10-tetraacethyl (DOTA), cyclic anhydride DTPA or cyclic anhydride DTPA.
In the above-mentioned inner complex, the polypeptide of said specific recognition test substance is Sostatin or RGD peptide.
Another object of the present invention provides a kind of molecular image probe.
Molecular image probe provided by the present invention is above-mentioned arbitrary described inner complex.
The application of above-mentioned arbitrary described inner complex in the preparation molecular image probe also belongs to protection scope of the present invention.
Cyclic anhydride DTPA (being cDTPA) is the anhydride form of DTPA, is linear DTPA through hydrolysis, and cDTPA is as acid anhydrides, and its activity is high than lienar for.
The molecular formula of DTPA and DOTA is respectively suc as formula shown in I, the formula II
1, the principle of probe of the present invention:
In the probe of the present invention, polypeptide is and target receptor specificity bonded small molecules, is the important component part of probe; 153Gd
3+Be SPECT imaging core and MR imaging core; Sequestrant connects polypeptide and 153Gd
3+
Ri 153Gd
3+240.4 ± 10 days transformation period, the gamma-rays peak value of emission is respectively 41 and 102keV, is very good nucleus medical imaging agent.If two atom proton numbers are identical, but the neutron number is different, then they still have identical ordination number, are the elements of same position at periodictable, so both just are isotropic substance.Active isotropic substance is called " ri ", does not have radioactive then be called " stable isotope ", and not all isotropic substance all has radioactivity.Ri (radiosotlope) is unsettled, its meeting " change ".Radioisotopic nucleus is very unstable, can incessantly, spontaneously radiate ray, and until becoming another kind of stable isotope, Here it is so-called " nuclear disintegration ".Ri can radiate alpha-ray, β ray, gamma-rays and electron capture etc., but ri might not radiate these several kinds of rays simultaneously when carrying out nuclear disintegration when carrying out nuclear disintegration.The speed of nuclear disintegration does not receive the influence of ambient conditionss such as temperature, pressure, EM field, does not receive the influence of element state of living in yet, and is only relevant with the time.The speed of ri decay is represented with " transformation period " usually.Transformation period (half-life) is that some amount ri atom number reduces to its needed time of initial value one half.Transformation period like phosphorus-32 is 14.3 days, in other words, if 1,000,000 phosphorus-32 atoms were arranged originally, after 14.3 days, only remaining 500,000.Transformation period is long more, explains and decays slowly more; Transformation period is short more, explains and decays soon more.Transformation period is a radioisotopic characteristic constant, and different ri has the different transformation period, and the kind that radiates ray in the time of decay is also different with quantity.Because radioactivity is the inherent nature of nuclear, does not receive the influence of outside temperature, pressure, chemical environment, pH value etc., so radioactivity 153Gd
3+Can become SPECT imaging core.Gd causes that through bound water molecule the surrounding tissue relaxation changes, thereby causes that the MR signal changes, because a kind of isotropic substance of element has identical chemical property, therefore, radioactivity 153Gd
3+Can both cause with non radioactive isotope 157Gd and to organize relaxation, so radioactivity 153Gd
3+Can become MR imaging core.Radioactivity 153Gd
3+The ability that has MR imaging and SPECT imaging as central ion.
Sequestrants such as DOTA, NOTA, DTPA, MAG3, HYNIC can be connected with covalent linkage with polypeptide; Radioactivity 153Gd
3+Nuclear is outer to have the unoccupied orbital that number does not wait, and by a plurality of coordination N atoms on the sequestrant electronics is provided, and forms co-ordination bond, is the stable chelate at center thereby form with the metals ion.
So probe of the present invention can be realized SPECT imaging and MR imaging simultaneously.
Bifunctional linking reagent has multiple choices, and is specific as follows: DTPA is linear, and inner complex stability is a bit weaker; Hydrazino-nicotinamide (hydrazinonictinamide; HYNIC) biological activity is the highest; Can combine with any polypeptide; And ethylenediamine-N,N'-diacetic acid(EDDA) (EDDA), trishydroxymethyl glycocoll (Tricine), three sulfonate sodiums of triphenylphosphine (TPPTS), BP (3-benzoylpyridine), AP (3-acetylpridine), NGA collaborative parts such as (galactosy 1-neoglycoalbumin) are used, and can form highly stable mixture; Sulfydryl acetyl three glycyl-N-maloyl imines ester (NHS-MAG3; N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycline) also be a kind of BFCA commonly used, need do not assist part, mark is simple; Consuming time few, the factor that influences mark rate is less; And the 7-triazacyclononane nitrilotriacetic (1,4,7-triazacyclononane triacetic acid; NOTA) and tetraazacyclododecanand (1,4,7; 10-tetraazacyclododecane, DOTA) and verivate be cave nest shape compound, its inner complex is the most stable; Wherein DOTA is because have more chelating site, thereby becomes chelating Gd
3+Ionic is first-selected.
2, the advantage of probe of the present invention:
(1) probe of the present invention is owing to be that a kind of imaging core is exercised two kinds of imaging functions simultaneously, but not two kinds of imaging cores constitute combination probes, and therefore, probe molecule amount of the present invention is very little, and stability of molecule is high, is prone in vivo that homogeneous microstructure distributes, and the target-seeking ability is strong.
(2) use polypeptide in the probe of the present invention, the preparation of need not desensitizing greatly makes things convenient for clinical position; The molecular weight of polypeptide quasi-molecule is little, and it is strong in in-house penetrance and perviousness, generally in injection back about the 4 hours best visualization time behind the injection probe.Can accomplish inspection on the same day of injected molecules probe like this; Polypeptide quasi-molecule stability in vivo is high, minimum to the spinoff of human body in vivo; Polypeptide class specificity small molecules connects with bifunctional chelating agent easily, makes the synthetic and preparation process of probe significantly oversimplify convenient research and clinical position; The polypeptide class is because molecule is little, thus have very high target and background is organized ratio, so that obviously improve the pathological tissues picture contrast.
(3) adopt the small molecules sequestrant can eliminate sequestrant to the active influence of polypeptide functional group; Sequestrants such as DOTA, NOTA, DTPA, MAG3, HYNIC reduce synthetic cost of probe and synthetic difficulty; Whole probe molecule amount is less, can improve the penetrance of the organism of probe, has improved the biology performance of probe.
(4) probe structure of the present invention is simple, and construction step is few, and it is low to make up difficulty, and construction process is stable, is easy to clinical practice and uses.Construction process with conventional MR probe is the same, uses 153Gd
3+Mark DOTA-OCT can obtain MR/SPECT bimodulus probe once going on foot.Based on this, the step that makes up the bimodulus probe has been simplified greatly.
(5) free Gd
3+Poisonous, must just can be applied to in-vivo imaging later on by chelating.Sequestrant DOTA is bigger than linear ligands chelating stability constants such as DTPA as a kind of annular part in the probe of the present invention, and Gd
3+Ri has very high stability after combining, and makes the Gd of chelating be difficult for dissociating, and has avoided free Gd
3+The shortcoming that toxicity is big.
(6) same a kind of imaging core of probe of the present invention has two kinds of imaging functions concurrently and has great inherent advantage.Under MR forms images clearly background; Can bring into play the responsive advantage of nuclear medicine; So as to exploring the enhancing degree and the mechanism and enhancement mechanism of MR probe; Make molecular probe experiment in vitro such as receptor-ligand combine in experiment, toxicity test and the body distribution experiment to become simple and accurately, make that insoluble always MR molecular probe can not quantitative problem be readily solved.And, according to similar thinking, can utilize radioactivity Fe to make up SPIO quasi-molecule probe as the SPIO core, can increase the susceptibility of MR imaging, help the progress and the clinical application of MR molecular image; Can also expand to the PET/CT field, utilize radioiodine partly to replace the on-radiation iodine in the present CT contrast medium, make up the special-purpose difunctional contrast medium of PET/CT.
Through synthesizing of this probe, for the structure of other similar probes is laid a good foundation; Can carry out the specificity imaging according to the corresponding molecular probe of different disease pattern layout, early diagnosis of tumor is provided and differentiate qualitative novel method for clinical.
Description of drawings
Fig. 1 is the binding substances synoptic diagram of sequestrant and polypeptide.
Fig. 2 is the probe structure synoptic diagram.
Fig. 3 Somatostatin and Sostatin amino acid are formed.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Sequestrant DOTA and sequestrant DTPA be available from lark prestige company, products catalogue be numbered T1875 (CAS number:
60239-18-1) and 114322500 (CAS number:
67-43-6); Sostatin (OCT) is available from Shanghai peptide bodyguard bio tech ltd, and products C AS number is 83150-76-9;
The aminoacid sequence of Sostatin: (D-Phe-c [Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-[ol]).
Common several kinds of RGD titles and structure:
C (RGDfC): cyclo (Arg-Gly-Asp-D-Phe-Cys), article No.: PCI-3686-PI
C (RGDfK): cyclo (Arg-Gly-Asp-D-Phe-Lys), article No.: PCI-3661-PI
Trifluoroacetate Salt:cyclo (Arg-Gly-Asp-D-Phe-Lys), article No.: PCI-3919-PI
C (RADfK): cyclo (Arg-Ala-Asp-D-Phe-Lys), article No.: PCI-3883-PI.
Above rgd peptide also all can replace Sostatin to carry out the probe preparation.
The structure of embodiment 1, molecular image probe
Sequestrant DOTA and Sostatin are connected to form binding substances (DOTA-OCT) through covalent linkage, and the structural formula of binding substances is as shown in Figure 1.
Binding substances and radioactive metal ion 153Gd
3+Be connected to form inner complex (153Gd through co-ordination bond
3+-DOTA-OCT), the structural formula of inner complex is as shown in Figure 2.
Co-ordination bond, promptly central metallic ions nuclear is outer has the unoccupied orbital that number does not wait, and by a plurality of ligating atoms electronics is provided, and forms co-ordination bond, and forming with the metals ion is the stable chelate at center.
The preparation of embodiment 2, molecular image probe
1, the binding substances of DOTA and Sostatin (DOTA-OCT)
Whether adopt the classical Hnatowitch method of improvement such as Fania to connect sequestrant and peptide molecule, it is complete to detect connection with the ITLC method, and with HPLC separation, purified product, product is finally identified with mass spectrum.
The binding substances of DOTA and Sostatin can be available from AnaSpec company, article No.: 24195, and network address:
Http:// www.anaspec.com/products/product.asp? Id=30878, name of product is DOTA-Octreotide.
2, radioactivity 153Gd
3+Inner complex (153Gd with binding substances formation
3+-DOTA-OCT)
Radioactivity 153Gd
3+(100 μ Ci) from Chinese Institute for Atomic Research or PerkinElmer company buy.
5 μ mol DOTA-OCT are dissolved in 10ml 0.2M Tris-HCl (pH6-7) damping fluid, and heating (60 ℃ of water-baths) 10 minutes is again to wherein adding 153Gd
3+5 μ mol, room temperature (25 degree) left standstill 120 minutes, obtained inner complex molecular probe 153Gd
3+-DOTA-OCT.
3, probe separates, purifying
The probe product separates with C-18Sep-Pak, before the last appearance, with 5ml ethanol and the activation in advance of 5ml water; After the last appearance, use the 5ml normal saline washing earlier, use then 80% EtOH (ethanol) aqueous solution as moving phase as elutriant, every pipe 1ml collects elutriant, detects each with gamma meter and manages.Collect ray counting largest tube elutriant (solution that wash-out first pipe and second pipe elute), promptly obtain the purifying probe.
Claims (7)
1. inner complex, for the polypeptide of sequestrant and specific recognition test substance is connected to form binding substances through covalent linkage, the inner complex that binding substances is connected to form through co-ordination bond with radioactive metal ion again.
2. inner complex according to claim 1 is characterized in that: said radioactive metal ion is 153Gd
3+Or other ri of Gd.
3. inner complex according to claim 1 and 2 is characterized in that: said sequestrant is linear sequestrant or annular sequestrant.
4. according to arbitrary described inner complex among the claim 1-3, it is characterized in that: verivate, NHS-MAG3 or hydrazino-nicotinamide that said linear sequestrant is diethylene triamine pentacetic acid (DTPA), diethylene triamine pentacetic acid (DTPA);
Said annular sequestrant is 1,4,7 7-triazacyclononane nitrilotriacetics, 1,4,7; 10-tetraazacyclododecanand-1,4,7,10-tetraacethyl, 1; 4,7 7-triazacyclononane nitrilotriacetics, 1,4,7; 10-tetraazacyclododecanand-1,4,7, the verivate of the verivate of 10-tetraacethyl, cyclic anhydride DTPA or cyclic anhydride DTPA.
5. according to arbitrary described inner complex among the claim 1-4, it is characterized in that: the polypeptide of said specific recognition test substance is Sostatin or RGD peptide.
6. molecular image probe is for profit requires arbitrary described inner complex among the 1-5.
7. profit requires the application of arbitrary described inner complex in the preparation molecular image probe among the 1-5.
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