CN100459932C - MRI molecular image probe and its preparing method - Google Patents

MRI molecular image probe and its preparing method Download PDF

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CN100459932C
CN100459932C CNB200710062715XA CN200710062715A CN100459932C CN 100459932 C CN100459932 C CN 100459932C CN B200710062715X A CNB200710062715X A CN B200710062715XA CN 200710062715 A CN200710062715 A CN 200710062715A CN 100459932 C CN100459932 C CN 100459932C
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molecular
mri
probe
chelating agen
image probe
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CN100998506A (en
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杜湘珂
霍天龙
张森
李绪斌
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Peking University Peoples Hospital
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Abstract

A MRI molecular image probe with high biologic performance and low cost is composed of central ion, specific molecule and chelating agent consisting of primary chelating agent chosen from HYNIC and secondary one chosen from Tricine and EDDA. Its preparing process is also disclosed.

Description

A kind of MRI molecular image probe and preparation method thereof
Technical field
The present invention relates to MRI molecular image probe and synthesis preparation method thereof.
Background technology
NMR (Nuclear Magnetic Resonance)-imaging (MRI) technology is one of medical science molecular imaging three big technology, and other also comprises PET (SPECT) and optical imagery.NMR (Nuclear Magnetic Resonance)-imaging molecular image technology is compared advantages such as having probe is "dead", image dissection resolution height with the PET technology; But weak point is a signal detect with specificity low.MR still belongs to anatomic form diagnosis category at present, functional MR and MR wave spectrum can only be included into indirect molecular image category in early stage, the MR molecular imaging is potential to become the most promising molecular imaging imaging means, and what lack now is the specific probe that direct targeting is arranged on the molecular level.
Probe is the basis of nmr molecular image technology (MRI).The MRI molecular image probe comprises the central ion that can be detected by MRI equipment, and can with the specific molecular (comprising part, specific antibody etc.) of object specific bond, in order to make the MRI molecular image probe have the stability of height and enough signal intensitys, this research is developed voluntarily and is made up MR targeting probe, specific molecular is connected on the chelating agen, central ion and chelating agen form chelate, carry out the targeted developing imaging of tumor or target pathological changes and detect.
In the MRI molecular image probe, central ion is selected the paramagnetic metal element usually for use, and the paramagnetic metal central ion of transition metal commonly used and lanthanide series metal (paramagnetic metal ion) has Fe 2+, Fe 3+, Mn 2+, Gd 3+And Dy 3+Deng.Several paramagnetic metal ions and chelating agen (part) binding ability relatively sees Table 1.
Table 1 paramagnetic metal ion and chelating agen (part) binding ability relatively
Paramagnetic metal ion Metal ion radius/nm Easily and the bonded coordination atom of metal ion Easily and the bonded coordinating group of metal ion
Gd 3+ 0.0938 Oxygen and nitrogen ligand Many many carboxylations of ammonia compounds
Fe 2+、Fe 3+ 0.076 The nitrogen ligand Carboxylic acid group, tyrosine, porphyrin
Mn 2+ 0.088 Oxygen and nitrogen ligand Carboxylate, phosphate
These metal ions all have water solublity, Gd 3+Be the most normal use paramagnetic metal central ion, Gd 3+7 unpaired electrons are arranged, and spin magnetic moment is big, the electric field symmetry, and relaxation efficient height, easily and the water coordination, and the water of coordination molecule is 8,9, is more satisfactory paramagnetic metal ion.But, free hydration Gd 3+Reaching most of coordination compounds can not be compatible with venous blood, and easily precipitation is separated out, and toxicity is big.This just requires to select to have very high stability after chelating agen and the metal ion combination.
Chelating agen (coordination compound) is chosen in MRI molecular probe preparation process and plays an important role.Chelating agen not only needs to form high stability chelate with paramagnetic metal ion, and needs and the specific molecular combination.So chelating agen plays important difunctional effect in the MRI molecular probe.
Chelating agen commonly used is generally divided into linearity and annular two big classes.No matter be that the sort of chelating agen all needs to form with paramagnetic metal ion and has high stability chelate, and can have stable combination, also need formed chelate not influence the biology performance of specific molecular with specific molecular.Because this type of chelating agen should connect with polypeptide, also will form chelate with metal ion, thus the bifunctional linking reagent that is otherwise known as (bifunctional conjugatingagent, BFCA).For the MRI specific probe, no matter be that linear chelating agen or annular chelating agen all belong to bifunctional linking reagent.
At present, linear chelating agen commonly used has diethylenetriamine pentaacetic acid (DTPA) and derivant thereof etc., and annular chelating agen has 1,4,7,10-tetraazacyclododecanand-1,4,7,10-tetraacethyl (DOTA), cyclic anhydride DTPA and ethylenediaminetetraacetic acid (EDTA) and their derivant etc.The molecular formula of DTPA and DOTA is respectively suc as formula shown in I, the formula II:
Figure C200710062715D00041
(formula I) (formula II)
In the MRI molecular image probe, specific molecular comprise can with enzyme, the bonded polypeptide of receptor-specific; Can with the competitive bonded micromolecule of enzyme acceptor; And have and bonded antibody of antigenic specificity etc.
Studies show that in the MRI of surface of cell membrane probe specificity Molecular Selection in conjunction with target spot for the MRI probe: polypeptide class and micromolecule class are better than the selection antibody class.This mainly be since antibody and monoclonal antibody specific class probe there are the following problems: antibody needs the preparation of desensitizing in use; Antibody comprises that specific monoclonal antibody specificity in vivo is still unstable, is difficult to reach its intended purposes; The antibody class molecular weight is penetrance and poor permeability in tissue greatly, so the best visualization time is long.Generally to about 24 hours, be unfavorable for clinical position like this; During the antibody class labelling to problems such as environmental requirement height.Although adopt the method for antibody fragment recent years for the antibody class video picture, still be difficult to reach the set goal.And, do not need desensitization to prepare for polypeptide class and micromolecule competitive inhibitor, greatly make things convenient for clinical position; The molecular weight of polypeptide quasi-molecule is little, and it is strong in in-house penetrance and permeability, generally in injection back about the 4 hours best visualization time behind the injection probe.Can finish inspection on the same day of injected molecules probe like this; Polypeptide quasi-molecule stability in vivo is high, minimum to the side effect of human body in vivo; Polypeptide class and specificity micromolecule class connect with bifunctional chelating agent easily, make 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.What enter clinical practice the earliest is that 8 peptides of somatostatin (Somatostatin) are intended like thing octreotide (Octreotide), can and the combination of somatostatin receptor (SSRT) specificity, the performance physiological action.Table 2 is specificity MRI molecular probes that several different paramagnetic metal ion chelates connect.
The specificity MRI molecular probe that table 2 adopts different paramagnetic metal ion chelates to connect
Figure C200710062715D00051
From present MRI molecular image probe, trend towards selecting DTPA, DOTA class chelating agen and peptide molecule to connect.But there are a lot of difficulties owing to there is following deficiency in DTPA, DOTA class chelating agen and make it connect with polypeptide:
1) DTPA and DOTA belong to multi-functional chemical compound, with DTPA, need be when DOTA class chelating agen is connected with the polypeptide class with chelating agen and polypeptide other close to roll into a ball and protect, in order to avoid influence polypeptide and chelating agen function.When needing behind the end of synthesis that the protecting group in the functional group is removed, to recover the function of chelating agen and polypeptide.Complexity is synthesized in so tangible increase, and the cost of the making of probe, makes the MRI probe lose the value that actual clinical is used.
2) multi-functional chelating agen will influence the biologic activity of polypeptide class.
3) the polyfunctional group chelating agen increases the volume of probe.
Summary of the invention
The purpose of this invention is to provide a kind of MRI molecular image probe and preparation method thereof.
MRI molecular image probe provided by the present invention, comprise central ion, specific molecular and chelating agen, described specific molecular is connected with chelating agen, central ion and chelating agen form chelate, wherein, described chelating agen comprises main sequestering agent and assistant sequestering agent, and described main sequestering agent is a diazanyl crosslinking amino nicotiamide, and described assistant sequestering agent is selected from a kind of in N-three (methylol) methylglycine and the EDDA.
In the MRI molecular image probe, when assistant sequestering agent was N-three (methylol) methylglycine, main sequestering agent diazanyl crosslinking amino nicotiamide can carry out chelating with N-three (methylol) methylglycine of 2 molecules; When assistant sequestering agent was EDDA, main sequestering agent diazanyl crosslinking amino nicotiamide can carry out chelating with the EDDA of 1 molecule.
In the present invention, central ion is preferably Gd 3+, Fe 2+, Fe 3+Or Mn 2+Specific molecular is preferably octreotide and/or RGD peptide.
The preparation method of MRI molecular image probe of the present invention comprises the steps:
1) preparing centre solion;
2) in being connected with the diazanyl crosslinking amino nicotinamide soln of specific molecular, add N-three (methylol) methylglycine or EDDA, mix, obtain chelating agent solution;
3) central ion solution is joined in the chelating agent solution, two kinds of solution are mixed, obtain described MRI molecular image probe.
Wherein, step 2) in, the mol ratio of diazanyl crosslinking amino nicotiamide and N-three (methylol) methylglycine is 1:2, perhaps, with the mol ratio of EDDA be 1:1.But in the actual fabrication process, the consumption of these two kinds of assistant sequestering agents is often excessive a little.
The present invention is a chelating agen with HYNIC and Tricine or EDDA, with Gd3 +Chelating (or Fe 2+, Fe 3+, Mn 2+), synthesize the MRI molecular image probe, have following advantage:
HYNIC has high degree of specificity and can combine with the polypeptide quasi-molecule;
Adopt the micromolecule chelating agen to eliminate of the influence of chelating agen functional group to polypeptide active, it is the important prerequisite of its physiologically active of performance that polypeptide keeps correct space structure, the macromole chelating agen can change the space structure of polypeptide owing to contain many functional groups, influences its physiologically active; And the micromolecule chelating agen does not have unnecessary nonessential functional group except that containing the functional group that is connected with the polypeptide end, thereby slight to the influence of polypeptide space structure.
HYNIC and Tricine and EDDA are that chelating agen reduces the synthetic cost of probe;
Reduce the penetrance that the probe molecule amount improves the organism of probe, improved the biology performance of probe.Studies have shown that the molecular weight of complex is when 50kd is above, penetrance in vivo descends obviously, is not suitable for the requirement of specificity video picture substantially; And probe molecule amount of the present invention still has good result of use below 2000.
Description of drawings
Fig. 1 is the T1WI image of Gd-Tricine-HYNIC-Octreotide molecular probe different time in the rabbit body.
Fig. 2 is the T1WI image of Gd-DTPA molecular probe different time in the rabbit body.
The specific embodiment
Diazanyl crosslinking amino nicotiamide (HYNIC), the molecular structure of N-three (methylol) methylglycine (Tricine) or EDDA (EDDA) is respectively shown in formula III, formula IV and formula V:
Figure C200710062715D00071
(formula III) (formula IV) (formula V)
HYNIC can be specifically connects with polypeptide, and has good stability, and HYNIC also can form highly stable chelate with metal ion and EDDA or Tricine, can be used as the MRI probe.
The MRI probe of embodiment 1, HYNIC and Tricine and paramagnetic metal Gd
1, GdCl 3Synthetic: as to get 36.3mg Gd 2O 3, add among the 10ml0.1mol/L HCl, be heated to 60 ℃.Sustained response 10min, to dissolving fully, the HCl of heating 100 degree evaporating surplus gets final product.
2, preparation chelating agent solution: with 3mg[HYNIC-D-Phe 1, Tyr 3]-Octreotide (HYNIC-TOC, Shanghai gill biochemical corp is synthetic) is dissolved in 8ml0.2M Na 2HPO 4In PH6-7 buffer, fully get 20mgTricine after the dissolving, 50mg mannitol is dissolved in the above-mentioned solution, heating in water bath 10 minutes.
3, preparation MRI probe: get the synthetic GdCl of the first step 3(3-4 μ mol) joins and carries out chelatropic reaction in the above-mentioned chelating agent solution, room temperature jog mixing, and product separates with HPLC, obtains the Gd-Tricine-HYNIC-Octreotide molecular probe.
This molecular probe molecular weight is below 2000.
With synthetic MRI probe-Gd-Tricine-HYNIC-Octreotide (this probe T1WI is high signal mutually) molecular probe solution, slowly inject through auricular vein and to have anaesthetized in the healthy completely new zealand white rabbit body, after observing no abnormal phenomenon, beginning MR scanning, overlap identical sequence multiple scanning with one per half an hour, observe liver, spleen, the signal change of kidney and bladder and other internal organs, the result is shown in Figure 1A-Fig. 1 F, wherein, Figure 1A is rabbit liver T1WI image before the administration, Figure 1B is an administration 0min rabbit liver T1WI image, Fig. 1 C is an administration 30min rabbit liver T1WI image, Fig. 1 D is an administration 60min rabbit liver T1WI image, and Fig. 1 E is an administration 90min rabbit liver T1WI image, and Fig. 1 F is an administration 90min rabbit bladder T1WI image photograph; Fig. 1 G scans the dirty T1WI image photograph of rabbit kidney immediately after the administration.Simultaneously, with conventional contrast medium Gd-DTPA (Magnevist, Germany is spirit earlier) in contrast, its result as shown in Figure 2, Fig. 2 A is a rabbit liver T1WI image before the administration, Fig. 2 B is an administration 0min rabbit liver T1WI image, Fig. 2 C is an administration 30min rabbit liver T1WI image, Fig. 2 D is an administration 60min rabbit liver T1WI image, and Fig. 2 E is an administration 60min rabbit bladder T1WI image, and Fig. 2 F is an administration 90min rabbit bladder T1WI image photograph; Fig. 2 G scans the dirty T1WI image photograph of rabbit kidney immediately after the administration.
The result shows that MRI probe of the present invention is different from conventional non-specific contrast medium, and following characteristics are arranged:
1, after the injection, liver, splenic T 1WI signal are high before than injection, with 60 minutes significantly (Fig. 1 D), illustrate that normal hepatocytes, spleen have probe distribution of the present invention; Conventional contrast medium (Magnevist) then is the passing along with the injection time, and signal descends.
2, the signal of gallbladder is very low before administration and during 0min, and manifest high signal later on gradually at 30min, this prompting: this probe is mainly drained through liver and gall; Conventional contrast medium (Magnevist) is mainly through renal excretion.
3, in whole checking process, do not see obvious blood vessel reinforcement shadow (may be slowly relevant) with this experiment injection.Probe specificity of the present invention is the non-vascular contrast medium, is different from conventional contrast medium.
4,90 minutes the time, bladder is not seen tangible enhancing phenomenon (Fig. 1 F).And this moment, conventional contrast medium (Magnevist) is the highest in bladder concentration, signal the strongest (Fig. 2 F).
5, kidney T1 does not see obvious enhancing (Fig. 1 G); And conventional contrast medium (Magnevist) kidney strengthens obviously (Fig. 2 G).
Comprehensive above-mentioned observed result, as can be seen, Gd-Tricine-HYNIC-Octreotide has possessed metabolism and the body internal characteristic that is different from conventional contrast medium (Magnevist), is a species specificity contrast medium.
The MRI probe of embodiment 2, HYNIC and EDDA and paramagnetic metal Gd
1, GdCl 3Synthetic: as to get 36.3mg Gd 2O 3, add among the 10ml0.1mol/L HCl, be heated to 60 ℃.Sustained response 10min, to dissolving fully, the HCl of heating 100 degree evaporating surplus gets final product.
2, prepare chelating agent solution: with 3mg[HYNIC-D-Phe 1, Tyr 3]-Octreotide (HYNIC-TOC) is dissolved in 8ml 0.2M Na 2HPO 4In PH6-7 buffer, fully get 40mg EDDA after the dissolving, 50mg mannitol is dissolved in above-mentioned solution, heating in water bath 10 minutes.
3, preparation MRI probe: get the synthetic GdCl of the first step 3(3-4 μ mol) adds and carries out chelatropic reaction in the above-mentioned chelating agent solution, room temperature jog mixing, and product separates with HPLC, obtains the Gd-EDDA-HYNIC-Octreotide molecular probe.
Through zoopery, has identical performance with the Gd-Tricine-HYNIC-Octreotide molecular probe of embodiment 1.

Claims (7)

1.MRI molecular image probe, comprise central ion, specific molecular and chelating agen, described specific molecular is connected with chelating agen, central ion and chelating agen form chelate, it is characterized in that: described chelating agen comprises main sequestering agent and assistant sequestering agent, described main sequestering agent is a diazanyl crosslinking amino nicotiamide, and described assistant sequestering agent is selected from a kind of in N-three (methylol) methylglycine and the EDDA.
2. MRI molecular image probe according to claim 1 is characterized in that: the mol ratio of diazanyl crosslinking amino nicotiamide and N-three (methylol) methylglycine is 1:2; Perhaps, the mol ratio of diazanyl crosslinking amino nicotiamide and EDDA is 1:1.
3. MRI molecular image probe according to claim 1 and 2 is characterized in that: described central ion is Gd 3+, Mn 2+, Fe 2+Or Fe 3+
4. MRI molecular image probe according to claim 1 and 2 is characterized in that: described specific molecular is octreotide or RGD peptide.
5. the preparation method of the described MRI molecular image probe of claim 1 comprises the steps:
1) preparing centre solion;
2) at the Na that is dissolved in pH6-7 2HPO 4Buffer is connected with in the diazanyl crosslinking amino nicotinamide soln of specific molecular, adds N-three (methylol) methylglycine or EDDA and mannitol, mixes, and obtains chelating agent solution;
3) chelating agent solution is mixed with central ion solution, obtain described MRI molecular image probe.
6. preparation method according to claim 5 is characterized in that: central ion is Gd 3+, Mn 2+, Fe 2+Or Fe 3+
7. preparation method according to claim 5 is characterized in that: described specific molecular is octreotide or RGD peptide.
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CN102558291B (en) * 2010-12-17 2014-03-12 北京大学人民医院 Dual mode molecule image probe
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