CN102766188B - Cholesterol derivative, inner complex, rHDL and uses thereof - Google Patents
Cholesterol derivative, inner complex, rHDL and uses thereof Download PDFInfo
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- CN102766188B CN102766188B CN201210257894.3A CN201210257894A CN102766188B CN 102766188 B CN102766188 B CN 102766188B CN 201210257894 A CN201210257894 A CN 201210257894A CN 102766188 B CN102766188 B CN 102766188B
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Abstract
The invention discloses a kind of cholesterol derivative, inner complex, rHDL and uses thereof.The present invention by cholesterol being connected to, nitrogenous many carboxyl ligand obtains containing the derivative ligand of one or two cholesterol; This cholesterol derivative and metal ion carry out obtaining corresponding inner complex after chelating; In high-density lipoprotein (HDL), assemble this inner complex prepare rHDL; This rHDL is used for as contrast medium.Particle diameter, the form of this contrast medium are similar to the high-density lipoprotein (HDL) in human body with biological activity, and relaxivity is higher than commercial contrast medium magnevist (Gd-DTPA); Its active targeting by high-density lipoprotein (HDL) can realize liver region specificity radiography, and is realized bile duct and duodenal nuclear magnetic resonance by the metabolic process of cholesterol at liver.
Description
Technical field
The present invention relates to the paramagnetism magnetic resonance contrast agent of liver function diagnosis, be specifically related to a kind of cholesterol derivative, inner complex, rHDL and the purposes as contrast medium thereof.
Background technology
Nuclear magnetic resonance is harmless without wound as one, and imaging orientation is flexible, and the method that spatial resolution is high, is widely used in clinical diagnosis.Along with new contrast agent application, mr imaging technique has and uses widely in liver imaging and the imaging of courage pancreas.Liver MR Imaging can be used for making a definite diagnosis vascular tumor, Focal nodular hyperplasia and tumour etc., and does not need examination of living tissue, operation or follow-up examination etc.And liver MR Imaging can be used for checking liver cirrhosis, steatosis or liver hemochromatosis etc., can check liver cancer or common hepatic duct cancer to the patient of liver cirrhosis, diagnoses the patient receiving transplantation.Diagnosis of hepatic metastases usually need to judge by stages with prognosis target, liver MR Imaging can check the degree of diagnosis of hepatic metastases preferably, thus for excision, radio frequency eliminates or chemoembolization provides foundation, and can monitor the recurrence of diagnosis of hepatic metastases.In nuclear magnetic resonance clinical diagnosis, contrast medium, by changing the relaxation time of tissue local, reaches and forms certain difference with the relaxation time of surrounding tissue, thus realizes the object strengthening radiography.But the contrast medium used clinically mostly at present is extracellular fluid contrast medium, and this kind of contrast medium, after intravenous injection, penetrates into intercellular substance from blood pond fast, and mainly remove at kidney, do not have organ specificity mr to strengthen.And have report to show that the extracellular fluid contrast medium based on gadolinium chelate compound can cause the patient of serious kidney injury to produce kidney source sexual system fibrotic disease in recent years.Because the dosage that this kind of contrast medium exists is high and the problem such as toxic side effect, so the organ targeting magnetic resonance contrast agent of development of new becomes the important step improving clinical diagnosis accuracy and security.
High-density lipoprotein (HDL), as the one of lipoprotein, is present in the spherical macromolecular lipid carrier of solubility in blood plasma.High-density lipoprotein (HDL) forms hydrophobic kernel by the non-polar lipid such as triglyceride and cholesteryl ester, outer wrap up by Lipid monolayer, phospholipid layer is inlayed several lipophorin such as cholesterol and apolipoprotein A-1.High-density lipoprotein (HDL) mainly take part in reverse cholesterol transport process in vivo, key step is cholesterol that first place of high-density lipoprotein (HDL) picked-up liver peripheral tissues is more than needed and is transformed into cholesteryl ester in circulation to be in vivo stored in fat core, and then cholesterol and Cholesterol ester transfer are carried out metabolism to liver by high-density lipoprotein (HDL).Utilize the approach of this reverse cholesterol transport to build contrast medium thus the liver specific target tropism of magnetic resonance contrast agent can be improved.High-density lipoprotein (HDL) has the following advantages as carrier: (1) HDL is as a kind of endogenous material, can degradation in vivo, without immune response, and avoids being engulfed system identification and removing by monocyte; (2) HDL can conduct drugs in target cell by receptor-mediated endocytosis or selectivity picked-up, has higher targeting; (3) the structure of HDL can make hydrophobic drug enter nonpolar lipid core, reduces the contact of medicine and external environment, avoids medicine to be destroyed.Through finding the literature search of prior art, US Patent No. 7211248, the DTPA of chelating gadolinium ion is proposed to be connected on lipid PA, detect atherosclerotic plaque after being assembled into high-density lipoprotein (HDL), and the document delivered major part using high-density lipoprotein (HDL) as contrast medium to detect atherosclerotic plaque.And be assembled into high-density lipoprotein (HDL) after not having document and patent report to use cholesterol to connect paramagnetic ion inner complex and carry out nuclear magnetic resonance, especially to liver and gall and duodenal nuclear magnetic resonance.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art, a kind of cholesterol derivative, inner complex, rHDL and the purposes as contrast medium thereof are provided.Based on the magnetic resonance contrast agent of cholesterol derivative of the present invention, be that a kind of high magnetic resonance imaging performance, bio-compatibility are good, the contrast medium of liver specificity target, achieve the FMRI to liver and relevant organ.This contrast medium transformation period is in vivo longer, can realize, to the lasting imaging in liver, bile duct and duodenum region, can reducing the consumption of contrast medium, reduces the toxicity of contrast medium, improves the imaging effect of contrast medium.This contrast medium can make up the weak point of existing hepatobiliary magnetic resonance imaging technique, improves the accuracy of magnetic resonance image-forming diagnose.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the present invention relates to a kind of cholesterol derivative, and its structural formula is as shown in formula I:
Wherein, R1 is the structure shown in formula II, and R2 is the structure shown in hydroxyl or formula II:
Preferably, described R ' be alkyl, the group in succession of polyalkylene glycol type or the group in succession of polyether type.
Further preferably, described R ' is the structure shown in formula III:
wherein, n=2 ~ 16.
Further preferably, described R ' is the structure shown in formula IV:
wherein, n=1 ~ 16.
Further preferably, described R ' is the structure shown in formula (V):
wherein, n=1 ~ 16.
Further preferably, described R ' is the structure shown in formula VI:
wherein, n=1 ~ 16.
Further preferably, described R ' is the structure shown in formula (VII):
wherein, n=1 ~ 16.
Second aspect, the present invention relates to a kind of inner complex, and described inner complex is formed by aforesaid cholesterol derivative and metal ion-chelant.
Preferably, described metal ion is lanthanide metal ion.
The third aspect, the present invention relates to a kind of rHDL, and described rHDL is made up of apolipoprotein A-1, dimyristoyl phosphatidyl choline, aforesaid inner complex.
Preferably, the scope of the mol ratio of described apolipoprotein A-1, dimyristoyl phosphatidyl choline, inner complex is 1:400:(20 ~ 400).
Preferably, described rHDL adopts the preparation of Sodium cholic acid surfactant method and obtains, and comprises the steps: described apolipoprotein A-1, dimyristoyl phosphatidyl choline (DMPC), inner complex and Sodium cholic acid to mix, hatches, dialysis removing Sodium cholic acid, to obtain final product.
Fourth aspect, the present invention relates to the purposes of a kind of aforesaid restructuring high density lipoprotein level as the magnetic resonance contrast agent of hepato-biliary function target.
Preferably, described magnetic resonance contrast agent is reflection cholesterol becomes the process of cholic acid contrast medium at liver intracellular metabolite.
Preferably, described magnetic resonance contrast agent is the contrast medium that reflection cholesterol is directly excreted to the metabolic process of bile duct in liver body.
Compared with prior art, the present invention has following beneficial effect:
1, the longitudinal relaxation efficiency of magnetic resonance imaging contrast of the present invention is better than the contrast medium of Clinical practice, can reduce using dosage.
2, contrast medium of the present invention can be selectively targeted to liver, and drain from the pathways metabolism of cholesterol from bile duct, thus can strengthen the nuclear magnetic resonance effect of bile duct and duodenal site.
3, contrast medium of the present invention can the magnetic resonance image (MRI) of functional enhancing liver, and the paramagnetic ion inner complex being connected with different quantities cholesterol can reflect different cholesterol metabolic approach.
4, the inner complex being connected with single cholesterol in the present invention can strengthen the magnetic resonance signal of liver the long period, then strengthened the magnetic resonance signal of bile duct and duodenum enclosure region by Bile metabolism, reflect cholesterol becomes cholic acid process at liver internal metabolism.
5, the inner complex of two cholesterol is connected with in the present invention compared with being connected with the inner complex of single cholesterol, the time that liver signal strengthens is shorter, but still there are enough imaging time windows, strengthen the magnetic resonance signal of bile duct and duodenum near zone at short notice, mainly reflect the metabolic process that cholesterol is directly excreted to bile duct in liver body.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of the gadolinium chelate compound of cholesterol derivative;
Fig. 2 is the structure iron of the DTPA part that different cholesterol linking groups is formed;
Fig. 3 is the NMR of single cholesterol derivative DTPA-chol
1h spectrogram;
Fig. 4 is the NMR of single cholesterol derivative DTPA-chol
13c spectrogram;
Fig. 5 is two cholesterol derivative DTPA-(chol)
2nMR
1h spectrogram;
Fig. 6 is two cholesterol derivative DTPA-(chol)
2nMR
13c spectrogram;
Fig. 7 is the grain size distribution of the rHDL Gd-chol-HDL of the gadolinium chelate compound Gd-DTPA-chol being assembled with single cholesterol;
Fig. 8 is the gadolinium chelate compound Gd-DTPA-(chol) being assembled with two cholesterol
2rHDL Gd-(chol)
2the grain size distribution of-HDL;
Fig. 9 is transmission electron microscope (TEM) picture of the rHDL Gd-cholHDL of the gadolinium chelate compound GdDTPA-chol being assembled with single cholesterol;
Figure 10 is the gadolinium chelate compound Gd-DTPA-(chol) being assembled with two cholesterol
2rHDL Gd-(chol)
2transmission electron microscope (TEM) picture of-HDL;
Figure 11 is prepared contrast medium longitudinal relaxation efficiency (1/T in aqueous
2) relative to the rectilinear of gadolinium concentration matching;
Figure 12 be Gd-chol-HDL in rat body to liver and duodenal T
1weighted imaging figure;
Figure 13 is Gd-(chol)
2-HDL in rat body to liver and duodenal T
1weighted imaging figure;
Figure 14 is the liver of two kinds of contrast medium in rat body and the magnetic resonance signal enhancing scale map in duodenum region.
Embodiment
Below in conjunction with accompanying drawing, embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
the preparation of embodiment 1, cholesterol derivative and inner complex thereof
1.1, cholesterol derivative is prepared
1.1.1, the preparation of cholesterol derivative I
As shown in Figure 1,500mmol quadrol is dissolved in 100ml anhydrous methylene chloride, joins in dried 100ml there-necked flask, add 10mmolN simultaneously, N-diisopropylethylamine, stir and make it mixing.Be dissolved in by 5mmol cholesteryl chloroformate in 20ml anhydrous methylene chloride, dropwise slowly join in the solution of quadrol under ice-water bath, continue half an hour, the temperature of reaction system rises to room temperature.Nitrogen is passed in there-necked flask, the lower reaction system of sealing at room temperature stirs 2 days, after reaction terminates, reaction solvent is joined in 250ml eggplant-shape bottle, vacuum pump is used to pump methylene dichloride until minimum volume, add the deionized water of 10ml precooling, stir and make it half an hour to dissolve quadrol and hydrochloride.With 20ml dichloromethane extraction twice, 15ml deionized water wash twice, use anhydrous sodium sulfate drying to spend the night, cross and filter sodium sulfate precipitation, use vacuum pump to pump methylene dichloride supernatant, obtain crude product.Use silica gel to be separated and obtain cholesterol-quadrol, separatory developping agent is methylene chloride/methanol/ammoniacal liquor (92:7:1, v/v).
2mmol diethylene triamine pentacetic acid (DTPA) bisgallic acid acid anhydride (structural formula is " 4 " in Fig. 1) is dissolved in 50ml anhydrous dimethyl formamide, join in 250ml there-necked flask, add 2mmolN, N-diisopropylethylamine, heats a little until diethylene triamine pentacetic acid (DTPA) bisgallic acid acid anhydride all dissolves the cooling of rear use ice-water bath.There-necked flask is sealed; pass into nitrogen protection; add 1mmol cholesterol-quadrol (structural formula is " 3 " in Fig. 1); be dissolved in 20ml anhydrous methylene chloride; use syringe slowly to join in solution, every 15 minutes injection 1ml, injection terminates rear temperature of reaction and is slowly raised to room temperature; continue stirring reaction, react 24 hours.Reaction terminates rear use oil pump and pumps reaction solvent, in vacuum drying oven, dry crude product.Crude product is joined in 20ml deionization, at room temperature stir 1 hour, then be warmed up to 60 DEG C of stirrings 3 hours, make diethylene triamine pentacetic acid (DTPA) dissolve completely thus remove, collect filter residue after filtration, in vacuum drying oven, dry product.Silica gel is used to isolate single cholesterol derivative DTPA-chol(structural formula for " 5 " in Fig. 1) and two cholesterol derivative DTPA-(chol)
2(structural formula is " 6 " in Fig. 1).
The DTPA-chol part (structural formula is " 5 " in Fig. 1) synthesized is white powdery solids, water insoluble, can be dissolved in the mixed solvent of methyl alcohol and chloroform, methylene dichloride, can not singly be dissolved in chloroform, methylene dichloride and methyl alcohol; DTPA-(chol)
2part (structural formula is " 6 " in Fig. 1) is white powdery solids, and water insoluble and methyl alcohol, can be dissolved in methyl alcohol, methylene dichloride, stable in the air, and long-term physico-chemical property of placing does not change.
The molecular formula of Gd-DTPA-chol is C
44h
73n
5o
11, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum qualification, in the negative ion mode, molecular formula is C
44h
72n
5o
11, molecular weight calculated value is 846.5228, and measured value is 846.5253, ppm is 3.0.
Fig. 3 is the NMR1H spectrogram of single cholesterol derivative DTPA-chol, 1HNMR (400MHz, CDCl3+dropsofCD3OD): δ=0.64 (s, 3H, H-18 ', CH3), 0.82 (d, 3H, H-27 ', CH
3), 0.83 (d, 3H, H-26 ', CH
3), 0.87 (d, 3H, H-21 ', CH
3), 0.96 (s, 3H, H-19 ', CH
3), 1.01-1.69 (m, 21H, 1-CH
2, 9-CH, 11-CH
2, 12-CH
2, 14-CH, 15-CH
2, 16-CH
2, 17-CH, 20-CH, 22-CH
2, 23-CH
2, 24-CH
2, 25-CH), 1.71-2.04 (m, 5H, 2-CH
2, 7-CH
2, 8-CH), and 2.17-2.30 (m, 2H, H-4 ', CH
2), 2.54-2.79 (m, 4H, 2xN-CH
2), 3.13-3.27 (m, 4H, 2XN-CH
2), 3.36-3.53 (brs, 14H, 5xN-CH
2-CO, 2xNH-CH
2), 5.31 (s, 1H, H-6 '), 4.38 (s, 1H, H-3 '),
Fig. 4 is the NMR of single cholesterol derivative DTPA-chol
13c spectrogram,
13cNMR (400MHz, CDCl
3+ dropsofCD
3oD): δ=11.78,18.64,19.23,20.99,22.45,22.71,23.81,24.22,27.94,28.16,29.62,31.81,35.75,36.14,36.50,36.92,38.55,39.45,39.68,42.26,49.98,56.14,56.65,74.45,122.50,139.69
Gd-DTPA-(chol)
2molecular formula be C
74h
123n
7o
12, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum qualification, in the negative ion mode, molecular formula is C
74h
122n
7o
12, molecular weight calculated value is 1300.9151, and measured value is 1300.9171, ppm is 1.5.
Fig. 5 is two cholesterol derivative DTPA-(chol)
2nMR
1h spectrogram, DTPA-(chol)
2nMR be:
1hNMR (400MHz, CDCl
3+ dropsofCD
3oD): δ=0.64 (s, 6H, H-18 ', CH
3), 0.82 (d, 6H, H-27 ', CH
3), 0.83 (d, 6H, H-26 ', CH
3), 0.87 (d, 6H, H-21 ', CH
3), 0.96 (s, 6H, H-19 ', CH
3), 1.01-1.69 (m, 21H, 1-CH
2, 9-CH, 11-CH
2, 12-CH
2, 14-CH, 15-CH
2, 16-CH
2, 17-CH, 20-CH, 22-CH
2, 23-CH
2, 24-CH
2, 25-CH), 1.71-2.04 (m, 10H, 2-CH
2, 7CH
2, 8-CH), and 2.17-2.34 (m, 4H, H-4 ', CH
2), 2.56-2.70 (brs, 4H, 2xN-CH
2), 3.1-3.2 (s, 4H, 2xN-CH
2), 3.10-3.20 (s, 8H, 2xN-CH
2), 3.22-3.33 (brs, 18H, 5xN-CH
2-CO, 4xNH-CH
2), 5.31 (s, 2H, H-6 '), 4.38 (s, 2H, H-3 ');
Fig. 6 is two cholesterol derivative DTPA-(chol)
2nMR
13c spectrogram,
13cNMR (400MHz, CDCl
3+ dropsofCD
3oD): δ=11.78,14.02,18.63,19.23,20.98,22.47,22.63,22.73,23.81,24.22,27.95,28.17,29.64,31.79,35.76,36.13,36.50,36.92,38.52,39.45,39.68,42.25,49.97,56.12,56.64,74.56,122.50,139.71
1.1.2, the preparation of cholesterol derivative II
According to the method that 1.1.1 is similar, 250mmol2-(aminooxy)-ethylamine dihydrochloride is dissolved in 100ml anhydrous methylene chloride, joins in dried 100ml there-necked flask, add 10mmolN simultaneously, N-diisopropylethylamine, stirs and makes it mixing.Be dissolved in by 5mmol cholesteryl chloroformate in 20ml anhydrous methylene chloride, dropwise slowly join in the solution of 2-(aminooxy)-ethylamine dihydrochloride under ice-water bath, continue half an hour, the temperature of reaction system rises to room temperature.Nitrogen is passed in there-necked flask, the lower reaction system of sealing at room temperature stirs 2 days, after reaction terminates, reaction solvent is joined in 250ml eggplant-shape bottle, vacuum pump is used to pump methylene dichloride until minimum volume, use silica gel to be separated and obtain product 2-(aminooxy)-ethamine-cholesterol, separatory developping agent is methylene chloride/methanol/ammoniacal liquor (90:10:1, v/v).
According to the method that 1.1.1 is similar, 2-(aminooxy)-ethamine-cholesterol and diethylene triamine pentacetic acid (DTPA) bisgallic acid acid anhydride react, use silica gel to carry out separation and purification, obtain DTAP monoamide part (structural formula is " 1 " in Fig. 2) and DTPA bisamide product (structural formula is " 2 " in Fig. 2)
The DTPA-chol part (structural formula is " 1 " in Fig. 2) synthesized is white powdery solids, water insoluble, can be dissolved in the mixed solvent of methyl alcohol and chloroform, methylene dichloride; DTPA-(chol)
2part (structural formula is " 2 " in Fig. 2) is white powdery solids, and water insoluble and methyl alcohol, can be dissolved in methyl alcohol, methylene dichloride, stable in the air, and long-term physico-chemical property of placing does not change.
The molecular formula of DTPA-chol part (structural formula is " 1 " in Fig. 2) is C
44h
73n
5o
12,use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum qualification, in the negative ion mode, molecular formula is C
44h
72n
5o
12, molecular weight calculated value is 862.5177, and measured value is 862.5198, ppm is 2.4.
DTPA-(chol)
2the molecular formula of part (structural formula is " 2 " in Fig. 2) is C
74h
123n
7o
14, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum qualification, in the negative ion mode, molecular formula is C
74h
122n
7o
14, molecular weight calculated value is 1332.9050, and measured value is 1332.9115, ppm is 4.9.
1.1.3, the preparation of cholesterol derivative III
According to the method that 1.1.1 is similar, by 500mmol1,3-diamino-acetone solution in 100ml anhydrous methylene chloride, join in dried 100ml there-necked flask, add 10mmolN simultaneously, N-diisopropylethylamine, stir and make it mixing.Be dissolved in by 5mmol cholesteryl chloroformate in 20ml anhydrous methylene chloride, dropwise slowly join in the solution of 1,3-diamino-acetone under ice-water bath, continue half an hour, the temperature of reaction system rises to room temperature.Nitrogen is passed in there-necked flask, the lower reaction system of sealing at room temperature stirs 2 days, after reaction terminates, reaction solvent is joined in 250ml eggplant-shape bottle, vacuum pump is used to pump methylene dichloride until minimum volume, use silica gel to be separated and obtain product 2-(aminooxy)-ethamine-cholesterol, separatory developping agent is methylene chloride/methanol/ammoniacal liquor (80:20:1, v/v).
According to the method that 1.1.1 is similar, 2-(aminooxy)-ethamine-cholesterol and diethylene triamine pentacetic acid (DTPA) bisgallic acid acid anhydride react, use silica gel to carry out separation and purification, obtain DTAP monoamide part (structural formula is " 3 " in Fig. 2) and DTPA bisamide product (structural formula is " 4 " in Fig. 2)
The DTPA-chol part (structural formula is " 3 " in Fig. 1) synthesized is white powdery solids, water insoluble, can be dissolved in the mixed solvent of methyl alcohol and chloroform, methylene dichloride; DTPA-(chol) 2 part (structural formula is " 4 " in Fig. 1) is white powdery solids, and water insoluble and methyl alcohol, can be dissolved in methyl alcohol, methylene dichloride, stable in the air, and long-term physico-chemical property of placing does not change.
The molecular formula of DTPA-chol part (structural formula is " 3 " in Fig. 2) is C
45h
73n
5o
12, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum qualification, in the negative ion mode, molecular formula is C
45h
72n
5o
12, molecular weight calculated value is 874.5177, and measured value is 874.5210, ppm is 3.8.
DTPA-(chol)
2the molecular formula of part (structural formula is " 4 " in Fig. 2) is C
76h
123n
7o
14, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum qualification, in the negative ion mode, molecular formula is C
76h
122n
7o
14, molecular weight calculated value is 1356.9050, and measured value is 1357.5405, ppm is 4.7.
1.2, the inner complex that cholesterol derivative and metal ion are formed is prepared
Described metal ion can be any one in the lanthanide series metals such as gadolinium, protactinium, europium, terbium, dysprosium, ytterbium; Gadolinium is selected in the present embodiment.
Claim DTPA-chol (360mg, 0.425mmol) to join in 100ml flask, add 30ml deionized water, add 2N sodium hydroxide solution (425ul) stirring simultaneously and make it to dissolve, solution presents opalescence.Six chloride hydrate gadolinium (158mg, 0.425mmol) be dissolved in 2ml pure water, slowly join in DTPA-chol solution, while stirring, maintain about pH6.8 by the pH value of 2N sodium hydroxide solution regulator solution, along with adding of Gadolinium trichloride solution, solution becomes oyster white gradually.Stirred solution 3 hours under room temperature condition, is then slowly heated to 60 DEG C and stirs 3 hours, after reaction terminates, add 180ml acetonitrile precipitation product.By product collected by filtration, dried overnight in vacuum drying oven, collecting product Gd-DTPA-chol(structural formula is " 7 " in Fig. 1).
The gadolinium chelate compound Gd-DTPA-(chol) of two cholesterol derivative
2(structural formula is " 8 " in Fig. 1) prepares according to the synthetic method of Gd-DTPA-chol.
Gd-DTPA-chol is white powdery solids, Gd-DTPA-(chol)
2for white waxy solid.Two kinds of inner complexs are insoluble in water; Dissolve in the mixed solvent of methyl alcohol and chloroform, methylene dichloride.Stable in the air, without photosensitivity, long-term physico-chemical property of placing does not change, and wherein, chemical formula and the output of the inner complex formed by cholesterol derivative I and metal ion are as shown in table 1 below.In Gd-DTPA-chol, the content of free gadolinium ion is lower than 0.3%, Gd-DTPA-(chol)
2in the content of free gadolinium ion lower than 0.5%, two kinds of gadolinium chelate compound purity >99.5%,
The physical properties of table 1 two kinds of inner complexs
| Chemical formula/molecular formula | Mole | Productive rate (%) | Color |
| Gd-DTPA-chol/C 44H 69N 5O 11NaGd | 1024 | 21.6 | White powder |
| Gd-DTPA-(chol) 2/C 74H 120N 7O 12Gd | 1456 | 34.5 | White waxy |
the preparation of embodiment 2, contrast medium
Use Sodium cholic acid surfactant method to be assembled in high-density lipoprotein (HDL) the gadolinium chelate compound Gd-DTPA-chol of the single cholesterol derivative in cholesterol derivative I and obtain rHDL Gd-chol-HDL(as contrast medium).The molar ratio of each component of high-density lipoprotein (HDL) is: Gd-DTPA-chol:DMPC: Sodium cholic acid=1:1:2 (mol/mol); By described high-density lipoprotein (HDL) and inner complex mixing, incubated at room temperature, dialysis removing Sodium cholic acid, obtains rHDL.Be specially: claim Gd-DTPA-chol (5mg, 5 μm of ol), DMPC (3.4mg, 5 μm of ol), by chloroform/methanol (3/1, v/v) mixed solvent makes it to dissolve, dry up chloroform with nitrogen, in vacuum drying oven, remove remaining solvent, add Sodium cholic acid 4.30mg, use 1mlTBS buffer solution, within ultrasonic 30 minutes, make it to dissolve.According to apolipoprotein AI: DMPC=1:400 (mol/mol), take out the apolipoprotein AI sample being kept at-20 DEG C, place and make it recovery temperature to room temperature in 10 minutes, the apolipoprotein A-1 solution getting 700ug adds in the sodium cholate solution of Gd-DTPA-chol, ultrasonic 3s, at 4 DEG C of vibration 12-16 hour.Solution being installed to molecular weight is in the dialysis tubing of 10000, uses 2LTBS damping fluid (20mMTris, 150mMNaCL, pH8.0) to dialyse 2 days under 4 DEG C of conditions, and changes dialyzate three times.Collect sample, be placed on 4 DEG C of preservations.
The gadolinium chelate compound Gd-DTPA-(chol) of two cholesterol derivative
2use identical Sodium cholic acid surfactant method to be assembled in high-density lipoprotein (HDL), obtain rHDL Gd-(chol)
2-HDL(is used as contrast medium).
Fig. 7 is grain size distribution when using dynamic light scattering determination rHDL to be assembled with single cholesterol derivative Gd-DTPA-chol, result shows that median size is 21.7 ± 5.8nm, particle size dispersion index (PdI) is 0.445, particle diameter is similar to wild-type high-density lipoprotein (HDL), and particle current potential is-59 ± 26mV;
Fig. 8 is assembled with two cholesterol derivative Gd-DTPA-(chol) for using dynamic light scattering determination
2time grain size distribution, result shows that median size is 25.5 ± 6.8nm, and particle size dispersion index (PdI) is 0.293, and particle diameter is similar to wild-type high-density lipoprotein (HDL), and particle current potential is-30 ± 6mV.And two kinds of rHDLs can keep the long period under 4 degree.
Fig. 9 is the transmission electron microscope picture of rHDL Gd-chol-HDL, can find out that the Gd-chol-HDL particle obtained is discoid high-density lipoprotein (HDL) by this figure, and calculating the median size that 200 particles obtain is 22.0 ± 3.8nm, similar to using the median size of dynamic light scattering determination.
Figure 10 is rHDL Gd-(chol)
2the transmission electron microscope picture of-HDL, can be found out the Gd-(chol) obtained by this figure
2-HDL particle is discoid high-density lipoprotein (HDL), and to calculate the median size that 200 particles obtain be 25.1 ± 3.7nm, similar to using the median size of dynamic light scattering determination.
The contrast medium of Figure 11 prepared by embodiment 2 longitudinal relaxation efficiency (1/T2) is in aqueous relative to the rectilinear of gadolinium concentration matching, straight slope is longitudinal relaxation rate r1, from this figure, the longitudinal relaxation efficiency r1 of Gd-chol-HDL is 7.67mM-1s-1, Gd-(chol)
2the longitudinal relaxation efficiency r1 of-HDL is 5.16mM-1s-1, higher than the relaxivity of commercial contrast medium magnevist (Gd-DTPA).
Figure 12 be Gd-chol-HDL in rat body to liver and duodenal T
1weighted imaging figure, enters in the SD rat body of 150g by prepared contrast medium by tail vein injections, and dosage is 10 μm of ol/kg.The instrument used is siemens MAGNETOMTrio3T, and use coil is mousecoil, diameter 5cm.Adopt FSE T1 weighted imaging sequence, the parameter of scanning is TR/TE=1120/24ms, and thickness is 2mm, number of plies 10-11, and the visual field is 60X60mm, and sweep time is about 5min.In different time points, MRI scan is carried out to rat, significantly can be strengthened the magnetic resonance signal intensity at liver and duodenum annex position from this figure, Gd-chol-HDL.
Figure 13 is Gd-(chol)
2-HDL in rat body to liver and duodenal T1 weighted imaging figure, be injected in rat body according to the method same with Gd-chol-HDL, carry out MRI scan in different time points.From this figure, Gd-(chol)
2-HDL significantly can strengthen the magnetic resonance signal intensity at liver and duodenum annex position.
Figure 14 is the liver of two kinds of contrast medium in rat body and the magnetic resonance signal enhancing scale map in duodenum region, from this figure, and contrast medium Gd-chol-HDL and Gd-(chol)
2-HDL represents cholesterol two kinds of pathways metabolisms respectively, and single cholesterol derivative of Gd-chol-HDL assembling reflects cholesterol in liver body, is metabolised to the process that then cholic acid is excreted to bile duct, and Gd-(chol)
2two cholesterol derivatives of-HDL assembling reflect the process being directly excreted to bile duct after cholesterol enters liver.Can show from this figure: the gadolinium chelate compound that the cholesterol of different quantities connects not only can carry out nuclear magnetic resonance enhancing to liver and gall and duodenum, and can carry out FMRI to liver.
embodiment 3, particle diameter and longitudinal relaxation efficiency test
The formula of rHDL can have an impact to its character.According to the gadolinium chelate compound Gd-DTPA-(chol) of method in embodiment 2 by the two cholesterol derivatives in cholesterol derivative II
2make rHDL, adopt different liquid formulation, after making rHDL, measure its particle diameter and longitudinal relaxation efficiency.Particle diameter uses dynamic light scattering determination, and result is shown as the particle size results under intensity.Following table 2 shows as DMPC: when the molar ratio of inner complex improves, the particle diameter corresponding increase of direct ratio thereupon of rHDL, and longitudinal relaxation efficiency but reverse proportionality reduces.
Table 2
In sum, cholesterol is connected to the part nitrogenous many carboxyl ligand obtaining containing one or two cholesterol by the present invention, carries out obtaining corresponding inner complex after chelating to metal ion; Obtain rHDL by inner complex being assembled into high-density lipoprotein (HDL), this rHDL is used as contrast medium.Particle diameter, the form of this contrast medium are similar to the high-density lipoprotein (HDL) in human body with biological activity, and the relaxivity of contrast medium is higher than commercial contrast medium magnevist (Gd-DTPA).This contrast medium can be realized liver region specificity radiography by the active targeting of high-density lipoprotein (HDL), and is realized bile duct and duodenal nuclear magnetic resonance by the metabolic process of cholesterol at liver.Inner complex containing different number cholesterol in this contrast medium can by the functional detection of different cholesterol metabolic approach realization to liver.
Claims (7)
1. a cholesterol derivative, is characterized in that, structural formula is as shown in formula I:
Wherein, R1 is the structure shown in formula II, and R2 is the structure shown in hydroxyl or formula II:
Described R ' is the structure shown in formula III:
wherein, n=2.
2. an inner complex, is characterized in that, described inner complex is formed by cholesterol derivative according to claim 1 and gadolinium ion chelating.
3. a rHDL, is characterized in that, described rHDL is made up of apolipoprotein A-1, dimyristoyl phosphatidyl choline, inner complex according to claim 2.
4. rHDL as claimed in claim 3, it is characterized in that, the scope of the mol ratio of described apolipoprotein A-1, dimyristoyl phosphatidyl choline, inner complex is 1:400:(20 ~ 400).
5. a rHDL as claimed in claim 3 is preparing the purposes of magnetic resonance contrast agent of hepato-biliary function target.
6. purposes as claimed in claim 5, is characterized in that, described magnetic resonance contrast agent is reflection cholesterol becomes the process of cholic acid contrast medium at liver intracellular metabolite.
7. purposes as claimed in claim 5, is characterized in that, described magnetic resonance contrast agent is the contrast medium that reflection cholesterol is directly excreted to the metabolic process of bile duct in liver body.
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| CN104163915B (en) * | 2013-05-16 | 2016-09-28 | 沈阳药科大学 | Cholesterol-poloxamer-cholesterol triblock copolymer and its preparation method and application |
| CN104987395A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof |
| CN105001323A (en) * | 2015-07-14 | 2015-10-28 | 上海拜豪生物科技有限公司 | Nickel-very low density lipoprotein chelate, and preparation method and application thereof |
| CN104987396A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Lead-very low density lipoprotein chelate as well as preparation method and application thereof |
| CN104987398A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Chromium-low density lipoprotein chelate as well as preparation method and application thereof |
| CN104987393A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Cadmium-very low density lipoprotein chelate as well as preparation method and application thereof |
| CN104987397A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Mercury-low density lipoprotein chelate as well as preparation method and application thereof |
| CN105001322A (en) * | 2015-07-14 | 2015-10-28 | 上海拜豪生物科技有限公司 | Chromium-high density lipoprotein (HDL) chelate and preparation method therefor and application thereof |
| CN105037528A (en) * | 2015-07-14 | 2015-11-11 | 上海拜豪生物科技有限公司 | Nickel-low density lipoprotein chelate and preparation method therefor and application thereof |
| CN104987394A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Mercury-very low density lipoprotein chelate as well as preparation method and application thereof |
| CN105061586A (en) * | 2015-07-14 | 2015-11-18 | 上海拜豪生物科技有限公司 | Lead-low-density lipoprotein chelate as well as preparation method and application thereof |
| CN104987399A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Arsenic-very low density lipoprotein chelate as well as preparation method and application thereof |
| CN108543081A (en) * | 2018-03-19 | 2018-09-18 | 西北师范大学 | A kind of T1/T2 double-mode magnetic resonances image-forming contrast medium and its preparation method and application |
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