CN102766188A - Cholesterol derivative, chelate, recombinant high density lipoprotein and application thereof - Google Patents
Cholesterol derivative, chelate, recombinant high density lipoprotein and application thereof Download PDFInfo
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- CN102766188A CN102766188A CN2012102578943A CN201210257894A CN102766188A CN 102766188 A CN102766188 A CN 102766188A CN 2012102578943 A CN2012102578943 A CN 2012102578943A CN 201210257894 A CN201210257894 A CN 201210257894A CN 102766188 A CN102766188 A CN 102766188A
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Abstract
The invention discloses a cholesterol derivative, chelate, recombinant high density lipoprotein and application thereof. According to the invention, a derivative ligand containing one or two cholesterol is obtained by connecting cholesterol to a nitrogen-containing polycarboxylic ligand; the cholesterol derivative chelates with metal ion to obtain the corresponding chelate; recombinant high density lipoprotein is prepared by assembling the chelate in high density lipoprotein; and the recombinant high density lipoprotein can be used as a contrast agent. The particle size, morphology and biological activity of the contrast agent is similar to those of the high density lipoprotein in the human body and the relaxation efficiency is higher than that of commercial contrast agent magnevist (Gd-DTPA). The liver specific contrast can be achieved by the active targeting of the high density lipoprotein, and magnetic resonance imaging of bile duct and duodenum can be realized in the metabolic process of the cholesterol in liver.
Description
Technical field
The present invention relates to the paramagnetism magnetic resonance contrast agent of liver function property diagnosis, be specifically related to a kind of cholesterol derivative, inner complex, reorganization RHDL and as the purposes of contrast medium.
Background technology
Nuclear magnetic resonance is as a kind of harmless nothing wound, and the imaging orientation is flexible, and the method that spatial resolution is high is widely used in the clinical diagnosis.Along with new contrast agent application, mr imaging technique has use widely in liver imaging and the imaging of courage pancreas.The liver nuclear magnetic resonance 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 the liver nuclear magnetic resonance can be used for checking liver cirrhosis, steatosis or liver blood pigmentation disease etc., can the patient liver cancer or the common hepatic duct cancer of liver cirrhosis be diagnosed the patient who receives transplantation.The hepatic metastases knurl need judge usually by stages and the prognosis target that the liver nuclear magnetic resonance can be checked the degree of hepatic metastases knurl preferably, thereby eliminates or chemoembolization provides foundation for excision, radio frequency, and can monitor the recurrence of hepatic metastases knurl.In the nuclear magnetic resonance clinical diagnosis, contrast medium is through changing the relaxation time of tissue local, and the relaxation time that reaches with surrounding tissue forms certain difference, thereby realizes strengthening the purpose of radiography.But mostly the contrast medium that uses clinically at present is the extracellular fluid contrast medium, and this type contrast medium is penetrated into the intercellular substance from the blood pond fast after intravenous injection, and mainly removes at kidney, does not have the organ specificity mr to strengthen.And to have report to show with the gadolinium chelate compound in recent years be that main extracellular fluid contrast medium can cause the patient of serious kidney injury to produce kidney source property system fiber disease.Because problems such as dosage height that this type contrast medium exists and toxic side effect, so the organ target property magnetic resonance contrast agent of development of new becomes the important step that improves clinical diagnosis accuracy and security.
RHDL is a kind of as lipoprotein, is present in the spherical macromolecular lipid carrier of solubility in the blood plasma.RHDL is formed hydrophobic kernel by non-polar lipid such as triglyceride and cholesteryl esters, and skin is wrapped up by the phosphatide unimolecular layer, on phospholipid layer, is inlaying several kinds of lipophorins such as SUV and apolipoprotein A-1.RHDL has mainly been participated in SUV antiport process in vivo; Key step is for the picked-up of RHDL at first liver peripheral tissues place SUV more than needed and be transformed into cholesteryl ester in the circulation in vivo and be stored in the fat nuclear, and RHDL is transported to liver with SUV and cholesteryl ester and carries out metabolism then.Thereby utilize the approach of this SUV antiport to make up the liver specific target tropism that contrast medium can improve magnetic resonance contrast agent.RHDL has the following advantages as carrier: ⑴ HDL is as a kind of endogenous material, can degradation in vivo, and no immunoreation, and avoid being engulfed system identification and removing by monocyte; ⑵ HDL can conduct drugs in the target cell through receptor-mediated endocytosis or selectivity picked-up, and higher target property is arranged; ⑶ the structure of HDL can be so that hydrophobic drug gets into nonpolar lipid kernel, and the minimizing medicine contacts with external environment, avoids medicine to be destroyed.Literature search through to prior art is found; U.S. Pat 7211248; Proposition is connected to the DTPA of chelating gadolinium ion on the lipid PA; Detect atherosclerotic plaque after being assembled into RHDL, and the document major part of having delivered detects atherosclerotic plaque with RHDL as contrast medium.And do not have document and patent report to use SUV to connect to be assembled into RHDL behind the paramagnetic ion inner complex to carry out nuclear magnetic resonance, especially to liver and gall and duodenal nuclear magnetic resonance.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned prior art, a kind of cholesterol derivative, inner complex, reorganization RHDL are provided and as the purposes of contrast medium.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, realized FMRI to liver and relevant organ.This contrast medium transformation period in vivo is longer, can realize the lasting imaging to liver, bile duct and duodenum zone, can reduce the consumption of contrast medium, reduces the toxicity of contrast medium, improves the imaging effect of contrast medium.This contrast medium can remedy the weak point of existing hepatobiliary magnetic resonance imaging technique, improves the accuracy of magnetic resonance image-forming diagnose.
The objective of the invention is to realize through following technical scheme:
First aspect the present invention relates to a kind of cholesterol derivative, and its structural formula is shown in formula I:
Wherein, R1 is the structure shown in the formula II, and R2 is the structure shown in hydroxyl or the formula II:
Preferably, said R ' is the group in succession of alkyl, polyalkylene glycol type or the group in succession of polyether type.
Further preferably, said R ' is the structure shown in the formula III:
Further preferably, said R ' is the structure shown in the formula IV:
Further preferably, said R ' is the structure shown in the formula (V):
Further preferably, said R ' is the structure shown in the formula VI:
Further preferably, said R ' is the structure shown in the formula (VII):
Second aspect the present invention relates to a kind of inner complex, and said inner complex is by aforesaid cholesterol derivative and metal ion-chelant and form.
Preferably, said metals ion is a lanthanide metal ion.
The third aspect the present invention relates to a kind of reorganization RHDL, and said reorganization RHDL is made up of apolipoprotein A-1, dimyristoyl phosphatidyl choline, aforesaid inner complex.
Preferably, the scope of the mol ratio of said apolipoprotein A-1, dimyristoyl phosphatidyl choline, inner complex is 1:400: (20～400).
Preferably; Said reorganization RHDL adopts the preparation of Sodium cholic acid surfactant method and gets, and comprises the steps: said apolipoprotein A-1, dimyristoyl phosphatidyl choline (DMPC), inner complex and Sodium cholic acid are mixed, and hatches; Sodium cholic acid is removed in dialysis, promptly gets.
Fourth aspect the present invention relates to the purposes of a kind of aforesaid reorganization high density lipoprotein level as the magnetic resonance contrast agent of hepato-biliary function property target.
Preferably, said magnetic resonance contrast agent becomes the contrast medium of the process of cholic acid at the liver intracellular metabolite for the reflection SUV.
Preferably, said magnetic resonance contrast agent directly is excreted to the contrast medium of the metabolic process of bile duct in the liver body for the reflection SUV.
Compared with prior art, the present invention has following beneficial effect:
1, the longitudinal relaxation efficient of magnetic resonance imaging contrast of the present invention is better than the contrast medium of clinical use, can reduce using dosage.
2, contrast medium of the present invention can be selectively targeted to liver, and drain from bile duct from the pathways metabolism of SUV, thereby can strengthen the nuclear magnetic resonance effect at bile duct and duodenum position.
3, the MRI that contrast medium of the present invention can functional enhancing liver, the paramagnetic ion inner complex that is connected with the different quantities SUV can reflect different cholesterol metabolic approach.
4, the inner complex that is connected with single SUV among the present invention can strengthen the magnetic resonance signal of liver the long period; Strengthen the magnetic resonance signal in bile duct and duodenum annex zone then through the bile metabolism, reflected that SUV becomes the process of cholic acid at the liver internal metabolism.
5, the inner complex that is connected with two SUV among the present invention is compared with the inner complex that is connected with single SUV; The liver signal enhanced time is shorter; But enough imaging time windows are arranged still; Strengthen the magnetic resonance signal of bile duct and duodenum near zone at short notice, reflected that mainly SUV directly is excreted to the metabolic process of bile duct in the liver body.
Description of drawings
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 forms of different SUV linking group;
Fig. 3 is the NMR of single cholesterol derivative DTPA-chol
1The H spectrogram;
Fig. 4 is the NMR of single cholesterol derivative DTPA-chol
13The C spectrogram;
Fig. 5 is two cholesterol derivative DTPA-(chol)
2NMR
1The H spectrogram;
Fig. 6 is two cholesterol derivative DTPA-(chol)
2NMR
13The C spectrogram;
Fig. 7 is the size distribution figure of reorganization RHDL Gd-chol-HDL that is assembled with the gadolinium chelate compound Gd-DTPA-chol of single SUV;
Fig. 8 is the gadolinium chelate compound Gd-DTPA-(chol) that is assembled with two SUV
2Reorganization RHDL Gd-(chol)
2The size distribution figure of-HDL;
Fig. 9 is transmission electron microscope (TEM) picture of reorganization RHDL Gd-cholHDL that is assembled with the gadolinium chelate compound GdDTPA-chol of single SUV;
Figure 10 is the gadolinium chelate compound Gd-DTPA-(chol) that is assembled with two SUV
2Reorganization RHDL Gd-(chol)
2The transmission electron microscope of-HDL (TEM) picture;
Figure 11 is the prepared longitudinal relaxation efficient (1/T of contrast medium in the aqueous solution
2) with respect to the rectilinear of gadolinium concentration match;
Figure 12 is that Gd-chol-HDL is intravital to liver and duodenal T rat
1Be weighted to image pattern;
Figure 13 is Gd-(chol)
2-HDL is intravital to liver and duodenal T rat
1Be weighted to image pattern;
Figure 14 is that the magnetic resonance signal of two kinds of contrast medium in intravital liver of rat and duodenum zone strengthens scale map.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
The preparation of embodiment 1, cholesterol derivative and inner complex thereof
1.1, the preparation cholesterol derivative
1.1.1, the preparation of cholesterol derivative I
As shown in Figure 1, the 500mmol quadrol is dissolved in the 100ml anhydrous methylene chloride, join in the dried 100ml there-necked flask, add 10mmol N simultaneously, the N-diisopropylethylamine stirs and makes it mixing.5mmol SUV chloro-formic ester is dissolved in the 20ml anhydrous methylene chloride, under ice-water bath, dropwise slowly joins in the solution of quadrol, continue half a hour, the temperature of reaction system rises to room temperature.Feed nitrogen in the there-necked flask; Sealing reaction system was down at room temperature stirred 2 days; Reaction joins reaction solvent in the 250ml eggplant-shape bottle after finishing, and uses vacuum pump to take out methylene dichloride until minimum volume; The deionized water that adds the 10ml precooling stirs and makes it to dissolve quadrol and hydrochloride half a hour.With 20ml dichloromethane extraction twice, 15ml deionized water wash twice uses anhydrous sodium sulfate drying to spend the night, and removes by filter the sodium sulfate deposition, uses vacuum pump to take out methylene dichloride supernatant, obtains crude product.Use silica gel to separate and obtain SUV-quadrol, separatory developping agent be methylene chloride/ammoniacal liquor (92:7:1, v/v).
2mmol diethylene triamine pentacetic acid (DTPA) bisgallic acid acid anhydride (structural formula is " 4 " among Fig. 1) is dissolved in the 50ml anhydrous dimethyl formamide; Join in the 250ml there-necked flask; Add 2mmol N; The N-diisopropylethylamine is heated a little and is all dissolved the cooling of back use ice-water bath until diethylene triamine pentacetic acid (DTPA) bisgallic acid acid anhydride.With the there-necked flask sealing, feed nitrogen protection, add 1mmol SUV-quadrol (structural formula is " 3 " among Fig. 1); Be dissolved in the 20ml anhydrous methylene chloride; Use syringe slowly to join in the solution, per 15 minutes injection 1ml, injection finishes the afterreaction temperature and slowly is raised to room temperature; Continue stirring reaction, reacted 24 hours.Reaction finishes the back and uses oil pump to take out the dereaction solvent, in vacuum drying oven, dries crude product.Crude product is joined in the 20ml de-ionized, at room temperature stirred 1 hour, be warmed up to 60 ℃ again and stirred 3 hours, remove, filter the back and collect filter residue, in vacuum drying oven, dry product thereby diethylene triamine pentacetic acid (DTPA) is dissolved fully.Use silica gel to isolate single cholesterol derivative DTPA-chol (structural formula is " 5 " among Fig. 1) and two cholesterol derivative DTPA-(chol)
2(structural formula is " 6 " among Fig. 1).
The DTPA-chol part that synthesizes (structural formula is " 5 " among Fig. 1) is for the white powder solid, and is 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 " among Fig. 1) is the white powder solid, and water insoluble and methyl alcohol can be dissolved in methyl alcohol, the methylene dichloride, and is stable in the air, places physico-chemical property for a long time and 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 and identify that under negative ion mode, molecular formula is C
44H
72N
5O
11, the molecular weight calculated value is 846.5228, and measured value is 846.5253, and ppm is 3.0.
Fig. 3 is the NMR 1H spectrogram of single cholesterol derivative DTPA-chol, 1H NMR (400MHz, CDCl3+drops of CD3OD): and 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), 2.17-2.30 (m, 2H, H-4 ', CH
2), 2.54-2.79 (m, 4H, 2x N-CH
2), 3.13-3.27 (m, 4H, 2X N-CH
2), 3.36-3.53 (brs, 14H, 5xN-CH
2-CO, 2x NH-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
13The C spectrogram,
13C NMR (400MHz, CDCl
3+ drops of CD
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 and identify that under negative ion mode, molecular formula is C
74H
122N
7O
12, the molecular weight calculated value is 1300.9151, and measured value is 1300.9171, and ppm is 1.5.
Fig. 5 is two cholesterol derivative DTPA-(chol)
2NMR
1The H spectrogram, DTPA-(chol)
2NMR be:
1HNMR (400MHz, CDCl
3+ drops of CD
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), 2.17-2.34 (m, 4H, H-4 ', CH
2), 2.56-2.70 (brs, 4H, 2x N-CH
2), 3.1-3.2 (s, 4H, 2x N-CH
2), 3.10-3.20 (s, 8H, 2x N-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
13The C spectrogram,
13C NMR (400MHz, CDCl
3+ drops of CD
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 1.1.1 similar methods, 250mmol2-(aminooxy)-ethamine dihydrochloride is dissolved in the 100ml anhydrous methylene chloride, join in the dried 100ml there-necked flask, add 10mmol N simultaneously, the N-diisopropylethylamine stirs and makes it mixing.5mmol SUV chloro-formic ester is dissolved in the 20ml anhydrous methylene chloride, under ice-water bath, dropwise slowly joins in the solution of 2-(aminooxy)-ethamine dihydrochloride, continue half a hour, the temperature of reaction system rises to room temperature.Feed nitrogen in the there-necked flask; Sealing reaction system was down at room temperature stirred 2 days, and reaction joins reaction solvent in the 250ml eggplant-shape bottle after finishing; Use vacuum pump to take out methylene dichloride until minimum volume; Use silica gel to separate and obtain product 2-(aminooxy)-ethamine-SUV, separatory developping agent be methylene chloride/ammoniacal liquor (90:10:1, v/v).
According to the 1.1.1 similar methods; 2-(aminooxy)-ethamine-SUV 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 " among Fig. 2) and DTPA bisamide product (structural formula is " 2 " among Fig. 2)
The DTPA-chol part that synthesizes (structural formula is " 1 " among Fig. 2) is for the white powder solid, and is water insoluble, can be dissolved in the mixed solvent of methyl alcohol and chloroform, methylene dichloride; DTPA-(chol)
2Part (structural formula is " 2 " among Fig. 2) is the white powder solid, and water insoluble and methyl alcohol can be dissolved in methyl alcohol, the methylene dichloride, and is stable in the air, places physico-chemical property for a long time and does not change.
The molecular formula of DTPA-chol part (structural formula is " 1 " among Fig. 2) is C
44H
73N
5O
12,Use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum and identify that under negative ion mode, molecular formula is C
44H
72N
5O
12, the molecular weight calculated value is 862.5177, and measured value is 862.5198, and ppm is 2.4.
DTPA-(chol)
2The molecular formula of part (structural formula is " 2 " among Fig. 2) is C
74H
123N
7O
14, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum and identify that under negative ion mode, molecular formula is C
74H
122N
7O
14, the molecular weight calculated value is 1332.9050, and measured value is 1332.9115, and ppm is 4.9.
1.1.3, the preparation of cholesterol derivative III
According to the 1.1.1 similar methods, with 500mmol 1,3-diamino--acetone solution joins in the dried 100ml there-necked flask in the 100ml anhydrous methylene chloride, adds 10mmol N simultaneously, and the N-diisopropylethylamine stirs and makes it mixing.5mmol SUV chloro-formic ester is dissolved in the 20ml anhydrous methylene chloride, under ice-water bath, dropwise slowly joins 1, in the solution of 3-diamino--acetone, continue half a hour, the temperature of reaction system rises to room temperature.Feed nitrogen in the there-necked flask; Sealing reaction system was down at room temperature stirred 2 days, and reaction joins reaction solvent in the 250ml eggplant-shape bottle after finishing; Use vacuum pump to take out methylene dichloride until minimum volume; Use silica gel to separate and obtain product 2-(aminooxy)-ethamine-SUV, separatory developping agent be methylene chloride/ammoniacal liquor (80:20:1, v/v).
According to the 1.1.1 similar methods; 2-(aminooxy)-ethamine-SUV 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 " among Fig. 2) and DTPA bisamide product (structural formula is " 4 " among Fig. 2)
The DTPA-chol part that synthesizes (structural formula is " 3 " among Fig. 1) is for the white powder solid, and is water insoluble, can be dissolved in the mixed solvent of methyl alcohol and chloroform, methylene dichloride; DTPA-(chol) 2 parts (structural formula is " 4 " among Fig. 1) are the white powder solid, and water insoluble and methyl alcohol can be dissolved in methyl alcohol, the methylene dichloride, and is stable in the air, place physico-chemical property for a long time and do not change.
The molecular formula of DTPA-chol part (structural formula is " 3 " among Fig. 2) is C
45H
73N
5O
12, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum and identify that under negative ion mode, molecular formula is C
45H
72N
5O
12, the molecular weight calculated value is 874.5177, and measured value is 874.5210, and ppm is 3.8.
DTPA-(chol)
2The molecular formula of part (structural formula is " 4 " among Fig. 2) is C
76H
123N
7O
14, use quadrupole flight time mass spectrum combined instrument (q-TOF) to do high resolution mass spectrum and identify that under negative ion mode, molecular formula is C
76H
122N
7O
14, the molecular weight calculated value is 1356.9050, and measured value is 1357.5405, and ppm is 4.7.
1.2, the inner complex of preparation cholesterol derivative and metals ion formation
Said metals ion can be in the lanthanide series metals such as gadolinium, protactinium, europium, terbium, dysprosium, ytterbium any one; Select gadolinium in the present embodiment for use.
(360mg 0.425mmol) joins in the 100ml flask, adds the 30ml deionized water, adds 2N sodium hydroxide solution (425ul) stirring simultaneously and makes it dissolving, and solution presents opalescence to claim DTPA-chol.Six hydration Gadolinium trichloride (158mg; 0.425mmol) be dissolved in the 2ml pure water, slowly joining in the DTPA-chol solution, the pH value with 2N sodium hydroxide solution regulator solution in the time of stirring maintains about pH6.8; Along with the adding of Gadolinium trichloride solution, solution becomes oyster white gradually.Stirred solution is 3 hours under the room temperature condition, slowly is heated to 60 ℃ then and stirs 3 hours, after reaction finishes, adds 180ml acetonitrile precipitation product.Product is filtered collecting precipitation, and dried overnight in the vacuum drying oven is collected product Gd-DTPA-chol (structural formula is " 7 " among Fig. 1).
The gadolinium chelate compound Gd-DTPA-(chol) of two cholesterol derivatives
2(structural formula is " 8 " among Fig. 1) is according to the compound method preparation of Gd-DTPA-chol.
Gd-DTPA-chol is the white powder solid, Gd-DTPA-(chol)
2Be 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, no photosensitivity is placed physico-chemical property for a long time and is not changed, and wherein, the chemical formula and the output of the inner complex that is formed by cholesterol derivative I and metals ion are as shown in table 1 below.The content of free gadolinium ion is lower than 0.3%, Gd-DTPA-(chol) among the Gd-DTPA-chol
2In the content of free gadolinium ion be lower than 0.5%, two kind of gadolinium chelate compound purity; 99.5%,
The physical properties of two kinds of inner complexs of table 1
| 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 | The white wax shape |
The preparation of embodiment 2, contrast medium
Use the Sodium cholic acid surfactant method to be assembled into the RHDL Gd-chol-HDL that obtains in the RHDL recombinating (as contrast medium) the gadolinium chelate compound Gd-DTPA-chol of the single cholesterol derivative in the cholesterol derivative I.The molar ratio of each component of RHDL is: Gd-DTPA-chol:DMPC: Sodium cholic acid=1:1:2 (mol/mol); Said RHDL and inner complex are mixed, hatch under the room temperature, Sodium cholic acid is removed in dialysis, obtains the RHDL of recombinating.Be specially: claim Gd-DTPA-chol (5mg, 5 μ mol), DMPC (3.4mg; 5 μ mol), (3/1, v/v) mixed solvent makes it dissolving with chloroform/methanol; Dry up chloroform with nitrogen, in vacuum drying oven, remove remaining solvent, add Sodium cholic acid 4.30mg; With the dissolving of 1ml TBS damping fluid, made it to dissolve in ultrasonic 30 minutes.According to apolipoprotein AI: DMPC=1:400 (mol/mol); Taking-up is kept at-20 ℃ apolipoprotein AI sample; Place and made it recovery temperature to room temperature in 10 minutes; The apolipoprotein A-1 solution of getting 700ug adds in the Sodium cholic acid solution of Gd-DTPA-chol, and ultrasonic 3s was 4 ℃ of vibrations 12-16 hour.It is in 10000 the dialysis tubing that solution is installed to molecular weight, uses 2L TBS damping fluid (20mMTris, 150mM NaCL, pH8.0) dialysis 2 days under 4 ℃ of conditions, and change dialyzate three times.Collect sample, be placed on 4 ℃ of preservations.
The gadolinium chelate compound Gd-DTPA-(chol) of two cholesterol derivatives
2Use identical Sodium cholic acid surfactant method to be assembled in the RHDL, RHDL Gd-(chol) obtains recombinating
2-HDL (as contrast medium).
Size distribution figure when Fig. 7 is assembled with single cholesterol derivative Gd-DTPA-chol for using dynamic light scattering determination reorganization RHDL; The result shows that median size is 21.7 ± 5.8nm; Particle size dispersion index (PdI) is 0.445; Particle diameter is similar with the wild-type RHDL, and the particle current potential is-59 ± 26mV;
Fig. 8 is assembled with two cholesterol derivative Gd-DTPA-(chol) for using dynamic light scattering determination
2The time size distribution figure, the result shows that median size is 25.5 ± 6.8nm, particle size dispersion index (PdI) is 0.293, particle diameter is similar with the wild-type RHDL, the particle current potential is-30 ± 6mV.And two kinds of reorganization RHDLs can keep the long period under 4 degree.
Fig. 9 is the transmission electron microscope picture of reorganization RHDL Gd-chol-HDL; Can find out that by this figure the Gd-chol-HDL particle that obtains is discoid RHDL; And calculating the median size that 200 particles obtain is 22.0 ± 3.8nm, similar with the median size of using dynamic light scattering determination.
Figure 10 is reorganization RHDL Gd-(chol)
2The transmission electron microscope picture of-HDL can be found out the Gd-(chol) that obtains by this figure
2-HDL particle is discoid RHDL, and to calculate the median size that 200 particles obtain be 25.1 ± 3.7nm, and is similar with the median size of using dynamic light scattering determination.
Figure 11 is the rectilinear of the prepared longitudinal relaxation efficient (1/T2) of contrast medium in the aqueous solution of embodiment 2 with respect to the match of gadolinium concentration; Straight slope is longitudinal relaxation rate r1; Visible by this figure, the longitudinal relaxation efficient r1 of Gd-chol-HDL is 7.67mM-1s-1, Gd-(chol)
2The longitudinal relaxation efficient r1 of-HDL is 5.16mM-1s-1, and is higher than the relaxation efficient of commercial contrast medium magnevist (Gd-DTPA).
Figure 12 is that Gd-chol-HDL is intravital to liver and duodenal T rat
1Be weighted to image pattern, prepared contrast medium is injected into through tail vein in the SD rat body of 150g, dosage is 10 μ mol/kg.Employed instrument is siemens MAGNETOM Trio 3T, uses coil to be mouse coil, diameter 5cm.Adopt FSE T1 weighted imaging sequence, the parameter of scanning is TR/TE=1120/24ms, and bed thickness is 2mm, number of plies 10-11, the visual field is 60X60mm, sweep time about 5min.In different time points rat is carried out MRI scan, visible by this figure, Gd-chol-HDL can significantly strengthen the magnetic resonance signal intensity at liver and duodenum annex position.
Figure 13 is Gd-(chol)
2-HDL is weighted to image pattern to liver and duodenal T1 in that rat is intravital, according to being injected in the rat body with the same method of Gd-chol-HDL, carries out MRI scan in different time points.Visible by this figure, Gd-(chol)
2-HDL can significantly strengthen the magnetic resonance signal intensity at liver and duodenum annex position.
Figure 14 is that the magnetic resonance signal of two kinds of contrast medium in intravital liver of rat and duodenum zone strengthens scale map, and is visible by this figure, contrast medium Gd-chol-HDL and Gd-(chol)
2-HDL has represented two kinds of pathways metabolisms of SUV respectively, and single cholesterol derivative of Gd-chol-HDL assembling has reflected that SUV is metabolised to the process that cholic acid is excreted to bile duct then in the liver body, and Gd-(chol)
2Two cholesterol derivatives of-HDL assembling have reflected the process that directly is excreted to bile duct behind the SUV entering liver.Can show from this figure: the gadolinium chelate compound that the SUV of different quantities connects not only can carry out the nuclear magnetic resonance enhancing to liver and gall and duodenum, and can carry out FMRI to liver.
The prescription of reorganization RHDL can exert an influence to its character.According to the gadolinium chelate compound Gd-DTPA-(chol) of method among the embodiment 2 with the two cholesterol derivatives in the cholesterol derivative II
2Make the reorganization RHDL, adopt different lipid prescriptions, measure its particle diameter and longitudinal relaxation efficient behind the making reorganization RHDL.Particle diameter uses dynamic light scattering determination, and the result is shown as the particle diameter result under intensity.Following table 2 shows as DMPC: when the molar ratio of inner complex improves, and the particle diameter corresponding increase of direct ratio thereupon of reorganization RHDL, longitudinal relaxation efficient but reverse proportionality reduces.
Table 2
In sum, the present invention is connected to the part that obtains containing one or two SUV on nitrogenous many carboxyls part with SUV, carries out obtaining corresponding inner complex behind the chelating with metals ion; Make the reorganization RHDL through inner complex being assembled into RHDL, this reorganization RHDL is as contrast medium.The particle diameter of this contrast medium, form and biological activity are similar with the intravital RHDL of people, and the relaxation efficient of contrast medium will be higher than commercial contrast medium magnevist (Gd-DTPA).This contrast medium can be realized liver site specific radiography through the active target of RHDL, and realize bile duct and duodenal nuclear magnetic resonance through the metabolic process of SUV at liver.The inner complex that contains different number SUV in this contrast medium can be realized the functional detection to liver through different cholesterol metabolic approach.
Claims (15)
1. cholesterol derivative is characterized in that structural formula is shown in formula I:
Wherein, R1 is the structure shown in the formula II, and R2 is the structure shown in hydroxyl or the formula II:
2. cholesterol derivative as claimed in claim 1 is characterized in that, said R ' is the group in succession of alkyl, polyalkylene glycol type or the group in succession of polyether type.
3. cholesterol derivative as claimed in claim 2 is characterized in that, said R ' is the structure shown in the formula III:
4. cholesterol derivative as claimed in claim 2 is characterized in that, said R ' is the structure shown in the formula IV:
wherein, n=1～16.
5. cholesterol derivative as claimed in claim 2 is characterized in that, said R ' is the structure shown in the formula (V):
6. cholesterol derivative as claimed in claim 2 is characterized in that, said R ' is the structure shown in the formula VI:
wherein, n=1～16.
7. cholesterol derivative as claimed in claim 2 is characterized in that, said R ' is the structure shown in the formula (VII):
wherein, n=1～16.
8. an inner complex is characterized in that, said inner complex is by the described cholesterol derivative of claim 1 and metal ion-chelant and form.
9. inner complex as claimed in claim 8 is characterized in that, said metals ion is a lanthanide metal ion.
10. a reorganization RHDL is characterized in that said reorganization RHDL is made up of apolipoprotein A-1, dimyristoyl phosphatidyl choline, the described inner complex of claim 8.
11. reorganization RHDL as claimed in claim 10 is characterized in that, the scope of the mol ratio of said apolipoprotein A-1, dimyristoyl phosphatidyl choline, inner complex is 1:400: (20～400).
12. reorganization RHDL as claimed in claim 10; It is characterized in that; Said reorganization RHDL adopts the preparation of Sodium cholic acid surfactant method and gets, and comprises the steps: said apolipoprotein A-1, dimyristoyl phosphatidyl choline, inner complex and Sodium cholic acid are mixed, and hatches; Sodium cholic acid is removed in dialysis, promptly gets.
13. a reorganization high density lipoprotein level as claimed in claim 10 is as the purposes of the magnetic resonance contrast agent of hepato-biliary function property target.
14. purposes as claimed in claim 13 is characterized in that, said magnetic resonance contrast agent becomes the contrast medium of the process of cholic acid at the liver intracellular metabolite for the reflection SUV.
15. purposes as claimed in claim 13 is characterized in that, said magnetic resonance contrast agent directly is excreted to the contrast medium of the metabolic process of bile duct in the liver body for the reflection SUV.
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