CN103159842B - Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof - Google Patents
Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof Download PDFInfo
- Publication number
- CN103159842B CN103159842B CN201310087314.5A CN201310087314A CN103159842B CN 103159842 B CN103159842 B CN 103159842B CN 201310087314 A CN201310087314 A CN 201310087314A CN 103159842 B CN103159842 B CN 103159842B
- Authority
- CN
- China
- Prior art keywords
- annexin
- cys
- medicine box
- mark
- tin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the development technical field of single photon emission computed tomography (SPECT) and in particular relates to a Cys-Annexin V kit used for 99mTc labeling, a preparation method and application thereof in apoptosis detection. Each kit provided by the invention is prepared as follows: 25-150ug of Cys-Annexin V, 0.5-6mg of edetate, 5-40ug of tin salt and 0.1mmol of pH buffer agent. When the kit provided by the invention carries out 99mTc labeling for Cys-Annexin V, the operation is simple, the labeling rate is stable and larger than 95%, and the labeled product can be obviously distinguished from the impurities possibly appearing during the reaction. Compared with the labeling method in the existing technology, the method provided by the invention has obvious advantages.
Description
Technical field
The invention belongs to single photon emission computed tomography (SPECT) imaging technique field, be specifically related to a kind of for
99mthe Cys-Annexin V medicine box of Tc mark, and compound method and its detecting the application in apoptosis.
Background technology
Apoptosis (Apoptosis), also known as apoptosis (Programmed Cell Death, PCD), is the important component part in cell life cycle, be different from downright bad and unexpected death by the cell initiative death process of gene regulating.Apoptosis relates to the change of a series of biomolecules and morphocytology, wherein, phosphatidylserine (PS) in cell membrane lipid bilayer turn to skin by film inner layer, be exposed to cell surface, being apoptosis earliest events, before occurring in apoptotic morphologic change, is also the primary event of apoptosis cascade reaction, as apoptotic cell by the mark of phagocytic cell identification, thus cause the shrinkage of endochylema further, chromatinicly to concentrate and nuclear dna degraded etc.Therefore, the PS turned up is current most study and is hopeful most to detect target spot with the apoptosis of application prospect, can early detection apoptosis, has higher ageing, normal utilizes the medicine with PS specific binding to detect apoptosis both at home and abroad.
Annexin V (Annexin V, also known as Annexin A5 and AnxA5) is a kind of human endogenous's property albumen, belongs to Ca
2+dependency cardiolipin binding protein family is separate from placenta the earliest, has the effect of the solid and anti-inflammatory of some anti-freezings, is once studied by as a kind of antithrombotics.Chromosomal localization analysis shows, and Annexin V is by the Annexin V genes encoding be positioned on the Article 4 karyomit(e) 4q26-q28 of people, and containing 319 each amino acid, comprise 70-80 amino acid repeating unit, molecular weight is about 35kD.Research confirm Annexin V can with PS specific binding, avidity is up to 10
-9mol/L.Therefore, carry out apoptosis imaging in body using radioisotope labeling Annexin V as developer, realize apoptotic live body without wound video picture, with the generation of apoptosis in Real-Time Monitoring body, become the research emphasis in monitoring active somatic cell apoptosis field, and achieve greater advance.
The synthesis key of Annexin V class developer is the mark of radionuclide, should obtain higher radiochemical purity, can not damage again the binding activities of Annexin V simultaneously.At present, with
99mtc marks Annexin V most study,
99mthe advantage of Tc is that the transformation period is 6h, can video picture in time, and the dosage making again patient be subject to is less, and can be obtained easily by Mo/Tc producer.But,
99mtc directly marks that Annexin V is more difficult, and mark rate is low, and can produce some metaproteins in labeling process, causes the radioactive uptake of liver to increase.Therefore, Annexin V
99mtc mark is based on indirect labelling, and conventional difunctional intercalating agent has N-1-imino--4-mercaptobutyl (Imino), quadrol halfcystine (EC), disulfide dinitrogen (N
2s
2, also known as BTAP) etc., the most common with hydrazino-nicotinamide (HYNIC), mark obtains
99mtc-HYNIC-Annexin V specific activity is higher, and radiochemical purity is greater than 90%, and in apoptosis animal model, obtains good assessment.But these indirect labeling methods process more complicated, require strict to technical qualification, product needs purifying, especially not easily makes medicine box, and Clinical practice is subject to a definite limitation.
Up-to-date research shows, Annexin V can be conducive to using in DNA recombinant expression after structural modification
99mtc directly marks.The three-dimensional structure display of Annexin V, its N-holds and C-holds the opposite being positioned at molecular film binding site, therefore in regrouping process, recombinant protein is not affected in the expression of Escherichia colli or folding to the modification of Annexin V, do not change the affinity that it is combined with film PS yet.As foreign scholar Tait JF etc. with the addition of 7 amino acid at N-end, wherein containing 1-2 cysteine residues, the three kinds of mutant obtained, called after respectively: Annexin V-116, Annexin V-117 and Annexin V-118, domestic scholars Hua Zichun professor etc. hold interpolation 10 continuous print Histidines, called after His10-Annexin V at the N of Annexin V, the binding activities of Annexin V variant and the film of these restructuring follows that natural A nnexin V's is consistent, and with
99mtc-HYNIC-Annexin V has more similar bio distribution.These show that to the protein engineering transformation of Annexin V be feasible.
Chinese patent CN102690345A further discloses again and a kind ofly to clone in intestinal bacteria, express and the new Annexin V variant that obtains of purifying, and this variant only with the addition of 1 halfcystine (Cys-Annexin V) at the C-end of natural A nnexin V.Experimental result shows, when SP column purification, relative to independent Annexin V, Cys-Annexin V is combined more firm with SP post, need just can be eluted by higher ionic strength, this has stronger metal ion binding capacity from a side illustration Cys-Annexin V than Annexin V.And adopt direct labelling method to carry out Cys-Annexin V
99mthe experiment of Tc mark also confirms
99mtc-Cys-Annexin V has performance akin with Cys-Annexin V, and mark rate is up to about 95%.
But applicant finds under study for action, record in this patent documentation with in prior art
99mwhen the method for Tc mark marks Cys-Annexin V, the marked product obtained
99mtc-Cys-Annexin V and other radioimpurity radioactive impurities are (as raw material Na
99mtcO
4and by product
99mtc colloid etc.) be difficult to distinguish, affect follow-up research and utilization.
Summary of the invention
For this reason, technical problem to be solved by this invention is in prior art
99mtc marks marked product and radioimpurity radioactive impurity in the method for Cys-Annexin V and is difficult to the problem be separated, and then provides that a kind of step is simple, mark rate is high, can be used for
99mthe medicine box of the Cys-Annexin V variant of Tc mark;
Further, present invention also offers the compound method of above-mentioned medicine box and it is detecting the application in apoptosis.
For solving the problems of the technologies described above, of the present invention for
99mthe Cys-Annexin V medicine box of Tc mark, is characterized in that, often prop up described formula and comprise:
Cys-Annexin V 25-150μg；
Edetate 0.5-6mg;
Tin salt 5-40 μ g;
PH6.0-8.0 adjusted by pH buffer reagent.
Described edetate is sodium ethylene diamine tetracetate or ethylenediamine tetraacetic acid (EDTA) potassium.
Described tin salt is the mixture of one or more in tin protochloride, tin protofluoride, stannous tartrate, tin protobromide or stannous citrate.
Described pH buffer reagent buffer reagent is the mixture of one or more of phosphoric acid salt, Citrate trianion, ammonium salt, acetate, carbonate, borate, barbiturate, phthalate.
Present invention also offers a kind of prepare above-mentioned for
99mthe method of the Cys-Annexin V medicine box of Tc mark, compound method of the present invention can directly dissolve according to the content of each component the reaction system being hybridly prepared into 1ml, directly injects 10ml cillin bottle and carries out lyophilize again to powdered form.But in view of reaction system less solution preparation tolerance range problem more rambunctious, compound method of the present invention is selected with 100ml reaction system for preparation unit, i.e. the reaction system of preparation 100 bottles of described medicine boxs, then carries out the mode of packing.
The preparation method of 100 bottles of Cys-Annexin V medicine boxs is as follows, take pH buffer reagent 10mmol, be dissolved in 90mL distilled water, add the edetate of 50-600mg, add the tin salt solution 0.25-2mL of 2mg/mL again, finally add the Cys-Annexin V solution 0.5-3mL of 5mg/mL, then to add distilled water to liquid cumulative volume be 100mL, be sub-packed in by 1.0mL/ bottle again in the cillin bottle of 10mL specification, after lyophilize, namely obtain Cys-Annexin V medicine box.
Further, described lyophilize step carries out lyophilize realization by gradient control lyophilize temperature to described cillin bottle.
Preferably, to be actual conditions be described lyophilize step: control described freezing temp and be-40 DEG C, freezing 4h; And vacuumize and make vacuum pressure reach 1-2Pa, more freezing 2h; Temperature is adjusted to-20 DEG C, freezing 3h; Again temperature is adjusted to-10 DEG C, freezing 13h; Subsequently temperature is adjusted to 10 DEG C, dry 2h; Again temperature is adjusted to 25 DEG C, dry 4h, after gland, namely obtains described Cys-Annexin V medicine box.
What preparation obtained often props up containing 25-150 μ g Cys-Annexin V in medicine box, the tin salt of 5-40 μ g, the edetate of 0.5-6mg.After medicine box adds 1mL distilled water, after powder dissolution, the pH value range of whole system is 6.0-8.0.
The present invention selects that described tin salt is dissolved in concentration is in the dilute hydrochloric acid of 1%, then adds in reaction system and react.
Present invention also offers one
99mtc marks the method for Cys-Annexin V medicine box, in above-mentioned medicine box, namely add the Na of new drip washing
99mtcO
4solution, puts 35-37 DEG C of water-bath 15min and namely obtains marked product
99mtc-Cys-Annexin V.
Further, described Na
99mtcO
4the volume that adds of solution is 1ml.
Described Na
99mtcO
4solution can adopt any means well known to those skilled in the art in prior art to obtain.
Medicine box of the present invention is adopted to mark, not only mark rate is higher, and detect between each mark impurity that may exist of discovery and marked product and can be separated preferably, described detecting step carries out with HPLC method, and described purification step adopts HPLC method, and actual conditions comprises: select TSKGEL gel column, take concentration as the phosphoric acid buffer of 0.05M, pH7.0 be moving phase, flow velocity is 0.8mL/min, and ultraviolet detection wavelength is 278nm, described in
99mthe retention time of Tc-Cys-Annexin V is 9.8min.
Present invention also offers a kind of labelled protein, the marked product namely obtained by above-mentioned medicine box and marking method.
And a kind of above-mentioned for
99mthe Cys-Annexin V medicine box of Tc mark is detecting the application in apoptosis.
Technique scheme of the present invention has the following advantages compared to existing technology:
1, of the present invention for
99mthe medicine box of Cys-Annexin V of Tc mark, achieves that to be undertaken carrying out froze-dried kit to Cys-Annexin V by ligand exchange method direct first
99mtc marks, not only simple to operate, and mark rate higher (reaching more than 95%), and by HPLC method detection display, marked product
99mother radioimpurity radioactive impurities that may exist in Tc-Cys-annexin V and labeled reactant are (as raw material Na
99mtcO
4and by product
99mtc colloid etc.) obviously distinguish, be conducive to promoting the use of clinical from now on;
2, method steps of the present invention is simple, and mark visual result is accurate, simultaneously because labeling effciency is up to more than 95%, can directly use without the need to specific purification step, therefore be easy to prepare medicine box, solve in prior art marked product that other direct marking methods exist and the problem that impurity is easily obscured;
3, the labelled protein adopting medicine box of the present invention to mark to obtain and Cys-annexin V have akin performance, after mark
99mtc-Cys-annexin V can be used as developer and is applied to the apoptosis situation detecting cell, and the experiment of rat liver apoptosis proves, labelled protein of the present invention intuitively can reflect the apoptosis situation of cell, and image results and situ end labeling to detect apoptotic result consistent, result is accurate, and medicine box of the present invention can be applicable to apoptotic detection.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is the HPLC color atlas of labelled precursor Cys-Annexin V of the present invention;
Fig. 2 is embodiment 2 marker protein
99mthe HPLC color atlas of Tc-Cys-Annexin V;
Fig. 3 is in embodiment 3
99mthe HPLC color atlas of Tc-EDTA;
Fig. 4 is in embodiment 4
99mthe HPLC color atlas of Tc colloid;
Fig. 5 is Na in embodiment 4
99mtcO
4hPLC color atlas;
Fig. 6 is in embodiment 5
99mthe distribution plan of Tc-Cys-Annexin V in normal mouse body;
Fig. 7 is in embodiment 5
99mthe blood medicine clearance curve of Tc-Cys-Annexin V in normal mouse body;
Fig. 8 is rat liver Apoptosis Model in embodiment 6
99mtc-Cys-Annexin V video picture figure;
Fig. 9 is the apoptosis situation that embodiment 7 situ end-labelling detects rat apoptosis liver cell;
Figure 10 is the impact of content on mark rate of Cys-Annexin V in EXAMPLE Example 8;
Figure 11 be in embodiment 9 tin salt content on the impact of mark rate;
Figure 12 is that the content of edetate (EDTA) in embodiment 10 is on the impact of mark rate;
Figure 13 is the HPLC figure of reaction solution in embodiment 11.
Embodiment
Embodiment 1
The concrete process for preparation of medicine box described in the present embodiment is as follows: take SODIUM PHOSPHATE, MONOBASIC 1.9mmol, Sodium phosphate dibasic 8.1mmol, be dissolved in 90mL distilled water, add 150mg sodium ethylene diamine tetracetate, add tin protochloride dilute hydrochloric acid (1% hydrochloric acid) the solution 1mL of 2.00mg/mL, finally add the Cys-Annexin V solution 1.5mL of 5mg/mL, sterile filtration after mixing, be sub-packed in 10mL cillin bottle by 1.00mL/ bottle again, described cillin bottle is put into Freeze Drying Equipment, described Freeze Drying Equipment temperature is set and is-40 DEG C, carry out freezing 4h, open vacuum pump evacuation subsequently (after vacuum pump unlatching, whole process all completes vacuumizing under state, until medicine box freeze-drying completes), vacuum pressure is made to reach 1-2Pa, freezing 2h again, temperature is adjusted to-20 DEG C, freezing 3h, subsequently temperature is adjusted to-10 DEG C, freezing 13h, again temperature is adjusted to 10 DEG C, dry 2h, again temperature is adjusted to 25 DEG C, dry 4h, namely required froze-dried kit is obtained after gland, in medicine box, material is white powdery solids.
Often prop up medicine box containing 75.0 μ g Cys-Annexin V, the tin protochloride of 20.0 μ g and the sodium ethylene diamine tetracetate of 1.50mg, after adding 1mL distilled water in medicine box, pH value is 6.0-8.0.
Described Cys-Annexin V is provided by Jiangsu Babiao Biopharmaceutical Research Institute Co., Ltd..Using gene engineering technique utilizes escherichia coli cloning, expresses and purifying, holds interpolation 1 halfcystine, purify with metal affinity chromatography at the C of Annexin V, restructuring preparation Cys-Annexin V.Concrete preparation process and feature are shown in Chinese patent CN102690345A.
Embodiment 2
With
99mtc marks the method for Cys-Annexin V medicine box
The medicine box prepared in Example 1, adds the Na of the new drip washing of 1ml in described medicine box
99mtcO
4solution, puts 35-37 DEG C of water-bath 15min, obtains marked product
99mtc-Cys-Annexin V, described Na
99mtcO
4solution is provided by Nuclear Medicine of clinical portion, Jiangsu Inst of Atomic Medical Sciences, and exit dose is 18.5-555MBq.
HPLC method is adopted to detect described marked product, adopt TSK GEL gel column (SWG2000swxL), with the phosphoric acid buffer of concentration 0.05M, pH7.0 for moving phase, flow velocity 0.8mL/min, ultraviolet detection wavelength 278nm, and be provided with isotope detector.Detect unlabelled Cys-Annexin V with identical testing conditions simultaneously.As shown in Figure 1-2, record
99mretention time (the t of Tc-Cys-Annexin V
r) be 9.8min, consistent with not having the retention time of markd Cys-Annexin V.By HPLC chromatogram, calculating mark rate is 98.5%.Visible, medicine box of the present invention not only can realize using
99mtc marks Cys-Annexin V, and the efficiency of mark is higher.
Embodiment 3
Prepare described medicine box according to the method described in embodiment 1, its difference is only to incite somebody to action
99mtc-Cys-Annexin V and other radioimpurity radioactive impurities make a distinction, Cys-Annexin V is not added when preparing described medicine box, make not containing target-marking Cys-Annexin V in the medicine box obtained, and the operation of other preparations and mark is constant, the marked product obtained mainly
99mtc-EDTA, carries out HPLC detection with the testing conditions of HPLC purifying in embodiment 2, as shown in Figure 3,
99mretention time (the t of Tc-EDTA
r) be 13.4min.
Embodiment 4
Prepare described medicine box according to the method described in embodiment 1, its difference is only to incite somebody to action
99mtc-Cys-Annexin V and other radioimpurity radioactive impurities make a distinction, Cys-Annexin V and sodium ethylene diamine tetracetate is not added when preparing described medicine box, make in the medicine box obtained not containing target-marking Cys-Annexin V and sodium ethylene diamine tetracetate, and the operation of other preparations and mark is constant, the marked product obtained mainly
99mtc colloid, carries out HPLC detection with the testing conditions of HPLC purifying in embodiment 2, as shown in Figure 4,
99mretention time (the t of Tc colloid
r) be 12.5min.
HPLC detection is carried out, Na with the testing conditions of HPLC purifying in embodiment 2
99mtcO
4retention time (the t of solution
r) be 15.9min, as shown in Figure 5.
Explanation in Fig. 1-5
99mtc-Cys-Annexin V and other radioimpurity radioactive impurities (as
99mtc colloid,
99mtc-EDTA and Na
99mtcO
4) can separate completely, calculated by HPLC
99mthe mark rate of Tc-Cys-AnnexinV, not only feasible and intuitively accurate.
Embodiment 5
Pass through
99mthe bio distribution of Tc-Cys-Annexin V in Mice Body, illustrates that the biodistribution feature of this tagged compound meets the requirement as developer.
Get ICR mouse 35, be divided into 7 groups at random, often organize 5, through tail vein injection 0.2mL(3.7MBq) medicine box described in embodiment 2 carries out the marked product that marking operation obtains
99mtc-Cys-Annexin V, respectively at 5,15,30,60,120,180,240min totally 7 time point sacrificed by decapitation after injection.Take out blood, brain, the heart, liver, spleen, lung, kidney, stomach, small intestine, bladder, the internal organs such as muscle and bone, measure radiocounting with γ calculating instrument after weighing, calculate every gram of internal organs percentage injection dose rate (%ID/g, the radiocounting of every gram of internal organs/be injected into gross activity counting × 100% in rat body), as shown in Figure 6, the radioactivity absorbed in kidney is the highest, next is bladder, liver and lung, and the picked-up in brain is very low.With the matching of DAS2.0 software, analyze
99mthe pharmacokinetic curve of Tc-Cys-AnnexinV in normal mouse body, as shown in Figure 7, meet two-compartment model feature, medicine for formula is: C=5.972e
-0.123t+ 0.877e
-0.005t, distribution phase transformation period (T
1/2 α) be 5.619min, remove phase transformation period (T
1/2 β) be 129.465min, average blood plasma clearance rate (CL) is 0.084L/min/kg, area (AUC under drug-time curve
0 → ∞value) be 238.123%ID/g*min.
Embodiment 6
Rat liver Apoptosis Model
99mtc-Cys-Annexin V video picture
Select SD rat 4 (Ministry of Health's nuclear medicine emphasis experiment provides), body weight 280-290g, with the cycloheximide dosimetric system of 5mg/kg for rat liver Apoptosis Model, 3 rats are respectively through tail vein injection cycloheximide solution, and another 1 rat injecting normal saline compares.After 3 hours, 4 rats are respectively through tail vein injection 0.2mL(18.5MBq) embodiment 2 acceptance of the bid remembers
99mtc-Cys-AnnexinV, after injection developer, 3h carries out the Static planar video picture of forward and backward position, energy peak 140KeV, energy window 20%, image array 128 × 128, acquisition counter 1000K.As shown in Figure 8, left side is model mouse, and arrow place is the video picture of apoptosis liver, and right side is normal control mouse, and in the anteposition Static planar video picture figure of left side model mouse, can be observed liver region has obvious radioactivity dense poly-.
After video picture terminates, rat is put to death, take out liver immediately, part formalin solution is fixed, another part γ calculating instrument measures radiocounting, and weigh, calculate every gram of internal organs percentage injection dose rate (%ID/g), the radioactivity (ID%/g) of model mouse liver picked-up is 2.83 times of control group.
Embodiment 7
The situ end labeling (TUNEL) of rat apoptosis liver detects
TUNEL method is used for the inspection of individual cell level apoptosis immunohistochemical methods and quantitative analysis, and its principle is marker DNA fracture position, and detailed step is as follows: (1) rat liver sample is fixed after 24h through formalin solution, paraffin embedding, section, every sheet thickness 4-5 μm; (2) dimethylbenzene dewaxing 30min, after graded ethanol aquation, PBS rinses, each 1min, (3) drip Proteinase K, 37 DEG C hatch 30min after, PBS washs 3 times, (4) drip TUNEL and detect liquid (purchased from green skies biotechnology research institute), 37 DEG C of lucifuges hatch 1h, and PBS washs 3 times, (5) with after anti-fluorescent quenching mounting fluid-tight sheet, 100 times of fluorescence microscopy Microscopic observations.As shown in Figure 9, left side is model mouse, and right side is control group, and in figure, arrow indication is fluorescigenic apoptotic body, the fluorescigenic apoptotic body that the liver of model mouse is more as seen, and in the liver of control group fluorescigenic apoptotic body is just seldom.Medicine box described in this result and the embodiment of the present invention 2 marks and obtains
99mthe result of the rat liver apoptosis imaging of Tc-Cys-Annexin V is consistent, explanation
99mtc-Cys-Annexin V tagged compound can show the apoptosis situation of rat liver as developer.
Following each embodiment is studied and is affected situation on medicine box mark rate when each component concentration changes within the specific limits.
Embodiment 8
Keep constant by embodiment 1 of other component concentrations, the content changing Cys-Annexin V in medicine box is 10-200 μ g, by embodiment 2 method and measure mark rate.As shown in Figure 10, the content of Cys-Annexin V is within the scope of 25-150 μ g, and medicine box mark rate reaches more than 90% for result.
Embodiment 9
Keep constant by embodiment 1 of other component concentrations, changing tin protochloride in medicine box is tin protobromide, and its content is 1-100 μ g, measures mark rate by the method for embodiment 2.As shown in figure 11, the content of tin protochloride is within the scope of 5-40 μ g, and medicine box mark rate reaches more than 90% for result.
Embodiment 10
Keep constant by embodiment 1 of other component concentrations, changing edetate in medicine box is ethylenediamine tetraacetic acid (EDTA) potassium, and its content is 0.1-10mg, by embodiment 2 method mark and measure mark rate.As shown in figure 12, the content of sodium ethylene diamine tetracetate is within the scope of 0.5-6mg, and medicine box mark rate reaches more than 90% for result.
Embodiment 11
With art methods, Cys-Annexin V is marked:
Get 3.5mg tin protochloride and be dissolved in the SnCl making 3.5mg/ml in the PBS damping fluid of 10ml pH7.4
2for subsequent use.Under room temperature, get 100 μ L SnCl
2the Cys-Annexin V20 μ L that solution adds 0.6 μ g/ μ L wherein reacts.Add volume wherein and be less than 0.1ml, exit dose is the Na of 185MBq
99mtcO
4solution, then instills the static 30min reaction of 10mg/mL Vc10 μ L.With the condition detection reaction liquid of HPLC described in embodiment 2, result as shown in figure 13.Visible, carry out Cys-Annexin V mark with method of the prior art, the impurity existed in method is difficult to distinguish with labelled protein, easily affects the detection of Product Labeling rate on the one hand, also makes the purity of product be affected simultaneously.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (4)
1. one kind for
99mthe Cys-Annexin V medicine box of Tc mark, is characterized in that: often prop up described medicine box formula and comprise:
Cys-Annexin V 25-150μg；
Edetate 0.5-6mg; Described edetate is sodium ethylene diamine tetracetate or ethylenediamine tetraacetic acid (EDTA) potassium;
Tin salt 5-40 μ g; Described tin salt is the mixture of one or more in tin protochloride, tin protofluoride, stannous tartrate, tin protobromide or stannous citrate;
PH6.0-8.0 adjusted by pH buffer reagent; Described pH buffer reagent is the mixture of one or more of phosphoric acid salt, Citrate trianion, ammonium salt, acetate, carbonate, borate, barbiturate, phthalate.
2. one kind preparation according to claim 1 for
99mthe method of the Cys-Annexin V medicine box of Tc mark, it is characterized in that: take pH buffer reagent 10mmol, be dissolved in 90mL distilled water, add the edetate of 50-600mg, add the tin salt solution 0.25-2mL of 2mg/mL again, finally add the Cys-Annexin V solution 0.5-3mL of 5mg/mL, mixing and adding distilled water to cumulative volume is 100mL, be sub-packed in the cillin bottle of 10mL specification by 1.0mL/ bottle, and described cillin bottle is carried out lyophilize to its inner material by gradient control temperature is white powder, described lyophilize step is actual conditions: control described freezing temp and be-40 DEG C, freezing 4h, and vacuumize and make vacuum pressure reach 1-2Pa, more freezing 2h, temperature is adjusted to-20 DEG C, freezing 3h, again temperature is adjusted to-10 DEG C, freezing 13h, subsequently temperature is adjusted to 10 DEG C, dry 2h, again temperature is adjusted to 25 DEG C, dry 4h, after gland, namely obtains described Cys-Annexin V medicine box.
3. preparation according to claim 2 is used for
99mthe method of the Cys-Annexin V medicine box of Tc mark, is characterized in that: with concentration for tin salt described in the diluted hydrochloric acid dissolution of 1%.
4. one kind
99mtc marks the method for Cys-Annexin V, it is characterized in that: the Na adding new drip washing in medicine box according to claim 1
99mtcO
4solution, puts 35-37 DEG C of water-bath 15min, obtains marked product
99mtc-Cys-Annexin V.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310087314.5A CN103159842B (en) | 2013-03-18 | 2013-03-18 | Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310087314.5A CN103159842B (en) | 2013-03-18 | 2013-03-18 | Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103159842A CN103159842A (en) | 2013-06-19 |
| CN103159842B true CN103159842B (en) | 2015-07-01 |
Family
ID=48583406
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310087314.5A Active CN103159842B (en) | 2013-03-18 | 2013-03-18 | Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103159842B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669855B (en) * | 2016-02-04 | 2020-02-11 | 江苏省原子医学研究所 | A kind of 18Method for rapidly marking Cys-Annexin V by using F and application thereof |
| IT201600101875A1 (en) | 2016-10-11 | 2018-04-11 | Advanced Accelerator Applications Int S A | Lyophilized compositions comprising rhAnnexin V-128, process for their preparation and their use for the preparation of formulations containing 99mTc-rhAnnexin V-128 |
| CN115429904A (en) * | 2022-09-23 | 2022-12-06 | 江苏省原子医学研究所 | 99m Tc marked freeze-dried product medicine box and its preparation method and use |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004013303A2 (en) * | 2002-08-02 | 2004-02-12 | Surromed, Inc. | Modified annexin proteins and methods for treating vaso-occlusive sickle-cell disease |
| CN101080240A (en) * | 2004-12-17 | 2007-11-28 | 皇家飞利浦电子股份有限公司 | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
| WO2008059489A2 (en) * | 2006-11-13 | 2008-05-22 | Spectrum Dynamics Llc | Radioimaging applications of and novel formulations of teboroxime |
| CN102690345A (en) * | 2011-03-24 | 2012-09-26 | 江苏靶标生物医药研究所有限公司 | Human annexin V variant and its expression, preparation method and use |
-
2013
- 2013-03-18 CN CN201310087314.5A patent/CN103159842B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004013303A2 (en) * | 2002-08-02 | 2004-02-12 | Surromed, Inc. | Modified annexin proteins and methods for treating vaso-occlusive sickle-cell disease |
| CN101080240A (en) * | 2004-12-17 | 2007-11-28 | 皇家飞利浦电子股份有限公司 | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
| WO2008059489A2 (en) * | 2006-11-13 | 2008-05-22 | Spectrum Dynamics Llc | Radioimaging applications of and novel formulations of teboroxime |
| CN102690345A (en) * | 2011-03-24 | 2012-09-26 | 江苏靶标生物医药研究所有限公司 | Human annexin V variant and its expression, preparation method and use |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103159842A (en) | 2013-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7011816B2 (en) | Labeling targeting agents with gallium-68 and gallium-67 | |
| Xavier et al. | Site-specific labeling of His-tagged Nanobodies with 99m Tc: a practical guide | |
| Mukherjee et al. | Development of single vial kits for preparation of 68 Ga-labelled peptides for PET imaging of neuroendocrine tumours | |
| Yang et al. | Synthesis and evaluation of 68Ga-labeled DOTA-2-deoxy-D-glucosamine as a potential radiotracer in μPET imaging | |
| Velikyan et al. | Synthesis and preclinical evaluation of 68Ga-labeled collagelin analogs for imaging and quantification of fibrosis | |
| CN103228298A (en) | Apoptosis PET imaging agents | |
| CN103159842B (en) | Cys-Annexin V kit used for 99mTc labeling and preparation method and application thereof | |
| CN106581700A (en) | Novel HER2-targeting polypeptide radioactive medicine and preparation method and application thereof | |
| WO2020238795A1 (en) | Rk polypeptide radiopharmaceutical targeting her2, and preparation method therefor | |
| Tanzey et al. | Gallium-68: Methodology and novel radiotracers for positron emission tomography (2012–2017) | |
| RU2568888C1 (en) | METHOD AND FORMULATION FOR PRODUCING 99m TC LABELLED 5-THIO-D-GLUCOSE AGENT FOR RADIONUCLIDE DIAGNOSIS | |
| CN103159841B (en) | Method for marking Cys-Annexin V by use of 99mTc and application of method | |
| Wang et al. | Synthesis and evaluation of 99mTc–2-[(3-carboxy-1-oxopropyl) amino]-2-deoxy-D-glucose as a potential tumor imaging agent | |
| CN106492237B (en) | A kind of isoDGR polypeptide radiopharmaceuticals and its preparation method and application | |
| Vats et al. | Single vial cold kits optimized for preparation of gastrin releasing peptide receptor (GRPR)-radioantagonist 68Ga-RM2 using three different 68Ge/68Ga generators | |
| Park et al. | Imaging of the third gasotransmitter hydrogen sulfide using 99mTc-labeled alpha-hydroxy acids | |
| CN107847617A (en) | Metal tricarbonyl compound containing substituted iminodiacetic acid part and the purposes as radioisotopic tracer | |
| CN104193820B (en) | Method for labeling Cys-Annexin V with ＜18＞F and application of ＜18＞F-FBEM-Cys-Annexin V | |
| WO2011033112A2 (en) | 68ga-labeled human serum albumin | |
| Garnuszek et al. | Comparison of chromatographic methods for quality control of DMSA complexes with 99mTc and 188Re at (III) and (V) oxidation states | |
| Badbarin et al. | Optimized preparation and evaluation of 99mTc-Streptomycin | |
| TWI745616B (en) | Radioactive labeling for a long-acting peptide targeting pharmaceutical and the production method thereof | |
| BR102018006679A2 (en) | PHARMACEUTICAL FORMULATION AND ITS METHOD OF PREPARATION | |
| US20200129646A1 (en) | Radioactive labeled long-acting peptide targeted pharmaceutical and production method thereof | |
| Daruwati et al. | Profile of MIBI Liquid Phase Radiopharmaceutical for Myocardial Imaging |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |