CN105561330B - A kind of human calcitonin-Cucurbituril compound formulation and preparation method thereof - Google Patents
A kind of human calcitonin-Cucurbituril compound formulation and preparation method thereof Download PDFInfo
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- 238000009472 formulation Methods 0.000 title claims abstract description 58
- 239000000203 mixture Substances 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 101000741445 Homo sapiens Calcitonin Proteins 0.000 claims abstract description 173
- 229940045644 human calcitonin Drugs 0.000 claims abstract description 170
- MSBXTPRURXJCPF-DQWIULQBSA-N cucurbit[6]uril Chemical compound N1([C@@H]2[C@@H]3N(C1=O)CN1[C@@H]4[C@@H]5N(C1=O)CN1[C@@H]6[C@@H]7N(C1=O)CN1[C@@H]8[C@@H]9N(C1=O)CN([C@H]1N(C%10=O)CN9C(=O)N8CN7C(=O)N6CN5C(=O)N4CN3C(=O)N2C2)C3=O)CN4C(=O)N5[C@@H]6[C@H]4N2C(=O)N6CN%10[C@H]1N3C5 MSBXTPRURXJCPF-DQWIULQBSA-N 0.000 claims abstract description 50
- 150000001875 compounds Chemical class 0.000 claims abstract description 31
- 239000003125 aqueous solvent Substances 0.000 claims abstract description 12
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 20
- 239000011575 calcium Substances 0.000 claims description 20
- 229910052791 calcium Inorganic materials 0.000 claims description 20
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- 239000007853 buffer solution Substances 0.000 description 15
- 102000055006 Calcitonin Human genes 0.000 description 14
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- 239000007924 injection Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
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- 230000028993 immune response Effects 0.000 description 5
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavine T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 description 5
- 229960003773 calcitonin (salmon synthetic) Drugs 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
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- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- QGZCUOLOTMJILH-UHFFFAOYSA-N 2h-tetrazol-2-ium;bromide Chemical compound [Br-].C1=N[NH+]=NN1 QGZCUOLOTMJILH-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 208000037147 Hypercalcaemia Diseases 0.000 description 2
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- 229910019142 PO4 Inorganic materials 0.000 description 2
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- 239000012153 distilled water Substances 0.000 description 2
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- VSHJAJRPRRNBEK-LMVCGNDWSA-N eel calcitonin Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CS)[C@@H](C)O)C(C)C)CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C1=CN=CN1 VSHJAJRPRRNBEK-LMVCGNDWSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 238000000111 isothermal titration calorimetry Methods 0.000 description 2
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- 208000013076 thyroid tumor Diseases 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 210000003771 C cell Anatomy 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101000741447 Gallus gallus Calcitonin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
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- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/23—Calcitonins
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a kind of human calcitonin Cucurbituril compound formulation, which includes human calcitonin Cucurbituril supramolecular complex, human calcitonin and Cucurbituril, its preparation method and is：Human calcitonin and Cucurbituril are dissolved under conditions of 0~40 DEG C in the aqueous solvent that pH value is 7~8 and are uniformly mixed to get to human calcitonin Cucurbituril compound formulation；Or human calcitonin and Cucurbituril are dissolved in the aqueous solvent that pH value is 7~8 and are uniformly mixed under conditions of 0~40 DEG C, it is then freeze-dried to get to human calcitonin Cucurbituril compound formulation.Compound formulation of the present invention is using process to be not susceptible to fibrosis, and the bioactivity higher of the more individual human calcitonin of bioactivity of the compound formulation, and the present invention helps to promote the clinical practice of human calcitonin.
Description
Technical field
The invention belongs to biomedicine field, more particularly to a kind of human calcitonin-Cucurbituril compound formulation and its preparation side
Method.
Background technology
Calcitonin (calcitonin, CT) is the linear pattern peptide hormone containing 32 amino acid, it is moved by vertebra
The postbranchial body of object and thyroid parafollicular cell (parafollicular cells, also known as C cells) secretion of mammal
Hormone, have its corresponding receptor in bone and kidney, play an important role of to adjust Ca,P metabolism, be currently used primarily in and Low BMD
The treatment of relevant disease.The secretion of calcitonin is related with flowing through the calcium concentration in thyroid blood, therefore blood calcium concentration
Increase can cause calcitonin secretion to increase and inhibit bone information, reduce the blood calcium concentration of hypercalcemia patient.
The amino acid sequence of the calcitonin of separate sources is different, and bioactive, wherein salmon calcitonin are all shown to people
(sCT), the active highest of eel calcitonin (eCT) and chicken calcitonin (cCT), and human calcitonin (hCT) is due to meeting faster fibres
Change and activity it is minimum.Due to non-Genus Homo source the amino acid sequence of calcitonin and the amino acid sequence differences of human calcitonin compared with
Greatly, for example, the homology of sCT and hCT is only 50%, for human body, the calcitonin in non-Genus Homo source is a kind of immune response
Original, the immune response that human body generates it can influence its clinical value.Although current clinical treatment osteoporosis, hypercalcemia
Mostly using sCT during disease, but since it is poor with the homology of hCT, immune response and antibody that when application generates can influence treatment effect
Fruit.
Although by hCT for treating osteoporosis, hypercalcinemia, human body will not generate it immune response, hCT ratios
SCT is easier fibrosis, and in aqueous solvent, hCT molecules are easy to mutually assemble and occur fibrosis, form long fibre precipitation
Object, the long fibre sediment of calcitonin easily trigger thyroid tumors (Hazard JB, Hawk WA, Crile G.J Clin
Endocr Metab.1959；19:152-61.)；The mutual aggregation of calcitonin molecule can also cause the reduction of its bioactivity.It is above-mentioned
Problem causes the clinical practice of hCT to be faced with very big predicament, and hCT not yet realizes clinical practice at present.Therefore, if can develop
The method of hCT fibrosis can effectively be inhibited, prepare and be not easy fibrosis and the hCT products with compared with high bioactivity, for
Important meaning will be generated by promoting the clinical practice of hCT.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of human calcitonin-Cucurbituril compound formulation and
Its preparation method occurs fibrosis to inhibit human calcitonin, while improves the bioactivity of human calcitonin in use, pushes away
Into the clinical practice of human calcitonin.
Human calcitonin of the present invention-Cucurbituril compound formulation, including human calcitonin-Cucurbituril supramolecular complex, people
Calcitonin and Cucurbituril, the Cucurbituril are Cucurbituril 7 or Cucurbituril 8.
The preparation method of human calcitonin of the present invention-Cucurbituril compound formulation, operation are as follows：
It is 1 according to human calcitonin and the molar ratio of Cucurbituril:(10~50) human calcitonin and Cucurbituril are measured, calcium is dropped into people
Element and Cucurbituril are dissolved in the aqueous solvent that pH value is 7~8 and are uniformly mixed under conditions of 0~40 DEG C to drop to get to people
Calcium element-Cucurbituril compound formulation；
Or according to human calcitonin and the molar ratio of Cucurbituril it is 1:(10~50) human calcitonin and Cucurbituril are measured, by people
Calcitonin and Cucurbituril are dissolved in the aqueous solvent that pH value is 7~8 under conditions of 0~40 DEG C and are uniformly mixed, Ran Houleng
It is lyophilized dry to get to human calcitonin-Cucurbituril compound formulation；
The dosage of the aqueous solvent should make that the concentration of human calcitonin is 100~200 μm of ol/L, the concentration of Cucurbituril is 1
~10mmol/L, the Cucurbituril are Cucurbituril 7 or Cucurbituril 8.
In the above method, it according to the molar ratio of human calcitonin and Cucurbituril is 1 that preferred technical solution, which is,:(10~25) count
Measure human calcitonin and Cucurbituril.
In the above method, the aqueous solvent is deionized water, physiological saline or phosphate buffer.
Human calcitonin-Cucurbituril the compound formulation prepared according to above-mentioned first method can be injected after concentration is regulated
The mode of administration uses, and the human calcitonin-Cucurbituril compound formulation prepared according to above-mentioned second method is using aqueous solvent
It dissolves and can be used after regulating concentration in a manner of drug administration by injection.
Human calcitonin provided by the invention-Cucurbituril compound formulation can inhibit human calcitonin fibrosis and improve its biology work
The main reason for property, is as follows：Cucurbituril 7 or Cucurbituril 8 are cyclic macromolecular, and annular interior cavity is hydrophobic, and Cucurbituril 7
And the size of the ring-shaped cavity of Cucurbituril 8 is matched with the size of the amino acid containing phenyl ring on human calcitonin polypeptide chain, aqueous molten
In agent, the hydrophobic internal cavities of Cucurbituril 7 or Cucurbituril 8 can include the amino acid containing phenyl ring on human calcitonin polypeptide chain and pass through hydrophobic
Interaction makes the conformation of human calcitonin change, and after conformation change, the mutual aggregation between human calcitonin molecule is reduced, from
And effectively inhibit human calcitonin and fibrosis occurs when mutually assembling.Meanwhile the reduction mutually assembled between human calcitonin molecule
It can increase the quantity of human calcitonin monomer in aqueous solvent and keep Stable conformation, and the bioactivity of human calcitonin monomer is high
In the bioactivity of human calcitonin aggregation, therefore human calcitonin of the present invention-Cucurbituril compound formulation can improve people and drop calcium
The bioactivity of element.
Compared with prior art, the invention has the advantages that：
1. human calcitonin provided by the invention-Cucurbituril compound formulation is a kind of new human calcitonin preparation, aqueous
In solvent, Cucurbituril ring-type hydrophobic internal cavities, which can include on human calcitonin polypeptide chain the amino acid containing phenyl ring and pass through hydrophobic effect, to be made
The conformation of human calcitonin changes, and then reduces the chance mutually assembled between human calcitonin molecule, inhibits human calcitonin
Fibrosis occurs due to intermolecular mutual aggregation, experiment shows human calcitonin and after Cucurbituril interaction, human calcitonin is gathered
Collection form is changed, and does not re-form ripe fiber, therefore, compound formulation of the present invention can reduce the length of human calcitonin
Fibrillar precipitate triggers the possibility of thyroid tumors, than the drug safety higher of individual human calcitonin.
2. in human calcitonin provided by the invention-Cucurbituril compound formulation, due to Cucurbituril and the phase interaction of human calcitonin
With so that the intermolecular chance mutually assembled of human calcitonin is reduced, so as to add the people of Stable conformation in compound formulation drop
The quantity of calcium element monomer, the increase of human calcitonin amount of monomer can improve its bioactivity, therefore compound formulation of the present invention
There is higher bioactivity than individual human calcitonin.Cell experiment shows compared with individual human calcitonin, institute of the present invention
The bioactivity higher of human calcitonin-Cucurbituril compound formulation is stated, and without apparent cytotoxicity.Zoopery shows this
It is considerably higher to invent the more individual human calcitonin of drop blood calcium ability of the human calcitonin-Cucurbituril compound formulation, identical dense
Drop blood calcium ability under conditions of degree with salmon calcitonin approaches, and human calcitonin of the present invention-Cucurbituril compound formulation is not
Can because the reason for the homology in clinical practice when generate immune response due to influence therapeutic effect.From more than content, this hair
The bright human calcitonin-Cucurbituril compound formulation has potential applicability in clinical practice, and the present invention helps to promote human calcitonin
Clinical practice.
3. the preparation method of human calcitonin of the present invention-Cucurbituril compound formulation is simple, process conditions are mildly easy to push away
Wide application.
Description of the drawings
Fig. 1 is change of each sample with the fluorescence intensity after the effect of ThT dyestuffs with mixing time in embodiment 1 and comparative example 1
Change curve.
Fig. 2 is that the transmission electron microscope photo of each sample that 20h is obtained is stirred in embodiment 1 and comparative example 1, in figure
Scale is 100nm.
Fig. 3 is the result figure that embodiment 6 is interacted using identical titration calorimetry test human calcitonin with Cucurbituril 7.
Fig. 4 is the cytotoxicity test result figure in embodiment 7.
Fig. 5 is the blood calcium versus time curve of rat in embodiment 8.
Specific embodiment
Below by embodiment combination attached drawing to human calcitonin of the present invention-Cucurbituril compound formulation and preparation method thereof
It is specifically described, it is necessary to which indicated herein to be, embodiment is served only for that the present invention is further illustrated, it is impossible to be interpreted as
Limiting the scope of the invention, person skilled in art can be made some with content according to the present invention and nonessential changed
It is embodied into adjustment, but such specific implementation should still fall within protection scope of the present invention.
In following each embodiments, the preparation method of phosphate buffer (PBS buffer solution) is：Prepare 0.2mol/L's respectively
Na2HPO4With the NaH of 0.2mol/L2PO4Mother liquor takes the NaH of 19mL2PO4With the Na2HPO of 81mL4Mother liquor is added in container, so
After add in NaCl, add distilled water or deionized water to 700~900mL and stirring be completely dissolved sodium chloride, adjust pH value to 7.0
1L is settled to up to PBS buffer solution with distilled water or deionized water after~7.4, wherein, the concentration of NaCl is 100mmol/L.
By the following examples 1 and comparative example 1 illustrate individual human calcitonin and human calcitonin-Cucurbituril of the present invention
The fibrosis situation of human calcitonin in compound formulation.
Embodiment 1
Human calcitonin (hCT) 2.0mg, Cucurbituril 7 (CB [7]) 6.9mg addition are had been loaded in the 1# glass tube vials of stirrer,
HCT 2.0mg, CB [7] 17.4mg addition are had been loaded in the 2# glass tube vials of stirrer, hCT 2.0mg, CB [7] 34.8mg are added
In the 3# glass tube vials for entering to have been loaded with stirrer, in 1~3# glass tube vials, the molar ratio of hCT and CB [7] is respectively 1:10、1:25、1:
50。
The PBS buffer solution 3mL that pH value is 7.4 is separately added into 1~3# glass tube vials, be placed in ice bath ultrasound to hCT and
CB [7] is completely dissolved with mixing to get 1~3# glass tube vials to hCT-CB [7] compound formulation, to be then placed in 40 DEG C of water
It in bath, is stirred using constant temperature blender with magnetic force with the rotating speed of 200r/min, from 0~4h of stirring is started at interval of 0.5h
It is sampled, is sampled from the 4~20h for starting stirring at interval of 1h, stop stirring after stirring 20h.
After each sample is taken out, Thioflavin T (ThT dyestuffs) and sample effect 15min are added in immediately, and it is strong then to test fluorescence
Degree, the results are shown in Figure 1.After stirring 20h, transmission electron microscope photo such as Fig. 2 (B) of gained sample in 1~3# glass tube vials
Shown in~(D).
Comparative example 1
HCT2.0mg additions are had been loaded in the glass tube vial of stirrer, add in the PBS buffer solution 3mL that pH value is 7.4, then
It is placed in ultrasound in ice bath to be completely dissolved to obtain hCT solution to hCT, then the glass tube vial is placed in 40 DEG C of water-bath, uses perseverance
Warm magnetic stirring apparatus is stirred with the rotating speed of 200r/min, is sampled from the 0~4h for starting stirring at interval of 0.5h, from
4~the 20h for starting stirring is sampled at interval of 1h, stops stirring after stirring 20h.
After each sample is taken out, ThT dyestuffs and sample effect 15min are added in immediately, then tests fluorescence intensity, as a result as schemed
Shown in 1.It stirs obtained by 20h shown in transmission electron microscope photo such as Fig. 2 (A) of sample.
Since ThT dyestuffs can be combined specifically with the hCT after fibrosis and its fluorescence is made drastically to enhance, by Fig. 1
The fluorescence intensity of middle each sample can determine whether the fibrosis situation of hCT in each sample.As shown in Figure 1, in comparative example 1, hCT is in PBS
Fibrosis can occur in buffer solution, and in embodiment 1, in hCT-CB [7] compound formulation the fibrosis of hCT be substantially reduced or
Fibrosis no longer occurs for person.
From Fig. 2 (A), hCT stirs 20h in PBS solution, generates the fiber of a large amount of maturations, and hCT fiber meetings
It flocks together to form thicker fiber, the diameter for the individual fibers that arrows go out is about 8nm in Fig. 2 (A).By Fig. 2 (B)~
(D) understand, the form and Fig. 2 (A) of hCT-CB [7] compound formulation prepared by embodiment 1 are significantly different, illustrate prepared by embodiment 1
HCT-CB [7] compound formulation in, after hCT and CB [7] interact, the accumulation shape of hCT is changed, no longer
Mutually aggregation forms ripe fiber.In Fig. 2 (B), according to molar ratio=1 of hCT and CB [7]:Prepared by 10 additive amount
In hCT-CB [7] compound formulation, after hCT and CB [7] interactions, there is no ripe fiber generations, only generate a small amount of fibre
The preceding aggressiveness of sample is tieed up, and forms the nanoparticle that grain size is 10~100nm；In Fig. 2 (C) (D), according to hCT's and CB [7]
Molar ratio=1:25 and 1:In hCT-CB [7] compound formulation prepared by 50 additive amount, after hCT and CB [7] interactions, no
There are ripe fiber and fibroid preceding aggressiveness production again, form the nanoparticle that a large amount of grain sizes are 10~100nm.
From Fig. 1 and Fig. 2, after hCT and CB [7] interactions, the accumulation shape of hCT is changed, institute of the present invention
Fibrosis is less likely to occur the human calcitonin stated in human calcitonin-Cucurbituril compound formulation.
Embodiment 2
According to molar ratio=1 of hCT and CB [7]:10 metering hCT and CB [7], hCT and CB [7] additions are had been loaded with stirring
In the glass tube vial of son, deionized water is added in into glass tube vial in room temperature, concussion glass tube vial is completely dissolved hCT and CB [7], go from
The concentration that the dosage of sub- water should make the concentration of hCT be 100 μm of ol/L, CB [7] is 1mmol/L, is then stirred at 25 DEG C using magnetic force
It mixes device and 20min is stirred with the rotating speed of 500r/min, be freeze-dried to get hCT-CB [7] compound formulation.
Embodiment 3
According to molar ratio=1 of hCT and CB [7]:30 metering hCT and CB [7], hCT and CB [7] additions are had been loaded with stirring
In the glass tube vial of son, the PBS buffer solution that pH value is 7.4 is added in into glass tube vial in room temperature, concussion glass tube vial makes hCT and CB [7]
It is completely dissolved, the concentration that the dosage of PBS buffer solution should make the concentration of hCT be 100 μm of ol/L, CB [7] is 3mmol/L, then will
The glass tube vial is placed in 40 DEG C of water-bath stirs the glass tube vial using constant temperature blender with magnetic force with the rotating speed of 100r/min
30min is freeze-dried to get hCT-CB [7] compound formulation.
Embodiment 4
According to molar ratio=1 of hCT and CB [7]:50 metering hCT and CB [7], hCT and CB [7] additions are had been loaded with stirring
In the glass tube vial of son, the PBS buffer solution that pH value is 7.4 is added in into glass tube vial in room temperature, concussion glass tube vial makes hCT and CB [7]
It is completely dissolved, the concentration that the dosage of PBS buffer solution should make the concentration of hCT be 200 μm of ol/L, CB [7] is 10mmol/L, Ran Hou
25 DEG C are stirred the glass tube vial 40min to get hCT-CB [7] compound system using constant temperature blender with magnetic force with the rotating speed of 100r/min
Agent.
Embodiment 5
According to molar ratio=1 of hCT and Cucurbituril 8 (CB [8]):25 metering hCT and CB [8], hCT and CB [8] is added in
It has been loaded in the glass tube vial of stirrer, physiological saline is added in into glass tube vial in room temperature, concussion glass tube vial makes hCT and CB [8] complete
Fully dissolved, the concentration that the dosage of physiological saline should make the concentration of hCT be 100 μm of ol/L, CB [8] is 2.5mmol/L, then by institute
Glass tube vial is stated to be placed in ice bath, it is compound to get hCT-CB [8] with the rotating speed stirring 1h of 50r/min using constant temperature blender with magnetic force
Preparation.
Embodiment 6
In the present embodiment, using identical titration calorimetry test human calcitonin and the situation of the interaction of Cucurbituril 7.
(1) hCT 2.0mg and CB [7] 6.9mg is separately added into two test tubes, is then separately added into two test tubes
PH value is 7.4 PBS buffer solution 3mL, and rocking test tube makes hCT and CB [7] be completely dissolved to obtain hCT solution and CB [7] solution, is adjusted
The pH value of hCT solution and CB [7] solution is saved, the difference of the pH value of the two is made to be no more than 0.1, then operation is de-gassed to the two.
(2) hCT solution is taken to add in sample cell, CB [7] solution is taken to add in syringe, by CB [7] solution with 2 μ L every time
Amount be added dropwise in sample cell and with the solution in the rotating speed stirred sample pond of 750r/min, control entire body in titration process
The constant temperature of system measures the thermal change in titration process, the results are shown in Figure 3, from the figure 3, it may be seen that CB [7] solution at 25 DEG C
Exothermic reaction has occurred after being added dropwise in hCT solution, with the increase of dripping quantity, thermal discharge gradually tends towards stability, and passes through it
The matched curve of exothermic process understands that human calcitonin is interacted with Cucurbituril 7, and Δ G ＜ 0, illustrates hCT and CB
[7] interaction can spontaneously occur and form hCT-CB [7] supramolecular complex, and the binding constant both in the compound is
6.73×104, the combination of the two is than about 1.
Embodiment 7
In the present embodiment, the cytotoxicity of hCT and hCT-CB of the present invention [7] compound formulation is measured using mtt assay.
(1) hCT is 1. taken, hCT dissolvings are configured to the hCT solution of 100mg/L in room temperature cell culture medium, then it is dilute successively
It releases to 50,25,10,1mg/L and filtration sterilization, is denoted as hCT/CB [7]=1:0；
2. it is 1 by the molar ratio of hCT and CB [7]:10 four groups of hCT of metering and CB [7] sample, in room temperature cell culture medium
By the dissolving of each group sample and mixing, be made hCT concentration be respectively 1,10,25,50, hCT-CB [7] compound formulation of 100mg/L simultaneously
Filtration sterilization is denoted as hCT/CB [7]=1:10；
3. it is 1 by the molar ratio of hCT and CB [7]:25 four groups of hCT of metering and CB [7] sample, in room temperature cell culture medium
By the dissolving of each group sample and mixing, be made hCT concentration be respectively 1,10,25,50, hCT-CB [7] compound formulation of 100mg/L simultaneously
Filtration sterilization is denoted as hCT/CB [7]=1:25；
4. it is 1 by the molar ratio of hCT and CB [7]:50 four groups of hCT of metering and CB [7] sample, in room temperature cell culture medium
By the dissolving of each group sample and mixing, be made hCT concentration be respectively 1,10,25,50, hCT-CB [7] compound formulation of 100mg/L simultaneously
Filtration sterilization is denoted as hCT/CB [7]=1:50.
In the step, the formula of the cell culture medium is：The high glycoform DMEM of volume fraction 90%, 10% tire ox blood
Each component is first used 450nm micropore filtering film mistakes by the clearly, streptomysin of the penicillin of 100 μ/mL and 100 μ g/mL after mixing
Filter, then with being used after 220nm micro porous filtration membrane filtrations.
(2) each sample that step (1) is prepared is separately added into 96 orifice plates for having spread uniform L929 cells, be subsequently placed in
In sterile incubator, in 5%CO2Concentration, 95% relative humidity, 37 DEG C of cultures add in tetrazolium bromide (MTT) afterwards for 24 hours, in sterile incubator
According to aforementioned condition continue cultivate 5h, then into each hole of 96 orifice plates add in dimethyl sulfoxide (DMSO) (DMSO) dissolve living cells generate
Crystal violet, then detect its OD value with microplate reader and calculate cytoactive, the results are shown in Figure 4.
As shown in Figure 4, when hCT concentration is 1mg/mL, hCT/CB [7]=1:10 and hCT/CB [7]=1:25 with
HCT/CB [7]=1:0 cytotoxicity is suitable, when hCT concentration is 10~100mg/mL, hCT/CB [7]=1:10 and
HCT/CB [7]=1:25 compared with hCT/CB [7]=1:0 cytotoxicity is substantially reduced, when hCT concentration is 25~100mg/mL,
HCT/CB [7]=1:50 compared with hCT/CB [7]=1:0 cytotoxicity is substantially reduced.As shown in the above, it is provided by the invention
HCT-CB [7] compound formulations are compared with individual hCT, cytotoxicity smaller, bioactivity higher.
Embodiment 8
The present embodiment compares hCT by zoopery, and salmon calcitonin (sCT) and hCT-CB of the present invention [7] are multiple
The drop blood calcium ability of preparation is closed, step is as follows：
(1) sCT is 1. taken, is dissolved to form sCT solution with PBS buffer solution in room temperature；
2. taking hCT, hCT solution is configured in the dissolving of room temperature PBS buffer solution；
3. it is 1 by the molar ratio of hCT and CB [7]:10 metering hCT and CB [7], then in room temperature PBS buffer solution by hCT
HCT-CB [7] compound formulation is made with CB [7] dissolvings and mixing；
4. it is 1 by the molar ratio of hCT and CB [7]:25 metering hCT and CB [7], then in room temperature PBS buffer solution by hCT
HCT-CB [7] compound formulation is made with CB [7] dissolvings and mixing；
Step 1.~4. in each solution and compound formulation of middle preparation, the concentration of sCT or hCT are 50 μ g/mL.
(2) 8 weeks healthy male SD rats 20 that weight is 220~240g are only randomly divided into 5 groups, every group 4, each group is equal
Drug administration by injection, by Rat Fast 18h before injection, by rat body weight, injection volume 0.2mL/100g.
First group is blank control group, injects PBS buffer solution；
The sCT solution that second group of injecting step (1) is 1. prepared；
The hCT solution that 3rd group of injecting step (1) is 2. prepared；
HCT-CB [7] compound formulation that 4th group of injecting step (1) 3. prepares；
HCT-CB [7] compound formulation that 5th group of injecting step (1) 4. prepares；
Respectively before the injection, 0.5h, 1h, 2h, 3h and blood is taken to rat tail for 24 hours after injection, is then tried using calcium ion
Agent box tests the blood calcium of each blood sample, obtains the blood calcium versus time curve of each group rat, as shown in Figure 5.
As shown in Figure 5, inject the rat of hCT solution after injection 0~interior for 24 hours blood calcium and blank control group it is basic
Unanimously, hCT is almost without blood calcium effect is dropped, and compared with hCT, the drop blood calcium ability of hCT-CB [7] compound formulation is substantially more preferable, and
And the drop blood calcium ability of hCT-CB [7] compound formulation that step (1) is 4. prepared is approached with the drop blood calcium ability of salmon calcitonin, is said
It is bright that human calcitonin is prepared into human calcitonin of the present invention-Cucurbituril compound formulation, human calcitonin drop blood calcium ability can be improved.
Claims (4)
1. a kind of human calcitonin-Cucurbituril compound formulation, it is characterised in that the compound formulation includes human calcitonin-Cucurbituril oversubscription
Sub- compound, human calcitonin and Cucurbituril, the Cucurbituril are Cucurbituril 7 or Cucurbituril 8.
2. the preparation method of human calcitonin described in claim 1-Cucurbituril compound formulation, it is characterised in that operation is as follows：
It is 1 according to human calcitonin and the molar ratio of Cucurbituril:(10~50) human calcitonin and Cucurbituril are measured, by human calcitonin and
Cucurbituril be dissolved under conditions of 0~40 DEG C pH value be 7~8 aqueous solvent in and be uniformly mixed to get to human calcitonin-
Cucurbituril compound formulation；
Or according to human calcitonin and the molar ratio of Cucurbituril it is 1:(10~50) human calcitonin and Cucurbituril are measured, calcium is dropped into people
Element and Cucurbituril are dissolved in the aqueous solvent that pH value is 7~8 and are uniformly mixed under conditions of 0~40 DEG C, and then freezing is dry
It is dry to get to human calcitonin-Cucurbituril compound formulation；
The dosage of the aqueous solvent should make the concentration of human calcitonin be 100~200 μm of ol/L, the concentration of Cucurbituril be 1~
10mmol/L, the Cucurbituril are Cucurbituril 7 or Cucurbituril 8.
3. the preparation method of human calcitonin-Cucurbituril compound formulation according to claim 2, it is characterised in that drop calcium according to people
The molar ratio of element and Cucurbituril is 1:(10~25) human calcitonin and Cucurbituril are measured.
4. the preparation method of human calcitonin-Cucurbituril compound formulation according to Claims 2 or 3, it is characterised in that the water
Property solvent be deionized water, physiological saline or phosphate buffer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6204256B1 (en) * | 1996-11-01 | 2001-03-20 | Polymed | Acylated cyclodextrin derivatives |
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Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100554156B1 (en) * | 2003-07-26 | 2006-02-22 | 학교법인 포항공과대학교 | Nano-particles comprising curcurbituril derivatives, pharmaceutical composition containing the same, and process for the preparation thereof |
| KR20050102295A (en) * | 2004-04-21 | 2005-10-26 | 학교법인 포항공과대학교 | Liposome and process for the preparation thereof |
| KR100750320B1 (en) * | 2006-01-04 | 2007-08-17 | 학교법인 포항공과대학교 | Gel comprising cucurbitril |
| KR100853172B1 (en) * | 2007-04-04 | 2008-08-20 | 포항공과대학교 산학협력단 | Liposome sensitive to ph or reductive condition and processes for the preparation thereof |
| KR101118587B1 (en) * | 2009-08-17 | 2012-06-12 | 포항공과대학교 산학협력단 | Responsive polymer capsule, and method for preparing thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6204256B1 (en) * | 1996-11-01 | 2001-03-20 | Polymed | Acylated cyclodextrin derivatives |
| CN102369018A (en) * | 2009-03-12 | 2012-03-07 | 北欧生物科技公司 | Treatment of diabetes and metabolic syndrome |
Non-Patent Citations (4)
| Title |
|---|
| Cucurbituril Homologues and Derivatives:New Opportunities in Supramolecular Chemistry;Jae Wook Lee，等;《Accounts of Chemical Research》;20030523(第36期);第621-630页 * |
| Supramolecular Chemistry at Interfaces:Host-Guest Interactions for Fabricating Mutifunctional Biointerfaces;Hui Yang,等;《Accounts of Chemical Research》;20140425(第47期);第2106-2115页 * |
| 葫芦脲超分子包结物的构建;张洪源;《中国博士学位论文全文数据库工程科技Ⅰ辑》;20140415(第4期);第B014-36页 * |
| 超分子组装体作为蛋白质药物载体的研究进展;曹叔琴，等;《化工新型材料》;20150831;第43卷(第8期);第10-12页 * |
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