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Cyclosporine conjugate and preparation method and application thereof

A technology for cyclosporine and conjugates, which is applied in the field of cyclosporine conjugates and their preparation, can solve the problems of high price, organic solvent pollution, time-consuming and other problems, save detection time, and make up for time-consuming problems. long effect

Inactive Publication Date: 2018-11-13
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the instrument method is expensive, time-consuming, and causes organic solvent pollution, requiring large-scale equipment and specialized technical personnel

Method used

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  • Cyclosporine conjugate and preparation method and application thereof
  • Cyclosporine conjugate and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Preparation of solution A: 50 mg of cyclosporine (0.042 mmol) was added to a 25 mL round bottom flask, and dissolved in 10 mL of dichloromethane. Add 14 mg m-chloroperoxybenzoic acid (MCPBA) to it, stir overnight at room temperature, then add aqueous sodium bicarbonate solution, extract with dichloromethane, combine the organic phases, and rotate under reduced pressure to obtain the product dissolved in dilute hydrochloric acid aqueous solution and stir at room temperature After 5h, adjust the pH to 7, add succinic anhydride, hydroxysuccinimide, ethyl [3-(dimethylamino) propyl] carbodiimide (EDC) at a molar ratio of 1:2 to 8:5 ~15 was dissolved in DMF-water two-phase mixed solvent, and reacted to generate active intermediates of cyclosporine analogs and ethyl[3-(dimethylamino)propyl]carbodiimide, which was solution A.

[0041](2) Preparation of cBSA: Dissolve 18.0mg of ethylenediamine in 20ml of phosphate buffer solution with a pH of 7.40 and a concentration of 0.01...

Embodiment 2

[0047] (1) Preparation of solution A: 50 mg of cyclosporine (0.042 mmol) was added to a 25 mL round bottom flask, and dissolved in 10 mL of dichloromethane. Add 14 mg m-chloroperoxybenzoic acid (MCPBA) to it, stir overnight at room temperature, then add aqueous sodium bicarbonate solution, extract with dichloromethane, combine the organic phases, and rotate under reduced pressure to obtain the product dissolved in dilute hydrochloric acid aqueous solution and stir at room temperature After 5h, adjust the pH to 7, add succinic anhydride, hydroxysuccinimide, ethyl [3-(dimethylamino) propyl] carbodiimide (EDC) at a molar ratio of 1:2 to 8:5 ~15 was dissolved in DMF-water two-phase mixed solvent, and reacted to generate active intermediates of cyclosporine analogs and ethyl[3-(dimethylamino)propyl]carbodiimide, which was solution A.

[0048] (2) Preparation of cBSA: Dissolve 18.0mg of ethylenediamine in 20ml of phosphate buffer solution with a pH of 7.40 and a concentration of 0.0...

Embodiment 3

[0054] Preparation, purification and detection of antibodies

[0055] 1. Antibody Preparation

[0056] The cyclosporine conjugate prepared in Example 2 above was selected as the immunogen to carry out animal immunization experiments to prepare antibodies.

[0057] Take 1ml of the solution of the conjugate of cyclosporine at 1mg / ml, add an equal volume of Freund's complete adjuvant, fully emulsify, and inject into four male healthy New Zealand white rabbits with a body weight of 2kg at multiple points subcutaneously, 1ml / Only, 15 days later, the same amount of antigen was fully emulsified with Freund's incomplete adjuvant for the second immunization. After the second immunization, a booster immunization was performed every 15 days, and the amount of antigen was halved, and a total of 5 immunizations were given. Seven days after the last immunization, blood was collected from the heart, left at room temperature for 1 hour, overnight at 0-4°C, centrifuged at 13,000 rpm for 15 m...

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Abstract

The invention discloses a cyclosporine conjugate and a preparation method and application thereof. The cyclosporine conjugate is as shown in general formula (I), wherein n is the molecular number of cyclosporine combined with one bovine serum albumin molecule and is an integer ranging from 1 to 20, BSA is bovine serum albumin, and the molecular weight range is 6.6-6.9KDa. The theophylline conjugate is formed by coupling cyclosporine hapten with carrier substance bovine serum albumin or ovalbumin generating immunogenicity. The preparation method includes: connecting and combining cyclosporine with the carrier substance generating immunogenicity to form the cyclosporine conjugate capable of inducing animal systems to generate antibodies. The cyclosporine conjugate immunizes New Zealand whiterabbits to prepare antiserum with the titer reaching 50,000, and the lowest detection limit of the antiserum is 20ppb. The cyclosporine conjugate is simple in method, fast, accurate and high in specificity and lays a foundation for the enzyme linked immunosorbent assay kit of cyclosporine.

Description

technical field [0001] The present invention relates to a conjugate of cyclosporine and its preparation method and application, in particular to a conjugate of cyclosporine and its preparation method and application. The invention belongs to the field of immune monitoring of blood drug concentration of immunosuppressant drugs. Background technique [0002] The following titles involved in the present invention apply to the entire specification and claims: [0003] BSA: serum albumin (Bovine Serum Albumin), product of Sigma company [0004] PBS: Phosphate Buffered Saline (0.01M, pH=7.40) [0005] Sephadex-G75: dextran gel, product of Sigma [0006] cBSA: bovine serum albumin modified with ethylenediamine [0007] Dialysis Membrane: Solaibao Technology Co., Ltd. [0008] Cyclosporine: a product of Sigma [0009] EDC: Ethyl[3-(dimethylamino)propyl]carbodiimide, referred to as EDC, a product of Sigma [0010] Cyclosporine is a western medicine with the molecular formula C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K16/44C07K1/107
CPCC07K1/1077C07K14/765C07K14/77C07K16/44C07K19/00
Inventor 郗日沫李小刚刘玮孟萌尹永梅张丽莎许坤龙浩
Owner NANKAI UNIV
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