Novel chemotherapeutic drug nano ADC based on antibody conjugation and preparation method and application thereof

A technology of chemotherapeutic drugs and conjugates, applied in the field of medicine, can solve problems affecting the effective combination of antibodies and antigens, reduce killing, and reduce ADC activity, and achieve the effects of improving bioavailability, good targeting effect, and enhancing affinity

Active Publication Date: 2015-04-08
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Although antibody-drug conjugates have certain advantages over single antibodies and chemotherapeutic drugs in terms of killing effect and reducing the toxicity of chemotherapeutic drugs, due to the different abundance of antigens expressed on the surface of different cells, a single antibody-drug conjugate The therapeutic effect of cells with relatively insufficient antigen expression is not satisfactory; and studies have shown that the number of effector molecules coupled to ADC affects the aggregation of ADC, antigen binding activity, clearance rate in blood circulation, activity and resistance. Receptivity, the number of coupled effector molecules is too small, which reduces the activity of ADC; on the contrary, too much will reduce the half-life and tolerance of ADC drugs in the blood, and at the same time affect the effective combination of antibodies and antigens; in addition, a single antibody- The accumulation of drug conjugates in tumors is low, chemically conjugated drugs are not stable enough in blood circulation, and th

Method used

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  • Novel chemotherapeutic drug nano ADC based on antibody conjugation and preparation method and application thereof
  • Novel chemotherapeutic drug nano ADC based on antibody conjugation and preparation method and application thereof
  • Novel chemotherapeutic drug nano ADC based on antibody conjugation and preparation method and application thereof

Examples

Experimental program
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Example Embodiment

[0048] Example 1 Synthesis of doxorubicin with disulfide bond (DOX-PDPH)

[0049] Weigh 27.25 mg of doxorubicin (ADR) and 11.78 mg of hydrazino-4-(2-pyridinedithio)butyric acid (PDPH), dissolve them in 3 ml of methanol; stir the reaction in the dark at room temperature for 6 days , under different stirring speeds (100rpm, 500rpm, 1000rpm, 3000rpm); the fast halving speed can prevent the precipitation of doxorubicin, which is beneficial to the synthesis of ADR-PDPH. After completion of the reaction, an orange-yellow product can be obtained. A schematic diagram of the synthesis of ADR-PDPH is shown in figure 1 .

Example Embodiment

[0050] Example 2 Preparation of thiolated Her2

[0051] According to the ratio of 1 mg of her2 and 75 μg of 2-IT (2-iminothiolane·HCI (Traut's reagent, Shanghai Qianchen Biotechnology Co., Ltd.)) (the molar ratio of Anti-Her2-Fab and 2-IT is 1:100 or according to the actual situation to increase the ratio of the two) to mix the two. Seal with parafilm to prevent 2-IT from being oxidized. The reaction was carried out at room temperature for 2h. The reaction system can be scaled up. Thiylated Her2 was dialyzed (PBS, 5mM EDTA, pH=7.4) to remove excess Traut reagent. Change the dialysate every 6-8 hours and dialyze 2 to 3 times.

Example Embodiment

[0052] Example 3 Antibody thiolation standard curve

[0053] Ellman's test measures the amount of -SH of thiolated antibodies, and DTNB is Ellman's reagent. It is used for colorimetric determination of sulfhydryl groups in biological samples. It is easily soluble in water. In the presence of sulfhydryl compounds, colorless DTNB will be converted into yellow 5-mercapto-2-nitrobenzoic acid. Since 5-mercapto-2-nitrobenzoic acid has an absorption maximum at 412 nm, the absorption spectrum of DTNB does not interfere with the determination of sulfhydryl groups.

[0054] Configuration of reagents:

[0055] (1) Tris-HCl buffer (0.25M): After accurate preparation of DDW (deuterium depleted water), adjust pH=8.3 with hydrochloric acid, and prepare 300ml.

[0056] (2) Cysteine ​​standard solution (1 mM): 0.017563 g of L-cysteine ​​(molecular weight: 175.63) was accurately weighed, dissolved in 1 ml of formic acid, and made up to 100 ml with DDW.

[0057] (3) DTNB (molecular weight: ...

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Abstract

The invention discloses nanoparticles of an antibody chemotherapeutic drug such as doxorubicin and maytansine (DOX, DM1) based on an antibody conjugation new technology and a preparation method and an application thereof. Experimental results show that compared with a single antibody and a single drug, nanoparticles formed by self-assembling antibody-chemotherapeutic drug conjugated conjugates has a targeting property significantly enhanced, and has the drug toxic and side effects on bodies significantly reduced; the formed nanoparticles use enhanced permeability and retention effect (EPR) of tumor tissues in a composite mode to enhance intratumoral accumulation, enhances the bioavailability of the drug, at the same time, reduces the use dosage of the drug, and provides a high-efficiency and low-toxicity nano treatment mode for tumor targeted therapy.

Description

【Technical field】 [0001] The invention relates to the technical field of medicine, in particular to a novel antibody-coupled chemotherapeutic drug nano-ADC and its preparation method and application. 【Background technique】 [0002] Tumor is one of the three major diseases that seriously threaten human health today. The incidence of cancer in my country has reached 2 / 1,000, and the mortality rate is 1.5 / 1,000. Among them, lung cancer has the highest mortality rate, liver cancer ranks second, and gastric cancer ranks third. The treatment effect of malignant gastric cancer is poor, the late metastasis rate is high, and the prognosis is often poor. Conventional treatments such as radiotherapy, chemotherapy and surgery, which are commonly used in clinical practice, relieve the pain to a great extent and prolong the survival time; however, chemotherapy or radiotherapy cannot change the survival period of patients, even for cancers that are sensitive to the first chemotherapy For...

Claims

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Application Information

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IPC IPC(8): A61K47/38A61K31/704A61K31/5365A61K9/14A61K9/107A61P35/00
Inventor 李威
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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