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A54-GFLG-DOX conjugate as well as coupling method and application thereof

A technology of conjugates and liver cancer, which is applied in the direction of drug combinations, pharmaceutical formulas, and medical preparations containing active ingredients, etc., which can solve the limitations of doxorubicin, doxorubicin myelosuppressive myocardial toxicity and neurotoxicity, and endanger the health of patients and other issues to achieve the effect of effective killing and reducing the killing effect

Inactive Publication Date: 2011-07-20
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest problem in clinical application of doxorubicin is that doxorubicin has obvious side effects such as myelosuppression, myocardial toxicity and neurotoxicity.
Acute myocardial toxicity often occurs at the initial stage of medication, which may be relieved quickly, but when the cumulative dosage of doxorubicin in the human body reaches 500mg / m 2 Obvious symptoms of chronic myocardial intoxication will appear when the time comes, seriously endangering the patient's health, so that the clinical application of doxorubicin is greatly limited

Method used

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  • A54-GFLG-DOX conjugate as well as coupling method and application thereof
  • A54-GFLG-DOX conjugate as well as coupling method and application thereof
  • A54-GFLG-DOX conjugate as well as coupling method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Chemical synthesis of A54-GFLG-DOX conjugate

[0057] The structure of polypeptide A54 (hepatoma specific targeting peptide A54, amino acid sequence: AGKGTPSLETTP) contains two carboxyl groups -COOH, and four amino acid GFLGs are connected to the terminal carboxyl group to obtain A54-GFLG. Using Fmoc to protect the -COOH in the middle of the liver cancer-specific targeting peptide A54, exposing the GFLG terminal -COOH, HoSu activates the polypeptide to neutralize the terminal carboxyl group with triethylamine (Et3N) and doxorubicin hydrochloride- After the NH2 reaction, piperidine removes Fmoc, and the product is purified and separated by preparative HPLC.

[0058] The specific steps are as follows:

[0059] 1. Using conventional peptide synthesis technology, four amino acids G, F, L, and G are sequentially connected to the terminal carboxyl group of polypeptide A54 to form polypeptide A54-GFLG;

[0060] 2. Take 5.8 mg (0.01 mmol) of doxorubicin hydrochloride and di...

Embodiment 2

[0065] Example 2: Cytotoxicity detection of A54-GFLG-DOX and DOX-A54H

[0066] In order to detect whether the synthesized A54-GFLG-DOX maintains the killing effect of doxorubicin on liver cancer cells, a cytotoxicity experiment was used to detect the changes in cell viability of the cells after the drug was applied compared to the untreated control group. The specific steps are as follows:

[0067] 1. 0.25% trypsin digests adherent cells BEL-7402, and collects the cells in a serum-containing medium;

[0068] 2. Count the cells, centrifuge, resuspend in the medium, and adjust the cell concentration to 1×10 5 Pcs / ml;

[0069] 3. Inoculate cells in a 96-well cell culture plate and add 90ul of cell suspension to each well. Leave 3 blank holes and add 100ul cell-free medium as a blank control;

[0070] 4. Dilute the drugs A54-GFLG-DOX, DOX-A54H, DOX-A54M and DOX with the culture medium to make a series of 6 low concentrations. Add 10ul of the drug to each hole and 3 replicate holes for eac...

Embodiment 3

[0076] Example 3: Targeted detection of A54-GFLG-DOX and DOX-A54H

[0077] As a classic antibiotic broad-spectrum anti-tumor drug, doxorubicin molecules inhibit DNA replication by cross-linking with tumor cell DNA, and block the action of RNA polymerase to inhibit RNA synthesis, thereby achieving the purpose of inhibiting tumor growth. And doxorubicin itself has red fluorescence. According to the action mechanism and characteristics of doxorubicin, the present invention adds two synthetic target drugs to liver cancer cell lines. After incubation, observe the red fluorescence of doxorubicin to determine the present invention. The fluorescence intensity of A54-GFLG-DOX conjugate and DOX-A54H is used to determine and compare the activity of the two drugs.

[0078] The specific steps are as follows:

[0079] 1. Seed cells in a 96-well cell culture plate to make the number of cells 1×10 4 Pcs / hole;

[0080] 2. At 37℃, saturated humidity, 5% CO 2 Cultivate in a cell incubator until the cel...

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Abstract

The invention discloses an A54-GFLG-DOX conjugate which has the structure disclosed in a formula (1), wherein A54 is liver cancer specificity targeted peptide with the amino acid sequence of NH2-AGKGTPSLETTP-COOH, and the A54 is coupled with adriamycin by virtue of a linker peptide GFLG. The invention also discloses a preparation method of the A54-GFLG-DOX conjugate and an application of the A54-GFLG-DOX conjugate in preparing medicaments capable of treating liver cancer. The A54-GFLG-DOX conjugate has the beneficial effect of administrating the medicaments in a targeted mode, enhancing the medical effect, lightening the toxic and side effect and the like.

Description

Technical field [0001] The invention relates to a coupled targeted drug for treating tumors in the technical field of biomedicine, and a preparation method and application thereof, in particular to an A54-GFLG-DOX conjugate and a preparation method and application thereof. Background technique [0002] Liver cancer occurs in the liver and can be divided into primary liver cancer and metastatic liver cancer. We usually refer to liver cancer as primary liver cancer (hepatocellular carcinoma, HCC). Primary liver cancer is one of the common malignant tumors in my country. It is the second cause of tumor mortality. The incidence rate is increasing year by year. The number of people who die from liver cancer each year accounts for 45% of the world's total liver cancer deaths, which is a serious threat To protect people’s health and lives. How to effectively diagnose and treat liver cancer has always been a focus of research and exploration by scientists all over the world, especially ...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P35/02
CPCY02P20/55
Inventor 杜冰岳苗苗钱旻任华
Owner EAST CHINA NORMAL UNIVERSITY
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