Ivermectin preparation as well as preparation method and application thereof

A technology of ivermectin and preparation, applied in the field of biopharmaceuticals, can solve the problem of high cost, achieve the effects of accelerated internalization and enrichment, stable structure, and long-term preservation and use

Active Publication Date: 2022-02-01
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires the preparation of antibodies with high binding efficiency and strong targeting, and the cost is extremely high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of ACE2 positive exosomes

[0032] Human bronchial epithelial cells 16HBE cells (Wuhan Pu Nuo Sai Life Technology Co., Ltd.) were washed twice in PBS (pH7.2), and then cultured in 90% high glucose DMEM+10% fetal bovine serum. After 3 days, the conditioned medium was collected and exosomes were isolated by centrifugation. During the separation, the buffer was PBS (pH 7.2). Process conditioned medium by centrifugation at 1000xg for 10 minutes to remove cells and large debris; centrifuge at 10,000xg for 20 minutes; discard the pellet and then ultracentrifuge at 110,000xg for 75 minutes to further process the supernatant; discard the supernatant and resuspend the pellet, Obtain exosomes.

Embodiment 2

[0033] Embodiment 2 Preparation of ivermectin exosome preparation

[0034] 10 prepared by embodiment 1 5 Dilute exosomes in PBS (pH7.2) to 200μl in three parts, add ivermectin (Sigma-Aldrich) respectively to make the final concentrations 1μg / mL, 2μg / mL, 3μg / mL, and then add Equal volume of cold 2x electroporation buffer (PBS pH 7.2, 600 mM sucrose). Electroporation was performed at 250 V and 125 μF in a 4 mm cuvette in a Gene Pulser II Electroporator (BioRad). Exosomes were recovered at 37 °C for 20 min. Unincorporated ivermectin was removed by 3 consecutive spin desalting columns (ThermoFisher Scientific), where in the last desalting step the sucrose concentration in the samples was reduced stepwise to 150 mM, 50 mM and 10 mM. The content of ivermectin in exosomes was detected by high performance liquid chromatography. The results are shown in Table 1. When ivermectin is in the range of 1 μg / mL to 3 μg / mL, the recovery rate of ivermectin exosome dosage forms is above 50%,...

Embodiment 3

[0037] Example 3 Validation of Ivermectin Exosome Targeting

[0038] In order to detect that the ACE2-positive exosomal ivermectin preparation can enrich ACE2-positive cells, we applied the ACE2-positive exosomal ivermectin preparation (ivermectin content: 1.56 μg / mL) prepared in Example 2 to at 10 7 Personal umbilical cord mesenchymal stem cells (Saiye (Suzhou) Biotechnology Co., Ltd.; ACE2 low expression) and 10 7 Individual bronchial epithelial cells in 16HBE cells (Wuhan Pu Nuosai Life Technology Co., Ltd.; ACE2 highly expressed). at 37°C, 5% CO 2 Incubate for 48 hours. After washing with PBS 3 times, use TrypLE TM Express digested for 5 minutes, cells were collected and washed by centrifugation. The harvested cells were dissolved in 0.5ml methanol, vortexed for 3 minutes, then centrifuged at 2500 rpm for 15 minutes, and the supernatant was collected. The precipitate was dissolved in 0.5ml of methanol again, and the extraction was repeated once. The supernatants wer...

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Abstract

The invention discloses an ivermectin preparation as well as a preparation method and an application thereof. The ivermectin preparation is prepared by taking an exosome as a drug carrier to load ivermectin. According to the invention, the exosome generated by ACE2 positive human cells is used as a carrier, so that a virus taking ACE2 as a binding target can be bound with the carrier, on one hand, the binding degree of the virus and the human cells can be partially closed, and the virulence of the virus is reduced, and on the other hand, after the virus is bound with the ACE2 positive exosome preparation, when infection occurs, Ivermectin can enter cells together with virus genetic materials to prevent virus replication in target cells. According to the invention, infection of viruses such as SARS-CoV-2 and the like on ACE2 positive cells can be effectively inhibited.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to an ivermectin preparation and a preparation method and application thereof. Background technique [0002] Ivermectin is an antiparasitic drug widely used in the field of human, livestock and plant protection. However, many studies in recent years have confirmed that ivermectin can inhibit the recognition and binding of the import protein (IMP) a / b1 heterodimer to the viral integrase (IN), thereby inhibiting the nuclear entry process of RNA viruses and inhibiting viral infection. . At present, relevant studies have proved that ivermectin can control the infection of various viruses that rely on IMPa / b1 to achieve nuclear proliferation during infection through the above mechanism, including HIV-1, DENV serotypes 1-4, Influenza viruses and the recently reported novel coronavirus SARS-CoV-2. [0003] Different drug formulations and delivery systems have been...

Claims

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Application Information

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IPC IPC(8): A61K47/46A61K31/7048A61P31/14C12N5/071
CPCA61K47/46A61K31/7048A61P31/14C12N5/0688C12N2509/00Y02A50/30
Inventor 孙博
Owner SOUTHEAST UNIV
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