WO2022000492A1

WO2022000492A1 - Use of artemisinin or derivatives artesunate and dihydroartemisinin

Info

Publication number: WO2022000492A1
Application number: PCT/CN2020/100235
Authority: WO
Inventors: 李延兵; 郭衍
Original assignee: 中山大学附属第一医院
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2022-01-06

Abstract

The present invention relates to the technical field of biomedicine, relates to a new use of artemisinin or a derivative (artesunate) and dihydroartemisinin artemisinin chloroquine or derivatives (artesunate and dihydroartemisinin) in drugs, and more specifically relates to a use of artemisinin or a derivative (artesunate) and dihydroartemisinin artemisinin chloroquine or derivatives (artesunate and dihydroartemisinin) in drugs for treating Graves eye diseases. The present invention proves that: the artemisinin or derivatives (artesunate and dihydroartemisinin) have obvious inhibitory effects on excessive proliferation, adipogenic differentiation, hyaluronic acid secretion, and fibrosis of orbital fibroblasts; the effects are superposed to display physiopathologic effective regulation and control of the artemisinin chloroquine or derivatives (artesunate and dihydroartemisinin) on pathological dominant factors of Graves eye diseases, i.e., the orbital fibroblasts. The artemisinin chloroquine or derivatives (artesunate and dihydroartemisinin) are shown to have potential treatment values on Graves eye diseases from the physiopathologic level, and relate to a new use of drugs, so that social and scientific research resources can be saved.

Description

A kind of artemisinin or derivatives artesunate, the purposes of dihydroartemisinin

technical field

The invention belongs to the technical field of biomedicine, and relates to new uses of artemisinin or derivatives artesunate and dihydroartemisinin, more particularly, relates to a kind of artemisinin or derivatives artesunate, dihydroartemisinin Use of artemisinin in a medicament for the treatment of Graves' eye disease.

Background technique

Graves ophthalmopathy (GO) is the most common extrathyroidal manifestation of Graves disease, accounting for about 50%, manifesting as eyelid contracture, exophthalmos, pain, redness, and even optic nerve compression, causing blindness and seriously affecting the health and quality of life of patients . At present, the effective rate of clinical application of glucocorticoid is 63% (oral) to 88% (intravenous pulse), but it is often only for acute or moderately severe patients, and cannot reverse the infiltrative proptosis of GO, with side effects such as edema, obesity, bone It also limits its application due to sclerosis and other diseases, while surgery and radiotherapy are only used as supplementary treatments. Therefore, it is urgent to explore new treatment strategies for the cause. Orbital fibroblasts (OFs) excessively proliferate, differentiate into adipocytes, secrete hydrophilic mucopolysaccharides, increase the orbital contents, increase the intraorbital pressure, and secrete inflammatory factors to recruit inflammatory cells, causing exophthalmos and exophthalmos. A central factor in inflammatory infiltration, but current drugs targeting orbital fibroblasts to slow proptosis are limited.

Studies have found that autophagy plays a crucial regulatory role in the proliferation and early differentiation of orbital fibroblasts, so inhibition of autophagy may be a new strategy for orbital fibroblasts to treat exophthalmos in the inactive stage of Graves ophthalmopathy. Goodall et al. screened a variety of antimalarial drugs such as artemisinin (ARS) using tumor cell lines expressing fluorescent LC3 and found that antimalarial drugs all inhibited autophagy to varying degrees, indicating that inhibiting autophagy may be antimalarial One of the common mechanisms of medicine. Artemisinin and its derivatives are an old drug widely used in clinical practice and have good safety and tolerance. We used orbital fibroblasts isolated from the surgically resected periorbital adipose tissue of patients with Graves ophthalmopathy as the research object, and confirmed that artemisinin (ARS) and its derivatives artesunate (ART) and dihydrocyanine Artemisinin (dihydroartemisinin, DHA) significantly inhibits various pathological mechanisms of orbital fibroblasts including proliferation, differentiation, hyaluronic acid secretion and fibrosis.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the above-mentioned defects and technical deficiencies in the prior art, and to provide a new use of artemisinin or derivatives artesunate and dihydroartemisinin in medicines for treating Graves ophthalmopathy.

The technical scheme of the present invention is as follows:

A kind of purposes of artemisinin or derivatives artesunate, dihydroartemisinin, the purposes are artemisinin or derivatives artesunate, dihydroartemisinin in the preparation treatment and/or alleviation of Graves eye disease application in medicines.

A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation The application of proliferative drugs.

A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation Application of Adipogenic Differentiation Drugs.

A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation Application of hyaluronic acid in the synthesis and secretion of drugs.

A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation Fibrotic drug application.

In addition, in the above scheme, artemisinin or its derivatives artesunate and dihydroartemisinin are prepared into pharmaceutical preparations, and the preparations are in the form of tablets or injections.

In the described pharmaceutical preparation, the content of artemisinin in the preparation is 62.5 mg/tablet of artemisinin, the content of dihydroartemisinin in the preparation is 20 mg/tablet of dihydroartemisinin, and the content of artesunate in the preparation is 20 mg/tablet of dihydroartemisinin. The content of the medicine is artesunate 50mg/tablet and injection 60mg/piece.

The invention takes the fibroblasts isolated and extracted from the periorbital adipose tissue surgically removed from patients with Graves' ophthalmopathy as the research object. Over-proliferation, adipogenic differentiation, hyaluronic acid secretion and fibrosis of cells were significantly inhibited. These effects superimposed on artemisinin or its derivatives, artesunate and dihydroartemisinin, are the leading factors in the pathology of Graves ophthalmopathy—— Efficient regulation of fibroblast pathophysiology.

The present invention has the following beneficial effects:

The present invention is different from the existing therapy for hyperthyroid exophthalmos, in that artemisinin or its derivatives artesunate and dihydroartemisinin play a role in the pathophysiological mechanism of orbital fibroblasts, and have a certain immunoregulatory effect. It is not immunosuppressive, and has been used clinically for many years, with high safety and low price.

The present invention regulates all pathogenic mechanisms of Graves ophthalmopathy exophthalmos, and the raw materials are readily available, and has been widely used in other clinical diseases, and it is of great significance to save social resources and the like as an old drug for new use.

Description of drawings

Figure 1 shows that artemisinin and its derivatives inhibit the proliferation of orbital fibroblasts in patients with Graves' ophthalmopathy.

Figures 2A-2B show that artemisinin and its derivatives inhibit the adipogenic differentiation of orbital fibroblasts from patients with Graves' ophthalmopathy.

Figures 3A-3C show that artemisinin and its derivatives inhibit the synthesis and secretion of hyaluronic acid in orbital fibroblasts of patients with Graves' ophthalmopathy.

Figures 4A-4B show that artemisinin and its derivatives inhibit the fibrosis of orbital fibroblasts in patients with Graves' ophthalmopathy.

detailed description

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

Materials and kits used:

1) Proliferation medium: DMEM-F12 (Gibco Laboratories, USA) containing 10% (vol/vol) fetal bovine serum.

2) Differentiation medium and Oil Red O staining solution: commercial induced adipogenic medium (Celeira, Guangzhou, China).

3) Proliferation-related experimental kit: cell cycle detection kit (Keygen, Guangzhou, China).

4) RNA reverse transcription kit (Promega, USA);

5) Hyaluronic acid ELISA kit (Echelon Bioscience Inc., USA).

6) Artemisinin raw material (sigma, USA): take 56.5 mg of powder and add 1.0 mL of dimethyl sulfoxide (DMSO) in a sterile environment to prepare a 200.0 mM stock solution (1000×) in a ratio of 1:4 Diluted to 50.0mM stock solution (1000×), the latter was diluted to 10.0mM stock solution (1000×) at a ratio of 1:5, and stored at -20°C in separate packages, and in the corresponding medium at a ratio of 1:1000 Dilute to 200.0 μM, 50.0 μM and 10.0 μM to treat cells.

7) Artesunate raw material (APExBIO, USA): Take 19.0 mg of powder and 1.0 mL of DMSO in a sterile environment to prepare a stock solution with a concentration of 50.0 mM, and dilute with DMSO at a ratio of 1:5, 1:25, and 1:100, respectively. Three 1000× stock solutions: 10.0 mM, 2.0 mM and 0.5 mM, which were separately packaged and stored at -20 °C, and were diluted in the corresponding medium at a ratio of 1:1000 to 10.0 μM, 2.0 μM and 0.5 μM to treat cells .

8) Dihydroartemisinin raw material (APExBIO, USA): take 12.4 mg of powder and 1 mL of DMSO in a sterile environment to prepare a 50.0 mM stock solution, and dilute it with DMSO at the ratios of 1:2.5, 1:12.5, and 1:50 respectively. Three 1000× stock solutions: 20.0 mM, 5.0 mM and 1.0 mM, which were aliquoted and stored at -20°C, and were diluted 1:1000 in the corresponding medium to 20.0 μM, 5.0 μM and 1.0 μM to treat cells.

Example 1: Artemisinin and its derivatives inhibit the proliferation of orbital fibroblasts in patients with Graves' ophthalmopathy

Method steps:

A) Orbital fibroblasts (n=5) from patients with Graves' ophthalmopathy were extracted and passaged to the third passage in proliferation medium for experiments;

B) After synchronization treatment with basal medium, divided into control group (DMSO), different concentrations of artemisinin (ARS) (50.0 and 200.0 μM), artesunate (ART) (2.0 and 10.0 μM) and dual Hydroartemisinin (DHA) (5.0 and 20.0 μM) treatment group, treated for 20 hours, digested cells in each group, and fixed overnight in 75% ethanol;

C) After centrifugation at 4°C to remove the supernatant, the cells were resuspended in PBS and stained with PI, and the ratio of each cell cycle was detected by flow cytometry.

Results: As shown in Figure 1, orbital fibroblasts from patients with Graves' eye disease were treated with different concentrations of artemisinin and its derivatives in the proliferation medium, and the cell cycle was detected by flow cytometry, and it was found that 200.0 μM artemisinin Compared with the control group, the proportion of cells in G0/1 phase and the proportion of cells in S phase were significantly different in the group treated with artesunate, 10.0 μM artesunate and 20.0 μM dihydroartemisinin, indicating that artemisinin and its derivatives inhibited the Orbital fibroblast proliferation in patients with Graves' eye disease.

Example 2: Artemisinin and its derivatives inhibit the adipogenic differentiation of orbital fibroblasts from patients with Graves' ophthalmopathy

Method steps:

A) Orbital fibroblasts (n=5) from patients with Graves' ophthalmopathy were passaged to the third passage in proliferation medium;

B) After the cell density reached 100% contact inhibition for 48 hours, the induction medium was changed, and divided into control group (DMSO), different concentrations of artemisinin (ARS) (10.0, 50.0 and 200.0 μM), artesunate (ART) ) (0.5, 2.0 and 10.0 μM) and dihydroartemisinin (DHA) (1.0, 5.0 and 20.0 μM) treatment groups;

C) Oil red O staining quantitative analysis, real-time fluorescence quantitative PCR experiment and Western blotting experiment were performed after induction of differentiation to detect the effect of drugs on the adipogenic differentiation of orbital fibroblasts.

Results: As shown in Figure 2A-2B, the orbital fibroblasts of patients with Graves' ophthalmopathy were induced to differentiate and cultured, and were treated with different concentrations of artemisinin and its derivatives. Oil red O staining and quantitative experiments, real-time quantitative PCR Experiments and Western blotting experiments showed that artemisinins inhibited the adipogenic differentiation of orbital fibroblasts.

Example 3: Artemisinin and its derivatives inhibit the synthesis and secretion of hyaluronic acid in orbital fibroblasts of patients with Graves' ophthalmopathy

Method steps:

A) Orbital fibroblasts from patients with Graves' ophthalmopathy were passaged to the third passage in proliferation medium for experiments;

B) Divided into IL-1β treated group and non-treated group, further divided into control group (DMSO), different concentrations of artemisinin (ARS) (10.0, 50.0 and 200.0 μM), artesunate (ART) (0.5, 2.0 and 10.0 μM) and dihydroartemisinin (DHA) (1.0, 5.0 and 20.0 μM) treatment groups;

C) After the cells were treated, the hyaluronic acid concentration and the expression of hyaluronic acid synthase 2 and hyaluronidase 3 were detected by hyaluronic acid enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) kit respectively.

Results: As shown in Figure 3A-3C, artemisinin and its derivatives significantly inhibited the secretion of hyaluronic acid in a concentration-dependent manner, and significantly inhibited the expression of hyaluronic acid synthase 2, but did not affect the expression of hyaluronidase 3. Express.

Example 4: Artemisinin and its derivatives inhibit the fibrosis of orbital fibroblasts in patients with Graves' ophthalmopathy

Method steps:

B) Under transforming growth factor-β1 treatment, it was further divided into control group (DMSO), different concentrations of artemisinin (ARS) (10.0, 50.0 and 200.0 μM), artesunate (ART) (0.5, 2.0 and 10.0 μM) μM) and dihydroartemisinin (DHA) (1.0, 5.0 and 20.0 μM) treatment groups;

C) After the cells were treated, the expression of fibrosis markers was detected by real-time fluorescent quantitative PCR experiment and western blotting experiment.

Results: As shown in Figures 4A-4B, artemisinin and its derivatives significantly inhibited the expression of fibrosis markers in a concentration-dependent manner.

The present invention discovers for the first time that artemisinin and its derivatives can simultaneously act on various pathological mechanisms of orbital fibroblasts of patients with hyperthyroidism, including proliferation, differentiation, hyaluronic acid formation and fibrosis, which determines that artemisinin and its derivatives Potential therapeutic effects in future clinical applications.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims

A kind of purposes of artemisinin or derivatives artesunate, dihydroartemisinin, the purposes are artemisinin or derivatives artesunate, dihydroartemisinin in the preparation treatment and/or alleviation of Graves eye disease application in medicines.
A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation The application of proliferative drugs.
A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation Application of Adipogenic Differentiation Drugs.
A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation Application of hyaluronic acid in the synthesis and secretion of drugs.
A use of artemisinin or derivatives artesunate, dihydroartemisinin, characterized in that: the artemisinin or derivatives artesunate, dihydroartemisinin inhibit orbital fibroblasts in preparation Fibrotic drug application.
The use of artemisinin or derivatives artesunate and dihydroartemisinin according to claims 1-4, characterized in that: artemisinin or derivatives artesunate and dihydroartemisinin are prepared into Pharmaceutical preparations, which are in the form of tablets or injections.
The use of artemisinin or its derivatives artesunate and dihydroartemisinin according to claim 5, characterized in that, in the pharmaceutical preparation, the content of artemisinin in the preparation is artemisinin 62.5 mg/tablet.
The use of artemisinin or derivatives artesunate and dihydroartemisinin according to claim 5, wherein in the pharmaceutical preparation, the content of dihydroartemisinin in the preparation is a tablet 20mg/tablet.
The use of artemisinin or its derivatives artesunate and dihydroartemisinin according to claim 5, characterized in that, in the pharmaceutical preparation, the content of artesunate in the preparation is 50 mg of tablet / tablet, injection 60mg / support.