WO2015135306A1

WO2015135306A1 - Uses of artemisinin and derivatives thereof in manufacture of medicaments for prevention and treatment of vascular diseases in ophthalmology and pharmaceutical compositions

Info

Publication number: WO2015135306A1
Application number: PCT/CN2014/085736
Authority: WO
Inventors: 高前应; 宗瑶
Original assignee: 中山大学中山眼科中心
Priority date: 2014-03-11
Filing date: 2014-09-02
Publication date: 2015-09-17
Also published as: CN104906084A

Abstract

Disclosed are uses of artemisinin and derivatives thereof in the manufacture of medicaments for inhibition of ocular vascular endothelial cell proliferation, or the prevention and treatment of related diseases of ocular vascular system exudation, edema, new blood vessels formation and proliferation, and pharmaceutical compositions comprising artemisinin and derivatives thereof. Artemisinin and derivatives thereof of the present invention can be used for the prevention and treatment of ocular vascular related diseases, such as age-related macular degeneration, diabetic retinopathy, retinal central vein occlusion and retinal branch vein occlusion etc.

Description

Application of artemisinin and its derivatives in preparing medicine for preventing and treating ophthalmic vascular diseases and pharmaceutical composition

The present invention relates to the field of ophthalmic vascular diseases, and more particularly to the use of artemisinin and its derivatives in the preparation of a medicament for the prevention and treatment of ophthalmic vascular related diseases and a pharmaceutical composition. Background technique

Artemisinin (Q), also known as artemisinin, flavonoids, and artemisinin, is a sesquiterpene lactone drug extracted from the plant Artemisia annua L., and its common derivatives are: Hydrogen artemisinin (DI), artesunate (ART), artemether (J), arteether (Arteether), artemisone (Artemisone), and the like. Due to the presence of a peroxide bridge in its molecular structure, it is mediated by free iron produced by hemoglobin decomposition, producing free radicals. In the prior art, artemisinin and its derivatives are mainly used to kill malaria parasites. It is used for the treatment of various types of malaria, especially against chloroquine and sinister malaria.

At present, pathological processes such as ocular vascular exudation, edema, neovascularization and proliferation are the main causes of many blinding diseases in ophthalmology. Among them, retinal choroidal vascular disease is the most serious, and the most important disease is age-related macular degeneration. ( age-related macular degeneration, AMD), diabetic retinopathy (DR), central retinal vein occlusion (C VO ), branch retinal vein occlusion (B VO ), premature infant Retinopathy of prematurity (OP) and sickle cell retinopathy, among which AMD and DR are the main causes of blindness in adults.

The main strategies for the treatment of retinal choroidal neovascularization are: intravitreal injection, laser and photodynamic therapy, and gene therapy. The currently clinically available drugs are: Lucentis, Avastin, Pegaptanib, and Trap Eye, KH902, and Sima-027, which are undergoing clinical trials. Beimizumab and bevacizumab were mainly injected into the vitreous cavity. Ranibizumab and bevacizumab are currently the most comprehensive and evidence-based medical evidence, in clinical In the treatment, it plays a good role in preventing vision loss, improving the patient's vision and inhibiting the function of new blood vessels. But with the increasing use of the application, its own problems have gradually emerged:

1. As a antibody fragment with a molecular weight of 48 kDa, ranibizumab has a maximum concentration of 1 day in the vitreous cavity after intravitreal injection, a half-life of about 3 days, and a biological activity of about one month. Therefore, the average annual injection was 6.9 times. Repeated injections may produce a variety of complications: retinal detachment, retinal tears, elevated intraocular pressure, cataracts, and even endophthalmitis;

2, 10-20% of patients still have ineffective treatment, and some patients have drug tolerance after repeated treatment, and the therapeutic effect is reduced;

3, the price is expensive, which limits its application to some extent.

Laser and photodynamic therapy are also effective treatments that inhibit neovascularization and promote the regression of new blood vessels, and can delay vision loss. However, as a form of traumatic treatment, laser treatment causes irreversible damage to the surrounding vision, and some patients can relapse. Summary of the invention

Based on this, one of the objects of the present invention is to provide an artemisinin and a derivative thereof for use in the preparation of a medicament for the prevention and treatment of ophthalmic vascular diseases.

The specific technical solutions to solve the above technical problems are as follows:

The use of artemisinin and its derivatives for the preparation of a medicament for inhibiting the proliferation of ocular endothelial cells or preventing and treating ocular vasculature exudation, edema, neovascularization, and proliferation.

In some of the embodiments, the diseases related to prevention of ocular vascular system exudation, edema, neovascularization, and proliferation are age-related macular degeneration (AMD), diabetic retinopathy (diabetic retinopathy). DR), central retinal vein occlusion

(central retinal vein occlusion, CRVO), branch retinal vein occlusion (BRVO), premature delivery JL» retinopathy of prematurity (ROP), Coats disease (retinal telangiectasia) Disease, macular edema (Macular edema), retinal vein inflammation (young recurrent vitreous hemorrhage, Eales disease), polypoid choroidal vasculopathy

( olypoidal choroidal vasculopathy , PCV ), neovascular glaucoma ( NVG ), corneal neovascularization ( vaginal neovascularization) (iris neovascularization), choroidal neovascularization, retinal neovascularization, orbital tumor, inflammatory pseudotumor, 腺^ gland-related eye disease (thyroid-associated ophthalmopathy), Thyroid related immune orbitopathy, central exudative chorioretinopathy, neovascularization and hemorrhage caused by high myopia, uveitis ( Uveitis ), proliferative vitreoretinopathy (P VR) and traumatic proliferative vitreoretinopathy ( PVR ), retinal hemangioma and sickle cell retinopathy.

In some of these embodiments, the neovascularization-related disease is a retinal choroidal neovascular disease.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting proliferation of ocular endothelial cells or preventing and treating ocular vasculature exudation, edema, neovascularization, and proliferation, and the pharmaceutical composition is effective The composition is artemisinin and its derivatives.

In some of the embodiments, the pharmaceutical composition is administered by: dissolving artemisinin and its derivative in a 0.9% sodium chloride solution or a pharmaceutically acceptable solubilizing solubilizing agent; Release technology makes artemisinin and its derivatives into nanoparticles, micelles, nanotubes or dendrimers.

In some of these embodiments, the solubilizing solubilizing agent is: sodium hydrogencarbonate solution, dimercapto sulfoxide, ethanol, lecithin, lactic acid, glycerin, stearic acid polyethylene, polyvinyl alcohol, soya lecithin or polan At least one of sam.

In some of these embodiments, the pharmaceutical composition is in the form of an eye drop or an injection.

In some of the embodiments, the administration route of the injection is: an extra-balloon injection, an implantation treatment, or an intraocular injection; the extra-spherical injection is a subconjunctival injection, a peribulbar injection, a post-ball injection, or a sieve plate. Injection; the intraocular injection is an injection of the anterior chamber and the vitreous cavity.

In some of these embodiments, the eye drops are administered through the conjunctiva.

The above artemisinin derivatives are dihydroartemisinin, artesunate, artemether, arteether or artemisinone. Application of artemisinin and its derivative according to the invention in preparing medicine for treating ophthalmic vascular diseases The invention has the following advantages and beneficial effects: According to a large number of experiments and studies by the inventors of the present invention, it is concluded that artemisinin and its derivatives inhibit the proliferation of ocular endothelial cells or prevent ocular vascular exudation, edema, neovascularization, It has a significant effect on the proliferation of related diseases; and its long-lasting effect not only reduces the number of intraocular injections, but also reduces the occurrence of complications. DRAWINGS

1 is a graph showing the results of HUVEC apoptosis induced by artemisinin, dihydroartemisinin, artesunate and artemisinic acid in Example 2;

2 is a graph showing the results of RF/6A apoptosis induced by artemisinin, dihydroartemisinin, artesunate, and artemether in Example 2;

Figure 3 is a comparison diagram of the results of the fundus photography of each group in the in vivo test in Example 2;

Fig. 4 is a graph showing the results of fundus fluorescein angiography in each group in the in vivo test in Example 2. detailed description

The invention will be further described below in conjunction with specific embodiments. Example 1

The application of artesunate in the preparation of a medicament for treating retinal choroidal neovascularization includes the following contents:

(1) Solution: the artesunate and 0.05% sodium bicarbonate solution are formulated into a gradient solution of 0.1-6 mg/ml;

(2) dose: 0.1-0.5ml;

(3) Operation steps:

A. Surgical area disinfection: disinfect the eyelids and the skin around the eyes with 10% povidone iodine. Dissolve 3 drops of 0.5% povidone in the conjunctiva for a few minutes, rinse the conjunctival sputum with tobramycin solution, and use antibiotic drops. Eye drops prevent corneal dryness and abrasion.

B. Local anesthesia: Local anesthesia is performed by dropping 3 to 4 drops of Aerkain eye drops into the conjunctiva.

C, intravitreal injection: the injection site should be located 3.5-4 mm behind the limbus, the syringe is tilted through the surface of the sclera with a 30 gauge needle, and then vertically penetrated, the penetration depth is between 5-7 mm, so that the needle tip is in the middle The vitreous between the cells gently injects the drug into the vitreous cavity; when repeating the injection, move one hour at a time. Example 2

(1) In vitro test

First, the purpose of the experiment

The effects of artemisinin (Q) and its derivatives (artesunate (ART), dihydroartemisinin (DI) and artemether (J)) on HUVEC and RF/6A were analyzed by in vitro experiments. Analysis of artemisinin and its derivatives inhibited HUVEC and RF/6A proliferation, induced apoptosis of HUVEC and RF/6A, and further screened the most effective artemisinin derivative monomers to calculate IC50 to find suitable concentration.

Second, the practical approach

(1) Human umbilical vein endothelial cells (HUVEC) were extracted for primary culture, and cells of passage 2-4 were used.

(2) Artemisinin and its derivative monomers induce HUVEC apoptosis experiment

Subject: HUVEC, rhesus monkey retinal choroidal endothelial cells (RF/6A);

The experimental method is as follows, Apoptosis Test (MTT), repeated 3 times.

Specific experimental steps:

1. Collect log phase cells, adjust the cell suspension concentration to 50000 / ml, and add lOOul cell suspension per well (5000 cells per well). (The edge holes are filled with sterile PBS;).

2. Incubate at 5% C0 ₂ at 37 °C until the cells grow to 80% confluence (96-well flat bottom plate), add a concentration gradient of the drug, 100 ul per well, and set 5 replicate wells for each concentration.

3. Incubate for 24 hours at 5% C0 ₂ at 37 ° C and observe under an inverted microscope.

4. Suck the inner treatment solution, add 200μ1ΜΤΤ solution (0.5mg/ml) to each well, and continue to culture for 4h.

5. Stop the culture and carefully remove the MTT solution from the well.

6. Add 150 μl of dimercaptosulfoxide (DMSO) to each well and shake at low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at OD490nm by an enzyme-linked immunosorbent assay.

Third, the experimental results

Artesunate (ART), Dihydroartemisinin (DI), Artemisinin, and Artemether (J) induce apoptosis of HUVEC and RF/6A. See Figures 1 and 2.

As can be seen from Figure 1: Artesunate (ART) IC50: 28.7 ± 2.53 _g / ml, dihydroartemisinin (DI) IC50: 2.1 ± 0.37 _g / ml, artesunate and dihydroartemisinin in this When the concentration used in the experiment can significantly inhibit the proliferation of HUVEC and induce the apoptosis of HUVEC, the inhibition of HUVEC proliferation by dihydroartemisinin is better than that of the experiment: Artemisinin can induce HUVEC withering at a concentration above 50 (^g/ml). The effect of death, and the artemether needs to be higher than the lmg/ml oil solution to induce apoptosis.

It can be seen from Figure 2 that artemisinin and its derivatives show the same trend as HUVEC in inducing RF/6A apoptosis.

In addition, since artesunate is water-soluble and the metabolite in the body is dihydroartemisinin, subsequent animal experiments were carried out with artesunate.

(two) in vivo test

First, the purpose of the experiment

The effect of artesunate on retinal choroidal neovascularization was analyzed by in vitro experiments.

Second, the practical approach

(1) To create a model of retinal neovascularization in rabbit eyes: Intravitreal injection of 2 _g VEGF solution 40 μl into the right eye of rabbits.

(2) Treatment plan: After successful modeling, follow the following experiments to treat:

Model group: intravitreal injection of normal saline ΙΟΟμΙ;

Artesunate treatment group: intravitreal injection of artesunate solution 0.1-0.5ml (obtained in Example 1);

Avastin treatment group: intravitreal injection of 125 _g / ml Avastin solution 0.15 ml (refer to clinical dosage);

Normal control group: the left eye of all rabbits;

(3) Postoperative evaluation indicators:

Fundus photography, time point: 1 week, 2 weeks, 4 weeks;

Fundus fluorescein angiography, time point: 2 weeks, 4 weeks;

Third, the experimental results The results are shown in Figures 3 and 4.

It can be seen from Fig. 3 and Fig. 4: Retinal choroidal neovascularization model: After injection of 2 g VEGF solution 40 μl, the retinal vasodilation of rabbit eyes is distorted, new blood vessels appear around the optic disc, and fluorescein angiography shows obvious leakage of retinal choroid and neovascularization around the optic disc. .

Analysis of treatment effects: Retinal vasodilation and retinal choroidal leakage continued to worsen in the untreated model group, and traction retinal detachment occurred after 4 weeks. Intravitreal injection of artesunate injection can significantly inhibit choroidal leakage and neovascularization in rabbit eyes, retinal vasocondylar dilation is significantly reduced, vascular exudation and neovascularization around the optic disc are also significantly reduced, after 4 weeks of treatment The retina still calmed down, no retinal traction detachment was seen; Avastin had a significant effect of inhibiting retinal choroidal vascular exudation, edema and neovascularization within 2 weeks after intravitreal injection, but the effect began 2 weeks after injection. Attenuated, retinal choroid recurrence of vascular exudation, edema and neovascularization. This result indicates that artesunate can significantly inhibit retinal choroidal vascular exudation, edema and neovascularization, and its effect of inhibiting vascular exudation, edema and neovascularization is more durable than that of Avastin; Therefore, the number of intraocular injections can be significantly reduced, and complications can be reduced.

However, it is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

claims

1. The application of artemisinin and its derivatives in the preparation of drugs that inhibit the proliferation of ocular vascular endothelial cells or prevent and treat ocular vascular system exudation, edema, neovascularization, and proliferation-related diseases.

2. Application according to claim 1, characterized in that the diseases related to preventing and treating ocular vasculature exudation, edema, neovascularization, and proliferation are age-related macular degeneration, diabetic retinal disease, and central retinal vein occlusion. , branch retinal vein occlusion, retinopathy of prematurity, retinal telangiectasia, macular edema, periretinal vein inflammation, polypoidal choroidal vasculopathy, neovascular glaucoma, corneal neovascularization, iris neovascularization, choroidal neovascularization, retinal neovascularization Blood vessels, eye and orbital tumors, inflammatory tumors, thyroid-related eye disease, thyroid-related immune orbitopathy, central exudative chorioretinopathy, neovascularization and bleeding caused by high myopia, uveitis, Proliferative vitreoretinopathy, traumatic proliferative vitreoretinopathy, retinal hemangioma, or sickle cell retinal disease.

3. The application according to claim 1, characterized in that the disease related to neovascularization is retinal choroidal neovascular disease.

4. The application according to any one of claims 1 to 3, characterized in that the artemisinin derivative is dihydroartemisinin, artesunate, artemether, arteether or artemisinone.

5. A pharmaceutical composition for inhibiting the proliferation of ocular vascular endothelial cells or preventing and treating diseases related to ocular vascular system exudation, edema, neovascularization, and proliferation, characterized in that the active ingredient of the pharmaceutical composition is Artemisinin and its derivatives.

6. The pharmaceutical composition according to claim 5, characterized in that the drug-carrying method of the pharmaceutical composition is: using sustained-release technology to prepare artemisinin and its derivatives into nanoparticles, nanomicelles, Nanotubes or dendrimers; or artemisinin and its derivatives dissolved in 0.9% sodium chloride solution or a pharmaceutically acceptable solubilizer.

7. The pharmaceutical composition according to claim 6, wherein the solubilizing and solubilizing agent is: sodium bicarbonate solution, dimethyl sulfoxide, ethanol, lecithin, lactic acid, glycerin, stearic acid polyethylene , polyvinyl alcohol, soy lecithin or at least one poloxamer.

8. The pharmaceutical composition according to claim 5, characterized in that, the dosage of the pharmaceutical composition Available as eye drops or injections.

9. The pharmaceutical composition according to claim 8, characterized in that the administration route of the injection is: extrabulbar injection, implantation treatment or intraocular injection; the extrabulbar injection is subconjunctival injection, bulbar injection Peripheral injection, retrobulbar injection or pericribriform injection; the intraocular injection is anterior chamber and vitreous cavity injection.

10. The pharmaceutical composition according to any one of claims 5 to 9, characterized in that the artemisinin derivative is dihydroartemisinin, artesunate, artemether, arteether or artemisinin ketone.