Use of Beaubellin in the manufacture of drugs to inhibit angiogenesis

Abstract

This invention provides the use of beuverisin in the manufacture of a pharmaceutical for inhibiting angiogenesis, wherein the therapeutically effective concentration of beuverisin is in the range of 0.1 μM to 5 μM. This can be used in the manufacture of a pharmaceutical for the treatment or prevention of retinal disorders, or in combination with an anticancer drug in the manufacture of a pharmaceutical composition or pharmaceutical combination for treating cancer.

Description

Technical Field

[0001] The present invention provides the use of bevacizumab in the manufacture of a medicament for inhibiting angiogenesis, which can be used for developing a medicament for treating or preventing retinal disorders, or a pharmaceutical composition or combination agent for treating cancer.

[0002] Cancer is a disease caused by abnormal hypertrophy and proliferation of cells, which migrate and infiltrate other tissues through the circulatory or lymphatic system of the subject, resulting in organ dysfunction or failure. A strong correlation has been demonstrated between cancer cells and angiogenesis. Angiogenesis is the process by which existing blood vessels grow new blood vessels. Tumor cells require large amounts of nutrients and oxygen for growth and diffusion, which are supplied through blood vessels. Tumors usually promote angiogenesis in surrounding tissues to form more extensive vascular networks to meet the needs of growth and metabolism. Therefore, inhibition of angiogenesis is effective in suppressing the development of cancer to a certain extent.

[0003] Angiogenesis is also involved in the development of related eye diseases. When blood circulation in the eye is poor, hypoxia in the retinal tissue and secretion of vascular endothelial growth factor (VEGF) occur. Normally, VEGF induces compensatory angiogenesis to counteract the hypoxic environment, but in many pathological conditions, the new blood vessels are thin and densely packed. These do not contribute to an increase in blood flow supply, but rather increase the permeability and leakage of the blood vessel wall. The accumulated fluid further inhibits blood flow, forming a vicious cycle.

[0004] Bevacizumab compounds and their pharmaceutical compositions have been disclosed to be useful for improving eye diseases caused by angiogenesis, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR).

[0005] However, it is still desirable to develop a medicament for inhibiting angiogenesis that can be used for preventing or treating retinal disorders or treating cancer.

Summary of the Invention

[0006] Therefore, the present invention relates to the use of a compound in the manufacture of a pharmaceutical for inhibiting angiogenesis, the compound being known as GYT-088, having a therapeutically effective concentration range of 0.1 μM to 5 μM. [ka] This provides a viewercin having the structure of the provided material.

[0007] In one embodiment of the present invention, a pharmaceutical agent for inhibiting angiogenesis can be used in the development of pharmaceutical agents for treating or preventing retinal disorders.

[0008] In one example of the present invention, the retinal disorder is selected from the group consisting of age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy.

[0009] In one embodiment of the present invention, a pharmaceutical agent for inhibiting angiogenesis can be used in the development of pharmaceutical agents for treating or preventing retinal disorders.

[0010] In one example of the present invention, the retinal disorder is selected from the group consisting of age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy.

[0011] According to an example of the present invention, the therapeutically effective concentration of viewbericin in humans is preferably 0.5 μM to 5 μM.

[0012] According to an example of the present invention, the therapeutically effective dose range for treating or preventing retinal disorders is 2.0 to 5.5 μg per eye.

[0013] In another embodiment, the present invention provides a pharmaceutical composition for inhibiting angiogenesis, comprising beuvericin having a therapeutically effective concentration range of 0.1 μM to 5 μM and a pharmaceutically acceptable carrier.

[0014] According to an example of the present invention, the therapeutically effective concentration of viewbericin is preferably 0.5 μM to 5 μM.

[0015] In one example of the present invention, the pharmaceutical composition can be combined with an anticancer drug in the manufacture of a pharmaceutical composition or combination of pharmaceuticals for treating cancer.

[0016] It should be understood that the general description above and the detailed description below are for illustrative and explanatory purposes only and do not limit the invention. [Brief explanation of the drawing]

[0017] The above summary and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. To illustrate the invention, the drawings show a currently preferred embodiment.

[0018] [Figure 1] Figure 1 provides a schematic diagram showing the timeline and flow of the experimental process.

[0019] [Figure 2] Figure 2 provides images and analysis data showing the results of fundus photography (FP) and fluorescein angiography (FFA). Figure 2(A) shows the results on day 0 after laser injury. Figure 2(B) shows the results on day 7 after laser injury. Figure 2(C) shows the results on day 14 after laser injury. Figure 2(D) shows the quantified values ​​of the detection results from fluorescein angiography. *p<0.05 means there is a significant difference on day 7 between the high-dose GYT088 treatment group (5.12 ng / eye) and the other groups;**p<0.01 means there is a significant difference on day 7 between the Eylea treatment group and the other groups;##p<0.01 means there is a significant difference on day 14 between the intermediate-dose and high-dose GYT088 treatment groups (2.56 ng / eye, 5.12 ng / eye) and the other groups;###p<0.001 means there is a significant difference on day 14 between the Eylea treatment group and the other groups.

[0020] [Figure 3] Figure 3 provides images and analysis data showing the observation of laser damage in the retinal layer by spectral domain optical coherence tomography (SD-OCT). Figure 3(A) shows the results on day 0 after laser damage. Figure 3(B) shows the results on day 7 after laser damage. Figure 3(C) shows the results on day 14 after laser damage. Figure 3(D) provides the quantification values of the images showing the observation by SD-OCT for evaluating the structural inhibition in the retinal layer. *p<0.05 means that a significant difference was observed between the Allergan treatment group and other groups on day 7 of administration; p<0.001 means that there is a significant difference between the Allergan, low-dose, medium-dose, and high-dose GYT088 treatment groups (1.28 ng / eye, 2.56 ng / eye, 5.12 ng / eye) and other groups on day 14.

[0021] In the following description, details of one or more embodiments of the present invention will be described. Other features or advantages of the present invention will become apparent from the detailed description of some specific embodiments and the appended claims.

[0022] Embodiments of the present invention

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] The present invention is further illustrated by the following embodiments provided by way of example and not intended to limit the present invention.

[0025] As used herein, the article "a" refers to one or more (i.e., at least one) grammatical objects. For example, the term "a component" refers to one or more components.

[0026] As used herein, the term "angiogenesis" refers to the process of formation of new blood vessels that occurs when vascular endothelial growth factor (VEGF) promotes the endothelial growth or migration of vascular cells for tissue growth, healing or repair. This physiological process is essential for the normal development and maintenance of tissue physiology, but when the balance of angiogenesis is disrupted, it can cause diseases, especially in diseases such as cancer and chronic inflammation.

[0027] As used herein, the term "cancer" means a disease that develops when abnormal cells grow and divide uncontrollably. These abnormal cells are called tumors. Cancer is usually a malignant tumor that divides through the body's circulation and invades other normal cells and tissues, causing dysfunction and organ failure.

[0028] As used herein, the term "retinal disease" or "retinopathy" means various diseases that affect the retinal tissue of the eye. The retina is an internal tissue of the eye that receives light and converts it into nerve signals, which are sent to the brain for visual recognition. Damage or disease to the retina leads to visual dysfunction or vision loss. Common retinal diseases include age-related macular degeneration (AMD), diabetic retinopathy, glaucomatous optic neuropathy, retinal detachment and retinal vascular diseases.

[0029] As used herein, the term "therapeutically effective concentration" means a concentration that results in an effect in the treatment, cure, prevention or improvement of a disease, disorder or side effect, or a reduction in the rate of progression of a disease or disorder, compared to a corresponding patient not receiving such concentration. This term also includes an amount effective to enhance normal physiological functions within that range.

[0030] As used herein, the term "buergericin" means a compound produced by fungi of the genus "Buergeria" as a secondary metabolite. It belongs to a group of compounds known as cyclic peptides and has the following special structure:

Chemical formula

[0031] The structural features of beuverisin include an alternating arrangement of three D-hydroxyvaleric acid molecules and three N-methylphenylalanine molecules. Beuverisin possesses a wide range of biological activities, including antimicrobial, insecticidal, and anticancer activity against various tumor cell lines.

[0032] In this invention, it has been discovered that a predetermined low dose of beaubellicin is effective in inhibiting angiogenesis, and this can be used to develop pharmaceuticals for preventing or treating retinal disorders, or in combination with anticancer drugs in the manufacture of pharmaceutical compositions or combinations of pharmaceuticals for treating cancer.

[0033] According to the present invention, the therapeutically effective dose (amount) for humans is calculated to be 2.56 μg / eye to 5.12 μg / eye based on experiments using mice. Therefore, the effective concentration range of beuvericin for inhibiting angiogenesis can be evaluated as 0.1 μM to 5 μM, preferably 0.5 μM to 5 μM.

[0034] For therapeutic use, compounds at therapeutically effective concentrations are formulated into pharmaceutical compositions for administration. Accordingly, the present invention further provides a pharmaceutical composition comprising beuvericin having a therapeutically effective concentration range of 0.1 μM to 5 μM and a pharmaceutically acceptable carrier.

[0035] As used herein, the term "pharmaceutically acceptable carrier" means a carrier, diluent, or excipient that is compatible with other components of the formulation and is not harmful to the subject to which it is administered together with the pharmaceutical composition. Depending on the requirements of the pharmaceutical formulation, any carrier, diluent, or excipient that is generally known or used in the art may be used in the present invention.

[0036] According to the present invention, pharmaceutical compositions can be adapted for administration by any suitable route, including but not limited to oral, rectal, nasal, topical, vaginal, or non-enteral routes. In a specific example of the present invention, the pharmaceutical composition is formulated for oral administration or injection. Such formulations can be prepared by any method known in the field of pharmacy.

[0037] The technical features of the present invention are further illustrated by the following embodiments. These embodiments are provided solely for illustrative purposes and are not intended to limit the scope of the invention. [Examples]

[0038] Example: Experiment with Beaubellin to improve retinopathy

[0039] 1. Experimental materials

[0040] The beuvericin (BEA) (hereinafter referred to as GYT088) according to the present invention was supplied by Shiyuantang Biotechnology Co. 5 mg of BEA (molecular weight 783.96) was dissolved in DMSO at a stock concentration of 50 mM, and then diluted to 100 μM with physiological saline. Before injection, the sample was diluted with physiological saline to 1.6 μM, 3.2 μM, and 6.4 μM, and based on a mouse vitreous volume of 4 μl (injection volume was 1 μl), the low-dose, medium-dose, and high-dose groups corresponded to 1.28 ng / eye, 2.56 ng / eye, and 5.12 ng / eye, respectively. Assuming a human vitreous volume of 4-4.5 mL, the levels were converted to 1.28 μg / eye, 2.56 μg / eye, and 5.12 μg / eye, respectively, for the low-dose, medium-dose, and high-dose levels.

[0041] 2. Experimental Design

[0042] This study used male C57BL / 6 mice from BioLASCO Taiwan CO., Ltd., aged 7-8 weeks with an average weight of 17-20g. The mice were housed in the animal laboratory of Taipei Medical University in China and Taiwan for at least 5 days prior to the start of the experiment. The incubation temperature was set to 23±1℃, the humidity to 39-43%, and an automated light control system maintained 12 hours of light and 12 hours of dark. The mice were fed rodent feed 5001 (Purina, MO, USA), and drinking water was supplied indefinitely.

[0043] After the adaptation period, the retina of each mouse was ablated with laser light (day 0), and the degree of retinal damage was confirmed on the same day by fundus photography (FP), fluorescein angiography (FFA), and frequency-domain optical coherence tomography (SD-OCT) (Figure 1). The mice were randomly divided into five groups: (1) control group, (2) positive control group: Eylea (40 μg / eye / μL), and (3-5) GYT088 treatment groups with low dose (1.28 ng / eye), intermediate dose (2.56 ng / eye), and high dose (5.16 ng / eye). Intravitreal injection of the corresponding drug was administered. Mice in each group were clinically observed daily, and retinal damage repair was examined on days 7 and 14.

[0044] 3. Method

[0045] 3.1 Laser-induced choroidal angiogenesis in mice

[0046] C57BL / 6 mice were administered a dissociative rapid-acting general anesthetic via intraperitoneal (IP) injection. After the mice reached deep anesthesia, they were placed on a stand and pupillary dilation was induced with atropine. Thirty minutes later, a contact gel (OmniVision) was applied, and retinal laser damage was induced using a dual-frequency Nd:YAG laser (Laser LP352; Lumenis Inc., Salt Lake City, UT). The irradiation time was 0.1 seconds, and the laser energy was set to 250-300 mW, creating a damaged area with a diameter of approximately 100 μm. Two to four laser burns were applied between retinal blood vessels within the same field of view. On the same day, fundus fluorescein angiography (FFA) was performed to record the fluorescein leakage site. After recovery from anesthesia, the mice were returned to their cages and observed.

[0047] 3.2 Fluorescein angiography (FFA) and analysis

[0048] Mice were deeply anesthetized and their pupils dilated. Using a Micron III retinal angiography microscope (Phoenix, San Ramon, CA, USA), the focal length was adjusted, and the fovea of ​​the mouse was placed in the center of the fundus lens to capture fundus photographs (FP). Next, a 10% sodium fluorescein solution was injected intraperitoneally, and retinal angiography was performed with a 520 nm filter to capture and analyze sequential images. After drug administration, the results of FP, FFA, and SD-OCT were observed weekly on day 0 and recorded until day 14. Clear images were selected for analysis and imported into a 16-pixel format using ImageJ. The software automatically quantified the pixel values ​​and area of ​​the fundus region by removing background values. Due to differences in individual retinal structures, post-laser wound healing varies, and excessive signal exposure may occur in those areas. If fluorescence leakage occurs outside the direct laser-damaged area, it is considered a phenomenon of fundus neovascularization. Therefore, three fixed regions of fluorescence leakage outside the laser-damaged area were randomly selected and quantified.

[0049] 3.3 Spectral-domain optical coherence tomography (SD-OCT) and analysis

[0050] The OCT (Optical Coherence Tomography) system used in this experiment (Phoenix Research Laboratories, San Ramon, CA) is based on the principle of frequency-domain optical homodyne tomography. Computed tomography (CT) scans were divided into three layers: the first layer covered the nerve fiber layer (NFL) to the inner retinal layer (INL), the second layer covered the INL to the internal / outer segment (IS / OS), and the third layer covered the IS / OS to the retinal pigment epithelium (RPE). In a double-blind study, damage was scored according to the underlying retinal structure, and the average score after scoring for three participants was used as the index value for damage. The scoring criteria were as follows:

[0051] [Table 1]

[0052] statistical analysis

[0053] All data are presented as mean ± standard deviation (SEM) and statistically analyzed using one-way ANOVA (Turkish test). A p<0.05 value indicates a statistically significant difference.

[0054] 4.Results

[0055] 4.1 Image Analysis of Fluorescein Angiography (FFA)

[0056] FFA images were analyzed using ImageJ image analysis, and the fluorescence leakage area was quantified. Since there was no significant leakage on day 0, the obtained fluorescence mainly originated from new blood vessels. Three laser-induced wounds were observed in the fundus of each group of mice on day 0, and the relative mean fluorescence leakage ratios on days 0, 7, and 14 after laser injury were quantified (Figure 2). The angiogenesis index on day 7 for the low-dose to high-dose GYT088 administration groups was 1.92±0.21 times, 1.59±0.22 times, and 1.53±0.17 times, respectively, compared to day 0, and a statistically significant difference in angiogenesis inhibition was observed in the high-dose administration group (p<0.05). The angiogenesis index on day 14 in the low-dose to high-dose GYT088 administration groups was 2.46±0.18 times, 1.79±0.17 times, and 1.63±0.16 times, respectively, compared to day 0, and a statistically significant difference in angiogenesis inhibition was observed in the medium-dose and high-dose GYT088 administration groups (p<0.01). Since the inhibition of GYT088 showed a dose- and time-dependent trend, intravitreal injection of GYT088 (2.56 and 5.12 ng / eye) was considered to have an effect in inhibiting laser-induced angiogenesis. The human equivalent dose range was 25.6 and 51.2 μg / eye.

[0057] 4.2 Analysis of the Retinal Sublayer Structure

[0058] Using OCT images, we observed the structural integrity of the retinal sublayer and several substructural changes, such as some high-reflectivity nodules, which may be caused by the aggregation of immune cells or migration of pigment epithelial cells. Retinal cysts were also observed on OCT images, and to compare the degree of retinal damage in each group from fundus photographs, each group of fundus photographs was assigned a scale from 0 to 4 according to the severity of the disease, and the results of the blinded three-group trail were quantified and statistically analyzed (Figure 3). The results showed that the damage scores for each group on day 7 ranged from 0.80 to 1.62, and the damage score in the Eylea group on day 7 (0.80 ± 0.13) was significantly lower than that of the control group (1.38 ± 0.17), indicating that Eylea can protect the retinal sublayer. On day 14, the damage scores for each group ranged from 0.93 to 2.62, and the damage score in the high-dose GYT088 (5.12 ng / eye) treatment group was significantly lower than that of the control group, demonstrating that GYT088 can maintain the retinal sublayer and reduce the degree of cell aggregation.

[0059] Although the present invention has been described above with reference to the embodiments described above, this does not limit the invention. Any modifications or changes that can be made by those skilled in the art without departing from the spirit and scope of the invention are permitted. Accordingly, the scope of protection of the present invention shall be defined by the claims appended herein.

Claims

1. Use of a compound for manufacturing a pharmaceutical product for inhibiting angiogenesis, wherein the compound has a therapeutically effective concentration range of 0.1 μM to 5 μM, formula: 【Chemistry 1】 Viewbellin has the structure of [unclear] and is used.

2. The use of Beuvericin according to claim 1, wherein the therapeutically effective concentration of Beuvericin is 0.5 μM to 5 μM.

3. The use according to claim 1, wherein a pharmacopoeia for inhibiting angiogenesis is used in the development of a pharmaceutical composition for the prevention or treatment of retinal disorders.

4. The use according to claim 3, wherein the retinal disorder is selected from the group consisting of age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy.

5. A pharmaceutical composition for inhibiting angiogenesis, comprising beuvericin having a therapeutically effective concentration range of 0.1 μM to 5 μM and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, wherein the therapeutically effective concentration is 0.5 μM to 5 μM.

7. The pharmaceutical composition according to claim 5, for use in the treatment or prevention of retinal disorders.

8. The pharmaceutical composition according to claim 5, which is used in combination with an anticancer drug in the manufacture of a pharmaceutical composition or pharmaceutical combination for treating cancer.

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