阅读说明：本技术 治疗眼疾的方法 (Method for treating eye diseases ) 是由 李水盛 苏铭嘉 林隆志 徐兆民 于 2020-04-01 设计创作，主要内容包括：本发明提供一种治疗例如老年性黄斑部病变(age-related macular degeneration,AMD)、糖尿病性视网膜病变(diabetic retinopathy,DR),或黄斑部水肿(macular edema,ME)等眼疾的方法,该方法包含对一有此需求的个体施用包含一治疗有效量的白僵菌素的组合物。(The present invention provides a method of treating an eye condition such as age-related macular degeneration (AMD), Diabetic Retinopathy (DR), or Macular Edema (ME), the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of beauvericin.)

1. Use of beauvericin (beauvericin) in the preparation of a medicament for the treatment of ocular diseases.

2. The use of claim 1, wherein beauvericin has a structure of:

3. the use as in claim 1, wherein the ocular disease is caused by ocular degeneration with neovascularization.

4. The use of claim 1, wherein the eye disease is age-related macular degeneration (AMD), Diabetic Retinopathy (DR), or Macular Edema (ME).

5. The use of claim 4, wherein the age related macular degeneration (AMD) is wet age related macular degeneration (wet AMD).

6. The use of claim 4, wherein the Diabetic Retinopathy (DR) is non-proliferative diabetic retinopathy (NPDR).

7. A pharmaceutical composition for treating an ocular condition comprising in combination with one or more pharmaceutically acceptable carriers and a therapeutically effective amount of beauvericin.

8. The pharmaceutical composition of claim 7, wherein the ocular disease is caused by ocular degeneration with neovascularization.

9. The pharmaceutical composition of claim 7, wherein the eye disease is age-related macular degeneration (AMD), Diabetic Retinopathy (DR), or Macular Edema (ME).

10. The pharmaceutical composition of claim 9, wherein the age related macular degeneration (AMD) is wet age related macular degeneration (wet AMD).

11. The pharmaceutical composition of claim 9, wherein the Diabetic Retinopathy (DR) is non-proliferative diabetic retinopathy (NPDR).

Technical Field

The present invention relates to a method of treating eye diseases such as age-related macular degeneration, diabetic retinopathy, or macular edema.

Background

Age-related macular degeneration (AMD) and Diabetic Retinopathy (DR) are the leading causes of acquired blindness in developed countries, characterized by pathological Posterior Segment Neovascularization (PSNV). Pathological ocular angiogenesis, including pathological Posterior Segment Neovascularization (PSNV), occurs as a series of events that progress from an initial stimulus to the formation of abnormal new microvessels. The major causes of age-related macular degeneration (AMD) and Diabetic Retinopathy (DR) are still unknown, however, the production of various angiogenic growth factors appears to be a common stimulus. Found in tissues and fluids of patients with pathological ocular angiogenesis. After initiation of the angiogenic cascade, the basement membrane degrades with extracellular matrix microvasculature and microvascular endothelial cells proliferate and migrate. The endothelium grows out of the anastomotic forming vessel and subsequently forms the cross lumen. New microvessels often have increased vascular permeability or leakage due to unformed barrier function, which can lead to tissue edema. The presence of a continuous basement membrane and normal endothelial junctions between other endothelial cells and the outer coating cells indicate differentiation towards mature microvessels; however, this differentiation process is often compromised in pathological situations.

Macular Edema (ME) is the leading cause of visual loss in diabetic patients, while preretinal neovascularization (PDR) is the leading cause of actual blindness. Diabetes is characterized by persistent hyperglycemia, which causes reversible and irreversible pathological changes in the microvasculature of various organs. Thus, Diabetic Retinopathy (DR), a retinal microvascular disease, is characterized by a cascade of stages with increasing severity and worsening vision prediction. The major risk factors for diabetic retinopathy include the duration of diabetes, glycemic control, and the presence of systemic hypertension. Diabetic Retinopathy (DR) is generally classified in two major clinical stages: non-proliferative diabetic retinopathy (NPDR) and Proliferative Diabetic Retinopathy (PDR), wherein the term "proliferation" refers to the formation of preretinal neovascularization as described above.

Macular degeneration, also known as age-related macular degeneration (AMD or ARMD), is a medical condition that can lead to blurred or poor vision in the center of the field of vision. Early stages are usually free of any symptoms; however, over time, some people have gradually deteriorated vision, possibly affecting one or both eyes. There are several preventive measures, including exercise, good eating habits, and no smoking, but no cure or treatment can restore vision that has been lost. anti-Vascular Endothelial Growth Factor (VEGF) drugs or, less commonly, laser coagulation or photodynamic therapy, injected into the eye in wet form, can slow the progression. Supplements may slow the disease for those already suffering from the disease.

There are currently no approved pharmacological therapies for the treatment of age-related macular degeneration (AMD), Diabetic Retinopathy (DR), and/or macular edema. There is still a need to develop drugs for the treatment of said diseases.

Disclosure of Invention

In the present invention it has been unexpectedly found that compounds such as beauvericin (named GYT-088 in the present invention) are effective in treating eye diseases.

In one aspect, the present invention provides a method of treating an eye condition, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of beauvericin having the following structural formula:

in another aspect, the invention also provides the use of beauvericin in the manufacture of a medicament for the treatment of an eye disease.

In another aspect, the present invention provides a pharmaceutical composition for treating an ocular disease comprising in combination with one or more pharmaceutically acceptable carriers and a therapeutically effective amount of beauvericin.

In one embodiment, the eye disease is caused by ocular degeneration with neovascularization.

In some embodiments of the invention, the eye disease is age-related macular degeneration (AMD), Diabetic Retinopathy (DR), or Macular Edema (ME). In a preferred embodiment of the invention, the eye disease is nonproliferative diabetic retinopathy (NPDR) or wet age-related macular degeneration (wet AMD).

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.

In the drawings:

FIG. 1 provides images showing angiogenesis in matrigel embolization in groups treated with beauvericin (GYT-088, 2.4mg/kg i.p.) and sorafenib (30 mg/kg i.p.), respectively.

Figure 2 provides a comparison of heme levels for each group, showing that beauvericin provides greater efficacy in inhibiting VEGF-induced angiogenesis.

FIG. 3 shows that after oral administration of GYT-008 beauvericin (GYT-088), a test drug according to the invention, was observed in the eyes of animals, indicating that GYT-088 can cross the blood-brain barrier (BBB), especially the blood-retinal barrier (BRB), into the extravascular space of the retinas and the vitreous of the animals.

Figure 4 provides an image showing the measurement of the fovea and macular center of the animal.

Detailed Description

Unless defined otherwise, 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.

In the present invention, it was first discovered that some compounds have a significant effect in treating eye diseases such as age-related macular degeneration (AMD), Diabetic Retinopathy (DR), or Macular Edema (ME).

The present invention provides a method of treating an eye condition comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of beauvericin, which is a cyclohexapeptide coupled from three L-N-methylphenylanilines and three D-2-hydroxyisovaleric acid and has the following structural formula:

the compounds of the present invention, such as beauvericin, may be synthesized or isolated from fungi such as Cordyceps sobolifera (Cordyceps cicadae), Cordyceps sobolifera (Cordyceps cicalifornia), and Isaria fumosorosea (Isaria fumosorosea) according to conventional techniques or methods.

In the present invention, the compounds are shown to be effective in treating eye diseases by inhibiting VEGF-induced angiogenesis.

In one embodiment, the eye disease is caused by ocular degeneration with neovascularization. Some examples of such eye diseases include age-related macular degeneration (AMD), Diabetic Retinopathy (DR), and Macular Edema (ME). In a preferred embodiment of the invention, the eye disease is nonproliferative diabetic retinopathy (NPDR) or wet age-related macular degeneration (wet AMD).

As used herein, the term "therapeutically effective amount" refers to an amount of a compound or agent that results in the treatment, cure, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving the amount. The term also includes within its scope an amount effective to enhance normal physiological function.

For use in therapy, the therapeutically effective amount of the compound is formulated into a pharmaceutical composition for administration. Accordingly, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the compound beauvericin and one or more pharmaceutically acceptable carriers.

The term "pharmaceutically acceptable carrier" as used herein refers to a carrier, diluent or excipient that is acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the individual with whom the pharmaceutical composition is administered. Any carrier, diluent or excipient commonly known or used in the art may be used in the present invention, depending on the requirements of the pharmaceutical formulation.

According to the present invention, the pharmaceutical composition may be adapted for administration by any suitable route, including but not limited to oral, rectal, nasal, topical, vaginal, or parenteral routes. In a particular embodiment of the invention, the pharmaceutical composition is formulated for oral administration. Such formulations may be prepared by any method known in the art of pharmacy.

The present invention is further illustrated by the following examples, which are provided by way of illustration and not by way of limitation.

Examples

Example 1

In vivo matrigel embolization angiogenesis test

Matrigel plugs were made from a gel-like protein mixture secreted by Engelbreth-Holm-swarm (EHS) mouse sarcoma cells (produced and sold by Corning Life Sciences, Inc. and BD Biosciences, Inc.). Matrigel resembles the complex extracellular environment found in many tissues and is used by cell biologists as the matrix for culture testing (basement membrane matrix).

The test animals were 5 to 8 weeks old B57l/6 mice. Matrigel was mixed with PBS at a ratio of 4: 1 to obtain matrigel plugs. Mice were administered matrigel plugs containing 30IU heparin and VEGF150ng/ml (500 ul/mouse) and then divided into four groups (four mice per group), intraperitoneally or orally with beauvericin (GYT-088 group), sorafenib (sorafenib group) as a positive control, VEGF alone (vehicle group), and without any treatment (control group). One day later, animals were given a 30mg/kg intraperitoneal dose of sorafenib, or a 2.4mg/kg intraperitoneal dose of beauvericin (0.1 c.c./mouse/day), or a 24mg/kg oral dose of beauvericin, once a day for 6 days. On day 7, animals were sacrificed and matrigel plugs were removed to observe angiogenesis. And hemoglobin was measured for each mouse with the Drabkin's reagent kit.

The results are provided in fig. 1 and 2. As shown in fig. 1, VEGF-induced angiogenesis was inhibited in the group injected intraperitoneally with beauvericin (GYT-088 group) or the group with sorafenib (sorafenib group) as a positive control. As shown in fig. 2, showing the measured heme content, beauvericin provides higher efficacy in inhibiting VEGF-induced angiogenesis.

In conclusion, beauvericin provides efficacy in inhibiting angiogenesis, even better than sorafenib.

Example 2

The curative effect of beauvericin with different doses on improving diabetic macular edema of rhesus macaque for 60 days is evaluated, and a basis is provided for clinical trial dosage design and safety research.

Rhesus Macaque (Rhesus Macaque) was used in the evaluation.

Primary end point:

retinal thickness was measured with OCT to quantify retinal thickness before and after treatment.

Retinal thickness was evaluated by two methods using Optical interference tomography (OCT): (1) 1.5mm on the center recess and on either side of the center recess (three-point method), and (2) using a 52-point grid to determine the thickness of the yellow spot portion in different thickness groupings (250-. This method was developed by Primed team collaborating with the expert Timothy Kern, doctor diabetic retinopathy, famous by Case Western Reserve University. Please refer to ARVO 2018 poster: assessment of macular thickening in spontaneously diabetic rhesus macaques (Association of pathological thickening in cationic bile rhesus monkeys).

Secondary endpoint and security parameters:

(1) fundus Photography (FP). Detecting a change in bleeding, exudate or vasculopathy before and after treatment; and

(2) the influence of the medicine on metabolism, fat profile, and liver and kidney functions.

Non-human primates (NHPs) are very similar to humans in their physiological functions and ocular structures. Primed has a unique large population of non-human primate (NHP) populations (n about 1500) with 5 years of historical clinical/metabolic data, with idiopathic metabolic disease, including Fasting Plasma Glucose (FPG), insulin resistance, Body Mass Index (BMI), Triglyceride (TG), Total Cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c). Color doppler ultrasound, Kowa fundus camera, Heidelberg optical interference tomography (Heidelberg OCT), slit lamps, and ophthalmoscopes are used to diagnose the diabetic state of non-human primates (NHPs), showing that the disease characteristics of rhesus macaque idiopathic Diabetic Macular Edema (DME) are very similar to those of clinical patients, showing vascular leakage, retinal thickening, exudation, or hemorrhage. Rhesus macaques with idiopathic Diabetic Macular Edema (DME) provide an ideal model for human Diabetic Macular Edema (DME)/Diabetic Retinopathy (DR) research and new drug development.

Inclusion criteria

(1) Female, age 13-18 years (equivalent to a human adult of 40-60 years), weighing 6-7 kg;

(2) the course of diabetes mellitus: more than 3 years;

(3) diabetic Macular Edema (DME) in one/both eyes;

(4) diabetic Macular Edema (DME): retinal thickness measured by Heidelberg spectral optical interference tomography (Heidelberg Spectralis OCT) in the last half year and diagnosed with Kowa VX-20 Fundus camera by Fundus Photography (FP)/Fluorescein Fundus Angiography (FFA);

(5) the Foveal Thickness (CRT) is 260 μm or more and/or the average Thickness of the Early Diabetic Retinopathy Treatment Study (ETDRS) area is 319 μm or more. (retinal thickening is defined by the CRT mean and Standard Deviations (SD) and ETDRS thickness of normal macaques). Retinal thickening is defined as thickness that exceeds the normal mean by 2 standard deviations. Non-diabetic cynomolgus monkey: the retinas of the non-diabetic animals had a mean foveal thickness of 192. + -.11 μm, measured at 1.5mm from the temporal and nasal fovea of 307. + -.15 and 328. + -.13, respectively. This method was developed by Primed team collaborating with the doctor Timothy Kern, a diabetic retinopathy expert known by Case Western Reserve University. Please refer to ARVO 2018 poster: evaluation of macula thickening of spontaneous diabetic rhesus macaques; and

(6) with/without vascular leakage or non-perfused areas.

Exclusion criteria

(1) Hypertension of the third and above stage;

(2) progressive ocular or systemic inflammation or infection; intraocular high pressure: intraocular pressure (IOP) >21 mmHg;

(3) laboratory examination shows that kidney or liver function is obviously impaired;

(4) any other history that may affect the efficacy of the treatment.

Group size

6 spontaneous Diabetic Macular Edema (DME) rhesus macaques (with medical history in one year) were provided, and 4 of the 6 monkeys were subjected to Fundus Angiography (FA), Fundus Photography (FP), optical interference tomography (OCT), and slit lamp examination, and blood biochemical examination prior to treatment in the early stages of the study (4 weeks). Optical interference tomography (OCT) will divide these 4 monkeys into two groups according to their retinal thickness:

(1) first, vehicle, N ═ 1;

(2) group 2, trial, N ═ 3;

(route of administration: oral, once daily; dose: Total Basal Dose (TBD)).

And (4) measuring the result:

the main efficacy parameters are as follows:

retinal thicknesses include Central Subfield Thickness (CST) and ETDRS thickness. Once before and once on day 14 (D14), day 30 (D30), and day 60 (D60) post-dose; whether the test was increased on day 45 (D45) was determined from the results of D14 and D30.

Secondary efficacy and safety parameters:

(1) fundus photography: 3 times, with pre-dose as baseline, once on each of day 30 (D30) and day 60 (D60) post-dose;

(2) fluorescein Fundus Angiography (FFA): baseline was taken prior to dosing;

(3) intraocular pressure and blood pressure measurement: 2 times before and after administration;

(4) clinical signs of the eyes were assessed by daily cage-side observation;

(5) measurement of metabolic parameters and liver and kidney functions: FPG, FRA, LDL, HDL, TC, TG: once before and once on day 14 (D14), day 30 (D30), day 45 (D45), and day 60 (D60) post-dose;

(6) a slit lamp: 1 time before administration, 1 time after administration, and 2 times in total;

(7) weight: once before and once on day 30 (D30) and day 60 (D60) after dosing;

(8) food intake/clinical observations: including abnormal symptoms, and behaviors, once a day.

Fundus Photography (FP) and Fluorescein Fundus Angiography (FFA)

(1) Frequency: fluorescein Fundus Angiography (FFA) was performed once prior to dosing.

(2) The instrument comprises the following steps: KOWA VX-20.

(3) The purpose is as follows: retinal blood vessels were examined for damage and leakage.

(4) FFA image acquisition program:

prior to image acquisition, ketamine was injected intramuscularly: the animals were anesthetized with a xylazine mixture (1:1, 8mg/kg ketamine). After anesthesia, 2 drops of Tropicamide (Tropicamide) phenylephrine eye drops were administered to each eye to dilate the pupil. After anesthesia, the animals were placed in a dark room until the pupil diameter was greater than 6 mm. Self-preserved eyelid spectra were placed in the eye. The fundus posterior pole of the dominant eye of photography focuses well.

(5) The time was started when 10% sodium fluorescein (Alcon Laboratories, Inc., USA) was injected through the femoral vein at a dose of 0.075 mL/kg. A series of back polar photographs were taken of the dominant eye every second for the first 30 seconds and every 2-3 seconds for the next 30 seconds. Both eyes were photographed at 5 minutes and 10-15 minutes.

Results

Vehicle group: #6189 shows mild non-proliferative diabetic retinopathy (NPDR) prior to administration. As shown in table 1, no significant change was observed before and after the administration as analyzed by Fundus Photography (FP). GYT-088 group: #251, #2003, and #3536 showed mild or moderate non-proliferative diabetic retinopathy (NPDR). Analysis by Fundus Photography (FP) on day 28 post-dose (D28) improved the non-proliferative diabetic retinopathy (NPDR) characteristics in # 2003. No significant changes were seen before and after dosing to other animals at any time point by Fundus Photography (FP) analysis, see table 1.

TABLE 1 post-treatment non-proliferative diabetic retinopathy (NPDR) associated changes

The results are provided in fig. 4. On day 14 after administration, in #2003 experimental animals (NPDR group), it was found that the Central Macular Thickness (CMT) was changed from 204 μm and 210 μm in the left and right eyes to 197 μm and 202 μm, which were decreased by 7 μm and 8 μm, respectively; the fovea thickness was reduced from 257 μm and 260 μm for the left and right eyes to 249 μm and 251 μm, respectively, by 8 μm and 9 μm. On the other hand, it was found that in vehicle-administered animal #6189 (vehicle group), the change in central macula thickness or fovea thickness was within a range of ± 2 μm as normally found in normal animals. On day 56 post-dose, the central macula thickness was reduced by 6 μm and 8 μm, and the left and right eye macula thicknesses were reduced by 3 μm and 5 μm, respectively. Thus, it can be concluded that GYT-088 provides efficacy in diabetic macular edema.

TABLE 2 changes in Diabetic Macular Edema (DME) thickness

While this specification contains many specifics, these should not be construed as limitations on the scope of the invention or of what may be claimed, but rather as descriptions of specific embodiments or example specific features of the invention. Certain features that are described in this specification in the context of separate embodiments or examples can also be implemented in combination in a single embodiment.