KR102501677B1 - Vascular leakage blocking effect of primaquine diphosphate and its targets usp1 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating vascular leakage disease containing a USP1 inhibitor and a method for screening a USP1 inhibitor, wherein the USP1 inhibitor and promaquine diphosphate inhibit apoptosis of vascular endothelial cells and actin stress induced by VEGF It was confirmed that vascular leakage was inhibited by suppressing the formation of fibers and increasing the outer sheath actin ring structure.
Accordingly, the composition according to the present invention containing the USP1 inhibitor can prevent or treat vascular leakage and various diseases caused by vascular leakage.

Description

Vascular leak blocking effect of USP1 with Primaquine diphosphate and its target {VASCULAR LEAKAGE BLOCKING EFFECT OF PRIMAQUINE DIPHOSPHATE AND ITS TARGETS USP1}

The present invention relates to a composition for preventing or treating vascular leakage disease containing a USP1 inhibitor and a method for screening a USP1 inhibitor.

A cross section of the blood vessel wall shows the inner layer (tunica intima) composed of endothelium, the connective tissue composed of elastin and collagen fibers, the tunica media composed of smooth muscle, and the tunica adventitia composed of a collagen fiber layer. It is known that the asymmetric structure of blood vessels suppresses the generation of blood vessels, prevents blood vessel leakage, and imparts fluidity to blood vessels. In particular, in the inner layer composed of endothelial cells of blood vessels, sugar polymers accumulate on the surface to form a glucocortical layer, which directly regulates the flow of blood and prevents direct contact between blood and endothelial cells. It prevents blood components from entering endothelial cells. In addition, it is known to be involved in various physiological activities such as blood vessel elasticity regulation, blood and tissue retention and solute exchange, leukocyte migration, hemostasis and blood coagulation, and inflammatory response.

It is known that when such a glucocortical layer is damaged, the function of blood vessels is primarily impaired, and secondarily, physiological activities related to blood vessels are inhibited. What is most affected by the damage of the glucocortical layer is the damage to the function of blood vessels. The glucocortical layer is damaged by mechanical stimulation such as wounds and surgeries, and components in the blood can escape to the outside of the blood vessels through endothelial cells. . A symptom in which components in the blood escape outside the blood vessels is called vascular leakage.

In addition, as a result of conducting a study on diabetic patients with frequent vascular leakage, it was found that vascular leakage is caused by overexpression of vascular endothelial growth factor (VEGF). In other words, the overexpression of VEGF induced at the onset of diabetes degrades VE-cadherin, which plays an important role in maintaining the bond between endothelial cells at the junction of endothelial cells, thereby reducing the bond between endothelial cells and reducing the carbohydrate cortex. It is known that it causes damage to the layer, and thereby vascular leakage occurs. In addition, VEGF plays an important role in disruption of the vascular-retinal barrier by changing the integrity of tight junctions and structuring the cytoskeleton of endothelial cells, which is known to increase vascular permeability during the development of diabetic retinopathy. .

Therefore, there is a growing need for a treatment targeting the early and reversible stages of diseases caused by vascular leakage.

Chemistry & Biology 18, 1390-1400, November 23, 2011, "Selective and Cell-Active Inhibitors of the USP1/ UAF1 Deubiquitinase Complex Reverse Cisplatin Resistance in Non-small Cell Lung Cancer Cells", Junjun Chen, et al. Frontiers in Pharmacology, September 2018, Vol.9, Article 1080, "Inhibition of Ubiquitin-Specific Proteases as a Novel Anticancer Therapeutic Strategy", Tao Yuan, et al. Villamil, M. A., et al., “Serine phosphorylation is critical for the activation of ubiquitin-specific protease 1 and its interaction with WD40-repeat protein UAF1.” Biochem. 51, 9112-9113 (2012) Zhang X.-Y., et al., "The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression." Mol. Cell 29:102-111 (2008)

The present inventors have tried to find a substance capable of preventing or treating diseases caused by vascular leakage caused by damage to blood vessel integrity. As a result, the present inventors found that primaquine diphosphate, a drug currently used for malaria treatment, and ubiquitin specific peptidase 1 (USP1) inhibitor inhibit the death of vascular endothelial cells, By suppressing the formation of actin stress fibers induced by VEGF and increasing the structure of cortical actin ring, it was confirmed that vascular leak-related diseases can be prevented or treated, thereby completing the present invention.

Accordingly, an object of the present invention includes a USP1 inhibitor, wherein the USP1 inhibitor is at least one blood vessel selected from the group consisting of an antibody to the USP1 protein, an antisense oligonucleotide to the USP1 gene, siRNA, and primaquine diphosphate. To provide a pharmaceutical composition for preventing or treating vascular leakage disease.

Another object of the present invention is to include at least one USP1 inhibitor selected from the group consisting of an antibody to the USP1 protein, an antisense oligonucleotide to the USP1 gene, siRNA, and primaquine diphosphate, and to prevent cancer cells due to vascular leakage. It is to provide a pharmaceutical composition for preventing metastasis.

Another object of the present invention is to provide a method for screening an inhibitor of USP1, comprising contacting a USP1 protein or USP1 gene with a candidate substance, and analyzing the resulting change in activity of the USP1 protein or expression of the USP1 gene.

Another object of the present invention is to measure the expression level of USP1 protein or USP1 gene in a sample isolated from a subject of interest; and comparing the expression level of the USP1 protein or USP1 gene with the expression level of the USP1 protein or USP1 gene in a control sample; And to provide a method for providing information for diagnosing a vascular leak disease, including indicating that the risk of developing a vascular leak disease is high when the expression level of the USP1 protein or the USP1 gene is increased compared to the control group.

In order to achieve the above object, one aspect of the present invention includes a USP1 inhibitor, wherein the USP1 inhibitor is a group consisting of an antibody to the USP1 protein, an antisense oligonucleotide to the USP1 gene, siRNA, and primaquine diphosphate It provides a composition for preventing or treating one or more vascular leakage diseases selected from.

As used herein, the term "vascular leakage or vascular leakage" may be used interchangeably, and refers to a symptom in which blood components escape to the outside of blood vessels through endothelial cells due to damage to the cortical layer. .

The vascular leakage or vascular leakage disease is diabetes, inflammation, retinopathy, diabetic retinopathy, macular degeneration, glaucoma, stenosis, restenosis, arteriosclerosis, atherosclerosis, cerebral edema, arthritis, arthropathy, uveitis, inflammatory bowel disease, macular edema , hyperlipidemia, ischemic disease, diabetic foot ulcer, corpulent hypertension, acute lung injury, myocardial ischemia, heart failure, acute lower limb ischemia, myocardial infarction, stroke, ischemia or reperfusion injury, vascular leak syndrome, edema, transplant rejection, burns, acute or It may be adult respiratory distress syndrome, sepsis or an autoimmune disease, but is not limited thereto.

According to one embodiment of the present invention, diphosphate, siUSP1 and USP1 inhibitors inhibit apoptosis of vascular endothelial cells (Figs. 1, 6 and 9), inhibit cell permeability (Figs. 2, 7 and 10), and cytoskeleton The effect on change was confirmed (FIG. 3, 8 and 11), and it was confirmed that the effect of suppressing the level of vascular leakage induced by processing VEGF at the animal level (FIGS. 4 and 5) was confirmed.

According to one embodiment of the present invention, an antibody against the USP1 protein, an antisense oligonucleotide against the USP1 gene, siRNA, and one or more USP1 inhibitors selected from the group consisting of primaquine diphosphate (primaquine diphosphate) to prevent vascular leakage Provided is a pharmaceutical composition for preventing metastasis of cancer cells.

In the present invention, when the composition is administered in combination with an anticancer agent, it can give a synergistic effect to prevent metastasis of cancer cells, inhibit growth of cancer cells, and induce apoptosis.

In the pharmaceutical composition of the present invention, the type of anticancer agent that is co-administered with the composition may be used without limitation as long as it is a conventional anticancer agent that has been generally used for cancer treatment. Examples of such anti-cancer agents include alkylating agents, microtubule inhibitors, antimetabolites, and topoisomerase inhibitors as chemo-anticancer agents, monoclonal antibodies and signal transduction inhibitors as target anti-cancer agents, and immune checkpoint inhibitors and immune checkpoint inhibitors as immuno-anticancer agents. Cell therapy (CAR-T), antibody-drug conjugate (ADC), bispecific antibodies, anti-cancer viruses and anti-cancer vaccines may be included, and hormonal anti-cancer agents may include male hormone inhibitors and female hormone inhibitors.

The components of the alkylating agent include mechlorethamine, cyclophosphamaide, chlorambucil, thiotepa, altretamine, procarbazine, and busulfan. (busulfan), streptozocin, carmustine, lomustine, dacabazine, cisplatin, carboplatin and oxaliplatin.

Components of the microtubule inhibitor include taxotere, velban, oncovin, and navelbine, and components of the antimetabolite include fluorouracil, capecitabine ( capecitabine, cytarabine, gemcitabine, fludarabine, methotrexate, pemetrexed and mercaptopurin.

Components of the topoisomerase include hycamtin, camptosar, vepesid, taxol, blenoxane, adriamycin and cerubidine do.

Components of the monoclonal antibody include trastuzumab, pertuzumab, panitumumab, and cetuximab that target EGFR; bevacizumab, ramucirumab and aflibercept, which target VEGFR; rituximab and obinutuzumab, which target CD20; daratumumab, which targets CD38; and denosumab, which targets RNAK-L.

Components of the signal transduction inhibitor include ibrutinib targeting BTK, dasatinib targeting Bcr-abl, nilotinib, imatinib, and bosutinib (bosutinib); osimertinib, erlotinib, and gefitinib, which target EGFR; nintedanib, sunitinib, sorafenib, cabozantinib, lenvatinib, regorafenib, etc targeting the PDGFR, VEGFR and FGFR families axitinib, pazopanib and cabozantinib; trametinib and dabrafenib targeting MEK/RAF, abemaciclib and palbociclib targeting CDKs, lenalidomide targeting ubiquitin ( lenalidomide), JAK-targeting ruxolitinib, ALK-targeting alectinib and crizotinib, PARP-targeting olaparib, and proteasome-targeting. These include lxazomib, which targets Bcl-2, and venetoclax, which targets Bcl-2.

The components of the immune checkpoint inhibitor are ipilimumab, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab and durvalumab ( duralumab), and the components of the immune cell therapy include tisagenlecleucel and axicabtagene ciloleucel.

The components of the ADC are gemtuzumab-ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, and Eri Includes eribulin mesylate.

The component of the double antibody includes blinatumomab, the component of the anti-cancer virus includes talimogene laherparepvec, and the component of the anti-cancer vaccine includes sipulecucel-T -T), and the components of the testosterone inhibitor include bicalutamide and enzalutamide.

Components of the female hormone inhibitor may include tamoxifen, anastrozole, letrozole, exemestane, and fulvestrant.

In addition, vitamin A and its metabolites, including tretinoin and alitretinoin, which inhibit the abnormal differentiation of cancer cells, and thalidomide and lenalidomide related to the regulation of tumor necrosis factor-α may be included.

In addition, radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and γ-ray irradiation.

Therefore, the anticancer agent inhibits the proliferation of cancer cells for one or more carcinomas selected from the group consisting of osteosarcoma, melanoma, uterine cervical cancer, gastric cancer and glioblastoma, or It may be to promote apoptosis, but is not limited thereto.

USP1/UAF1 inhibitors related to USP1 of the present invention act synergistically with cisplatin to inhibit cell proliferation of cisplatin-resistant non-small cell lung cancer (NSCLC) (Chemistry & Biology 18, 1390-1400, November 23, 2011, " Selective and Cell-Active Inhibitors of the USP1/ UAF1 Deubiquitinase Complex Reverse Cisplatin Resistance in Non-small Cell Lung Cancer Cells", Junjun Chen, et al.). In addition, an examination of tumor microarray expression data in the Oncomine Research Edition database revealed that USP1 mRNA expression levels change markedly in several malignancies, including osteosarcoma, melanoma, and gastric cancer. USP1 is aberrantly overexpressed in gastric or cervical cancer, melanoma and sarcoma, as well as osteosarcoma. Considering the frequent overexpression of USP1 in various tumors and the important role of USP1 in DNA damage response, USP1 inhibition may be a novel anti-cancer therapy (Frontiers in Pharmacology, September 2018, Vol.9, Article 1080, "Inhibition of Ubiquitin- Specific Proteases as a Novel Anticancer Therapeutic Strategy", Tao Yuan, et al.).

The term "combined administration" used in the present invention can be achieved by simultaneously, sequentially, or individually administering the individual components of the therapy. A combination treatment effect is obtained by administering two or more drugs simultaneously or sequentially, or by alternately administering them at regular or unspecified intervals. , efficacy measured by time to disease progression or survival can be defined as being therapeutically superior to and capable of providing a synergistic effect over the efficacy obtainable by administering one or the other of the components of a combination therapy at conventional doses. can

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. may be administered.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. It can be. The daily dose of the pharmaceutical composition of the present invention is, for example, 0.05 to 100 mg/kg, in terms of mouse versus human.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

According to one embodiment of the present invention, the primaquine diphosphate of the present invention is applied to the target prediction site (http://carlsbad.health.unm.edu/tomcat/carlsbad/carlsbadone, https://pubchem.ncbi.nlm.nih .gov/compound/6135 and https://www.ebi.ac.uk/chembl/compound_report_card/CHEMBL43128/), candidate genes were derived, and while identifying genes with high expression in endothelial cells, USP1 confirmed to be affected. Thereafter, the inhibitory effect of siUSP and USP1 inhibitors on vascular leakage was confirmed.

It has been reported that about 100 deubiquitinating enzymes (DUBs) exist in humans, and the deubiquitinating enzymes are largely UCH (Ub C-terminal hydrolase), USP (Ub-specific processing protease), Otubain (OTU- domain Ub-aldehyde-binding protein), Machado-Joseph disease (MJD), and Jab1/Pad1/MPN domain metallo-enzyme (JAMM) families. peptidase, USP) form the largest group. In the case of USP, it has an active-domain consisting mainly of 350 amino acids and is a proteolytic enzyme (cysteine protease). Although the degree of amino acid conservation of these USP active domains is not high overall, the regions surrounding active cysteine and histidine residues, that is, the cysteine box & histidine box, are well conserved. there is. In the case of USP, in addition to these active domains, it contains a number of domains whose functions have not yet been identified, and are thought to be involved in substrate recognition, intracellular localization, and protein-protein interactions.

The composition and role of the above USP differs depending on its type. Ubiquitin itself contains seven lysine (Lys) residues, all of which can be ubiquitinated, resulting in polyubiquitin chains of different linkages. Depending on these differently linked ubiquitin chains, cells perform their respective functions, including processing of polyubiquitinated proteins by proteasome-associated DUBs, histone deubiquitination, RNAi revealing the role of DUBs in cell cycle regulation and DNA repair; DUBs have functions such as regulating kinase cascades of signal transduction pathways and endocytosis. Representatively, USP1 (NCBI Gene ID: 7398) is an enzyme encoded by the USP1 gene in humans. The gene encodes a member of the ubiquitin-specific processing (UBP) protease family, which is a deubiquitination enzyme (DUB) with histidine (His) and cysteine (Cys) domains. The protein is located in the cytoplasm and cleaves the ubiquitin moiety from the ubiquitin fusion precursor and the ubiquitinated protein. Proteins specifically deubiquitinate proteins in the Fanconi anemia (FA) DNA repair pathway. Alternative transcript splice variants have been characterized. In addition, the USP1 is composed of 785-amino acids including a catalytic triad consisting of Cys90, His593 and Asp751 (Villamil, MA, et al., "Serine phosphorylation is critical for the activation of ubiquitin-specific protease 1 and its interaction with WD40-repeat protein UAF1.” Biochem. USP22 (NCBI Gene ID: 23323) is composed of 520 amino acids, including Cys185 and His479. In addition, the USP22 is located in the USP22 gene of chromosome 17 in humans. The USP22 is a histone DUB component of the transcriptional regulatory histone acetylation complex SAGA, and serves to catalyze the deubiquitination of histones H2A and H2B (Zhang X.-Y., et al., "The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression. " Mol. Cell 29:102-111(2008)).

According to one embodiment of the present invention, it includes a USP1 inhibitor, wherein the USP1 inhibitor is an antibody to the USP1 protein, an antisense oligonucleotide to the USP1 gene, siRNA, and primaquine diphosphate to vascular endothelial growth factor (vascular endothelial growth factor) endothelial growth factor, VEGF) activity was confirmed (Figs. 1 to 11).

The present invention provides a method for screening a vascular leakage inhibitor, comprising contacting a USP1 protein or USP1 gene with a candidate substance and analyzing the resulting change in activity of the USP1 protein or expression of the USP1 gene.

In addition, if the candidate substance inhibits the activity of the USP1 protein or the expression of the USP1 gene, it provides a screening method for a vascular leak blocker comprising selecting a vascular leak blocker, and whether the candidate substance inhibits the activity of VEGF It provides a screening method for a vascular leak blocker further comprising analyzing.

The present invention includes the steps of measuring the expression level of USP1 protein or USP1 gene in a sample isolated from a subject of interest; and comparing the expression level of the USP1 protein or USP1 gene with the expression level of the USP1 protein or USP1 gene in a control sample; and a method for providing information for diagnosing a vascular leak disease, including indicating that the risk of developing a vascular leak disease is high when the expression level of the USP1 protein or the USP1 gene is increased compared to the control group.

The term "candidate" used while referring to the screening method of the present invention refers to an unknown substance used in screening to examine whether or not it affects the expression level of a gene or affects the expression or activity of a protein. . The sample includes, but is not limited to, an antibody against the USP1 protein, an antisense oligonucleotide against the USP1 gene, siRNA, and a USP1 inhibitor selected from primaquine diphosphate.

Accordingly, the present invention provides a pharmaceutical composition and screening method wherein the USP1 inhibitor inhibits VEGF activity.

The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.

It includes the USP1 inhibitor according to the present invention, wherein the USP1 inhibitor is an antibody to the USP1 protein, an antisense oligonucleotide to the USP1 gene, siRNA, or primaquine diphosphate for various uses for the prevention or treatment of vascular leakage diseases can be utilized

Figure 1 shows the results confirming the death and proliferation of vascular endothelial cells of primaquine diphosphate.
Figure 2 shows the result of confirming the cell permeability inhibitory effect of primaquine diphosphate through TEER assay (Fig. 2A) and FITC-Dextran Permeability assay (Fig. 2B).
3 shows the effect of promaquine diphosphate on improving the stability of VE-cadherin (FIG. 3A) and the effect of promaquine diphosphate on inhibiting the formation of VEGF-induced actin stress fibers and increasing the sheath actin ring structure ( Figure 3B) shows the result of checking.
Figure 4 shows the vascular leakage blocking effect of promaquin diphosphate by VEGF on shaved mice (FIG. 4A) (FIG. 4B), histamine Shows the results of confirming the vascular leakage blocking effect (FIG. 4C) and the quantitative value (FIG. 4D).
Figure 5 is the effect of inhibiting retinal vascular permeability induced in a diabetic mouse model by a group administered with promaquine diphosphate (Figs. 5A and 5B) and quantitative values expressed as the product of the number of leaks and the amount of leaks found in retinal blood vessels ( GRADE) shows the results of in vivo analysis of the effect of promaquine diphosphate on the vascular permeability of diabetic retinopathy.
Figure 6 shows the results of confirming cell death and proliferation through MTT assay when USP1 is knocked down.
Figure 7 shows the result of confirming the cell permeability inhibitory effect of siUSP1 through TEER assay (Fig. 7A) and FITC-Dextran Permeability assay (Fig. 7B).
8 shows the effect of siUSP1 on improving the stability of VE-cadherin (FIG. 8A) and the effect of siUSP1 inhibiting the formation of VEGF-induced actin stress fibers and increasing the sheath actin ring structure (FIG. 8B). Results shows
Figure 9 shows the results of confirming cell death and proliferation of ML-323, an inhibitor of USP1 (FIG. 9A), and confirming cell death and proliferation of SJB2-043 (B) (FIG. 9B).
10 shows the result of confirming the cell permeability inhibitory effect of ML-323 and SJB2-043 through TEER assay (FIGS. 10A and 10B) and FITC-Dextran Permeability assay (FIGS. 10C and 10D).
11 shows the effect of ML-323 and SJB2-043 on improving the stability of VE-cadherin (FIG. 11A), and ML-323 and SJB2-043 inhibit the formation of actin stress fibers induced by VEGF and integumentary actining The result of confirming the effect of increasing the structure (FIG. 11B) is shown.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Experimental example

Culture example: vascular endothelial cell culture

Human retinal endothelial cells (HREC) were purchased from Cell-Systems (USA) and 20% (w/v) fetal bovine serum (FBS, HyClone, Canada), 100 units / ml of penicillin (penicillin, Invitrogen, USA), 100 μg / ml of streptomycin (streptomycin, Invitrogen, USA), 3 ng / ml of fibroblast growth factor (bFGF; basic fibroblast growth factor, USPtate Biotechnology, USA) and After inoculation into a 100 mm culture dish containing EGM medium (Life Technologies, USA) containing 5 units/ml of heparin, the cells were cultured in a 5% CO ₂ incubator at 37°C.

Experimental Example 1: Confirmation of cell death and proliferation of primaquine diphosphate

MTT assay was performed to confirm cell death and proliferation of primaquine diphosphate. Vascular endothelial cells were cultured for 24 hours using 20% M199 containing FBS at (3.0±0.2) × 10 ⁴ cells/well in a collagen-coated 24-well plate. When the cell confluency reached about 70%, primaquine diphosphate was treated with SFM at concentrations of 1, 5, 10, and 20 μM, followed by incubation for 2 days. After 2 days, the cells were incubated in MTT solution for 4 hours and the degree of apoptosis was measured.

As a result, it was confirmed that cells proliferated significantly in the groups treated with 5 and 10 μM (FIG. 1). (P<0.0001)

Experimental Example 2: Confirmation of cell permeability inhibitory effect of primaquine diphosphate

In order to confirm the cell permeability inhibitory effect of primaquine diphosphate, permeability assay was performed using TEER assay and FITC-dextran permeability assay. TEER is a value representing the electrical resistance of membranes and can be used to measure the resistance formed by the tight junctions of cells. It is an experiment to find out the cell permeation by the amount of leakage of FITC. Vascular endothelial cells (5.5±0.5) × 10 ⁴ cells/well were cultured for 2 days using 10% EGM on a transwell coated with collagen to form a confluent monolayer. After starvation using SFM for 2 hours, treatment with 5 μM of primaquine diphosphate and pre-incubation for 30 minutes, VEGF 30 ng/ml was added for 30 minutes to induce permeability of the HUVEC monolayer. Liver treatment.

As a result, the electrical resistance value decreased because the permeability of the cell wall increased when VEGF was treated compared to the control group, whereas the electrical resistance value significantly increased when VEGF and primaquine diphosphate were treated together compared to the VEGF only treated group. It was confirmed that (Fig. 2A). (P<0.05)

In addition, it was confirmed that the amount of FITC leakage increased when VEGF was treated compared to the control group, whereas the amount of FITC leakage decreased when VEGF and primaquine diphosphate were treated together compared to the group treated with only VEGF (Fig. 2B). (P<0.05)

Experimental Example 3: Checking the effect of primaquine diphosphate on cytoskeletal changes

It is known that vascular permeability is affected by the stability of anchoring junctions between vascular cells. Human umbilical vein endothelial cells (HUVECs) were cultured in a 35 mm dish using 10% EGM for 2 days to become confluent. After starvation with SFM for 2 hours, treatment with 5 μM of primaquine diphosphate and pre-incubation for 30 minutes, VEGF 50 ng/ml was treated for 30 minutes to induce cell permeability. Cells were then fixed with 4% PFA for 20 min at room temperature and permeabilized in 0.1% Triton X-100/PBS. Then, the cells were reacted with VE-cadherin (SC-9989) antibody for 2 hours.

As a result, it was confirmed that primaquine diphosphate not only prevents VE-cadherin from being destabilized by VEGF, but also improves the stability of VE-cadherin between cells.

In addition, it is known that changes in the structure of actin constituting the cytoskeleton are closely related to the permeability of vascular endothelial cells. When the permeability of vascular endothelial cells increases, the formation of actin stress fibers increases and the outer cortical actin ring structure decreases (FIG. 3A).

Using this, the inhibitory effect of primaquine diphosphate on vascular endothelial permeability was investigated. Saturated HUVECs were treated with 5 μM of primaquine diphosphate and pre-incubated for 30 minutes, and then treated with VEGF 50 ng/ml for 30 minutes to induce cell permeability. Cells were then fixed with 4% PFA for 20 minutes at room temperature and washed three times with PBS (pH 7.4). Cells were then permeabilized in 0.1% Triton X-100/PBS and reacted with F-actin (invitrogen R415) for 30 minutes. Cells were then observed under a confocal microscope (Carl Zeiss).

As a result, it was confirmed that primaquine diphosphate inhibited the formation of actin stress fibers induced by VEGF and increased the outer sheath actin ring structure (FIG. 3B).

Experimental Example 4: in vivo Confirmation of vascular leakage inhibitory effect of primaquine diphosphate in

In order to investigate the vascular leakage inhibitory effect of primaquine diphosphate in vivo , it was tested using Miles assay. 2 to 3 days prior to the experiment, the backs of BALB/C mice are shaved. After anesthesia with 2.5% avertin, 100λ IV of evans blue (in PBS) is injected. After 10 minutes, a control group, a group treated with VEGF and VEGF and primaquine diphosphate, and a group treated only with primaquine diphosphate were injected intradermally into the back of the shaved mouse. After 30 minutes, the skin pieces were dissolved in formide and quantified. In the same way, experiments were conducted not only with VEGF but also with histamine, which induces vascular leakage.

After intradermal injection, the entire back was seen (Fig. 4-A), and the epidermis was peeled off and confirmed (Figs. 4B and 4C).

As a result, it was confirmed that vascular leakage was induced in the group treated with VEGF and histamine, whereas vascular leakage was suppressed in the group treated with primaquine diphosphate. A significant difference was also obtained in the quantified value (Fig. 4D). (P <0.001)

Experimental Example 5: Confirmation of the effect of primaquine diphosphate on vascular permeability of diabetic retinopathy

To analyze the effect of primaquine diphosphate on vascular permeability in diabetic retinopathy in vivo, a diabetic mouse model (DM) was created by inducing hyperglycemia in C57BL/6J mice using streptozotocin. Primaquine diphosphate 0.5, 1, 5 mpk was orally administered to these mice for 10 days. 24 hours after the last oral administration, 70 kDa FITC-dextran (Sigma (30 mg/ml in FDW)) was injected into the left ventricle. FITC-Dextran was allowed to circulate for about 5 minutes and then the eyes were enucleated and immediately fixed in 4% PFA. Then, the retina was removed, flat mounted, and observed under a fluorescence microscope.

As a result, it was confirmed that the group orally administered with 1mpk of primaquine diphosphate inhibited DM-induced retinal vascular permeability very effectively (FIG. 5A).

This was also confirmed in the quantified values (FIG. 5B). (P<0.01) Quantitative value (Grade) was expressed as the product of the number of leaks and the amount of leaks found in the retinal blood vessels.

Experimental Example 6: Confirmation of cell death and proliferation of knockdown USP1

When primaquine diphosphate was added and predicted at the target prediction site, several candidate genes were derived, and among them, it was confirmed that USP1 (Ubiquitin specific peptidase 1) was affected while identifying genes with high expression in endothelial cells. MTT assay was performed to confirm cell death and proliferation when USP1 was knocked down. Control vascular endothelial cells and USP1-knockdown vascular endothelial cells were cultured for 24 hours using 20% M199 containing FBS at (3.2±0.2) × 10 ⁴ cells/well in a collagen-coated 24-well plate. When the saturation of the cells reached about 70%, they were divided into a group treated with nothing, a group treated with 5 μM primaquine diphosphate in SFM, and a group treated with VEGF, and cultured for 2 days. After 2 days, the cells were incubated in MTT solution for 4 hours and the degree of apoptosis was measured.

As a result, it was confirmed that cells proliferated in USP1-knockdown vascular endothelial cells compared to control vascular endothelial cells, and in the VEGF-treated group, the role of VEGF in USP1-knockdown vascular endothelial cells compared to control vascular endothelial cells It was confirmed that it blocked (FIG. 6). (**P<0.01, ***P<0.0001)

Experimental Example 7: Confirmation of cell permeability inhibitory effect of siUSP1

In order to confirm the cell permeability inhibitory effect of small interfering USP1 (siUSP1), permeability assay was performed using TEER assay and FITC-dextran permeability assay. Control vascular endothelial cells and USP1-knockdown vascular endothelial cells (6.0±0.5) × 10 ⁴ cells/well were cultured for 2 days using 10% EGM on a collagen-coated transwell to form a saturated monolayer. After starvation using SFM for 2 hours, VEGF 30 ng/ml was treated for 30 minutes to induce the permeability of the HUVEC monolayer.

As a result, the electrical resistance value decreased because the permeability of the cell wall increased when treated with VEGF compared to the control group. It was confirmed that the value increased (FIG. 7A). (*P<0.05, ***P<0.001)

In addition, FITC leakage increased when USP1 was knocked down compared to control vascular endothelial cells, whereas FITC leakage decreased when USP1 knocked down vascular endothelial cells were treated with VEGF compared to VEGF-untreated vascular endothelial cells. could (Fig. 7B). (*P<0.05, ****P<0.0001)

Experimental Example 8: Confirmation of the effect of siUSP1 on cytoskeletal changes

It is known that vascular permeability is affected by the stability of adhesion junctions between vascular cells. siCON HUVECs and siUSP1 HUVEcs were cultured in 35 mm dishes using 10% EGM for 2 days to saturation. After starvation with SFM for 2 hours, VEGF 50 ng/ml was treated for 30 minutes to induce cell permeability.

As a result, siUSP1 not only prevented VEGF from destabilizing VE-cadherin, but also improved the stability of VE-cadherin between cells (FIG. 8A).

In addition, structural changes of actin constituting the cytoskeleton of siUSP1 were investigated.

As a result, it was confirmed that siUSP1 suppressed the formation of actin stress fibers induced by VEGF and increased the outer actin ring structure (FIG. 8B).

Experimental Example 9: Blocking effect of USP1 inhibitors on vascular leakage

Since the vascular leakage blocking effect of siUSP1 was previously confirmed, an experiment was conducted to see whether the same effect was shown when USP1 was knocked down as well as when an inhibitor of USP1 was used. MTT assay was performed to confirm cell death and proliferation of USP1 inhibitors ML-323 and SJB2-043. Vascular endothelial cells were cultured for 24 hours using 20% M199 containing FBS at (3.0±0.2) × 10 ⁴ cells/well in a collagen-coated 24-well plate. When the cell confluency reached about 70%, ML-323 and SJB2-043 were treated with SFM at 1, 5, 10, and 20 µM and cultured for 2 days. After 2 days, the cells were incubated in MTT solution for 4 hours and the degree of apoptosis was measured.

As a result, it was confirmed that cells proliferated significantly in the group treated with 1 μM of both USP1 inhibitors (FIG. 9). (P<0.0001)

Experimental Example 10: Confirmation of cell permeability inhibitory effect of USP1 inhibitor

To confirm the cell permeability inhibitory effect of SJB2-043 and ML-323, permeability assay was performed using TEER assay and FITC-dextran permeability assay. Vascular endothelial cells (5.5±0.5) × 10 ⁴ cells/well were cultured for 2 days using 10% EGM on a transwell coated with collagen to form a saturated monolayer. After starvation using SFM for 2 hours, treatment with 5 μM primaquine diphosphate and pre-incubation for 30 minutes, VEGF 30 ng/ml was treated for 30 minutes to induce permeability of the HUVEC monolayer.

As a result, when only VEGF was treated compared to the control group, the electrical resistance value decreased because the permeability of the cell wall increased, whereas when VEGF and SJB2-043 were treated together, the electrical resistance value significantly increased compared to the VEGF only treated group. was confirmed (FIG. 10A). (P<0.05)

Also, ML-323 showed the same result (FIG. 10B). (P<0.005)

In addition, it was confirmed that the amount of FITC leakage increased when only VEGF was treated compared to the control group, whereas the amount of FITC leakage decreased when VEGF and SJB2-043 and ML-323 were treated together compared to the VEGF only treated group (FIG. 10C and 10D). (****P<0.0001, *P<0.05)

Experimental Example 11: Confirmation of the effect of USP1 inhibitor on cytoskeletal change

It is known that vascular permeability is affected by the stability of vascular cell-to-vascular adhesion junctions (AJ). HUVECs were cultured in 35 mm dishes using 10% EGM for 2 days to saturation. After starvation with SFM for 2 hours, ML-323 and SJB2-043 were treated with 1 uM and pre-incubated for 30 minutes, followed by treatment with VEGF 50 ng/ml for 30 minutes to induce cell permeability.

As a result, it was confirmed that the two USP1 inhibitors not only prevented VEGF-induced destabilization of VE-cadherin, but also improved the stability of VE-cadherin between cells (FIG. 11A).

In addition, it is known that changes in the structure of actin constituting the cytoskeleton are closely related to the permeability of vascular endothelial cells. When the permeability of vascular endothelial cells increases, the formation of actin stress fibers increases and the outer actin ring structure decreases. Saturated HUVECs were treated with 1 μM of ML-323 and SJB2-043 and pre-incubated for 30 minutes, then treated with VEGF 50 ng/ml for 30 minutes to induce cell permeability. Cells were then observed under a confocal microscope (Carl Zeiss).

As a result, it was confirmed that the USP1 inhibitors ML-323 and SJB2-043 inhibited the formation of actin stress fibers induced by VEGF and increased the sheath actin ring structure (FIG. 11B).

Claims (1)

A pharmaceutical composition for preventing or treating diabetic retinopathy, comprising one or more USP1 inhibitors selected from the group consisting of antisense oligonucleotides and siRNAs for the USP1 gene, wherein the USP1 inhibitor inhibits VEGF activity.

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