Application of Cpd1 in preparation of medicine or combined medicine for treating diabetic retinopathy

Abstract

The invention discloses an application of Cpd1 in preparation of a medicine or a combined medicine for treating diabetic retinopathy, the agonist Cpd1 of GPR61 increases the retinal thickness of a mouse with diabetic retinopathy and reduces formation of microaneurysm, and the Cpd1 reduces activation and proliferation of retinal Muller cells induced by high glucose and release of proinflammatory cytokines, so that the effect of treating diabetic retinopathy is achieved. Compared with the existing anti-VEGF drug therapy, the Cpd 1 does not need to be injected repeatedly, so that the economic burden of NPDR patients and the trouble of re-diagnosis are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an application of Cpd1 in the preparation of a medicine or a combination medicine for treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is one of the most serious and common complications of diabetes, which may eventually lead to irreversible visual impairment and is the leading cause of blindness among the working-age population (15-64 years old). 1 DR can be roughly divided into two stages, including nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). The first-line treatment for severe NPDR includes regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs. 2 Although early treatment of NPDR with anti-VEGF drugs can significantly reduce the incidence of PDR and subsequent blindness 3 However, treatment is usually initiated only after NPDR patients develop visual impairment. 4 , as anti-VEGF therapy is associated with high levels of economic burden, patient-reported anxiety, and an uncommon but serious risk of endophthalmitis 5 Therefore, it is imperative to explore better treatment strategies for NPDR.

[0003] G protein-coupled receptor 61 (GPR61) is an orphan receptor that is closely related to biogenic amine receptors and is mainly expressed in the pituitary and appetite regulation centers of the hypothalamus and brainstem. 6,7 Mutational and human genome-wide association studies have linked GPR61 to type 2 diabetes and body mass index-related phenotypes 8,9 , making it a potential target for regulating appetite and body weight. However, the role of GPR61 in CNV is still unclear. GPR61 inverse agonist 1 (GPR61Inverse agonist 1), also known as compound 1 (Cpd 1), is a newly discovered GPR61 agonist. 10 .

[0004] The present application found that Cpd1 increased retinal thickness in diabetic retinopathy mice and reduced the formation of microaneurysms. In addition, Cpd1 reduced high glucose-induced retinal Müller cell activation, proliferation, and the release of proinflammatory cytokines.

[0005] References

[0006] 1.Kropp, M. et al. Diabetic retinopathy as the leading cause of blindness and early predictor of cascading complications-risks and mitigation. EPMA J 14, 21–42 (2023).

[0007] 2. Simó, R. & Hernández, C. New Insights into Treating Early and Advanced Stage Diabetic Retinopathy. Int J Mol Sci 23, (2022).

[0008] 3.Ding,

[0009] 4. Chauhan, MZ, Rather, PA, Samarah, SM, Elhusseiny, AM & Sallam, ABCurrent and Novel Therapeutic Approaches for Treatment of Diabetic MacularEdema. Cells 11, 1950 (2022).

[0010] 5.Rennie,C.,Lotery,A.,Payne,J.,Singh,M.&Ghanchi,F.Suboptimal outcomesand treatment burden ofanti-vascular endothelial growth factor treatment fordiabetic macular oedema in phakic patients.Eye 38,215–223(2024).

[0011] 6.Lee,D.K.et al.Identification offour novel human G protein-coupledreceptors expressed in the brain.Brain Res Mol Brain Res 86,13–22(2001).

[0012] 7.Cikos,S.,Gregor,P.&Koppel,J.Cloning of a novel biogenic aminereceptor-like G protein-coupled receptor expressed in human brain1.Biochimicaet Biophysica Acta(BBA)-Gene Structure and Expression 1521,66–72(2001).

[0013] 8.Yuan,W.et al.An integrated epigenomic analysis for type 2diabetessusceptibility lociin monozygotic twins.Nat Commun 5,5719(2014).

[0014] 9.Felix,J.F.et al.Genome-wide association analysis identifies threenew susceptibility loci for childhoodbody mass index.Hum Mol Genet 25,389–403(2016).

[0015] 10. Lees, JA et al. An inverse agonist oforphan receptor GPR61 acts by a G protein-competitive allosteric mechanism. Nat Commun 14, 5938 (2023). Summary of the invention

[0016] Technical issues solved:

[0017] The present application aims at the deficiencies of the prior art, solves the technical problems such as the lack of effective and economical treatment methods for NPDR, and provides an application of Cpd1 in the preparation of a drug or a combination drug for treating diabetic retinopathy.

[0018] Technical solution:

[0019] To achieve the above objectives, this application is implemented through the following technical solutions:

[0020] A use of Cpd1 in preparing a medicine or a combination medicine for treating diabetic retinopathy.

[0021] Furthermore, the drug or combination drug is in an injectable or non-injectable dosage form.

[0022] Furthermore, the drug or drug combination is a solution, suspension, injection or spray.

[0023] Furthermore, the drug or combination drug for treating diabetic retinopathy is administered via intravenous, transdermal, mucosal or intravitreal injection.

[0024] Furthermore, the drug or combination drug for treating diabetic retinopathy is administered once a day, once every two days, once every three days, once a week, once a month or once a year during a period of one day to one year.

[0025] Furthermore, the dosage range of Cpd1 is 5-20 μg / individual body weight (kg).

[0026] Beneficial effects:

[0027] The present application provides an application of Cpd1 in the preparation of a drug or a combination drug for treating diabetic retinopathy, which has the following beneficial effects compared with the prior art:

[0028] 1. The GPR61 agonist Cpd1 increases retinal thickness and reduces microaneurysm formation in mice with diabetic retinopathy;

[0029] 2. Cpd1 reduces high glucose-induced retinal Müller cell activation, proliferation, and release of proinflammatory cytokines;

[0030] 3. Compared with existing anti-VEGF drug treatments, Cpd 1 does not require repeated injections, reducing the economic burden and trouble of follow-up visits for NPDR patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing that Cpd 1 alleviates DR-induced reduction in mouse retinal thickness in this application, wherein A is a HE staining graph of mouse retinal tissue, and B is a statistical analysis graph of mouse retinal tissue thickness;

[0032] Figure 2 In this application, Cpd 1 reduces the formation of microaneurysms in the retina of mice induced by DR;

[0033] Figure 3 This is a diagram of Cpd 1 reducing HG-induced mouse RMCs activation in this application, wherein A is a WB detection of GFAP protein levels in mouse retinal tissue, and B is a statistical analysis of GFAP protein levels;

[0034] Figure 4 The figure of the present application shows that Cpd 1 inhibits HG-induced proliferation of mouse RMCs, wherein A is a proliferation figure of mouse RMCs detected by EdU experiment, and B is a statistical analysis figure of the ratio of the number of EdU-positive cells to the number of DAPI-positive cells;

[0035] Figure 5 This is a diagram of the application showing that Cpd 1 inhibits the release of proinflammatory cytokines in mouse RMCs induced by HG, wherein A, B, and C are the concentrations of IL-6 (A), IL-8 (B), and TNF-α (C) in the culture supernatant of mouse RMCs detected by ELISA, respectively. DETAILED DESCRIPTION

[0036] The present invention will be described below in conjunction with specific embodiments. The described embodiments are only a part of the embodiments of the present invention rather than all of them, and these embodiments are only used to illustrate the present invention and are not used to limit the protection scope of the present invention, which is defined by the claims.

[0037] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0038] Cpd1 is an agonist of G protein-coupled receptor 61 (GPR61).

[0039] Example 1

[0040] High-fat diet and streptozotocin-induced type 2 diabetes model in mice and treatment:

[0041] Male C57BL / 6J mice (6-8 weeks) were fed a low fat diet (LFD; rodent diet, containing 10 kcal% fat; D12450B, Research diets, USA) or a high fat diet (HFD; 20% protein, 60% fat and 20% carbohydrate; D12492, Research diets). The mice fed with LDF were used as the normal group. The mice fed with HFD were intraperitoneally (IP) injected with 100 mg / kg of streptozotocin (STZ) dissolved in 0.1 M citrate buffer (pH 4.5) after 5 weeks of feeding. Blood glucose levels were measured one week after STZ injection, and mice with random blood glucose levels higher than 16.7 mmol / l were considered to be type 2diabetes mellitus (T2DM) mice. 17 weeks after STZ injection, mice were intravitreally injected with 5, 10 or 20 μg of Cpd1 (HY-156354, MedChemExpress, USA) once a day for one week. One week after Cpd1 injection, mice were anesthetized with 2% isoflurane inhalation and killed by cervical dislocation.

[0042] Hematoxylin-eosin (HE) staining of mouse retinal tissue:

[0043] Mouse eyeballs were fixed in 4% paraformaldehyde (PFA), embedded in paraffin and cut into 4 μm sections. After dewaxing, HE staining was performed and the slides were observed under an optical microscope. Retinal thickness was measured using Image-Pro Plus 6.0 software.

[0044] Immunofluorescence staining of mouse retinal tissue:

[0045] Mouse eyeballs were fixed in 4% PFA at room temperature for 40 minutes. Then, the retina was dissected under a dissecting microscope and incubated with 4% PFA overnight at 4°C. Next, the retina was removed and incubated in 70% ethanol / PBS for 20 minutes, and then incubated in 1% TritonX-100 / PBS for 20 minutes. The retina was then incubated overnight at 4°C in PBS containing 5mg / ml AlexaFluor-488-coupled isolectin B4 (isolectin B4, IB4, vascular marker; I21411, Thermo Fisher Scientific, USA) antibody, followed by sealing and examination under a confocal microscope.

[0046] Isolation, culture, identification and processing of primary mouse retinal Müller cells (RMCs):

[0047] Primary RMCs were isolated from 6-8 week old C57BL / 6 mice. The mice were anesthetized and sacrificed, and their retinas were dissected under sterile conditions. The shredded retinas were digested with 2% neutral protease and then incubated in 5% CO. 2 Incubate at 37°C in an incubator for 1 hour. Then transfer the digested cells to a 75 cm 2 flask and continue to culture in the incubator for 3-4 days. Mouse RMCs were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100U / mL penicillin-streptomycin, 1mM sodium pyruvate, 44mM sodium bicarbonate and 25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid, HEPES]. After cell purification, the cell morphology was observed under a phase contrast microscope, and RMCs were labeled with glutamine synthetase (GS) antibody fluorescent staining, and the fluorescent signal was photographed with a confocal microscope. In a 24-well plate, 2×10 4 RMCs / well for 24 hours. Different glucose concentrations were prepared by adding D-glucose (108337, Millipore, USA) to serum-free and glucose-free DMEM. RMCs were treated with 10 μg, 20 μg or 40 μg Cpd1 for 24 hours.

[0048] Western blot (WB) was used to detect the expression of GFAP in RMCs:

[0049] RMCs were collected, and the cells were lysed by adding radioimmunoprecipitation assay (RIPA) lysis buffer, and the total cell protein was extracted. The protein concentration was measured by bicinchoninic acid (BCA) method. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electrotransferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with Tris-buffered saline with 0.1% Tween 20 Detergent (TBST) containing 0.5% skim milk powder for 1 hour at room temperature. Primary antibodies including GFAP (sc-33673, Santa Cruz Biotechnology, USA) and GAPDH (sc-32233, Santa Cruz Biotechnology) antibodies were added and incubated overnight at 4°C. The primary antibody incubation solution was discarded, and TBST buffer containing horseradish peroxidase-conjugated goat anti-mouse IgG (ab97023, Abcam) was added and incubated at room temperature for 2 hours. The images were detected using the ImageLab chemiluminescence system.

[0050] 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay:

[0051] After culturing RMCs of different groups for 24 h, the chamber was removed from the 37°C CO2 incubator. According to the instructions of the EdU kit, 50 μM EdU culture medium was added to each well and incubated at 37°C for 2 h, and the culture medium was discarded. The cells were washed 3 times with PBS, 5 min each time. They were fixed with 4% PFA for 30 min, and the fixative was discarded. 2 mg / mL glycine was added to neutralize the excess aldehyde groups, incubated for 5 min, and the solution was discarded. Washed once with PBS, 5 min; washed once with PBST, 10 min; washed once with PBS, 5 min. Apollo stain was added, incubated for 30 min, and the stain was discarded. Washed 3 times with PBST, 10 min each. DAPI stain was added, incubated for 30 min, and the stain was discarded. Washed 3 times with PBST, 10 min each. DAPI stain was added, incubated for 30 min, and the stain was discarded. Washed 3 times with PBS, 10 min each. The slides were sealed with anti-fluorescence quenching sealing agent and stored at 4°C. Images were taken using a fluorescence microscope. The average ratio of EdU-positive cells (red) to DAPI-positive cells (blue) was analyzed as an indicator of cell proliferation activity.

[0052] Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of IL-6, IL-8 and TNF-α in the culture supernatant of RGCs:

[0053] Mouse IL-6 (A43656, Thermo Fisher Scientific, USA), IL-8 (EK732214, AFGBioscience, USA) and TNF-α (ab208348, Abcam, USA) ELISA detection kits were used to detect the protein concentrations of IL-6, IL-8 and TNF-α in the culture supernatant of RGCs according to the manufacturer's instructions.

[0054] Statistical methods:

[0055] Each data is expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments; the comparison between two groups of data was performed using the two-sided Student-t test, while the comparison between multiple groups of data was performed using one-way analysis of variance and Bonferroni post hoc test. P < 0.05 was considered statistically significant.

[0056] Example 2

[0057] Cpd 1 alleviates DR-induced reduction in retinal thickness in mice:

[0058] HE staining was used to measure the retinal thickness of mice. Compared with the normal group, the retinal thickness of mice in the DR group was reduced. Compared with the DR group, the retinal thickness of mice in the DR+5μg Cpd 1, DR+10μg Cpd 1 or DR+20μg Cpd 1 groups increased, indicating that 5, 10 or 20μg Cpd 1 can alleviate the reduction of retinal thickness in mice induced by DR. Figure 1 As shown in A and B, the present application Cpd1 alleviates the reduction of DR-induced mouse retinal thickness, wherein A is a HE staining image of mouse retinal tissue, and B is a statistical analysis image of mouse retinal tissue thickness.

[0059] *** P<0.001, compared with the normal group. ## P < 0.001, ### P<0.001, compared with the DR group.

[0060] Example 3

[0061] Cpd 1 reduces the formation of microaneurysms in the retina of DR-induced mice:

[0062] IB4 staining of the whole mouse retina showed that compared with the normal group, the IB4 positive signal in the DR group mouse retina was aggregated, indicating that DR induced the formation of microaneurysms in the mouse retina. Compared with the DR group, the aggregation of IB4 signals in the retina of the mouse in the DR+10μg Cpd 1 or DR+20μg Cpd 1 group was reduced, indicating that 10 or 20μg Cpd 1 can reduce the formation of microaneurysms in the mouse retina induced by DR, such as Figure 2 shown.

[0063] Figure 2 Figure 1 shows that Cpd 1 reduces the formation of microaneurysms in the mouse retina induced by DR. IB4 fluorescent staining of mouse retinal flat mounts. White arrows indicate microaneurysms.

[0064] Example 4

[0065] Cpd 1 reduces HG-induced activation of RMCs in mice:

[0066] WB detected the expression of GFAP (RMCs activation marker) in mouse RMCs. Compared with the normal group, the expression of GFAP in the RMCs of the HG group mice increased, indicating that HG induced the activation of mouse RMCs. Compared with the HG group, the expression of GFAP in the RMCs of the HG+10μg Cpd 1, HG+20μg Cpd 1 or HG+40μg Cpd 1 groups mice decreased, indicating that 10, 20 or 40μg Cpd1 can reduce HG-induced mouse RMCs activation, such as Figure 3 As shown in A and B.

[0067] Figure 3 Figure 2 shows that Cpd 1 reduces HG-induced RMCs activation in mice, where A is WB detection of RMCs in mouse retinal tissue

[0068] A is the protein level graph of GFAP, B is the statistical analysis graph of GFAP protein level, *** P<0.001, compared with the normal group. ## P<

[0069] 0.001, ### P<0.001, compared with the HG group.

[0070] Example 5

[0071] Cpd 1 inhibits HG-induced proliferation of mouse RMCs:

[0072] The EdU experiment detected the proliferation of mouse RMCs. Compared with the normal group, the proportion of EdU-positive cells in the RMCs of mice in the HG group increased, indicating that HG induced the proliferation of mouse RMCs. Compared with the HG group, the proportion of EdU-positive cells in the RMCs of mice in the HG+10μg Cpd 1, HG+20μg Cpd 1 or HG+40μg Cpd 1 groups decreased, indicating that 10, 20 or 40μg Cpd 1 can reduce the proliferation of mouse RMCs induced by HG, such as Figure 4 As shown in A and B.

[0073] Figure 4 This is a diagram of Cpd 1 inhibiting HG-induced mouse RMCs proliferation in this application, wherein A is a diagram of EdU experiment detecting mouse RMCs proliferation, EdU (red) and DAPI (blue) double labeling; B is a statistical analysis diagram of the ratio of the number of EdU-positive cells to the number of DAPI-positive cells, *** P<0.001, compared with the normal group, ## P < 0.001, ### P<0.001, compared with the HG group.

[0074] Example 6

[0075] Cpd 1 inhibits HG-induced release of proinflammatory cytokines from mouse RMCs:

[0076] ELISA results showed that compared with the normal group, the protein concentrations of proinflammatory cytokines IL-6, IL-8, and TNF-α in the culture supernatant of RMCs in the HG group were increased, indicating that HG induced the release of proinflammatory cytokines in RMCs in mice. Compared with the HG group, the protein concentrations of IL-6, IL-8, and TNF-α in the culture supernatant of RMCs in the HG+10μg Cpd 1, HG+20μg Cpd 1, or HG+40μg Cpd 1 groups were decreased, indicating that 10, 20, or 40μg Cpd 1 could reduce the release of proinflammatory cytokines in RMCs induced by HG, as shown in Figure 3. Figure 5 As shown in A, B and C.

[0077] Figure 5 This is a diagram of the inhibition of HG-induced release of pro-inflammatory cytokines in mouse RMCs by Cpd 1 in this application, wherein A, B, and C are the concentrations of IL-6 (A), IL-8 (B), and TNF-α (C) in the culture supernatant of mouse RMCs detected by ELISA, respectively. *** P<0.001, compared with the normal group, ## P < 0.001, ### P<0.001, compared with the HG group.

[0078] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A use of Cpd1 in the preparation of a drug or a combination drug for treating diabetic retinopathy.

2. The use of Cpd1 according to claim 1 in the preparation of a drug or a combination drug for treating diabetic retinopathy, characterized in that: The drug or combination drug is in an injectable or non-injectable dosage form.

3. The use of Cpd1 according to claim 1 in the preparation of a medicament or a combination medicament for treating diabetic retinopathy, characterized in that: The medicine or combined medicine is a solution, suspension, injection or spray.

4. The use of Cpd1 according to claim 1 in the preparation of a medicament or a combination medicament for treating diabetic retinopathy, characterized in that: The drug or combination drug for treating diabetic retinopathy is administered by intravenous, transdermal, mucosal or intravitreal injection.

5. The use of Cpd1 according to claim 1 in the preparation of a medicament or a combination medicament for treating diabetic retinopathy, characterized in that: The drug or combination drug for treating diabetic retinopathy is administered once a day, once every two days, once every three days, once a week, once a month or once a year during a period of one day to one year.

6. The use of Cpd1 according to claim 1 in the preparation of a medicament or a combination medicament for treating diabetic retinopathy, characterized in that: The dosage range of Cpd1 is 5-20 μg / individual body weight (kg).

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