CN117797148A

CN117797148A - Use of GNE-495 in the treatment of autosomal dominant polycystic kidney disease

Info

Publication number: CN117797148A
Application number: CN202410233936.2A
Authority: CN
Inventors: 李清刚; 陈香美; 李孟飞; 尚顺来; 肖昀明; 孙铭阳
Original assignee: First Medical Center of PLA General Hospital
Current assignee: First Medical Center of PLA General Hospital
Priority date: 2024-03-01
Filing date: 2024-03-01
Publication date: 2024-04-02

Abstract

The invention relates to the technical field of biomedicine, in particular to application of GNE-495 in treating autosomal dominant hereditary polycystic kidney disease. The invention researches and screens to obtain a medicine with the function of treating autosomal dominant hereditary polycystic kidney disease, GNE-495.GNE-495 can inhibit damage of diseases to kidney structures by inhibiting vesicle formation and growth in the kidneys of patients, so that the effect of effectively treating polycystic kidney disease is achieved. The application of the GNE-495 provided by the invention in the treatment of autosomal dominant genetic polycystic kidney disease has important significance in the field of kidney disease treatment.

Description

Use of GNE-495 in the treatment of autosomal dominant polycystic kidney disease

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of GNE-495 in treating autosomal dominant hereditary polycystic kidney disease.

Background

Autosomal Dominant Polycystic Kidney (ADPKD) is one of the common hereditary kidney diseases with a morbidity of 1/400 to 1/1000. About half of ADPKD patients progress to End Stage Renal Disease (ESRD) before age 60, requiring renal replacement therapy. The pathology of ADPKD changes to a hyperproliferation of tubular epithelial cells, while including hyperproliferation of macrophages and fibroblasts, leading to inflammation and fibrosis, ultimately exacerbating ADPKD progression. Related genetic studies indicate that 85% of ADPKD is derived from PKD1 gene mutations and 15% is derived from PKD2 gene mutations.

However, the pathogenesis of ADPKD is still unknown, and similar pathological characteristics and signal disorders exist between ADPKD and tumors, such as continuous activation of proliferation signals, escape of growth inhibition, cell death resistance and the like. Thus, cystic disease has been studied in recent years in the context of tumor biology, and preclinical and clinical studies suggest that antitumor drugs have preventive and therapeutic prospects for ADPKD.

Tolvaptan is a currently approved drug for the treatment of ADPKD that delays cyst growth by antagonizing AVPR2 (vasopressin receptor) and inhibiting cAMP signaling in the cyst epithelium. Although studies have demonstrated that tolvaptan can significantly improve kidney function, patients are at risk of liver injury after prolonged administration. There is therefore an urgent need to find therapeutic agents that are effective against ADPKD with few side effects.

Disclosure of Invention

In order to solve the problems existing in the prior art, the invention provides the application of GNE-495 in treating autosomal dominant genetic polycystic kidney disease.

There are studies showing that ADPKD development and progression is associated with excessive activation of multiple downstream signaling pathways (e.g., ras/MAPK, mTOR, wnt, etc.), which lead to epithelial hyperproliferation, abnormal electrolyte trafficking, and resultant cyst formation and expansion. However, the more specific pathogenesis is not yet fully defined, and although studies in the prior art focus on these pathways, and these signaling pathways are the main targets for the discovery of new drug intervention targets, due to the complexity of ADPKD disease mechanisms, there is no clear indication of how intervention of these pathways can have an effect of effectively treating ADPKD.

In a first aspect, the invention provides the use of GNE-495 in the treatment of autosomal dominant polycystic kidney disease.

The invention further provides the use of GNE-495 in the manufacture of a medicament for the treatment of autosomal dominant polycystic kidney disease.

Further, the application includes: GNE-495 treats autosomal dominant polycystic kidney disease by inhibiting vesicle production and growth.

Further, the autosomal dominant polycystic kidney disease is triggered by the Pkd gene.

Further, the application includes:

for patients with autosomal dominant polycystic kidney disease, 20-1000 mg/kg GNE-495 is administered.

In a second aspect, the invention provides a medicament for treating autosomal dominant polycystic kidney disease comprising GNE-495.

Further, the method further comprises the following steps: a pharmaceutically acceptable carrier.

Further, the concentration of GNE-495 in the medicine is 20-800 nM.

Further, the dosage form of the medicament comprises: one or more of tablets, capsules, granules, oral liquid, injection powder injection, spray or suppository.

The invention has the following beneficial effects:

the invention researches and screens to obtain the medicine GNE-495 with effective treatment effect on autosomal dominant hereditary polycystic kidney disease. GNE-495 can ensure the integrity of kidney tissue results and improve by inhibiting the formation and growth of vesicles, and has remarkable treatment effect on autosomal dominant genetic polycystic kidney disease. The GNE-495 provided by the invention has important application value in the field of treating autosomal dominant genetic polycystic kidney disease and has important significance in the field of treating kidney related diseases.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is the effect on renal cell of the blank, control, yang, and various experimental groups provided in example 1 of the present invention.

FIG. 2 shows the results of the changes in the renal tissue structures of mice in the experimental and control groups provided in example 2 of the present invention; wherein the experimental group results are the treatment results of GNE-495 of 50 mg/kg.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental methods referred to in the examples below, unless otherwise indicated, may be carried out by methods conventional in the art. The experimental materials referred to in the examples below are commercially available unless otherwise specified.

Some of the reagents involved in the following experimental examples:

forskolin (Forskolin, also known as Coleonol) is an inducer of intracellular cAMP formation, available from MedChemExpress biotechnology company, usa, cat: HY-15371.

Rapamycin (Sirolimus) is a potent and specific mTOR inhibitor available from MedChemExpress biotechnology, usa under the designation: HY-10219 acts on HEK293 cells to inhibit mTOR with an IC50 of 0.1nM.

GNE-495 is a selective MAP4K4 inhibitor available from MedChemExpress Biotech, inc., USA under the trade designation HY-100343, IC50 value 3.7 nM, and packaging amount 5mg.

PF-06260933 is an orally active, highly selective MAP4K4 small molecule inhibitor available from MedChemExpress Biotech, inc., USA under the trade designation: HY-19562, IC50 values of 3.7 and 160 nM in kinase and cell experiments, respectively, and packaging amount of 10 mg.

Upprosertib (GSK 2141795) is an effective selective broad-spectrum inhibitor of Akt, available from MedChemExpress Biotech, inc. of America under the trade designation HY-15965, inhibits the activity of Akt1/Akt2/Akt3, has an IC50 value of 180/328/38 nM, respectively, and is packaged in a quantity of 10 mg.

DS-6051b is a new generation of selective ROS1/NTRK inhibitors, available from Selleck, inc., cat#: s8901, ic50 for ROS1, trkA, trkB and TrkC were 0.207 nM,0.622 nM,2.28 nM and 0.980 nM, respectively, in 5mg pack.

BAY1125976 is a selective allosteric AKT1/2 inhibitor with a strong efficacy against AKT signal dependent tumor growth in a mouse model. Purchased from Selleck corporation under the trade designation: s8500, BAY1125976 with respect to AKT1, AKT2 and AKT3 with respect to ic50 at 10 μm ATP are respectively denoted by 5.2 nM, 18 nM and 427 nM, BAY1125976 with respect to AKT1 and AKT2 with respect to ic50 at 2 mM ATP are respectively denoted by 44 nM and 36 nM, and the packaging amount is 25 mg.

The preparation method of the GNE-495 injection for animal experiments comprises the following steps: first, GNE-495 powder 1mg was prepared by adding DMSO to 20. Mu.l to prepare a 50mg/ml mother solution. Then, 10. Mu.l of mother solution is taken, 200. Mu.l of PEG300 and 25. Mu.l of Tween-80 stock solution are added, and 265. Mu.l to 500. Mu.l of physiological saline is added to prepare 100. Mu.g/100. Mu.l of GNE-495 injection.

Example 1

1. Construction of kidney-like organs using human pluripotent stem cells ((hiPSCs)

1. 1ml of matrigel (Basement Membrane Matrix, purchased from Beijing Seebeck Biotechnology Co., ltd.) was sucked by a pipette, transferred into a cell culture dish, and shaken well to uniformly distribute the matrigel over the bottom of the dish.

2. The matrigel in the new dish used in step 1 was removed, 2ml PSC-Easy complete medium was added, and the mixture was placed in a pre-heat at 37 ℃. Old dishes (cells in which the state of the cells was observed well under a microscope, the cell confluency was 80 to 90%) of human pluripotent stem cells (hiPSCs, experimental primordial cells purchased from beijing seebec biotechnology limited) which had been continuously cultured were removed, medium without feeder layer in the dishes was removed (pipetted, discarded), 1ml of trypsin EDTA (0.25%) was pipetted into the cell dishes, and the cells were left at 37 ℃ for 3min. Then, the trpsin EDTA was removed by blotting and removing the remaining trpsin EDTA as thoroughly as possible. 1ml of feeder-free medium was aspirated with a pipette, transferred to a cell culture dish, and the cells were blown. The human pluripotent stem cells separated from the original culture surface are obtained and inoculated into a new culture dish according to the ratio of 1:8, and are uniformly shaken horizontally. The majority of the cell clusters were microscopically confirmed to be about 15 cells in size and about 18% in cell density. Culturing at 36-37.5 ℃, replacing the culture medium without the feeder layer for 1 time every day, and carrying out passage once in 4 days.

3. And when the cell reaches 40-50% confluence, the day 0 is defined, the feeder layer-free culture medium is removed, and a basic culture medium (STEMdiff APEL Medium) containing cytokines is added to culture and differentiate the human pluripotent stem cells. Basal medium containing 8. Mu.M of CHIR99021 was used on day 0, basal medium containing 8. Mu.M of CHIR99021 was changed once on day 2, basal medium containing 200ng/ml of FGF9+1. Mu.g/ml of heparin was used starting on day 4, and medium was changed every 2 days (containing 200ng/ml of FGF9+1. Mu.g/ml of heparin). The amount of medium used was about 2ml each time the medium was changed.

4. On day 7, 1ml of trypsin EDTA (0.25%) was pipetted with a pipette, transferred into a cell culture dish and left at 37℃for 3min. And then sucking the trypsin EDTA, removing the residual trypsin EDTA as much as possible, and avoiding sucking up white cell clones in the sucking process. 1ml of culture medium without a feeder layer is sucked by a pipette and transferred into a cell culture dish, and cells are blown, wherein the blowing action is gentle and slow, and the total times are less than 10 times. Cells were collected in 15ml centrifuge tubes and centrifuged (400 g,3 min).

5. The medium was removed (as depleted as possible) until only one cell sphere remained. Cells were resuspended in 1ml basal medium. Cell counts were performed by taking 10. Mu.l of cell suspension. Each kidney-like body has a size of about 5X 10 ⁵ The number of cells in a cell is one,the desired cell suspension was aliquoted into 1.5 ml Eppendorf tubes. The Eppendorf tube (400 g,2 min) was centered. 1.2ml of basal medium containing 5. Mu.M CHIR99021 was added to the lower chamber of a 6 well Transwell polyester film cell culture apparatus. The Transwell chamber (upper chamber) was attached to the surface of the medium. The cell spheres were picked up at the large caliber end of the P1000 or P200 gun head and placed on a Transwell chamber. Incubating the cell spheres for 1h at 36-37.5 ℃. After 1h, the basal medium containing 5. Mu.M CHIR99021 was removed and 1.2ml basal medium containing 200ng/ml FGF9+1. Mu.g/ml heparin was used. Basal medium containing 200ng/ml FGF9+1. Mu.g/ml heparin was changed every 2 days during the culture. The culture is continued by changing the basal medium without cytokines every 2 days from the 12 th day, and the co-culture is carried out for more than 18 days. After construction of kidney-like bodies, cell type verification was performed using immunofluorescent staining methods. Adding proper volume of frozen stock solution into mature kidney-like body, transferring organ-like suspension into frozen stock tube, placing into gradient program cooling box, placing into refrigerator at-80deg.C overnight, and transferring into liquid nitrogen container for long-term storage the next day.

Through the steps, the hiPSC cell strain is differentiated to obtain the kidney progenitor cells and finally matured into kidney, and the kidney organoids are formed for the next drug screening.

2. Inhibition of the kidney organoids of polycystic kidney by GNE-495

1. Cell resuscitation

100mL of DMEM/F12 medium (available from Biological Industries, cat# 01-172-1 ACS) containing 3% FBS,0.75 mu g l-1 gamma-interferon (available from Sigma-Aldrich, cat# I4777), 1g l-1 insulin, 0.67 mg l-1 sodium selegite, 0.55 g l-1 transferin (available from Thermo Fisher, cat# 41400045), 0.1 mu M3, 5-triedo-l-thronine (available from Sigma-Aldrich, cat# T6397) and 1% penicillin-streptomycin (available from Gibco, cat# 15140-122) was prepared. After the preparation, the culture medium is placed in a 37 ℃ water bath kettle, and after the culture medium is in a water bath for a moment, the culture medium is moved into 8mL of a centrifuge tube and is moved into 10mL of each of 3 culture bottles. The freezer tube was removed from the liquid nitrogen and rapidly placed in a 37℃water bath for thawing, and the cells were removed into the centrifuge tube as soon as possible (about 1 min) after thawing. After centrifugation 1000bmp for 5min, the supernatant was discarded and the bottom of the centrifuge tube was flicked to loosen the cell pellet. 6mL of culture medium is slowly added, shaking is carried out while adding, after the culture medium is uniformly distributed, 2mL of each of 3 culture flasks is added, and the culture flasks are slowly and uniformly shaken. Cells were observed for uniformity of distribution and then incubated in a 37℃incubator.

2. Organoid drug screening

Control groups (blank, 20. Mu.M Forskolin control), 5 experimental groups (PF-06260933, GNE-495, uprosertib, DS-6051b, BAY 1125976), yang reference group (20 nM Rapamycin) were established and tested for half-inhibitory concentration (IC 50):

(1) Cell culture in DMEM (# 11965-092) contains 10% FBS,10 mM HEPES (# 15630-080), 1 XMEM NEAA (# 11140-050), 600 μg/mL Geneticin (# 10131-027) and 1000U/mL Penicillin/Streptomycin (# 15140-122,Life Technologies).

(2) For culturing and seeding, cells were washed with non-cationic DPBS (available from Lonza, cat# 17-512Q) and incubated with Versen (available from Life Technologies, cat# 15040-066) at 37℃for 5min at room temperature. 5mL of 0.25% Trypsin-EDTA (available from Life Technologies, cat# 25200-056) was added and the flask was gently shaken to loosen the cells. Cells were passaged at 1:15 in complete medium.

(3) Transient transfection: 35000 cells per well were seeded into 96-well plates with a volume of 100 μl per well. After 15 minutes at room temperature, they were transferred to 37℃with 5% CO ₂ The incubator was maintained for 6 hours. For each well of cells, 25. Mu.L of FuGENE HD transfection reagent (available from Promega, cat# E2311) was added to 500. Mu.L of OptiMEM medium (available from Life Technologies, cat# 31985-070) pre-warmed to 37℃and incubated for 5 minutes at room temperature. 8.75 μg of pcDNA3.1-traf2-FLAG tag (NM-001242559.1) and 3.5 μg of pcDNA3.1-MAP4K4-myc-Histag (NM-021138.3) were added to the OptiMem mixture, gently mixed, and incubated for 15 minutes at room temperature. The DNA-Fugene mixture was diluted with 2.625 mL OptiMem, and 25 μl was added to each well. Shaking the plate back and forth, gently mixing, and returning to 37℃with 5% CO ₂ Incubators were incubated for 48 hours. Cells were treated with drugs dissolved in DMSO, diluted in complete mediumThe final DMSO concentration was 0.25%. Culture plate 37 ℃,5% CO ₂ Is cultured in an incubator for 1.5 hours. The medium was then aspirated and washed with 150. Mu.L/well of cation-free PBS. 150 μl/well HNGT Buffer (50 mM HEPES pH 7.5, 150 mM NaCl,10% glycerol, 1% triton-X) was then added, followed by 1X haltproterase/Phosphatase Inhibitor Cocktail (available from Pierce, cat# 78447). The cell plates were heat sealed and placed on a rotating plate shaker for 15 minutes. The cell plates were centrifuged at 3000 rpm for 5min and placed in a-80 ℃ freezer until an enzyme-linked immunosorbent assay was performed.

(4) Preparing an ELISA plate the day before detection; anti-FLAG antibody (Sigma #f3165) was formulated at 5 μg/mL in 0.5M carbonate/bicarbonate buffer (pH 9.6) and added to 96-well plates at 75 μl/well. Incubate overnight at 4 ℃. On the day of the experiment, cell plates were placed and washed on an ELX-405 plate washer using a wash buffer (1 XTBS/0.05% Tween-20buffer; 5X 100. Mu.L/well). At room temperature, the cells were blocked with 75. Mu.L/well of 5% FBS in the wash solution for 1 hour. The reaction solution in the wells was blotted and 50. Mu.L/well of thawed cell lysate was added thereto. Incubate for 1 hour at room temperature. After washing the cell plates 5 times with 100. Mu.L/well of wash solution, 50. Mu.L/well of anti-Phospho-Thronine antibody (from Cell Signaling Technology, cat. No. 9381 s) diluted 1:1500 in wash solution was added; incubate for 1 hour at room temperature and wash as before. Add donkey anti-rabbit antibody (ex Jackson, cat# 711-036-152) diluted 1:4000 in wash solution at 50. Mu.L/well; incubate for 1 hour at room temperature. Washing is the same as before. Add 1x SuperSignal ELISA Pico Substrate (available from Pierce, cat# 37069) 50 μl/well. After 5 minutes incubation, the microplate reader reads.

Percent inhibition was calculated from sample wells containing DMSO alone (0% inhibition) or standard compound (100% inhibition). The compound concentration response was in accordance with a four parameter equation to determine compound IC50 values. Dosing was performed according to the results of the concentration test used for Forskolin-induced vesicle production and Rapamycin-inhibited vesicle production. The experimental group was set with 2 concentrations of each agent, with a low concentration of 10 times the drug IC50 and a high concentration of 50-100 times the drug IC50, and the groupings are shown in table 1. The repeated 3 duplicate wells of each group, with fresh drug replacement every 24h, were continuously applied for 8 days, and the structure of each group of kidney units was observed under the mirror every day, and the results were shown in FIG. 1, in which PF-06260933 (especially 20. Mu.M) and Utroservib (especially 4. Mu.M) were optimal in the inhibition of vesicles, but had a significant effect on the differentiation structure. DS-6051b and BAY1125976 inhibited vesicles less effectively than the other groups. Only GNE-495 has better vesicle inhibition effect, and meanwhile, the differentiation structure is not influenced, and the 400nM GNE-495 effect is better than 40nM GNE-495.

Table 1 specific drug amounts for each group

Example 2

The present example further constructs a PKD mouse model to determine the in vivo effect of GNE-495 in inhibiting vesicle growth, comprising the following steps:

1. establishing a polycystic kidney mouse model:

for this study, C57BL6 Pkd cond/cond mice were crossed with C57BL6 tamoxifen-Cre (B6.Cg-Tg [ Cre/Esr1 ]) 5Amc/J mice (available from Jackson Laboratories, cat# 004682). To establish an early-onset polycystic kidney mouse model, cre recombinase activity was induced by intraperitoneal injection of tamoxifen (10 mg/kg) (Sigma, st. Louis, MO) on day 10 (P10) after birth of the mice. To establish a chronic PKD mouse model, mice were induced for Cre recombinase activity by injection of tamoxifen (total dose: 300 and 100 mg/kg, 3 consecutive days) on day 30 post-natal (P30) of the mice.

2. Inhibition of vesicle growth in a polycystic kidney mouse model by GNE-495

Preparation of GNE-495 stock: GNE-495 was dissolved in DMSO solution. Storage mode and period of stock solution: -80 ℃ for 6 months, -20 ℃ for 1 month.

The working solution is prepared by adding 10% DMSO stock solution and 90% corn oil, mixing and clarifying. The specific dosage may be calculated using a formulation calculator. The working solution is prepared immediately before use and is used in the same day. The clear solution obtained in the previous step must be ensured before the next step, and physical methods such as vortex, ultrasonic or water bath heating and the like can be adopted for dissolution assistance. Pkd1f/f-Cre transgenic polycystic kidney mice model was divided into three groups (n=7) and GNE-495 (50 mg/kg) and DMSO (control group) were administered, respectively. Treatment was initiated on day 11 for 3 weeks with intraperitoneal injections every week for five consecutive days, two days apart. WT mice were equally divided into three groups (n=7) and received the same treatment as described above. A total of 6 groups.

After the treatment, the kidney tissues are subjected to histopathological observation, 3 groups of Pkd f/f-Cre mouse kidney tissues treated by DMSO or GNE-495 are taken, the kidney tissues are fixed for 24 hours by using a 10% neutral formaldehyde solution in volume fraction, then ethanol is dehydrated, paraffin is embedded, and the kidney tissues are sectioned, HE staining and PAS staining are carried out, the histopathological changes of the kidney tissues are observed under an optical microscope, and the staining results of the sections are shown in figure 2: in the control mice, there were a large number of vesicles in the kidneys of the mice, the tissue structure was severely destroyed, the kidneys of the mice were significantly reduced by treatment with GNE-495, and the kidney tissue structure was improved. In addition, the results of the weight test performed on the mice in each group showed that there was no significant difference in the weight of the mice between the groups.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

Use of gne-495 for the treatment of autosomal dominant polycystic kidney disease.
Use of gne-495 for the manufacture of a medicament for the treatment of autosomal dominant polycystic kidney disease.
3. The use according to claim 1 or 2, wherein said treating autosomal dominant polycystic kidney disease comprises: GNE-495 treats autosomal dominant polycystic kidney disease by inhibiting vesicle production and growth.
4. The use according to claim 1 or 2, wherein the autosomal dominant polycystic kidney disease is triggered by the Pkd gene.
5. The application according to claim 1 or 2, characterized in that it comprises:

for patients with autosomal dominant polycystic kidney disease, 20-1000 mg/kg GNE-495 is administered.
6. A medicament for treating autosomal dominant polycystic kidney disease comprising GNE-495.
7. The medicament according to claim 6, further comprising: a pharmaceutically acceptable carrier.
8. The medicament of claim 6, wherein the concentration of GNE-495 in the medicament is 20nM to 800 nM.
9. The medicament according to any of claims 6 to 8, wherein the dosage form of the medicament comprises: one or more of tablets, capsules, granules, oral liquid, injection powder injection, spray or suppository.