CN110201171A - Application of the Fak inhibitor in treatment autosomal dominant polycystic kidney disease - Google Patents

Abstract

The present invention uses the MDCK vesicle model to prove that the FAK inhibitor can inhibit the formation and growth of vesicles, and confirms the pharmacological activity of the FAK inhibitor in the kidney through the in vitro embryonic kidney vesicle model, which can significantly inhibit the development of vesicles in the kidney Finally, it was further proved in the polycystic kidney mouse model that FAK inhibitors also have the effect of inhibiting the development of vesicles in vivo, and the above-mentioned vesicle inhibition in vitro and in vivo has a dose-effect relationship. FAK inhibitors did not affect the viability of kidney cells, indicating that the inhibitory effect of FAK inhibitors on polycystic kidney disease has nothing to do with its cytotoxicity; the regulation of intracellular signaling pathways by FAK inhibitors may be one of the important mechanisms for inhibiting the development of renal vesicles 1. The FAK inhibitor is VS4718, and it is concluded that VS4718 can be used for the treatment of autosomal dominant polycystic kidney disease.

Description

Application of FAK inhibitors in the treatment of autosomal dominant polycystic kidney disease

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of a focal adhesion kinase inhibitor, that is, a FAK inhibitor, in the treatment of autosomal dominant polycystic kidney disease.

Background technique

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic hereditary disease with an incidence rate of about 1/1000-1/400, ranking first among hereditary kidney diseases. ADPKD is characterized by multiple progressive fluid-filled vesicles in bilateral kidneys, excessive proliferation of vesicular epithelial cells, abnormal secretion of cyst fluid, and interstitial fibrosis. The gradually enlarged fluid-filled vesicles constantly squeeze the surrounding normal kidney tissue, leading to the destruction of normal kidney structure and loss of function, and eventually develop into end-stage renal failure (ESRF). The fourth cause of renal failure. Patients with ADPKD usually experience marked progressive decline in renal function in middle age, and the glomerular filtration rate decreases year by year. About 50% of ADPKD patients enter end-stage renal failure by the age of 60. At present, there is a lack of specific drugs for the treatment of ADPKD. Patients with end-stage renal failure mainly rely on dialysis or kidney transplantation to maintain their lives, which has brought a huge burden to society and families. Therefore, screening ADPKD-specific therapeutic drugs is an urgent problem to be solved. The problem.

Similar pathological features and signaling disturbances exist between ADPKD and tumors, such as: persistent activation of proliferative signals, escape from growth inhibition, and resistance to cell death. Therefore, cystic diseases have been studied in the context of tumor biology in recent years, and preclinical and clinical studies have suggested that antineoplastic agents have promise for the prevention and treatment of ADPKD. Studies have found that multiple signaling pathways are "co-activated" in tumors and ADPKD, including cAMP-PKA, PI3K-Akt, mTOR, Wnt, growth factors (EGF, TGF-β, PDGF, etc.), integrin (Integrin) and other signals, These signaling pathways play a very important role in the abnormal proliferation, apoptosis, regulation of cystic fluid secretion, interstitial accumulation and remodeling of vesicular epithelial cells. Many researchers at home and abroad regard the intervention of the above signaling pathways as the main goal of discovering new drug intervention targets. For example, the activation of the cAMP-PKA signaling pathway is considered to be an important "driving force" to promote the progression of PKD, and a large number of studies have focused on this signaling pathway. At present, tolvaptan, a vasopressin V2 receptor blocker, is the first and only drug successfully marketed for the treatment of ADPKD. It inhibits the cAMP-PKA signaling pathway and delays the progression of renal vesicle, interstitial Progression of qualitative lesions and decline in renal function. Moreover, in vitro stimulation of canine collecting duct epithelial MDCK cells or embryonic kidney with forskolin (FSK) or 8-Br-cAMP, an AC agonist of adenylyl cyclase, resulted in the formation of a large number of fluid-filled vesicles similar to those in PKD mice.

In the pathogenesis of ADPKD, in addition to eventually leading to renal failure, a variety of extrarenal organs are often involved, including cystic degeneration of the liver, spleen, etc., and some cardiovascular and cerebrovascular diseases, especially early-onset hypertension (HTN) , more than 60% of PKD patients suffer from premature hypertension, so ADPKD is considered a systemic disease, and the corresponding cardiovascular disease is likely to be an early warning of end-stage renal failure. However, there is currently no effective treatment for ADPKD, except that the end-stage renal failure mainly relies on dialysis or kidney transplantation. Due to the complexity of ADPKD disease, it is an urgent problem to be solved that combined medication can preserve normal renal function and effectively avoid the occurrence of end-stage renal failure, and on the other hand, maximize its effect on slowing down the development of the disease and reducing side effects.

Contents of the invention

The technical problem to be solved by the present invention is how to treat autosomal dominant polycystic kidney disease (autosomaldominant polycystic kidney disease, ADPKD). In order to achieve the above object, the technical scheme provided by the invention is:

The FAK inhibitor is used in the treatment and/or prevention of autosomal dominant polycystic kidney disease.

Also provided: the application of FAK inhibitor in the treatment and/or prevention of autosomal dominant polycystic kidney disease caused by Pkd1 gene and Pkd2 gene.

At the same time, it is provided that the FAK inhibitor is used in the preparation of products for inhibiting the production of kidney vesicles.

Also provided is the application of the FAK inhibitor in inhibiting the production of renal vesicles.

Also provided is the application of the FAK inhibitor in the preparation of products inhibiting the FAK-Src signaling pathway.

At the same time, it is provided that the FAK inhibitor is used in inhibiting the FAK-Src signaling pathway.

As an improvement, the kidney cells of the kidney are canine kidney cells.

As an improvement, the FAK inhibitor is VS4718.

As an improvement, the product includes a pharmaceutically acceptable carrier, which is a solid carrier or a liquid carrier.

As an improvement, the kidney diseases are all achieved by inhibiting the production of renal vesicles and inhibiting the FAK-Src signaling pathway.

As an improvement, the dosage of the FAK inhibitor is 40-200nM.

Beneficial effects: the present invention uses the MDCK vesicle model to prove that the FAK inhibitor can inhibit the formation and growth of vesicles, and confirms the pharmacological activity of the FAK inhibitor in the kidney through the in vitro embryonic kidney vesicle model, which has a positive effect on the development of vesicles in the kidney Significant inhibitory effect. Finally, it was further proved in the polycystic kidney mouse model that FAK inhibitors also have the effect of inhibiting vesicle development in vivo. The above in vitro and in vivo vesicle inhibitory effects are in a dose-effect relationship. FAK inhibitors did not affect the viability of kidney cells, indicating that the inhibitory effect of FAK inhibitors on polycystic kidney disease has nothing to do with its cytotoxicity; the regulation of intracellular signaling pathways by FAK inhibitors may be one of the important mechanisms for inhibiting the development of renal vesicles 1. The FAK inhibitor is VS4718, and it is concluded that VS4718 can be used for the treatment of autosomal dominant polycystic kidney disease.

Focal adhesion kinase (FAK) is an intracellular non-receptor protein tyrosine kinase, which is the "junction" of multiple signal transduction pathways and can integrate growth factors (such as EGF, PDGF, etc.) , TGF-β, etc.), Integrin, receptor tyrosine kinases, G protein-coupled receptors, and mechanical stimulation. The high correlation between FAK1 and tumor invasion and metastasis has been confirmed in a large number of studies. Integrin-FAK/Src signaling is considered to be the key signaling pathway mediating tumor invasion and migration; Also plays a role in the proliferation and apoptosis of tubular epithelial cells. The phosphorylation levels of total FAK protein and FAK-Y397 allelic sites are increased in various types of tumor cells, and multiple FAK inhibitors with different mechanisms of action are in the stage of preclinical research and clinical trials.

FAK1 mainly functions in two ways, kinase-dependent and kinase-independent ("scaffolding" protein function), especially the phosphorylation activation of its multiple phosphorylation sites (FAK-pY397, Y576, Y577, Y861, and Y925, etc.). FAK participates in the regulation of biological processes such as embryonic development, cell proliferation, differentiation, apoptosis, adhesion and migration by regulating multiple signaling pathways closely related to cell proliferation, such as Ras-MAPKs, P53 and PI3K-Akt-mTOR in cells . FAK2 (PYK2) is another member of the FAK subfamily, which has high sequence homology with FAK1. FAK2 and FAK1 share many common downstream signals, such as Src. Several studies have shown that inhibition of FAK1 activation leads to compensatory activation of FAK2.

This study found that the phosphorylation level of FAK was abnormal in patients with ADPKD, suggesting that FAK may play an important role in the occurrence and development of ADPKD.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Figure 1 is the structural formula of the FAK inhibitor.

Figure 2 is a schematic diagram of MDCK cell colonies and vesicles.

Figure 3 shows the inhibitory effect of FAK inhibitors on the growth of MDCK vesicles.

Fig. 4 is a schematic diagram of a mouse embryonic kidney vesicle model.

Figure 5 shows the inhibitory effect and dose effect of FAK inhibitors on the growth of embryonic kidney vesicles.

Figure 6 is the photos of the kidneys and body weight statistics of the mice after administration of the Pkd1 flox/flox; Ksp-Cre mouse model.

Figure 7 shows the statistics of liver weight index and kidney weight index of Pkd1 flox/flox; Ksp-Cre mouse model administered.

Figure 8 is the HE staining diagram of the kidneys of the Pkd1 flox/flox; Ksp-Cre mouse model administered.

Figure 9 shows the cytotoxicity of VS4718 on MDCK cells detected by CCK-8 assay.

Figure 10 shows the effects of forskolin stimulation on FAK1 and Src phosphorylation in MDCK cells at different time points.

Figure 11 shows the regulatory effect of VS4718 on the FAK-Src signal transduction pathway of MDCK cells stimulated by forskolin.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The canine kidney cells (Madin-Darby canine kidney cells, MDCK) in the following examples are products of ATCC cell bank, and the number is CCL-34.

Example 1

Vesicle growth inhibition experiments were performed.

Canine kidney cells (Madin-Darby canine kidney cells, MDCK) were cultured in three-dimensional Matrigel (Purecol Collagen, Inamed Biomaterials Fremont Company, Cat. No. 5409) in vitro. The culture medium 1 used was to add three-dimensional Matrigel, HEPES, penicillin and streptomycin obtained the three-dimensional matrigel concentration of 2.9mg/ml, HEPES concentration of 10mM, penicillin concentration of 100U/ml, streptomycin concentration of 100μg/ml culture solution, pH 7.4. Culture solution 2 is a culture solution with FBS concentration of 10% and forskolin concentration of 10 μM by adding FBS and forskolin (FSK, forskolin, Sigma company, product number F6886) to DMEM/F12 culture solution. DMEM/F12 culture solution It is a liquid obtained by mixing equal volumes of DMEM medium (Invitrogen Company of the United States, catalog number 12100-046) and F12 medium (Invitrogen Company of the United States, catalog number 21700-075).

Take a 24-well plate, mix MDCK cells in 0.4ml of cold culture solution 1, add to one well of the 24-well plate, and the number of cells in each well is the same. Place the 24-well plate in a 37°C cell culture incubator for about 90 minutes. After the three-dimensional matrix gel is solidified, add 1.5ml of culture solution 2 to each well, and place it in a 5% _CO2 incubator at 37°C for 4 days. The single-layered epithelial-coated single-vesicle vesicles can be observed under the microscope; then add VS4718 with a final concentration of 0.625 μM, 1.25 μM, 2.5 μM and 5 μM in the cell culture wells to continue the culture, and each dose is repeated 3 holes. Replace the fresh medium containing VS4718 and forskolin every 12 hours, track and take photos every two days to record each vesicle and measure the diameter of the vesicles to evaluate the inhibitory effect of different concentrations of VS4718 on the growth of vesicles. Observe for 8 days and count 10 per well. more than vesicles, the vesicle growth curve is drawn. The inhibitory effect curve of VS4718 on vesicle growth is shown in Figure 3. The growth curve on the right side of Figure 3: the black ball curve represents the culture medium containing forskolin from day 5 to 12, the diamond curve represents the culture medium containing 0.625 μM VS4718 and forskolin from day 5 to 12, and the inverted triangle curve represents the culture medium containing 0.625 μM VS4718 and forskolin on day 5. -12 days were cultured with the culture medium containing 1.25 μM VS4718 and forskolin, the positive triangle curve represented the culture medium containing 2.5 μM VS4718 and forskolin on the 5th-12th day, and the square curve represented the culture medium containing 5 μM VS4718 and forskolin on the 5th-12th day culture medium culture. The inhibitory effect of VS4718 on vesicle growth is shown in Figure 3, and the control group in Figure 3 is the treatment group with a VS4718 concentration of 0. Among them, the first row indicates that the culture solution containing only forskolin was cultured on the 5th to 12th day, the second row indicates that the culture medium containing 0.625 μM VS4718 and forskolin was co-cultured on the 5th to 12th day, and the third row indicates the 5th to 12th day Days were co-cultured with culture medium containing 1.25 μM VS4718 and forskolin, the fourth row indicated days 5-12 were co-cultured with culture medium containing 2.5 μM VS4718 and forskolin, the fifth row indicated days 5-12 were cultured with culture medium containing 5 μM VS4718 Forskolin culture medium co-cultivation. It can be seen that the total triterpenes of Ganoderma lucidum significantly inhibited the growth of vesicles, indicating that the inhibitory effect of VS4718 on vesicles was enhanced with the increase of concentration, indicating that the inhibitory effect of VS4718 on the growth of MDCK vesicles had a dose effect.

Example 2

In the evening of the first day, C57BL/6 mice over 6 weeks old (Experimental Animal Center, Peking University Health Science Center) were mated with males and females in the same cage at a ratio of 1:1. The plug indicates that the female mouse has been pregnant for half a day. The mice without the vaginal plug are divided into cages first, and then closed in the cage at night, and then observed on the second day; the pregnant female mice are fed alone for 13 days, and the transwell plate is used for the embryonic kidney on the 13th day (Corning Company, Cat. No. 3401) for cultivation.

The 13.5-day-old mouse embryonic kidney was taken and placed in the upper chamber of the transwell, and DMEM culture solution containing 8-Br-cAMP (Sigma Company, product number B-5386) with a final concentration of 100 μM was added to the lower culture well for cultivation. Under the action of cAMP, multiple and progressively growing renal vesicles will be formed in the kidney tissue, which can be used as an in vitro whole organ model to evaluate the prevention and/or treatment of ADPKD by VS4718. A schematic diagram of the embryonic kidney vesicle model (polycystic kidney) is shown in FIG. 7 .

During the culture of embryonic kidneys, as shown in Figure 8, the embryonic kidneys in the first line were added with 100 μM 8-Br-cAMP and continued to culture until the 6th day, and the second, third, and fourth lines were added with 100 μM 8-Br-cAMP. On the basis of Br-cAMP stimulation, 8nM, 40nM, and 200nM VS4718 were added for treatment, cultured until the 6th day, and fresh corresponding culture medium was replaced every 12h. Follow up and take pictures every day to record the condition of the kidneys, and the experiment is repeated three times. The results showed that the administration of 40nM and 200nM VS4718 significantly inhibited the development of kidney vesicles, indicating that VS4718 can inhibit the development of embryonic kidney vesicles in a dose-dependent manner.

Example 3

The mice used were obtained as follows: Pkd1 flox ^/ flox mice were mated with Ksp-Cre mice to obtain a generation of Pkd1 ^+/- ; Ksp-Cre mice, Pkd1 ^+/- ; male mice of Ksp-Cre mice Mating with female mice to obtain wild-type mice Pkd1 ^+/+ ; Ksp-Cre and Pkd1 flox ^/ flox; Ksp-Cre mice (PKD mice). Among them, the genetic backgrounds of Pkd1 flox ^/ flox mice and Ksp-Cre mice are both C57BL/6 mice, both of which are recorded in the literature (...Wang W, Li F, Sun Y, et al. Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling. FASEB J. 2015; 29(4):1551-1563.). Pkd1 flox ^/ flox mice are mice obtained by knocking out the Pkd1 gene specifically for the whole kidney in the background of C57BL/6 mice, so that the mice can develop rapidly progressive ADPKD after birth. After surviving for about 7-10 days, genetic identification was carried out on the first day after the mouse was born to determine the genotype of the mouse. The wild-type C57BL/6 mice corresponding to Pkd1 flox ^/ flox mice were designated as Pkd1 ^+/+ mice. Because this part of the content is not involved in the claims, the source may or may not be provided, and the color markings on the above comply with the regulations.

Wild-type mice (Pkd1 ^+/+ ; Ksp-Cre) and PKD mice (Pkd1 flox ^/ flox; Ksp-Cre) were randomly divided into two groups, blank control group (empty solvent group, that is, injection of DMSO aqueous solution, DMSO The aqueous solution is composed of normal saline and DMSO, the volume ratio of DMSO and normal saline is 1:500) and the administration group (10 mg VS4718 per kilogram of body weight per day), with no less than 5 mice in each group. From the third day after birth, each mouse was administered by intraperitoneal injection with an insulin syringe every 12 hours (each injection volume was 20 μl), each mouse in the blank control group was injected with 20 μl DMSO aqueous solution each time, and the administration group Each mouse was injected with 20 μl of VS4718 solution (VS4718 solution is a solution obtained by dissolving commercial VS4718 powder in DMSO aqueous solution), and the administration continued until the seventh day after birth. Weigh, kill, and take tissues. Judging from the size and body weight of the mice (Figure 6: the photo of the individual mice on the top, and the statistical chart of the weight of the mice in each group on the bottom), there is no significant difference among the groups. From the perspective of kidney size (the lower left side of Figure 8 is the photo of the mouse kidney), on the 7th day after birth, in the gene knockout PKD mice, obvious polycystic kidneys occurred, and after VS4718 treatment, the kidney volume was significantly reduced. However, VS4718 had no significant effect on normal kidney size. There was no significant difference in liver weight index (liver weight/body weight) among the mice in each group (statistical graph on the right side of Figure 7), but in PKD mice, VS4718 treatment significantly reduced the kidney weight index of polycystic kidney mice ( Bilateral kidney weight/body weight) (statistical graph on the left side of Figure 7). The results of HE staining of mouse kidney sections showed that in PKD mice, there were a large number of vesicles in the mouse kidneys, and the mouse kidneys were significantly smaller and the kidney tissue structure was improved after administration of VS4718 (Figure 8 HE staining pictures of kidneys). In Figure 11, "wild-type mice + Ganoderma lucidum total triterpenes" means wild-type mice injected with Ganoderma lucidum total triterpenes solution, "WT" means wild-type mice injected with DMSO aqueous solution, "WT+10mg/kg/d" Denotes wild-type mice injected with VS4718 solution. "PKD" means Pkd1 flox ^/ flox; Ksp-Cre mice injected with DMSO aqueous solution, "PKD mice+10mg/kg/d" means Pkd1 flox ^/ flox; Ksp-Cre mice injected with VS4718 solution.

Example 4

1. Determine the cytotoxicity of VS4718 by CCK-8 method

The MDCK cell suspension in the logarithmic phase was inoculated in a 96-well culture plate, each well contained 1× ¹⁰³ cells, and each well was given 100 μl of DMEM medium containing 10% fetal bovine serum (FBS, Gibco Fisher Scientific, Netherlands) (U.S. Invitrogen Company, catalog number 12100-046), and placed in a 5% CO ₂ incubator at 37° C. for 24 hours. FBS was removed and serum starved for 24 hours. Afterwards, the Ganoderma lucidum total triterpene solution was added to the cell culture wells (administration wells), and the volume added to each well was the same. 50μM, and 100μM, one concentration per well, cultured for 24 hours. Remove the supernatant, add DMEM culture solution containing 10% CCK-8 reagent, continue culturing in a 5% _CO2 incubator at 37°C for 1 hour, detect the OD value of each well with a microplate reader (detection wavelength 450nm), set to zero Wells (containing equal amounts of medium, CCK-8 and DMSO, without VS4718) and control wells (containing equal amounts of cells, medium, CCK-8 and DMSO, without VS4718, that is, VS4718 was given at 0 μg/ml Medicine hole), each group set at least 5 replicate holes. Cell viability was calculated according to the following formula, cell viability=(administration well-zero adjustment well)]/(control well-zero adjustment well)×100%. The experiment was repeated three times. The experimental results are shown in Figure 9. There was no significant difference between the different concentration administration groups and the control group (0 μg/ml). VS4718 less than 25 μM did not affect the cell viability of MDCK cells and had no toxic effect on MDCK cells, indicating that VS4718 inhibited The role of vesicles is independent of their cytotoxicity.

2. Targets and mechanisms of total triterpenes of Ganoderma lucidum

Western blotting was used to detect the effect of VS4718 on the signal transduction pathway of vesicle epithelial cells. The specific experimental process is as follows:

MDCK cells were cultured to 70%-80% confluence using DMEM medium containing 10% fetal bovine serum, replaced with serum-free medium and cultured for 24 hours, and then given forskolin (FSK) stimulation, the final concentration of forskolin in the medium was 10 μM , stimulated for different times, respectively 0, 15, 30, 45, 60, 120, 240, and 480 minutes, and detected the phosphorylation levels of pFAK1 and pSrc, important molecules of the FAK-Src signaling pathway, at different time points. The results are shown in FIG. 10 , which indicated that after forskolin stimulation, the phosphorylation levels of pFAK1 and pSrc, important molecules of the FAK-Src signaling pathway, reached the peak at 60 minutes.

Then serum-starved MDCK cells for 24 hours were added forskolin and VS4718 for stimulation, the final concentration of forskolin in the medium was 10 μM, and the final concentration of VS4718 in the medium was 0 nM, 8 nM, 40 nM and 200 nM, each VS4718 The concentration of forskolin in the culture medium was 10 μM, and the MDCK cells stimulated without forskolin were used as the control (Ctr), and the above groups were stimulated for 60 minutes.

The total protein of the above-mentioned groups of cells was extracted, and Western blot experiments were performed to detect changes in the important molecular levels of the signaling pathway FAK-Src signaling pathway in PKD diseases. As shown in Figure 11, it was found that VS4718 can significantly down-regulate the elevated FAK-Src signaling pathway. When adding FSK but not adding Ganoderma lucidum total triterpenes, it showed down-regulation of the phosphorylation levels of pFAK1 and pSrc.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1.FAK inhibitor is treating and/or is preventing to apply in autosomal dominant polycystic kidney disease.

The autosomal dominant inheritance polycystic kindey that Pkd1 gene and Pkd2 gene cause is being treated and/or prevented to 2.FAK inhibitor Application in disease.

3.FAK inhibitor inhibits to apply in kidney vesica generation product in preparation.

4.FAK inhibitor is applied in inhibiting the generation of kidney vesica.

5.FAK inhibitor inhibits to apply in FAK-Src signal path product in preparation.

6.FAK inhibitor is applied in inhibiting FAK-Src signal path.

7. any one of -4 application according to claim 1, it is characterised in that: the kidney cell of the kidney is canine kidney cells.

8. any one of -6 application according to claim 1, it is characterised in that: the Fak inhibitor is VS4718.

9. application according to claim 5, it is characterised in that: the product includes pharmaceutically acceptable carrier, the carrier For solid carrier or liquid-carrier.

10. application according to claim 1 or 2, it is characterised in that: the nephrosis, which passes through, inhibits kidney vesica to generate, inhibit FAK-Src signal path is realized.