Application of verofenib in preparation of medicine for treating renal fibrosis diseases

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to application of verofenib in preparation of medicines for treating renal fibrosis diseases. It is found for the first time that the vilorafenib is combined with the EIF4A1 protein to inhibit the activity of renal fibers, the vilorafenib can be used as a potential anti-renal-tissue-fibrosis medicine, and a new thought is provided for research of anti-organ or tissue-fibrosis medicines.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the use of vemurafenib in the preparation of drugs for treating renal fibrosis. Background Technology

[0002] EIF4A1 (eukaryotic translation initiation factor 4A1) is a member of the DEAD-box RNA helicase family. As the core subunit of the eIF4F complex, it mediates the initiation of mRNA translation. This protein eliminates the secondary structure of the mRNA 5'-UTR through ATP-dependent unwinding activity, assisting ribosomes in locating the start codon and playing a pivotal role in regulating gene expression. Its activity is positively regulated by eIF4B / 4H and inhibited by PDCD4, and it is linked to upstream metabolic states through the mTOR signaling pathway. In the field of tumorigenesis, EIF4A1 has been shown to drive malignant phenotypes by preferentially unwinding oncogene mRNAs (such as c-MYC and Cyclin D1). Its overexpression is highly correlated with the progression and drug resistance of various cancers, including breast cancer and lymphoma. The research team has developed inhibitors derived from natural products (such as silvestrol and curcumin derivatives) and synthetic small molecules (eFT226) to selectively block cancer cell proliferation by stabilizing the RNA-enzyme complex or competitively binding to ATP sites. These compounds have shown specific inhibitory effects on oncogene translation in preclinical models.

[0003] Recent structural biology studies have revealed the functional mechanism of EIF4A1: conformational rearrangement of its N-terminal and C-terminal domains synergistically hydrolyzes ATP, driving the dissociation of the RNA double helix. Its dynamic interactions with eIF4E and eIF4G further refine the assembly model of the translation initiation complex. Furthermore, EIF4A1 is involved in viral infection mechanisms; for example, hepatitis C virus promotes its own genome translation by hijacking this protein, suggesting its potential as an antiviral target.

[0004] Vemurafenib is a small molecule kinase inhibitor targeting BRAF V600 mutations. It was approved by the FDA in 2011 for the treatment of unresectable or metastatic melanoma positive for BRAF V600E or V600K mutations. BRAF gene mutations can lead to abnormal activation of the MAPK signaling pathway, promoting tumor proliferation. Vemurafenib, by selectively inhibiting mutant BRAF kinases, blocks downstream signal transduction and significantly inhibits tumor growth. Clinical trials have shown that compared with traditional chemotherapy, vemurafenib reduced patient mortality by 63%, significantly shrunk tumors in 48% of patients, and significantly prolonged progression-free survival and overall survival. Further research is needed to expand the application of vemurafenib in other pharmaceutical applications. Summary of the Invention

[0005] The purpose of this invention is to provide the use of vemurafenib in the preparation of a medicament for treating renal fibrosis.

[0006] This invention provides the use of vemurafenib in the preparation of medicaments for treating fibrotic diseases.

[0007] Vemurafenib is a small molecule kinase inhibitor targeting BRAF V600 mutations; by selectively inhibiting mutant BRAF kinases and blocking downstream signal transduction, it significantly inhibits tumor growth. This invention is the first to discover that vemurafenib improves renal fibrosis by binding to the EIF4A1 protein, providing a new drug candidate for the clinical treatment of fibrotic diseases.

[0008] Furthermore, vemurafenib is the sole active ingredient in the drug.

[0009] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the excipients include any one or more of non-toxic fillers, stabilizers, diluents, and adjuvants.

[0011] Furthermore, the diluent is either water or physiological saline.

[0012] The present invention also provides a medicament for treating renal fibrosis, the medicament being a mixture of vemurafenib and excipients, wherein the content of vemurafenib in the medicament is 0.1 wt% to 99 wt%.

[0013] Furthermore, the solid dosage forms include granules, tablets, capsules, pills, and drop pills, and the solution dosage forms include oral liquid preparations, oral enemas, and injectable dosage forms.

[0014] Furthermore, the solution formulation is a solution composed of water and vemurafenib, or a solution composed of physiological saline and vemurafenib.

[0015] The beneficial effects of this invention are as follows: This invention selected vemurafenib from a clinically approved FDA small molecule drug library, finding it to have anti-fibrotic activity. Results showed that vemurafenib exhibited strong anti-renal fibrosis activity both in vitro and in vivo, and also demonstrated anti-liver fibrosis activity in in vitro experiments. Knockdown of the target protein eukaryotic translation initiation factor 4A1 confirmed that vemurafenib targets this protein to inhibit renal fibrosis. Therefore, vemurafenib can be considered a potential anti-fibrotic drug, providing a new source for research on anti-organ fibrosis drugs. Attached Figure Description

[0016] Figure 1The expression of fibronectin, type I collagen, α-smooth muscle actin and EIF4A1 in an obstructed kidney model (UUO) under high-dose (20 mg / kg) and low-dose (10 mg / kg) vemurafenib conditions.

[0017] Figure 2 This figure shows the protein expression of smad2, smad3, and phosphorylated smad2 and smad3 in the TGF-β / smad signaling pathway in a vemurafenib-treated obstructed kidney (UUO) model.

[0018] Figure 3 This is a real-time PCR analysis of fibronectin mRNA in a vemurafenib-treated obstructed kidney model.

[0019] Figure 4 This is a real-time PCR analysis of type I collagen mRNA in a vemurafenib-treated obstructed kidney model.

[0020] Figure 5 This is a real-time PCR analysis of α-smooth muscle actin mRNA in a vemurafenib-treated obstructed kidney model.

[0021] Figure 6 This image shows the effect of vemurafenib treatment on reducing renal interstitial fibrosis in an obstructed kidney model with urinary tract infection (UUO).

[0022] Figure 7 This is a quantitative diagram of representative Masson staining collagen fiber deposition analysis in a vemurafenib-treated obstructed kidney model.

[0023] Figure 8 The figure shows the results of the anti-renal fibrosis activity of vemurafenib in NRK-52E cells under EIF4A1-siRNA interference. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1 Twenty-four male C57BL / 6J mice, weighing approximately 30g, were used. Six mice were divided into three groups: a sham operation (Control) group, a UUO model group, a low-dose UUO + vemurafenib group (10mg / kg, dissolved in 0.5% sodium carboxymethyl cellulose solution), and a high-dose UUO + vemurafenib group (20mg / kg, dissolved in 0.5% sodium carboxymethyl cellulose solution). The UUO model procedure was as follows: After general anesthesia, the mice were fixed. An incision was made on the left dorsal side of the mouse. After opening the incision, the left ureter was ligated once near the kidney and once near the kidney. The incision was then sutured closed. In the Control group, only a dorsal incision was made, without ureteral ligation, and the incision was then sutured closed. UUO + Vemurafenib low-dose group: Starting from day 1 of UUO modeling, each mouse was administered 10 mg / kg of vemurafenib by gavage every other day; UUO + Vemurafenib high-dose group: Starting from day 1 of UUO modeling, each mouse was administered 20 mg / kg by gavage every other day; UUO model group: Starting from day 1 of UUO modeling, each mouse was administered 0.5% sodium carboxymethyl cellulose solution by gavage, the same volume as the drug-treated group, every other day, and body weight was recorded. Fourteen days post-surgery, all mice were uniformly sacrificed. At sacrifice, blood was collected from the eyeballs, and 20 mL of ice-cold PBS was administered into the ventricle until the kidneys turned pale.

[0026] Take 50 mg of kidney tissue and place it in a 2.0 mL EP tube. Add 0.5 mL of PIRA lysis buffer, then add two steel balls and use a homogenizer to thoroughly pulverize the homogenate. Let it stand on ice for 10 min to fully lyse. Centrifuge at 4 °C and 12000 rpm for 10 min. Keep the supernatant and transfer it to a new EP tube. After dilution, determine the protein concentration using a BCA kit. Add 5× loading buffer and boil to denature the protein for 10 min to obtain the protein sample.

[0027] Prepare SDS-PAGE polyacrylamide gels, and prepare the necessary separating and concentrating gels for the experiment. Place the gels in the electrophoresis tank, taking care not to tear the sample wells of the polyacrylamide gel when removing the comb. After removing the comb, pour electrophoresis buffer into the assembled tank. Take 30 μg of protein sample from each well. After turning on the instrument, perform electrophoresis at a constant voltage of 80V for 30 minutes until the sample moves from the concentrating gel to the separating gel. Adjust the voltage to 130V and continue electrophoresis at a constant voltage for 1 hour until the marker reaches the bottom of the gel. Prepare a PDVF membrane of appropriate size in advance, activate it in anhydrous methanol for 30 seconds, and then equilibrate it in transfer buffer to pre-cool the transfer buffer. Place the (anode) sponge-filter paper-membrane-gel-filter paper-sponge (cathode) in the transfer clamp in the following order, remove all air bubbles, clamp the transfer clamp, and place it in the transfer tank. Fill the tank with pre-cooled transfer buffer and perform transfer at a constant current of 250mA at low temperature. After transfer, wash with TBST, then transfer the membrane to a 5% (v / v) skim milk solution (dissolved in TBST) and block on a shaker at room temperature for 1 hour. After blocking, wash away excess skim milk with TBST, place the membrane in the target antibody working solution (prepared according to the instructions), and incubate overnight at 4°C. First, recover the primary antibody, then wash with TBST for 10 minutes, repeating three times as needed. Pour in the secondary antibody working solution (prepared according to the target antibody instructions) and incubate on a shaker at room temperature for 1 hour. After incubation, wash the target band three times with TBST, 10 minutes each time on a shaker. Prepare the ECL chemiluminescence solution according to a 1:1 volume ratio of solution A to solution B, and use in the dark. First, remove excess liquid from the target band, then place it in the prepared ECL chemiluminescence solution container for reaction. After 1 minute, develop and photograph the membrane in a developing apparatus.

[0028] The results are as follows Figure 1 As shown, the expression levels of fibrotic proteins FN (Fibronectin, ab2413, Abcam), COL (Collagen I, ab270993, Abcam), and α-SMA (A2547, Sigma) were significantly increased in the UUO model group, and the expression level of EIF4A1 protein was also increased. Compared with the model group, the levels of fibrotic proteins FN, COL, and α-SMA in the vemurafenib administration group were significantly reduced, and the expression level of EIF4A1 protein in the administration group was also reduced. Moreover, the anti-renal fibrosis effect of the high-dose vemurafenib group was better than that of the low-dose group, which to some extent indicates that vemurafenib has a good anti-renal fibrosis effect.

[0029] Example 2 Fresh tissue protein samples were taken, and the Western Blot experiment method in Example 1 was followed as usual, including gel preparation, electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and development, to investigate whether vemurafenib treatment affected the Smad signaling pathway in the UUO renal fibrosis model.

[0030] according to Figure 2 The results showed that both the UUO model group and the vemurafenib treatment group expressed Smad2 and Smad3 proteins normally. The expression levels of p-Smad2 and p-Smad3 proteins were significantly increased in the UUO model group. In the vemurafenib treatment group, under both low and high dose conditions, the expression levels of p-Smad2 and p-Smad3 proteins were significantly decreased compared to the UUO model group. Therefore, it is preliminarily believed that the anti-renal fibrosis effect of vemurafenib is related to the regulation of the Smad signaling pathway.

[0031] Example 3 Take an appropriate amount of fresh tissue, sterile and enzyme-free 2 mL, add 1 mL of Trizol reagent and 2 magnetic beads, homogenize the tissue thoroughly for 2 min using a homogenizer, and let it stand on ice at low temperature for 10 min to allow the tissue to fully lyse. The entire process is carried out at a low temperature of 4℃.

[0032] RNA isolation: Add chloroform (0.2 mL chloroform per 1 mL Trizol), shake vigorously for 15 seconds. Incubate at room temperature for 3 min. Centrifuge at 12000 rpm for 15 min at 4℃. At this point, the mixture separates into three layers, with the upper colorless aqueous phase containing RNA. RNA precipitation and washing: Transfer the upper aqueous phase to a new sterile enzyme-free centrifuge tube, add an equal volume of isopropanol, gently invert to mix, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4℃. A white RNA precipitate is visible at the bottom of the centrifuge tube. Discard the supernatant, add 1 mL of 75% ethanol, and gently invert to wash the precipitate. Centrifuge at 7500 rpm for 5 min at 4℃, discard the supernatant, and repeat the washing once. Air dry the RNA precipitate at room temperature for 5 min until there is no ethanol odor. Add an appropriate amount of DEPC water to the dried RNA precipitate and gently pipette to fully dissolve the RNA. Take a small amount of RNA solution and measure its absorbance at 260 nm and 280 nm wavelengths using a spectrophotometer to assess the concentration and purity of the RNA (A). 260 / A 280The ratio should be between 1.8 and 2.0. RNA was reverse transcribed into cDNA using the Novizan Reverse Transcription Kit (R323-01). The cDNA samples from each group were then quantitatively analyzed using a 2×Taq Pro Universal SYBR qPCR MasterMix (Q712-03). Primer sequences are shown in Table 1. The gene expression levels of Fibronectin, Collagen I, and α-SMA in each experimental group were determined.

[0033] Table 1: PCR primer sequences The results are as follows Figures 3-5 As shown, the expression levels of fibrosis genes FN (Fibronectin), COL (Collagen I), and α-SMA were significantly increased in the UUO model group. Compared with the model group, the expression levels of fibrosis genes FN, COL, and α-SMA in the vemurafenib administration group were significantly decreased, and the anti-renal fibrosis effect of the high-dose vemurafenib group was better than that of the low-dose group, indicating that vemurafenib has a good anti-renal fibrosis effect at the gene level.

[0034] Example 4 Kidney tissue was harvested and fixed in 4% paraformaldehyde for 24 hours, then dehydrated, cleared, and impregnated with paraffin before being embedded in paraffin blocks. The paraffin blocks were then sliced ​​into 4μm thick sections using a microtome, placed on glass slides, and incubated at 60°C.

[0035] Baking the slides for 2 hours ensures firm adhesion. Dewaxing the slides sequentially in xylene I and xylene II for 10 minutes each, then hydrating them with a gradient of alcohols (100%, 95%, 90%, 80%, 70%) for 5 minutes each, until finally settling in distilled water. Stain the slides in hematoxylin solution for 5 minutes, rinse with tap water, then differentiate with 1% hydrochloric acid alcohol for a few seconds, immediately rinsing with tap water to restore blue color. Stain the slides in eosin solution for 2 minutes, then dehydrate them with a gradient of alcohols (80%, 90%, 95%, 100%) for 5 minutes each. Clear the slides by immersing them in xylene I and xylene II for 5 minutes each, then add neutral resin and cover with a coverslip. Finally, observe and photograph the slides under an optical microscope.

[0036] Results of HE staining of mouse kidney tissue sections as follows Figure 6The study showed that the Control group had no obvious histopathological changes, with tightly packed renal tubules, full glomeruli, and close adhesion between the glomeruli and Bowman's capsule. Compared with the Control group, the model group mice exhibited severe renal parenchyma atrophy and thinning, reduced and atrophied glomeruli, cystic dilation of some renal tubules, and interstitial fibrosis. The vemurafenib treatment group significantly reduced renal tubular dilation, showed relatively full glomeruli, and cystic dilation of a small number of renal tubules, significantly improving renal fibrosis.

[0037] Example 5 The fixation, clearing, paraffin embedding, dewaxing, and hydration of kidney tissue samples were performed in the same manner as in Example 4. During the staining stage, sections were stained with hematoxylin for 5 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, and then rinsed with tap water to regain blue color. Sections were then treated with Masson's blue solution for 3 minutes and rinsed with water. Sections were stained with Ponceau S and Acidic Fuchsin for 10 minutes and rinsed with water. Sections were then treated with phosphomolybdic acid solution for 10 minutes, observing periodically until collagen fibers were clearly differentiated. Then, sections were directly stained with aniline blue solution for 5 minutes. After graded alcohol destaining, xylene clearing, and mounting with neutral resin, the sections were observed under an optical microscope. Collagen fibers appeared blue, cell nuclei appeared blue-black, and muscle fibers and erythrocytes appeared red.

[0038] Masson staining results of mouse kidney tissue sections are as follows Figure 7 The study showed that the Control group had no significant histopathological changes, and no fibrosis was observed in the tubular basement membrane and peritubular tissue of the kidneys. Compared with the Control group, the model group mice showed significant proliferation of tubular basement membrane and peritubular fibrosis in their kidneys, and a large amount of collagen fiber deposition was found in the tubulointerstitium. The vemurafenib treatment group significantly reduced collagen fiber deposition in the tubulointerstitium. This indicates that both low-dose and high-dose vemurafenib treatments are effective in treating renal fibrosis.

[0039] Example 6 NRK-52E cells were seeded into 12-well plates and allowed to reach 100% confluence after 72 hours. After cell adhesion, the cells were starved and transfected with si-EIF4A1 (Rat) for 10 hours. Then, the cells were treated with vemurafenib for 48 hours, with or without TGF-β1 (10 ng / mL). Proteins were then extracted, and the expression levels of four proteins—fibronectin, collagen I, α-smooth muscle actin (α-SMA), and EIF4A1—were detected by Western blotting. The results are shown below. Figure 8As shown in Table 2, the specific si-EIF4A1(Rat) sequence used in this embodiment is shown in Table 3. The consumables used in the Western Blot experiment in the above embodiment are shown in Table 3.

[0040] Table 2: siRNA sequence information Table 3: Western Blot Reagents It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0041] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0042] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of vemurafenib in the preparation of a medicament for treating renal fibrotic diseases.

2. Use according to claim 1, characterized in that, The vemurafenib is the only effective component in the medicament.

3. Use according to claim 1, characterized in that, The medicament further comprises pharmaceutically acceptable adjuvants.

4. Use according to claim 3, characterized in that, The adjuvants comprise any one or more of fillers, stabilizers, diluents, adjuvants.

5. Use according to claim 4, characterized in that, The diluents are any one of water and normal saline.

6. A medicament for treating a renal fibrotic disease, characterized by, The medicament is prepared by mixing the vemurafenib and the adjuvants as described in claim 1, and the content of the vemurafenib in the medicament is 0.1wt% to 99wt%.

7. The medicament according to claim 6, characterized in that, The dosage form of the medicament is a solid dosage form or a solution dosage form, the solid dosage form comprises granules, tablets, capsules, pills, dripping pills, and the solution dosage form comprises oral liquid preparations, intragastric administration preparations, injection administration dosage forms.

8. The medicament according to claim 7, characterized in that, The solution dosage form is a solution of water and the vemurafenib, or a solution of normal saline and the vemurafenib.

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