Application of pimozide 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 pimozide in preparation of medicines for treating renal fibrosis diseases. It is found for the first time that pimozide inhibits renal fiber activity by combining with EIF4A1 protein, pimozide can be used as a potential anti-renal fibrosis drug, and a new thought is provided for research of anti-organ or tissue fibrosis drugs.

Description

Technical Field

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

[0002] EIF4A1 (eukaryotic translation initiation factor 4A1) is an ATP-dependent RNA helicase belonging to the DEAD-box protein family. Its name comes from the conserved amino acid sequence "Asp-Glu-Ala-Asp" (DEAD) of this family. It is a crucial subunit of the eIF4F complex, essential for cap recognition and facilitating the binding of mRNA to ribosomes. This protein is highly conserved in eukaryotes, with a total length of 406 amino acids. It primarily participates in the mRNA unwinding process during translation initiation and is a core component of the eIF4F complex. The eIF4F complex also includes eIF4E (mRNA 5' cap-binding protein) and eIF4G (scaffold protein), whose function is to mediate ribosome binding to mRNA.

[0003] EIF4A1 provides energy through ATP hydrolysis, unwinding the complex secondary structures (such as stem-loop structures) of the 5' untranslated region (UTR) of mRNA, promoting the scanning of the small ribosomal subunit (40S) to the start codon (AUG), thereby initiating protein synthesis. This process is particularly important for the translation of mRNAs with high GC content or highly structured structures. EIF4A1 activity is regulated by multiple factors, including eIF4B and eIF4H (which enhance its helicase activity) and PDCD4 (which inhibits its function). Furthermore, the mTOR signaling pathway indirectly affects EIF4A1 activity by regulating the assembly of the eIF4F complex through phosphorylation of downstream effector molecules.

[0004] In summary, EIF4A1 plays a central role in gene expression regulation, and its functional abnormalities are closely related to diseases, making it an important target for translational medicine research.

[0005] Pimozide is a long-acting antipsychotic drug belonging to the dibenzopyrene class of compounds. It is primarily used to treat acute and chronic schizophrenia, Tourette syndrome, monosymptomatic hypochondria, and paranoid states. The drug is absorbed approximately 50% after oral administration, undergoing significant first-pass metabolism, with peak plasma concentrations reached in 4–12 hours. Its half-life is as long as 55 hours, and in some patients, it can even reach 150 hours, thus requiring only once-daily dosing. The recommended starting dose is 1–2 mg daily, gradually adjusted according to the patient's condition, with a maximum daily dose not exceeding 20 mg. However, pimozide has significant side effects, commonly including extrapyramidal symptoms (such as tremor and muscle rigidity), dry mouth, fatigue, and insomnia. In severe cases, it can cause ventricular arrhythmias or QT interval prolongation, and even sudden death. Therefore, it is contraindicated in patients with a history of cardiac conduction abnormalities, and regular electrocardiogram monitoring is necessary during treatment. Furthermore, concomitant use of pimozide with certain antibiotics, such as macrolides, or drugs that prolong the QT interval may increase the risk of death and must be strictly avoided.

[0006] Currently, the pharmacological effects of pimozide are mainly attributed to its antagonism of dopamine D2 and D3 receptors, inhibiting dopaminergic activity in the central nervous system and thus improving psychotic symptoms such as hallucinations, delusions, and thought disorders. In addition, pimozide also exhibits some calcium channel antagonism, which may be related to its long-acting nature. Further research is needed to expand the application range of pimozide and explore its other pharmaceutical uses. Summary of the Invention

[0007] The purpose of this invention is to provide the use of pimozide in the preparation of medicaments for treating fibrotic diseases.

[0008] This invention provides the use of pimozide in the preparation of medicaments for treating fibrotic diseases.

[0009] Pimozide is a D2 dopamine receptor antagonist; it binds with high affinity to the clonal 5-HT7 receptor; it is also a Ca2+ receptor. 2+ Channel antagonist, antipsychotic drug. This invention is the first to discover that pimozide improves renal fibrosis by binding to the EIF4A1 protein, providing a new drug candidate for the clinical treatment of fibrotic diseases.

[0010] Furthermore, the fibrotic diseases include renal fibrosis and liver fibrosis.

[0011] Furthermore, pimozide is the sole active ingredient in the drug.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include any one or more of non-toxic fillers, stabilizers, diluents, and adjuvants.

[0014] Furthermore, the diluent is either water or physiological saline.

[0015] The present invention also provides a medicament for treating renal fibrosis, the medicament being a mixture of pimozide and excipients, wherein the pimozide content in the medicament is 0.1 wt% to 99 wt%.

[0016] 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.

[0017] Furthermore, the solution formulation is a solution composed of water and pimozide, or a solution composed of physiological saline and pimozide.

[0018] The beneficial effects of this invention are as follows: This invention selected pimozide from the FDA's clinically approved small molecule drug library, which exhibits antifibrotic activity. The results showed that pimozide has strong anti-renal fibrosis activity both in vitro and in vivo, and its anti-liver fibrosis activity was also found in in vitro experiments. By knocking down the target protein eukaryotic translation initiation factor 4A1, it was verified that pimozide targets this protein to inhibit renal fibrosis. Therefore, pimozide can be considered a potential anti-fibrotic drug, providing a new source for the research of anti-organ fibrosis drugs. Attached Figure Description

[0019] Figure 1 The expression of fibronectin, type I collagen, α-smooth actin and EIF4A1 in an obstructed kidney model (UUO) after treatment with high-dose (10 mg / kg) and low-dose (5 mg / kg) pimozide.

[0020] 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 pimozide-treated obstructed kidney model (UUO).

[0021] Figure 3 A real-time PCR analysis of fibronectin mRNA expression in a pimozide-treated obstructed kidney model.

[0022] Figure 4 This is a real-time PCR analysis of type I collagen mRNA expression in a pimozide-treated obstructed kidney model.

[0023] Figure 5 This is a real-time PCR analysis of α-smooth muscle actin mRNA expression in a pimozide-treated obstructed kidney model.

[0024] Figure 6 This image shows the effect of pimozide treatment on reducing renal interstitial fibrosis in an obstructed kidney model with ulcerative colitis (UUO).

[0025] Figure 7 This is a quantitative diagram of representative Masson staining collagen fiber deposition analysis in a pimozide-treated obstructed kidney model.

[0026] Figure 8 The figure shows the anti-renal fibrosis activity of pimozide in NRK-52E cells with and without EIF4A1 siRNA interference.

[0027] Figure 9 The figure shows the anti-hepatic fibrosis activity of pimozide in LX-2 cells with and without EIF4A1 siRNA interference. Detailed Implementation

[0028] 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.

[0029] 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 + pimozide group (5mg / kg, dissolved in 0.5% sodium carboxymethyl cellulose solution), and a high-dose UUO + pimozide group (10mg / kg, dissolved in 0.5% sodium carboxymethyl cellulose solution). UUO model procedure: 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 proximal to the kidney. The incision was then sutured closed. In the Control group, only a dorsal incision was made, without ureteral ligation. The incision was then sutured closed. UUO + pimozide low-dose group: Starting on day 1 of UUO modeling, each mouse was administered pimozide by gavage at a dose of 5mg / kg every other day. UUO + Pimozide High-Dose Group: Starting from day 1 of UUO modeling, each mouse was administered 10 mg / kg of pimozide via gavage, once every other day. UUO Model Group: Starting from day 1 of UUO modeling, mice were administered 0.5% sodium carboxymethyl cellulose solution via gavage, the same volume as the treatment group, once 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.

[0030] 50 mg of kidney tissue was placed in an EP tube, 0.5 mL of PIRA lysis buffer was added, followed by two steel balls. The homogenate was then thoroughly pulverized using a homogenizer. The homogenate was incubated on ice for 10 min to achieve complete lysis. The homogenate was then centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was retained and transferred to a new EP tube. After dilution, the protein concentration was determined using a BCA assay kit. 5× loading buffer was added, and the mixture was boiled to denature the protein for 10 min to obtain the protein sample. 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 80 V for 30 min until the sample moves from the concentrating gel to the separating gel. Adjust the voltage to 130 V 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 (anodide) 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 250 mA at low temperature. After transfer, wash with TBST, then transfer the membrane to a 5% skim milk (TBST dissolved) solution and block on a shaker at room temperature for 1 hour. After blocking, wash away excess skim milk with TBST, then place the membrane in the target antibody working solution (the target antibody is prepared according to the instructions and stored at 4℃ or -20℃), and incubate overnight at 4℃. First, recover the primary antibody, then wash with TBST for 10 min, repeating 3 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 min each time on a shaker. Prepare the ECL chemiluminescence solution according to a 1:1 volume ratio of solution A to solution B. Protect from light during use. First, remove excess liquid from the target band, then place it in the prepared ECL chemiluminescence solution container for reaction. After 1 min, develop and photograph it in a developing apparatus.

[0031] The results are as follows Figure 1As 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 pimozide-treated group were significantly reduced, and the expression level of EIF4A1 protein in the treated group was also reduced. Moreover, the anti-renal fibrosis effect of the high-dose pimozide group was better than that of the low-dose group, which to some extent indicates that pimozide has a good anti-renal fibrosis effect.

[0032] 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 pimozide treatment had any effect on the Smad signaling pathway in the UUO renal fibrosis model.

[0033] according to Figure 2 The results showed that both the UUO model group and the pimozide treatment group normally expressed Smad2 and Smad3 proteins. The expression levels of p-Smad2 and p-Smad3 proteins were significantly increased in the UUO model group. In the pimozide 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 pimozide may be related to the regulation of the Smad signaling pathway.

[0034] 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℃.

[0035] RNA isolation: Add chloroform (0.2 mL chloroform per 1 mL Trizol), shake vigorously for 15 seconds. Incubate at room temperature for 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. 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, incubate at room temperature for 10 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C. A white RNA precipitate will be 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 minutes at 4°C, discard the supernatant, and repeat the washing once. Air dry the RNA precipitate at room temperature for 10 minutes 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 280 The 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.

[0036] 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 levels of fibrosis genes FN, COL, and α-SMA in the pimozide-treated group were significantly reduced, and the anti-renal fibrosis effect of the high-dose pimozide group was better than that of the low-dose group, indicating that pimozide has a good anti-renal fibrosis effect at the gene level.

[0037] 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.

[0038] Baking the slides for 3 hours ensures firm adhesion. Dewaxing is done by sequentially immersing the slides in xylene I and xylene II for 10 minutes each, followed by hydration with a gradient of alcohols (100%, 95%, 90%, 80%, 70%) for 5 minutes each, until finally settling in distilled water. The slides are then stained with hematoxylin for 5 minutes, rinsed with tap water, and differentiated with 1% hydrochloric acid alcohol for a few seconds, immediately rinsed with tap water to regain blue color. The slides are then stained with eosin for 2 minutes, followed by dehydration with a gradient of alcohols (80%, 90%, 95%, 100%) for 5 minutes each. The slides are then cleared by immersing them in xylene I and xylene II for 5 minutes each, and finally, neutral resin is added and a coverslip is placed on top. Finally, the slides are observed and photographed under an optical microscope.

[0039] Results of HE staining of mouse kidney tissue sections as follows Figure 6 The 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 pimozide treatment group significantly reduced renal tubular dilation, showed relative glomerular filling, and cystic dilation of a small number of renal tubules, significantly improving renal fibrosis.

[0040] 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, and then differentiated in 1% hydrochloric acid alcohol for a few seconds before rinsing with tap water to regain blue color. Sections were then treated in 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 in phosphomolybdic acid solution for 10 minutes, observing periodically until collagen fibers were clearly differentiated. Finally, sections were directly stained in 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.

[0041] 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 pimozide treatment group significantly reduced collagen fiber deposition in the tubulointerstitium. This indicates that both low-dose and high-dose pimozide treatments are effective in treating renal fibrosis.

[0042] Example 6 NRK-52E cells were seeded into 12-well plates and allowed to confluence for 72 hours until 100% confluence was achieved. After cell adhesion, NRK-52E cells were transfected with siRNA-EIF4A1 (Rat) for 10 hours, followed by treatment with pimozide for 48 hours in the presence or absence of TGF-β1. 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 8 As shown in Table 2, the specific sequence of the siRNA-EIF4A1(Rat) used is shown in Table 2.

[0043] Table 2: siRNA sequences To further verify whether pimozide's anti-liver fibrosis effect targets the EIF4A1 protein, LX-2 cells were used, and siRNA interference experiments were performed as described above. The specific siRNA-EIF4A1 (Human) sequence used is shown in Table 2. The consumables used in the Western Blot experiments in the above examples are shown in Table 3.

[0044] 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.

[0045] 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.

[0046] 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. Application of pimozide in the preparation of drugs for the treatment of renal fibrosis.

2. The application according to claim 1, characterized in that, Pimozide is the only active ingredient in the drug.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 4, characterized in that, The excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

5. The application according to claim 5, characterized in that, The diluent is either water or physiological saline.

6. A drug for treating renal fibrosis, characterized in that, The drug is composed of pimozide as described in claim 1 and excipients, wherein the pimozide content in the drug is 0.1 wt% to 99 wt%.

7. The drug according to claim 6, characterized in that, The drug is available in either a solid dosage form or a solution dosage form. The solid dosage forms include granules, tablets, capsules, pills, and drop pills, while the solution dosage forms include oral liquid preparations, oral enemas, and injectable dosage forms.

8. The medicament according to claim 7, characterized in that, The solution formulation is a solution composed of water and pimozide, or a solution composed of physiological saline and pimozide.

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