Application of BTK inhibitor in preparation of medicine for treating acute kidney injury

Abstract

The invention discloses application of a BTK inhibitor in preparation of a medicine for treating acute kidney injury and complications thereof. The invention provides a new strategy for treating acute kidney injury and complications thereof by using a BTK inhibitor, and particularly relates to application of the BTK inhibitor as an acute kidney injury medicine. The invention finds that serum creatinine and urea nitrogen levels can be effectively reduced by knocking out mouse BTK, and kidney injury is relieved and conversion from AKI to CKD is delayed or prevented by recovering kidney pathological structure and reducing apoptotic cell generation, which indicates that BTK is an effective target for treating acute kidney injury. Meanwhile, through treatment of an acute renal ischemia-reperfusion model, it is found that the BTK inhibitor such as ibrutinib can relieve ischemia-reperfusion induced renal function impairment and improve the renal function of a mouse, and it is indicated that the BTK inhibitor can serve as a treatment scheme for acute renal injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. More specifically, this invention relates to novel applications of Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib, in the treatment of acute kidney injury (AKI) and its related complications, and also to the application of BTK as a target in the preparation and / or screening of drugs for the prevention and treatment of acute kidney injury and its complications. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function. Its pathological mechanisms involve inflammation, oxidative stress, and apoptosis. It is a serious clinical syndrome posing a significant threat to global public health. The incidence of AKI is increasing annually, and it is particularly common in patients in intensive care units, those undergoing major surgery, and those with multiple underlying diseases. The causes of AKI are diverse, including ischemia-reperfusion injury, drug or toxin exposure, infection, and surgical complications. Once AKI occurs, the mortality rate is significantly increased. Even those who survive often face long-term adverse consequences, with progression to chronic kidney disease (CKD) being a prominent issue, severely impacting patients' quality of life and prognosis.

[0003] Currently, treatments for acute kidney injury (AKI) have many limitations. Clinically, the focus is mainly on early identification and prevention of AKI triggers. However, for patients with existing kidney damage, there are no specific drugs that can effectively reverse or significantly slow this pathological progression. Current clinical practice primarily involves supportive care, hoping for natural kidney recovery, but this is clearly insufficient to effectively restore kidney function and carries a high risk of complications. Common complications related to acute kidney injury include AKI recurrence, progression to chronic kidney disease (CKD), progression to end-stage renal disease (ESRD), cardiovascular events, and death. Therefore, the development of novel and effective AKI treatments is urgently needed.

[0004] BTK is a member of the cytoplasmic non-receptor tyrosine kinase TEC family and an important enzyme involved in the B cell receptor signaling pathway. BTK's role is not limited to B cell development; it can be expressed by other immune cell populations and is involved in the immune regulation and function of bone marrow cells. BTK inhibitors are mainly used for the treatment of tumors, especially hematologic malignancies. However, whether BTK is associated with the occurrence, development, and treatment of acute kidney injury (AKI) has not yet been reported. Summary of the Invention

[0005] This invention addresses the shortcomings of existing AKI treatment strategies and aims to explore effective AKI treatment drugs. This invention has discovered that BTK can serve as a therapeutic target for acute kidney injury.

[0006] The first objective of this invention is to provide the use of BTK inhibitors in the preparation of medicaments for treating acute kidney injury and its related complications.

[0007] A second objective of this invention is to provide the application of BTK as a target in the preparation of drugs for the prevention and treatment of acute kidney injury and its complications.

[0008] A third objective of this invention is to provide the application of BTK as a target in screening drugs for the prevention and treatment of acute kidney injury and its complications.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: The main characteristics of acute kidney injury (AKI) are elevated serum creatinine and blood urea nitrogen levels; significant inflammatory cell infiltration and structural damage in the tissues stained with hematoxylin and eosin (HE); aggravated renal tubular damage stained with pase smear; and increased Kim-1 staining. The main characteristic of worsening chronic renal fibrosis is increased Masson staining. This invention investigates an acute ischemia-reperfusion kidney injury model constructed using BTK knockout mice, using serum creatinine and blood urea nitrogen levels as indicators, and detecting the aforementioned pathological and molecular characteristics. The results show that BTK knockout mice can alleviate kidney damage by improving ischemia-reperfusion-induced renal function, restoring renal pathological structure, and reducing apoptotic cell production, thus delaying or preventing the conversion of AKI to CKD, indicating that BTK is an effective therapeutic target for acute kidney injury. Meanwhile, BTK inhibitors, such as ibrutinib, acalatinib, telatinib, orelabrutinib, pitobrutinib, nemabrutinib, veribrutinib, and lissobradib, have yielded similar experimental results in the treatment of acute ischemia-reperfusion kidney injury models, indicating that BTK inhibitors can be used as a treatment option for acute kidney injury.

[0010] Therefore, this invention claims protection for the following application schemes: Application of BTK inhibitors in the preparation of drugs for the treatment of acute kidney injury and its related complications.

[0011] Preferably, the BTK inhibitor refers to an active molecule that inhibits BTK, including but not limited to ibrutinib, acalabrutinib, tirabrutinib, orelabrutinib, pirtobrutinib, nemtabrutinib, vecabrutinib, and lissobrba.

[0012] This invention also provides the application of BTK as a target in the preparation and screening of drugs for the prevention and treatment of acute kidney injury and its related complications.

[0013] This invention also provides the application of BTK as a target in screening drugs for the prevention and treatment of acute kidney injury and its related complications.

[0014] Experiments have shown that when ibrutinib is used as a BTK inhibitor to treat mice with acute ischemia-reperfusion kidney injury, the pathological structure of the kidney is restored better and fewer apoptotic cells are produced, thereby significantly reducing kidney damage and effectively delaying or preventing the conversion of AKI to CKD.

[0015] Specifically, in the above applications, BTK inhibitors can be administered parenterally or orally at a dose of 1 mg / kg / day to 10 mg / kg / day. In this embodiment, the dose is 5 mg / kg / day.

[0016] Specifically, the BTK inhibitor can reduce serum creatinine levels.

[0017] Specifically, the BTK inhibitor can reduce urea nitrogen levels.

[0018] Specifically, the BTK inhibitor can reduce the production of apoptotic cells.

[0019] Specifically, the BTK inhibitor can delay or prevent the conversion of AKI to CKD.

[0020] Specifically, in the above applications, the relevant complications are recurrence of AKI, progression to chronic kidney disease, progression to end-stage renal disease, cardiovascular events, and death.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides novel applications of BTK inhibitors in the preparation of drugs for treating acute kidney injury and its complications, as well as novel applications of BTK as a target in screening drugs for the prevention and / or treatment of acute kidney injury and its complications. This invention establishes an acute renal ischemia-reperfusion model using BTK gene knockout mice and finds that BTK knockout in mice can alleviate ischemia-reperfusion-induced renal function damage and improve renal function, indicating that BTK is an effective target for the treatment of acute kidney injury. Simultaneously, studies have found that BTK inhibitors, such as ibrutinib, acalatinib, tillatinib, orelabrutinib, pitobrutinib, nelmabrutinib, veribrutinib, and lisobreb, can effectively reduce serum creatinine and blood urea nitrogen levels, alleviate kidney damage by restoring renal pathological structure and reducing apoptotic cell production, and delay or prevent the conversion of AKI to CKD, indicating that BTK inhibitors can be used as a treatment option for acute kidney injury. Attached Figure Description

[0022] Figure 1 The serum creatinine and blood urea nitrogen levels in BTK knockout mice after ischemia-reperfusion treatment, **P <0.01, *** P <0.001, **** P <0.0001.

[0023] Figure 2 HE and PAS staining results of pathological damage to kidney tissue of BTK knockout mice after ischemia-reperfusion treatment.

[0024] Figure 3 The image shows the results of the immunofluorescence (Kim-1) experiment on the kidney tissue of BTK knockout mice.

[0025] Figure 4 This image shows the Masson staining results of the kidneys of BTK knockout mice. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] Ibrutinib, CAS number 936563-96-1.

[0029] Acalabrutinib, CAS number 1420477-60-6.

[0030] Tirabrutinib, CAS number 75747-14-7.

[0031] Orelabrutinib, CAS number 1655504-04-3.

[0032] Pirtobrutinib, CAS number 2101700-15-4.

[0033] Nemtabrutinib, CAS number 2095393-15-8.

[0034] Vecabrutinib, CAS number 1510829-06-7.

[0035] Rilzabrutinib, CAS number 1575596-29-0.

[0036] All of the above-mentioned drugs were purchased from MCE Company.

[0037] Six-week-old male C57BL / 6 mice, weighing approximately 20g, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0038] Example 3: The role of BTK in acute kidney injury To investigate the role of BTK in renal ischemia-reperfusion injury, this embodiment uses BTK knockout mice to construct an acute kidney injury model of ischemia-reperfusion.

[0039] 1. Experimental Methods (1) Laboratory animals and grouping The experiment was designed with three groups of mice: Experimental Group 1: Sham Surgery Group; Experimental group 2: Control group (model group); Experimental group 3: BTK knockout group (BTK-KO group).

[0040] Thirty-six C57BL / 6 mice were acclimatized for one week and then divided into three groups of 12 mice each. Six mice in each group were collected during the acute phase (day 3 post-surgery), and six mice were collected during the chronic phase (day 21 post-surgery). The BTK knockout group consisted of C57BL / 6 mice with the BTK gene knocked out.

[0041] (2) Ischemia-reperfusion surgery to establish an acute kidney injury model: Anesthetized with isoflurane on a 37°C constant temperature heating pad, the kidneys were exposed, and both renal pedicles were clamped with non-traumatic vascular clamps for 30 minutes. The color change of the kidneys was observed. The kidneys turned black within 1 minute after clamping and returned to normal color within about 10 seconds after the clamps were removed.

[0042] The mice in the sham surgery group only underwent abdominal surgery and did not undergo renal pedicle clamping (i.e., renal ischemia-reperfusion surgery).

[0043] (3) Experimental materials: Kidney and serum samples were collected from mice in the acute phase on day 3 post-surgery, and from mice in the chronic phase on day 21 post-surgery.

[0044] (4) Detection indicators and methods: 1) Renal function index detection: After drug administration, blood was collected from the orbital sinus of mice, and serum was separated by centrifugation. The degree of renal function impairment was assessed using serum creatinine (SCr) and blood urea nitrogen (BUN) level kits. The serum volume was calculated, and the samples were added sequentially according to the manufacturer's instructions. The system in 1.5 mL EP tubes was thoroughly mixed, and 200 μL was pipetted into each well of a 96-well plate and incubated at 37°C for 30 min. The absorbance value of each well was measured at a wavelength of 546 nm. A standard curve was constructed, and the serum creatinine and blood urea nitrogen concentrations of each well were calculated.

[0045] 2) Kidney histopathological examination: Mouse kidney tissue was taken, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE), PAS (Periodic Acid-Schiff) stain, and Masson stain. The specific steps for HE staining are as follows: Paraffin sections were dewaxed to water: Sections were sequentially immersed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min. After rinsing with tap water, sections were treated with high-resolution constant staining pretreatment solution for 1 min, then sequentially stained with hematoxylin and eosin staining solutions. Finally, the sections were dehydrated, mounted, examined under a microscope, and the images were acquired and analyzed. The specific steps for PAS staining are as follows: The dewaxing process for paraffin sections was the same as for HE. Sections were then sequentially stained with PAS staining solutions B, A, and C, protected from light. After staining, the sections were rinsed with tap water, differentiated with hydrochloric acid solution, and then blued with ammonia solution. Finally, the sections were rinsed with running water, dehydrated, mounted, and examined under a microscope. The standardized procedure for Masson staining can be summarized as follows: After dewaxing paraffin sections to water, the cell nuclei are typically stained with Weigert iron hematoxylin for about 5-10 minutes, followed by washing with water to allow differentiation to return to blue; then, the sections are stained with Ponceau S and acidic fuchsin for 5-10 minutes to make muscle fibers, etc., appear red; next, they are treated with 1% phosphomolybdic acid solution for 1-3 minutes to promote differentiation and inhibit red staining; then, without washing, they are directly transferred to aniline blue solution for 1-2 minutes to make collagen fibers appear blue; finally, they are briefly rinsed with a weak acid solution to optimize the hue, and then quickly dehydrated, cleared with xylene, and mounted with neutral resin.

[0046] 3) Immunofluorescence staining: On the first day, paraffin sections of mouse kidneys were obtained, dewaxed, and hydrated. The sections were then immersed in citrate buffer (pH 6.0) at 90-95°C for 30 minutes for antigen retrieval. After natural cooling, the sections were placed in 3% hydrogen peroxide solution to block endogenous peroxidase. 3% BSA was added to the histochemistry zone for blocking at room temperature for 60 minutes. Primary antibody prepared in PBS at a specific ratio was added to the sections, and the sections were incubated overnight at 4°C in a humidified chamber. On the second day, the sections were removed and brought to room temperature. After washing with PBST, secondary antibody was added and incubated for 60 minutes. Finally, the nuclei were stained with DAPI, mounted with anti-fluorescence quenching mounting medium, and examined under a microscope.

[0047] 2. Experimental Results: Figure 1Serum levels of creatinine and blood urea nitrogen (BUN) were measured in mice after ischemia-reperfusion treatment by collecting serum from each group. The results showed that serum creatinine and BUN were significantly elevated in the control group, being (4.1 ± 0.3) times higher than in the sham-operated group, indicating successful model establishment. Compared with the control group, the BTK knockout group showed significantly lower serum creatinine and BUN levels after ischemia-reperfusion treatment. This result indicates that BTK knockout can alleviate ischemia-reperfusion-induced renal function damage, thereby improving renal function in mice.

[0048] Figure 2 HE and PAS staining results showed that in the control group mice, after ischemia-reperfusion treatment, the kidney tissue exhibited significant pathological changes such as renal tubular epithelial cell necrosis and shedding, luminal dilation, interstitial edema, and inflammatory cell infiltration. However, the pathological damage in the kidney tissue of the BTK knockout group mice after ischemia-reperfusion treatment was significantly reduced. Specifically, the renal tubular epithelial cell structure of the BTK knockout group mice was relatively intact, the degree of luminal dilation was reduced, and interstitial edema and inflammatory cell infiltration were decreased. This result indicates that BTK knockout can improve the pathological structure of the kidney induced by ischemia-reperfusion, thereby reducing the degree of kidney tissue damage.

[0049] Figure 3 Immunofluorescence assays showed that, compared with the control group, the expression of Kim-1 (red fluorescence) in the kidney tissue of BTK knockout mice was decreased. This result further confirms that BTK knockout has a positive effect on alleviating ischemia-reperfusion-induced renal tubular injury in mice.

[0050] Figure 4 Masson staining results showed that, compared with the control group, BTK gene knockout reduced the degree of chronic tissue fibrosis, confirming that BTK inhibition slows the transformation of acute injury into chronic injury.

[0051] 3. Conclusion: BTK knockout was used to treat mice with acute kidney injury. The results showed that BTK knockout could alleviate ischemia-reperfusion-induced kidney function damage, improve kidney function, and slow the progression of AKI to CKD. This further suggests that BTK is a target for the treatment of acute kidney injury, and BTK inhibitors can be used as a treatment option for acute kidney injury. Advancing the research and development of BTK inhibitors can help improve acute kidney injury.

[0052] Example 2: Protective effect of BTK inhibitors on acute kidney injury To investigate the role of BTK inhibitors such as ibrutinib in acute kidney injury, this example uses C57BL / 6 mice to construct an acute ischemia-reperfusion kidney injury model. The specific operation for constructing the model is the same as in Example 1.

[0053] 1. Experimental Methods (1) Animal grouping and drug treatment plan: The experiment was designed with three groups of mice: Experimental Group 1: Sham Surgery Group (the sham surgery group in Example 1); Experimental Group 2: Modeling Group; Experimental group 3: BTK inhibitor treatment group (dosage was 5 mg / kg / day).

[0054] After acclimatizing for one week, 12 mice were randomly divided into experimental group 2 and experimental group 3, with 6 mice in each group.

[0055] The BTK inhibitor powder was dissolved in DMSO and stored at -20°C. Following ischemia-reperfusion surgery, mice in the BTK inhibitor treatment group were administered the corresponding dose via gavage, diluted with physiological saline, in 100 μL volumes. Mice in the sham-operated and model groups were administered the same volume of physiological saline via gavage daily for 3 consecutive days.

[0056] The BTK inhibitors selected were ibrutinib, acalatinib, telatinib, orelabrutinib, pitobrutinib, nemabrutinib, veribrutinib, and lissobradib, to investigate the therapeutic effects of different BTK inhibitors on a mouse model of ischemia-reperfusion.

[0057] (2) Detection indicators and methods: The main tests include kidney function indicators and a kidney damage score.

[0058] The detection methods for renal function indicators (serum creatinine and blood urea nitrogen) are the same as in Example 1.

[0059] Kidney Injury Scoring Criteria: Kidney tissue was stained with hematoxylin and eosin, and the degree of renal tubular injury was assessed according to the method described in Example 1. Renal tubular injury was classified into 6 grades based on the absence of the brush border, tubular dilation, cast formation, tubular necrosis, and neutrophil infiltration. Ten high-power fields (original magnification × 200) were randomly selected, five located in the renal cortex and five in the cortico-medulloeal junction. Each field was scored from 0 to 5, specifically as follows: 0: Normal; 1: Mild injury, affecting 0%~10%; 2: Moderate injury, affecting 11% to 25%; 3: Severe injuries, affecting 26% to 49%; 4: Highly severe injuries, affecting 50% to 75% of cases; 5: Extensive damage, involving >75%.

[0060] 2. Experimental Results The results of different BTK inhibitors on renal function indicators and renal injury scores in mice are shown in Table 1.

[0061] Table 1

[0062] As shown in Table 1, compared with the model group, the serum creatinine and blood urea nitrogen levels, as well as the kidney injury score, were significantly reduced in mice treated with different BTK inhibitors. Ibrutinib showed the best effect in reducing serum creatinine and blood urea nitrogen levels, while telatinib had a similar effect on reducing serum creatinine levels, but a slightly lower effect on reducing blood urea nitrogen levels. Meanwhile, ibrutinib and telatinib showed similar effects on reducing kidney injury scores, indicating that the pathological structure of the kidneys was significantly improved.

[0063] 3. Conclusion: Through experimental research, the inventors discovered that BTK inhibitors such as ibrutinib can alleviate kidney damage by improving ischemia-reperfusion-induced renal function damage in mice and restoring the pathological structure of the kidneys, thereby delaying or preventing the conversion of AKI to CKD.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of BTK inhibitors in the preparation of drugs for the treatment of acute kidney injury and its related complications.

2. The application according to claim 1, characterized in that, The BTK inhibitor is selected from at least one of the following drugs or their pharmaceutically acceptable salts: ibrutinib, acalabrutinib, telatinib, orelabrutinib, pitobrutinib, nemabrutinib, veribrutinib, and lissobradib.

3. Application of BTK as a target in the preparation of drugs for the prevention and treatment of acute kidney injury and its related complications.

4. Application of BTK as a target in screening drugs for the prevention and treatment of acute kidney injury and its related complications.

5. The application according to any one of claims 1 to 4, characterized in that, The drug has the function of lowering serum creatinine levels.

6. The application according to any one of claims 1 to 4, characterized in that, The drug has the function of reducing blood urea nitrogen levels in patients with kidney damage.

7. The application according to any one of claims 1 to 4, characterized in that, The drug has the function of reducing the production of apoptotic cells.

8. The application according to any one of claims 1 to 4, characterized in that, The drug can delay or prevent the transformation of acute kidney injury into chronic kidney disease.

9. The application according to any one of claims 1 to 4, characterized in that, The drug can delay or prevent the progression of acute kidney injury to end-stage renal disease.

10. The application according to any one of claims 1 to 4, characterized in that, The drug can delay or prevent the recurrence of acute kidney injury.

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