A drug targeting the CK1ε gene or protein to treat the intestinal toxicity of duveliximab.

Abstract

This invention discloses a drug targeting the CK1ε gene or protein for treating duveliximab intestinal toxicity, belonging to the field of pharmaceutical technology. The drug reverses duveliximab intestinal toxicity by downregulating CK1ε gene expression, inhibiting CK1ε protein function, or inhibiting CK1ε protein accumulation. This invention provides a novel therapeutic drug target for intervening intestinal toxicity caused by duveliximab. This invention proposes that the E3 ubiquitin ligase NEDD4L can ubiquitinate CK1ε and subsequently degrade it through the ubiquitin-proteasome pathway, providing a new direction for current intervention strategies for drug-induced intestinal toxicity and addressing, to some extent, the current situation of limited available intervention drugs and their singular mechanisms. The intervention drug reverses duveliximab intestinal toxicity by downregulating CK1ε, expanding the clinical application value of duveliximab.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of the CK1ε gene or CK1ε protein as a drug target in the preparation of drugs for treating the intestinal toxicity of duveridine. Background Technology

[0002] Duvelisib, a typical representative of PI3K inhibitors, can simultaneously inhibit the activity of PI3K-δ and PI3K-γ. It is suitable for the treatment of relapsed or refractory chronic lymphocytic leukemia and two types of indolent non-Hodgkin lymphoma. Patients have a long progression-free survival (16.4 months vs 9.1 months) and a high overall response rate (73.8% vs 45.3%), demonstrating excellent therapeutic effects and making it irreplaceable in clinical practice. However, Duvelisib has been widely used in clinical practice with serious and even fatal intestinal toxicity. 77.8% of patients experienced intestinal toxicity after using Duvelisib, with the incidence of grade 3 or higher adverse reactions reaching as high as 22.2% (Patel K, et al. Duvelisib for CLL / SLL and follicular non-Hodgkin lymphoma. Blood 2019; 134:1573-7.; Davids MS, et al. Efficacy and Safety of Duvelisib Following Disease Progression on Ofatumumab in Patients with Relapsed / Refractory CLL or SLL in the DUO Crossover Extension Study. ClinCancer Res 2020; 26:2096-103.). Therefore, intestinal toxicity has been marked with a "black box warning" by the US FDA.

[0003] Currently, there are no effective means to intervene in the intestinal toxicity of duveliximab in clinical practice. The main reason for this problem is that the molecular mechanism by which duveliximab causes intestinal toxicity has not been fully elucidated. Given the important and irreplaceable role of this drug in the clinical treatment of patients with relapsed or refractory hematological malignancies, in-depth research into the molecular mechanism of duveliximab-induced intestinal toxicity, the discovery of interventional targets, and the search for effective intervention strategies are of great significance for the clinical application of duveliximab and other PI3K inhibitors.

[0004] Casein kinase 1ε (CK1ε) regulates cellular processes under various physiological and pathological conditions by phosphorylating multiple protein substrates, playing a multi-functional role in cell cycle regulation. CK1ε can stabilize β-catenin by phosphorylating Disheveled (DVL), thereby activating the classical Wnt signaling pathway and promoting the transcription of cell proliferation-related genes (Janovska P, et al. Targeting Casein Kinase 1 (CK1) in Hematological Cancers. Int J Mol Sci 2020; 21.). CK1ε can also promote the binding of the cell cycle positive regulator protein cdc25A to SCF / β-TrCP by phosphorylating multiple inhibitory sites, inducing its degradation via the ubiquitin-proteasome pathway, thereby inhibiting cell cycle progression (Piao S, et al. CK1 epsilon targets Cdc25A for ubiquitin-mediated proteolysis under normal conditions and in response to checkpoint activation. Cell Cycle 2011; 10:531-7.). However, there are currently no literature reports on the relationship between CK1ε protein and the intestinal toxicity of duvelixib, which requires further research.

[0005] β,β-Dimethacryloxyshikonin (ALCAP2) is the main active ingredient in shikonin root and belongs to the naphthoquinone class of compounds. Existing studies have shown that β,β-dimethacryloxyshikonin has anti-tumor effects. However, there are currently no reports of β,β-dimethacryloxyshikonin treating CK1ε-related diseases or injuries. Summary of the Invention

[0006] The purpose of this invention is to explore genes / proteins related to the intestinal toxicity induced by duveliximab, thereby using these genes / proteins as targets for the prevention and treatment of duveliximab intestinal toxicity, screening drugs for the treatment of duveliximab intestinal toxicity side effects, solving the intestinal toxicity side effects of duveliximab, and expanding the clinical application value of duveliximab.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides the application of targeting the casein kinase 1ε gene (CK1ε) or casein kinase 1ε (CK1ε) in the preparation of a drug for treating intestinal toxicity caused by duveliximab, wherein the intestinal toxicity caused by duveliximab includes G0 / G1 phase arrest of small intestinal epithelial cells, and the drug is a drug that downregulates the expression of the casein kinase 1ε gene or targets and inhibits the function of casein kinase 1ε or inhibits the accumulation of casein kinase 1ε.

[0009] The pathological manifestations of intestinal toxicity caused by Duvelixib also include: inhibition of intestinal epithelial cell proliferation, shortening and disordered arrangement of small intestinal villi, increased spacing between colonic glands, and thickening of the basal layer.

[0010] Specifically, the nucleotide sequence of the human casein kinase 1ε gene is shown in SEQ ID No. 1, and the amino acid sequence of casein kinase 1ε is shown in SEQ ID No. 2.

[0011] This study found that duveliximab inhibits the degradation of CK1ε protein in small intestinal epithelial cells, leading to its accumulation. Both duveliximab action and CK1ε overexpression cause G0 / G1 phase arrest in small intestinal epithelial cells, suggesting that CK1ε protein accumulation is a key cause of duveliximab-induced intestinal toxicity. CK1ε may be a potential drug target for preventing or treating duveliximab-induced intestinal toxicity. Therefore, inhibiting CK1ε gene expression, targeting and inhibiting CK1ε function, or inhibiting CK1ε protein accumulation could be used as a means to intervene in duveliximab intestinal toxicity.

[0012] This invention employs RNA interference technology to downregulate the CK1ε protein level in small intestinal epithelial cells, thereby reversing the G0 / G1 phase arrest induced by duvelixisib. Specifically, after using siRNA, the increased proportion of small intestinal epithelial cells in the G0 / G1 phase and the elevated levels of the cell cycle arrest-related protein p27 induced by duvelixisib were significantly downregulated, revealing that the CK1ε gene is a key gene in duvelixisib-induced intestinal damage. Therefore, this invention provides a novel drug target for intervening intestinal toxicity caused by duvelixisib.

[0013] A drug formulation targeting the CK1ε gene to knock down its expression was developed. The drug aims to treat the intestinal toxicity side effects caused by duveridine by downregulating the expression of the CK1ε gene.

[0014] Preferably, the drug comprises siRNA targeting the casein kinase 1ε gene. The siRNA targeting the CK1ε gene can inhibit CK1ε expression, thereby reversing dovelixib-induced intestinal toxicity.

[0015] This invention also provides the application of the CK1ε gene or CK1ε protein as a drug target in screening drugs for the treatment of intestinal toxicity of duveridine.

[0016] Specifically, cell or animal models are used to screen for drugs that promote CK1ε degradation or drugs that target and inhibit CK1ε function, and the activity of the test drug is evaluated by measuring CK1ε protein activity or expression level.

[0017] This invention has found that the combined use of CK1ε inhibitors or CK1ε ubiquitination promoters can significantly downregulate CK1ε accumulation induced by duveliximab and reverse cell cycle arrest in small intestinal epithelial cells induced by duveliximab. Therefore, this invention provides a novel use of CK1ε inhibitors or CK1ε ubiquitination promoters in intervening in duveliximab-induced enterotoxicity.

[0018] Specifically, the drug includes a casein kinase 1ε inhibitor or a casein kinase 1ε ubiquitination promoter. Targeted inhibition of CK1ε or CK1ε ubiquitination promoters can significantly reduce the excessive activation of CK1ε function in small intestinal epithelial cells induced by duvelixib.

[0019] Preferably, the CK1ε inhibitor is PF-4800567.

[0020] Preferably, the casein kinase 1ε ubiquitination promoter is a NEDD4-like E3 ubiquitin ligase (NEDD4L) or a NEDD4L promoter. This invention has found that NEDD4L is an E3 ubiquitin ligase of CK1ε, which ubiquitinates CK1ε and subsequently degrades it via the ubiquitin-proteasome pathway, thereby reducing the accumulation of CK1ε protein and achieving the goal of treating the enterotoxic side effects caused by duvelixib.

[0021] Another object of the present invention is to provide the use of NEDD4-like E3 ubiquitin ligase or NEDD4-like E3 ubiquitin ligase promoter in the preparation of medicaments for treating diseases or damage caused by abnormal expression of casein kinase 1ε protein, wherein the NEDD4-like E3 ubiquitin ligase or its promoter promotes casein kinase 1ε ubiquitination thereby reducing the accumulation of casein kinase 1ε.

[0022] Furthermore, the disease or injury is a duveliximab-induced intestinal toxicity. The NEDD4L promoter can downregulate duveliximab-induced CK1ε protein accumulation by being used in combination with duveliximab.

[0023] Preferably, the NEDD4L promoter can be, but is not limited to, β,β-dimethylacryloylshikonin or a pharmaceutically acceptable salt thereof.

[0024] This invention provides an effective treatment for intestinal toxicity caused by duveliximab. Animal experiments show that, compared with duveliximab monotherapy, the combination of β,β-dimethacryloylshikonin can reverse the downregulation of NEDD4L protein levels, upregulation of CK1ε and p27 expression, and G0 / G1 cell cycle arrest induced by duveliximab in intestinal epithelial cells, thus restoring intestinal damage.

[0025] Preferably, the mass ratio of β,β-dimethylacryloylshikonin to duveliximab in the drug is 1.5-3:1.

[0026] The drug also includes pharmaceutically acceptable excipients, including fillers, wetting agents, binders, disintegrants, or lubricants. The drug may be formulated as a solid or liquid dosage form. Preferably, the drug is formulated as an oral dosage form.

[0027] The present invention also provides an antitumor combination pharmaceutical composition comprising a first formulation formed of duveliximab and a pharmaceutically acceptable carrier, and a second formulation formed of β,β-dimethylacryloylshikonin or a salt thereof and a pharmaceutically acceptable carrier.

[0028] The present invention demonstrates that the combined use of β,β-dimethylacryloylshikonin or its salt with duveliximab has good biocompatibility, not only reversing the intestinal toxicity caused by duveliximab but also enhancing the antitumor effect of duveliximab.

[0029] The present invention also provides the use of the described pharmaceutical composition in the preparation of a drug for treating lymphoma. The lymphoma may be, but is not limited to, relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).

[0030] The beneficial effects of this invention are as follows:

[0031] (1) This invention provides the application of the CK1ε gene or CK1ε protein as a drug target in the preparation of a drug for treating duveliximab intestinal toxicity. The drug reverses duveliximab intestinal toxicity by downregulating the expression of the CK1ε gene, inhibiting the functional activity of the CK1ε protein, or inhibiting the accumulation of the CK1ε protein. This invention provides a novel therapeutic drug target for intervening in the intestinal toxicity caused by duveliximab.

[0032] (2) This invention proposes for the first time that NEDD4L is the E3 ubiquitin ligase of CK1ε, which ubiquitinates CK1ε and then degrades it through the ubiquitin-proteasome pathway. This provides a new direction for finding intervention strategies to address drug-induced intestinal toxicity and, to some extent, solves the current situation of limited available intervention drugs and single mechanisms in clinical practice. Attached Figure Description

[0033] Figure 1 The effects of doveliximab and CK1ε overexpression plasmid on CK1ε protein and IEC-6 intestinal epithelial cells were investigated. A shows the cell cycle of doveliximab at different concentrations and durations using flow cytometry; B shows the expression level of CK1ε protein using Western blot; and C shows the effect of CK1ε overexpression plasmid using Western blot.

[0034] Figure 2 The study investigated the effect of CK1ε gene knockdown on the inhibition of IEC-6 intestinal epithelial cell proliferation caused by doveliximab. In the study, A was the detection of CK1ε protein and p27 expression levels by Western blot; B was the detection of cell cycle arrest by flow cytometry; and C was the detection of the effect of in vivo specific knockdown of CK1ε on the inhibition of intestinal epithelial cell proliferation caused by doveliximab by immunohistochemical staining.

[0035] Figure 3 The effect of CK1ε inhibitors on the inhibition of intestinal epithelial cell proliferation caused by duvelixib.

[0036] Figure 4 The effects of duveliximab on the transcriptional, protein, and ubiquitination levels of CK1ε in intestinal epithelial cells were investigated, where A represents the transcriptional level, B represents the protein level, and C represents the ubiquitination level.

[0037] Figure 5 The effect of NEDD4L on CK1ε protein expression level is shown in Figure A, where Western blot was used to detect CK1ε protein and p27 expression levels, and immunoprecipitation assay was used to detect ubiquitination level.

[0038] Figure 6 To investigate the effect of the combined use of CK1ε ubiquitination promoter β,β-dimethylacryloylshikonin on duveliximab-induced cell cycle arrest in intestinal epithelial cells IEC-6, A was the detection of CK1ε protein and p27 expression levels by Western blot; B was the detection of cell cycle arrest by flow cytometry.

[0039] Figure 7 The effect of combined use of CK1ε ubiquitination promoter β,β-dimethylacryloylshikonin on duveliximab-induced intestinal injury in mice was investigated.

[0040] Figure 8 The effects of combined β,β-dimethylacryloxyshikonin on intestinal epithelial cell proliferation and CK1ε protein accumulation were detected by immunohistochemical staining of intestinal sections. In this study, A represents the expression level of PCNA detected by immunohistochemistry, and B represents the expression level of CK1ε protein detected by immunohistochemistry.

[0041] Figure 9The effect of combined use of β,β-dimethylacryloylshikonin on mouse body weight.

[0042] Figure 10 To investigate the effect of combined use of β,β-dimethylacryloylshikonin on the antitumor effect of duveliximab. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0045] C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; 2-hydroxypropyl-β-cyclodextrin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; rat small intestinal epithelial cells (IEC-6) were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; CK1ε antibody was purchased from Origene Corporation; GAPDH antibody was purchased from Hangzhou Daige Biotechnology Co., Ltd.; p27 antibody was purchased from Santa Cruz Biotechnology Co., Ltd.; PCNA antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd.; siRNA was purchased from Shanghai Gemma Pharmaceutical Co., Ltd., with the negative control (NC) positive strand sequence being 5'-ACGUGACACGUUCGGAGAATT-3'; the siCK1ε positive strand sequences were 5'-GCUAUGUGC UCAUGUACUUTT-3' and 5'-GCAAUCUGGUAUACAUCAUTT-3'; transfection reagents were also used. Purchased from Polyplus Transfection; AAV9-NC and AAV9-sh CK1ε were purchased from Shanghai Jikai Gene Technology Co., Ltd.

[0046] Duveliximab, CAS No. 1201438-56-3, chemical name (S)-3-(1-((9H-purin-6-yl)amino)ethyl)-8-chloro-2-phenyl-1(2H)-isoquinolinone, molecular formula C 22 H 17 ClN6O, with a molecular weight of 416.86, was purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., and its structural formula is as follows:

[0047]

[0048] β,β-Dimethylacryloylshikonin (ALCAP2), CAS No. 24502-79-2, molecular formula C 21 H 22 O6, with a molecular weight of 370.396, was purchased from Nantong Jingwei Biotechnology Co., Ltd., and its structural formula is as follows:

[0049]

[0050] PF-4800567, CAS number 1188296-52-7, molecular formula C 17 H 18 ClN5O2, with a molecular weight of 359.81, was purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., and its structural formula is as follows:

[0051]

[0052] Example 1

[0053] Rat small intestinal epithelial cells (IEC-6) were seeded at a density of 80,000 cells / well in 12-well plates. After 24 hours of stable adhesion, the cells were treated with gradients of doveliximab (0, 2, 4, 8, and 16 μM) for 24 hours or with 8 μM doveliximab for different durations (0, 3, 6, 9, 12, and 24 hours). Cells were harvested, and cell cycle analysis was performed using PI staining combined with flow cytometry. Proteins were extracted and quantified, and then Western blot was used to determine protein levels.

[0054] Rat small intestinal epithelial cells IEC-6 were seeded at a density of 120,000 per well in 12-well plates. After 24 hours of stable adhesion, the CK1ε overexpression plasmid (original vector: pCDNA3.0-3'HA, CK1ε coding sequence Gene ID: 58822) and the negative control vector were introduced into IEC-6 cells using jetPRIME transfection reagent. Cells were harvested 24 hours later, proteins were extracted and quantified, and then the protein levels were detected by Western blot.

[0055] The results are as follows Figure 1 As shown, the flow cytometry results indicate that, compared to the control group, with increasing concentration and duration of dovexib treatment, the proportion of cells in the G0 / G1 phase significantly increased. Figure 1 A). Protein level detection using Western blot showed that, compared to the control group, duvelixib significantly upregulated CK1ε protein levels in IEC-6 with increasing concentration and time. Figure 1 B) suggests that duvelixib induces cell cycle arrest and CK1ε protein accumulation. After intracellular overexpression of CK1ε, the level of the cell cycle arrest-related protein p27 significantly increased (B). Figure 1C) suggests that CK1ε may be a key protein in the cell cycle arrest caused by duvelixib.

[0056] Example 2

[0057] Rat small intestinal epithelial cells (IEC-6) were seeded at a density of 80,000 cells / well in 12-well plates. After 24 hours of stable adhesion, siRNA targeting CK1ε (5'-GCUAUGUGCUCAUGUA CUUTT-3', 5'-GCAAUCUGGUAUACAUCAUTT-3') and negative control NC (5'-ACGUGACACGUUCGGAGAATT-3') were introduced into IEC-6 cells using jetPRIME transfection reagent. After stable expression, the cells were treated with 8 μM dovexib for 24 hours, and then the cells were harvested. Cell cycle was detected by PI staining combined with flow cytometry. Proteins were extracted and quantified, and then the protein levels were detected by Western blot.

[0058] Twenty male C57BL / 6 mice were randomly divided into two groups: the AAV9-NC group and the AAV9-sh CK1ε group, with ten mice in each group. Each mouse was administered 1 × 10¹² dextrose via enema. 11 The viral load was 100 μL. After 3 weeks of stable culture, mice from the AAV9-NC group and the AAV9-sh CK1ε group were randomly assigned to the following groups: AAV9-NC-vehicle group; AAV9-NC-duvelisib group; AAV9-sh CK1ε-vechile group; and AAV9-sh CK1ε-duvelisib group. Five mice were assigned to each group. The treatment groups received 10 mg / kg duvelisib via gavage daily, while the control group received 10 μL / g of standard 20% cyclodextrin via gavage daily for 8 weeks. Intestinal tissue was dissected, and PCNA expression levels were detected by immunohistochemical staining.

[0059] like Figure 2 As shown, knocking down CK1ε in IEC-6 cells followed by administration of duveliximab reversed the drug-induced increase in p27 and CK1ε protein levels. Figure 2 A), G0 / G1 cell cycle arrest ( Figure 2 B) indicates that siRNA targeting CK1ε can alleviate cell cycle arrest by reversing CK1ε protein accumulation induced by duveliximab. Specific knockdown of CK1ε in the mouse gut followed by administration of duveliximab reversed drug-induced inhibition of intestinal epithelial cell proliferation. Figure 2 C).

[0060] Example 3

[0061] Rat small intestinal epithelial cells IEC-6 were seeded at a density of 80,000 per well in 12-well plates. After 24 hours of stable adhesion, the cells were treated with a combination of CK1ε inhibitor PF-4800567 (0.5 μM) and duveliximab (8 μM) for 24 hours before being harvested. Proteins were extracted and quantified, and then Western blot was used to detect protein levels.

[0062] like Figure 3 As shown, the CK1ε inhibitor PF-4800567 can reverse the increase in p27 levels, a cell cycle arrest-related protein, induced by duvelixib.

[0063] Example 4

[0064] Rat small intestinal epithelial cells IEC-6 were seeded in 6-well plates at a density of 160,000 cells / well. After 24 hours of stable adhesion, the cells were treated with a concentration gradient of duveliximab (0, 2, 4, 8 and 16 μM) for 24 hours. RNA was then extracted from the cells and the transcription level was detected by qRT-PCR.

[0065] Human embryonic kidney cells 293T were seeded at a density of 2 million per dish in cell culture dishes. After 24 hours of stable adhesion, CK1ε and ubiquitin (original vector: pCDNA3.0-3'His, Gene ID of the Ubc coding sequence: 50522) were exogenously overexpressed. After treatment with duvelixib, CK1ε was enriched by immunoprecipitation, and the ubiquitination status of CK1ε was detected.

[0066] like Figure 4 As shown, there was no significant difference in CK1ε transcription levels among IEC-6 cells. Figure 4 A) Under the action of the protein synthesis inhibitor actinomycete, it was found that doveliximab could prolong the half-life of CK1ε. Figure 4 B) suggests that duveliximab can inhibit the degradation of CK1ε. Immunoprecipitation results indicate that duveliximab can significantly reduce the ubiquitination level of CK1ε. Figure 4 C). The above results suggest that duvelixib may upregulate CK1ε protein levels by inhibiting CK1ε degradation via the ubiquitin-proteasome pathway.

[0067] Example 5

[0068] Rat small intestinal epithelial cells IEC-6 were seeded at a density of 120,000 per well in 12-well plates. After 24 hours of stable adhesion, the NEDD4L overexpression plasmid (original vector: pCDNA3.0-3'Flag, Gene ID of NEDD4L coding sequence: 291553) and the negative control vector were introduced into IEC-6 cells using jetPRIME transfection reagent. Cells were harvested after 24 hours, proteins were extracted and quantified, and then Western blot was used to detect protein levels.

[0069] Using liposome transfection technology, 293T cells overexpressing NEDD4L, ubiquitin, and CK1ε were constructed. Immunoprecipitation assays were used to detect the interaction between the two and the changes in CK1ε ubiquitination levels.

[0070] like Figure 5 As shown, overexpression of NEDD4L in IEC-6 cells alleviated the increase in p27 and CK1ε protein levels induced by dovexib. Immunoprecipitation results showed that CK1ε polyubiquitination was enhanced in the presence of NEDD4L. These studies collectively demonstrate that NEDD4L can promote CK1ε polyubiquitination and its degradation.

[0071] Example 6

[0072] Rat small intestinal epithelial cells IEC-6 were seeded at a density of 80,000 per well in 12-well plates. After 24 hours of stable adhesion, the cells were treated with β,β-dimethylacryloylshikonin (1, 2 μM) and duveliximab (8 μM) for 24 hours before harvesting. Cell cycle was detected by PI staining combined with flow cytometry. Proteins were extracted and quantified, and then Western blot was used to detect protein levels.

[0073] Sixty male C57BL / 6J mice were randomly divided into four groups: a control group, a dovelixiceb group, a low-dose β,β-dimethacryloxyshikonin group, a dovelixiceb + low-dose β,β-dimethacryloxyshikonin group, a high-dose β,β-dimethacryloxyshikonin group, and a dovelixiceb + high-dose β,β-dimethacryloxyshikonin group, with one mouse in each group. The mice were administered the drugs via gavage. The dosage of dovelixiceb was 10 mg / kg / day, the dosage of low-dose β,β-dimethacryloxyshikonin was 15 mg / kg / day, and the dosage of high-dose β,β-dimethacryloxyshikonin was 30 mg / kg / day. The control group received a 20% cyclodextrin solution. The administration was repeated for 8 weeks. Mice were weighed, and small and colonic tissues were dissected, their lengths measured, and the small and colonic tissues were embedded, sectioned, stained with hematoxylin and eosin (HE), and immunohistochemically stained.

[0074] like Figure 6As shown, β,β-dimethacryloxyshikonin can reverse the downregulation of NEDD4L protein levels and the upregulation of CK1ε and p27 expression induced by dovelixib. Flow cytometry data showed that β,β-dimethacryloxyshikonin can reverse dovelixib-induced G0 / G1 cell cycle arrest.

[0075] like Figure 7 As shown, HE staining of intestinal tissue revealed that the damage induced by administration of duvelixib to mice, including shortened and disordered villi in the small intestine, increased interglandular spacing, and thickened base of the colon, was restored to normal after combined administration of β,β-dimethylacryloylshikonin.

[0076] like Figure 8 As shown, immunohistochemical analysis of intestinal tissue sections revealed PCNA expression. The inhibition of intestinal epithelial cell proliferation induced by dovexib was significantly improved upon combined administration of β,β-dimethylacryloxyshikonin. Figure 8 A). Immunohistochemical staining of intestinal sections was performed to detect changes in CK1ε protein levels. Combined administration of β,β-dimethacryloylshikonin significantly reduced the increase in CK1ε protein levels induced by dovelixib. Figure 8 B).

[0077] like Figure 9 As shown, the combined use of β,β-dimethylacryloylshikonin had no significant effect on the body weight of mice.

[0078] Example 7

[0079] Human chronic lymphocytic B-cell leukemia cells MEC-1 were seeded at a density of 8000 cells / well in 96-well plates. After stabilization for 24 hours, the cells were treated with β,β-dimethylacryloxyshikonin (1, 2 μM) and dovelixibuvir (4 μM) for 48 hours. Then, 20 μL of CCK-8 reagent was added to each well, and the cells were incubated for 2-3 hours. The absorbance was measured at 450 nm, and the cell viability was calculated.

[0080] like Figure 10 As shown, the combined use of 1 μM β,β-dimethacryloylshikonin had no effect on the antitumor effect of duveliximab, while the combined use of 2 μM β,β-dimethacryloylshikonin enhanced the antitumor effect of duveliximab.

Claims

1. The use of NEDD4-like E3 ubiquitin ligase NEDD4L or NEDD4-like E3 ubiquitin ligase promoter in the preparation of a drug for treating intestinal toxicity caused by duveliximab, characterized in that, The intestinal toxicity caused by Duvelixib includes G0 / G1 phase arrest of small intestinal epithelial cells; the NEDD4-like E3 ubiquitin ligase promoter is β,β-dimethylacryloylshikonin or a pharmaceutically acceptable salt thereof.

2. The application as described in claim 1, characterized in that, The intestinal toxicity caused by Duvelixib also includes inhibition of intestinal epithelial cell proliferation, shortening and disordered arrangement of small intestinal villi, increased spacing between colonic glands, and thickening of the basal layer.

3. A combination antitumor drug composition, characterized in that, The formulation comprises a first formulation of doveliximab and a pharmaceutically acceptable carrier, and a second formulation of β,β-dimethylacryloylshikonin or a salt thereof and a pharmaceutically acceptable carrier.

4. The use of the pharmaceutical composition according to claim 3 in the preparation of a drug for treating lymphoma.

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