Application of verapamil and tetrandrine in prevention and treatment of P-glycoprotein mediated drug-resistant leukemia

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of verapamil and tetrandrine in prevention and treatment of P-glycoprotein mediated drug-resistant leukemia. In particular to application of verapamil and tetrandrine in preparation of a product for inhibiting the drug efflux function of P-glycoprotein and application of verapamil and tetrandrine in preparation of a product for resisting P-glycoprotein mediated tumor multidrug resistance. The invention relates to application of verapamil and tetrandrine in preparation of a product for preventing and treating P-glycoprotein mediated drug-resistant leukemia and application of verapamil and tetrandrine in preparation of a product for assisting daunorubicin in tumor treatment. In the present disclosure, the combination of verapamil and tetrandrine can enhance the inhibitory effect on the drug efflux function of P-glycoprotein. The combination of the verapamil and the tetrandrine can play a role in treating the P-glycoprotein mediated multidrug resistance diseases, and particularly, the combination of the verapamil and the tetrandrine overcomes the problem that side effects are easily caused when the tetrandrine is independently used.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the use of verapamil combined with tetrandrine in preventing and treating P-glycoprotein-mediated drug-resistant leukemia. Background Art

[0002] P-glycoprotein is a 170kD transmembrane protein encoded by the multidrug resistance gene 1 (MDR-1), also known as the ATP-binding cassette B1 gene (ABCB1). P-glycoprotein is widely expressed in different tissues such as the intestine, liver, kidney, placenta and blood-brain barrier. It is also present in B and T lymphocytes and bone marrow cells in peripheral blood. P-glycoprotein transports a variety of endogenous substrates to protect organs and tissues from toxic compounds. However, increased P-glycoprotein activity or elevated P-glycoprotein levels are associated with unsuccessful treatment of many diseases as well as disease exacerbation and adverse clinical outcomes because P-glycoprotein acts as a molecular "hydrophobic vacuum cleaner" to pull drug molecules out of the membrane and expel them from target cells, promoting multidrug resistance.

[0003] Verapamil is a phenylalkylamine ( Figure 1 A), originally developed as a use-dependent blocker of voltage-dependent L-type calcium channels. It can be used for angina pectoris, hypertension, and supraventricular tachycardia. In 1981, it was first reported that verapamil overcomes vincristine resistance in P388 leukemia by inhibiting P-glycoprotein-mediated efflux function. Due to its clinical availability, many trials have been initiated to study the clinical efficacy of verapamil as a chemosensitizer in different refractory cancers. However, verapamil has also been shown to have strong drug toxicity when used alone, limiting the application prospects of verapamil as a tumor treatment drug.

[0004] Tetrandrine is a bis-benzylisoquinoline alkaloid ( Figure 1 In China, it has been approved for the treatment of silicosis and rheumatoid arthritis patients. It is also registered as (NSC-77037) is an oral P-glycoprotein inhibitor in the U.S. It is currently being evaluated in combination with doxorubicin for the treatment of patients with metastatic, unresectable sarcoma who have progressed after doxorubicin (ClinicalTrials.gov Identifier: NCT03002805).

[0005] However, there is no relevant research on the effect of tetrandrine combined with verapamil on P-glycoprotein-mediated multidrug-resistant diseases, especially human multidrug-resistant leukemia. Summary of the invention

[0006] In view of the above problems, the present invention provides the use of verapamil combined with tetrandrine in preventing and treating P-glycoprotein-mediated drug-resistant leukemia, mainly to solve the problem that verapamil cannot be effectively used to treat P-glycoprotein-mediated multidrug-resistant diseases, especially human multidrug-resistant leukemia.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] Application of verapamil combined with tetrandrine in the preparation of products for inhibiting the drug efflux function of P-glycoprotein.

[0009] In specific applications, the application conditions can be selected as follows:

[0010] Among them, the drug efflux function of P-glycoprotein is the function of P-glycoprotein to expel anti-tumor drugs. The drug efflux function of P-glycoprotein will expel anti-tumor drugs and affect the effect of anti-tumor drugs at the tumor site. When the drug efflux function of P-glycoprotein is inhibited, anti-tumor drugs can play a better role at the tumor site, thereby improving the therapeutic effect on the tumor.

[0011] Among them, for the combination of verapamil and tetrandrine, generally speaking, the concentrations of the two are 0.5 μM verapamil and 0.3 μM tetrandrine. In addition, other ratios are not excluded.

[0012] Application of Verapamil combined with Tetrandrine in the preparation of a cell model that inhibits the drug efflux function of P-glycoprotein. Verapamil combined with Tetrandrine is mainly used to inhibit the drug efflux function of P-glycoprotein in a cell model.

[0013] The cell model is generally a MOLT-4 cell model. The drug efflux function of P-glycoprotein in the MOLT-4 cell model is controlled by a combination of verapamil and tetrandrine. By adjusting the drug efflux function of P-glycoprotein, model variables can be adjusted, for example, the effects of other anti-tumor drugs in the MOLT-4 cell model can be verified when the drug efflux function of P-glycoprotein changes.

[0014] Application of Verapamil combined with Tetrandrine in the preparation of products for resisting P-glycoprotein-mediated tumor multidrug resistance. The combination of Verapamil and Tetrandrine can treat tumor multidrug resistance mediated by P-glycoprotein and reduce tumor treatment resistance caused by P-glycoprotein.

[0015] Among them, the P-glycoprotein-mediated drug-resistant leukemia is at least human multidrug-resistant leukemia, and at least should be understood to mean any one of them or others.

[0016] Among them, most of the products against P-glycoprotein-mediated tumor multidrug resistance are products against human multidrug-resistant leukemia resistance, which are commonly used in the treatment of human multidrug-resistant leukemia and can have better effects, and they mainly play an anti-resistance role.

[0017] Application of Verapamil combined with Tetrandrine in the preparation of products for preventing and treating P-glycoprotein-mediated drug-resistant leukemia. Here, the combination of Verapamil and Tetrandrine is mainly used to treat P-glycoprotein-mediated drug-resistant leukemia, and directly treats P-glycoprotein-mediated drug-resistant leukemia on the basis of reducing drug resistance, such as treating multidrug-resistant leukemia in humans.

[0018] The application of verapamil combined with tetrandrine in the preparation of products for assisting daunorubicin in treating tumors. The combination of verapamil and tetrandrine can improve the therapeutic effect of daunorubicin on tumors and enhance the toxicity of daunorubicin to tumor cells.

[0019] Wherein, the tumor is a drug-resistant leukemia mediated by P-glycoprotein, and in particular can be a human multidrug-resistant leukemia.

[0020] Application of tetrandrine in the preparation of a product that reduces the cytotoxicity of a tumor therapeutic drug containing verapamil. In this application, tetrandrine mainly plays a role in reducing the cytotoxicity of verapamil, so that verapamil can effectively treat tumors while reducing side effects. Verapamil is generally used as a chemotherapy sensitizer in tumor treatment.

[0021] In the present disclosure, the combination of verapamil and tetrandrine can enhance the inhibitory effect on the drug efflux function of P-glycoprotein. The combination of verapamil and tetrandrine can play a therapeutic role in multidrug resistance diseases mediated by P-glycoprotein, and the effect is significantly improved compared with the use of any component alone, especially the combination of verapamil and tetrandrine overcomes the problem that tetrandrine alone is prone to side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Middle: Chemical structures of verapamil (A) and tetrandrine (B).

[0023] Figure 2 Middle: Effects of verapamil and tetrandrine on P-glycoprotein function. Verapamil (A) or tetrandrine (B) alone can induce the accumulation of Rh123 in MOLT-4 / DNR cells in a dose-dependent manner; (C) Representative results of Rh123 fluorescence intensity after intervention with verapamil (blue line) and tetrandrine (red line).

[0024] Figure 3Middle: Effect of Verapamil combined with Tetrandrine on P-glycoprotein function. Effect of 0.3 μM Tetrandrine on Rh123 accumulation in MOLT-4 / DNR cells in the presence or absence of 5 μM Verapamil, with typical fluorescence intensity shown in (A) and Rh123 accumulation (%) shown in (B). The effect of the combination was better than that of either alone.

[0025] Figure 4 Middle: Effects of verapamil and tetrandrine on the cytotoxicity of daunorubicin to MOLT-4 / DNR cells. MOLT-4 / DNR cells (5×10 5 Cells / mL) were seeded into 96-cell plates, and cell viability was determined by WST-8 assay 48 h after drug intervention; data are expressed as mean ± SEM and statistically analyzed by Bonferroni's multiple comparisons (n ​​= 4); ***P < 0.001 compared with the vehicle group, #P < 0.05 compared with 0.5 μM verapamil.

[0026] Figure 5 Middle: Computer simulation of the binding of verapamil and tetrandrine to humanized P-glycoprotein (PDB ID: 6QEX). (A) Rainbow cartoon view of humanized P-glycoprotein 6QEX; (B) 3D view of the binding of verapamil and tetrandrine to humanized P-glycoprotein (6QEX), with verapamil and tetrandrine shown in red and black, respectively; (C) 2D view of the interaction between verapamil and P-glycoprotein groups. (D) 2D view of the interaction between tetrandrine and P-glycoprotein groups; (E) Venn diagram of the interaction groups between verapamil and tetrandrine and P-glycoprotein.

[0027] Figure 6 Middle: Computer simulation of the binding of verapamil and tetrandrine to mouse P-glycoprotein (PDB ID: 5KPI). (A) Rainbow cartoon view of mouse P-glycoprotein 5KPI; (B) 3D view of the binding of verapamil and tetrandrine to mouse P-glycoprotein (5KPI), with verapamil and tetrandrine shown in red and black, respectively; (C) 2D view of the interaction between verapamil and P-glycoprotein groups; (D) 2D view of the interaction between tetrandrine and P-glycoprotein groups; (E) Venn diagram of the interaction groups between verapamil and tetrandrine and P-glycoprotein. DETAILED DESCRIPTION

[0028] The following is a further analysis based on specific research projects.

[0029] 1. Materials and Methods

[0030] 1.1. Materials

[0031] RPMI 1640 and FBS were purchased from Gibco BRL (Grand Island, NY, USA). Tetrandrine, verapamil and daunorubicin (DNR) were purchased from Sigma Aldrich (St. Louis. Mo., USA). The drugs were dissolved in ethanol (Wako Pure Chemical Industries, Ltd., Japan) at a concentration of 15 mM, filtered through a 0.2 μm membrane filter (Advantec Co., Japan), and then stored at 4 ° C until use. Working concentrations were prepared by dilution with ethanol. Rhodamine 123 (Rh 123) was purchased from Sigma Aldrich (St. Louis. Mo., USA). WST-8 kit was purchased from Dojindo Molecular Technologies, Inc., Japan. All other reagents were of the highest quality available from commercial suppliers.

[0032] Cell culture

[0033] MOLT-4 / DNR cells were derived from the MOLT-4 cell line by exposing parental cells to increasing concentrations of DNR for more than 3 months. MOLT-4 / DNR cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100,000 IU / L penicillin and 100 mg / L streptomycin at 37°C and 5% CO2.

[0034] 1.3. P-glycoprotein function evaluation

[0035] Cellular P-glycoprotein function was determined by Rh 123 assay. 5×10 5 The cells were co-cultured with 2 μM Rh 123 dye at 37°C and 5% CO2 for 1 hour with or without drug addition. After PBS was cleared, FACSCanto TM Ⅱ Measure the fluorescence intensity of intracellular Rh 123.

[0036] Cytotoxicity assay

[0037] MOLT-4 / DNR cells (1.5×10 5 Cells / mL) were inoculated into 96-cell plates. Drugs were then added to the corresponding wells. Ethanol was added to the control wells. After incubation for 48 hours, 10 μL of WST-8 solution was added to each well and then incubated for another 3 hours at 37°C. Optical density values ​​were measured at 450nm absorbance (reference 650nm). Relative cell viability was expressed as the ratio of the absorbance of each treatment group to the absorbance of the untreated control group.

[0038] 1.5. Molecular docking simulation

[0039] Molecular docking was performed using PyRx-Python Prescription 0.8 and AutoDockVina software. 2D and 3D visualization of ligand-receptor interactions was performed using Discovery Studio 2016 and UCSF ChimeraX software. Software parameters were set to default values.

[0040] Statistical analysis

[0041] Statistical analysis of the data was performed using GraphPad PRISM 5.0 (GraphPad Software Inc., San Diego, CA). Two-sided p values ​​< 0.05 were considered significant.

[0042] 2. Results

[0043] This study explored the effects of tetrandrine and verapamil on P-glycoprotein function, and used tetrandrine and verapamil in vitro to act on human multidrug-resistant leukemia MOLT-4 / DNR cells with a large amount of P-glycoprotein. The binding sites of tetrandrine and verapamil in the human P-glycoprotein pocket were also analyzed by computer graphics.

[0044] 2.1. Verapamil and tetrandrine inhibit P-glycoprotein function alone

[0045] like Figure 2 As shown in A and B, both verapamil and tetrandrine induced intracellular Rh 123 accumulation with good dose correlation, and nM concentration also had a significant inhibitory effect (p<0.001). Compared with verapamil, P-glycoprotein had a more obvious effect on tetrandrine, because 1μM tetrandrine increased the fluorescence intensity of Rh 123 more significantly than 5μM verapamil ( Figure 2 Middle C).

[0046] 2.2. The combination of verapamil and tetrandrine exhibits a stronger ability to inhibit P-glycoprotein function

[0047] like Figure 3 As shown in Figure A, the fluorescence intensity of Rh 123 after combined intervention (green line) increased more significantly than that after intervention with 5 μM verapamil (blue line) or 0.3 μM tetrandrine (red line). Figure 3 As shown in B, the combination showed a significantly enhanced ability to inhibit the efflux function of P-glycoprotein than either tetrandrine or verapamil alone (p<0.001).

[0048] 2.3. The combination of verapamil and tetrandrine significantly enhances the cytotoxicity of daunorubicin

[0049] When MOLT-4 / DNR cells were treated with DNR alone, DNR IC 50 When DNR was combined with 0.5μM verapamil or 0.3μM tetrandrine to treat MOLT-4 / DNR cells, the DNR IC 50 The values ​​were 22.66±11.59 and 10.65±3.03 nM, respectively. The combination of DNR with 0.5 μM verapamil and 0.3 μM tetrandrine, DNR IC 50 The value was significantly reduced to 2.44±0.98nM (p<0.001). This experimental result is consistent with the experimental results of P-glycoprotein function determination in 2.2.

[0050] 2.4. Docking model Effect of verapamil powder and tetrandrine on P-glycoprotein

[0051] We used computer-aided drug design software to simulate their binding sites on P-glycoprotein ( Figure 5 and Figure 6 The protein crystal structure of human P-glycoprotein was obtained from the Protein Data Bank ( Figure 5 A in Figure 1, PDB ID: 6QEX). The best binding affinities for verapamil and tetrandrine provided by AutodockVina were -5.4 and -7.4 kcal / mol, respectively. When interacting with human P-glycoprotein, tetrandrine shares most of the same residues with verapamil ( Figure 5 E). However, the same residues interact with verapamil or tetrandrine in slightly different ways. For example, Glu243 and 255 interact with verapamil through the π-anion system, while interacting with tetrandrine through carbon-hydrogen bonds. ALA819 interacts with tetrandrine through the amide-π system, while interacting with verapamil through the π-alkyl system ( Figure 5 C and D). Verapamil specifically interacts with ALA823, GLY251, and THR240. However, tetrandrine specifically interacts with GLY812 ( Figure 5 Middle E).

[0052] 5KPI mouse P-glycoprotein is a 1276 residue polypeptide with 87% sequence identity to human P-glycoprotein. It consists of two homologous halves connected by a flexible linker of approximately 75 residues ( Figure 6 A). The spacing of the nucleotide binding domains is 45.50 to 59.64. The best binding affinities of verapamil and tetrandrine provided by Autodock Vina are -4.5 and -7.3 kcal / mol, respectively. Figure 6As shown in Figures C and D, the binding of verapamil or tetrandrine to P-glycoprotein occurs mainly through specific interactions such as hydrogen bonding and nonspecific interactions such as hydrophobic bonding at the drug binding site of P-glycoprotein. However, the conventional binding sites of tetrandrine and verapamil appear to be completely different. The oxygen atoms at the 3,4-dimethoxy residue of verapamil interact with the ARG925 and GLA921 residues in the 5KPI chain through hydrogen bonds. The oxygen atoms at the 12-methoxy and 11,12'-oxygen bridges of tetrandrine interact with the LYS891 residue on the 5kp B chain through hydrogen bonds. The interaction between verapamil and the 5KPI drug binding site is stabilized by van der Waals forces or hydrophobicity, mainly through residues in the B chain such as MET915, ILE892, LYS891, THR478, THR479, GLU482, ALA477, ARG523, and LEU519, including two residues in one chain, GLN918 and ILE922 ( Figure 6 (C). The van der Waals or hydrophobic interactions between tetrandrine and 5KPI are through residues in chains A and B (A: SER919, GLN918, GLN921, ILE922, and ARG925; B: SER919, ILE892, GLY888, ALA477, LEU519, and Arg523; Figure 6 Therefore, tetrandrine and verapamil can enter P-glycoprotein through different residues in the large drug binding pocket of 5KPI ( Figure 6 (B).

[0053] Discussion

[0054] Overexpression of P-glycoprotein is considered to be the main cause of chemotherapy failure in different types of cancer. Meanwhile, increased P-glycoprotein activity on normal immune cells has been reported to be associated with unsuccessful treatment of autoimmune diseases as well as disease exacerbation and poor clinical outcomes. Therefore, P-glycoprotein is an attractive target for the treatment of some refractory diseases. To our knowledge, the combination of verapamil and tetrandrine showed a stronger ability to inhibit P-glycoprotein function through different binding sites, which is a new finding.

[0055] Verapamil is one of the recognized first-generation P-glycoprotein inhibitors. Although verapamil has been observed to increase the survival of patients with anthracycline-resistant metastatic breast cancer, it has significant dose-limiting toxicities such as hypotension, heart block, and fluid retention. The development of fourth-generation P-glycoprotein inhibitors from natural products has attracted considerable attention from scholars, including Figure 2 As shown in C, 1 μM tetrandrine has a stronger inhibitory effect on P-glycoprotein function than 5 μM verapamil. Tetrandrine seems to be a suitable candidate drug for P-glycoprotein inhibitors.

[0056] Interestingly, the combination of 0.3 μM tetrandrine and 5 μM verapamil showed a stronger inhibitory effect on P-glycoprotein efflux function than either tetrandrine or verapamil alone, and the difference was significant (p<0.001, Figure 3 In addition, the combination of 0.3 μM tetrandrine and 0.5 μM verapamil reduced the DNR IC 50 The value is greater than that of tetrandrine or verapamil alone ( Figure 4 ). The above information suggests that the molecular mechanisms of tetrandrine and verapamil may be different. Generally, P-glycoprotein inhibitors block the efflux function by binding in a pocket of the protein, which leads to the obstruction of the conformational change between the inward and outward directions. Therefore, we attempted to analyze the drug binding sites of verapamil and tetrandrine in the P-glycoprotein pocket. Computer graphics P-glycoprotein consists of two homologous halves connected by a peptide fragment of approximately 75 amino acids in length. Verapamil and tetrandrine may enter the inner cavity of one homologous half ( Figure 5 A, PDB ID: 6QEX). Figure 5 As shown in Figure 2B, verapamil and tetrandrine share many of the same residues in different interaction modes ( Figure 5 C and D). Meanwhile, verapamil interacted specifically with the residues of ALA823, GLY251, and THR240. However, tetrandrine interacted specifically with the residue of GLY812 ( Figure 5 On the other hand, verapamil and tetrandrine may enter the cavity between the two homologous halves ( Figure 6 A and B in the figure, PDB ID: 5KPI). Figure 6 As shown in Figures C to E, although verapamil and tetrandrine have many identical residues, they also interact with some residues separately. For example, verapamil specifically binds to GLU482, MET915, THR478, and Thr479 of the B chain of P-glycoprotein through van der Waals forces ( Figure 6 (C). Tetrandrine specifically binds to GLY888 and SER919 of the B chain, and SER919 binds to the B chain through van der Waals forces ( Figure 6 In short, verapamil and tetrandrine can enter P-glycoprotein through different residues in the large drug binding pocket, which may help the combination of verapamil and tetrandrine to exert a stronger inhibitory effect on P-glycoprotein function ( Figure 3 and Figure 4 ).

[0057] IV. Conclusion

[0058] The combination of verapamil and tetrandrine showed a stronger potency than either tetrandrine or verapamil in inhibiting P-glycoprotein function. Tetrandrine significantly enhanced the inhibitory effect of verapamil on P-glycoprotein in MOLT-4 / DNR cells (p<0.001). Therefore, the combination of verapamil and tetrandrine significantly reduced the IC 50 The results showed that the combination of verapamil and tetrandrine has a stronger inhibitory effect on P-glycoprotein function than verapamil alone. The combination of verapamil and tetrandrine has a stronger inhibitory effect on P-glycoprotein function than verapamil alone. The combination of verapamil and tetrandrine may benefit patients with P-glycoprotein-mediated multidrug resistance diseases, although the dose-limiting toxicity of verapamil excludes its use as an effective drug for inhibiting P-glycoprotein function in vivo, especially in clinical practice.

[0059] It is clear to those skilled in the art that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention, and reference can be made to the prior art for any details not detailed herein. All of them fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.

Claims

1. Application of verapamil combined with tetrandrine in the preparation of products for inhibiting the drug efflux function of P-glycoprotein.

2. The use according to claim 1; wherein The drug efflux function of the P-glycoprotein is the function of the P-glycoprotein to efflux tumor drugs; verapamil 0.5 μM, tetrandrine 0.3 μM.

3. Application of verapamil combined with tetrandrine in preparing a cell model for inhibiting the drug efflux function of P-glycoprotein.

4. The use according to claim 3; wherein: The cell model is a MOLT-4 cell model.

5. Application of verapamil combined with tetrandrine in the preparation of products for combating P-glycoprotein-mediated tumor multidrug resistance.

6. The use according to claim 5; wherein: The product against P-glycoprotein-mediated tumor multidrug resistance is a product against human multidrug-resistant leukemia resistance.

7. Use of verapamil combined with tetrandrine in the preparation of products for preventing and treating P-glycoprotein-mediated drug-resistant leukemia; wherein: P-glycoprotein-mediated drug-resistant leukemia is at least multidrug-resistant leukemia in humans.

8. Application of Verapamil combined with Tetrandrine in the preparation of products for assisting daunorubicin in treating tumors.

9. The use according to claim 8; wherein: The tumor is a drug-resistant leukemia mediated by P-glycoprotein; the drug-resistant leukemia mediated by P-glycoprotein is at least human multidrug-resistant leukemia.

10. Use of tetrandrine in the preparation of products for reducing the cytotoxicity of tumor therapeutic drugs containing verapamil.

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