CN116942687A - Pharmaceutical composition for resisting chronic myelogenous leukemia drug resistance and application thereof - Google Patents

Abstract

The invention provides a medicine composition for resisting chronic myelogenous leukemia drug resistance and application thereof. The pharmaceutical composition comprises ouabain and imatinib or pharmaceutically acceptable salts thereof. Ouabain has proliferation and apoptosis inhibiting effects on imatinib-resistant CML cell line K562/G01 cells, and ouabain is more remarkable in combination with IM than in combination with IM on K562/G01 cells, which indicates that ouabain is a potential drug for increasing sensitivity of CML-resistant cells to IM. Moreover, the ouabain has no obvious toxicity to normal mice after long-term application.

Description

Pharmaceutical composition for resisting chronic myelogenous leukemia drug resistance and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical compositions, in particular to a pharmaceutical composition for resisting chronic myelogenous leukemia drug resistance and application thereof.

Background

Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell malignancy characterized by philadelphia chromosome, and the reported cases in 2017 (Lin et al 2020) estimated 34179 and 24054 deaths. CML is often accompanied by fusion of the ABL1 gene on chromosome 9 and the BCR gene on chromosome 22, thereby producing a BCR-ABL1 fusion protein, which has high tyrosine kinase activity and is a key driver of CML. Although tyrosine kinase inhibitors (tyrosine kinase inhibitors, TKIs) such as Imatinib Mesylate (IM) can significantly increase survival in early patients, about 40% of patients in chronic phase have to discard IM due to failure and/or intolerance, and IM-related resistance cannot prevent recurrence and progression of the disease, and has become one of the most important causes of death in CML patients. Therefore, there is an urgent need for a new drug that can reverse IM resistance.

Ouabain (G-ouabain) is a quick-acting Cardiac Glycoside (CG) isolated from seeds of strongylon spica. The debate about CGs safety has long been due mainly to its narrow therapeutic window and cardiotoxicity. Ouabain is prepared by inhibiting Na ⁺ /K ⁺ ATPase increases intracellular Ca ²⁺ Concentration, thereby improving myocardial contractility. In addition to the traditional effects, recent researches have proved that ouabain has anti-inflammatory, anti-coronavirus, anticancer, and drug resistance reversing effects. Ouabain has been reported to target human leukemia cells, including interfering with intracellular redox homeostasis of lymphocytic leukemia cells and modulating immune cell activity in vivo. Nevertheless, the therapeutic effect of ouabain on IM-resistant CML has not been reported.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a medicine composition for resisting chronic myelogenous leukemia drug resistance and application thereof, and solves the problem of IM drug resistance existing in the prior art.

In one aspect of the invention, a pharmaceutical composition for resisting chronic myelogenous leukemia drug resistance is provided, which comprises the following components: ouabain, imatinib, or a pharmaceutically acceptable salt thereof.

Further, the molar ratio of ouabain to imatinib or a pharmaceutically acceptable salt thereof is from 0.01 to 1:10, preferably from 0.05 to 1:10, more preferably 1:10.

Further, the pharmaceutical composition is in the form of a formulation.

Further, the ouabain, imatinib, or pharmaceutically acceptable salts thereof are each dissolved in a solvent comprising DMSO, water to form a formulation.

Further, in the preparation, the concentration of ouabain is 0.01-1. Mu.M, preferably, the concentration of ouabain is 0.05-1. Mu.M, and more preferably, the concentration of ouabain is 1. Mu.M; the concentration of imatinib or a pharmaceutically acceptable salt thereof is 10 μm.

Further, the pharmaceutical composition further comprises an adjuvant.

In another aspect of the present invention, a method for preparing a pharmaceutical composition is provided, comprising mixing ouabain, imatinib, or pharmaceutically acceptable salts thereof.

Further, the method comprises the following steps:

s1: respectively dissolving ouabain, imatinib or pharmaceutically acceptable salts thereof in a solvent to obtain ouabain solution and imatinib solution;

s2: mixing ouabain solution and imatinib solution;

preferably, the solvent is DMSO, water.

In a further aspect of the invention, there is provided the use of said pharmaceutical composition or ouabain in the manufacture of a medicament having the following effect:

(1) Drug resistance for treating chronic myelogenous leukemia;

(2) Promoting apoptosis of imatinib-resistant leukemia cells;

(3) Inhibiting proliferation of imatinib-resistant leukemia cells;

preferably, the imatinib-resistant leukemia cells are K562/G01 cells.

Further, the molar ratio of ouabain to imatinib or a pharmaceutically acceptable salt thereof is 0.01-1:10, preferably 0.05-1:10;

the pharmaceutical composition is in the form of a formulation;

preferably, the ouabain, imatinib, or pharmaceutically acceptable salts thereof are each dissolved in a solvent to form a formulation, the solvent comprising DMSO, water;

more preferably, the concentration of ouabain in the formulation is from 0.01 to 1. Mu.M, preferably the concentration of ouabain is from 0.05 to 1. Mu.M, more preferably the concentration of ouabain is 1. Mu.M; the concentration of imatinib or a pharmaceutically acceptable salt thereof is 10 μm.

The technical principle of the invention is as follows: in the prior art, ouabain can play an anti-leukemia role by inducing apoptosis of leukemia cells, affecting redox homeostasis and targeting related signaling pathways, but the role of ouabain in imatinib-resistant CML is not clear. Through repeated and extensive experimental study, the inventor finds that ouabain can inhibit the cell viability in a time and dose dependent manner, and even low-concentration ouabain (0.01 mu M and 0.1 mu M) can generate cytotoxicity on IM-resistant CML cell strain K562/G01 cells, which indicates that the ouabain has obvious capability of inhibiting the proliferation of K562/G01 cells. Further, the inventors compared the effect of single and combined administration on K562/G01 cell proliferation and apoptosis, and found that high concentration (1. Mu.M) of ouabain combined IM inhibited cell proliferation significantly more than IM and ouabain alone, and induced apoptosis. This suggests that ouabain and IM have a synergistic effect in promoting apoptosis. Furthermore, the resistance reversal factor RF increased with increasing concentration, indicating that ouabain can inhibit IM resistance of K562/G01 cells in a concentration-dependent manner.

Compared with the prior art, the invention has the following beneficial effects: in the invention, ouabain has antiproliferative and pro-apoptotic effects on imatinib-resistant CML cell lines K562/G01 cells, and the combined use of ouabain and IM has more remarkable growth inhibition and pro-apoptotic effects on K562/G01 cells than the combined use of IM alone, which shows that ouabain is a potential drug for increasing the sensitivity of drug-resistant cells to IM, and the combined use of ouabain and IM has a synergistic effect. Moreover, the ouabain has no obvious toxicity to normal mice after long-term application.

Drawings

FIG. 1 shows the effect of ouabain and its combined IM on K562/G01 cell proliferation, wherein a is the chemical structure of ouabain, b is the concentration-effect curve of the CCK-8 assay after 24h and 48h of ouabain action on K562/G01 cells, c is the effect of CCK-8 cytotoxicity assay after 24h on K562/G01 cell proliferation of ouabain combined IM, data expressed as mean+ -SD (n=3)..p <0.001 compared to control or 10. Mu.M IM group.

FIG. 2 shows the effect of ouabain in combination with IM on apoptosis and cell cycle of K562/G01 cells, wherein a is the effect of flow cytometry on apoptosis of K562/G01 cells after 24h of ouabain combination with IM, and b is the effect of ouabain alone or in combination with IM on apoptosis of K562/G01 cells after 24h, p <0.05.

FIG. 3 shows the effect of ouabain on normal mouse body weight, where a is the male KM mouse body weight ratio and b is the female KM mouse body weight ratio.

FIG. 4 shows the effect of ouabain on normal mouse blood convention, where a is white blood cell count (WBC), b is red blood cell count (RBC), c is hemoglobin (Hb), d is mean red blood cell volume (MCV), e is mean red blood cell hemoglobin (MCH), f is mean red blood cell hemoglobin concentration (MCHC), and g is platelet count (PLT). Values are expressed as mean±sd (n=6), p >0.05, compared to the control group.

Fig. 5 shows the effect of ouabain on glutamic pyruvic transaminase (ALT) and Creatinine (CREA) in normal mice, wherein a is glutamic pyruvic transaminase (ALT) and b is Creatinine (CREA), and the values are mean±sd (n=6), p >0.05, compared with control group.

Fig. 6 shows the effect of ouabain on organ index in normal mice, wherein a is heart organ index, b is liver organ index, c is kidney organ index, d is lung organ index, e is spleen organ index, and the values are mean±sd (n=6), <0.05, <0.01, compared to control group.

FIG. 7 shows the effect of ouabain on normal mouse organs, wherein a is cardiac morphology change; b is a kidney morphology change; c is a morphological change of the liver; d is the morphological change of the lung; e is spleen morphology change.

FIG. 8 is a photograph showing the effect of ouabain on histological examination of normal mouse organs, wherein a is a heart HE staining micrograph; b is a liver HE staining micrograph; c is a kidney HE staining micrograph; d is a lung HE staining micrograph; e is a photomicrograph of spleen HE staining.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. All data of the present invention were tested using IBM SPSS 26.0 software and expressed as mean ± Standard Deviation (SD). The comparison between the two groups adopts t distribution test, the comparison between the multiple groups adopts single factor analysis of variance, and p <0.05 is taken as the difference to have statistical significance.

EXAMPLE 1 preparation of pharmaceutical compositions

10mg of IM was dissolved in 1.6958mL of dimethyl sulfoxide (DMSO) (Solarbio Corp., china) and 15.2622mL of triple distilled water to prepare a 1mM stock solution of IM. Ouabain was dissolved in DMSO to make up a stock solution at a concentration of 10 mM. After filtration and sterilization, the cells were stored at-20 ℃. The chemical structure of ouabain is shown in FIG. 1 a.

Example 2 in vitro action of pharmaceutical compositions

1. Cell culture

K562/G01 cells were cultured in RPMI-1640 medium (Gibco Co., USA) containing 10% fetal bovine serum (Gibco Co., USA), 100. Mu.g/mL streptomycin (Hyclone Co., USA), 100U/mL penicillin (Hyclone Co., USA), and placed at 37℃in 5% CO ₂ In a saturated humidity incubator. Selecting growthCells in good and logarithmic growth phase were tested.

2. Cytotoxicity test

(1) About 5X 10 per well ³ The individual cells were seeded in 96-well plates and treated with ouabain at concentrations of 0.01, 0.1, 1, 10 μm for 24 and 48h, respectively. 10 mu L Cell Counting Kit-8 (CCK-8) solution was added to each well and incubated at 37℃for 3h. Absorbance was measured at a wavelength of 450 nm. Cell viability (%) = [ (OD) _{Sample of} -OD _{Blank space} )/(OD _Control -OD _{Sample of} )]X 100%. The results are shown in FIG. 1 b.

From the results, ouabain significantly reduced proliferation of K562/G01 cells in a dose-and time-dependent manner.

(2) To assess the effect of ouabain on the IM sensitivity of K562/G01 cells, cells were treated with IM alone or in combination with ouabain for 24h and then examined for cell proliferation with CCK-8.

About 5X 10 per well ³ The individual cells were seeded in 96-well plates and treated with ouabain at a concentration of 0.5. Mu.M, ouabain at 1. Mu.M, ouabain at 10. Mu.M, ouabain at 0.5. Mu.M+10. Mu.M, ouabain at 1. Mu.M+10. Mu.M for 24h, respectively, and the control group was treated with the same amount of medium. 10 mu L Cell Counting Kit-8 (CCK-8) solution was added to each well and incubated at 37℃for 3h. Absorbance was measured at a wavelength of 450 nm. Cell viability was calculated according to the method of step (1). The results are shown in FIG. 1 c.

From the results, it was found that the cell viability was significantly different from that of the IM-treated group alone and that of the ouabain-treated group alone at 1. Mu.M after the combined action of ouabain and IM, and the cell viability was significantly reduced. It was shown that high concentrations of ouabain can promote sensitivity of IM to K562/G01 cells, whereas 0.5 μm ouabain and IM had no statistical significance for cell growth inhibition (fig. 1 c).

(3) Ouabain concentration changes affect the effect of IM on K562/G01 cells

About 5X 10 per well ³ The cells were inoculated in 96-well plates, ouabain at 0.01, 0.1, 1, 10. Mu.M and IM at different concentrations were added, and the cells were treated for 24h, respectively, with the untreated ouabain as control. mu.L of Cell counter was added per wellng Kit-8 (CCK-8) solution, incubated at 37℃for 3h. Absorbance was measured at a wavelength of 450 nm. Cell viability (%) = [ (OD) _{Sample of} -OD _{Blank space} )/(OD _Control -OD _{Sample of} )]X 100%. IC for estimating IM using Prism 5 (GraphPad Software, USA) software ₅₀ . The results are shown in Table 1.

TABLE 1 IC50 of ouabain on K562/G01 cells

As can be seen from Table 1, as the concentration of ouabain increased, the IC50 value of IM for K562/G01 cells decreased, and at the lowest concentration (0.01. Mu.M), ouabain decreased the IC50 of IM in K562/G01 cells from 94.92 to 69.56. Mu.M, with an RF value of 1.36. Furthermore, when cells were treated with 10 μm ouabain, the RF value reached 131.83. These results indicate that ouabain has the potential to reverse the resistance of K562/G01 cells to IM.

3. Flow cytometry to detect apoptosis

About 5X 10 per well ⁵ The cells were inoculated into 6-well plates and cultured in combination with IM (10. Mu.M), ouabain (0.5. Mu.M, 1. Mu.M), and the control group was treated with the same amount of medium. After 24h, the cells were washed 2 times with Phosphate Buffered Saline (PBS), and incubated with 5. Mu.L of Annexin-V FITC labeling solution and Propidium Iodide (PI) for 15min. Apoptosis was analyzed using a flow cytometer. The results are shown in FIG. 2 a.

As a result, it was found that 0.5. Mu.M or 1. Mu.M ouabain or 10. Mu.M IM slightly promoted apoptosis of K562/G01 cells, and the apoptosis rates were 9.94.+ -. 4.18%, 17.97.+ -. 2.2% and 8.64.+ -. 2.59%, respectively. In contrast, when 10. Mu.M IM was used in combination with 0.5. Mu.M or 1. Mu.M ouabain, the apoptosis rates were 20.11.+ -. 0.16 and 25.81.+ -. 1.4%, respectively. Thus, ouabain enhances IM-induced apoptosis.

4. Flow cytometry to detect cell cycle

To explore the mechanism of ouabain to inhibit K562/G01 cell viability, cell cycle was examined by flow cytometry.

About 5X 10 per well ⁵ Individual cells were seeded in 6-well platesThe cells were cultured with IM (10. Mu.M), ouabain (0.5. Mu.M, 1. Mu.M), and the two drugs, respectively. After 24h, cells were washed 2 times with PBS, and then the cell suspension was added to 75% cold ethanol and stored overnight at 4 ℃. After washing the cells with PBS, RNase and PI dye solution were added. The sample was examined using a flow cytometer. The results are shown in FIG. 2 b.

As can be seen from the results, there was no statistical difference in the ratio of cells in the G0/G1, S and G2/M phases of the experimental group, although the ratio of cells in the G2/M phase was increased, after 24 hours of either ouabain single drug treatment or combined IM action on K562/G01 cells, as compared with the 10. Mu.M IM group.

EXAMPLE 3 toxicity test of pharmaceutical compositions

1. Experimental animals: healthy Kunming (KM) mice (n=24), 6 weeks old, weighing 18-22 g, SPF grade. Mice were housed in separate cages (3 mice/cage) for 1w. The room temperature and humidity are strictly controlled, and the food and water can be eaten and drunk freely.

Grouping and treating animals:

mice (n=12 females; 12 males) were randomly divided into 4 groups (n=3/group): high dose group (1.0 mg/kg ouabain), medium dose group (0.5 mg/kg ouabain), low dose group (0.1 mg/kg ouabain) and control group (equal amount of physiological saline). Mice were intraperitoneally injected every other day for 30 days. After administration, mice were anesthetized, and hematological, biochemical, and histological examinations were taken to detect the toxic effects of ouabain on normal mice.

2. General sign observation of mice:

daily activities, mental states, feeding and defecation of the mice were observed and recorded daily, and the mice were weighed every 2d to represent weight changes in weight ratio (weight ratio=weight/initial weight). The results are shown in FIG. 3 and Table 2.

TABLE 2 Effect of ouabain on mouse body weight (g)

From the detection results, the daily activities, the mental states, the eating and defecation of the mice in each group are normal during the administration period. At a concentration of 0.5mg/kg, the body weight gain was maximized in both female and male mice, whereas the body weight gain was slowest in the 0.1mg/kg ouabain-treated mice (FIG. 3). However, there was no statistical difference (p > 0.05) between each treatment group compared to the control group (table 2), and the growth trend was substantially consistent for each group (fig. 3).

3. Blood routine detection:

after administration, the tail was cut, blood was taken out, and the blood was mixed with a diluent, and red blood cells, mean red blood cell hemoglobin (MCH), white blood cells, mean red blood cell volume (MCV), mean red blood cell hemoglobin concentration (MCHC), hemoglobin, and Platelets (PLT) were measured by a full-automatic blood analyzer. The results are shown in FIG. 4.

As shown by the results, the conventional indexes of the blood of the mice in the groups with low ouabain (0.1 mg/kg), medium (0.5 mg/kg) and high dose (1.0 mg/kg) have no significant difference (p > 0.05) compared with the control group.

4. Glutamic pyruvic transaminase and creatinine detection:

after the end of the administration, serum from mice was collected by centrifugation at 3000r/min for 20min and levels of glutamic pyruvic transaminase (ALT) and Creatinine (CREA) were detected using the kit. The results are shown in FIG. 5.

Glutamic pyruvic transaminase (ALT) and Creatinine (CREA) were used as sensitive liver and kidney injury markers, respectively, to evaluate the effect of ouabain on liver and kidney function in KM mice. From the results, no significant difference (p > 0.05) was seen for each treatment group compared to the control group, whether ALT or CREA.

5. Organ index and histological examination:

mice were sacrificed, and heart, liver, kidney, lung, spleen were collected, and related measurements were performed and expressed as organ indexes. The organs were dehydrated, paraffin embedded and sectioned after fixation in 4% paraformaldehyde for 24 h. Finally, paraffin-embedded sections were stained with hematoxylin and eosin for further histological examination. The results are shown in FIGS. 6-8.

Organ index is a common index for evaluating organ effects in toxicology studies. From the results, the heart organ index increased with increasing dose, and both the medium dose group (p < 0.05) and the high dose group (p < 0.01) were significantly higher than the control group (fig. 6 a), suggesting that the heart of the mice may become engorged or hypertrophic. Since the difference between the kidney organ index (p < 0.01) and the lung organ index (p < 0.05) is only shown in the low dose group (fig. 6c, 6 d), experimental errors may be caused. No significant abnormalities were seen in the treated organs, both macroscopic and microscopic (fig. 7, 8), compared to the control. The KM mice showed no pathological reactions such as edema, inflammatory infiltration, cell degeneration, necrosis, etc. in heart, liver, spleen, kidney, lung tissues (FIG. 8).

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims (10)

1. A pharmaceutical composition for resisting chronic myelogenous leukemia drug resistance, which is characterized in that: comprises the following components: ouabain, imatinib, or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for resistance to chronic myelogenous leukemia according to claim 1, wherein: the molar ratio of ouabain to imatinib or a pharmaceutically acceptable salt thereof is 0.01-1:10, preferably 0.05-1:10, more preferably 1:10.

3. A pharmaceutical composition for resistance to chronic myelogenous leukemia according to claim 1, wherein: the pharmaceutical composition is in the form of a formulation.

4. A pharmaceutical composition according to claim 3 for the treatment of chronic myelogenous leukemia, which comprises: the ouabain, imatinib or pharmaceutically acceptable salts thereof are dissolved in solvents to form a preparation, wherein the solvents comprise DMSO and water.

5. A pharmaceutical composition according to claim 4 for resistance to chronic myelogenous leukemia, wherein: in the preparation, the concentration of ouabain is 0.01-1 mu M, preferably, the concentration of ouabain is 0.05-1 mu M, and more preferably, the concentration of ouabain is 1 mu M; the concentration of imatinib or a pharmaceutically acceptable salt thereof is 10 μm.

6. The pharmaceutical composition of claim 1, wherein: the pharmaceutical composition further comprises an adjuvant.

7. A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 6, characterized in that: comprises mixing ouabain, imatinib or pharmaceutically acceptable salts thereof.

8. A method of preparing a pharmaceutical composition according to claim 7, wherein: the method comprises the following steps:

s1: respectively dissolving ouabain, imatinib or pharmaceutically acceptable salts thereof in a solvent to obtain ouabain solution and imatinib solution;

s2: mixing ouabain solution and imatinib solution;

preferably, the solvent is DMSO, water.

9. Use of a pharmaceutical composition according to any one of claims 1 to 6 or ouabain in the manufacture of a medicament having the following effect:

(1) Drug resistance for treating chronic myelogenous leukemia;

(2) Promoting apoptosis of imatinib-resistant leukemia cells;

(3) Inhibiting proliferation of imatinib-resistant leukemia cells;

preferably, the imatinib-resistant leukemia cells are K562/G01 cells.

10. The use according to claim 9, wherein: the molar ratio of ouabain to imatinib or a pharmaceutically acceptable salt thereof is 0.01-1:10, preferably 0.05-1:10;

the pharmaceutical composition is in the form of a formulation;

preferably, the ouabain, imatinib, or pharmaceutically acceptable salts thereof are each dissolved in a solvent to form a formulation, the solvent comprising DMSO, water;

more preferably, the concentration of ouabain in the formulation is from 0.01 to 1. Mu.M, preferably the concentration of ouabain is from 0.05 to 1. Mu.M, more preferably the concentration of ouabain is 1. Mu.M; the concentration of imatinib or a pharmaceutically acceptable salt thereof is 10 μm.