Use of birch sap in the treatment of chronic myeloid leukemia with imatinib mesylate

Abstract

The present invention relates to the field of drug treatment for chronic myeloid leukemia (CML), and specifically to the application of birch sap combined with imatinib mesylate (Gleevec) in the treatment of chronic myeloid leukemia. Birch sap contains rich birch polysaccharides and pentacyclic triterpenoids such as betulinic acid and betulin, which can effectively induce apoptosis of leukemia drug-resistant cells, enhance the sensitivity of drug-resistant cells to drugs, reverse the secondary drug resistance generated by imatinib mesylate during treatment, and reduce the levels of arginine vasopressin (AVP) and lactic acid in the blood, increase the content of human liver glycogen, and effectively alleviate the common treatment side effects such as water retention and systemic edema, physical decline, fatigue, nausea, and vomiting caused by taking imatinib mesylate. Compared with the use of drugs alone, the combined use of birch sap can effectively enhance the therapeutic effect of imatinib mesylate on chronic myeloid leukemia and reduce drug side effects.

Description

Technical Field

[0001] The invention relates to the technical field of drugs for treating chronic myeloid leukemia, and in particular to application of birch sap in the process of treating chronic myeloid leukemia with imatinib mesylate. Background Art

[0002] Chronic myeloid leukemia (CML) is a malignant blood disease derived from hematopoietic stem cells, accounting for 15% to 20% of leukemia patients. Imatinib mesylate (Gleevec), as a tyrosine kinase inhibitor, is a commonly used therapeutic drug for the treatment of chronic myeloid leukemia. However, since imatinib mesylate requires continuous use to maintain its efficacy, secondary drug resistance is prone to occur during clinical use, which seriously affects the effectiveness of clinical treatment. At the same time, during the treatment of leukemia with imatinib mesylate, a series of drug side effects such as water retention, systemic edema, fatigue, easy fatigue, nausea, and vomiting are often accompanied.

[0003] Birch sap is rich in birch polysaccharides and pentacyclic triterpenoid compounds such as betulinic acid and betulin, which have obvious proliferation inhibitory effects on various tumor cells such as melanoma cells, prostate cancer cells, cervical cancer cells, leukemia cells, etc. It can induce apoptosis of leukemia resistant cells, increase the sensitivity of resistant cells to drugs, and effectively reverse the secondary drug resistance generated during drug treatment; reduce the concentration of arginine vasopressin (AVP) in the blood, slow down the antidiuretic effect, promote urine excretion, relieve water retention and systemic edema, increase the content of liver glycogen in the body, reduce the level of lactic acid in the blood, improve fatigue and fatigue symptoms, and shorten fatigue recovery time.

[0004] Currently, there are no literature or patents reporting and research on the combined use of birch sap and imatinib mesylate in the treatment of chronic myeloid leukemia. Birch sap has important clinical application significance in solving imatinib mesylate resistance and drug side effects. Summary of the invention

[0005] In response to existing clinical problems, the present invention provides the use of birch sap in the treatment of chronic myeloid leukemia with imatinib mesylate. Compared with monotherapy, the combined use of birch sap and imatinib mesylate improves the therapeutic effect on chronic myeloid leukemia while effectively reducing the drug toxicity and side effects associated with the use.

[0006] The birch sap ingredients involved in the patent can effectively induce apoptosis of leukemia-resistant cells and enhance the drug sensitivity of leukemia-resistant cells to imatinib mesylate.

[0007] At the same time, it relieves typical drug side effects such as water retention, systemic edema, fatigue, nausea and vomiting.

[0008] The birch sap disclosed in the patent can be provided by birch sap stock solution or by further processing the birch sap stock solution through concentration, ultrafiltration, purification, etc.

[0009] Furthermore, the birch sap involved can be provided in the form of any mixture containing birch sap ingredients, and the mixture form includes but is not limited to solid preparations, gel preparations and liquid preparations.

[0010] In addition to the birch sap component, the solid preparation may also contain one or more combinations of starch, sucrose, dextrin, microcrystalline cellulose, lactose, pregelatinized starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, polyvinyl propionyl pyrrolidone, cellulose derivatives, magnesium stearate, and micro-powdered silica gel.

[0011] The liquid preparations involved may contain, in addition to birch sap ingredients, one or more of water, ethanol, glycerol, propylene glycol, polyethylene glycol, fatty oil, liquid paraffin, sodium stearate, calcium stearate, oleic acid, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, lecithin, soybean lecithin, sorbitan fatty acid, polysorbate, polyoxyethylene-polyoxypropylene copolymer, benzoic acid, sodium benzoate, sorbic acid, sodium sorbate, benzalkonium bromide, and parahydroxybenzoates.

[0012] In addition to the birch juice component, the gel may also contain one or more of carbomer, polyethylene glycol, chitosan, polyvinyl pyrrolidone, polyvinyl alcohol, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, glycerol, propylene glycol, ethanol, Tween-80, sulfite, cysteine, di-tert-butylhydroxytoluene (BHT), and potassium sorbate. DETAILED DESCRIPTION

[0013] The present invention is specifically described by the following examples, which are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention.

[0014] Example 1 Effect of birch sap on apoptosis of leukemia cells resistant to imatinib mesylate

[0015] 1. Imatinib mesylate-resistant leukemia cell culture.

[0016] Leukemia cells K562 / G01 were provided by the Institute of Hematology, Chinese Academy of Medical Sciences.

[0017] All cells were cultured in RPMI1640 medium containing 10% calf serum in a 37°C, 5% CO2, saturated humidity incubator. 1 μg / ml imatinib mesylate was added to the cell culture medium to maintain cell resistance.

[0018] 2. Preparation of mouse serum containing birch sap: 18 SPF Kunming mice were randomly divided into 3 groups and gavaged with birch sap at doses of 18 ml / kg, 9 ml / kg and 3 ml / kg respectively for 21 consecutive days, twice a day. 2 hours after the last gavage, femoral artery blood was collected and serum was separated and collected. The sera in the same group were mixed and filtered and sterilized using a 0.22 μm microporous filter membrane to prepare mouse sera containing high, medium and low birch sap contents, which were then stored in a -20°C refrigerator for later use.

[0019] 3. Detection of apoptosis of leukemia cells resistant to imatinib mesylate

[0020] The K562 / G01 resistant cells in the logarithmic growth phase were divided into 5 groups, namely, blank control group (K562 / G01 resistant cells), imatinib mesylate monotherapy group, and imatinib mesylate combined with birch sap (serum with high, medium, and low birch sap content) groups.

[0021] K562 / G01 in the logarithmic growth phase was cultured in 5×10 4 The cell suspensions of 1 μg / mL were inoculated in 24-well culture plates. 20 μl of imatinib mesylate at a concentration of 1 μg / ml was added to the single-drug group, and 20 μl of mouse serum with different birch sap contents and 20 μl of imatinib mesylate at a concentration of 1 μg / ml were added to the combined drug groups, and a blank control group was set up.

[0022] The five groups of cells were placed in a 37°C incubator and cultured for 24, 48, and 72 h. The cells were then collected and transferred to EP tubes. The cells were centrifuged (2000 r / min, 15 min), the supernatant was discarded, and the cells were washed three times with PBS at 4°C. The density was adjusted to 5×10 6 / mL, and 5 μL Annexin V reagent was added to each, and the mixture was reacted at room temperature in the dark for 30 min. Cell apoptosis was detected by flow cytometer.

[0023] The experimental results showed that compared with the control group, the apoptosis rate of drug-resistant cells in the imatinib mesylate monotherapy group and the birch sap combination group was significantly increased; at 48h and 72h, the apoptosis rate of drug-resistant cells in the birch sap combination group was significantly higher than that in the imatinib mesylate monotherapy group, among which the cell apoptosis rate in the group combined with high-dose birch sap mouse serum was the highest, indicating that the combination of birch sap and imatinib mesylate can effectively promote the apoptosis of leukemia resistant cells, significantly improve the therapeutic effect of imatinib mesylate on chronic myeloid leukemia, and is dose-dependent.

[0024] Table 1 Changes in apoptosis rate of K562 / G01 drug-resistant cells after drug treatment

[0025] Group 24h apoptosis rate 48h apoptosis rate 72h apoptosis rate Blank control group 8.34±1.22 10.27±1.43 13.43±0.40 Imatinib mesylate alone 9.75±0.81 24.31±0.64 36.77±0.74 Birch sap combined with medication (low dose) 10.72±0.24 41.81±1.56 50.63±1.36 Birch sap combined with medication group (medium dose) 15.96±0.37 54.64±0.73 69.86±1.21 Birch sap combined with medication (high dose) 21.08±0.40 61.36.±1.27 72.04±1.53

[0026] Example 2 Experiment on reversal of drug resistance of leukemia cells resistant to imatinib mesylate by birch sap

[0027] Referring to the method in Example 1, imatinib mesylate-resistant leukemia cells and mouse sera with different birch sap contents were cultured.

[0028] The cells in the logarithmic growth phase were taken and the cultured K562 / G01 resistant cells were divided into 5 groups, namely, blank control group (K562 / G01 resistant cells), imatinib mesylate monotherapy group, and imatinib mesylate combined with birch sap (mouse serum with high, medium and low birch sap content) group.

[0029] K562 / G01 in the logarithmic growth phase was cultured in 5×10 4 / mL cell suspension was inoculated in 96-well culture plates for culture. 20μl of imatinib mesylate at a concentration of 1μg / ml was added to the single-drug group, and 20μl of mouse serum with different birch sap contents and 20μl of imatinib mesylate at a concentration of 1μg / ml were added to the combined drug groups, and a blank control group was set up.

[0030] After culturing the five groups of cells in a 37°C incubator for 72 hours, 20 μL of 5 mg / mL MTT solution was added respectively. After incubation for 4 hours, the cells were centrifuged at 3600 r / min for 10 minutes, and the supernatant was discarded. The precipitate was dissolved with 200 μL DMSO. After sufficient dissolution, the absorbance of each group of bacterial suspension was measured at 570 nm, and the cell proliferation inhibition rate was calculated according to the absorbance value according to the following formula.

[0031] Cell proliferation inhibition rate (%) = (1-experimental group value / control group absorbance value) × 100%

[0032] SPSS software was used to calculate the IC of each group of cells 50 The resistance multiple is calculated using the following formula:

[0033] Resistance multiple = Imatinib mesylate monotherapy group cell IC 50 / birch sap combined with histocyte IC 50

[0034] The experimental results showed that the mouse serum prepared by different doses of birch sap increased the IC of imatinib mesylate on K562 / G01 drug-resistant cells. 50 The values ​​decreased from 43.52±1.26μg / ml to 27.77±0.81μg / ml, 8.35±1.40μg / ml, and 8.35±1.40μg / ml, which increased the sensitivity of K562 / G01 cells to imatinib mesylate by 1.57 times, 5.21 times, and 14.18 times, respectively. The experimental results showed that birch juice can effectively enhance the drug sensitivity of leukemia-resistant cells to imatinib mesylate.

[0035] Table 2 Effect of birch sap on drug resistance of imatinib mesylate-resistant cells

[0036] Group <![CDATA[IC 50 (μg / ml)]]> Resistance reversal factor Blank control group —— —— Imatinib mesylate alone 43.52±1.26 —— Birch sap combined with medication (low dose) 27.77±0.81 1.57 Birch sap combined with medication group (medium dose) 8.35±1.40 5.21 Birch sap combined with medication (high dose) 3.07±0.69 14.18

[0037] Example 3 Experiment on the effect of birch sap on water retention in rats induced by imatinib mesylate

[0038] Animal grouping.

[0039] Thirty-six male SD rats were selected and randomly divided into 6 groups, namely blank control group, water retention model group, positive control group and birch sap high-, medium- and low-dose groups, with 6 rats in each group. Except for the blank control group, rats in each group were injected with imatinib mesylate to establish the rat water retention model.

[0040] Establishment of imatinib mesylate-induced water retention model in rats.

[0041] Imatinib mesylate was diluted with normal saline to an injection solution of 0.5 mg / ml, and injected into the tail vein of rats at a dose of 9 ml / kg for 13 days. On the 14th day, the rats were injected with the same method at a dose of 18 ml / kg to complete the establishment of the rat water retention model induced by imatinib mesylate.

[0042] Experimental groups and drug administration.

[0043] After the modeling was completed, the positive control hydrochlorothiazide group was gavaged with 10 mg / kg hydrochlorothiazide every day, the high, medium and low dose groups of birch sap were gavaged with 18 ml / kg, 9 ml / kg and 3 ml / kg birch sap every day, respectively, and the blank control group and the model group were gavaged with the same volume of normal saline once a day for 30 consecutive days.

[0044] Determination of urine volume in mice.

[0045] After the last intragastric administration, the 24-hour urine of rats in each group was collected, and the urine volume of the rats was measured and recorded.

[0046] Determination of AVP (arginine vasopressin) content in rats.

[0047] 5 ml of blood from the right abdominal aorta of the rat was collected into a vacuum collection tube at 60000 r / min. -1 After centrifugation for 15 min, the upper serum was collected and the AVP content of rats was determined by ELISA.

[0048] The experimental results showed that the AVP level of rats in the model group was significantly higher than that in the blank control group, while the urine volume was lower than that in the blank control group; the AVP levels in the blood of rats in the high, medium and low birch sap groups were significantly lower than those in the model group, and the urine volume was similar to that of rats in the control group, indicating that birch sap can promote urination by lowering the blood AVP level, thereby alleviating water retention and systemic edema caused by the use of imatinib mesylate.

[0049] Table 3 Effects on plasma AVP concentration and mouse urine

[0050] Group <![CDATA[Dose / ml·kg -1 > AVP (ng / l) Urine volume (ml / 100g) Blank control group 273.15±16.20 10.8±0.7 Model Group 477.22±11.47 1.9±0.4 Hydrochlorothiazide 10 288.67±13.65 9.4±0.2 Birch sap low dose group 3 369.53±10.88 3.2±0.2 Birch sap medium dose group 9 328.19±16.40 5.7±0.9 High-dose birch sap 18 279.03±12.81 8.7±0.5

[0051] Example 4 Experiment on the effect of birch sap on exercise-induced fatigue in mice treated with imatinib mesylate

[0052] Experimental groups and drug administration.

[0053] A total of 72 Kunming mice were selected and randomly divided into 6 groups, including blank control group, imatinib mesylate model group, positive control American ginseng group, and high-, medium-, and low-dose birch juice groups, with 12 mice in each group.

[0054] Imatinib mesylate was diluted with normal saline to an injection solution of 0.5 mg / ml. Except for the blank group, the rest groups of mice were injected through the tail vein at a dose of 9 ml / kg for 30 days.

[0055] After 30 days, the positive control American ginseng group was gavaged with 200 mg / kg American ginseng granules every day, the high, medium and low doses of birch juice groups were gavaged with 18 ml / kg, 9 ml / kg and 3 ml / kg of birch juice respectively every day, and the blank control group and the model group were gavaged with the same volume of normal saline once a day for 30 consecutive days.

[0056] Weighted swimming test for mice.

[0057] One hour after the last administration, the mice were placed in a swimming box at a depth of 30 cm and a water temperature of 25°C for weighted swimming, and a lead block of 5% of the mouse's body weight was tied to the base of the mouse's tail. The time from the start of swimming to the time when the mouse's body was completely submerged in water and did not return to the surface for 10 seconds was recorded, which was the exhaustive swimming time of the mouse.

[0058] Determination of serum lactate in mice.

[0059] After the weighted swimming, blood was collected from the mouse's orbital cavity, and the blood lactate level of the mouse was detected according to the instructions of the lactate detection kit.

[0060] Determination of mouse liver glycogen.

[0061] After blood was collected from the mouse orbits, the mice were killed by cervical dislocation, the mice were dissected and the fresh livers were separated. After rinsing with saline, the water was dried and the liver glycogen content of the mice was detected according to the instructions of the liver glycogen detection kit.

[0062] The experimental results showed that compared with the blank control group, the exhaustive swimming time of the mice in the model group administered with imatinib mesylate was significantly shortened, the blood lactate level was significantly increased, and the liver glycogen content was decreased. Compared with the mice in the model group, the exhaustive swimming time and liver glycogen content of the mice in each birch sap dose group were significantly increased, and the blood lactate level was significantly decreased. The effect was significantly enhanced with the increase in the dose of birch sap. The experimental results show that birch sap can effectively improve the exercise and fatigue resistance of mice by increasing the liver glycogen content and reducing the blood lactate level, and shorten the fatigue recovery time after exercise.

[0063] Table 4 Comparison of exhaustive swimming time, serum lactate and liver glycogen levels in mice of each group

[0064] Group Exhaustive swimming time / (min) Blood lactate level / (mmol / L) Liver glycogen / (mg / g) Blank control group 12.18±1.13 12.43±0.29 5.45±1.72 Model Group 7.32±0.97 17.22±0.16 4.01±1.28 Positive control group 18.45±1.46 11.75±0.49 8.36±2.44 Birch sap low dose group 12.37±0.71 12.01±0.33 7.84±1.35 Birch sap medium dose group 17.02±1.24 9.93±0.50 8.95±0.27 High-dose birch sap 21.89±0.85 8.26±0.24 10.24±1.04

[0065] Example 5 Experiment on the effect of birch sap on nausea and vomiting in mice administered with imatinib mesylate

[0066] Grouping and dosing.

[0067] Thirty-six Kunming mice were randomly divided into 6 groups, namely normal control group, model group, positive control ondansetron group, low-, medium- and high-dose birch sap groups, with 6 mice in each group. Mice in the positive control ondansetron group were intraperitoneally injected with 2 ml / kg of ondansetron hydrochloride injection, and the high-, medium- and low-dose birch sap groups were intragastrically gavaged with 18 ml / kg, 9 ml / kg and 3 ml / kg of birch sap, respectively, every day. The blank control group and the model group were gavaged with the same volume of normal saline, once a day, for 7 consecutive days.

[0068] 2 hours after the last intragastric administration, imatinib mesylate was diluted with normal saline to a 0.5 mg / ml injection solution. Except for the blank group, the remaining groups of mice were injected through the tail vein at a dose of 9 ml / kg.

[0069] After the injection of imatinib mesylate, the number of retching and vomiting of the mice was observed within 0-24, 24-48 and 48-72 h.

[0070] The experimental results showed that compared with the blank control group, the number of dry heaves in the model group mice increased significantly; compared with the model group, the number of dry heaves in the high, medium and low dose groups of birch sap decreased significantly at all time periods, with the high dose group of birch sap having the most significant effect. The experimental results showed that birch sap can effectively alleviate the nausea and dry heaves symptoms of mice caused by the administration of imatinib mesylate, and effectively reduce the number of vomiting.

[0071] Table 5 Number of dry retching in mice in each group

[0072] Group <![CDATA[Dose / ml·kg -1 > 0～24 24～48 48～72h Blank control group 0 0 0 Model Group 182.45±19.26 143.74±12.51 125.36±14.63 Ondansetron group 2 70.27±12.68 55.62±16.93 38.55±21.37 Birch sap low dose group 3 102.62±20.43 77.93±14.52 52.69±16.10 Birch sap medium dose group 9 68.34±11.06 52.48±17.31 40.32±13.56 High-dose birch sap 18 55.02±12.53 42.43±16.09 28.95±11.03

Claims

1. The use of birch sap in the preparation of a drug for improving the side effects of imatinib mesylate in the treatment of chronic myeloid leukemia, characterized in that: Birch sap can improve the side effects of imatinib mesylate treatment, including water retention, systemic edema, fatigue, nausea and vomiting.