LU505195B1 - Use of Carvedilol in the Preparation of Drugs for the Treatment of Leukaemia - Google Patents
Use of Carvedilol in the Preparation of Drugs for the Treatment of Leukaemia Download PDFInfo
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- LU505195B1 LU505195B1 LU505195A LU505195A LU505195B1 LU 505195 B1 LU505195 B1 LU 505195B1 LU 505195 A LU505195 A LU 505195A LU 505195 A LU505195 A LU 505195A LU 505195 B1 LU505195 B1 LU 505195B1
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- NPAKNKYSJIDKMW-UHFFFAOYSA-N carvedilol Chemical compound COC1=CC=CC=C1OCCNCC(O)COC1=CC=CC2=NC3=CC=C[CH]C3=C12 NPAKNKYSJIDKMW-UHFFFAOYSA-N 0.000 title claims abstract description 80
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention discloses the use of carvedilol in the preparation of drugs for the treatment of leukaemia, which belongs to the field of pharmaceutical technology, the present invention provides the use of carvedilol in the preparation of drugs for the treatment of leukaemia, through the research of the present invention it was found that: carvedilol has a strong inhibitory effect on the proliferation of leukaemia cell lines, carvedilol inhibits the cell cycle progression through the blocking of the cell cycle in the G0/G1 phase of the leukaemia cell lines, and at the same time promoting apoptosis of leukaemia cells. In conclusion, carvedilol, a third-generation β-adrenergic receptor blocker, has important potential significance in the therapeutic field of leukaemia.
Description
Use of Carvedilol in the Preparation of Drugs for the Treatment of Leukaemia
The present invention relates to the field of pharmaceutical technology, and specifically to the use of carvedilol in the preparation of drugs for the treatment of leukaemia.
Background Technology
Leukaemia is a malignant tumour of the haematopoietic system and is the most common malignant disease in children. The incidence of leukaemia is increasing every year. However, the disease is associated with many factors and its etiology is still unknown. B-Adrenergic receptor blockers are mainly used for the treatment of cardiovascular diseases, such as heart failure and hypertension. Huang Q et al. proposed that abnormal activation of the sympathetic nervous system leads to an increase in the secretion of epinephrine and norepinephrine, which activates the B-AR signalling pathway, which in turn promotes the development of tumours. Therefore, we speculate that B-adrenergic receptor blockers may become novel drugs for the treatment of leukaemia. The anti-tumour effects of B-adrenergic receptor blockers have been reported in a number of studies; however, most of these studies have focused on the first-generation B-blocker, propranolol, which inhibits the proliferation and promotes apoptosis of some tumour cells. For example, propranolol is anti-gastric and anti-breast cancer, and propranolol is also effective in the treatment of infantile hemangiomas. In addition, propranolol has been found to inhibit the proliferation of leukaemia cell lines. However, a dose-dependent cytotoxic effect of propranolol on leukaemia cell lines (Molt-4, BALL-1 and U937) was only possible at concentrations of 2200 pM.
Our previous study also confirmed that propranolol inhibited the proliferation of the acute lymphoblastic leukaemia cell line CEM-C1, but at a semi-inhibitory concentration with an IC50 of >100 pM. The concentration of this drug that inhibited the proliferation of leukaemia cells was much higher than that of the clinical dose of 2 mg/kg/d (2 mg/kg/d is equivalent to the in vitro cellular experimental dose of 67.61 uM). Therefore, this result suggests that propranolol has a weak inhibitory effect on the proliferation of leukaemia cells.
Carvedilol, as a non-selective third-generation B-adrenergic receptor blocker, is mainly used for clinical treatment and prevention of heart failure after chemotherapy. There are fewer studies 019° on the antitumour effects of carvedilol, e.g. carvedilol has been shown to inhibit the proliferation of a few solid malignant tumours, e.g. melanoma and cervical cancer cell lines, with a semi-inhibitory concentration of IC50 > 30 uM. However, the pathogenesis and development of leukaemia is different from that of other malignant solid tumours, and the question of whether carvedilol has any inhibitory effect on the proliferative activity of leukaemia cells, whether it has anti-leukaemia effects, and how strong or weak its effect is, is not reported. For this reason, the present invention provides the use of carvedilol in the preparation of drugs for the treatment of leukaemia.
Contents of the Invention
The present invention provides the use of carvedilol in the preparation of drugs for the treatment of leukaemia by effectively inhibiting the proliferation of leukaemia cells, blocking the cell cycle of leukaemia cells in the GO/G1 phase and promoting the apoptosis of leukaemia cells (including the human lymphoblastic leukaemia cell lines CEM-C1, CEM-C1, and BALL-1 and the human myeloid leukaemia cell lines THP-1, MV4-11, and OCI- AML2), inhibited their cellular development process.
The present invention provides the use of carvedilol in the preparation of drugs for the treatment of leukaemia.
Preferably, the use of carvedilol in the preparation of a drug promoting apoptosis in leukaemia cells.
Preferably, the use of carvedilol in the preparation of a drug that blocks the GO/G1 phase cell cycle of said leukaemia cells.
Preferably, said leukaemia cells comprise lymphoblastic leukaemia cell lines, myeloid leukaemia cell lines.
Preferably, said lymphoblastic leukaemia cell line comprises CEM-C1, CEM-C1, BALL-1.
Preferably, said myeloid leukaemia cell line comprises THP-1, MV4-11, OCI-AML2.
Preferably, said drug is in the form of a solid, semi-solid or liquid.
Preferably, the formulation of said drug is an aqueous solution, non-aqueous solution,
suspension, ingot, capsule, tablet, granule, pill or powder. LUS05195
Preferably, the route of administration of said drug is injection administration or oral administration.
The beneficial effects of the present invention compared to the existing technology are:
The present invention provides the use of carvedilol in the preparation of drugs for the treatment of leukaemia, through the research of the present invention, it was found that carvedilol had a strong inhibitory effect on the proliferation of leukaemia cell lines, and carvedilol inhibited the process of cell cycle and promoted the apoptosis of leukaemia cells by blocking the cell cycle of the leukaemia cell lines in the G0/G1 phase. In conclusion, carvedilol, a third-generation B-adrenergic receptor blocker, has important potential significance in the therapeutic field of leukaemia.
FIGs. 1 - 6: the graphs of carvedilol of the present invention inhibiting the activity of CEM-C1,
CEM-C7, BALL-1, THP-1, MV4-11, and OCI-AML2 cell lines;
FIG. 7: a graph of propranolol inhibition of CEM-C1 cell line activity of the present invention;
FIG. 8: the half inhibitory concentration values of propranolol of the present invention on
CEM-C1 over carvedilol graph;
FIG. 9: the results of staining of the CEM-C1, CEM-C7, and BALL-1 cell lines YF
R647A-Annexin V/PI of the present invention;
FIG. 10: the results of YF R647A-Annexin V/PI staining of THP-1, MV4-11, and OCI-AML2 cell lines of the present invention;
FIG. 11: the flow-through results of the apoptosis-promoting CEM-C1, CEM-C7, and BALL-1 cell lines of the present invention with carvedilol;
FIG. 12: the flow-through results of the apoptosis promotion of THP-1, MV4-11, and
OCI-AML2 cell lines by carvedilol of the present invention;
FIG. 13: that carvedilol of the present invention has a strong apoptosis-promoting effect on leukaemia cells in a concentration-dependent manner;
FIG. 14: a flow-through result graph of carvedilol of the present invention blocking CEM 199
CEM-C7, and BALL-1 cell cycle in GO/G1 phase;
FIG. 15: a flow-through result graph of carvedilol of the present invention blocking THP-1,
MV4-11, and OCI-AML2 cell cycle in GO/G1 phase;
FIG. 16: a comparison of the volume and weight of leukaemia transplant tumours in nude mice in the CVD group and the CON group of the present invention;
FIG. 17: the apoptosis map of H&E staining of leukaemia transplant tumours in the CVD group of the present invention;
FIG. 18: a map of leukaemic infiltration in the bone marrow of mice in the CVD group of the present invention;
FIG. 19: a comparison of the percentage of leukaemia cells in nude mice in the CVD group and the CON group of the present invention
FIG. 20: a comparison of the number of leukocytes in nude mice in the CVD group and the
CON group of the present invention.
Specific Embodiments
In order to enable the technical personnel in the field to better understand the technical solution of the present invention to be implemented, the following combines specific embodiments and the accompanying drawings to further illustrate the present invention, but the cited embodiments are not to be taken as a limitation of the present invention. The following test methods and detection methods are conventional methods if not otherwise specified; the reagents and raw materials mentioned are commercially available if not otherwise specified.
Materials and methods
Materials
Drugs and reagents: fetal bovine serum (FBS) was from BI (Israel), RPMI-1640 medium was purchased from Gibco (USA), Cell Counting Kit-8 (CCK8) was purchased from dojindo (China), Cell
Culture Plates were purchased from NEST (China), pipette tips were purchased from Biosharp (China), YF R647A-Annexin V /PI Apoptosis Kit was purchased from American Corporation (China),
DNA content quantitative assay (cell cycle) was purchased from Solarbio (China), carvedilol 5599519 propranolol were purchased from Topo Science (China).
Cell lines: human lymphoblastic leukaemia cell lines CEM-C1 and CEM-C7 were obtained from West China Hospital of Sichuan University, BALL-1 was purchased from DSMZ- German 5 Collection of Microorganisms and Cell Cultures GmbH (Leibniz Institute, Germany); and human myeloid leukaemia cell lines THP-1, MV-4-11 and OCI-AML2 were purchased from Fuheng Cell
Centre (Shanghai, China).
Experimental Methods
Drug preparation: Carvedilol and propranolol were dissolved in a small amount of DMSO and then dissolved again in RPMI-1640 medium and stored at -80°C. The two drugs mentioned above were diluted in the medium and made into a concentration of 0-80 uM, respectively.
Cell culture: each of the six cell lines was kept in RPMI-1640 medium containing 10% fetal bovine serum and incubated with different concentrations of carvedilol (0-80 uM) for 24h and 48h at 37°Cin a 5% CO2 incubator.
Inhibitory effect of carvedilol on the proliferative activity of leukaemia cells:
The inhibitory effect of carvedilol on the viability of leukaemia cells was detected using the cell counting kit (CCK8) method, in which six types of cells were inoculated in 96-well plates (20,000 cells per well) and then treated with carvedilol and propranolol. The concentrations of carvedilol were 0 pM, 10 uM, 20 uM, 40 pM, 60 pM, and 80 uM, and the concentrations of propranolol were 0 uM, 20 uM, 40 pM, 80 uM, 120 pM, and 160 uM, respectively. 10 ul of CCK8
Activity Detector was added to each well after incubation for 24 h and 48 h. Cells were incubated in the absence of light at 37°C, and after 2 h, the plates were placed on an enzyme labeller (BIO-BRI Chengdu, China) at 450 nm.
Apoptosis experiments:
The effect of carvedilol (20 uM and 40 pM) on apoptosis of leukaemia cells was detected using Annexin V/PI fluorescence staining. Cells were inoculated in 12-well plates at a seeding density of 2x105 cells/ml, and after 24 h of intervention with carvedilol (20 uM and 40 uM), cells were collected and gently washed three times with PBS. Then, 2 x 105 cells were collected BAS 199 resuspended with 100 uL 1xAnnexin V binding buffer. Next, 5 ul YFR647A Annexin V and 5 pl propidium iodide (Pl) were incubated with the cells at room temperature in the dark for 15 min. apoptosis was detected using an inverted microscope (Leica, Germany) and flow cytometry.
Cell cycle experiments:
Flow cytometry was used to detect the effect of carvedilol (20 uM) on the leukaemia cell cycle. Cells were inoculated in 12-well plates at a seeding density of 2x105 cells/ml. After 24 h of intervention with carvedilol (20 uM), the cells were collected and gently washed twice with PBS.
Then, 1x106 cells were collected and resuspended with 500 pl of pre-cooled ethanol solution at a concentration of 75% for 2 h. After 2 h, the cells were washed once using pre-cooled PBS and resuspended using 100 ul of RNase A solution for another 0.5 h at 37°C. Then, 400 ul of propidium iodide (Pl) was added into the tubes and incubated in darkness at 4°C for 0.5 h. Finally, the cells were incubated with a flow cytometry instrument (BD FACSCanto TM, USA) to determine the cell cycle.
Construction of an in vivo model of leukaemia:
Four-week-old female nude mice, weighing 17.0-20.0 g, were purchased from Changzhou
Cavens Laboratory Animal Co. Ltd (strain: BALB/c-nu, batch number: 202210375, licence: SCXK (Su) 2021-0013). These mice were kept in the SPF laboratory of Zunyi Medical University. One week later, 5 x 106 of CEM-C1 cells (in logarithmic growth phase) were injected subcutaneously into the posterior right axilla of each nude mouse. One week after transplantation, leukaemia graft tumours were formed and the nude mice were randomly divided into two groups (3 mice per group): control group (CON group) and carvedilol group (CVD group). When the leukaemia transplant tumour reached about 0.5 cm, each nude mouse in the carvedilol group was gavaged with CVD (10 mg/kg/day) for a fortnight; while the control group was given the same amount of water. Changes in the size of the transplanted tumour were observed, and H&E staining was used to detect apoptosis of leukaemia cells in the transplanted tumour and the degree of infiltration of leukaemia cells in the bone marrow, as well as to detect the percentage of leukaemia cells and the number of leukaemia cells in the peripheral blood. The animal experiment protocol was approved by Zunyi Medical University (Ethical approval number: ZMU21-2204-001). LUS05195
Statistical analyses:
SPSS 18.0 (Zunyi Medical University) was used for statistical analysis. Data were expressed as mean + standard deviation. Differences were analysed using one-way ANOVA or unpaired t-test, with P<0.05 representing a statistically significant difference.
Experimental results 1. Inhibitory effect of carvedilol on the proliferative activity of leukaemia cells
Carvedilol was used to treat 6 types of human leukaemia cells for 24 h and 48 h. As shown in
FIGs. 1-6, carvedilol inhibited the proliferation of leukaemia cells in a dose- and time-dependent manner. 24 h later, the semi-inhibitory concentrations of carvedilol on CEM-C1, CEM-C7 and
BALL-1 lymphoblastic leukaemia cell lines were (13.80+4.51) uM, (7.93+0.91) uM and (23.78+2.32) uM. After 48 h, the semi-inhibitory concentrations of carvedilol on the appealing three lymphoblastic leukaemia cell lines were (3.58+1.02) pM, (1.76+0.40) uM, and (15.84+2.00) pM, respectively. At the same time, after 24 h of incubation, the effects of carvedilol on THP-1,
MV4-11 and OCI -AML2 myeloid leukaemia cell lines were (14.36+0.56) uM, (13.78+0.20) uM and (18.72+0.42) uM, respectively. After 48h, the semi-inhibitory concentrations of carvedilol on the appealing three myeloid leukaemia cell lines were (13.27+0.59) uM, (12.96+0.38) pM and ( 16.82+0.60) pM. It can be seen that the semi-inhibitory concentration of carvedilol on all leukaemia cells had an IC50 <24 pM, and this concentration was less than the maximum clinically administered dose (1 mg/kg/d, which is equivalent to the cellular experimental dose of 24.6 uM), therefore, this indicates that carvedilol has a strong inhibitory effect on the proliferative activity of leukaemia. In addition, as a control, we also investigated the inhibitory effect of propranolol (0, 20, 40, 80, 120 and 160 pM) on the proliferation of CEM-C1 cells. As shown in Fig. 7, the semi-inhibitory concentrations of propranolol on CEM-C1 after 24 h and after 48 h were (100.17 + 23.95) uM and (114.54 + 25.73) uM, respectively. Therefore, carvedilol had a stronger inhibitory effect on the proliferation of leukaemia cells compared to propranolol, as shown in Fig. 8. 2. Carvedilol promotes apoptosis in leukaemia cells
Carvedilol promotes apoptosis in leukaemia cells. 6 types of human leukaemia cell lines were > 19° intervened with carvedilol (20 uM).After 24 h, the effect of carvedilol on apoptosis of leukaemia cells was investigated using the YF R647A-Annexin V/PI dual fluorescence assay. All the cells were collected according to the instructions of the YF R647A-Annexin V/PI Apoptosis Kit. Leukaemia cells were stained with Annexin V and PI avoiding light for 5 min. YF R647A-Annexin V stained the cell membranes of early apoptotic cells with green fluorescence, and PI stained the nuclei of late apoptotic cells with red colour. As shown in FIGs. 9 and 10, carvedilol promoted apoptosis in all leukaemia cells. In addition, 6 types of human leukaemia cell lines were intervened with carvedilol 20 pM and 40 uM, respectively, and the apoptosis rate was detected by flow assay after 24 h. As can be seen from FIG. 11 and FIG. 13, carvedilol promoted apoptosis of all leukaemia cells in a dose-dependent manner. 3. Carvedilol blocks the cycle of leukaemia cells
We performed cell cycle analysis on 6 types of human leukaemia cell lines treated with 20
HM carvedilol for 24 hours. As shown in FIGs. 14 and 15, exposure of leukaemia cells to carvedilol resulted in prolongation of the GO/G1 phase (P<0.05) and correlated with a decrease in the S phase. These data suggest that carvedilol inhibits the proliferation of leukaemia cells by inhibiting the GO/G1 phase of the cell cycle. 4. In vivo antileukaemic effects of carvedilol
In the nude mouse leukaemia transplantation model, CEM-C1 cells in the logarithmic growth phase were injected subcutaneously into the posterior aspect of the right axilla of 5-week-old nude mice; after successful modelling of the leukaemia transplantation tumour, the mice were divided into CON and CVD groups (10 mg/kg/d by continuous gavage for 2 weeks). As shown in
FIG. 16, the volume and weight of the transplanted tumour in the control group increased rapidly (P<0.05) compared to the carvedilol (CVD) group. After 2 weeks of administration, as shown in
FIG. 17, H&E staining of the transplanted tumours showed apoptosis and coagulative necrosis of some leukaemia cells in the transplanted tumours in the CVD group. H&E staining of bone marrow (BM) was performed, and as shown in FIG. 18, leukaemic infiltration was lower in the BM of mice in the CVD group. Meanwhile, we identified CEM-C1 surface antigen, and found that CD4 was the main antigenic marker of CEM-C1 cells, and detected leukaemia cells CEM-C1 in Fhe 019° peripheral blood of nude mice, and the results as shown in FIG. 19, showed that the percentage of leukaemia cells in the peripheral blood of nude mice in the CVD group was significantly lower (P<0.05). In addition, as shown in FIG. 20, the number of leukocytes in the peripheral blood of nude mice in the CVD group was also significantly reduced (P<0.05).
Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their technical equivalents, the present invention is intended to encompass these modifications and variations as well.
Claims (9)
1. The use of carvedilol in the preparation of drugs for the treatment of leukaemia.
2. Use according to claim 1, characterised by the use of carvedilol in the preparation of a drug promoting apoptosis in leukaemia cells.
3. Use according to claim 2, characterised by the use of carvedilol in the preparation of a drug that blocks the GO/G1 phase cell cycle of said leukaemia cells.
4. Use according to claim 3, characterised in that said leukaemia cells comprise lymphoblastic leukaemia cell lines, myeloid leukaemia cell lines.
5. Use according to claim 4, characterised in that said lymphoblastic leukaemia cell line comprises CEM-C1, CEM-C1, BALL-1.
6. Use according to claim 4, characterised in that said myeloid leukaemia cell line comprises THP-1, MV4-11, OCI-AML2.
7. Use according to claim 1, characterised in that said drug is in the form of a solid, semi-solid or liquid.
8. Use according to claim 1, characterised in that the preparation of said drug is an aqueous solution, non-aqueous solution, suspension, ingot, capsule, tablet, granule, pill or powder.
9. Use according to claim 1, characterised in that the route of administration of said drug is injection administration or oral administration.
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