CN110141664B - Pharmaceutical composition for treating acute myeloid leukemia - Google Patents

Pharmaceutical composition for treating acute myeloid leukemia Download PDF

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CN110141664B
CN110141664B CN201910452580.0A CN201910452580A CN110141664B CN 110141664 B CN110141664 B CN 110141664B CN 201910452580 A CN201910452580 A CN 201910452580A CN 110141664 B CN110141664 B CN 110141664B
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pharmaceutical composition
homoharringtonine
leukemia
kinase inhibitor
wee1 kinase
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CN110141664A (en
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陆滢
叶佩佩
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Hangzhou Health Online Information Technology Co ltd
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Ningbo Yinzhou People's Hospital
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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • 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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  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

The invention relates to a pharmaceutical composition for treating leukemia, which comprises homoharringtonine and a Wee1 kinase inhibitor as active ingredients, wherein the weight ratio of homoharringtonine to the Wee1 kinase inhibitor is 1:10-10: 1. The pharmaceutical composition has remarkable curative effect on treating leukemia, particularly chronic myelogenous leukemia or acute myelogenous leukemia. The invention provides a brand new idea for developing a combined administration scheme for treating leukemia.

Description

Pharmaceutical composition for treating acute myeloid leukemia
Technical Field
The invention relates to the field of medicines, and in particular relates to a pharmaceutical composition for treating leukemia.
Background
Leukemia is a bone marrow-derived cancer, which refers to the uncontrolled, immortal accumulation of large numbers of immature blood cell precursors and infiltration of other tissues and organs, while inhibiting the production of normal blood cells. The leukemia patients are easy to ignore the initial symptoms because of unobvious, and the common symptoms comprise weakness, anemia, palpitation, short breath, enlargement of liver, spleen and lymph nodes, bone pain and the like. Meanwhile, leukemia patients often have complications caused by hematopoietic disorders, including: hemorrhage, fever, refractory infection, high white blood cell count, dyspnea, obnubilation or organ failure. The exact etiology of leukemia is not clear at present, and possible causes include viral factors, chemical factors, radiation factors, genetic factors, and the like, such as smoking, ionizing radiation, certain chemical agents (such as benzene), down syndrome, and the like, and the people with family leukemia history also belong to high risk groups.
Leukemia is classified into acute leukemia and chronic leukemia according to the speed of progression. Acute leukemia is characterized by a rapid increase in the number of immature blood cells, and as so many abnormal cells accumulate in the bone marrow, they prevent the bone marrow from making normal blood cells. With the rapid development of the disease, malignant cells accumulate and spread to the blood and gradually infiltrate other tissues and organs, and therefore, acute leukemia must be treated as soon as possible. Chronic leukemia is characterized by an excessive accumulation of relatively mature, but still abnormal, white blood cells, which typically progress for months or years resulting in a higher rate of abnormal white blood cell production than normal white blood cells, and thus, an increasing accumulation of abnormal cells. Leukemia can be classified into lymphoid leukemia and myeloid leukemia according to the source of pathogenic cells. In general, in combination with the cell types from which the condition is acute and mild, leukemias can be divided into four types: acute Lymphoid Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphoid Leukemia (CLL) and Chronic Myeloid Leukemia (CML).
Acute Myeloid Leukemia (AML) is a heterogeneous hematopoietic malignant proliferative disease, the most common type of blood malignant tumor in adults, and the incidence of AML has been increasing year by year due to air pollution and other causes in recent years. With the continuous development of modern hematology foundation and clinic, the diagnosis, typing and grading of AML are continuously perfected, the diagnosis and prognosis grading of AML are more accurate and detailed, the treatment strategy selected according to the prognosis grading is more individualized, but no new breakthrough is made in the treatment field.
In the last 30 years, the AML induction scheme based on cytarabine and anthracycline chemotherapeutics has been dominant, while the complete remission rate (CR) of chemotherapy has only been maintained at 50-70% and 5-year survival rate is only around 20-30%. Although a subset of patients can benefit from the increasingly sophisticated hematopoietic stem cell transplantation techniques, the limited source of transplant donors, transplant-related toxicities and complications, post-transplant rejection and relapse have limited the widespread use of transplantation techniques in AML patients, and refractory and relapsed AML remain a problem for countless hematologists. Therefore, research institutes at home and abroad have been constantly exploring and studying the optimal therapeutic strategy for AML.
The use of targeted drugs (all-trans retinoic acid (ATRA) and/or arsenous acid) in the particular AML type, Acute Promyelocytic Leukemia (APL), has in recent years shifted from the most lethal AML to the most clinically cured AML (over 90% CR and 85% long-term disease-free survival); in the chronic granulocytic leukemia, the targeted drug for the BCR-ABL fusion gene, namely the first generation and the second generation, is applied to clinic and obtains good curative effect; therefore, targeted drugs for genes become hot spots for research, but fusion genes with the characteristics are not found in AML of other types except APL, and the mechanism of the fusion genes often involves multiple karyotypes or genetic abnormalities, so that the research and development of the targeted drugs for AML genes are very difficult.
Meanwhile, research institutions at home and abroad continuously research new drug action targets of AML, and in view of the importance and increasingly clear mechanism of epigenetic change in the AML pathogenesis, effective AML resistance activity is also shown for small molecular compounds of epigenetic markers, such as synthesis of DNA methyltransferase inhibitors, histone low acetylation inhibitors and the like and clinical phase I or II experiments, a certain research result is obtained, and the research results show that the small molecular compounds have great application prospects, so that the treatment targets of the epigenetic markers are one of the focuses of research in recent years.
Homoharringtonine (HHT) is an antitumor drug extracted from Chinese plants. Homoharringtonine is present in Cephalotaxus plants, Cephalotaxus c.druupaceae sieb.etc. Cephalotaxus Zucc and Cephalotaxus c.fortunei hook.f. were first isolated in 1963 from Cephalotaxus japonica et al. In 1969, Powell and the like firstly separate homoharringtonine from cephalotaxus sinensis, determine that the homoharringtonine has the structure of (2R) -2-hydroxy-2- (4-hydroxy-4-methylpentyl) succinic acid (methyl), and find that the homoharringtonine has obvious activity on mouse lymphoid leukemia P388.
Homoharringtonine is easily soluble in methanol, ethanol or chloroform, and slightly soluble in water or diethyl ether, and is presumed to be poor in oral absorption, which suggests that the oral absorption problem of HHT should be improved by means of modifying main drug, adding adjuvants such as solubilizer, or making into suspension.
Homoharringtonine has been used in the treatment of AML for over 30 years in China. Homoharringtonine is mainly used in combination in treating AML, mainly in HA (HHT, cytarabine) scheme, HAA (HHT, cytarabine and aclacinomycin) scheme and HAD (HHT, cytarabine and daunorubicin) scheme. In addition, there are some unique combinations of medication, for example, the HIA (daunorubicin + HHT + cytarabine) regimen reduces cardiovascular toxicity and increases treatment tolerance; (ii) HIA in combination with decitabine for treatment of relapsing refractory AML; HAAG (HHT + aclarubicin + cytarabine + granulocyte colony stimulating factor) + Decitabine (DAC) for advanced acute myeloid leukemia; HAAG is combined with traditional Chinese medicine for treatment, so that adverse effects of chemotherapy of old patients can be relieved, and the pain degree of the patients can be reduced; the HHT and the ibutinib have obvious curative effect on FLT3-ITD mutant acute myelogenous leukemia.
Recently, the Zhejiang university institute of hematology combines other domestic research and clinical institutes to apply an induction scheme combining the HHT as the basis with the cytarabine and/or anthracycline antitumor drugs, obtains good curative effect on the treatment of AML, and has great application prospect due to lower economic and side effects.
The research on the action mechanism of homoharringtonine against AML shows that HHT can change the activity of telomerase, down-regulate MCL-1 and survivin, inhibit PI3K/AKT signals and the phosphorylation of JAK2-STAT5 pathway, and the gene expression analysis after the action of HHT on AMLHL-60 cell strain shows that HHT can also obviously down-regulate c-myc gene.
Wee1 kinase is one of the important members of the serine/threonine protein kinase family, and plays a variety of important roles throughout the cell cycle, such as ensuring DNA replication accuracy and chromatin integrity, blocking DNA replication initiation and G2 phase to M phase transition, etc. Ataxia telangiectasia mutated gene ATM and ATR and Rad3 related protein ATR are core response kinases for DNA damage response. Wee1 kinase plays a key role in the DNA damage response pathway described above. After DNA damage is recognized, phosphorylation-activated Wee1 kinase phosphorylates Thr15 on cdc2, thereby inhibiting cdc2 activity, halting the cell cycle for transient mitosis to complete DNA repair.
During the cell cycle, the p53 protein examines DNA damage at G1/S phase and G2/M phase through ATM-Chk2-p53 pathway and monitors the integrity of genome. When the cells are damaged, p53 activates the expression of p2 gene, and p21 protein acts with corresponding CDK-cyclins complex to block each check point and repair damaged cells. If the p53 gene is mutated or deleted, it is unable to control the proliferation of cells, resulting in the canceration of cells. Studies have shown that over 50% of tumors have p53 gene deletion or mutation, causing defects in the cell cycle G1/S checkpoint, making the process of tumor cell DNA replication and injury repair more dependent on the G2/M checkpoint. The Wee1 kinase, a key kinase of the G2/M checkpoint, is particularly important in the response to DNA damage in tumor cells, which makes the Wee1 kinase highly expressed in many tumors. After inhibiting the activity of Wee1 kinase, cdc2 does not generate phosphorylation inhibition, and DNA damage enters M phase without being repaired in time, so that genomic instability and chromosome deletion are caused, mitotic catastrophe is triggered, and tumor cell apoptosis is caused. On the other hand, the p53 gene of normal cells is normally functional, and can be examined for DNA damage at the check points of G1/S and G2/M phases, respectively, and when the activity of Wee1 kinase of normal cells is inhibited, its functional loss can be compensated by a p 53-dependent DNA damage repair mechanism, so that normal cells can survive. Therefore, inhibition of Wee1 kinase activity can kill p53 gene-deficient tumor cells selectively without affecting normal cells, and is an ideal tumor treatment approach.
Traditional chemotherapeutic drugs such as platinum, camptothecin and nucleoside analogues act on the DNA synthesis process to cause DNA synthesis damage, and radiotherapy can also cause direct damage of tumor cell DNA. Since Wee1 kinase is a key kinase in the DNA damage response pathway, inhibition of its activity could theoretically enhance the anti-tumor effects and radiation therapeutic effects of traditional chemotherapeutic drugs. In addition, PARP inhibitors are also active in DNA damage repair processes and in combination with Wee1 kinase inhibitors may enhance tumor therapy. On the other hand, the combination of other antitumor drugs (such as paclitaxel and monoclonal antitumor drugs) acting on non-DNA targets and Wee1 kinase inhibitor has been carried out in relevant clinical studies.
No reports are available in the literature on the combination of homoharringtonine and a Wee1 kinase inhibitor for treating leukemia.
Disclosure of Invention
The invention aims to solve the technical problems, provides a novel pharmaceutical composition for treating leukemia, and provides a brand new thought for clinically developing a combined administration scheme for treating leukemia.
The above object of the present invention is achieved by the following technical means.
The invention provides a pharmaceutical composition for treating leukemia, wherein the active ingredients in the pharmaceutical composition are homoharringtonine and a Wee1 kinase inhibitor.
Preferably, the Wee1 kinase inhibitor is selected from the following compounds:
(a) 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one;
(b) 2-ethyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one;
(c) 2-propyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one;
(d) 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one;
(e) 2-cyclopropyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one;
(f) 2-butyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one;
(g) 2-cyclobutyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one.
Preferably, the weight ratio of the harringtonine to the Wee1 kinase inhibitor in the pharmaceutical composition is 1:10-10: 1.
More preferably, the weight ratio of the harringtonine to the Wee1 kinase inhibitor in the pharmaceutical composition is 1:5-5: 1.
Further preferably, the weight ratio of harringtonine to Wee1 kinase inhibitor in the pharmaceutical composition is 4:1 or 3: 1.
Most preferably, the weight ratio of harringtonine to the Wee1 kinase inhibitor (a) 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one in the above pharmaceutical composition is 4: 1.
Most preferably, the weight ratio of harringtonine to the Wee1 kinase inhibitor (d) 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one in the above pharmaceutical composition is 3: 1.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
Preferably, the pharmaceutical composition is a solid preparation.
More preferably, the solid preparation is a tablet, a capsule or a lyophilized preparation.
The invention also provides application of the pharmaceutical composition in preparing a medicament for treating leukemia.
Preferably, the leukemia is chronic myelogenous leukemia or acute myelogenous leukemia.
The invention also provides application of the pharmaceutical composition in preparing a medicine for inhibiting K562 cell proliferation.
The invention also provides application of the pharmaceutical composition in preparing a medicament for inhibiting HL-60 cell proliferation.
The invention has the following beneficial effects:
the inventor finds in research that the combination of homoharringtonine and the Wee1 kinase inhibitor has a significant effect on treating leukemia. More surprisingly, the ratio of the two active ingredients in a proper weight range produces unexpected excellent effects for treating leukemia.
Detailed Description
The present invention will be further described with reference to the following examples, but the embodiments of the present invention are not limited thereto. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Example 1
1.6g of homoharringtonine, 0.4g of 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one and 1g of tartaric acid are respectively dissolved in a proper amount of hot water for injection, and the tartaric acid solution is slowly added into the homoharringtonine and the 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidine-3 (2H) -ketone mixed solution is continuously stirred to completely dissolve active ingredients, then 10g of lactose and 15g of mannitol are added, stirred and dissolved, the pH value is adjusted to 4 by 4% NaOH, water for injection is added to 1000ml, sterile filtration is carried out, filtrate is respectively filled into sterilized tubular penicillin bottles according to 1 ml/piece, the vials are placed into a freeze dryer to be dried for 24 hours, sterilized butyl rubber plugs are pressed, aluminum caps are rolled, inspection and packaging are carried out, and the compound preparation is obtained.
Example 2
1g homoharringtonine, 1g 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one and 1g tartaric acid are respectively dissolved in a proper amount of hot water for injection, and then the tartaric acid solution is slowly added into the homoharringtonine and the 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidine-3 (2H) -ketone mixed solution is continuously stirred to completely dissolve active ingredients, then 10g of lactose and 15g of mannitol are added, stirred and dissolved, the pH value is adjusted to 4 by 4% NaOH, water for injection is added to 1000ml, sterile filtration is carried out, filtrate is respectively filled into sterilized tubular penicillin bottles according to 1 ml/piece, the vials are placed into a freeze dryer to be dried for 24 hours, sterilized butyl rubber plugs are pressed, aluminum caps are rolled, inspection and packaging are carried out, and the compound preparation is obtained.
Example 3
1.8g of homoharringtonine, 0.2g of 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one and 1g of tartaric acid are respectively dissolved in a proper amount of hot water for injection, and then the tartaric acid solution is slowly added into the homoharringtonine and the 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidine-3 (2H) -ketone mixed solution is continuously stirred to completely dissolve active ingredients, then 10g of lactose and 15g of mannitol are added, stirred and dissolved, the pH value is adjusted to 4 by 4% NaOH, water for injection is added to 1000ml, sterile filtration is carried out, filtrate is respectively filled into sterilized tubular penicillin bottles according to 1 ml/piece, the vials are placed into a freeze dryer to be dried for 24 hours, sterilized butyl rubber plugs are pressed, aluminum caps are rolled, inspection and packaging are carried out, and the compound preparation is obtained.
Example 4
1.5g homoharringtonine, 0.5g 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one and 1g tartaric acid are respectively dissolved in a proper amount of hot water for injection, and then the tartaric acid solution is slowly added into the homoharringtonine and the 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidine-3 (2H) -ketone mixed solution is continuously stirred to completely dissolve active ingredients, then 10g of lactose and 15g of mannitol are added, stirred and dissolved, the pH value is adjusted to 4 by 4% NaOH, water for injection is added to 1000ml, sterile filtration is carried out, filtrate is respectively filled into sterilized tubular penicillin bottles according to 1 ml/piece, the vials are placed into a freeze dryer to be dried for 24 hours, sterilized butyl rubber plugs are pressed, aluminum caps are rolled, inspection and packaging are carried out, and the compound preparation is obtained.
Example 5
1g of homoharringtonine, 1g of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one and 1g of tartaric acid are respectively dissolved in a proper amount of water for hot injection, and then the tartaric acid solution is slowly added into the homoharringtonine and the 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidine-3 (2H) -ketone mixed solution is continuously stirred to completely dissolve active ingredients, then 10g of lactose and 15g of mannitol are added, stirred and dissolved, the pH value is adjusted to 4 by 4% NaOH, water for injection is added to 1000ml, sterile filtration is carried out, filtrate is respectively filled into sterilized tubular penicillin bottles according to 1 ml/piece, the vials are placed into a freeze dryer to be dried for 24 hours, sterilized butyl rubber plugs are pressed, aluminum caps are rolled, inspection and packaging are carried out, and the compound preparation is obtained.
Example 6
1.8g of homoharringtonine, 0.2g of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one and 1g of tartaric acid are respectively dissolved in a proper amount of hot water for injection, and then the tartaric acid solution is slowly added into the homoharringtonine and the 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidine-3 (2H) -ketone mixed solution is continuously stirred to completely dissolve active ingredients, then 10g of lactose and 15g of mannitol are added, stirred and dissolved, the pH value is adjusted to 4 by 4% NaOH, water for injection is added to 1000ml, sterile filtration is carried out, filtrate is respectively filled into sterilized tubular penicillin bottles according to 1 ml/piece, the vials are placed into a freeze dryer to be dried for 24 hours, sterilized butyl rubber plugs are pressed, aluminum caps are rolled, inspection and packaging are carried out, and the compound preparation is obtained.
Test example 1: inhibition of K562 cell proliferation by the pharmaceutical composition of the invention
1. Test method
(1) Cell culture and assay grouping
K562 cells were routinely cultured in RPMI 1640 medium containing 10% inactivated fetal bovine serum at 37 ℃ with 5% CO2And culturing in an incubator with saturated humidity. Liquid change and passage are carried out every two days, and cells in logarithmic growth phase are taken for subsequent experiments. During the test, the added drug concentrations of each test group are respectively as follows: homoharringtonine group and Wee1 kinase inhibitor group a (i.e., Wee1 kinase inhibitor (a), 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d]Pyrimidine-3 (2H) -one) drug concentrations were set to 2. mu. mol/L, combination 1 was 1.6. mu. mol/L homoharringtonine + 0.4. mu. mol/L Wee1 kinase inhibitor a (i.e., the concentration ratio of homoharringtonine to Wee1 kinase inhibitor a was 4:1), combination 2 was 1.8. mu. mol/L homoharringtonine + 0.2. mu. mol/L Wee1 kinase inhibitor a (i.e., the concentration ratio of homoharringtonine to Wee1 kinase inhibitor a was 9:1), combination 3 was 1. mu. mol/L homoharringtonine + 1. mu. mol/L Wee1 kinase inhibitor a (i.e., the concentration ratio of homoharringtonine to Wee1 kinase inhibitor a was 1:1), and cell growth status was observed after 24H and 48H incubation in each test group.
(2) Cell proliferation viability assay
Taking the cells of each experimental group, adjusting the number of the cells to 5 × 105Perml, inoculated in 96-well plates at 100. mu.l/well, 6 replicates per well, 37 ℃ with 5% CO2Incubated overnight under conditions, post-well10. mu.l of CCK-8 solution was added, incubation was continued in the incubator for 2 hours, and the absorbance value (A value) at a wavelength of 450nm of each well was measured with a microplate reader. The experiment was repeated 3 times, and the cell growth inhibition ratio (%) (1-average a value in experimental group/average a value in control group) × 100%.
2. Test results
The results of the experiments on inhibition of K562 cell proliferation in each experimental group are shown in table 1.
TABLE 1 Effect of the pharmaceutical compositions of the present invention on inhibiting K562 cell proliferation
Figure GDA0003016517310000121
Figure GDA0003016517310000131
The test results in Table 1 show that homoharringtonine and Wee1 kinase inhibitor a both have inhibition effect on the proliferation of K562 cells, and become more and more obvious with the time. It is noteworthy that homoharringtonine significantly enhanced the proliferation inhibitory effect on K562 cells when used in combination with Wee1 kinase inhibitor a, with the best inhibitory effect of combination group 1.
Test example 2: the pharmaceutical composition of the invention has the effect of inducing HL-60 cell apoptosis
1. Test method
(1) Cell culture and assay grouping
The cell culture solution is HL-60 cell trypan blue cultured by RPMI-1640 containing 10% calf serum, and the count is carried out after the staining, and the experiment is applicable to the case that the number of the dye-repellent living cells is more than or equal to 98%. Seeding of 24-well cell culture plates 1X 10 cells per well6HL-60 cells/mL, 5% CO at 37 ℃2Preculture for 24h under the condition. During the test, the added drug concentrations of each test group are respectively as follows: homoharringtonine group and Wee1 kinase inhibitor group d (i.e., Wee1 kinase inhibitor (d), 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d]Pyrimidine-3 (2H) -one) was set at 2. mu. mol/L in each caseCombination 1 was 1.5 μmol/L homoharringtonine +0.5 μmol/L Wee1 kinase inhibitor d (i.e., the concentration ratio of homoharringtonine to Wee1 kinase inhibitor d was 3:1), combination 2 was 1.8 μmol/L homoharringtonine +0.2 μmol/L Wee1 kinase inhibitor d (i.e., the concentration ratio of homoharringtonine to Wee1 kinase inhibitor d was 9:1), and combination 3 was 1 μmol/L homoharringtonine +1 μmol/L Wee1 kinase inhibitor d (i.e., the concentration ratio of homoharringtonine to Wee1 kinase inhibitor d was 1: 1). Each of the above test groups was tested at 37 ℃ with 5% CO2Culturing for 24h and 48h under the condition, collecting cells, washing with PBS for 2 times, resuspending, and adjusting cell concentration to 1 × 106mL, the above experiments were repeated 3 times.
(2) Apoptosis detection
By adopting an FIT C-annexin V/PI method, 100 mu L of cell suspension with different action time of each test group is taken, 10 mu L of FIT C-annexin V is added, incubation is carried out for 10min at 37 ℃, cells are resuspended after PBS washing, 50 mu L of PI stop solution is added, and detection is carried out by a flow cytometer. The HL-60 apoptosis rate of each test group acting for 24h and 48h is calculated.
2. Test results
The results of the experiments on the induction of HL-60 apoptosis in each experimental group are shown in Table 2.
TABLE 2 Effect of the pharmaceutical compositions of the present invention on inducing HL-60 apoptosis
Figure GDA0003016517310000141
The results of the experiments in Table 2 show that homoharringtonine and Wee1 kinase inhibitor d both have the effect of inducing apoptosis of HL-60 cells, and become more and more obvious with the time. It is noteworthy that homoharringtonine, when used in combination with Wee1 kinase inhibitor d, had a significantly enhanced induction of HL-60 apoptosis, with the best inhibitory effect of combination group 1.

Claims (6)

1. The pharmaceutical composition for treating leukemia is characterized in that the active ingredients in the pharmaceutical composition are homoharringtonine and a Wee1 kinase inhibitor, wherein the weight ratio of homoharringtonine to the Wee1 kinase inhibitor is 4:1 or 3:1, and the Wee1 kinase inhibitor is selected from the following compounds:
(a) 2-methyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one; or
(d) 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is a solid formulation.
4. The pharmaceutical composition of claim 3, wherein the solid formulation is a tablet, a capsule, or a lyophilized formulation.
5. Use of a pharmaceutical composition according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of leukemia.
6. The use according to claim 5, wherein the leukemia is chronic myelogenous leukemia or acute myelogenous leukemia.
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