CN112675197A - Pharmaceutical composition for driving tumor by Hedgehog signal pathway of children - Google Patents
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- CN112675197A CN112675197A CN202110087411.9A CN202110087411A CN112675197A CN 112675197 A CN112675197 A CN 112675197A CN 202110087411 A CN202110087411 A CN 202110087411A CN 112675197 A CN112675197 A CN 112675197A
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Abstract
The invention discloses a pharmaceutical composition for driving tumors by a Hedgehog signal pathway of children, which comprises the following components in parts by weight: 0.046 part of arsenic trioxide and 1-4500 parts of cyclophosphamide. The pharmaceutical composition provided by the invention is combined with therapeutic drugs such as arsenic trioxide and cyclophosphamide, has a good treatment effect on a tumor driven by a Hedgehog signal pathway of a child, and particularly has a good treatment effect on neuroblastoma, acinar rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing's sarcoma.
Description
Technical Field
The invention discloses a pharmaceutical composition for a Hedgehog signal path driving tumor of a child, in particular to a pharmaceutical composition for neuroblastoma, acinar rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing's sarcoma.
Background
The first discovery of the Hedgehog (HH) gene in Drosophila melanogaster was reported by the N ü sslein-Vollhard C team in 1980 in Nature, and the HH signal pathway mainly includes four parts, namely an HH signal polypeptide, a transmembrane protein Patched (PTCH) and Smoothened (SMO), and a nuclear transcription factor Gli 1/2/3. HH signal polypeptide is the initiation factor of the whole pathway, including Sonic hedgehog (SHH), Indian hedgehog (IHH) and Desert hedgehog (DHH); after different HH signal polypeptides are combined with a membrane protein receptor PTCH, the inhibition effect of PTCH molecules on a key membrane protein SMO of cell signal transduction is relieved, and the activation of SMO protein molecules can change the functional state of a nuclear transcription factor Gli in an HH signal path, so that the change of the transcription activity of downstream genes is controlled. In recent years, researches show that when the pathway components are abnormal, such as PTCH deletion or mutation, SMO mutation and the like, the pathway is abnormally activated, and the pathway is related to the occurrence, development and chemotherapy resistance of various malignant tumors. Abnormal activation of SMO and Gli1/2 of an HH signal pathway is a key factor for maintaining transformation and proliferation characteristics of tumor cells, and by taking the SMO and Gli1/2 in the HH signal pathway as targets, over-activation of the HH pathway is inhibited, and the proliferation of the tumor cells can be fundamentally inhibited; such tumors are called HH pathway-driven (HH pathway-driven) tumors, and in children HH pathway-driven tumors mainly include Neuroblastoma (NB), Alveolar Rhabdomyosarcoma (ARMS), Medulloblastoma (MB), osteosarcoma, ewing's sarcoma (EWS), and the like.
Neuroblastoma (NB) is the most common extracranial solid tumor of embryonic origin in children, originating from primitive neural crest cells, and may occur anywhere in the sympathetic nervous system. The morbidity of NB of children under 15 years old in the United states is 1.1/10 ten thousand, and accounts for 8-10% of malignant tumors of the children; in China, the incidence of NB is second only to lymphoid/hematopoietic malignancies, accounting for 16.8% of malignancies in children of 0-4 years of age. High-risk neuroblastoma (HR-NB) accounts for 40% of the total morbidity, distant metastasis easily occurs in the early stage, the malignancy degree is high, although an active combination treatment scheme is adopted, the complete remission rate of induced chemotherapy is about 45%, the five-year survival rate (OS) is still lower than 15%, and the 2-year recurrence rate reaches 80.0%.
Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma derived from the primitive mesodermal stromal tissue that differentiates into the striated muscle, occurring predominantly in children, the most common soft tissue sarcoma in children, with an annual incidence of about 4.6/million, accounting for about 3.5% to 4.5% of all malignancies in childhood. According to the classification of soft tissue and bone tumors established by the WHO in 2013, RMS can be classified pathologically into embryonal type (erm), acinar type RMS (arms), pleomorphic, and fusiform/sclerosing RMS. ARMS is the second largest class of RMS second only to ERMS, accounting for approximately 20% to 30% of all RMS; ARMS is far more invasive than ERMS, and the growth of the ARMS is rapid, the early transfer is easy to occur, the ARMS is easy to relapse and the ARMS is resistant to chemotherapy; this tissue type is of high risk RMS in the risk group of the International Rhabdomyosarcoma Study Group (IRSG), even without metastasis and other prognostic adverse factors. The ARMS treatment difficulty is high, even if comprehensive treatment means such as operation, radiotherapy, chemotherapy and the like are adopted, the 5-year survival rate is less than 30%, and the ARMS treatment difficulty and the prognosis are worse when the ARMS is generated at the position (such as a jaw face, a skull base and the like) which is difficult to be resected by the operation.
Medulloblastoma (MB) is a highly aggressive tumor that proliferates clonally from embryonic cells in the cerebellum, with an annual overall incidence of about 5/million, the most common type of malignant brain tumors in children, accounting for about 20% of all brain tumors, and over one-third of all children dying within 5 years after diagnosis. MB can be classified into the following molecular pathological types: SHH type, WNT type, group3 (group3) and group4 (group 4). SHH type MB is good for infants, accounts for 25% to 30% of all MBs, is generally high in prognosis, is associated with TP53 mutation and MYCN amplification, is a very high risk group, has poor prognosis, and lacks an effective treatment method. The current first-line treatment scheme of MB sick children is to receive chemotherapy after operation and radiation, and the heterogeneity of different types of MB prognosis is large; according to DNA methylation typing, the SHH alpha subtype is often combined with TP53 mutation or MYCN and GLI2 amplification, the overall survival rate of the SHH beta subtype is low, and the SHH beta subtype has frequent metastasis. Previous clinical trials demonstrated that use of SMO antagonists improved the progression free survival time (PFS) of SHH-type MB with an Objective Remission Rate (ORR) of about 37-55%, but over time, many patients treated with SMO inhibitors were susceptible to development of therapeutic resistance leading to tumor recurrence.
Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, and the annual total incidence rate is about 3-11/million; in the past, amputation is the standard treatment method, the 5-year survival rate is 20%, and with the application of the neoadjuvant chemotherapy combined limb protection surgery, the 5-year OS of osteosarcoma is improved to 60-75%; however, 15% -30% of children had developed metastases at the initial visit, and despite intensive chemotherapy, the OS in these children was not significantly improved, with only 20-30% of OS for long periods. Clinical researches show that the total effective rate of the drug for treating the patient with osteosarcoma lung metastasis by using ATO, gemcitabine and docetaxel in combination can reach 76.9%.
Ewing sarcoma (Ewing sarcoma) is a highly aggressive tumor of bone and soft tissue, highly developed in children and adolescents, with a second-only incidence of osteosarcoma in malignant bone tumors in children, an annual incidence of about 4.5 cases/million, and a peak incidence of 11 cases/million at 12 years of age; through comprehensive treatment such as high-intensity systemic chemotherapy, operation, local radiotherapy and the like, the survival rate of the low-risk children patients is more than 70%, and the EFS and OS of the widely metastatic high-risk cases are only about 20%; even though high-risk EWS responds well to intensive therapy in the early stage of onset, OS is almost zero in 5 years of recurrence.
Disclosure of Invention
The present invention aims to overcome the problems of the prior art and provide a pharmaceutical composition capable of effectively treating a Hedgehog signaling pathway-driven tumor in children. Meanwhile, the invention also provides application of the pharmaceutical composition in preparing a medicine for treating the tumor driven by the Hedgehog signal pathway of the children.
In order to achieve the purpose, the invention adopts the technical scheme that: a pharmaceutical composition for driving tumors through a Hedgehog signal pathway in children comprises the following components in parts by weight: 0.046 part of arsenic trioxide and 1-4500 parts of cyclophosphamide.
Through a large amount of literature research and experiments, the inventor of the application finds that the pharmaceutical composition has a good treatment effect on the Hedgehog signal channel-driven tumor of the children by adopting arsenic trioxide and combining with a chemotherapeutic drug comprising cyclophosphamide and the like, and provides a new drug selection and a new method for treating the Hedgehog signal channel-driven tumor of the children.
As a preferred embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 1800-4500 parts of cyclophosphamide, 51-99 parts of pyran doxorubicin, 0.019-2 parts of vincristine, 140-260 parts of cisplatin, 420-780 parts of etoposide and 4.2-7.8 parts of topotecan; as a more preferable embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 3000 parts of cyclophosphamide, 75 parts of pyran doxorubicin, 2 parts of vincristine, 200 parts of cisplatin, 600 parts of etoposide and 6 parts of topotecan. The inventor of the present application has found through experimental studies that, when the pharmaceutical composition is selected and used in the above-mentioned selection and dosage, the pharmaceutical composition has a good therapeutic effect on neuroblastoma, that is, the arsenic trioxide is used in combination with the chemotherapeutic agent selected and used in the above-mentioned selection and dosage, so that the pharmaceutical composition has a good therapeutic effect on neuroblastoma. In specific use, the dosage and the use method of the pharmaceutical composition are as follows: arsenic trioxide chemotherapy is given for two days, and the combination with the conventional chemotherapy scheme of the other drugs is started from the third day, wherein each course of treatment uses arsenic trioxide for 10 days, and the chemotherapy lasts for 8-9 courses. The dosage of the arsenic trioxide is 0.16mg/kg. d, 5% GS or 0.9% NS 250-500 ml is added, ivdrivp is added, and PI is more than 8 h.
Specifically, when the pharmaceutical composition is used for treating Neuroblastoma (NB), the dosage of each component in the pharmaceutical composition is as follows: arsenic trioxide 0.16mg/kg. d.x 10d, cyclophosphamide (900-1500) mg/m2D x (2-3) d, pyrane doxorubicin (17-33) mg/m2dX 3d, vincristine (0.022 mg/kg.d-0.67 mg/m)2D) x 3d, cis-platinum (35-45) mg/m2dX 4d, etoposide (140-260) mg/m2Dx3 d, topotecan (1.4-2.6) mg/m2.d×3d。
In addition, the inventor of the present invention found in practical studies that when the pharmaceutical composition of the present invention is used for treating neuroblastoma, the cell cycle of Neuroblastoma (NB) is blocked at G2/M by first administering arsenic trioxide, and then other chemotherapeutic drugs such as cyclophosphamide are added, the NB cell killing effect is better than that of the simultaneous administration.
As a preferred embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 1.03-1200 parts of cyclophosphamide, 0.025-1.5 parts of vincristine and 0.0007-0.0013 part of actinomycin. The inventor of the present application has found through experimental studies that when the pharmaceutical composition adopts the selection and the dosage as described above, the pharmaceutical composition has a better therapeutic effect on acinar-type rhabdomyosarcoma. Namely, the arsenic trioxide is combined with the chemotherapeutics with the drug selection and dosage for use, and has better treatment effect on acinar rhabdomyosarcoma. In specific use, the dosage and the use method of the pharmaceutical composition are as follows: arsenic trioxide chemotherapy is given for two days, and the combination with the conventional chemotherapy scheme of the other drugs is started from the third day, wherein each course of treatment uses arsenic trioxide for 10 days, and the chemotherapy lasts for 8-9 courses. The dosage of the arsenic trioxide is 0.16mg/kg. d, 5% GS or 0.9% NS 250-500 ml is added, ivdrivp is added, and PI is more than 8 h.
Specifically, when the pharmaceutical composition is used for treating alveolar rhabdomyosarcoma, the dosage of each component in the pharmaceutical composition is as follows: arsenic trioxide 0.16mg/kg. d.times.10 d, cyclophosphamide 36-1200 mg/m2dX 1d, vincristine (0.025-1.5) mg/m2dX1 d, actinomycin (0.025-0.045) mg/kg X1 d.
As a preferred embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 500-1000 parts of cyclophosphamide, 210-390 parts of etoposide and 60-120 parts of cisplatin. As a more preferable embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 800 parts of cyclophosphamide, 300 parts of etoposide and 90 parts of cisplatin. The inventor of the present application has found through experimental studies that, when the pharmaceutical composition is selected and used in the above-mentioned selection and dosage, the pharmaceutical composition has a good therapeutic effect on medulloblastoma, that is, the arsenic trioxide is used in combination with the chemotherapeutic agent selected and used in the above-mentioned selection and dosage, so that the pharmaceutical composition has a good therapeutic effect on medulloblastoma. In specific use, the dosage and the use method of the pharmaceutical composition are as follows: arsenic trioxide chemotherapy is given for two days, and the combination with the conventional chemotherapy scheme of the other drugs is started from the third day, wherein each course of treatment uses arsenic trioxide for 10 days, and the chemotherapy lasts for 8-9 courses. The dosage of the arsenic trioxide is 0.16mg/kg. d, 5% GS or 0.9% NS 250-500 ml is added, ivdrivp is added, and PI is more than 8 h.
Specifically, when the pharmaceutical composition is used for treating medulloblastoma, the dosage of each component in the pharmaceutical composition is 0.16mg/kg of arsenic trioxide, multiplied by 10d, and 500-1000 mg/m of cyclophosphamide2dX 1d, etoposide (70-130) mg/m2dX 3d, cisplatin (60-120) mg/m2.d×1d。
As a preferred embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 840-1560 parts of cyclophosphamide, 800-1200 parts of methotrexate, 60-120 parts of pyran doxorubicin, 0.8-1.6 parts of actinomycin D, 85-155 parts of cisplatin and 20-40 parts of bleomycin; as a more preferable embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components by weight: 0.046 part of arsenic trioxide, 1200 parts of cyclophosphamide, 900 parts of methotrexate, 90 parts of pyran doxorubicin, 1.2 parts of actinomycin D, 120 parts of cisplatin and 30 parts of bleomycin. The inventor of the present application finds, through experimental studies, that when the pharmaceutical composition adopts the selection and the dosage as described above, the pharmaceutical composition has a better therapeutic effect on osteosarcoma, that is, the arsenic trioxide is used in combination with the chemotherapeutic drug selected and used in the pharmaceutical composition, so that the pharmaceutical composition has a better therapeutic effect on osteosarcoma. In specific use, the dosage and the use method of the pharmaceutical composition are as follows: arsenic trioxide chemotherapy is given for two days, and the combination with the conventional chemotherapy scheme of the other drugs is started from the third day, wherein each course of treatment uses arsenic trioxide for 10 days, and the chemotherapy lasts for 8-9 courses. The dosage of the arsenic trioxide is 0.16mg/kg. d, 5% GS or 0.9% NS 250-500 ml is added, ivdrivp is added, and PI is more than 8 h.
In particular to the application of the pharmaceutical compositionWhen the osteosarcoma is treated, the dosage of each component in the pharmaceutical composition is respectively as follows: arsenic trioxide 0.16mg/kg. d.times.10 d, cyclophosphamide (420-780) mg/m2dX 2d, methotrexate (800-1200) mg/m2dX 1d, pyrane doxorubicin (20-40) mg/m2dX 3D, actinomycin D (0.4-0.8) mg/m2dX 2d, cisplatin (85-155) mg/m2dX 1d, bleomycin (10-20) mg/m2.d×2d。
As a preferred embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components by weight: 0.046 part of arsenic trioxide, 1700-3000 parts of cyclophosphamide, 51-99 parts of pyran doxorubicin, 1.5-2 parts of vincristine, 4800-11500 parts of ifosfamide, 350-650 parts of etoposide and 0.85-1.6 parts of actinomycin D. As a more preferable embodiment of the pharmaceutical composition for driving tumor by Hedgehog signaling pathway in children of the present invention, the pharmaceutical composition comprises the following components by weight: 0.046 part of arsenic trioxide, 2400 parts of cyclophosphamide, 75 parts of pyran doxorubicin, 1.8 parts of vincristine, 8100 parts of ifosfamide, 500 parts of etoposide and 1.25 parts of actinomycin D. The inventor of the present application has found through experimental studies that when the pharmaceutical composition adopts the selection and the dosage as described above, the pharmaceutical composition has a better therapeutic effect on ewing sarcoma, i.e. the arsenic trioxide is used in combination with the chemotherapeutic drug selected and used in the pharmaceutical composition, so that the pharmaceutical composition has a better therapeutic effect on ewing sarcoma. In specific use, the dosage and the use method of the pharmaceutical composition are as follows: arsenic trioxide chemotherapy is given for two days, and the combination with the conventional chemotherapy scheme of the other drugs is started from the third day, wherein each course of treatment uses arsenic trioxide for 10 days, and the chemotherapy lasts for 8-9 courses. The dosage of the arsenic trioxide is 0.16mg/kg. d, 5% GS or 0.9% NS 250-500 ml is added, ivdrivp is added, and PI is more than 8 h.
Specifically, when the pharmaceutical composition is used for treating ewing's sarcoma, the dosage of each component in the pharmaceutical composition is as follows: arsenic trioxide 0.16mg/kg. d.x 10d, cyclophosphamide (850-1500) mg/m2D.times.2 d, A.pyransPlain (17-33) mg/m2dX 3d, vincristine (1.5-2) mg/m2dX 1d, ifosfamide (1200-2300) mg/m2D x (4-5) d, etoposide (70-130) mg/m2dX 5D, actinomycin D (0.85-1.6) mg/m2.d×1d。
As a preferable embodiment of the pharmaceutical composition for driving the tumor by the Hedgehog signal pathway of the children, the pharmaceutical composition further comprises 500-1000 parts by weight of vitamin C. The inventor of the application finds that after vitamin C is further added into the pharmaceutical composition, the killing effect of arsenic trioxide on tumor cells can be obviously enhanced, the arsenic trioxide and the tumor cells can play a synergistic effect in ways of inducing cytotoxic oxidative stress, activating caspase-3 and caspase-1 to trigger apoptosis and the like, and meanwhile, the addition of the vitamin C can reduce the blood toxicity, heart toxicity and liver toxicity caused by arsenic. When the vitamin C is used specifically, the vitamin C and the arsenic trioxide are used synchronously, so that adverse reactions of patients are reduced, the in-vivo curative effect of the patients is improved, and the dosage of the vitamin C is 0.5-1.0 g/m2D, adding 100-250 ml of 5% GS, and synchronizing the obtained product with the arsenic trioxide by ivdrip.
As a preferred embodiment of the pharmaceutical composition for the children Hedgehog signaling pathway-driven tumors according to the present invention, the children Hedgehog signaling pathway-driven tumors include neuroblastoma, alveolar rhabdomyosarcoma, medulloblastoma, osteosarcoma, ewing's sarcoma. The inventor of the present application finds that the pharmaceutical composition has better therapeutic effect on neuroblastoma acinar rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing sarcoma, but the children Hedgehog signaling pathway driving tumor includes but is not limited to neuroblastoma, acinar rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing sarcoma.
In addition, the invention also provides application of the pharmaceutical composition in preparation of a medicine for treating the Hedgehog signal pathway driven tumor of the children. The pharmaceutical composition can effectively treat the Hedgehog signal pathway-driven tumor of the child, and particularly has a good treatment effect on neuroblastoma, acinar rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing sarcoma.
The HH pathway drives the abnormal expression of HH signaling pathway components prevalent in the clinical pathology of tumors or tumor cell lines, and aberrant activation of this pathway is associated with the development, progression, and resistance to chemotherapy of a variety of malignancies. Arsenic Trioxide (ATO) specifically inhibits Gli protein, the downstream most component of the HH signal pathway, to inactivate the HH signal pathway, and besides ATO can kill tumor cells by inducing them to produce excessive ROS, destroying mitochondrial membrane potential, regulating calcium channels, blocking cell cycle, and the like. The ATO does not increase the expression of drug-resistant protein while killing tumor cells, and can enhance the anti-tumor effect of chemotherapeutic drugs and reduce the incidence rate of drug resistance by combining with common clinical chemotherapeutic drugs; is a new direction for clinically treating the Hedgehog signal path driving tumor of the refractory children.
The pharmaceutical composition provided by the invention is used by combining arsenic trioxide and cyclophosphamide and other therapeutic drugs, has a good treatment effect on tumors driven by a Hedgehog signal pathway of children, and particularly has a good treatment effect on neuroblastoma, acinar rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing's sarcoma. The pharmaceutical composition provided by the invention provides a new drug selection and a new treatment method for treating the pathway-driven tumors of children, particularly neuroblastoma, acinar-type rhabdomyosarcoma, medulloblastoma, osteosarcoma and Ewing's sarcoma.
Drawings
FIG. 1 is a graph comparing the effect of different drugs on the proliferation activity of DAOY cells in example 3 of the present invention.
FIG. 2 is a graph comparing the effect of different drugs on the proliferation activity of Saos2 cells in example 4 of the present invention.
FIG. 3 is a graph showing the effect of different drugs on the proliferation activity of RD-ES cells in example 5 of the present invention.
FIG. 4 is a comparative graph showing that vitamin C in the pharmaceutical composition of the present invention can synergistically kill tumor cells.
FIG. 5 is a comparative graph showing that vitamin C in the pharmaceutical composition of the present invention can reduce cardiotoxicity caused by arsenic trioxide.
FIG. 6 is a comparative graph showing that vitamin C in the pharmaceutical composition of the present invention can reduce the hepatotoxicity caused by arsenic trioxide.
FIG. 7 is a comparative graph showing that vitamin C in the pharmaceutical composition of the present invention can reduce the toxicity of blood system caused by arsenic trioxide.
Detailed Description
To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
Test of therapeutic Effect of one embodiment of the pharmaceutical composition of the present invention on Neuroblastoma (NB)
The pharmaceutical composition described in this example comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 3000 parts of cyclophosphamide, 75 parts of pyran doxorubicin, 2 parts of vincristine, 200 parts of cisplatin, 600 parts of etoposide and 6 parts of topotecan. When in specific use, the dosage of each component in the pharmaceutical composition is respectively as follows: arsenic trioxide 0.16mg/kg. d.x 10d, cyclophosphamide (900-1500) mg/m2D x (2-3) d, pyrane doxorubicin (17-33) mg/m2dX 3d, vincristine (0.022 mg/kg.d-0.67 mg/m)2D) x 3d, cis-platinum (35-45) mg/m2dX 4d, etoposide (140-260) mg/m2Dx3 d, topotecan (1.4-2.6) mg/m2.d×3d。
The test method comprises the following steps: the study was a non-randomized, multicenter, open clinical trial enrolled in new onset NB patients 14 years and younger and who volunteered to receive traditional chemotherapy or ATO combination chemotherapy. In this study, a total of 53 patients were currently enrolled, including 13 patients who received indirect chemotherapy from 1/2011 to 9/31/2019 (control group) and 40 patients who received indirect arsenic trioxide-combined chemotherapy from 1/2018 to 8/31/2020 (test group). We used the Objective Remission Rate (ORR) of two groups of infants 4 weeks after induction of chemotherapy as the primary observation, while monitoring and grading adverse events occurring during chemotherapy. Statistical processing was analyzed using SPSS 25.0 software, and the counting data between the two groups (positive rate of amplification of N-myc gene, rate of induction remission, incidence of adverse events, etc.) were examined using Fisher's exact probability method, and the counting data between the two groups (age composition between the two groups) were examined non-parametrically.
And (3) test results: 40 infants (Table 1) of the study group who received Arsenic Trioxide (ATO) combined chemotherapy in the cooperative group unit from 2018, month 1 to 2020, month 8, and day 31 were collected, wherein 21 (52.5%) of males, 19 (47.5%) of females, 3.8(0.7-16.4) of median age, all were confirmed by examination of pathological, imaging, and tumor markers, wherein the initial site was the most of the infants with adrenal gland (19, 47.5%), followed by retroperitoneum (12, 30.0%), mediastinum (6, 15.0%), bone (2, 5.0%), and neck (1, 2.5%). In 24 cases (60.0%) of the children with the onset of the disease, bone marrow metastasis was confirmed by bone marrow puncture, and in 34 cases (85.0%) of the children, amplification of the N-myc gene in the bone marrow or the surgical specimen was detected, and 16 cases (40.0%) were positive. 13 control infants who received conventional chemotherapy in the pediatrics of the university of Zhongshan, Sunzhai and Hakka disease (as shown in Table 1 below) at 1/2019/9/31 were collected, wherein 10 (79.6%) had males, 3 (23.1%) had females, 4.0(0.1-8.0) had the median age, the first site was the most children of the adrenal gland (9, 69.2%), the second was bone (3, 23.1%) and retroperitoneum (1, 7.7%). In the onset of the disease, 9 (69.2%) of the children had bone marrow metastases, and 5 (38.4%) of the children had been tested for amplification of the N-myc gene in bone marrow or surgical specimens, of which 4 (30.7%) were positive. In the evaluation of the efficacy after the end of induction, 13 infants in the control group were in Complete Remission (CR) in 3 (23.1%), Partial Remission (PR) in 3 (23.1%), and disease Progression (PD) in 7 (53.8%), and in total 6 cases induced remission was achieved with an ORR of 46.2%; in 40 children, 17 (42.5%) CR, 18 (45.0%) PR, 5 (12.5%) PD, 35 of the patients achieved induction remission with an ORR of 87.5%, which was significantly higher than the control (as shown in table 2 below) and statistically significant (87.5% vs 46.2%, P ═ 0.005).
TABLE 1 case characteristics of the test and control groups
TABLE 2 comparison of induction effects of test group and control group
From the test results, the induction remission rate of 4/M-stage high-risk neuroblastoma children can be remarkably improved by combining Arsenic Trioxide (ATO) with cyclophosphamide and other chemotherapies, so that the chance of subsequent transplantation can be won for more children, and the long-term survival rate of the children can be prolonged. We found in our studies that Arsenic Trioxide (ATO) in combination with a pharmacotherapeutic regimen such as cyclophosphamide is well tolerated in children patients, with only 5 cases showing slightly reversible cardiotoxicity. Compared with anti-GD 2 treatment, MIBG and proton treatment, the treatment scheme reduces the treatment cost to the maximum extent, greatly reduces the burden of families and society of patients while improving the induction remission rate, and provides the possibility of long-term survival for more children.
Example 2
Test on therapeutic Effect of one embodiment of the pharmaceutical composition of the present invention on Alveolar Rhabdomyosarcoma (ARMS) of childhood
The pharmaceutical composition described in this example comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 1.03-1200 parts of cyclophosphamide, 0.025-1.5 parts of vincristine and 0.0007-0.0013 part of actinomycin. When in specific use, the dosage of each component in the pharmaceutical composition is respectively as follows: arsenic trioxide 0.16mg/kg. d.times.10 d, cyclophosphamide 36-1200 mg/m2dX 1d, vincristine (0.025-1.5) mg/m2dX1 d, actinomycin (0.025-0.045) mg/kg X1 d.
The test method comprises the following steps: this example explores the clinical efficacy and safety of Arsenic Trioxide (ATO) in combination with classical VAC regimen chemotherapy in treating ARMS by developing a single-unit, multicenter, prospective clinical trial study, and search for new therapeutic approaches and options for AMRS patients. The method comprises the steps of enabling ARMS (acute respiratory syndrome) children with age less than or equal to 14 years to enter a group, carrying out chemotherapy on the ARMS children with age less than or equal to 8 years by using an Arsenic Trioxide (ATO) and VAC (vacuum alternating current) combination scheme in a classical RMS-CQ-2009 scheme for 8 courses of treatment, comparing the chemotherapy remission rate of the ARMS after induction chemotherapy of the single group of clinical tests and ARMS treatment reported in the literature by using an induced remission rate (ORR) of the Arsenic Trioxide (ATO) and VAC combination as a main observation index and using the current integral treatment level of the ARMS as an external control, and evaluating the overall survival rate (OS) and the non-event survival rate (EFS) of the ARMS by using the single group of clinical tests, and simultaneously monitoring and grading adverse reactions occurring during the chemotherapy to evaluate the curative effect and.
And (3) test results: at present, 7 patients with ARMS (shown in Table 3) are grouped, the infants are 3-14 years old (the median age is 5 years old), wherein the male/female ratio is 4/3, and the primary tumor parts comprise 3 limbs, 2 parameninges, 1 head and neck part and 1 abdominopelvic cavity; the risk groups comprise 5 cases of medium-risk groups and 2 cases of high-risk groups. A total of 3 patients with primary ARMS were treated, with 2 Complete Remissions (CR) and 1 Partial Remissions (PR) after 8 courses of ATO combined chemotherapy. There were 4 relapsed refractory ARMS, and tumor shrinkage was seen following chemotherapy with Arsenic Trioxide (ATO) in combination with chemotherapy, of which CR 3 and Stable Disease (SD) 1. Currently 3 of 7 patients survive disease-free, 3 with tumor (1 of them is a relapsing patient), and 1 relapsing death. Adverse chemotherapy reactions of 7 children patients mainly include myelosuppression (7/7, grade 2-4), emesis (grade 7/7, grade 1-3), transaminase increase (grade 1/7, grade 1), and dyspnea (grade 1/7, grade 3), wherein dyspnea is manifested by chest distress, short breath, no obvious organic lesion, and improvement after drug withdrawal.
TABLE 37 ARMS pediatric cases characteristics
From the test results, Arsenic Trioxide (ATO) combined with VAC regimen chemotherapy is primarily effective for primary treatment and relapse refractory children ARMS patients, the survival time of the patients can be prolonged, the adverse reaction is light, and the tolerance of the patients is good.
Example 3
Test of therapeutic Effect of one embodiment of the pharmaceutical composition of the present invention on medulloblastoma
The pharmaceutical composition described in this example comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 800 parts of cyclophosphamide and 300 parts of etoposide. When in specific use, the dosage of each component in the pharmaceutical composition is respectively as follows: arsenic trioxide 0.16mg/kg. d.x 10d, cyclophosphamide (500-1000) mg/m2dX 1d, etoposide (70-130) mg/m2dX 3d, cisplatin (60-120) mg/m2.d×1d。
The test method comprises the following steps: in this example, a blank control group, an Arsenic Trioxide (ATO) monotherapy group, a Cyclophosphamide (CTX) monotherapy group, and a combined therapy of arsenic trioxide and cyclophosphamide (ATO + CTX) were respectively provided, and the effects of each group on the proliferation activity of DAOY cells are shown in table 4 and fig. 1.
TABLE 4 Effect of different drugs on the proliferative Activity of DAOY cells
| OD average value | Difference P value between control group | |
| Blank control group | 0.0427±0.0082 | |
| ATO (4uM) single drug group | 0.0256±0.0047 | 0.028* |
| CTX (20uM) single drug group | 0.0305±0.0049 | 0.124 |
| ATO + CTX combined medicine adding set | 0.0204±0.0046 | 0.007* |
| P value difference between ATO + CTX groups | ||
| ATO (4uM) single drug group | 0.0256±0.0047 | 0.705 |
| CTX (0.2mM) single drug group | 0.0305±0.0049 | 0.219 |
*P<0.05，**P<0.01。
As can be seen from Table 4 and FIG. 1, the proliferation activity of DAOY cells of the medulloblastoma cell line is detected by an MTT method, and the DAOY cell activity can be obviously inhibited by 48h of single-drug treatment of ATO, and the inhibition effect of ATO and CTX on the DAOY cells is stronger.
Example 4
Test of therapeutic Effect of one embodiment of the pharmaceutical composition of the present invention on osteosarcoma
The pharmaceutical composition described in this example comprises the following components in parts by weight: 0.046 part of arsenic trioxide, 1200 parts of cyclophosphamide, 900 parts of methotrexate, 90 parts of pyran doxorubicin, 1.2 parts of actinomycin D, 120 parts of cisplatin and 30 parts of bleomycin. When in specific use, the dosage of each component in the pharmaceutical composition is respectively as follows: arsenic trioxide 0.16mg/kg. d.times.10 d, cyclophosphamide (420-780) mg/m2dX 2d, methotrexate (800-1200) mg/m2dX 1d, pyrane doxorubicin (20-40) mg/m2dX 3D, actinomycin D (0.4-0.8) mg/m2dX 2d, cisplatin (85-155) mg/m2dX 1d, bleomycin (10-20) mg/m2.d×2d。
The test method comprises the following steps: in this example, a blank control group, an Arsenic Trioxide (ATO) monotherapy group, a Cyclophosphamide (CTX) monotherapy group, and a combined therapy group of arsenic trioxide and cyclophosphamide (ATO + CTX) were provided, and the effects of each group on the cell proliferation activity of Saos2 are shown in table 5 and fig. 2.
TABLE 5 Effect of different drugs on the cell proliferation Activity of Saos2
*P<0.05，**P<0.01
As can be seen from Table 5 and accompanying figure 2, the WST-1 method is used to detect the cell proliferation activity of the human osteosarcoma cell line Saos2, and the results show that both ATO and CTX can obviously inhibit the cell proliferation of Saos2 after 24h treatment, and the combination of the two drugs can enhance the inhibition effect of a single drug.
Example 5
Test of the treatment effect of one embodiment of the pharmaceutical composition on Ewing's tumor
The pharmaceutical composition comprises, by weight, 0.046 part of arsenic trioxide, 2400 parts of cyclophosphamide, 75 parts of doxorubicin pyrane, 1.8 parts of vincristine, 8100 parts of ifosfamide, 500 parts of etoposide and 1.25 parts of actinomycin D. When in specific use, the dosage of each component in the pharmaceutical composition is respectively as follows: arsenic trioxide 0.16mg/kg. d.x 10d. Cyclophosphamide (850-1500) mg/m2dX 2d, pyrane doxorubicin (17-33) mg/m2dX 3d, vincristine (1.5-2) mg/m2dX 1d, ifosfamide (1200-2300) mg/m2D x (4-5) d, etoposide (70-130) mg/m2dX 5D, actinomycin D (0.85-1.6) mg/m2.d×1d。
The test method comprises the following steps: in this example, a blank control group, an Arsenic Trioxide (ATO) monotherapy group, a Cyclophosphamide (CTX) monotherapy group, and a combined medication group of arsenic trioxide and cyclophosphamide (ATO + CTX) were provided, and the effects of each group on the proliferation activity of RD-ES cells are shown in table 6 and fig. 3.
TABLE 6 Effect of different drugs on the proliferative Activity of RD-ES cells
*P<0.05，**P<0.01。
As can be seen from Table 6 and FIG. 3, the proliferation activity of the E.Ewing sarcoma cell line RD-ES cells was measured by MTT method, and it was found that the single-drug treatment of ATO for 48h significantly inhibited the proliferation of RD-ES cells, and that the combination of ATO and CTX enhanced the inhibitory effect of ATO on RD-ES cells.
Example 3
Synergistic effect test and side effect reduction test of vitamin C in pharmaceutical composition
After the vitamin C is added into the pharmaceutical composition, the combination of the vitamin C and the arsenic trioxide can obviously enhance the tumor cell killing effect of the arsenic trioxide, the vitamin C and the arsenic trioxide play a synergistic effect in a way of inducing cytotoxic oxidative stress, activating caspase-3 and caspase-1 to trigger apoptosis and the like, and meanwhile, the addition of the vitamin C can reduce the blood toxicity, heart toxicity and liver toxicity caused by arsenic agents.
1. The test of the synergistic effect of vitamin C and arsenic trioxide in the pharmaceutical composition
The test method comprises the following steps: pancreatic cancer cells are used, a blank control group, an ATO drug group, an ATO + vitamin C drug group and an ATO + isovitamin C drug group are set, after the drugs act for 48 hours, the apoptosis rate and the cell activity are measured by a flow cytometer, and the influence of the added vitamin C or the isomer isovitamin C on the ATO anti-tumor effect is verified.
The test results are shown in figure 4, and it can be seen from figure 4 that after arsenic trioxide and vitamin C are used in combination, the tumor cell death rate is increased by 45.7%, which is significantly higher than the cytotoxicity effect (29.7%) of two drugs, and the effect is shown as a synergistic effect.
2. Test for the Effect of vitamin C in the pharmaceutical composition of the present invention on reduction of side effects
The method comprises the steps of using normal myocardial cells, setting a blank control group, an ATO drug group and an ATO + vitamin C drug group, detecting the content of Reactive Oxygen Species (ROS) in cells by using a fluorescence immunoassay, and verifying the influence of the added vitamin C on the generation of ROS in ATO-induced cells. The test results are shown in FIG. 5.
As shown in the attached figure 5, vitamin C can reduce ROS in the myocardial cells after arsenic trioxide treatment, and protect the myocardial cells from oxidative damage.
Using 18 adult rats, the rats were randomly divided into 3 groups under strict control of growth environment and diet identity: the control group (single diet), the ATO group (oral arsenic agent) and the ATO + vitamin C group (oral arsenic agent + vitamin C) are killed after observing for 30 days, the liver tissues of the rats are taken for histopathological examination, the peripheral blood is taken for detecting the levels of red blood cells, white blood cells and hemoglobin, and the influence of the vitamin C on the liver injury and the hemogram inhibition of the rats caused by ATO is verified. The test results are shown in FIGS. 6 and 7.
In FIG. 6, FIG. A, B shows normal liver tissue in mice; C. graph D shows that mouse liver tissue hepatocyte necrosis and degenerative change occur after arsenic trioxide acts on the mouse liver tissue; E. panel F shows partial repair of liver tissue following vitamin C supplementation. As shown in FIG. 6, vitamin C can reduce the liver toxicity caused by arsenic trioxide.
As shown in the attached figure 7, after arsenic trioxide treatment, the red blood cells, white blood cells and hemoglobin of mice are obviously reduced, and the hemogram of the mice can be partially recovered after vitamin C is supplemented, namely the vitamin C can reduce the toxicity of the blood system caused by arsenic trioxide.
In conclusion, vitamin C can reduce the toxicity of the heart, liver and blood system caused by arsenic trioxide.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
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