CN114288303B

CN114288303B - Antineoplastic pharmaceutical composition containing piperazines and application thereof

Info

Publication number: CN114288303B
Application number: CN202210144360.3A
Authority: CN
Inventors: 岳欣; 彭振维; 王雪涔; 陈勇
Original assignee: First Affiliated Hospital of Sun Yat Sen University
Current assignee: First Affiliated Hospital of Sun Yat Sen University
Priority date: 2022-02-17
Filing date: 2022-02-17
Publication date: 2022-09-13
Anticipated expiration: 2042-02-17
Also published as: CN114288303A

Abstract

The invention provides an anti-tumor pharmaceutical composition containing piperazines and application thereof. According to the invention, on the basis of the original sunitinib, the peracetylazine is further added, so that a very obvious synergistic effect can be generated, the proliferation of cancer cells is obviously inhibited, and the sunitinib has very obvious killing efficiency on the cancer cells. The pharmaceutical composition provided by the invention can obviously improve the problems of serious drug resistance caused by the simple adoption of sunitinib in the prior art and the treatment effect obviously reduced when the conventional use amount of sunitinib is reduced, greatly improves the clinical treatment efficiency, reduces the toxic and side effects of patients, provides a new scheme for clinical treatment, and has very wide market prospect and extremely important social significance.

Description

Antineoplastic pharmaceutical composition containing piperazines and application thereof

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to an antitumor drug composition containing piperazines and application thereof.

Background

Renal cancer (Kidney cancer) is one of the three major malignancies of the urinary system. Among them, Renal Cell Carcinoma (RCC) is a malignant tumor derived from tubular epithelial cells, accounts for 90% of all kidney cancers, and is the most common type of Renal malignancy. Due to the application of screening means such as ultrasound and CT and the enhancement of health physical examination consciousness of people, the incidence rate of kidney cancer is increased, but early kidney cancer is treated by radical surgery, and the prognosis is better. The overall 5-year survival rate of kidney cancer is 49%, but the prognosis is very related to the clinical stage and pathological grade of the disease, and the prognosis of patients in clinical stage III and IV or pathological grade 3 and 4 is very poor.

Sunitinib (Sunitinib, trade name: Sutent) is an oral small molecule multi-target Receptor Tyrosine Kinase (RTK) inhibitor whose targets include vascular endothelial growth factor receptors 1,2,3(VEGF Receptor, VEGFR-1,2,3), platelet growth factor receptors alpha, beta (PDGF Receptor, PDGFR-alpha, beta), c-KIT, and the like. According to the existing research results, sunitinib exerts an antitumor effect mainly through three pathways: inhibiting tumor angiogenesis, destroying tumor blood vessels, and killing tumor cells directly. Sunitinib is currently approved for the treatment of advanced metastatic renal cell carcinoma and imatinib-resistant Gastrointestinal Stromal tumors (GIST), the first drug approved for the simultaneous treatment of both types of cancer. Corresponding research and clinical trials are also ongoing in other cancer species such as non-small cell lung cancer, intestinal cancer, osteosarcoma. In the field of kidney cancer, sunitinib has been the first line of use for metastatic kidney cancer since FDA approval in the united states in 2006.

Before the advent of targeted drugs, the treatment of advanced kidney cancer was dominated by cytokines. Sunitinib, after its advent, showed anti-tumor effects in both in vitro and in vivo preclinical studies. Moreover, the clinical efficacy of sunitinib has been demonstrated through observations over a decade. Although sunitinib slows the progression of metastatic renal cancer and improves patient prognosis, almost all patients eventually develop resistance and eventually progress again. According to the existing research reports, although the evaluation criteria of each research are slightly different, it can be seen that about 70% of patients are sensitive to sunitinib in the first line treatment, and 30% of patients show primary drug resistance to sunitinib. In sensitive patients, the effective period is usually only 6-15 months, after which acquired resistance and disease progression occurs. There are many studies on the mechanism of acquired drug resistance, and the mechanism can be generally classified into the following: activation of pro-angiogenic signals, alterations in the tumor microenvironment, lysosomal retention, action of non-coding RNAs, and activation of other signaling pathways.

Meanwhile, the results of clinical trials (NCT 00083889) with sunitinib in western patients showed that 98.13% of patients experienced Adverse reactions (overture Event, AE), with a incidence of treatment-related severe Adverse reactions (Serious overture Event, SAE) of 23.7% and Adverse reactions with incidence > 50% of diarrhea (65.6%), fatigue (62.4%) and nausea (57.6%) respectively. The results of clinical trials in chinese patients (NCT 00706706) showed that the occurrence rate of AE was 97.14%, the occurrence rate of SAE was 12.38%, and adverse reactions with an incidence rate > 50% were hand-foot syndrome (63.8%), leukopenia (52.4%), fatigue (51.4%) and thrombocytopenia (51.4%), respectively. From the research results, the sunitinib has more adverse reactions and high incidence, the adverse reactions seriously affect the life quality of patients, and although most of the adverse reactions can be relieved by reducing the drug dosage and changing the administration period, the adverse reactions are accompanied with the reduction of the anti-tumor effect, so that the sunitinib has larger application limitation. Therefore, how to reverse the drug resistance of sunitinib, improve the anti-tumor effect of sunitinib at low dose and reduce the toxic and side effects of sunitinib becomes an urgent technical problem to be solved.

Disclosure of Invention

In order to solve the problems and the defects, the invention provides an anti-tumor pharmaceutical composition containing peracetazine and an application thereof, wherein sunitinib and peracetazine are used in a combined manner, so that the problems of serious drug resistance caused by the use of conventional doses of sunitinib and obviously reduced treatment effect after the treatment dose is reduced in the prior art are solved, and a more obvious clinical treatment effect and reduced drug resistance and toxic and side effects of sunitinib are obtained.

The invention is realized by the following technical scheme:

the invention provides an anti-tumor pharmaceutical composition containing peracetazine in a first aspect, which comprises sunitinib or a pharmaceutically acceptable salt thereof and peracetazine or a pharmaceutically acceptable salt thereof.

Preferably, the tumor is one or more selected from renal cancer, lung cancer, intestinal cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer, leukemia, malignant lymphoma, nasopharyngeal cancer and pancreatic cancer.

Preferably, the tumor is selected from renal cancer.

In a second aspect, the present invention provides an anti-tumor pharmaceutical formulation comprising peracetazine, including sunitinib or a pharmaceutically acceptable salt thereof and peracetazine or a pharmaceutically acceptable salt thereof.

Preferably, the preparation dosage form is selected from one or more of tablets, capsules, pills, granules, injections, aerosols, sprays, films and suppositories.

Preferably, the pharmaceutically acceptable pharmaceutical excipients are selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants and coloring agents.

Preferably, the tumor is selected from renal cancer.

In a third aspect, the invention provides the use of a pharmaceutical composition comprising sunitinib or a pharmaceutically acceptable salt thereof and peracetylazine or a pharmaceutically acceptable salt thereof in the manufacture of a product for the treatment of tumours.

Preferably, the tumor is one or more selected from renal cancer, lung cancer, intestinal cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer, leukemia, malignant lymphoma, nasopharyngeal carcinoma and pancreatic cancer.

Preferably, the tumor is selected from renal cancer.

In a fourth aspect, the invention provides the use of peracetazine or a pharmaceutically acceptable salt thereof in the preparation of a product for reducing the anti-tumor drug resistance of sunitinib and/or improving the anti-tumor therapeutic effect of sunitinib.

Preferably, the tumor is selected from renal cancer.

Although sunitinib is widely used clinically for cancer treatment and can achieve relatively remarkable effect, sunitinib has been used as a first-line drug in the treatment of diseases such as kidney cancer. However, neither improvement in the patient's prognosis nor delay in the progression of the cancer prevents the development of resistance to sunitinib in the patient and ultimately leads to the disease progressing again; and when sunitinib is used in large quantities for a long time, serious toxic and side effects are often generated, so that the life quality of patients is greatly reduced. To date, no better treatment regimen has emerged to improve the severe resistance, reduced therapeutic efficacy, and severe toxic side effects of sunitinib, thereby compromising its clinical use and therapeutic efficacy in patients.

In this regard, the inventors have conducted extensive studies and have conducted screening using a compound library of the ceramic company in order to expect compounds that can improve clinical resistance to sunitinib and can achieve additive or even synergistic therapeutic effects therewith. In the screening process, the result shows that on the basis of the original sunitinib, the piperazines are further added and used in combination with the sunitinib, and a very remarkable synergistic effect (CI < 1) can be generated.

Piperazines (Piperacetazine, CAS: 3819-00-9) are phenothiazine derivatives, also known as: (ii) piperazines; aceperazine; acepiprazine and the like have similar pharmacological actions to chlorpromazine, belong to dopamine receptor antagonists and are mainly used for treating and improving diseases or symptoms such as psychosis, vomiting relieving, anesthesia and the like clinically. There is no evidence to date for its antitumor-related activity. According to the invention, researches show that the piperazines can obviously improve the sensitivity of sunitinib to tumor cells and greatly reduce the IC of sunitinib ₅₀ The combination of the two can produce very obvious synergistic effect.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention improves the existing clinical cancer treatment scheme, screens a large amount of compounds, and obtains the compound which can be combined with sunitinib to obviously reduce the drug resistance of the sunitinib.

(2) According to the invention, on the basis of the original sunitinib, the piperazines are further added, so that a very obvious synergistic interaction effect can be generated, the apoptosis of cancer cells is promoted in a combined manner, the proliferation of the cancer cells can be inhibited obviously, and the sunitinib has a very obvious killing effect on the cancer cells.

(3) The pharmaceutical composition provided by the invention can obviously improve the problem of serious drug resistance caused by singly adopting sunitinib in the prior art and the problem of obviously reduced treatment effect when the conventional use amount of sunitinib is reduced; the clinical dosage of the sunitinib is greatly reduced, the good treatment effect of the sunitinib can be always kept, and drug resistance caused by long-term use of the sunitinib is prevented; meanwhile, the serious adverse reaction caused by long-term large-dose sunitinib use can be avoided, the medication safety and the life quality of patients are improved, a new scheme is provided for clinical treatment of tumor diseases, and the sunitinib composition has a very wide market prospect and extremely important social significance.

Drawings

FIG. 1 is a diagram showing the results of an in vitro renal cancer 786-O cell combination index experiment.

FIG. 2 is a schematic diagram showing the experimental results of Caki-2 cell drug combination index of kidney cancer in vitro.

FIG. 3 is a diagram showing the results of in vitro renal cancer OS-RC-2 cell combination index experiments.

FIG. 4 is a schematic diagram of the in vivo in situ renal cancer mouse model construction and experimental procedures.

FIG. 5 is a graph showing the effect of in vivo drug combinations on tumor growth volume in mice.

FIG. 6 is a graph showing the effect of in vivo combination on mouse kidney and tumor mass.

FIG. 7 is a graph showing the effect of in vivo drug combination on the degree of improvement in mouse renal tumor burden.

FIG. 8 is a graph showing the effect of in vivo combination on body weight in mice.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Cell lines listed in the context of the present invention, including 786-O, Caki-2, OS-RC-2, Renca, etc., were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to ATCC guidelines, unless otherwise specified. All cell lines were identified by short tandem repeat analysis of the chinese centre for type culture collection (wuhan) and verified for the presence of mycoplasma contamination using a PCR assay kit (shanghai Biothrive Sci), while being cryopreserved in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention are commercially available.

Representative results from selection of biological replicates are presented in the context figures, and data are presented as mean ± SD and mean ± SEM as specified in the figure. All in vitro experiments were repeated at least three times and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 5.0 or SPSS 20.0 software. And comparing the difference of the mean values of two or more groups by using a t test or an analysis of variance.p< 0.05 was considered to be a significant difference.

Example 1 in vitro antitumor synergy experiments

(1) 786-O cells in the logarithmic growth phase are seeded in a 96-well plate according to a certain density (1500-;

(2) discarding the old culture medium after 24 hours, dividing the cells into 5 groups of 1-5 for use, adding culture mediums containing different drug concentrations, and arranging 3 multiple holes in each group, wherein sunitinib is added in group 1, piperazinone is added in group 2, sunitinib and piperazinone are added in group 3, DMSO is added in group 4, and group 5 is used as a blank group; group 1 sunitinib initial concentration was 100 μ M, after which each well was diluted in equal proportion in sequence; group 2 piperazines at an initial concentration of 100 μ M, after which each well was diluted in equal proportion in turn; group 3 sunitinib and piperazines were initially at 100 μ M each, after which each well was serially diluted in equal proportions.

(3) After 48 hours of culture, the liquid medicine is discarded, a culture medium containing 5 percent of CCK-8 is added, the mixture is placed in a 37 ℃ for incubation for 3 hours, and then an OD value of 450nm is detected by a microplate reader.

Based on the DMSO group, the cell survival rate = (the OD value of the drug-added group-the OD value of the blank group)/(the OD value of the DMSO group-the OD value of the blank group) × 100%, the survival conditions of the cells in each group are compared,

the results show that in group 1 with sunitinib alone, its IC is ₅₀ About 4.82. mu.M, and the IC of sunitinib after addition of peracetylazine ₅₀ Deserves to be remarkableThe reduction in tumor cell count was 1.48 μ M, which resulted in a reduction of sunitinib usage by about 69.3% compared to that achieved with single use.

The above experiment was then repeated using Caki-2 and OS-RC-2 cells. The results show that for Caki-2 cells, sunitinib usage was reduced by about 65.7% when it produced half inhibition of tumor cells compared to when used alone; for OS-RC-2 cells, the amount of sunitinib used when it produces half inhibition of tumor cells was reduced by about 59.6% compared to when used alone; therefore, the sensitivity of sunitinib to cancer cells can be obviously improved by adding the peracetylazine, so that the using amount of sunitinib is greatly reduced.

Further, on the basis of the above-mentioned in vitro cell proliferation inhibition experiment, a Fa-CI curve was generated by determining the concentration of the drug required for the drug alone (sunitinib or peracetazine) and the drug Combination (sunitinib + peracetazine) at 786-O cells reaching 10%, 30%, 50%, 70% and 90% inhibition using the formula to calculate the Combination Index (CI): wherein the CI calculation formula is as follows:

CI=D _1x /D ₁ +D _2x /D ₂ +（D _1x ·D _2x ）/（D ₁ ·D ₂ ）。

D _1x : the concentration of the 1 st drug at which a certain cytostatic rate is reached in the combination group;D _2x : the concentration of the 2 nd agent at which the combination achieves a certain cytostatic rate;D ₁ : the concentration of the 1 st drug when the single drug group reaches a certain cell inhibition rate;D ₂ : the concentration of the 2 nd drug when the single drug group reaches a certain cell inhibition rate.

When CI < 1 indicates that the two medicines jointly show synergistic action; when CI =1 indicates that the two drugs in combination exhibit additive effects; when CI > 1 indicates that the combination of the two drugs shows antagonism.

The results are shown in FIG. 1. The result shows that in 786-O cells, the piperazines and the sunitinib show a synergistic effect under various inhibition rates, and the synergistic effect is stable.

Subsequently, Caki-2 and OS-RC-2 cells were selected based on the original 786-O cells to repeat the above anti-tumor synergy experiment, and the results are shown in FIGS. 2-3. The results show that in Caki-2 cells, the piperazines show synergistic inhibition effect with sunitinib at almost every inhibition rate; for OS-RC-2 cells, the inhibition degree of the peracetylazine is 10% -70% and the inhibition degree of the peracetylazine and sunitinib show synergistic effect.

Therefore, the synergistic effect of the peracetylazine and the sunitinib is stable, the function is clear, and the synergistic effect of the peracetylazine and the sunitinib can realize a very obvious tumor cell synergistic inhibition effect.

Example 2 in vitro antitumor synergistic compatibility mode experiment

(1) 786-O, Caki-2 and OS-RC-2 cells in the logarithmic growth phase are planted in a 96-well plate according to a certain density (1500-;

(2) discarding the old culture medium after 24 hours, adding medicines containing different compatibility forms for continuous culture, and arranging 3 compound holes in each group;

(3) after 48 hours of culture, the cell culture medium was discarded, the medium containing 5% CCK-8 was added, incubated at 37 ℃ for 3 hours, and then the OD at 450nm was measured with a microplate reader.

And (3) performing corresponding synergistic effect evaluation on the inhibition rate by taking the DMSO group as a reference, and analyzing the effect of the combination drug by adopting a King's formula (Q value):

Q=E _(A+B) /(E _A +E _B -E _A ×E _B ) I.e. E _(A+B) : inhibition of combination, E _A : inhibition ratio of A-drug administration, E _B : and B, the inhibition rate of single use of the medicines. When Q is more than 1.15, the two medicines are synergistic; when Q is more than or equal to 0.85 and less than or equal to 1.15, the two medicines have additive action; when Q is less than 0.85, the two drugs are antagonistic.

TABLE 1 Effect of combination on tumor cell proliferation inhibition and combination index evaluation

As can be seen from table 1, sunitinib alone had only a limited inhibitory effect on the activity of three renal cancer cells, while peracetylazine alone had no significant inhibitory activity; however, when the peracetazine is further used in combination with sunitinib, the inhibition activity of sunitinib on renal cancer cells can be obviously improved.

Therefore, the piperazines can generate obvious anti-tumor synergistic effect with the sunitinib, so that the clinical dosage of the sunitinib is greatly reduced, the high-efficiency treatment effect of the sunitinib can be always kept, the drug resistance caused by long-term use of sunitinib is prevented, meanwhile, the serious adverse reaction caused by long-term use of large dosage of sunitinib is avoided, and the medication safety and the life quality of patients are improved. It should be understood herein that in the course of clinical practice, based on an understanding of the basic principles and concepts of the present invention, those skilled in the art will be able to make routine selections and adjustments to the dosage, administration mode, administration frequency, etc. of each drug, based on such factors as tumor type, patient age, complications, other underlying diseases, etc., and such factors as dosage, administration mode, administration frequency, etc. of each drug should not be taken as limitations of the present invention.

Example 3 in vivo antitumor assay

Balb/c mice about 5-6 weeks are selected to carry out in-vivo in-situ tumor growth inhibition experiments, and the specific experimental method is as follows:

(1) culturing murine renal carcinoma Renca cells in vitro, collecting cells in a logarithmic phase, centrifuging and suspending;

(2) injecting the cell suspension into the subcutaneous tissues of 3 Balb/c mice, taking out tumor bodies after the tumor bodies are formed, and dividing the tumor bodies into 5mm ³ Small tumor masses of left and right size;

(3) transplanting the obtained small tumor block to a plurality of new Balb/c mice under renal capsule;

(4) after the tumor formation, dividing 24 nude mice into 4 groups with the number of 1-4, and carrying out intragastric administration on 6 mice in each group; wherein group 1 is administered sunitinib-10 mg/kg-d, group 2 is administered peracetylazine-10 mg/kg-d, group 3 is administered sunitinib-5 mg/kg-d + peracetylazine-5 mg/kg-d, group 4 is administered with an equivalent amount of solvent, the administration is continued for 5 days, and the administration is stopped for 2 days, which is taken as an administration cycle, the administration is performed for 4 cycles in total, the tumor volume is measured once per week, the weight of the mouse is weighed after the treatment is finished, the kidney is taken out after the mouse is sacrificed, and the tumor volume and the kidney mass of the mouse are measured.

Experimental procedure the flow is shown in fig. 4, and the experimental results are shown in fig. 5-8.

The results show that the mean tumor volume of group 1 mice using sunitinib alone is 662.7cm ³ The tumor inhibition rate is 31.52%, (p< 0.01, vs group 4); group 2 mice using piperazines alone had a mean tumor volume of 1030.0cm ³ No significant tumor suppressive activity (N.S, vs group 4); in the case of group 3 in which sunitinib and piperazines were used in combination, the mean tumor volume of mice in the group was 252.4cm ³ The tumor inhibition rate is 73.9%, (p< 0.0001, vs group 4) (see FIG. 5).

Total mass analysis of kidney and tumor revealed that the mean mass of kidney + tumor was 0.742g and the tumor inhibition rate was 37.17% (1/37%) for sunitinib alone in group 1 micep< 0.05, vs group 4); the mean mass of kidney + tumor of mice in group 2 with piperazines alone was 1.181g, with no significant tumor-inhibiting activity (N.S, vs group 4); in the group 3 of sunitinib and piperazines, the average mass of the kidney and the tumor of the mice in the group is 0.307g, and the tumor inhibition rate is 74.01% (R) ((R))p< 0.0001, vs group 4) (see fig. 6), the trend of the results was essentially consistent with tumor volume.

Subsequent analysis of the kidney to mouse body weight ratio revealed that the kidney to body weight ratio of sunitinib alone in group 1 mice was 0.0491 and the degree of renal tumor burden was 38.08% ((38.08%) (p< 0.05, vs group 4); group 2 mice using peracetylazine alone had a kidney to body weight ratio of 0.0491 with no apparent tendency to reduce tumor burden (N.S, vs group 4); and for group 3, which used sunitinib and piperazines in combination, the kidney to body weight ratio of mice in the group was 0.0120 and the degree of renal tumor burden was 75.56% ((p＜0.0001，vs group 4) (see fig. 7), the resulting trend is substantially consistent with the results described above.

Further analysis of the body weight of the mice revealed (see fig. 8) that no significant reduction in the body weight occurred in each group of mice, indicating that no significant toxic side effects occurred in each group.

According to the results, the antitumor activity of sunitinib can be obviously improved, the tumor inhibition rate of sunitinib is increased, the load degree of the kidney in-situ tumor is obviously reduced, and the using amount of sunitinib is only half of that of group 1 by further combining the sunitinib with the peracetazine.

According to the invention, on the basis of the original sunitinib, the peracetylazine is further added, so that a very obvious synergistic effect can be generated, the proliferation of cancer cells is obviously inhibited, and the sunitinib has very obvious killing efficiency on the cancer cells. The pharmaceutical composition provided by the invention can obviously improve the problems of serious drug resistance caused by the simple adoption of sunitinib in the prior art and the treatment effect obviously reduced when the conventional use amount of sunitinib is reduced, greatly improves the clinical treatment efficiency, reduces the toxic and side effects of patients, provides a new scheme for clinical treatment, and has very wide market prospect and extremely important social significance.

The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.

Claims

1. The application of the piperazines or the medicinal salts thereof and the sunitinib or the medicinal salts thereof in preparing the pharmaceutical preparation for resisting renal cancer.

2. The use of claim 1, wherein the pharmaceutical preparation is in a dosage form selected from one or more of tablets, capsules, pills, granules, injections, aerosols, sprays, films and suppositories.