CN113662964A - Medicine for treating tumor - Google Patents

Medicine for treating tumor Download PDF

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CN113662964A
CN113662964A CN202010401405.1A CN202010401405A CN113662964A CN 113662964 A CN113662964 A CN 113662964A CN 202010401405 A CN202010401405 A CN 202010401405A CN 113662964 A CN113662964 A CN 113662964A
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cisplatin
zey
tumor
cells
sarcoma
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曹丽
杨淑贤
孙云方
李立勇
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Institute of Medicinal Plant Development of CAMS and PUMC
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Abstract

The invention discloses a medicine for treating tumors, which is a composition, and the active ingredients of the medicine are sanshool ketene and cisplatin. The Zey and cisplatin are combined to have obvious synergistic effect in resisting the proliferation of sarcoma cells. In sarcoma S180 cells, when used in combination with an onset dose of Zey, there was a strong synergistic effect and a cisplatin dose-reducing effect, and a reduction in IC50 values. This synergistic effect at high inhibition (> 80%) levels generally corresponds to higher therapeutic levels and development value.

Description

Medicine for treating tumor
Technical Field
The invention relates to a medicament, in particular to a medicament for treating tumors.
Background
In the drug treatment of tumors, acquired drug resistance and adverse reaction events are caused by using a single drug, the curative effect and the response rate of the single drug are limited, and even if the single drug is used for hot immunotherapy, the clinical response rate is only about 20 percent. Therefore, the NCCN guidelines, starting from enhancing drug action, improving patient quality of life, and reducing toxic and side effects, bring the combination into clinical drug practice, and indicate that it has irreplaceable effects. Therefore, the method fully analyzes and summarizes the characteristics of the medicaments, deeply excavates the combined medication strategy, is the key for further improving the curative effect and overcoming the drug resistance, and has important scientific research value and clinical use significance.
Cisplatin has the characteristics of wide anti-tumor spectrum, strong effect, definite curative effect and the like, is a first-line medicament for treating various solid tumors, is widely applied clinically, but has three serious problems: the three major problems of acquired drug resistance, narrow safety range and obvious tissue or organ toxicity, particularly liver and kidney toxicity, seriously jeopardize the life quality of patients, cause early treatment termination and cause the reduction of the use rate of the medicine, and are the troublesome problem of the current cisplatin application. There is therefore a need to develop a regimen for use in combination with cisplatin to synergistically sensitize, improve therapeutic efficacy, and reduce toxicity.
Disclosure of Invention
The invention aims to provide a scheme which can be used in combination with cisplatin to achieve synergistic sensitization, improve curative effect and reduce toxicity.
The invention provides a pharmaceutical composition for inhibiting tumors, which comprises the active ingredients of sanshool ketene and cisplatin.
Wherein the cisplatin is cisplatin for injection.
Wherein the pharmaceutical composition also comprises a solvent for dissolving the sanshool ketene and the cisplatin.
Wherein the solvent is normal saline.
Wherein the mass ratio of the sanshool ketene to the cisplatin in the combined medicine is 15-30: 1.
Wherein, the tumor inhibition is the inhibition of the growth of tumor cells.
Wherein the tumor is sarcoma.
Wherein inhibiting the growth of tumor cells is inhibiting the tumor weight and/or inhibiting the number of tumor cells.
The application of the sanshool and the cisplatin in the preparation of tumor inhibiting products also belongs to the protection scope of the invention.
The application of the composition in preparing tumor-inhibiting products is also within the protection scope of the invention.
Wherein the tumor is sarcoma.
Wherein inhibiting the growth of tumor cells is inhibiting the tumor weight and/or inhibiting the number of tumor cells.
The above medicament may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a lyoprotectant sugar, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as glycerol, mannitol; alginic acid; emulsifiers, such as Tween; phospholipids, such as lecithin, soya lecithin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, stearamide; cholesterol; macromolecular polymers such as polyethyleneimine, chitosan, hyaluronic acid; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer and the like; physiological saline, glycerol and phosphate buffered saline.
In the above application, the product may be a medicament.
In the above medicine and product, Zanthoxylum schinifolium ketene and cisplatin can be packaged separately or mixed together.
Above, the tumor may be a solid tumor or a non-solid tumor. The solid tumor can be a sarcoma.
In the invention, Zey and cisplatin are combined to have obvious synergistic effect in anti-sarcoma cell proliferation. In sarcoma S180 cells, when used in combination with an onset dose of Zey, there was a strong synergistic effect and a cisplatin dose-reducing effect, and a reduction in IC50 values. This synergistic effect at high inhibition (> 80%) levels generally corresponds to higher therapeutic levels and development value.
From the results of in vivo experiments, Zey and cisplatin alone have obvious tumor inhibition effects on a mouse sarcoma S180 transplantation tumor model, and when the Zey and cisplatin alone are combined, the tumor inhibition rate is greatly increased, and the synergy is obvious, so that the cisplatin treatment method is beneficial to reducing the dosage of cisplatin for patients with obvious clinical cisplatin toxicity, and obtaining longer cisplatin use time. Has high obvious consistency with in vitro experiments.
Meanwhile, in safety evaluation, it can be generally seen from weight data that Zey combined with cisplatin does not cause further weight reduction, and it shows that there is no possibility of superimposed toxicity, and in the related detection of liver and kidney, which are main accumulation organs of cisplatin, the combined scheme does not induce further damage to non-target tissues, and shows higher safety.
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FIG. 1 is a photograph of each group of tumors dissected at the end of the experiment in the example of the present invention.
FIG. 2 is a graph showing the change in body weight of each group of mice during the administration.
FIG. 3 is a pathological observation of liver tissues of each group of mice, in which the magnification is 200X and 400X, black arrows indicate hepatocyte necrosis and nuclear fragmentation; white arrows indicate inflammatory cell infiltration; the dark square box indicates that the central and portal veins are slightly distended with extravasated blood, and the lumen of the blood vessel has a small number of white blood cells.
FIG. 4 is a pathological observation of kidney tissues of various groups of mice, in which the magnification is 200X and 400X, and black arrows indicate the exudation of hemosiderin in renal cortical blood vessels; the white arrows indicate slight atrophy of the glomerular capsule cavity.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Description of materials:
sanshool ketene, english name Zeylenone, CAS: 193410-84-3, Zey for short, is white powder, and has dry storage and purity>97.5% (detected by HPLC method). Zey has a chemical structure of
Figure BDA0002489614580000031
Zey is a natural product with low toxic and side effects.
Cisplatin, structural formula is
Figure BDA0002489614580000032
CAS: 15663-27-1, the invention adopts cisplatin for injection, which is purchased from Qilu pharmaceutical Co., Ltd, and has the specification of 10 mg. When in use, the composition is dissolved in 10ml of normal saline to prepare an injection with the concentration of 1mg/ml, and the injection is diluted when in use.
S180 mouse sarcoma cells, grown in suspension, purchased from the cell center of the institute of basic medicine, Chinese academy of medical sciences, were cultured in complete medium containing 10% fetal bovine serum and 1% penicillin streptomycin mixture RPMI-1640(w/o Hepes).
Female ICR mice (18-20g) were housed in SPF-grade housing on a standard scale. Purchased from Sibefu (Beijing) Biotechnology Ltd, license number SCXK (Beijing) 2016-.
Description of the experimental reagents: RPMI-1640(w/o Hepes) medium, Corning Biotech, Inc.; ausbian extra grade Fetal Bovine Serum (FBS), shanghai wei zhengxiangyu biotechnology sijiqing company; CCK8 reagent, japan colleague reagent company; dimethylsulfoxide (DMSO), Sigma company; phosphate Buffered Saline (PBS), beijing solibao biotechnology limited; castor oil polyoxyethylene ether CAS: 61791-12-6, Aladdin reagents Inc.; absolute ethanol (analytical grade) and 4% formaldehyde, beijing chemical plant; 0.9% physiological saline, Shijiazhuang Siyao Co., Ltd; glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), Total Bilirubin (TBIL) and Urea (Urea) kit, Zhongsheng Bei Zhi Biotech limited; hematoxylin-eosin stain, bevacizumab biotechnology limited, beijing; xylene, hydrochloric acid, ammonia water and neutral gum, national drug group chemical reagents limited.
Formulation of 1, Zey
For in vitro studies, an appropriate amount of Zey was weighed and dissolved completely in DMSO to a concentration of 15mg/ml, which was the stock solution, dispensed, and placed in a-20 ℃ freezer for use. For administration, the medium is diluted to the desired concentration.
For in vivo studies, Zey was first dissolved in cremophor oil and ethanol (1:1, w/w) as solvents, placed in a refrigerator at 4 ℃ and diluted to the desired concentration with physiological saline at the time of administration.
2. Preparation of cisplatin
Cisplatin is dissolved in normal saline to make into injection with concentration of 1mg/ml, and diluted to desired concentration when used.
Example 1 measurement of cell proliferation inhibition by CCK8 method
1. The inhibition of S180 mouse sarcoma cells by cisplatin solutions at various concentrations was performed as follows.
1) S180 mouse sarcoma cells were cultured in complete medium containing 10% fetal bovine serum and 1% penicillin streptomycin cocktail RPMI-1640(w/o Hepes). After S180 cells in logarithmic growth phase are collected by centrifugation, cell suspension is prepared and counted, and the cell density is adjusted to 1 × 105Per mL;
2) sequentially adding the single cell suspension into a 96-well plate, wherein each well is 100 mu L, the edge is filled with PBS (edge effect is avoided) for one circle, after the cells are added, the edge of the 96-well plate is tapped by hands to ensure that the 96-well plate is uniformly paved and is prevented from aggregation, and then the 96-well plate is placed in an incubator; cells were randomly divided into 18 drug groups (6 cisplatin drug groups, 6 Zey groups, and 6 cisplatin + Zey groups) and 1 control group, each with 5 replicates.
3) Cisplatin solutions were prepared at 6 concentrations in the tube using the sesquidilution method, with the highest concentration of the 6 concentrations being 33.30 μ M, and the other concentrations being shown in column 1 of Table 1. Placing the prepared 6 cisplatin solutions on a tube frame according to the sequence of the concentration from small to large, adding 100 mu L of cisplatin solution with the corresponding concentration into each hole of 6 cisplatin drug groups, adding 100 mu L of culture medium into each hole of a control group, and lightly tapping the edge of a 96-hole plate to ensure that the drugs are as uniform as possible in the holes (5 multiple holes are arranged at each concentration);
4) after the medicine acts for 24 hours, adding 10 mu L of CCK8 reagent into each hole, and continuously culturing for 3 hours;
5) taking out the 96-well plate, observing the cell state, recording the phenomenon, and taking out the cells;
6) OD value was measured at 450nm using an Absorption plate of a microplate reader, and the cell proliferation inhibition ratio was%Drug group/ODControl group) X 100%, plotting the result with GraphPad Prism 5,and calculates the IC50 value with the CompuSyn software.
2. Inhibition of S180 mouse sarcoma cells by Zey solutions at various concentrations was performed as follows.
Replacing 3) in step 1 with the following procedure, keeping the other steps unchanged, and testing Zey solution for inhibiting S180 mouse sarcoma cells: zey solutions were prepared at 6 concentrations using a half-fold dilution method in the tube, with the highest concentration of the 6 concentrations of Zey being 26.16. mu.M, and the other concentrations being shown in column 3 of Table 1. The prepared 6 Zey solutions are put on a tube frame according to the sequence of the concentration from small to large, 100 mu L of Zey solution with the corresponding concentration is added into each hole of 6 Zey drug groups, 100 mu L of culture medium is added into each hole of a control group, the edge of a 96-hole plate is lightly tapped, and the drugs are enabled to be as uniform as possible in the holes (5 multiple holes are arranged at each concentration).
3. Various concentrations of cisplatin and one concentration of Zey solution and inhibition of S180 mouse sarcoma cells were performed as follows.
The procedure of 3) in step 1 was replaced with the following procedure, and cisplatin and Zey solutions were tested for inhibition of S180 mouse sarcoma cells, with the remaining steps being unchanged: cisplatin solutions of 6 concentrations were prepared in tubes using a sesquidilution method, the highest concentration of the cisplatin solution of 6 concentrations being 33.28 μ M, the other concentrations being shown in column 1 of table 1, and Zey stock solutions were added to the cisplatin solution of 6 concentrations to a Zey content of 1.6 μ M, resulting in 6 cisplatin + Zey solutions, the 6 cisplatin + Zey solutions all having a Zey content of 1.6 μ M, and the cisplatin contents were 33.28 μ M, 16.64 μ M, 8.32 μ M, 4.16 μ M, 2.08 μ M and 1.04 μ M, respectively. The prepared 6 cisplatin + Zey solutions are placed on a tube frame according to the sequence of the concentration from small to large, 100 mu L of cisplatin + Zey solution with the corresponding concentration is added into each hole of 6 cisplatin + Zey drug groups, 100 mu L of culture medium is added into each hole of a control group, and the edge of a 96-hole plate is lightly tapped to ensure that the drugs are as uniform as possible in the holes (5 multiple holes are arranged at each concentration).
4. Median Effect evaluation of the Effect of drug combinations
The international standard of calculation, the median effect method (Chou-Talalay), was chosen to analyze and evaluate Zey for its effect in combination with cisplatin. The obtained data were input into the international common CompuSyn software, and the Combination Index (CI) and Dose Reduction Index (DRI) which are main judgment indexes of the method were used for analysis, and the drug interaction of the Combination was evaluated.
Here, the CI value reflects the interaction between the two drugs. Formula is CI ═ D1/(Dx)1+D2/(Dx)2；(Dx)1And (Dx)2Is the concentration of the two compounds required alone to achieve a particular rate of inhibition; d1And D2Is the concentration required for the two compounds to achieve the same inhibition rate when used in combination as when used alone. The judgment method is as follows: CI>1.1, ═ 0.9 to 1.1 and<0.9 represents antagonism, additivity and synergy of the two drugs respectively.
The DRI value is reflected in a given level of effect, and the dose of each drug used synergistically can be reduced by a factor compared to the dose of each drug used alone. The DRI calculation formula of the combination of the two medicines is (DRI)1＝(Dx)1/(D)1The greater the DRI value, the greater the dose reduction indicating a therapeutic effect, but not necessarily always indicating a synergistic effect, and must be analyzed together with CI to reach a correct conclusion. DRI is clinically important, and reduced doses can reduce toxicity to the host while retaining therapeutic efficacy.
The CI analysis results are shown in table 1, and it can be seen from the data in table 1 that, when cisplatin and Zey act on sarcoma S180 cells individually, both have significant effect of inhibiting proliferation of sarcoma cells, indicating that both are effective when used alone, and from the inhibition rate, Zey has an anti-tumor effect equivalent to cisplatin; when a fixed concentration of Zey (effective dose, 1.6 μ M) and cisplatin was combined on sarcoma S180 cells, a more pronounced proliferation-inhibiting effect was observed than with cisplatin alone. Then, the CompuSyn software calculates the combination index, and the CI values are found to be less than 1, which indicates that the combination of the two medicines has a synergistic effect. In addition, when the inhibition rate of the combination group is 56% -89%, the combination group is strong synergistic effect.
TABLE 1 Combination Index (CI) analysis of the effects of 1 Zey on sarcoma S180 cells in combination with cisplatin for 24h
Figure BDA0002489614580000061
After analysis of the Combination Index (CI), further Dose Reduction (DRI) was evaluated, and the results are shown in Table 2. As can be seen from the DRI index analysis in Table 2, Zey had a dose-reducing effect on each of cisplatin in combination, with a DRI value of Zey ranging from 2.54 to 4.89 and a DRI value of cisplatin ranging from 1.60 to 17.98, and it can be seen that the dose-reducing effect on cisplatin was more pronounced than Zey when the two were used in combination. Therefore, when the same inhibition rate effect is achieved, the combination can realize obvious dose reduction, thereby being expected to improve the toxicity phenomenon of the cisplatin and having important value of further research.
TABLE 2 Dose Reduction Index (DRI) analysis of the 24h effect of 2 Zey in combination with cisplatin on S180 cells
Figure BDA0002489614580000062
Example 2
To further clarify the in vivo efficacy of this combination, the in vivo synergistic antitumor effect of Zey in combination with cisplatin was further investigated by establishing a mouse sarcoma S180 model.
1, establishment of S180 tumor-bearing mouse model
1.1 in vivo passages of S180
Collecting 10 female ICR mice with age of 4 weeks and weight of 18-20g, regulating activity, weight, food intake and water intake to normal, collecting sarcoma cells of S180 mice in logarithmic growth phase, and regulating cell density to 2.5 × 10 with physiological saline6One mouse/ml, 0.2 ml/mouse, after shaking, was intraperitoneally injected into the abdomen of ICR mice until the abdomen of the mice was raised to day 8, at which time, passage was required.
When passage is carried out, an ICR mouse with obvious abdominal bulge is killed by taking off the cervical vertebra, the ICR mouse is completely soaked into 75% alcohol solution for disinfection for a moment, the ICR mouse is taken into a super clean bench after being slightly drained, a sterilized small scissors is used for opening at the right side of the abdomen, 1ml of normal saline is injected into the abdomen of the mouse by a syringe for diluting the abdomen of the mouseWater, then, milky ascites was extracted with a pipette, placed in a 15ml centrifuge tube, diluted 100-fold with physiological saline, counted, and adjusted to a cell density of 2.5X 106One/ml, after shaking up, was again injected intraperitoneally at 0.2 ml/one of 10 ICR mice, this was the second generation. In the same manner, 4 passages were performed in vivo.
1.2S180 subcutaneous transplantation tumor model establishment
Collecting ascites of the S180 mouse sarcoma cells inoculated in 4 passages in vivo in the step 1.1, diluting with physiological saline 100 times, counting, and adjusting the content of the S180 mouse sarcoma cells to 5 × 106And (4) sealing the centrifuge tube and quickly taking the centrifuge tube to an animal room. Gently shake the cell suspension, and aspirate 0.2mL of cell suspension (1X 10) using a syringe under sterile conditions6One cell/one cell) was inoculated subcutaneously into the right axilla of ICR mice to obtain S180 tumor-bearing mouse models.
Grouping and administration of animals
The day after inoculation, animals were randomly assigned to 5 groups (10 per group) by body weight, with the following specific groups and drug injections:
(1) normal group (10 ICR mice, four weeks old, 22.3-24.8g in weight, not inoculated with S180 cells): drinking water in normal diet;
(2) model group (10 ICR mice aged four weeks and weighing 22.7-26.7g, inoculated with S180 cells according to the method in step 1): injecting normal saline into abdominal cavity of mouse for 1 time every day, and administering according to 0.1mL normal saline/10 g body weight;
(3) cis-platinum group (10 four weeks old, 22.3-26.4g weight ICR mice inoculated with S180 cells as in step 1): carrying out intraperitoneal injection of a cisplatin solution to the mice once every two days, wherein the using dose of the cisplatin is 2mg/kg/2 day;
(4) zey groups (10 ICR mice aged four weeks and weighing 22.0-25.5g, inoculated with S180 cells as in step 1): the mice are injected with Zey solution intraperitoneally for 1 time per day, and the use dose of Zey is 15 mg/kg/day;
(5) combination group (10 ICR mice aged four weeks and weighing 23.4-25.8g inoculated with S180 cells according to the method in step 1): the mice are injected intraperitoneally, Zey is injected 1 time per day, and the dosage of Zey is 15 mg/kg/day; cisplatin is injected every two days, and the using dose of the cisplatin is 2mg/kg/2 day;
the above groups are all administrated by intraperitoneal injection according to 0.1mL of medicament/10 g of body weight of a mouse for 10 days, the body weight of the mouse is measured and the tumor growth is observed, the tumor inhibition rate is calculated, and the synergistic effect of Zey on cisplatin is evaluated. As shown in table 3.
The preparation method of the Zey solution is as follows: zey the preparation method comprises dissolving Zey polyoxyethylene castor oil and ethanol (1:1, w/w) as solvent to obtain stock solution, and diluting with physiological saline to desired concentration to obtain Zey solution.
The preparation method of the cisplatin solution comprises the following steps: dissolving cisplatin in normal saline to obtain stock solution, and diluting with normal saline to desired concentration to obtain cisplatin solution.
The results are shown in table 3 and fig. 1. Statistical analysis was performed using GraphPad Prism 5.0 Software (GraphPad Software, La Jolla, Calif., USA), and the experimental data are expressed as mean. + -. standard deviation (means. + -. SD). Two-by-two comparisons between groups were performed using One-way analysis of variance (One-WayANOVA) followed by Tukey's post hoc test (t-test), and differences were considered statistically significant when p < 0.05.
The sarcoma S180 model is a very classical animal tumor model, has the functions of in vitro cell proliferation and in vivo transplantation, and is widely applied to screening of antitumor drugs. The data of table 3 and fig. 1 show that, among them, since no tumor grows in the mice of the normal group, table 3 and fig. 1 show only the data of the four groups of the model group, the cis-platinum group, Zey group and the combination group. Zey and cisplatin are respectively administrated, the tumor growth of S180 tumor-bearing mice is inhibited, when Zey and cisplatin are jointly used, the inhibition rate of S180 solid tumor is obviously improved, and the difference is obvious compared with that of cisplatin alone (p is less than 0.01); and also has synergistic effect in vivo after Chou analysis. Therefore, Zey can obviously enhance the anti-tumor effect of cisplatin when being used together with cisplatin. This result of in vivo experiments, in combination with the in vitro experimental results shown in the previous examples, further demonstrates that Zey increases the sensitivity of cisplatin to sarcoma synergistically inhibits the proliferation and growth of sarcoma in vitro.
TABLE 3 synergistic inhibition of tumor growth in S180 tumor-bearing mice by 3 Zey and cisplatin
Figure BDA0002489614580000081
Note:**p<0.01vs. the model set,##p<0.01vs. cisplatin group
Example 3
The main toxic target organs of cisplatin are liver, kidney and ear, which seriously affect the chemotherapy effect and the life quality of patients, so that the reduction of the toxicity of cisplatin and the enhancement of the chemotherapy effect through the synergistic sensitization are the hot spots of the current research. In conclusion, in this example 3, it is determined whether adverse reactions of liver and kidney, which are main toxic target organs of cisplatin, are aggravated after combined use by exploring in a sarcoma S180 mouse model, that is, it is determined that the scheme does not have potential safety hazards of additive toxicity while achieving synergistic effect.
3.1 weight Change analysis
Meanwhile, the weight data, statistical analysis and mapping of the five groups of mice in the example 2 in the drug test process are shown in fig. 2, and the results are shown in fig. 2, no death phenomenon occurs in all groups of mice in the drug administration process, and comparing the weight change trend in fig. 2, it can be seen that the weights of the mice in the normal group, the model group and the Zey group are gradually increased, the weights of the mice in the cis-platinum group and the combined group are slowly increased, and particularly, the weights of the two groups are different from the weights of the normal group on the 10 th day of drug administration, which shows that the weight of the mice is reduced due to platinum accumulation in the later period of drug administration, and the living state of the mice is influenced.
3.2 serum ALT, AST Activity, TBIL, Urea level assays
ALT and AST are the most important and sensitive indexes for detecting liver function, and the activity of the ALT and the AST reflects the degree of liver damage. Urea is a main component of low-molecular nitrogen-containing waste excreted by the kidney, has significance for the course of nephropathy, observation of disease condition and prognosis judgment, and is a main detection item of nephropathy.
The serum obtained in example 2 was taken out from-80 ℃ (serum was obtained by collecting blood from eyeball, removing cervical vertebra, killing, removing tumor, collecting liver and kidney after administration of five groups of mice in example 2, precipitating the blood at room temperature for 1h, centrifuging to obtain serum, storing the serum at-80 ℃), collecting appropriate amount of serum, subpackaging into EP tubes, and detecting ALT, AST activity, TBIL, and Urea level in serum according to the operation of glutamic-oxal transaminase (AST), glutamic-pyruvic transaminase (ALT), Total Bilirubin (TBIL) and Urea (Urea) kit (Shengbei Biotechnology Limited). The serum biochemical results are shown in table 4, and it can be seen from table 4 that the serum ALT, AST activity, TBIL and Urea levels of the mice in the cisplatin group are significantly increased, wherein the ALT, AST activity and TBIL levels are increased by 46.39%, 61.34% and 22.34% respectively compared with the normal group, and the Urea level is increased by 12.23% compared with the normal group, which indicates that cisplatin causes certain damage to the kidney and the liver, and the kidney has higher tolerance to cisplatin, and the liver has higher sensitivity to cisplatin than the kidney. In the combination group, the Urea change is not obvious, but the combination weakens the change caused by the cis-platinum on the indexes of ALT, AST and TBIL, improves the biochemical indexes to a certain extent and lightens the hepatotoxicity caused by the cis-platinum.
Table 4 effect of drug treatment on ALT, AST activity and TBIL, Urea levels in mouse serum (n ═ 10)
Figure BDA0002489614580000091
Note: is the abnormality of each group compared to the normal group, # is the ratio of the combination group to the cisplatin group, indicating whether attenuated.
*p<0.05,**p<0.01；#p<0.05,##p<0.01
3.3 pathological examination of liver and kidneys
Five groups of mice subjected to the drug test for 10 days in example 2 were randomly selected from 6 mice per group and subjected to pathological examination of liver and kidney, and the pathological section results of liver of five groups of mice are shown in fig. 3 and the pathological section results of kidney of five groups of mice are shown in fig. 4.
In FIG. 3, the upper panel is at 200X magnification, the lower panel is at 400X magnification, black arrows indicate hepatocyte necrosis, nuclear fragmentation; white arrows indicate inflammatory cell infiltration; the dark square box indicates that the central and portal veins are slightly distended with extravasated blood, and the lumen of the blood vessel has a small number of white blood cells. As can be seen from the pathological section results of the liver of fig. 3: inflammatory cell infiltration and extravasated blood appear in the model group, large-area hepatocyte necrosis, nuclear fragmentation and inflammatory cell infiltration are caused by the cisplatin group, the shapes of hepatocytes of the single Zey group and the combined group are basically normal, only trace white blood cells and extravasated blood expand, damage is slight, and the results are identical with the detection results of the biochemical indexes.
In FIG. 4, the upper panel is at 200X magnification, the lower panel is at 400X magnification, and black arrows indicate hemoxanthin exudation in renal cortical blood vessels; the white arrows indicate slight atrophy of the glomerular capsule cavity. The kidney is the major excretory organ for cisplatin, as can be seen from the pathological results of the kidney in figure 4: the kidney tissue structures of the model group and the combined group are normal, the boundaries of all structures are clear, the coloring is uniform, only renal vesicle capsule atrophy is shown, and the cisplatin group has an iron hemoxanthin exudation phenomenon and is accompanied with inflammatory cell infiltration.
The pathological detection in the steps is carried out step by step according to the conventional paraffin embedding, 4 mu m section, HE staining, microscopic examination, image acquisition and slide reading analysis.
In combination with the above examples, it was found from the results of in vitro experiments that Zey combined with cisplatin showed a significant synergistic effect in anti-sarcoma cell proliferation. In sarcoma S180 cells, when used in combination with an onset dose of Zey, there was a strong synergistic effect and a cisplatin dose-reducing effect, and a reduction in IC50 values. This synergistic effect at high inhibition (> 80%) levels generally corresponds to higher therapeutic levels and development value.
From the results of in vivo experiments, Zey and cisplatin alone have obvious tumor inhibition effects on a mouse sarcoma S180 transplantation tumor model, and when the Zey and cisplatin alone are combined, the tumor inhibition rate is greatly increased, and the synergy is obvious, so that the cisplatin treatment method is beneficial to reducing the dosage of cisplatin for patients with obvious clinical cisplatin toxicity, and obtaining longer cisplatin use time. Has high obvious consistency with in vitro experiments.
Meanwhile, in safety evaluation, it can be generally seen from weight data that Zey combined with cisplatin does not cause further weight reduction, and it shows that there is no possibility of superimposed toxicity, and in the related detection of liver and kidney, which are main accumulation organs of cisplatin, the combined scheme does not induce further damage to non-target tissues, and shows higher safety.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. A pharmaceutical composition for inhibiting tumors is characterized in that the active ingredients of the pharmaceutical composition are sanshool and cisplatin.
2. The pharmaceutical composition according to claim 1, wherein the cisplatin is cisplatin for injection.
3. The pharmaceutical composition of claim 1, further comprising a solvent for solubilizing the sanshool and cisplatin.
4. The pharmaceutical composition of claim 1, wherein the solvent is physiological saline.
5. The pharmaceutical composition according to claim 1, wherein the mass ratio of the sanshool ketene to the cisplatin in the combined medicine is 15-30: 1.
6. The pharmaceutical composition of claim 1, wherein said inhibiting a tumor is inhibiting the growth of a tumor cell.
7. The pharmaceutical composition of claim 1, wherein the tumor is a sarcoma.
8. Application of sanshool and cisplatin in preparing tumor inhibiting product is provided.
9. Use of a pharmaceutical composition according to any one of claims 1 to 5 for the preparation of a product for inhibiting tumors.
10. The use of claim 8 or 9, wherein the tumor is a sarcoma.
CN202010401405.1A 2020-05-13 2020-05-13 Medicine for treating tumor Pending CN113662964A (en)

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US20030008923A1 (en) * 2001-06-01 2003-01-09 Wyeth Antineoplastic combinations
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