CN110507653B

CN110507653B - Application of domperidone and combination of domperidone and paclitaxel in preparation of drugs for treating cancers

Info

Publication number: CN110507653B
Application number: CN201910713804.9A
Authority: CN
Inventors: 刘诗洋; 马骉; 孔双泉; 马胜楠; 宋梦薇; 赵光超; 郑计; 刘军; 梁东娜
Original assignee: Beijing Pharmaceutical Race And Co ltd
Current assignee: Beijing Pharmaceutical Race And Co ltd
Priority date: 2019-08-02
Filing date: 2019-08-02
Publication date: 2022-12-02
Anticipated expiration: 2039-08-02
Also published as: CN110507653A

Abstract

The invention relates to the technical field of medicines, and particularly discloses domperidone and application of the domperidone and paclitaxel in preparation of a medicine for treating cancer. The invention finds that domperidone can be independently applied to the medicaments for treating cancers to treat non-small cell lung cancer. The medicine obtained by combining domperidone and paclitaxel can be used for treating non-small cell lung cancer. The invention provides a new application of domperidone and the obtained combined medicament has an unexpected anticancer synergistic effect.

Description

Application of domperidone and combination of domperidone and taxol in preparation of drugs for treating cancers

Technical Field

The invention relates to the technical field of medicines. In particular to domperidone and application of the combination of the domperidone and paclitaxel in preparing a medicament for treating cancer.

Background

Cancer is a serious public health problem statistically, seriously threatens human life and quality of life, and is one of the diseases with the highest morbidity and mortality in the world. Curing cancer has been a struggle goal for researchers. In order to overcome cancer, researchers in various countries have made great efforts, and many new discoveries and treatment methods are continuously generated, but so far, the understanding of the development mechanism of cancer still remains limited. The effective antitumor drugs on the market are still rare at present.

Domperidone, 5-chloro-1- [1- [3- (2-oxo-1-benzimidazole) propyl ] 4-piperidinyl ] benzimidazol-2-one (formula: C22H24ClN 5O) was developed by Janson corporation of belgian as gastric motility and antiemetic. In 1978, the medicine is firstly listed in Germany, italian, english, japanese, french and other countries after being listed in Belgium, and is widely used in various countries. Domperidone serving as a peripheral dopamine receptor blocker can directly act on the gastrointestinal wall, increase the tension of sphincter at the lower part of an esophagus, prevent the reflux of stomach-esophagus, enhance the peristalsis of the stomach, promote the evacuation of the stomach, coordinate the motion of the stomach and duodenum, inhibit nausea and vomit, effectively prevent the reflux of bile and do not influence the secretion of gastric juice. And the dopamine is difficult to pass through a blood-cerebrospinal fluid barrier, and has no inhibition effect on dopamine receptors in the brain, so that no nervous and mental adverse reactions such as extrapyramidal systems and the like exist. The traditional Chinese medicine composition is widely used for treating vomit and nausea caused by various reasons in clinic and obtains satisfactory curative effect. However, no document report of domperidone anticancer is available at present.

Clinically, combination therapy with synergistic effects is expected due to: firstly, each component in the synergistic combination can be properly reduced in dosage, so that the risk of side effects of the medicine is reduced, and secondly, the synergistic effect can also obviously improve the overall effect of treatment. In addition, drug resistance is an important problem, and drug combinations are considered to be the best way to prevent or delay drug resistance. Therefore, the search for a drug combination with synergistic effect has very important significance for treating cancer.

The therapeutic effect of the drug combination is difficult to predict. For example, interactions between some drugs, which are often classified as antagonistic, can reduce the therapeutic effect or cause undesirable side effects; other drug combinations showed therapeutic efficacy of the sum of the individual drug potencies, which were classified as having additive effects. A combination of drugs is classified as synergistic only if it is greater in therapeutic index than the sum of the individual drugs.

At present, no literature report on the adoption of domperidone and chemotherapy drug combined for resisting cancers exists.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide an application of domperidone in preparation of a medicament for treating cancer.

The invention also aims to provide application of the combination of domperidone and paclitaxel in preparing a medicament for treating cancer.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

the invention provides an application of domperidone in preparing a medicament for treating cancer, wherein the cancer is non-small cell lung cancer.

Domperidone has been widely used for suppressing side effects such as nausea and vomiting in the treatment of cancer since its advent, but its effect on cancer cells itself has been neglected. The invention initially discovers that the single administration of domperidone has toxic effect on cancer cells, particularly non-small cell lung cancer cells, and can achieve the effects of controlling the proliferation of the cancer cells and inducing the apoptosis of the cancer cells. And the effect is verified in vitro and in vivo experiments of mice, and the application has clinical application prospect.

According to the invention, the effect of the dose concentration of domperidone on cancer cells is researched through a large number of experiments, and the effect of inhibiting the proliferation of the cancer cells is gradually enhanced along with the increase of the administration concentration of domperidone. In vitro experiments, the inhibition effect is ideal when the concentration is more than 4 uM.

In the use according to the invention, the active ingredient of the medicament is present in free form or in the form of a pharmaceutically acceptable salt.

The active ingredient or a pharmaceutically acceptable salt thereof may also be used in the form of a hydrate or other solvent, depending on the requirements of the application.

In the use of the present invention, the medicament comprises at least one pharmaceutically acceptable carrier.

In the application of the invention, the administration mode of the medicament is intravenous injection, oral administration or local administration.

The medicaments of the invention can be prepared in a manner that is conventional per se, e.g. by means of conventional mixing, granulating, sugar-coating, dissolving or lyophilizing processes. Which may contain a therapeutically effective amount of the pharmacologically active ingredient or may also contain one or more pharmaceutically acceptable carriers. The preferred route of administration of the agents of the invention is oral. The dosage form can be sugar-coated tablet, capsule or suppository. The unit content of active ingredient contained in a single dose in each dosage form does not necessarily per se need to constitute an effective amount, since the necessary effective amount can be reached by administration of a plurality of dosage units.

In preparing the medicament for oral dosage form, any of the conventional pharmaceutical media may be employed, such as water, glycols, oils or alcohols; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as powders, capsules and tablets. Tablets and capsules for ease of administration are preferred in the present invention, in which case solid pharmaceutical carriers are employed.

The invention also provides an application of the combination of domperidone and paclitaxel in preparing a medicament for treating cancer, wherein the cancer is non-small cell lung cancer.

In the existing method for treating cancer, paclitaxel is conventionally used as a chemotherapeutic agent to control cancer cells, domperidone is used as an antiemetic to relieve side effects brought by the chemotherapeutic agent, and the conventional method is a treatment scheme set by respectively utilizing the original effects of the domperidone and the paclitaxel. The invention breakthroughs the discovery that the two conventional medicaments can generate synergistic effect, breaks through the original medicament idea and provides a new alternative scheme for treating cancers, particularly non-small cell lung cancer. The scheme obtains a new medicine by combining domperidone which can stop vomit and play a synergistic effect with paclitaxel, the medicine can ensure the original treatment effect, realize the effect of reducing side effects by controlling the dosage of the paclitaxel which has stronger toxic and side effects on a hematopoietic system and an immune system, and also reduce the incidence rate of the side effects by integrally lower active ingredient dosage and/or lower application frequency, thereby relieving the pain of patients.

In the application of the invention, in the unit preparation of the medicine, the mass ratio of the domperidone to the paclitaxel is 100: (1-40); preferably 100:7.

the effective dosage of each of the active ingredients used in the combination of the invention may vary depending on the particular compound or pharmaceutical composition used, the mode of administration, the severity of the condition being treated. Preferably, the ratio of the total amounts of the two active ingredients to be administered in the combined preparation can be varied, for example according to the needs of the individual patient in need thereof.

In the use according to the invention, the active ingredients of the medicament are present in free form or in the form of a pharmaceutically acceptable salt and/or the medicament contains at least one pharmaceutically acceptable carrier.

In the use of the present invention, the domperidone and the paclitaxel may be administered in combination, separately or sequentially.

The active ingredients domperidone and paclitaxel of the present invention may be administered independently, or may be administered using a fixed combination with different contents of the respective ingredients, i.e. they may be administered simultaneously or sequentially at different time points, in any order. The parts of the kit may be administered simultaneously or chronologically staggered, i.e. any part of the kit of parts is administered at different time points with equal or unequal time intervals. Preferably, time intervals are used which maximize their synergistic effect.

In the application of the invention, the administration mode of the medicament is one or more of intravenous injection, oral administration or local administration.

The invention preferably administers the active ingredient domperidone through tablets or capsules, and the active ingredient taxol through intravenous injection.

The medicaments of the invention can also be prepared by conventional methods (which are described above) and can comprise a therapeutically effective amount of the pharmacologically active ingredient or can also comprise one or more pharmaceutically acceptable carriers. The unit content of active ingredient contained in a single dose in each dosage form does not necessarily need to constitute an effective amount per se, since the necessary effective amount can be achieved by administration of a plurality of dosage units.

The invention has the beneficial effects that:

based on the use of the domperidone as an antiemetic medicament to reduce side effects of chemotherapy in clinic, the invention also discovers that the domperidone has an anti-cancer effect, expands the indications of the domperidone and brings new hopes for cancer patients.

The invention relates to the use of a synergistic combination of domperidone and an antineoplastic chemotherapeutic selected from antineoplastic paclitaxel for the preparation of a medicament for the treatment of cancer.

Paclitaxel is widely accepted as a clinically common broad-spectrum antitumor drug because of long history and definite anticancer effect. But at the same time, the clinical application of the medicine is limited to a certain extent because of strong toxic and side effects on the hematopoietic system and the immune system. The invention adopts the domperidone and the drug obtained by combining the domperidone, which not only realizes the synergistic anticancer effect (the synergistic therapeutic cytotoxic effect is shown), improves the anticancer effect, but also makes the reduction of the dosage, the reduction of the side effect and the prevention or delay of the drug resistance possible while ensuring the original anticancer effect.

Drawings

FIGS. 1 to 3 are graphs showing the protocol of the cell Proliferation inhibitory effect test (including the component ratios and the dosages used) and the results of example 1 of the present invention, in which the ordinate represents the Proliferation Rate;

FIGS. 4 and 5 are graphs showing the results of H460 apoptosis test in example 2 of the present invention, wherein the ordinate of FIG. 5 shows the percentage of apoptosis;

FIGS. 6 and 7 are graphs showing the results of in vivo H460 cells in example 3 of the present invention, wherein the ordinate of FIG. 7 is weight (weight) in grams;

FIG. 8 is a graph showing the results of the cell proliferation inhibitory effect test of example 4 of the present invention, and the ordinate shows proliferation rate.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures and commercially available products unless otherwise specified.

The main experimental materials and instruments used in the invention are:

cell line: human lung cancer cell line H460 (basic medical institute of chinese medical science); domperidone (beijing and biopharmaceutical limited); DMEM/F12, RPMI-1640 medium, trypsin, PBS (Gibco, USA); paclitaxel (Sigma-Aldrich Co.); 96-well cell culture plates, 6-well cell culture plates (Corning, usa); cell Counting Kit-8 reagent (Homon chemical research institute); annexin V/PI apoptosis kit (American BD company)

Example 1

Domperidone and paclitaxel are applied to a non-small cell lung cancer cell line H460 by separate administration and combined administration, and the cell proliferation inhibition effect of the domperidone and the paclitaxel is tested.

Different concentrations of domperidone and paclitaxel were used alone and in combination to act on the non-small cell lung cancer cell line H460, respectively, in the concentrations and modes shown in fig. 1-3, and the administration unit was μ M (uM). The proliferation inhibitory effect on H460 was examined by a method of measuring cell viability by CCK-8, and the results are shown in FIGS. 1 to 3. Domperidone, when used alone, showed an effect of inhibiting proliferation of the non-small cell lung cancer cell line H460. And the inhibitory effect is gradually enhanced with the increase of the administration concentration of the domperidone.

When the dosage of the domperidone is unchanged (5 mu M and 7 mu M), the synergistic effect is shown corresponding to the paclitaxel with different concentrations, and the proliferation inhibition effect is greatly superior to that of the paclitaxel which is singly adopted.

The specific steps for detecting the cell proliferation effect are as follows:

i. cells in the logarithmic growth phase were taken and H460 cells were seeded in 96-well plates (96-well cell culture plates) at a cell concentration of 3X 104/ml, 100ul per well.

After 24 hours of adherent growth of the cells, different concentrations of domperidone and paclitaxel were added, 3-6 parallel wells per group.

And iii.48 hours later, preparing a CCK-8 mixed solution, namely a CCK-8 reagent: medium (phenol red-free medium DMEM/F12) =1:9 (vol/vol), the liquid in the 96-well plate was carefully pipetted and 100ul of CCK-8 mixture was added.

incubate at 37 ℃ for 1 hour.

v. put into a microplate reader for reading, and its optical density value [ OD (450) value ] is measured at 450 nM.

Statistical analysis.

Example 2

And detecting the condition that the domperidone and the paclitaxel induce H460 cell apoptosis under the single drug and the combined drug.

After cell inoculation, adding medicine, culturing for 48 hours, collecting cells, treating the cells according to the specification of an annexin V/PI apoptosis kit of BD company, and detecting apoptosis by flow analysis, wherein the annexin V +/PI-cells represent cells in early apoptosis stage, the annexin V +/PI + represent cells in late apoptosis stage, and the annexin V-/PI + represent necrotic cells. The results are shown in FIGS. 4 and 5, A: domperidone 5 μ M, B: paclitaxel 0.1uM, C: domperidone 5 μ M + paclitaxel 0.1uM, and the ordinate of FIG. 5 is the percentage of apoptosis (percentage of cells in early apoptosis + percentage of cells in late apoptosis). It can be seen that the single and combined administration can cause the increase of annexin V + cells to different degrees, and the single administration of domperidone can cause the apoptosis of H460 cells.

The effect of the further combined paclitaxel administration is far better than the result of the single paclitaxel administration, the apoptosis is increased from 13.95 percent to 39.47 percent when the single paclitaxel administration is adopted, and the remarkable synergistic effect of the domperidone and the paclitaxel is shown.

The method for detecting the apoptosis by using the annexinV/PI method comprises the following specific steps:

i. taking cells in logarithmic phase of growth to inoculate in a cell culture dish (6-hole cell culture plate), culturing for 24 hours, adding domperidone and paclitaxel according to a preset concentration (A: 5 mu M of domperidone; B: 0.1uM of paclitaxel; C: 5 mu M of domperidone + 0.1uM of paclitaxel) after the cells adhere to the wall, and treating for 48 hours.

And ii, diluting 10 x Binding Buffer in the apoptosis kit by deionized water according to the amount required by the experiment, namely the Binding Buffer: deionized water = 1.

Cell collection: the cells were digested with pancreatin without EDTA at a mass concentration of 0.25% and centrifuged at 1000 rpm for 5 minutes at room temperature.

Cell washing: resuspend cells with 4 ℃ pre-cooled PBS, centrifuge for 5 min at 1000 rpm, wash cells, repeat and wash with 1 × Binding Buffer.

Add 300ul 1x Binding Buffer to resuspend the cells.

Annexin V-FITC labeling: 5ul of annexin V-FITC was added thereto and mixed well, and then the mixture was protected from light.

Put into a rotator and incubate for 10 to 15 minutes at room temperature protected from light.

Pi labeling: 5ul of PI dye was added 5 minutes before loading.

And ix, adding 200ul of 1xBinding Buffer before the on-machine analysis.

x. in-machine (flow cytometer, bio-Rad Co.). (the sample is put on a machine for analysis within 4 hours after being prepared and is stored in a dark place at the temperature of 2-8 ℃).

Example 3

H460 tumor cells are inoculated to an immune-deficient SCID mouse (purchased from Beijing Wittingeri Hua laboratory animal technology Co., ltd.) to observe the tumorigenesis in vivo, so as to detect the in vivo effect of the single use and the combined use of the domperidone and the paclitaxel on the tumor cells.

First, H460 cells were cultured in a tumor cell culture medium DMEM or RPMI-1640 for 2 days, tumor cells were collected, digested (a medium of twice the volume was added immediately after 1 minute of pancreatin digestion with a mass concentration of 0.25%) was added), counted, cell density was adjusted according to the optimum cell inoculum amount found in the preliminary experiment and gradient-diluted to 5 × 105, and cell suspension and an artificial cell basement membrane (Matrialgel) were mixed in accordance with a ratio of 1:1 volume ratio, the total volume of inoculation is 100ul, and the inoculation is carried out to the underarm subcutaneous part of the mouse. The domperidone group (6) was administered daily at a dose of 0.25mg/g, and the paclitaxel group (6) was administered once every three days at a dose of 0.05mg/g. Domperidone + paclitaxel (6) was administered at 0.25mg/g domperidone per day and paclitaxel at 0.05mg/g every three days. After 20 days of administration (20 doses of domperidone, 7 doses of paclitaxel) the mice were sacrificed, and the tumors were dissected and removed, photographed, measured, and weighed. The results are shown in FIGS. 6 and 7.

From the figure, it can be clearly seen that the tumors of mice in the group administered with domperidone alone or in combination with paclitaxel are smaller than those in the control group (the average tumor weight of the control group is 0.9343g, the average tumor weight of domperidone alone is 0.7915g, the average tumor weight of paclitaxel alone is 0.6423g, and the average tumor weight of paclitaxel in combination with domperidone is 0.3225 g), which confirms the effect of domperidone alone and the synergistic effect of paclitaxel in combination.

The invention takes domperidone and the combination of domperidone and paclitaxel as research objects. The application effect of domperidone on the cancer treatment drug alone and the synergistic effect of the domperidone and paclitaxel in the cancer treatment drug are verified by detecting the proliferation inhibition effect, detecting the apoptosis condition and carrying out in vivo experiments on the axilla inoculated tumor cells of SCID mice.

Example 4

The cell proliferation inhibitory effect of domperidone alone was tested on the non-small cell lung cancer cell line H460 at different concentrations under the same experimental conditions using the test method of example 1.

As can be seen from fig. 8, the inhibitory effect was enhanced with the increase in the administered concentration of domperidone. When the concentration thereof is more than 4uM, a desired inhibitory effect can be achieved, preferably 4 to 10uM.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. Use of domperidone as the sole active ingredient for the preparation of a medicament for the treatment of cancer, said cancer being non-small cell lung cancer.

2. Use according to claim 1, characterized in that the active ingredient of the medicament is present in free form or in the form of a pharmaceutically acceptable salt.

3. Use according to claim 1 or 2, wherein the medicament comprises at least one pharmaceutically acceptable carrier.

4. The use according to claim 3, wherein the medicament is administered intravenously, orally or topically.

5. Use of domperidone in combination with paclitaxel for the manufacture of a medicament for the treatment of cancer, said cancer being non-small cell lung cancer; in the unit preparation of the medicine, the mass ratio of the domperidone to the paclitaxel is 5: (0.1-1) or 7: (0.1-1).

6. Use of domperidone in combination with paclitaxel for the manufacture of a medicament for the treatment of cancer, said cancer being non-small cell lung cancer; in the unit preparation of the medicine, the dosage of the domperidone is 5 mu M or 7 mu M, and the dosage of the paclitaxel is 0.03 mu M.

7. Use according to claim 5 or 6, wherein the active ingredient of the medicament is present in free form or in the form of a pharmaceutically acceptable salt and/or wherein the medicament comprises at least one pharmaceutically acceptable carrier.

8. The use according to claim 5 or 6, wherein domperidone is administered simultaneously or separately with the paclitaxel.

9. The use according to claim 8, wherein the medicament is administered by one or more of intravenous injection, oral administration or topical administration.