CN110743016A

CN110743016A - Combined pharmaceutical composition for resisting colorectal cancer and application thereof

Info

Publication number: CN110743016A
Application number: CN201911207465.3A
Authority: CN
Inventors: 朱浩; 朱永亮; 宋伟群; 徐佳怡
Original assignee: Suzhou Puruisen Gene Technology Co Ltd
Current assignee: Suzhou Puruisen Gene Technology Co Ltd
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2020-02-04
Also published as: CN112274645A

Abstract

The invention relates to a combined drug composition for resisting colorectal cancer and application thereof, wherein the combined drug composition comprises β -glucuronidase inhibitor and an anti-tumor drug, and the combined drug composition creatively combines β -glucuronidase inhibitor and the anti-tumor drug for use, has stronger anti-colorectal cancer activity than that of a single anti-tumor drug, and provides a new strategy and thought for treating the colorectal cancer.

Description

Combined pharmaceutical composition for resisting colorectal cancer and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a combined medicine composition for resisting colorectal cancer and application thereof.

Background

Colorectal cancer is a common malignant tumor of the digestive system, and has hidden morbidity and high mortality rate. Irinotecan is a first line of clinical use for the treatment of colorectal cancer. Irinotecan is metabolized in the liver, which causes great burden to the liver, and most of patients taking irinotecan have symptoms of nausea, appetite reduction, regurgitation, dyspepsia and the like and delayed diarrhea, so that the abdominal peristalsis of the patients occurs, and the death of the patients is possibly caused seriously; or cause neutropenia of the patient, resulting in reduced immunity.

Fluvoxamine Maleate (FM) is used as an antidepressant clinical drug, and selectively inhibits the reuptake of the central nervous presynaptic membrane on 5-hydroxytryptamine to increase the concentration of the 5-hydroxytryptamine in the presynaptic membrane gap, thereby playing a role in resisting depression and anxiety.

In the prior art, there are also reports of strategies related to colorectal cancer treatment, CN107158367A discloses a preparation method and application of a colorectal cancer stem cell vaccine, and a CD133+ colorectal cancer stem cell vaccine is prepared by using postoperative tumor tissue cells derived from a colorectal cancer patient, a human colorectal cancer cell line Lovo or a mouse colorectal cancer cell line CT26 through a repeated freeze thawing method.

On the basis, the invention also provides a novel gene vector targeting the AMPK α 1 gene and a method for wrapping interference gene fragments by the gene vector, and the obtained product can specifically silence and inhibit the expression of the AMPK α gene, so that the aims of inhibiting colorectal cancer recurrence and metastasis and treating colorectal cancer are fulfilled.

CN109589407A discloses mesoporous ruthenium nanoparticles for targeted therapy of colorectal cancer, and a preparation method and application thereof. Dissolving ruthenium trichloride into a perchloric acid solution, adding a nonionic surfactant, and mixing to obtain a mixed solution I; (2) adding the amino-modified colloidal silica nanoparticles into the mixed solution I, and mixing to obtain a mixed solution II; (3) adding a sodium borohydride solution into the mixed solution II, carrying out ultrasonic reaction, washing with water after the reaction is finished, and centrifuging to obtain an intermediate product; (4) and dispersing the intermediate product into a hydrofluoric acid solution, and then washing and drying to obtain the mesoporous ruthenium nano particle for the targeted therapy of the colorectal cancer. The ruthenium nano particle prepared by the invention has large specific surface area, can load ruthenium complex and coupling bispecific antibody, and is used for targeted anti-colorectal cancer combined photo-thermal and immunotherapy.

However, reports on colorectal cancer treatment strategies in the prior art are limited, and most of the treatment effects are not remarkable, so that the development of a new colorectal cancer treatment strategy is very meaningful.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an anti-colorectal cancer combined pharmaceutical composition and application thereof, wherein the combined pharmaceutical composition has stronger anti-colorectal cancer activity than irinotecan alone, and provides a new strategy and thought for the treatment of colorectal cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a pharmaceutical combination against colorectal cancer, comprising β -glucuronidase inhibitor and an anti-tumor drug.

The combined drug composition creatively combines the antitumor drug and the β -glucuronidase inhibitor, has stronger anti-colorectal cancer activity than that of a single antitumor drug, and provides a new strategy and thought for the treatment of colorectal cancer.

Preferably, the β -glucuronidase inhibitor comprises fluvoxamine maleate.

Preferably, the anti-tumor drug comprises irinotecan.

As a preferred technical scheme, the combined pharmaceutical composition combines irinotecan for first-line clinical colorectal cancer and fluvoxamine maleate for an antidepressant clinical drug, has higher biological safety, has stronger anti-colorectal cancer activity than that of irinotecan alone, and provides a new strategy and thought for the treatment of colorectal cancer, wherein fluvoxamine maleate inhibits the activity of β -glucuronidase so as to reverse the action of irinotecan originally promoting the activity of β -glucuronidase on β -glucuronidase, because β -glucuronidase can possibly reduce the drug effect of irinotecan and simultaneously trigger some side effects, and the composition finally promotes the effect of the combined pharmaceutical composition in the treatment of colorectal cancer through the reverse action.

Preferably, the pharmaceutical composition for combination is a single compound formulation.

Preferably, the combination pharmaceutical composition is a combination of two separate formulations, a fluvoxamine maleate formulation and an irinotecan formulation.

Preferably, the two separate formulations are administered simultaneously.

Preferably, the two separate formulations are administered sequentially.

The combined pharmaceutical composition can be in a single compound preparation form, and can also be a combination of two separate preparations; when two separate formulations are combined, the administration may be simultaneous or sequential, for example, irinotecan may be administered first, with the interval between fluvoxamine maleate, with irinotecan, or with the interval between fluvoxamine maleate and irinotecan, alternately.

In the present invention, the dosage form of the preparation includes any pharmaceutically acceptable dosage form. Such as tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or dropping pills and the like.

Preferably, the pharmaceutical composition for combination further comprises any one or the combination of at least two of pharmaceutically acceptable excipients.

The combined pharmaceutical composition can be administered alone or in combination with adjuvants to make into appropriate dosage forms, wherein the adjuvants comprise one or a combination of at least two of diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, pH regulator, antioxidant, bacteriostatic agent or buffer. Combinations of the at least two such as diluents and excipients, emulsifiers and co-solvents, fillers and binders and wetting agents, and the like.

In the present invention, the administration route of the combination pharmaceutical composition includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intraperitoneal injection.

Preferably, the pharmaceutical combination is a pharmaceutical combination carried on a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier comprises a liposome, micelle, dendrimer, microsphere or microcapsule.

In a second aspect, the present invention provides a use of the above anti-colorectal cancer combination pharmaceutical composition in the preparation of an anti-colorectal cancer drug.

In a third aspect, the invention provides an application of fluvoxamine maleate in preparing β -glucuronidase activity inhibitor.

In the fourth aspect, the invention also provides a novel anti-colorectal cancer combination therapy, which is a combination therapy of fluvoxamine maleate and irinotecan, the combination of fluvoxamine maleate and irinotecan has stronger anti-colorectal cancer activity than that of irinotecan singly used as a conventional medicament, can effectively prolong the survival period of tumor-bearing mice, provides a new strategy and thought for the treatment of colorectal cancer, and can remarkably inhibit the activity of β -glucuronidase and exert more effective anti-colorectal cancer activity.

Preferably, the route of administration of fluvoxamine maleate comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intraperitoneal injection.

Preferably, the route of administration of irinotecan comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intraperitoneal injection.

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

as a preferable technical scheme of the invention, the anti-tumor drug is irinotecan, the β -glucuronidase inhibitor is fluvoxamine maleate, and the fluvoxamine maleate enables the effect of irinotecan originally promoting the activity of β -glucuronidase to β -glucuronidase to be reversed by inhibiting the activity of β -glucuronidase, so that the effect of the combined drug composition in treating colorectal cancer is finally promoted.

Drawings

FIG. 1 is a standard graph in example 1;

FIG. 2 is a graph of the relative activity of β -glucuronidase in example 1 as a function of fluvoxamine maleate concentration;

FIG. 3 is a graph showing the comparison results of the activity of β -glucuronidase at the caecum site in each group in example 2;

FIG. 4 is a graph showing the results of comparing the activity of β -glucuronidase at ileum sites in each group in example 2;

FIG. 5 is a colon pathological section of each group of mice in example 3 (a is a normal group, b is a model group, c is a fluvoxamine maleate group, d is an irinotecan group, and e is a combination group).

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The experimental materials and reagents required for examples 1 and 2 are as follows:

test animals: male C57BL/6 mice, 18-22g in body weight, certification number: (Jing) scxk20060008, purchased from the laboratory animal center of the department of medicine, Beijing university.

Chemicals and reagents β -glucuronidase powder (Sigma, USA), 4-nitrophenyl β -D-glucuronide (PNPG) (Sigma, USA), and Azomethane (AOM)/dextran sulfate (DSS) (Sigma, USA).

Chemotherapeutic drugs: fluvoxamine maleate, purchased from the Lizhu group Lizhu pharmaceutical factory; irinotecan, purchased from belleck Chemicals, usa.

Example 1

Evaluation test of inhibition effect of fluvoxamine maleate on β -glucuronidase activity

The specific method comprises preparing β -glucuronidase powder (Sigma, USA) into 5U/. mu.L solution, adding 33. mu.L into 1.617mL Phosphate Buffer Solution (PBS), mixing, adding 100. mu.L of the above mixture into 33 wells of a 96-well plate, preparing fluvoxamine maleate into a concentration range of 0.0005-10. mu.M with PBS, adding 100. mu.L each of fluvoxamine maleate with different concentrations into a 96-well plate containing β -glucuronidase, making 3 duplicate wells for each concentration, adding 2. mu.L (25mM) of reaction substrate 4-nitrophenyl β -D-glucuronide (PNPG) (Sigma, USA), reacting at 37 ℃ in the dark for 1h, diluting 4-nitrophenol (PNP) with 1 XPBS, diluting with 1-200. mu.M, using standard solution of 100. mu.L each of the above standard solution with different concentrations, adding 150. mu.L of 0.2M Na, each₂CO₃Mixing for 1min, and measuring absorbance at 405nm to obtain standard curve, as shown in FIG. 1Shown in the figure. The equation for the standard curve obtained is y ═ 0.0042x +0.069, R²0.9948, and calculating the IC50, as shown in figure 2, the IC50 of the fluvoxamine maleate is 0.3023 mu M, which proves that the fluvoxamine maleate β -glucuronidase activity has obvious inhibition effect.

Example 2

In vivo test I

The specific method comprises the following steps: the fluvoxamine maleate is used to prepare the required concentration by using normal saline. The experimental animals are raised in cages with 8 animals per cage, the cages are cleaned once every three days, the experimental animals are adaptively raised for 1 week, the room temperature of a laboratory is 20-22 ℃, the relative humidity is 40% -60%, ventilation is carried out by a ventilation fan, and the natural light source is 12 h/day.

The colorectal cancer model was established using an Azomethane (AOM)/dextran sulfate (DSS) model induction method for 90 mice: after the injection is performed by using 12.5mg/kg AOM (argon oxygen decarburization) in an abdominal cavity, 3% DSS (sodium dodecyl sulfate) is used for drinking water for 7 days, and then normal drinking water is changed into drinking water for 14 days, so that 1 cycle is performed, and 2 cycles are performed in total; 2/5 mice developed colonic tubular adenomas starting at week 3, 3/5 mice developed adenocarcinomas at

week

4, and 100% of mice developed adenocarcinomas at week 6. 30 colon cancer model mice were selected and randomly divided into 3 groups of 10 mice each. The first group was injected intraperitoneally with normal saline, the second group with irinotecan (36.8mg/kg), and the third group with irinotecan (36.8mg/kg) + fluvoxamine maleate (2mg/kg) (co-injected), at an injection rate of 20mL/kg, once daily for 7 consecutive days.

Mice were sacrificed and dissected, equal amounts of cecum and ileum contents were taken, homogenized with 5mL of 1 x PBS solution, centrifuged at 4000rpm for 10min, the supernatant was placed in a sterile EP tube, 2mL of the supernatant to be tested was taken, and β -glucuronidase activity was tested, using the PNPG method.

The results are shown in FIGS. 3 and 4 (FIG. 3 is a graph showing the comparison results of the cecum β -glucuronidase activity of each group, and FIG. 4 is a graph showing the comparison results of the ileum β -glucuronidase activity of each group).

As can be seen from FIGS. 3 and 4, injection of irinotecan alone resulted in an increase in β -glucuronidase activity and combination of irinotecan with fluvoxamine maleate resulted in a significant decrease in β -glucuronidase activity relative to the control.

Example 3

In vivo test II

1. Experimental Material

1.1 Experimental instruments

Centrifuge (SiGMA), analytical balance (ME204 Mettler-Torledo apparatus (Shanghai) Co., Ltd.), PH meter, ophthalmic forceps, mouse gavage needle, and electrophoresis tank (BIO-RAD Co., Ltd.).

1.2 Experimental reagents

Aom (sigma), physiological saline, dss (sigma), irinotecan (fei china), fluvoxamine maleate (lizhu corporation), mouse TNF-a kit (nanjing institute of bioengineering), and mouse IL-6 kit (nanjing institute of bioengineering).

1.3 Experimental animals

SPF male C57BL/6 mice, weighing 18-22g, were purchased from the center of laboratory animals of the Jiangnan medical institute.

2. Experimental methods

2.1 mice grouping and dosing

The mice were kept in an SPF environment and were exposed to light for 12 h. After 1 week of adaptive feeding, 5 groups of 12 animals were randomly assigned to each group for a total of 60 animals, and the groups were respectively assigned to the normal group (group a): 0.9% normal saline is injected into the abdominal cavity. Model group (group b): injecting 0.9% normal saline into abdominal cavity; fluvoxamine maleate group (group c): injecting fluvoxamine into the abdominal cavity at 2 mg/kg; irinotecan group (group d): irinotecan is injected into the abdominal cavity at 80 mg/kg; irinotecan in combination with fluvoxamine maleate (group e): irinotecan 80mg/kg + fluvoxamine maleate 10mg/kg are injected intraperitoneally; the gavage amount is 20mL/kg, the administration is started 1 week after the molding, and the injection is performed once a day for 7 days continuously.

2.2 establishment of mouse colorectal cancer model

The method is induced by adopting an Azomethane (AOM)/dextran sulfate (DSS) model: after the injection, 3% DSS is used for 7 days, and then normal water is used for 14 days, 1 cycle is adopted, 2 times of cycles are carried out, and 6 weeks are spent on molding. Wherein the preparation of the DSS solution comprises the following steps: 10g DSS is dissolved in 500mL sterile drinking water and stored at 4 ℃ for use. Wherein the AOM solution is prepared by the following steps: dissolving AOM in sterile normal saline, storing at-80 deg.C to avoid repeated freeze thawing, and diluting with sterile normal saline.

2.3 determination of TNF-alpha and IL-6 content in Chamber serum

Continuously molding for 6 weeks, taking blood from eyeball 1 hr after the last administration, standing for a while, and centrifuging (3000rpm, 4 deg.C, 10 min). Centrifuging, collecting supernatant, and detecting content change of TNF-alpha and IL-6 by elisa method (according to kit instruction of Nanjing institute of bioengineering).

2.4 weighing of Colon tissue in mice

After the eyes were removed and blood was taken, the mice were sacrificed and placed on ice for dissection, and colon tissues of the mice were taken, colorectal tissues were washed with PBS solution, and weighed. Intestinal tract weight (CW/BW) was expressed as intestinal weight/body weight, while tumor volume was measured.

2.5 pathological section of mouse Colon tissue

Fixing the weighed colon tissue of the mouse in 10% formaldehyde for 24h, washing with PBS, dehydrating with ethanol, standing in xylene I and xylene II, embedding with paraffin, and slicing; and performing HE staining, staining the section with hematoxylin for 5min, differentiating with hydrochloric acid for 10s, washing with water twice, staining with eosin for 3min, dehydrating with ethanol, treating with xylene I and xylene II for 3min and 5min respectively, and observing under microscope.

2.6 statistical analysis

The experimental data were processed using SPSS24.0 for analysis.

3. Results of the experiment

3.1 detection results of TNF-alpha and IL-6 in serum of mice of each group

The inflammatory factors play an important role in the process of colon cancer, and the colon cancer onset condition of mice can be evaluated by detecting the inflammatory factors. The contents of TNF-alpha and IL-6 in each group of serum are detected by using the kit, and the following conclusion is obtained as shown in Table 1: the contents of TNF-alpha and IL-6 in the combined administration group are obviously lower than those of the illitegam group, the fluvoxamine maleate group and the model group, and the significant difference exists between the TNF-alpha and the IL-6 contents and the model group. The combined use of irinotecan and fluvoxamine maleate shows better effect of reducing inflammatory factors than irinotecan alone.

TABLE 1

Note: p <0.05 compared to model group; p <0.01, as compared to model groups.

3.2 results of Colon tissue weight and tumor volume in groups of mice

The data in Table 2 can reflect the inhibition of each group on the tumor, the weight of the colon is an index of the severity of the colon cancer, and the tumor volume can clearly reflect the inhibition of each administration group on the tumor. The results show that the tumor volume and weight of the combined administration group are obviously lower than those of the irinotecan group, the fluvoxamine maleate group and the model group, and the significant difference exists between the tumor volume and the weight of the combined administration group and the model group. Indicating that the combination of irinotecan and fluvoxamine maleate has a better effect in reducing the intestinal weight/body weight (CW/BW) index and tumor volume than irinotecan alone.

TABLE 2

Note: p <0.05 compared to model group; p <0.01, as compared to model groups.

3.3 Colon pathological section results of groups of mice

The results are shown in FIG. 5: the normal group has a complete structure, the model group has fibrosis expression and is damaged, the tumor malignancy is higher, the fibrosis of the combined administration group, the irinotecan group and the fluvoxamine maleate group is relieved, the tumor malignancy is lower than that of the model group, but the tumor malignancy of the combined administration group is lower than that of the irinotecan group and the fluvoxamine maleate group.

In conclusion, the experimental results show that the combined use of irinotecan and fluvoxamine maleate has better anti-colorectal cancer effect compared with the single use of irinotecan.

The applicant states that the present invention is illustrated by the above examples to show an anti-colorectal cancer combination pharmaceutical composition and its application, but the present invention is not limited to the above examples, i.e. it is not meant to imply that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. An anti-colorectal cancer combination pharmaceutical composition, which comprises β -glucuronidase inhibitor and an anti-tumor drug.

2. The anti-colorectal cancer combination according to claim 1, wherein the β -glucuronidase inhibitor comprises fluvoxamine maleate and the anti-neoplastic agent comprises irinotecan.

3. The anti-colorectal cancer combination according to claim 1, wherein the combination is a single combination formulation.

4. The anti-colorectal cancer combination according to claim 1, wherein the combination is a combination of two separate formulations, a fluvoxamine maleate formulation and an irinotecan formulation.

5. The anti-colorectal cancer combination according to claim 4, wherein the two separate formulations are administered simultaneously.

6. The anti-colorectal cancer combination according to claim 4, wherein the two separate formulations are administered sequentially.

7. The anti-colorectal cancer combination according to claim 1, further comprising any one or a combination of at least two of pharmaceutically acceptable excipients.

8. The anti-colorectal cancer combination according to claim 1, wherein the combination is a pharmaceutical combination supported on a pharmaceutical carrier.

9. Use of the anti-colorectal cancer combination pharmaceutical composition according to claim 1 for the preparation of an anti-colorectal cancer medicament.

10. Application of fluvoxamine maleate in preparing β -glucuronidase activity inhibitor is provided.