CN116115635A

CN116115635A - Application of low molecular citrus pectin alone or in combination with platinum drugs in preparation of anti-tumor products

Info

Publication number: CN116115635A
Application number: CN202310205562.9A
Authority: CN
Inventors: 郭秀丽; 孙超
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2023-03-06
Filing date: 2023-03-06
Publication date: 2023-05-16

Abstract

The invention belongs to the technical field of medicines, and discloses application of low-molecular citrus pectin in preparing an anti-tumor product by singly or jointly using platinum medicines. According to the invention, through researches, the MCP is reported to activate SOD3 activity of gastric cancer cells for the first time, the killing effect of oxaliplatin on gastric cancer cells can be enhanced, the risk of CIPN occurrence can be reduced by reducing the dosage of oxaliplatin, and simultaneously, the CIPN symptoms can be relieved by activating SOD3 activity and reducing ROS level. Lays an experimental foundation for researching the modified low molecular citrus pectin in the field of middle and late gastric cancer treatment, provides a new field of view, and has good practical application value.

Description

Application of low molecular citrus pectin alone or in combination with platinum drugs in preparation of anti-tumor products

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of low-molecular citrus pectin in preparing an anti-tumor product by singly or jointly using platinum medicines.

Background

The modified low molecular citrus pectin (Modified citrus pentin, MCP) mainly contains soluble dietary fiber pectin, is a compound polysaccharide containing rich galactose residues, is obtained by pH modification and high Wen Xiushi of citrus fruit components, has a molecular weight of about 8kDa, has low esterification degree, has no side chain, and is easy to be absorbed by gastrointestinal tracts to enter blood circulation. In recent years, it has anticancer effectThe effect is increasingly concerned. MCP is currently commercially available, e.g., GCS-100 from GlycoGenesys, ecoNugenics

And Centrax int.

Reactive Oxygen Species (ROS) play an important role in the development and progression of cancer. The resistance of cancer patients to oxaliplatin, or the appearance of oxaliplatin-induced peripheral neuropathy (CIPN) symptoms, is a significant cause of clinical necessity for replacement of oxaliplatin-based chemotherapy regimens. Oxaliplatin-induced ROS production is considered to be one of the important mechanisms of drug resistance, as well as the main mechanism by which oxaliplatin induces CIPN production. Potentiating oxaliplatin anti-tumor effects and reducing associated toxic effects by combination therapy is an important clinical therapeutic strategy. MCP can remarkably improve the superoxide dismutase (SOD) level and reduce the ROS level, has no obvious toxic or side effect, however, has not seen the relevant reports of the functions of enhancing the anti-tumor effect and reducing the toxic or side effect of chemotherapy in combination with the anti-tumor treatment related fields such as oxaliplatin and the like to achieve the sensitization and toxicity reduction effects of the chemotherapeutic drugs.

Disclosure of Invention

Aiming at the prior art, the invention provides the application of the modified low-molecular citrus pectin combined with oxaliplatin in anti-tumor treatment through long-term technical and practical exploration. According to the invention, researches show that the modified low-molecular citrus pectin can remarkably improve the in-vivo and in-vitro killing effect of oxaliplatin on gastric cancer cells, and relieve peripheral neuropathy (CIPN) symptoms caused by oxaliplatin by up-regulating SOD3 and reducing ROS level, so that the synergistic and toxicity-reducing effects are exerted.

The invention is realized by the following technical scheme:

in a first aspect of the invention, there is provided the use of low molecular citrus pectin in the preparation of a SOD activator product. Preferably, the SOD activator is specifically an SOD3 activator for gastric cancer, pancreatic cancer, breast cancer, lung adenocarcinoma or cardiovascular and cerebrovascular diseases. Further, the dosage of the low molecular citrus pectin for human use is 100-200 mug/kg.d.

In a second aspect, the invention provides the use of low molecular citrus pectin in the preparation of an anti-tumor co-product for alleviating the symptoms of platinum-induced peripheral neuropathy CIPN.

In a third aspect, the invention provides an application of a low molecular citrus pectin and platinum drug combination in preparing an anti-tumor product.

As a further illustration of the present invention, the low molecular citrus pectin is a modified low molecular citrus pectin MCP prepared separately from a citrus fruit extract.

As a further illustration of the invention, the products include pharmaceuticals, nutraceuticals and/or foods comprising carriers, excipients and diluents in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, or injections in the form of sterile injectable solutions. The product may be a medicament which may also contain suitable amounts of carriers, excipients and diluents commonly used. Further, the composition can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and sterile injectable solutions by a usual method. Non-pharmaceutically active ingredients such as carriers, excipients, and diluents which may be included in the present invention are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria. The carriers, excipients and diluents of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

As a further illustration of the present invention, the platinum-based drugs include, but are not limited to, oxaliplatin, cisplatin and carboplatin, with oxaliplatin being further preferred; the dosage range of the combined drug of the low molecular citrus pectin and the platinum antitumor drug is as follows: oral administration of MCP200 mug/kg.d+ intravenous drip oxaliplatin 65-100mg/m ² The method comprises the steps of carrying out a first treatment on the surface of the MCP200 μg/kg/oral administrationd+ intravenous drip cis-platinum 50-80mg/m ² The method comprises the steps of carrying out a first treatment on the surface of the 200 mu g/kg.d+ intravenous drip carboplatin 200-300mg/m for MCP oral administration ² . The pharmaceutical composition of the present invention may be administered to the body in a known manner. Such as by intravenous systemic delivery or local injection into tissue. Administration may optionally be via intravenous, transdermal, mucosal, or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

As a further illustration of the invention, the tumors include, but are not limited to, gastric cancer, colorectal cancer, esophageal cancer, non-small cell lung cancer, breast cancer, and renal cell carcinoma. The therapeutic effect of the anti-tumor product at least comprises: inhibit proliferation and/or metastasis of tumor cells.

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

the invention explores a new medical application of small molecular citrus pectin, opens up a new application field, and provides the application of the modified low molecular citrus pectin combined with oxaliplatin in anti-tumor treatment. According to the invention, through researches, the MCP is discovered for the first time to activate the activity of gastric cancer cell SOD 3; the anti-tumor effect of the oxaliplatin chemotherapeutic medicine can be increased, the toxic and side effects of chemotherapy can be reduced, the risk of CIPN occurrence can be reduced by reducing the dosage of oxaliplatin, and the CIPN symptoms can be relieved by reducing the ROS level. MCP is used as an anti-tumor synergistic agent and an auxiliary drug, and when the MCP and the auxiliary drug are used together, the MCP and the auxiliary drug play an anti-tumor role under the synergistic effect, so that the MCP has good practical application value.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 is a graph showing that MCP of example 1 of the present invention can up-regulate the activity of gastric cancer cells and SOD of the upper medium and can up-regulate the expression level of SOD 3mRNA of gastric cancer cells; FIG. 1 (a) is a graph showing the result of the SOD activity of MCP on two gastric cancer cells; FIG. 1 (b) is a graph showing the SOD activity of MCP on two gastric cancer cell upper culture media; FIG. 1 (c) is a graph showing the mRNA expression levels of MCP in MGC-803 and SGC-7901 cells SOD 3.

FIG. 2 is a graph showing that the in vivo imaging device of small animals detects MCP and oxaliplatin for combined treatment to inhibit the growth of gastric cancer nude mice transplantation tumor in example 2 of the present invention.

FIG. 3 (a) is a graph showing that MCP of example 3 of the present invention can inhibit the production of ROS induced by oxaliplatin.

FIG. 3 (b) is a graph showing the symptoms of CIPN caused by oxaliplatin in MCP in vivo experiments-Fengfu Rate experiments in which MCP of example 3 of the present invention was tested.

FIG. 3 (c) is a graph showing the symptoms of CIPN caused by oxaliplatin in MCP in vivo experiments-detection of thermal pain sensation experiments in accordance with example 3 of the present invention.

FIG. 3 (d) is a graph showing the effects of MCP in vivo experiments in animals in MCP-cold plate experiments in accordance with example 3 of the present invention to alleviate CIPN symptoms caused by oxaliplatin.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

The invention will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the invention. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.

In one embodiment of the present invention, modified low molecular citrus pectin is provided that upregulates gastric cancer MGC-803 and SGC-7901 cellular SOD activity.

In yet another embodiment of the present invention, modified low molecular citrus pectin is provided that upregulates gastric cancer MGC-803 and SGC-7901 cell upper layer culture medium SOD activity.

In yet another embodiment of the present invention, modified low molecular citrus pectin is provided that upregulates gastric cancer MGC-803 and SGC-7901 cell SOD 3mRNA expression levels.

In yet another embodiment of the present invention, a combination of modified low molecular citrus pectin and oxaliplatin is provided that reduces the IC of oxaliplatin against gastric cancer cells ₅₀ Values.

In yet another embodiment of the present invention, there is provided a combination of modified low molecular citrus pectin that enhances the growth inhibitory effect of oxaliplatin on gastric cancer transplants.

In yet another embodiment of the invention, modified low molecular citrus pectin is provided that can reverse oxaliplatin-induced elevated levels of ROS in gastric cancer cells.

In yet another embodiment of the invention, there is provided the use of a modified low molecular citrus pectin for alleviating symptoms of CIPN induced by oxaliplatin.

Example 1

MCP activates gastric cancer cell and SOD activity of culture medium

(1) 1X 106MGC-803 or SGC-7901 cells were seeded in 6-well plates and treated with MCP (MGC-803 cells given mg/mL, SGC-7901 cells given 6 mg/mL) and/or DETC (20. Mu.M), respectively, for 48 hours.

(2) The cells were transferred to EP tubes by washing 2 times with pre-chilled Phosphate Buffered Saline (PBS) and thoroughly ground on ice. Centrifuging 8000g of homogenate at 4 ℃ for 10 minutes, and taking the supernatant as a cell sample to be detected; transferring the upper cell culture solution to a 10kDa ultrafiltration tube, centrifuging at 4deg.C and 3000g, collecting 4 times of concentrated solution from MGC-803 cell culture solution, and collecting 8 times of concentrated solution from SGC-7901 cell culture solution as an upper culture medium sample to be detected.

(3) 158 mu L of SOD detection buffer solution, 1 mu L of NBT and 1 mu L of enzyme solution are uniformly mixed to prepare 160 mu L of NBT/enzyme working solution, and then a sample to be detected and a reaction starting working solution are added and fully and uniformly mixed. After incubation at 37℃for 30 minutes, the absorbance was measured at 560 nm.

The total SOD activity was calculated as follows:

% activity (cells) = (a) _{Blank 1} -A _{Sample of} )×100％/(A _{Blank 1} –A _{Blank 2} )

% activity (medium) = ((a) _{Blank 1} -A _{Blank 2} )-(A _{Sample of} –A _{Blank 3} ))×100％/(A _{Blank 1} –A _{Empty space} White color ₂ )

The result shows that: as shown in fig. 1 (a), MCP can up-regulate SOD activity of two gastric cancer cells, while DETC can inhibit SOD activity of gastric cancer cells in control group and MCP treated group. As shown in FIG. 1 (b), MCP can up-regulate the SOD activity of the gastric cancer cell upper medium, while DETC can inhibit the SOD activity of the gastric cancer cell upper medium in the control group and the MCP treatment group.

MCP can obviously improve the expression level of gastric cancer cell SOD 3mRNA

(1) 1X 106MGC-803 or SGC-7901 cells were seeded in 6-well plates and treated with MCP (MGC-803 cells given 5mg/mL, SGC-7901 cells given 6 mg/mL) and/or DETC (20. Mu.M), respectively, for 48 hours.

(2) Cellular RNA was extracted, cDNA was synthesized by reverse transcription, and the expression level of SOD 3mRNA was detected by PCR.

The result shows that: as shown in FIG. 1 (c), MCP can significantly increase the mRNA level of SOD3 in MGC-803 and SGC-7901 cells, but has no obvious effect on the mRNA expression level of SOD1 and SOD2, while DTEC can inhibit the expression of SOD1-3 mRNA.

Description: DETC, O-diethylthiophosphoryl chloride (Diethyl chlorothiophosphate), CAS:2524-04-1.Superoxide Dismutase an excellar (SOD 3) is one of the SOD isozymes, and the SOD3 is Cu ²⁺ 、Zn ²⁺ Is used as an auxiliary group, and the auxiliary group is a prosthetic group,belongs to Cu-Zn-SOD, can eliminate reactive oxygen species generated by in vivo metabolism, and can resist the damage of superoxide ions in the internal and external environments.

Example 2

MCP can enhance the killing effect of oxaliplatin on gastric cancer cells in vitro experiments

(1) Cells of 3X 103MGC-803 or SGC-7901 were seeded in 96-well plates and treated for 24 hours at various concentrations of oxaliplatin (0-80. Mu.M) and/or MCP (5 mg/mL for MGC-803 cells and 6mg/mL for SGC-7901 cells).

(2) Adding 20 μl MTT into each well, shaking, homogenizing, and adding 96-well plate at 37deg.C and 5% CO ₂ Incubate in incubator for 4 hours. After blotting the medium, 200 μl DMSO was added to each well of the 96-well plate and shaken well enough to measure absorbance (i.e. OD value) at 570nm using an enzyme-labeled instrument.

Growth inhibition% = (OD _{Blank group} -OD _{Administration group} )/OD _{Blank group}

(3) Calculating the growth inhibition rate of oxaliplatin and/or MCP with different concentrations to MGC-803 and SGC-7901 gastric cancer cells, and further calculating half Inhibition Concentration (IC) according to the obtained growth inhibition rate ₅₀ Values.

Table 1 shows the killing effect of oxaliplatin on gastric cancer cells by MCP according to example 2 of the present invention.

The result shows that: as shown in Table 1, the combination of MCP significantly reduces the IC of oxaliplatin against gastric cancer cells ₅₀ The value of the oxaliplatin enhances the killing effect of oxaliplatin on gastric cancer cells.

In vivo experiments with MCP in combination with oxaliplatin

(1) Taking 20 male mice, randomly dividing into 4 groups, establishing a nude mice armpit transplantation tumor model, and setting a control group, an oxaliplatin (15 mg/kg) group, an oxaliplatin (30 mg/kg) group and oxaliplatin (15 mg/kg) +MCP (200 mg/kg) when the tumor volume reaches 80-100 mm ³ At that time, packet processing is given.

(2) Control group, 5% glucose injection was intraperitoneally injected every 7 days for a total of 3 times, and tumor major and minor diameters were recorded every 3 days

(3) Oxaliplatin (15 mg/kg) groups, oxaliplatin 5mg/kg per 7 days, were intraperitoneally injected, and tumor long and short diameters were recorded every 3 days.

(4) Oxaliplatin (30 mg/kg) was used in groups, with oxaliplatin 10mg/kg per 7 days of intraperitoneal injection, and tumor long and short diameters were recorded every 3 days.

(5) In the combination group, 50. Mu.l of 4% MCP per day was lavaged, 5mg/kg oxaliplatin was intraperitoneally injected every seven days, and tumor long and short diameters were recorded every 3 days.

(6) On day 24, the proliferation of the transplanted tumor was detected by a small animal in vivo imager, and the result is shown in fig. 2 in detail.

The results show that: MCP can enhance the growth inhibitory effect of oxaliplatin on gastric cancer transplants.

Example 3

Mcp reversible oxaliplatin-induced elevation of gastric cancer cell ROS levels

(1) MGC-803 and SGC-7901 cells were seeded into 12-well plates and treated with oxaliplatin (15. Mu.M, 30. Mu.M) and/or MCP (5 mg/mL for MGC-803 cells and 6mg/mL for SGC-7901 cells).

(2) According to the instructions of the active oxygen detection kit, 2',7' -dichlorofluorescein diacetate (DCFH-DA) was diluted 1:1000 with serum-free medium to a final concentration of 10. Mu.M. The cell culture broth was aspirated, and a suitable volume of diluted DCFH-DA was added and incubated at 37℃for 20 minutes in the dark. Subsequently, photographs were taken under a fluorescence inverted microscope.

The result shows that: as shown in fig. 3 (a), oxaliplatin can up-regulate the production of ROS by gastric cancer cells and has a positive correlation with oxaliplatin concentration, while MCP can inhibit the production of ROS in gastric cancer cells of the control group and gastric cancer cells after oxaliplatin treatment.

In vivo experiments with MCP to alleviate symptoms of oxaliplatin-induced CIPN

(1) Mice of each group in example 2.

(2) Fengfu rad experiments, 24 hours after each oxaliplatin administration. Evaluation was performed using Von Frey (Fengfu rad) filaments to stimulate the midplantar region of the nude mice' forepaw, with the ambulation or licking of the stimulated paw being considered a positive response.

(3) Thermal pain was experienced by the experiment 48 hours after each oxaliplatin administration. The experimental nude mice were gently tethered, the hind paw soles of the test were exposed to a glass platform set at 50±1 ℃ and the time taken to first lift the licking paw or retraction reflex was recorded.

(4) The cold plate experiments were performed 72 hours after each oxaliplatin administration. During the whole test, unconstrained nude mice were exposed to cold plates with surface temperatures kept constant at 3±1 ℃ for 1 minute, and the number of tremors and/or licks was monitored.

The result shows that: as shown in fig. 3 (b-d), oxaliplatin reduces the anterior paw withdrawal threshold in mice, extends the time of hindpaw withdrawal after heating, and reduces the number of hindpaw tremors and/or licks after plantar cooling, suggesting that oxaliplatin reduces but enhances sensitivity to physiological pain in mice and that the effects on these CIPN-related symptoms are dose-dependent. The mice were significantly relieved of CIPN-related symptoms after MCP combination compared to oxaliplatin (15 mg/kg) group. These results suggest that MCP reduces/alleviates oxaliplatin-induced CIPN symptoms by up-regulating SOD3, reducing ROS levels, but does not diminish the ability to inhibit gastric cancer graft growth.

Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the invention, but rather, it is intended to cover all modifications or variations within the scope of the invention as defined by the claims of the present invention.

Claims

1. The application of low molecular citrus pectin in preparing SOD activator product is provided.

2. The use according to claim 1, wherein the SOD activator is in particular a SOD3 activator of gastric cancer, pancreatic cancer, breast cancer, lung adenocarcinoma or cardiovascular and cerebrovascular diseases.

3. Use according to claim 2, wherein the low molecular citrus pectin is administered in a dosage of 100-200 μg/kg-d for humans.

4. Application of low molecular citrus pectin in preparing antitumor auxiliary product for relieving peripheral neuropathy CIPN symptoms induced by platinum drugs is provided.

5. The application of low molecular citrus pectin and platinum drugs in preparing antitumor products.

6. The use according to any one of claims 1-5, wherein the low molecular citrus pectin is a modified low molecular citrus pectin MCP prepared separately from citrus fruit extract.

7. The use according to any one of claims 1 to 5, wherein the product comprises a pharmaceutical, nutraceutical and/or food product, the product comprising a carrier, an excipient and a diluent in the form of a powder, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations of sprays or injections in the form of sterile injectable solutions.

8. Use according to claim 4 or 5, characterized in that: such platinum-based agents include, but are not limited to, oxaliplatin, cisplatin, and carboplatin, with oxaliplatin being further preferred; the dosage range of the combined drug of the low molecular citrus pectin and the platinum antitumor drug is as follows: oral administration of MCP200 mug/kg.d+ intravenous drip oxaliplatin 65-100mg/m ² The method comprises the steps of carrying out a first treatment on the surface of the Oral administration of MCP200 mug/kg.d+ intravenous drip cis-platinum 50-80mg/m ² The method comprises the steps of carrying out a first treatment on the surface of the 200 mu g/kg.d+ intravenous drip carboplatin 200-300mg/m for MCP oral administration ² 。

9. The use according to claim 4 or 5, wherein the tumor includes, but is not limited to, gastric cancer, colorectal cancer, esophageal cancer, non-small cell lung cancer, breast cancer and renal cell carcinoma.

10. The use according to claim 5, wherein the therapeutic effect of the anti-tumor product comprises at least: inhibit proliferation and/or metastasis of tumor cells.