A lactic acid preparation capable of promoting tumor growth, preparation method and application thereof

Abstract

The present invention discloses a lactic acid preparation that can promote tumor growth, a preparation method and an application thereof. The reagent is composed of lactic acid in different proportions and commonly used pharmaceutical excipients hydroxypropyl-β-cyclodextrin, poloxamer 407 and normal saline. The application of the preparation can significantly promote the proliferation of cells such as breast cancer, lung cancer, liver cancer, colon cancer and leukemia, and the tumor formation and growth of transplanted tumors in mice. It shows that the preparation has the effect of significantly improving the efficiency and success rate of tumor formation in living tumor models, and has important market and application value in industries such as tumor research and anti-tumor drug development.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and specifically relates to a lactic acid preparation capable of promoting tumor growth, a preparation method and application thereof. Background Art

[0002] Tumors are one of the most important disease burdens in modern society and an important topic in medical research. To date, human beings have very limited knowledge of the precise mechanisms of tumor occurrence and development. In the process of scientific research, the lack of patient-derived tumor specimens for studying the growth, invasion, drug resistance and metastasis of tumors in the human body has greatly hindered human beings' in-depth research on the etiology and treatment of tumors. Therefore, the construction of animal xenograft tumor models of human tumor cells is an important topic facing domestic and international medical research. An ideal animal transplant tumor model is crucial for the screening and evaluation of anticancer drugs. Animal models commonly used in tumor research mainly include spontaneous animal tumor models, induced animal tumor models, transgenic animal tumor models and transplanted animal tumor models. At present, there are about 500 animal transplant tumor models in the world, but only less than 40 are suitable for drug screening. Due to the immune rejection in animals, the low tumorigenicity of some tumor cells and the separation from the source growth environment of the tumor, most tumor cells inoculated into animals will have problems such as large number of cells required for inoculation, long experimental cycle, high cost, slow tumor formation, uneven tumor volume, or even no tumor formation.

[0003] Tumor cells can evade the body's immune system and resist apoptosis through regulatory mechanisms at multiple levels, including genetics, epigenetics, and metabolism. Among them, metabolic reprogramming is an important "means" for tumor cells to escape the immune system, such as the famous "Warburg effect", which means that tumor cells will metabolize a large amount of glucose they take in to produce lactic acid through glycolysis even in the presence of sufficient oxygen. Existing studies have shown that lactic acid accumulated in the tumor microenvironment can promote tumor progression. For example, high concentrations of lactic acid can be transported into cells and metabolized as a fuel substrate. Lactic acid can promote tumor invasion and metastasis. Lactic acid plays an important role in promoting tumor angiogenesis and drug resistance. In addition, more and more evidence shows that lactic acid in the tumor microenvironment can play an immunosuppressive function, promoting tumor development by inducing and recruiting immunosuppressive-related cells and molecules.

[0004] Based on the role of lactic acid in the tumor microenvironment, the present application has formulated a new type of lactic acid preparation. Studies have shown that the preparation can promote the proliferation of various tumor cells, accelerate the formation of tumor cells in mice, and show significant activity in promoting tumor growth. Summary of the invention

[0005] The present invention provides a lactic acid preparation that can promote tumor growth by optimizing the preparation formula, and can be used for the construction and research of tumor models.

[0006] A lactic acid preparation capable of promoting tumor growth consists of hydroxypropyl-β-cyclodextrin, poloxamer 407, physiological saline and lactic acid, wherein the volume percentage of the lactic acid is 0.0001-15%.

[0007] Furthermore, the amount of hydroxypropyl-β-cyclodextrin added is 4-6 mg / mL, and the amount of poloxamer 407 added is 2-3 mg / mL.

[0008] Furthermore, the volume percentage of lactic acid is 2.5-15%.

[0009] The application of the above lactic acid preparation as a tumor cell proliferation reagent.

[0010] Furthermore, the tumor cells are breast cancer cells, liver cancer cells, non-small cell lung cancer cells, colorectal cancer cells, leukemia cells, lymphoma cells, myeloma cells, gastric cancer cells, prostate cancer cells, and pancreatic cancer cells.

[0011] Furthermore, breast cancer cells are specifically MBA-MD-468, MBA-MD-231, MCF-7, 4T1, liver cancer cells are specifically HepG2, SMMC-7721, Huh-7, non-small cell lung cancer cells are specifically HCC827, HCC827-ER, 1573, H1537, A549, PC9GR, PC9AR, H1299, H460, 1299, H1373, 1373, HCC827-AR, PC-9, NCI-H157, colorectal The cancer cells are specifically Lovo, HT-21, HCT-116, HT-29, and CT-26; the leukemia cells are specifically Jurkat, MOLT-13, K562, and TF-1; the lymphoma cells are specifically OCI-LY10 and U2932; the myeloma cells are specifically RPMI-8266, U266, MM1, and SH929; the gastric cancer cells are specifically SGC-7901 and MGC-803; the prostate cancer cells are specifically LNCap and PC3; and the pancreatic cancer cells are specifically BXPC-3.

[0012] Furthermore, the final concentration of lactic acid is (0.002~0.2) v%.

[0013] The application of the above lactic acid preparation in modeling of in vivo tumor models in experimental animals in cancer research.

[0014] Among them, the lactic acid preparation can promote the tumor formation and growth of breast cancer, liver cancer, non-small cell lung cancer, colorectal cancer, leukemia, lymphoma, myeloma, gastric cancer, prostate cancer, and pancreatic cancer cells in mice; the volume concentration of the lactic acid preparation is 2.5~10%, and when used in the modeling of the in vivo tumor model of experimental mice, the dosage volume is 0.1 mL / 20 g.

[0015] The invention discloses a method for preparing a lactic acid preparation capable of promoting tumor growth. The dosage of hydroxypropyl-β-cyclodextrin and poloxamer 407 is determined through a large number of preliminary experiments. The method specifically comprises the following steps: weighing a prescribed amount of hydroxypropyl-β-cyclodextrin and poloxamer 407, adding a prescribed amount of physiological saline, ultrasonicating for 15 minutes under an ultrasonic power of 240 W to dissolve the saline into a clear solution for standby use, then adding a prescribed amount of lactic acid, keeping the ultrasonic conditions unchanged, and continuing ultrasonicating for 15 minutes to obtain the lactic acid preparation.

[0016] The present invention aims to provide a type of lactic acid preparation that can improve the success rate and stability of establishing an animal transplant tumor model for tumor research and drug development. The application of the preparation can significantly promote the proliferation of breast cancer, lung cancer, liver cancer, colon cancer and leukemia cells and the formation and growth of transplanted tumors in mice. It shows that the preparation has the effect of significantly improving the efficiency and success rate of tumor formation in living tumor models, and has important market and application value in industries such as tumor research and anti-tumor drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The promoting effect of lactic acid preparations of different concentrations on the proliferation of various tumor cells in vitro;

[0018] a, b are the promoting effects of 0.002%, 0.06%, and 0.02% lactic acid on the proliferation of liver cancer cells;

[0019] c, d are the promoting effects of 0.002%, 0.06%, and 0.02% lactic acid on the proliferation of colon cancer cells;

[0020] e and f are the promoting effects of 0.002%, 0.06% and 0.02% lactic acid on the proliferation of lung cancer cells;

[0021] g, h are the promoting effects of 0.002%, 0.006%, and 0.02% lactic acid on the proliferation of gastric cancer cells;

[0022] i, j are the promoting effects of 0.002%, 006%, and 0.02% concentrations of lactic acid on the proliferation of prostate cancer cells;

[0023] k is the promoting effect of 0.002%, 006%, and 0.02% lactic acid on the proliferation of pancreatic cancer cells;

[0024] Figure 2Effects of various lactic acid preparations (LAP) and saline on the growth of transplanted tumors in experimental mice.

[0025] Lactic acid preparation 2.5% is a lactic acid preparation containing 2.5% lactic acid;

[0026] Lactic acid preparation 5% is a lactic acid preparation containing 5% lactic acid;

[0027] Lactic acid preparation 7.5% is a lactic acid preparation containing 7.5% lactic acid;

[0028] Lactic acid preparation 10% is a lactic acid preparation containing 10% lactic acid;

[0029] Lactic acid preparation 12.5% ​​is a lactic acid preparation containing 12.5% ​​lactic acid;

[0030] Normal saline is a solution containing 0.9% NaCl. DETAILED DESCRIPTION

[0031] The present invention is further described below by specific preparation examples. It should be understood that these implementation examples are only used for more detailed and specific descriptions and should not be construed as limiting the present invention in any form.

[0032] The present invention provides a general and / or specific description of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that, hereinafter, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art.

[0033] Example 1. Preparation of lactic acid preparation (1)

[0034]

[0035] Process: Weigh 5 mg of hydroxypropyl-β-cyclodextrin and 2.5 mg of poloxamer 407, add 0.975 mL of normal saline, and ultrasonicate for 15 min at an ultrasonic power of 240 W to dissolve them into a clear solution for later use. Add 0.025 mL of lactic acid and mix well to obtain a lactic acid preparation (1) with a concentration of 2.5%.

[0036] Example 2: Preparation of lactic acid preparation (2)

[0037]

[0038] Process: Weigh 5 mg of hydroxypropyl-β-cyclodextrin and 2.5 mg of poloxamer 407, add 0.95 mL of normal saline, and ultrasonicate for 15 min at an ultrasonic power of 240 W to dissolve them into a clear solution for use, add 0.05 mL of lactic acid, mix well, and obtain a lactic acid preparation (2) with a concentration of 5%.

[0039] Example 3: Preparation of lactic acid preparation (3)

[0040]

[0041] Process: Weigh 5 mg of hydroxypropyl-β-cyclodextrin and 2.5 mg of poloxamer 407, add 0.925 mL of normal saline, and ultrasonicate for 15 min at an ultrasonic power of 240 W to dissolve them into a clear solution for later use. Add 0.1 mL of lactic acid and mix well to obtain a lactic acid preparation with a concentration of 7.5% (3).

[0042] Example 4. Preparation of lactic acid preparation (4)

[0043]

[0044] Process: Weigh 5 mg of hydroxypropyl-β-cyclodextrin and 2.5 mg of poloxamer 407, add 0.9 mL of normal saline, and ultrasonicate for 15 min at an ultrasonic power of 240 W to dissolve them into a clear solution for use, add 0.1 mL of lactic acid, mix well, and obtain a lactic acid preparation with a concentration of 10% (4).

[0045] Example 5. Preparation of lactic acid preparation (5)

[0046]

[0047] Process: Weigh 5 mg of hydroxypropyl-β-cyclodextrin and 2.5 mg of poloxamer 407, add 0.875 mL of normal saline, and ultrasonicate for 15 min at an ultrasonic power of 240 W to dissolve them into a clear solution for use, add 0.125 mL of lactic acid, mix well, and obtain a lactic acid preparation with a concentration of 12.5% ​​(5).

[0048] Example 6: In vitro cell proliferation experiment

[0049] The CCK-8 method was used to determine the effect of different concentrations of lactic acid solutions on the proliferation of different cells. The basic principle is that the reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], which is reduced to a highly water-soluble yellow formazan product by dehydrogenase in cells under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells. Therefore, this property can be used to directly perform cell proliferation and toxicity analysis.

[0050] The method for CCK-8 to detect cell proliferation / toxicity is as follows:

[0051] (1) Preparation of cell suspension: cell counting;

[0052] (2) Inoculate into a 96-well plate: Depending on the appropriate number of cells to be plated, usually 5,000, add about 100 μl of cell suspension to each well, and make three replicate wells for the same sample;

[0053] (3) Culture in a 5% CO2 37℃ incubator: until the cell monolayer covers the bottom of the well, add lactic acid at a gradient concentration the next morning, with the final concentration of lactic acid being 0.002%, 0.006%, and 0.02%. Complete culture medium without lactic acid was used as a control.

[0054] (4) Incubate at 5% CO2, 37°C for 48 hours and observe under an inverted microscope;

[0055] (5) Add 10 ul of CCK-8. Since the amount of CCK8 added to each well is relatively small, add it as much as possible into the culture medium. If errors are caused by the reagent sticking to the well wall, it is recommended to gently tap the culture plate after adding the reagent to help mix it.

[0056] (6) Incubate the cells at 37°C in an atmosphere of 5% CO2 for 1-4 hours and measure the absorbance at 450 nm.

[0057] The test results are shown in Figure 1 ,Depend on Figure 1 It can be seen that low-concentration lactic acid (0.002%) preparations can promote the proliferation of HepG2, Lovo, HCT116, A549, H157, MGC-803, LNCap, PC3, BXPC-3 and other cells, and medium-concentration lactic acid (0.006%) preparations can promote the proliferation of SMMC-7721 and SGC-7901 cells. This shows that different cells have different tolerance to lactic acid, and the effect of promoting proliferation varies in different types of cells, which has a certain cell selectivity.

[0058] Example 7: Tumor formation experiment in mice

[0059] 1. Cell Culture

[0060] The cells were cultured according to the conventional cell culture method, and the culture method is as follows:

[0061] When the cell coverage in the culture dish reaches 80%-90% under a microscope, subculture is performed. The original culture medium is discarded in the biosafety cabinet, and 2 ml of trypsin is added and digested at 37°C for 2 min. After the cells become round, an equal volume of complete culture medium is added to terminate the digestion. The cells are blown with a pipette to make all the cells fall off, and then the cell suspension is placed in a 15 ml centrifuge tube and centrifuged at 900 r / min for 5 minutes. The supernatant is discarded, 2 ml of culture medium is added to resuspend the cells, and the mixture is mixed by blowing. The cells are evenly distributed into 3 100 mm cell culture dishes for further culture.

[0062] 2. Cell Collection

[0063] (1) Aspirate the old culture medium in the culture dish and wash once with PBS.

[0064] (2) Add 2 ml of trypsin and EDTA mixture to the culture dish.

[0065] (3) Place the culture flask in an incubator at 37°C for digestion. After 2 minutes, place the culture flask under an inverted microscope for observation. If the cytoplasm shrinks and the intercellular space increases, the digestion should be stopped immediately.

[0066] (4) Add complete culture medium to terminate digestion.

[0067] (5) Use a pipette to suck up the culture medium in the dish and repeatedly and gently blow the cells at the bottom of the dish to separate them from the bottom of the dish to form a cell suspension. Be gentle when blowing to avoid damaging the cells.

[0068] (6) After counting with a counting plate, adjust the cell suspension to 1×10 5 / mL, and place the collected 4T1 cells on ice for later use.

[0069] 3. Tumor Cell Inoculation

[0070] Select 60 BALB / c female mice aged 5-6 weeks and weighing 18-22g for tumor cell inoculation. Disinfect the skin at the inoculation site with alcohol cotton, mix the cell suspension and draw a certain amount of cell suspension with a syringe, and inoculate subcutaneously in the armpit at 100μL / mouse. The criterion for successful inoculation is: a nodule appears on the skin at the inoculation site. Return the inoculated animals to the original cage for breeding, keep the environment clean and avoid infection.

[0071] Observation of tumor inoculation site

[0072] One week after tumor cell inoculation, observe whether there is tumor mass formation at the inoculation site. If tumor mass formation is found, use a vernier caliper to measure the size (length and width) of the tumor and calculate the volume of the tumor using the formula:

[0073] Tumor volume = (long diameter × short diameter) 2 × 0.5), and wait until the tumor volume is about 100 mm 3 Start dosing.

[0074] 5. Dosage and Grouping

[0075] Select tumors with a volume of 70-100 mm 3 The 36 animals within the range were divided into 6 groups according to the size of the tumor and numbered; the drugs were administered according to the design in the table below, with a dosage volume of 0.1 mL / 20 g.

[0076]

[0077] The drug is injected intraperitoneally, with a frequency of once a day for 14 days.

[0078] 6. Testing indicators

[0079] I. General Clinical Observations

[0080] The mental state, behavioral activities, coat, skin, anus, eyelids, breathing and other conditions of each group of animals were recorded every day.

[0081] II. Tumor proliferation inhibition rate

[0082] The long and short diameters of the tumor were measured with a vernier caliper every other day, and the tumor volume was calculated according to the formula:

[0083] Tumor volume = (long diameter × short diameter) 2 ×0.5).

[0084] The relative tumor proliferation rate T / C (%) was used as the test evaluation index. The evaluation criteria were usually: T / C (%)>40% was invalid; T / C (%)≤40%, and P<0.05 after statistical processing was effective.

[0085] Note: Relative tumor proliferation rate T / C% = TRTV / CRTV*100%. (TRTV: RTV of treatment group; CRTV: RTV of negative control group, RTV relative to tumor volume).

[0086] The test results are as follows Figure 2Except for 12.5% ​​lactic acid preparation, injection of different concentrations of lactic acid preparations can promote the growth of tumor cells 4T1 in mice. The volume of transplanted tumors in this group of mice was significantly larger than that in the normal saline group. Among them, 5% lactic acid preparation had the most significant effect on promoting the growth of 4T1 transplanted tumors, and 12.5% ​​lactic acid preparation had no effect on promoting the formation of 4T1 transplanted tumors, indicating that high doses of lactic acid preparations failed to promote the formation of 4T1 transplanted tumors.

Claims

1. A lactic acid preparation capable of promoting tumor growth, characterized in that: It consists of hydroxypropyl-β-cyclodextrin, poloxamer 407, normal saline and lactic acid, wherein the volume percentage of lactic acid is 0.0001-15%; the added amount of hydroxypropyl-β-cyclodextrin is 4-6 mg / mL, and the added amount of poloxamer 407 is 2-3 mg / mL.

2. The lactic acid preparation capable of promoting tumor growth according to claim 1, characterized in that: The volume percentage of lactic acid is 2.5~15%.

3. The method for preparing the lactic acid preparation capable of promoting tumor growth according to claim 1 or 2, characterized in that: Weigh hydroxypropyl-β-cyclodextrin and poloxamer 407, add physiological saline to dissolve into a clear solution, set aside, add lactic acid, mix well, and obtain the solution.

4. Use of the lactic acid preparation according to claim 1 as a tumor cell proliferation reagent.

5. The use according to claim 4, characterized in that: The tumor cells are breast cancer cells, liver cancer cells, non-small cell lung cancer cells, colorectal cancer cells, leukemia cells, lymphoma cells, myeloma cells, gastric cancer cells, prostate cancer cells, and pancreatic cancer cells.

6. The use according to claim 5, characterized in that: Breast cancer cells are MBA-MD-468, MBA-MD-231, MCF-7, 4T1, liver cancer cells are HepG2, SMMC-7721, Huh-7, non-small cell lung cancer cells are HCC827, HCC827-ER, H1537, A549, PC9GR, PC9AR, H1299, H460, H1373, HCC827-AR, PC-9, NCI-H157, colorectal cancer cells are Lovo , HT-21, HCT-116, HT-29, CT-26, leukemia cells specifically include Jurkat, MOLT-13, K562, TF-1, lymphoma cells specifically include OCI-LY10, U2932, myeloma cells specifically include RPMI-8266, U266, MM1, SH929, gastric cancer cells specifically include SGC-7901, MGC-803, prostate cancer cells specifically include LNCap, PC3, and pancreatic cancer cells specifically include BXPC-3.

7. Use of the lactic acid preparation according to claim 1 in modeling tumor in vivo models of experimental animals in cancer research.

8. The use according to claim 7, characterized in that: The lactic acid preparation can promote the tumor formation and growth of breast cancer, liver cancer, non-small cell lung cancer, colorectal cancer, leukemia, lymphoma, myeloma, gastric cancer, prostate cancer, and pancreatic cancer cells in mice; the volume concentration of the lactic acid preparation is 2.5-10%.

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