CN116549596A

CN116549596A - Application of vinblastine derivative in preparation of medicines for treating lymph node metastasis

Info

Publication number: CN116549596A
Application number: CN202310663149.7A
Authority: CN
Inventors: 陈敏锋; 张冬梅; 叶文才; 齐明; 范舒然; 张君秋
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2023-06-06
Filing date: 2023-06-06
Publication date: 2023-08-08

Abstract

The invention belongs to the field of biological medicine, and in particular relates to application of a vinblastine derivative in preparing a medicine for treating tumor cell lymph node metastasis. The vinblastine derivative comprises vinblastine dipeptide and physiologically acceptable salts thereof; wherein, the vinblastine dipeptide is a compound obtained by condensing a hydrazinolysis vinblastine compound and N-benzyloxycarbonyl dipeptide; the hydrazinolysis vinblastine compound is a compound obtained by reacting a vinblastine compound with hydrazine hydrate; the vinblastine compound is preferably vinblastine. The vinblastine derivative provided by the invention can obviously inhibit proliferation, invasion and migration of tumor cells transferred to lymph nodes in vitro, inhibit in-situ growth of tumors and lymph node metastasis in vivo, and can be applied to treatment of patients suffering from tumor lymph node metastasis.

Description

Application of vinblastine derivative in preparation of medicines for treating lymph node metastasis

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application of a vinblastine derivative in preparation of a medicament for treating lymph node metastasis.

Background

Lymph node metastasis (LymphaticMetastasis, LNM) is one of the most important and common metastasis pathways of most solid tumors, including breast and colorectal cancers, and the presence of tumor cells in lymph nodes is an important basis for judging tumor staging and prognosis. Therefore, blocking lymph node metastasis of tumor cells can fundamentally prolong survival of tumor patients. Current strategies for treating tumor lymph node metastasis include lymph node cleaning and radiotherapy, but the treatment of side effects such as pain, joint movement disorder and lymphadenedema seriously affect the life quality of patients, and residual tumor cells are also highly likely to cause tumor recurrence and metastasis. Therefore, the development of the low-toxicity and high-efficiency tumor lymph node metastasis targeted therapeutic drug has important guiding significance and clinical application value for radical treatment of tumor surgery and improvement of chemotherapy curative effect.

The enzyme-activated prodrug can improve the targeting of the parent drug to the enzyme cells and reduce the toxic and side effects of the parent drug to organisms. The specific principle is as follows: the active group of the parent drug is blocked by the polypeptide to form an inactive form of the prodrug, which is a hydrolysis substrate for a specific enzyme, and when the prodrug encounters a specific enzyme highly expressed by a cell, the prodrug is hydrolyzed to release the active parent drug, thereby killing the cell expressing the enzyme. Fibroblast activation protein alpha (fibroblast activation protein alpha, FAP alpha) is a type II serine protease, which is one of the dipeptidyl peptidase family members, has dipeptidyl peptidase hydrolyzing activity, and uniquely has restriction endonuclease activity, and can specifically hydrolyze substrates coupled to N-terminal benzyloxy carbonyl (Z) -blocked Gly-Pro (Z-GP). The research shows that FAP alpha can be expressed on the surface of tumor cells, and the FAP alpha can be expressed on the surface of tumor cells of colorectal cancer, osteosarcoma and breast cancer to different degrees; furthermore, clinical studies demonstrate that in patients with various tumors, high expression of fapα in tumor tissue is positively correlated with clinical advanced stage, both metastatic positive and poor prognosis. The above studies indicate that fapα can be an effective target for treatment of lymphatic metastasis, and fapα enzyme-activated prodrugs are expected to be a good design strategy for drugs for treatment of lymphatic metastasis.

The vinblastine compounds are known bisindole alkaloids and derivatives thereof isolated from herba Catharanthi rosei of Apocynaceae, and include vinblastine, vinorelbine, vinflunine, vincristine, vinblastine or vindoline. Pharmacological studies have shown that vinblastine and its analogues or derivatives belong to cytotoxic drugs, mainly inhibiting the polymerization of tubulin, thus preventing the formation of spindle microtubules and blocking nuclear division in metaphase.

At present, vinblastine and derivatives thereof have adverse reactions such as bone marrow suppression and the like, and the long-term and large-dose application of the vinblastine and derivatives is limited. Therefore, it is extremely necessary to deeply develop the action of vinblastine for use in various studies.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides application of a vinblastine derivative in preparing a medicament for treating lymph node metastasis. According to the characteristic of high expression of FAP alpha by colorectal cancer cells of lymph node metastasis, the vinblastine compound is formed into the FAP alpha enzyme activated prodrug through hydrazinolysis and dipeptide derivatization, so that the toxic and side effects of the vinblastine and derivatives thereof are effectively reduced, the treatment effect of the vinblastine is enhanced, and the vinblastine compound is expected to be used for treating lymph node metastasis tumors in clinic.

Aiming at the technical effects, the technical scheme adopted by the invention is as follows:

the application of a vinblastine derivative in preparing a medicament for treating tumor cell lymph node metastasis is provided, wherein the vinblastine derivative has the following structural formula:

i is a kind of

Wherein Z-GP represents benzyloxycarbonyl glycyl prolyl.

Preferably, the vinblastine derivative comprises vinblastine dipeptides and physiologically acceptable salts thereof.

Preferably, the preparation process of the vinblastine dipeptide comprises the following steps: reacting the vinblastine compound with hydrazine hydrate to form a hydrazinolysis vinblastine compound, and condensing the hydrazinolysis vinblastine compound with N-benzyloxycarbonyl dipeptide.

The specific preparation process of the vinblastine dipeptide provided by the invention comprises the following steps:

s1, dissolving vinblastine in an organic solvent, adding 50% by weight of hydrazine hydrate, heating and stirring for reaction for 25 hours under the protection of nitrogen in a dark place, controlling the reaction temperature to 80 ℃, and separating and purifying after the reaction is finished to obtain a hydrazinolytic vinblastine compound;

s2, reacting the hydrazinolysis vinblastine compound obtained in the step S1, N-benzyloxycarbonyl dipeptide and a condensation reagent ethyl chloroformate (the feeding mole ratio of the N-benzyloxycarbonyl dipeptide to the condensation reagent ethyl chloroformate is 1:2:2) at the temperature of minus 20 ℃ in a dark place, stirring at the rotating speed of 60rpm for 10 hours, and then separating and purifying to obtain the vinblastine compound.

Wherein, the mol feeding ratio of the vinblastine to the hydrazine hydrate is 1:500.

Preferably, the hydrazinolysis vinblastine compound is hydrazinolysis vinblastine; the vinblastine compound is vinblastine

Preferably, the N-carbobenzoxy dipeptide is N-carbobenzoxy glycyl proline, and the structural formula is as follows:

preferably, the physiologically acceptable salt is selected from one of hydrochloride, sulfate, acetate, tartrate, citrate.

Preferably, the lymph node tumor cell metastasis sources include cells of colorectal, breast, and ovarian cancer cell lines.

Preferably, the colorectal cancer cell line comprises colorectal cancer cell line HCT116, colorectal cancer cell line SW620, colorectal cancer cell line DLD1; the breast cancer cell line comprises a breast cancer cell line MDA-MB-231, a breast cancer cell line MDA-MB-453 and a breast cancer cell line MCF-7; the ovarian cancer cell line comprises an ovarian cancer cell line A2780, an ovarian cancer cell line CoC1 and an ovarian cancer cell line SK-OV3.

Compared with the prior art, the invention has the technical advantages that:

(1) The vinblastine derivative provided by the invention can be used as FAP alpha enzyme activated prodrug, has good anti-tumor growth and lymphatic metastasis activity in vivo and in vitro, effectively reduces the toxic and side effects of vinblastine and the derivative thereof and enhances the treatment effect;

(2) The vinblastine derivative provided by the invention can obviously inhibit proliferation, invasion and migration capacity of tumor cells transferred to lymph nodes in vitro;

the vinblastine derivative provided by the invention can inhibit in-situ growth of tumor and metastasis to lymph node in vivo, and can be applied to treatment of patients suffering from tumor metastasis to lymph node.

Drawings

FIG. 1 is a synthetic route diagram of the vinblastine derivatives of the invention;

FIG. 2 is a graph showing the inhibition of cell migration capacity of targeted lymphotropic cells HCT116-LNM, SW620-LNM, DLD1-LNM and SW1116-LNM by vinblastine derivatives BX-CCJ and vinorelbine;

FIG. 3 shows the results of migration assays of vinblastine derivatives BX-CCJ and vinorelbine (BX-CCRB) on HCT116-LNM, SW620-LNM, DLD1-LNM and SW1116-LNM cells;

FIG. 4 is a graph showing the inhibition of the invasiveness of HCT116-LNM, SW620-LNM, DLD1-LNM and SW1116-LNM by vinblastine derivatives BX-CCJ and vinorelbine (BX-CCRB).

Detailed Description

The present invention will be further explained with reference to specific examples, but it should be noted that the following examples are only for explaining the present invention, and are not intended to limit the present invention, and all technical solutions identical or similar to the present invention are within the scope of the present invention. The specific techniques or conditions are not noted in this example and are practiced according to methods and apparatus conventional in the art; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The vinblastine compounds related in the embodiment of the invention are sequentially vinblastine (CCJ) and vinorelbine (CCRB), and the hydrazinolysis vinblastine compounds are obtained after hydrazinolysis; the hydrazinolysis vinblastine compounds are respectively as follows: vinblastine hydrazinolysis (JJ-CCJ), hydrazine Jie Changchun raylbine (JJ-CCRB); in the invention, lymph node metastasis (Lymphatic Metastasis, LNM) is carried out by injecting common tumor cell line into corresponding organ of nude mice, waiting for lymph node metastasis of tumor growth, and extracting tumor cells in lymph node, namely XX-LNM cells in test.

The vinblastine dipeptide is a hydrazinolysis vinblastine compound formed by reacting a vinblastine compound with hydrazine hydrate, and is condensed with N-benzyloxycarbonyl dipeptide (N-benzyloxycarbonyl glycyl proline (Z-GP-OH, formula II);

the structure and nomenclature of the compounds are shown below:

II (II)

Formula III

IV (IV)

EXAMPLE 1 tumor cells with directed lymphatic metastasis highly express FAP alpha

1. The experimental method comprises the following steps: cells in the logarithmic growth phase were inoculated into 6-well plates at an inoculation density of 3X 105 cells/well, 2mL of DMEM high-sugar complete medium (Gibco) was added to each well, and after overnight incubation in an incubator (37 ℃, 5% CO 2), the adherent cells were discarded from old medium and washed twice with PBS. In Western blotting immunoblotting experiments, 50. Mu.L of the cell lysate (Servicebio) was added, the cells were scraped off completely with a cell scraper, and collected and transferred into a clean 1.5mL EP tube. The mixture was lysed on ice for 40min, vortexed for 30s every 10min, and repeated 3 times. Then centrifuged at 12,000rpm at 4℃for 15min. The protein supernatant was transferred to another clean 1.5mL EP tube. Protein concentration was determined according to BCA protein quantification kit instructions, samples were prepared, electrophoresed, developed and protein banding results are shown in fig. 2.

2. Test results: from the experimental results shown in fig. 2, it can be seen that: human targeted lymphatic metastatic tumor cells highly express fapα compared to non-targeted lymphatic metastatic tumor cells.

EXAMPLE 2 inhibition of proliferation potency of vinblastine derivatives BX-CCJ on human-directed lymphatic metastasis tumor cells

1. The experimental method comprises the following steps: cells in the logarithmic growth phase were inoculated into 96-well plates by digestion, at a density of 5X 103 cells/well, 100. Mu.L of DMEM high-sugar complete medium (Gibco) was added to each well, after overnight incubation in an incubator, the old medium was removed, 1. Mu.M of half-diluted drug concentration (i.e., 1. Mu.M, 500nM,250nM,125nM,62.5nM,31.25nM, 15.245 nM,7.8125nM,3.90625nM,1.953125 nM) was added to each well, and an equal volume of PBS was additionally added as a negative control well, and an equal volume of DMEM high-sugar complete medium containing no drug of the inactivated serum was added as a positive control well. After 72h of cell culture, the old cell culture medium was removed, 30. Mu.L of MTT was added to each well, incubated at 37℃for 4h, the recovered MTT solution was removed, 100. Mu.L of DMSO was added to each well, and after complete dissolution of the formazan reaction product by shaking the shaker, the OD value of each well was measured using a fluorescent multifunctional microplate reader (wavelength 590, nm). The formula for calculating cell viability is as follows:

cell viability% = (mean OD value of dosing treatment group-mean OD value of blank wells)/(mean OD value of control group-mean OD value of blank wells) ×100%. The results are shown in Table 1.

2. Test results: from the experimental results shown in table 1, it can be seen that: the vinblastine derivative BX-CCJ can inhibit proliferation of human targeted lymphatic metastasis tumor cells, and has antiproliferative capacity superior to that of vinorelbine.

TABLE 1 statistical results of cell viability

EXAMPLE 3 inhibition of human-directed lymphatic metastatic tumor cell migration by vinblastine derivatives BX-CCJ

1. The experimental method comprises the following steps: cells in the logarithmic growth phase were centrifuged at 800 rpm for 3min after digestion, resuspended in DMEM high-sugar empty medium (containing no nutrients, but only 4.5 g/L glucose, L-glutamine and 110mg/L sodium pyruvate, gibco, cat# 11965092) (Gibco) and inoculated at 1X 104 cells/well into a Transwell-nested upper chamber with a pore size of 8.0. Mu.m, the inoculated cell suspension volume was 100. Mu.L, and 500. Mu.L of BX-CCJ or vinorelbine medium containing 10% FBS and specific concentrations (0 nmol/L, 2nmol/L, 4 nmol/L) was added to the lower chamber, and 3 multiplex wells were set per group. After incubation at 37℃for 48h in the incubator, all the medium in the upper and lower chambers was removed, the cells were fixed with 4% paraformaldehyde at room temperature for 50min, washed 2 times, and 500. Mu.L of 0.1% crystal violet solution was added to the lower chamber and stained for 3min. The cells were washed with PBS and gently rubbed with a cotton swab to remove cells that did not migrate to the lower chamber in the nested upper chamber. After the Transwell nested cells were naturally dried, the cells that had migrated to the lower layers of the nested cells were observed and photographed using an inverted microscope, 5 fields of view were randomly selected for each nest to photograph, and the number of migrated cells was counted using IPP software. The results are shown in FIG. 3.

2. Experimental results: from the experimental results shown in the figures, it can be seen that: compared with a blank control group, the vinblastine derivative BX-CCJ can obviously inhibit the migration capacity of human directional lymph node metastasis tumor cells, and the effect is superior to that of vinorelbine (see figure 3).

EXAMPLE 4 inhibition of the ability of vinblastine derivatives BX-CCJ to invade tumor cells in human-directed lymph node metastasis

1. The experimental method comprises the following steps: cells in the logarithmic growth phase were resuspended in DMEM medium at 800 rpm for 3min after digestion, inoculated at 1X 104 cells/well into a Transwell-nested upper chamber of 8.0 μm pore size, 20. Mu.L, 2.5mg/mL Matrigel (BD Co.), the cell suspension volume was inoculated at 100L, 500. Mu.L of BX-CCJ or BX-CCRB medium containing 10% FBS and specific concentrations (0 nmol/L, 2nmol/L, 4 nmol/L) was added to the lower chamber, and 3 multiplex wells were set per group. After incubation at 37℃for 48h in the incubator, all the medium in the upper and lower chambers was removed, the cells were fixed with 4% paraformaldehyde at room temperature for 50min, washed 2 times, and 500. Mu.L of 0.1% crystal violet solution was added to the lower chamber and stained for 3min. The cells were washed with PBS and gently rubbed with a cotton swab to remove cells that did not migrate to the lower chamber in the nested upper chamber. After the Transwell nested cells were naturally dried, the cells that had migrated to the lower layers of the nested cells were observed and photographed using an inverted microscope, 5 fields of view were randomly selected for each nest to photograph, and the number of migrated cells was counted using IPP software. The results are shown in FIG. 4.

2. Experimental results: from the experimental results shown in the figures, it can be seen that: compared with a blank control group, the vinblastine derivative BX-CCJ can obviously inhibit invasion capacity of human directional lymph node metastasis tumor cells, and the effect is better than that of vinorelbine.

EXAMPLE 5 inhibition of Changchun-base derivatives BX-CCJ on tumor lymph node metastasis in nude mice

1. The experimental method comprises the following steps: human directional lymph node metastasis tumor cells in the logarithmic growth phase were digested and collected, washed 3 times with PBS, centrifuged at 800 rpm for 3min, and the cell pellet was mixed with pre-chilled PBS and blown down uniformly to adjust the cell density to 1X 107 cells/mL. Cell suspensions were injected into the colorectal mesenteric wall, breast fat pad and ovary in situ, respectively, of 4-6 week old male BALB/C nude mice at a volume of 0.1 mL/mouse with islet injectors. After one week of tumor development, tumor-bearing mice were randomly divided into solvent control, BX-CCJ and vinorelbine dosing groups of 6 animals each. The administration mode is as follows: BX-CCJ or vinorelbine 1mg/kg was injected intravenously at the tail once a couple of days. The body weight of the nude mice was recorded and the results are shown in table 2. After 20 days of treatment, mice were sacrificed and tumor and lymph node tissues were dissected. Tumor tissue was weighed and photographed. Tumor and mesenteric lymph node tissues were fixed with 4% paraformaldehyde, embedded for sectioning, and histological analysis was performed after H & E staining. The results are shown in Table 3.

2. Experimental results: from the experimental results shown in table 2, it can be seen that: the vinblastine derivative BX-CCJ can remarkably inhibit the growth capacity of tumors, the tumor inhibition rates are 86.11%,80.65%,75.86%,87.50%,88.46% and 82.14%, the tumor inhibition rates of Yu Changchun Ruibian (BX-XXRB) are 47.22%, 54.84%, 44.83%,53.13%,50.00% and 53.57%, and the weight of nude mice is not obviously affected. BX-CCJ can significantly reduce the number of lymph node metastasis of colorectal cancer cells in human directional lymph node metastasis (counting the number of metastasis, namely quantifying the inhibition of lymph node metastasis, which is the number of lymph nodes invaded by tumors), and has better effect than vinorelbine (BX-CCRB).

TABLE 2 statistical results of tumor weights and tumor suppression rates

TABLE 3 statistical results of the number of lymph node metastases

* P <0.05, # P <0.01, # P <0.001 VS normal saline group, # P <0.01, # P <0.001 VS vinorelbine group

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The application of a vinblastine derivative in preparing a medicament for treating tumor cell lymph node metastasis is provided, wherein the vinblastine derivative has the following structural formula:

wherein Z-GP represents benzyloxycarbonyl glycyl prolyl.

2. The use according to claim 1, wherein the derivative of vinca-alkaloids comprises a dipeptide of the vinca-alkaloids and a physiologically acceptable salt thereof.

3. The use according to claim 2, wherein the preparation of the vinblastine dipeptide is: reacting the vinblastine compound with hydrazine hydrate to form a hydrazinolysis vinblastine compound, and condensing the hydrazinolysis vinblastine compound with N-benzyloxycarbonyl dipeptide.

4. The use according to claim 3, wherein the hydrazinolysis vinblastine compound is a hydrazinolysis vinblastine; the vinblastine compound is vinblastine.

5. The use according to claim 3, wherein the N-benzyloxycarbonyl dipeptide is N-benzyloxycarbonyl glycylproline having the formula:。

6. the use according to claim 2, wherein the physiologically acceptable salt is selected from one of hydrochloride, sulfate, acetate, tartrate, citrate.

7. The use of claim 1, wherein the source of lymph node tumor cell metastasis comprises cells of colorectal, breast and ovarian cancer cell lines.

8. The use according to claim 7, wherein the colorectal cancer cell line comprises colorectal cancer cell line HCT116, colorectal cancer cell line SW620, colorectal cancer cell line DLD1; the breast cancer cell line comprises a breast cancer cell line MDA-MB-231, a breast cancer cell line MDA-MB-453 and a breast cancer cell line MCF-7; the ovarian cancer cell line comprises an ovarian cancer cell line A2780, an ovarian cancer cell line CoC1 and an ovarian cancer cell line SK-OV3.