KR101942159B1 - Anti-cancer drug and diosgenin conjugates, preparation method thereof, and anti-cancer composition comprising the same - Google Patents
Anti-cancer drug and diosgenin conjugates, preparation method thereof, and anti-cancer composition comprising the same Download PDFInfo
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Abstract
The present invention relates to a conjugate of an anticancer agent and diosgenin, a method for producing the same, and an anticancer composition comprising the anticancer agent and the anticancer composition. The conjugate in which the anticancer agent and diosergen according to the present invention are linked by a linker, (DG) to conjugate with a target anticancer agent to overcome the anticancer drug resistance and improve the anticancer activity by enhancing the permeability of the cancer cell membrane.
Description
The present invention relates to a conjugate of an anticancer agent and diosgenin, a method for producing the same, and an anticancer composition containing the same.
Cancer is the most common disease in Korea with cardiovascular disease. Normally, cells divide by intracellular regulating function, grow, die and disappear, and maintain the balance of cell numbers. When these cells are damaged, abnormal cells that do not regulate their proliferation and inhibition become uncontrolled and not only proliferate but also invade surrounding tissues and organs, resulting in mass formation and normal tissue destruction. This condition is defined as cancer or cancer.
Tumors are benign tumors and malignant tumors. Benign tumors grow relatively slowly. They are tumors that can not be spread, metastasized, and removed in various parts of the body. Unless otherwise noted, most benign tumors are life-threatening It does not result. Malignant tumors, on the other hand, are those that cause rapid growth, invasive growth (penetrating or spreading), and spreading (spreading from one place to another) in each part of the body, resulting in life-threatening tumors.
The mechanism by which the cancer cells develop is that the cells have undergone growth, differentiation and programmed apoptosis, or have stopped growing, It is thought that the characteristics of the protein, which is the product of the gene, is changed, and as a result, an abnormality occurs in the regulation of cell growth, and cancer cells are formed.
Repeated administration of anticancer drugs causes many changes in side effects and in vivo efficacy of cancer patients. It is an important task to overcome the tolerance of the anticancer drug due to such repeated administration and to improve the permeability to cancer cells.
Accordingly, the present inventors have found that the use of diosgenin (DG), which is similar to the constituents of the cell membrane, can be conjugated with a target anticancer agent to overcome the anticancer drug resistance and improve the cell membrane permeability to improve the anticancer activity And completed the present invention. In addition, in the case of a hydrophilic anticancer agent, when it forms a conjugate with hydrophobic diosgenin (DG), it shows both hydrophilic properties, confirming that it enables self-assemble of stable nanoparticles, Completed.
The object of the present invention is to provide a conjugate in which an anticancer agent and diosgenin are linked by a linker, or a pharmaceutically acceptable salt thereof, which can improve the anticancer activity by overcoming the anticancer drug resistance and enhancing the permeability of the cancer cell membrane will be.
Another object of the present invention is to provide a method for producing a conjugate in which the anticancer agent and diosgenin are linked by a linker.
Yet another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a conjugate wherein the anticancer agent and Diosgenin are linked by a linker, or a pharmaceutically acceptable salt thereof.
It is another object of the present invention to provide a conjugated anti-cancer formulation in which the anticancer agent and diosergen are linked by a linker.
Another object of the present invention is to provide a nanoparticle wherein the conjugate in which the anticancer agent and Diosgenin are linked by a linker is formed by self-assembly.
In order to achieve the above object,
The present invention provides a conjugate in which an anticancer agent and diosgenin are linked by a linker, or a pharmaceutically acceptable salt thereof.
The present invention also relates to a process for producing a compound represented by the formula (1)
Adding carbonochloridic acid to diosgenin to obtain compound 4 (step 1);
Adding
Adding methotrexate (MTX) to Compound 6 to obtain Compound 1 (Step 3);
(1), wherein the anticancer agent is represented by the following formula (1a) and Diosgenin is linked with a linker.
[Reaction Scheme 1]
Further, the present invention relates to a process for the preparation of
Adding carbonochloridic acid to diosgenin to obtain compound 4 (step 1);
Adding
Adding methotrexate (MTX) to compound 8 to obtain compound 9 (step 3); And
Removal of the protecting group (Boc, tert-Butyloxycarbonyl) from compound 9 to obtain compound 2 (step 4);
(1b) and diosgenin are linked with each other by a linker.
[Reaction Scheme 2]
In the above Reaction Scheme 2, n is an integer of 0-2.
Also, as shown in the following
Adding succinic anhydride to diosgenin to obtain compound 10 (step 1);
Adding n-hydroxysuccinimide to compound 10 to obtain compound 11 (step 2); And
Adding Doxorubicin (DOX) to Compound 11 to obtain Compound 3 (Step 3);
, Wherein the anticancer agent represented by the following formula (1c) and diosgenin are linked by a linker.
[Reaction Scheme 3]
Further, the present invention relates to a process for preparing a compound represented by the formula
Adding succinic anhydride to diosgenin to obtain compound 10 (step 1);
Adding n-hydroxysuccinimide to compound 10 to obtain compound 11 (step 2); And
Adding cytarabine (Cyt) to compound 11 to obtain compound 1d (step 3);
(1d) and diosgenin are linked by a linker.
[Reaction Scheme 4]
The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a conjugate wherein the anticancer agent and Diosgenin are linked by a linker, or a pharmaceutically acceptable salt thereof.
Further, the present invention provides an anticancer formulation of a conjugate in which an anticancer drug and Diosgenin are linked by a linker.
The present invention also provides a nanoparticle in which a conjugate in which the anticancer agent and Diosgenin are linked by a linker is formed by self-assembly.
The conjugate in which the anticancer agent and diosergen according to the present invention are combined with the linker is conjugated with the target anticancer agent using diosgenin (DG) similar to the constituent components of the cancer cell membrane to overcome the anticancer drug resistance, There is an effect of significantly improving the anticancer activity by enhancing the permeability of the membrane. In the case of a hydrophilic anticancer agent, it exhibits both hydrophilicity when forming a conjugate with hydrophobic diosgenin (DG), which enables self-assemble of stable nanoparticles,
FIG. 1 (A) is a graph showing the cell survival rate after 48 hours of administration of DG, DSA, DOX and DOX-DG to HepG2 (human-derived liver cancer cells).
FIG. 1 (B) is a graph showing the cell survival rate after 48 hours of administration of DG, DSA, DOX, and DOX-DG to L929 cells (murine fibroblast cell line).
FIG. 2 is a graph showing a DOX-DG conjugate prepared in Example 3 measured for DOX emission according to pH environment change. FIG.
Figure 3 is a graph showing the activity of MTX, (b) Example 1 (compound 1a), (c) Example 1 (compound 1a) + GSH, (d) Example 2 (compound 1b3) Compound 1b3) + GSH.
4 is a fluorescence image obtained by confirming the cell uptake of Cyt-DG conjugated nanoparticles through a confocal scanning laser microscope.
5 is a graph showing the critical aggregation concentration (CAC) of the Cyt-DG conjugate by LC-MS spectrum.
FIG. 6 shows the results of (a) Dynamic light scattering (DLS) and (b) transmission electron microscopy (TEM) to examine the particle size distribution, zeta potential and morphology of the Cyt- DG conjugated nanoparticles prepared in Example 5 The results are analyzed.
FIG. 7 is a graph showing the change in diameter of Cyt-DG conjugated nanoparticles measured by DLS in (a) 0.1 M PBS and 5% glucose solution, (b) Cyt-DG conjugate in 1% and 10% And the change in diameter of the gate nano-particles is measured by DLS.
FIG. 8 is a schematic diagram showing a process in which Cyt-DG conjugated nanoparticles prepared in Example 5 are absorbed by cells.
Hereinafter, the present invention will be described in detail.
The present invention provides a conjugate in which an anticancer agent and diosgenin are linked by a linker, or a pharmaceutically acceptable salt thereof. Herein, the diosgenin is similar to the constituent components of the cancer cell membrane, so that the cancer cell membrane permeability is improved and the anticancer drug resistance is overcome.
Methotrexate (MTX), doxorubicin (DOX), cytarabine (Cyt) and the like may be used as the anticancer agent.
As the linker
, , , , Etc. may be used.
Preferable examples of the conjugate in which the anticancer agent according to the present invention and diosgenin are linked by a linker include conjugates represented by the following formulas (1a) to (1d).
[Formula 1a]
[Chemical Formula 1b]
(In the above formula (2), n is an integer of 0-2)
[Chemical Formula 1c]
.
≪ RTI ID = 0.0 &
The conjugate conjugated with an anticancer agent and diosergen according to the present invention is conjugated with a target anticancer agent using diosgenin (DG), which is similar to that of a cancer cell membrane, to increase the permeability of the cancer cell membrane, It is also effective to overcome the tolerance and to improve the anticancer activity due to the inherent anticancer activity of DG.
The anticancer drug-diosgenin conjugate of the present invention can be used in the form of a pharmaceutically acceptable salt, and the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. The expression pharmaceutically acceptable salt means a concentration that has a relatively non-toxic and harmless effective action to the patient, and that the side-effect caused by the salt is an anticancer agent that does not detract from the beneficial effects of the anticancer-diosgenin conjugate. Any of the diosgenin conjugates Organic or inorganic addition salts.
Examples of the inorganic acid include hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, maleic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, tartaric acid, succinic acid, malonic acid, succinic acid, malonic acid, glutamic acid, aspartic acid, oxalic acid, P-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid.
These salts also include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, the acid addition salt may be selected from the group consisting of acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camylate, citrate, eddylate, Hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, malate, glucoside, gluconate, gluconate, glucuronate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / Hydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydroxyacetate, Lactate, stearate, succinate, tartrate, tosylate, trifluoroacetate Diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like. Preferred is hydrochloride or trifluoroacetate.
The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving an anticancer agent-diosgenin conjugate in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, Followed by filtration and drying. Alternatively, the solvent and excess acid may be distilled off under reduced pressure, followed by drying or crystallization in an organic solvent.
In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).
Furthermore, the present invention encompasses the aforementioned anticancer drug-diosgenin conjugate and pharmaceutically acceptable salts thereof as well as possible solvates, hydrates, isomers and the like which can be prepared therefrom.
As shown in the following
Adding carbonochloridic acid to diosgenin to obtain compound 4 (step 1);
Adding
Adding methotrexate (MTX) to Compound 6 to obtain Compound 1 (Step 3);
(1), wherein the anticancer agent is represented by the following formula (1a) and Diosgenin is linked with a linker.
[Reaction Scheme 1]
In the
Specifically, as the solvent usable in
The reaction temperature may be from 0 to 30 ° C,
The reaction time can be 5-10 hours.
As the catalyst, triphosgene, phosgene, imidazolium chloride derivatives and anhydrous pyridine can be used.
In
Specifically, by using a solvent in Step 2 in dichloromethane (DCM), t- butanol, ethanol, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH 2 Cl 2, hexane, dimethylformamide (DMF), diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethylsulfoxide (DMSO), acetone and chlorobenzene may be used alone or in combination.
The reaction temperature may be from 0 to 30 ° C,
The reaction time may be 1-24 hours.
In
Specifically, the solvent that can be used in
The reaction temperature may be from 0 to 30 ° C,
The reaction time can be from 30 minutes to 5 days.
Production Method 2
As shown in the following Reaction Scheme 2,
Adding carbonochloridic acid to diosgenin to obtain compound 4 (step 1);
Adding
Adding methotrexate (MTX) to
Removal of the protecting group (Boc, tert-Butyloxycarbonyl) from compound 9 to obtain compound 2 (step 4);
(1b) and diosgenin are linked with each other by a linker.
[Reaction Scheme 2]
In the above Reaction Scheme 2, n is an integer of 0-2.
In Production Method 2 according to the present invention,
In Process 2 of the present invention, Step 2 is a step of adding
Specifically, by using a solvent in Step 2 in dichloromethane (DCM), t- butanol, ethanol, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH 2 Cl 2, hexane, dimethylformamide (DMF), diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethylsulfoxide (DMSO), acetone and chlorobenzene may be used alone or in combination.
The reaction temperature may be from 0 to 30 ° C,
The reaction time may be 1-24 hours.
In Process 2 of the present invention,
Specifically, the solvent that can be used in
The reaction temperature may be from 0 to 30 ° C,
The reaction time can be from 30 minutes to 5 days.
In Process 2 of the present invention,
Specifically, the solvent which can be used in
The reaction temperature may be from 0 to 30 ° C,
The reaction time may be 2-6 hours.
As shown in the following
Adding succinic anhydride to diosgenin to obtain compound 10 (step 1);
Adding n-hydroxysuccinimide to compound 10 to obtain compound 11 (step 2); And
Adding Doxorubicin (DOX) to Compound 11 to obtain Compound 3 (Step 3);
, Wherein the anticancer agent represented by the following formula (1c) and diosgenin are linked by a linker.
[Reaction Scheme 3]
In
Specifically, the solvent which can be used in
The reaction temperature may be 25-75 ° C,
The reaction time can be 10-30 hours.
In
Specifically, the solvent that can be used in step 2 is dichloromethane (DCM), t-butanol, ethanol, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH2Cl2, hexane, dimethylformamide (DMA), dimethylsulfoxide (DMSO), acetone, chlorobenzene, etc. may be used alone or in combination.
The reaction temperature may be from 0 to 30 ° C,
The reaction time can be 4-8 hours.
In
Specifically, the solvent that can be used in
The reaction temperature may be 10-40 ° C,
The reaction time can be 10-30 hours.
As shown in the following
Adding succinic anhydride to diosgenin to obtain compound 10 (step 1);
Adding n-hydroxysuccinimide to compound 10 to obtain compound 11 (step 2); And
Adding cytarabine (Cyt) to compound 11 to obtain compound 1d (step 3);
(1d) and diosgenin are linked by a linker.
[Reaction Scheme 4]
In
In
In
Specifically, the solvent that can be used in
The reaction temperature may be 10-40 ° C,
The reaction time can be 10-30 hours.
Pharmaceutical composition
The present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a conjugate in which an anticancer agent and diosgenin are linked by a linker, or a pharmaceutically acceptable salt thereof.
Herein, the cancer may be breast cancer, liver cancer, various types of leukemia (acute and chronic leukemia), lymphoma, colorectal cancer, rectal cancer, prostate cancer, stomach cancer and the like, preferably breast cancer or liver cancer.
The conjugate in which the anticancer agent and diosgenin are linked by a linker is characterized by inhibiting the resistance of the anticancer agent and further enhancing the permeability of the cancer cell membrane.
The anticancer agent-diosgenin conjugate according to the present invention may be administered in various formulations of oral or parenteral administration at the time of clinical administration. In the case of formulation, the fillers, extenders, binders, wetting agents, disintegrators, Diluents or excipients.
Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .
Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.
In addition, the effective dose of the anticancer agent-diosgenin conjugate of the present invention on the human body may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity, and is generally about 0.001 to 100 mg / kg / Day, preferably 0.01 to 35 mg / kg / day. It is generally 0.07 to 7000 mg / day, preferably 0.7 to 2500 mg / day, based on an adult patient weighing 70 kg, and may be administered once a day, It may be divided into several doses.
Anticancer formulation
The present invention provides an anticancer formulation of a conjugate in which the anticancer agent and diosgenin are linked by a linker. The anticancer formulation according to the present invention may be particularly advantageous for patients who have developed resistance to conventional anticancer agents.
Self-assembled nanoparticles
The present invention provides nanoparticles in which a conjugate in which a hydrophilic anticancer drug and hydrophobic diosgenin are linked by a linker is formed by self-assembly. The nanoparticles according to the present invention can be self-assembled into nanoparticles of 200 nm or less since the hydrophilic anticancer agent and hydrophobic diosgenin form a conjugate and exhibit both affinity.
For example, in Example 5, cytarabine (Cyt) was used as a hydrophilic anticancer agent to prepare nanoparticles. Cyt-DG conjugated nanoparticles have ideal pharmacological properties as drug delivery vehicles. Because many conventional studies have shown that nanoparticles with a particle size of less than 200 nm and negatively charged surface charge have a longer half-life after intravenous administration and have enhanced permeation and retention effects on cancerous tumor sites.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.
< Example 1 > MTX-DG Conjugate Preparation 1 (Compound 1a)
Step 1: Preparation of
Diosgenin (manufactured by Sigma-Aldrich) was subjected to dehydration reaction with chloroformic acid to prepare
Step 2: Preparation of compound 6
To a dry DCM solution (20 mL) in which diosgenyl chloroformate (
Step 3: Preparation of the desired compound 1a
DMF solution (5 mL) in which TBTU (70.6 mg, 0.44 mmol) was dissolved was dissolved in anhydrous DMF (5 mL) containing methotrexate (MTX) (100 mg, 0.22 mmol) and N-methylmorpholine (0.05 mL, 0.44 mmol) Was slowly added in an ice bath for 45 minutes. Next, Compound 6 (143.7 mg, 0.25 mmol) prepared in
Yield = 65%;
Analytical HPLC, retention time: 41.37 min;
1 H NMR (400 MHz, DMSO- d 6) δ 8.56 (s, 1H), 8.10 (s, 1H), 7.77 (s, 2H), 7.757.63 (d. 2H), 7.41 (s, 2H), 2H), 2.76 (t, 4H), 0.96 (d, 2H), 2.32 (s, 2H) (s, 3H), 0.90 (d, 3H), 0.73 (s, 6H);
13 C NMR (500 MHz, DMSO- d 6 ) δ 173.71, 172.86, 166.59, 165.83, 163.30, 163.17, 156.14, 155.64, 151.33, 149.61, 146.46, 140.23, 129.48, 128.93,122.34,122.13,121.90,111.63,108.87 , 80.65, 79.63, 73.52, 66.39, 62.26, 56.17, 55.34, 53.96, 49.87, 19.47, 17.55, 16.46, 15.12;
HRMS (ESI): m / z 1027.6061 (M-1), calculated (C 52 H 72 N 10 O 8 S 2 ) 1028.50.
< Example 2> MTX-DG Conjugate Preparation 2 (Compound 1b)
Example 2a: n = 0 (compound 1b1)
Example 2b: n = 1 (compound 1b2)
Example 2c: n = 2 (compound 1b3)
Step 1: Preparation of
Step 2: Preparation of
Step 3: Compound 9 or target compound 1b1 Produce
Compound 9 or the desired compound 1b1 was obtained in the same manner as in the
Compound 1b1:
Yield = 71%;
Analytical HPLC, retention time = 29.71 min;
1 H NMR (400 MHz, DMSO -d6, ppm) δ 8.55 (s, 1H), 7.91 (s, 1H), 7.77-7.75 (d, J = 8.87, 1H), 7.69-7.65 (d, J = 8.87 2H), 7.41 (s, 2H), 7.10 (s, 1H), 6.83-6.80 (dd, J = 9.10, 2H) , 2H), 3.42 (dd, 1H) 3.10 (t, 4H), 0.94 (s, 3H), 0.90 (d, 3H), 0.73 (s, 6H);
13 C NMR (500 MHz, DMSO- d 6) δ 173.51, 172.92, 166.66, 165.93, 163.19, 163.14, 156.19, 155.47, 151.21, 149.56, 146.58, 140.26, 129.49, 129.02, 122.28, 122.03, 121.90, 111.61, 108.87 , 80.64, 79.63, 73.40, 66.37, 62.24, 56.16, 55.29, 54.09, 49.87, 19.43, 17.52, 16.43, 15.09;
HRMS (ESI): m / z 935.6147 (M-1), calculated (C 50 H 68 N 10 O 8 ) 936.52.
Step 4: 1b2 And 1b3 Produce
To the anhydrous chloroform solution to which Compound 9 (n = 1) was added, TFA (10 eq.) Was slowly added in an ice bath. After 4 hours, the TFA was removed under vacuum, the sample was diluted with chloroform and then washed with water and brine. The obtained crude product was purified by HPLC to obtain objective compound 1b2 as a yellow solid.
Compound 1b2:
Yield = 54%;
Analytical HPLC, retention time = 25.91 min;
1 H NMR (400 MHz, DMSO- d 6, ppm) δ 8.57 (s, 1H), 8.15 (s, 1H), 7.86 (s, 2H), 7.757.63 (d. 2H), 7.42 (s, 1H 2H), 0.96 (s, 3H), 7.26 (s, IH), 6.82 (s, , 0.90 (d, 3H), 0.73 (s, 6H);
13 C NMR (600 MHz, DMSO- d 6) δ 174.86, 172.98, 167.31, 163.11, 156.26, 155.55, 151.23, 149.58, 146.44, 140.16, 131.97, 129.09, 128.98, 122.04, 121.86, 111.58, 108.85, 80.62, 73.63 , 66.34, 62.20, 56.11, 55.27, 49.80, 19.47, 17.50, 16.42, 15.09;
HRMS (ESI): m / z 978. 6692 (M-1), calculated (C 52 H 73 N 11 O 8 ) 979.56.
To the anhydrous chloroform solution to which Compound 9 (n = 2) was added, TFA (10 eq.) Was slowly added in an ice bath. After 4 hours, the TFA was removed under vacuum, the sample was diluted with chloroform and then washed with water and brine. The resulting crude product was purified by HPLC to obtain the desired compound 1b3 as a pale yellow solid.
Compound 1b3:
Yield = 51%;
Analytical HPLC, retention time: 18.45 min;
1 H NMR (300 MHz, DMSO- d 6, ppm) δ 8.64 (s, 1H), 8.27 (s, 1H), 8.16 (s, 2H), 7.81-7.74 (dd, 2H), 7.36-7.26 (s 2H), 6.82-6.80 (dd, 2H), 5.31 (s, IH), 4.83 (s, IH), 4.28 (m, 3H), 3.23 t, 2H), 0.96 (s, 3H), 0.91 (d, 3H), 0.73 (s, 6H);
13 C NMR (500 MHz, DMSO- d 6) δ 174.12, 172.99, 166.77, 163.09,156.55, 155.32, 151.55, 149.24, 144.94, 140.14, 129.61, 129.44, 122.53, 122.17, 121.99, 111.19,108.87, 80.61, 73.90 , 66.35, 62.19, 56.12, 55.94, 49.83, 19.42, 17.50, 16.40, 15.07;
HRMS (ESI): m / z 1021.7123 (M-1), calculated (C 54 H 78 N 12 O 8 ) 1022.61.
< Example 3> DOX -DG Conjugate Preparation (Compound 1c)
Step 1: Compound 10 ( DSA ) Preparation
(0.4 mmol), succinic anhydride (1 mmol) and DMAP (4-dimethylaminopyridine) (0.5 mmol) were dissolved in DCM (dichloromethane) and mixed thoroughly. Pyridine (0.5 mL) And refluxed. The reaction solution was checked by TLC. The final solution was cooled to room temperature, washed with water and hydrochloric acid solution, and then the organic solvent layer was dried over MgSO 4 and the organic solvent was evaporated to obtain a pale white powder. The residue was purified by silica
Step 2: Preparation of compound 11
Compound 10 (0.1 mmol), DCC (N, N'-dicyclohexylcarbodiimide) (0.3 mmol) and NHS (N-hydroxysuccinimide) (0.3 mmol) are dissolved in DCM in dichloromethane and mixed well for 6 hours. DCC in the mixture was removed by filtration while confirming the reaction solution by TLC (thin layer chromatography), and DCM was also distilled off to obtain Compound 11 as a white powder.
Step 3: Preparation of desired compound 1c
Doxorubicin (DOX) (0.02 mmol) and TEA (triethylamine) (10 mL) were dissolved in dimethylformamide (DMF) and mixed well. Compound 11 (0.05 mmol) was added. The mixture is stirred in a dark room at room temperature for 24 hours. After the reaction, dilute with DCM (dichloromethane), wash with water and saturated brine, and remove DMF. The organic layer was dried over Na 2 SO 4 , filtered, and distilled under reduced pressure to obtain a deep red powder, which was purified by silica gel column to give the final product, Compound 1c.
Yield = 47.5%;
1 H NMR (300 MHz, DMSO -d 6) δ 7.90 (d, J = 4.7 Hz, H3 & H1 of DOX), 7.68 - 7.56 (m, H2 of DOX), 5.57 (d, J = 8.0 Hz, H1 (D, 3'-CH of DOX), 4.94 (s, C'-OH of DOX), 5.47 (s, -NHC = O- of DOX-DG), 5.33 4.9 (s, H16 of DG), 3.98 (t, J = 5.9 Hz, 4'-OH of DOX) (m, 3H), 1.91 (dd, J = 26.8, 13.4 Hz), 2.98 (s, H4 of DOX), 2.98 2H), 1.63-1.46 (m, 11H), 1.37 (s, 4H), 1.24 (s, 3H) 9H), 1.22-1.09 (m, 12H), 1.09-0.94 (m, 13H), 0.93-0.71 (m, 18H), 0.70-0.58 (m, 3H).
< Example 4> Cyt -DG Conjugate Preparation (Compound 1d)
Step 1: Preparation of
Step 2: Preparation of compound 11
Compound 11 was obtained in the same manner as in Step 2 of Example 3.
Step 3: Preparation of desired compound 1d
Compound 11 obtained in Step 2 was added to a DMF solution in which Cytarabine was dissolved, and the mixture was stirred at room temperature for 48 hours under a nitrogen atmosphere. The solvent was removed in vacuo and purified by flash chromatography (1-5% methanol in CHCl 3) to give the desired compound 1d as white solid.
Yield = 32%;
Purity = 98%;
Analytic HPLC, retention time = 28.69 min;
1 H NMR (300 MHz, DMSO- d 6) δ 10.90 (s, 1H), 8.05 (d, 1H), 7.16 (d, 1H), 6.05 (d, 1H), 5.49 (s, 2H) 5.34 (d (S, 1H), 5.07 (s, IH), 4.45 (m, IH), 4.28 2H), 2.67 (t, 2H), 2.54 (t, 2H), 2.26 (d, 2H), 2.10-1.00 overlap peaks 0.97 (s, 3H), 0.91 (dd, 3H), 0.72 (m, 6H);
13 C-NMR (100 MHz, DMSO- d 6 )? 173.11, 172.01, 162.49, 155.04, 147.15, 139.90, 122.39, 108.92, 94.85, 87.46, 86.17, 80.63, 76.55, 74.93, 73.85, 66.35, 62.18, 61.43, , 49.80, 41.51, 40.20, 38.03, 31.19, 30.21, 28.87, 20.77, 19.33, 17.49, 16.40, 15.06;
HRMS (ESI): m / z 739.4809 (MH) - , calculated (C 40 H 57 N 3 O 10 ) 739.40.
< Example 5> Cyt -DG Conjugate Manufacture of nanoparticles
The Cyt-DG conjugated nanoparticles utilized a known nanoprecipitation method [Y. Jin, R. Xin, P. Ai, and D. Chen, Int J Pharm, 350, pp. 330 (2008)].
Specifically, while stirring the THF solution (0.5 mg, at 1 mg / mL) in which the Cyt-DG conjugate prepared in Example 4 was dissolved at 1000 rpm at room temperature, 1 mL of purified water containing 5% THF was added dropwise. The organic solvent was removed under vacuum at room temperature and the reaction (suspension) was purified with a dialysis tube (MWCO 2000) and deionized water for 1 day to remove residual organic solvent.
< Experimental Example 1 > MTX ( Methotrexate ) Evaluation of Anti-Cancer Resistance MTT assay)
Breast cancer patients who have experienced the anti-cancer drug, MTX, have developed resistance, and the MTX alone significantly reduces the membrane permeability of cancer cells. In Experimental Example 1, the following experiment was conducted to determine whether MTX-DG can reduce tolerance induced by MTX alone.
MCF-7 (human breast cancer cell line) and MDA-MB-231 (MTX resistant breast cancer cell line isolated from breast cancer patients who had experienced MTX treatment) were purchased from Korean Cell Line Bank, 10% FBS and 1% penicillin-streptomycin, and cultured in RPMI 1640 medium at humidified air conditions of 5% CO 2 and 37 ° C. The cultured cells were dispensed in 96-well plates at a concentration of 1 × 10 5 / well, and the cells were treated with the test compound in the wells. After 48 hours of incubation, 20 μL of MTT solution (5 mg / mL in phosphate-buffered saline (PBS)) was added and incubated for a further 4 hours. Next, the culture was discarded and replaced with 200 μL of isopropanol at room temperature. The absorbance of each well was measured with a microplate reader (570 nm), and the IC 50 value of each test compound was determined. As a result, the antiproliferative activity IC 50 values of MDA-MB-231 breast cancer cell lines are shown in Table 1 below.
(Diosgenin alone)
(Methotrexate alone)
(Example 1, compound 1a)
(Example 2, compound 1b1)
(Example 2, compound 1b2)
(Example 2, compound 1b3)
As shown in Table 1, the MTX-treated breast cancer cell line (MCF-7), which has not been treated with the anticancer agent MTX, exhibits excellent anticancer activity whereas the MTX-treated breast cancer cell line (MDA- MB-231) is resistant to MTX, and when treated with MTX alone, it shows almost no anti-activity. However, it was found that MTX-DG conjugate had the effect of overcoming MTX resistance as in Examples 1 and 2 according to the present invention. On the other hand, the compounds 1b1, 1b2, and 1b3 differ in the length of linkers connecting MTX and DG. As the length of the linker is longer, the effect of steric hindrance decreases and the anticancer activity is more excellent. Further, the linker of the compound 1b2 and 1b3 contained more amine groups than the linker of the compound 1b1, and it was found that the linker of the compound 1b2 and 1b3 was more absorbed into the cancer cell membrane. Furthermore, since the anticancer activity starts from the concentration of 65 μM or more even after diosgenin (DG) alone after the cancer cell membrane has permeated, it can be understood that the results of Table 1 are obtained.
Therefore, the anticancer agent-diosgenin conjugate according to the present invention can be useful as an anticancer agent formulation because it penetrates into cancer cells more well than the anticancer agent alone and overcomes the anticancer drug resistance and improves the anticancer activity.
< Experimental Example 2> DHFR ( Dihydrofolate reductase ) Inhibition evaluation
In Experimental Example 2, inhibitory effects of dihydrofolate reductase (DHFR) in vivo were evaluated using an enzyme assay to evaluate the inhibitory effects of dihydrofolate reductase (DHFR) DHFR) enzyme activity.
The DHFR inhibition assay was evaluated by monitoring NADPH oxidation in a microplate reader (340 nm) according to the instructions of the DHFR assay kit (Product Code CS0340, Saint Louis, Missouri 63103 USA).
Specifically, 0.002 units of DHFR and 6 μL of NADPH solution were added to the buffer solution together with the PBS solution (5 -10 μL) in which the test compound was dissolved (here, the control was buffer solution alone). The evaluation mixture was incubated at room temperature for 5 minutes, and 5 μL of dihydrofolic acid was added to initiate the reaction, and the change in absorbance at 340 nm was measured. Experimental results were expressed as percent inhibition of enzyme activity relative to control. The DHFR inhibition IC 50 values are shown in Table 2 below.
(Diosgenin alone)
(Methotrexate alone)
(Example 1, compound 1a)
(Example 2, compound 1b1)
(Example 2, compound 1b2)
(Example 2, compound 1b3)
As shown in Table 2, the DHFR inhibitory activity of the MTX-DG conjugate was lower than that of MTX alone (3.39 nM). This result was consistent with previous studies in which the affinity of the most conjugate products for the target enzyme was reduced after the chemical modification of the glutamate residues of MTX. The inhibitory activity of the MTX-DG conjugate tended to increase with increasing linker length (1b1 <1b2 <1b3) connecting MTX and DG in compound 1b1, 1b2 and 1b3 of Example 2. This result implies that the length of the linker is important to maintain affinity for the enzyme. On the other hand, although the linker lengths of Example 1 (compound 1a) and Example 2 (compound 1b3) were similar, the affinity to DHFR of Example 1 was higher, The presence of disulfide bonds in the linker is expected.
< Experimental Example 3> In the reducing environment, MTX-DG From a conjugate Evaluation of Separation of MTX
To evaluate the redox reaction-release of MTX from the MTX-DG conjugate prepared in Example 1 (Compound 1a) and Example 2 (Compound 1b3), 10 mM glutathione (GSH) Were used.
Figure 3 is a graph showing the activity of MTX, (b) Example 1 (compound 1a), (c) Example 1 (compound 1a) + GSH, (d) Example 2 (compound 1b3) Compound 1b3) + GSH.
As shown in Figure 3, Figure 3 (c) shows the appearance of a new HPLC peak at retention time 4.76 min, indicating rapid release of MTX from the MTX-DG conjugate of Example 1 (compound 1a) . The retention time of the MTX released from the MTX-DG conjugate in FIG. 3 (c) is slightly different from the retention time of the MTX in FIG. 3 (a) The released MTX is covalently linked to the cysteamine through an amide bond. In contrast, the MTX-DG conjugate of Example 2 (compound 1b3) in Figure 3 (e) did not show release of MTX in glatathione (GSH) treatment. This result was relatively stable under reducing conditions because there was no redox-reactive disulfide bond in the linker in Example 2 (compound 1b3).
On the other hand, in Fig. 3 (c), further mass analysis (UFLC-MS) was carried out for a retention time of 4.76 min. The mass spectrometry results (515.3023, M + 2) were in close agreement with the predicted mass of the MTX cysteamine conjugate (513.19), indicating that a peak at 4.76 min retention was released from the MTX-DG conjugate of Example 1 Gt; MTX < / RTI >
< Experimental Example 4> DOX -DG Conjugate Evaluation of antitumor activity
To investigate the anticancer activity of the DOX-DG conjugate prepared in Example 3 (Compound 1c), the following experiment was conducted.
Specifically, diosgenin (DG) alone, Compound 10 (DSA) prepared in
FIG. 1 (A) is a graph showing the cell survival rate after 48 hours of administration of DG, DSA, DOX and DOX-DG to HepG2 (human-derived liver cancer cells).
Fig. 1 (B) is a graph showing the cell survival rate after 48 hours of administration of DG, DSA, DOX, and DOX-DG to L929 (mouse fibroblast cell line).
As shown in Fig. 1, the DOX-DG conjugate of Example 3 showed the highest cancer cell toxicity in HepG2 (human-derived liver cancer cells) in Fig. 1 (A). In FIG. 1 (B), L929 (mouse fibroblast cell line; normal cell control group) showed similar tendency to DG, DSA, DOX and DOX-DG.
(Liver cancer cells)
(Fibroblast)
As shown in Table 3, the DOX-DG conjugate of Example 3 was the most toxic to liver cancer cells (HepG2) and the cytotoxicity of fibroblast (L929) was lower than that of DOX alone.
< Experimental Example 5> DOX -DG Conjugate Assessment of release of anticancer drugs by pH environment
To determine the degree of release of the anticancer drug according to the pH change of the DOX-DG conjugate prepared in Example 3, the pH was set to 5.0, 6.5 and 7.4 in vitro and then the DOX emission was detected. The results are shown in Fig.
FIG. 2 is a graph showing a DOX-DG conjugate prepared in Example 3 measured for DOX emission according to pH environment change. FIG.
As shown in FIG. 2, the relatively slow and low amount of DOX released at pH 7.4 indicates that DOX-DG is relatively stable in the blood of the body. The rapid and more excreted DOX release in acidic environments is selectively released in the endosomal / lysosomal environments of tumor cells.
Therefore, the anticancer agent-diosgenin conjugate according to the present invention may be useful as an anticancer agent formulation because it selectively releases an anticancer agent in the vicinity of a tumor cell and is not released in a general body environment.
< Experimental Example 6> Cyt -DG Conjugate Of nanoparticles in vitro Cell Absorption Assessment
In order to examine the in vitro cell uptake and dispersion tendency of the Cyt-DG conjugated nanoparticles prepared in Example 5, a confocal scanning laser microscope and HPLC (cell absorption quantitation) were performed using MCF-7 cell line (human breast cancer cell line) Were used.
Specifically, the MCF-7 cell line was divided into 12-well plates at a density of 1.0 x 10 4 cells on coverslips and cultured overnight. The cells were treated with 10 μg / mL rhodamine B (fluorescent dye) or rhodamine B-labeled Cyt-DG conjugated nanoparticles and cultured at 37 ° C. for 4 hours. Next, the culture medium containing rhodamine B or rhodamine B-labeled Cyt-DG conjugated nanoparticles was removed, washed twice with cold PBS, and lysed with 1% Triton x-100 for 5 minutes. After washing again 3 times with cold PBS, the cover glass was treated with DAPI-containing encapsulant (3 μL), the cells were placed on a microscope slide with the dispensed side facing down and sealed with a manicure. Confocal microscopy (Carl Zeiss LSM 510 system) was used to confirm the tendency of cell uptake and dispersion,
4 is a fluorescence image obtained by confirming the cell uptake of Cyt-DG conjugated nanoparticles through a confocal scanning laser microscope.
As shown in Fig. 4, the cell absorption efficiency of rhodamine B-labeled Cyt-DG conjugated nanoparticles was clearly higher than that of rhodamine B alone. Rhodamine B alone is absorbed into cells through passive diffusion, while rhodamine B labeled Cyt-DG conjugated nanoparticles are absorbed into cells by endocytosis (see FIG. 8).
The concentration of rhodamine B-labeled Cyt-DG conjugated nanoparticles was 2.5 times higher than that of rhodamine B alone. This result implies that the cytotoxicity of rhodamine B-labeled Cyt-DG conjugated nanoparticles is better.
< Experimental Example 7> Cyt -DG Conjugate Evaluation of cancer cell growth inhibition by nanoparticles
MTT assay was performed to evaluate the in vitro anticancer activity of the Cyt-DG conjugate prepared in Example 4 (Compound 1d).
Specifically, the cells were seeded at a concentration of 1.0 × 10 5 cells / well on a 96-well plate, and when the wells were about 80% full, the Cyt-DG conjugate was treated at various concentrations and cultured for 48 hours or 72 hours . Next, PBS buffer containing 10 μL of MTT (5 mg / mL) solution was added and further incubated for 4 hours. After incubation, the medium containing the drug was removed and replaced with 200 μL of isopropanol at room temperature. The absorbance of each well was quantitated with a microplate reader (570 nm). All of the PBS solution, diosgenin (DG) and mixture thereof (Cyt / DG, 1/1) dissolved in cytarabine (Cyt) as an anticancer agent were dissolved in 5% PEG 400 and their anticancer activities were measured by MTT assay Respectively. At this time, treatment with PBS or 0.5% PEG 400 was used as a control. The IC 50 (μM) values of each of the Cyt-DG conjugated nanoparticles, DG and Cyt were calculated and are shown in Table 4 below.
(Acute promyelocytic leukemia cell line)
(Non-metastatic human breast cancer cell line)
(Metastatic human breast cancer cell line)
As shown in Table 4, the anticancer activity of the Cyt-DG conjugated nanoparticles after culturing for 48 hours in the HL-60 cell line was higher than that of the DG and Cyt alone groups. The enhanced anti-cancer activity of the Cyt-DG conjugated nanoparticles Activity is expected to be due to increased accumulation in cancer cells. It is known that cytarabine (Cyt) does not generally respond to solid tumor treatment. Surprisingly, Cyt-DG conjugated nanoparticles showed an IC 50 of 30.05 μM in MCF-7 cell line (solid tumor). Similarly, the Cyt-DG conjugated nanoparticles showed an IC 50 of 81.86 μM in the MDA-MB-231 cell line (solid tumor).
< Experimental Example 8> Cyt -DG Conjugate Identification of nanoparticles
Cytarabine (Cyt) is a hydrophilic anticancer drug and Diosgenin (DG) is a hydrophobic steroidal saponin. The Cyt-DG conjugate exhibits both amphiphilic properties, which allows self-assemble of stable nanoparticles.
Determination of critical aggregation concentration of nanoparticles
The critical aggregation concentration (CAC) was measured using pyrene with a fluorescent probe. Specifically, 10 μL of a pyrene solution (5 μM) dissolved in acetone was added to the Cyt-DG conjugate solution (1.0 × 10 -3 to 0.4 mg / mL) prepared in Example 4 and ultrasonicated for 1 minute. The acetone was evaporated, and the mixed solution was allowed to equilibrate to the aqueous phase of pyrene in the dark at room temperature overnight. Fluorescence spectra of all solutions were measured with a spectrophotometer (FP-6200; JASCO) at a wavelength of 390 nm. I 336 / I 334 'of Cyt-DG conjugate concentration (log C) versus fluorescence intensity ratio was used for CAC determination.
5 is a graph showing the critical aggregation concentration (CAC) of the Cyt-DG conjugate by LC-MS spectrum.
As shown in FIG. 5, the Cyt-DG conjugate exhibited a very low critical aggregation concentration (12.5 μg / mL), indicating a high stability against dilution.
Particle size, Surface charge And form ( Morphology )
FIG. 6 shows the results of (a) Dynamic light scattering (DLS) and (b) transmission electron microscopy (TEM) to examine the particle size distribution, zeta potential and morphology of the Cyt- DG conjugated nanoparticles prepared in Example 5 The results are analyzed.
As shown in FIG. 6, the DLS analysis of FIG. 5 (a) shows that the particle size distribution is narrow in the region of 189.6 nm and shows a surface charge of -8.42 mV. The TEM image in FIG. 5 (b) shows that the nanoparticles exhibit spherical morphology, and the particle size has a diameter of approximately 75 nm smaller than the DLS result. This result is expected to be due to the reduced impact of nanoparticles on TEM sample preparation. When the labeling content of rhodamine B was less than 1%, the size of rhodamine B-labeled Cyt-DG conjugated nanoparticles did not increase.
Cyt-DG conjugated nanoparticles have ideal pharmacological properties as drug delivery vehicles. Because many conventional studies have shown that nanoparticles with a particle size of less than 200 nm and negatively charged surface charge have a longer half-life after intravenous administration and have enhanced permeation and retention effects on cancerous tumor sites.
Cyt -DG Conjugate Stability of nanoparticles
The physico-chemical stability of the Cyt-DG conjugated nanoparticles (Example 5) was evaluated through particle size change and PDI (polydiversity index) evaluation using DLS. Specifically, the particle size changes of Cyt-DG conjugated nanoparticles in 0.1M PBS solution, 5% glucose solution, PBS solution containing 1% FBS and PBS solution containing 10% FBS were measured using DLS.
FIG. 7 is a graph showing the change in diameter of Cyt-DG conjugated nanoparticles measured by DLS in (a) 0.1 M PBS and 5% glucose solution, (b) Cyt-DG conjugate in 1% and 10% And the change in diameter of the gate nano-particles is measured by DLS.
As shown in Fig. 7, Cyt-DG conjugated nanoparticles were stable in PBS solution at 4 占 폚 for one week (Fig. 7 (a)). However, as the FBS content in the PBS solution increased, the particle size tended to decrease significantly (Fig. 7 (b)).
≪ Formulation Example 1 > Preparation of pharmaceutical preparation
<1-1> Preparation of powder
The conjugate 2 g of the present invention
Lactose 1 g
After mixing the above components, the mixture was packed in an airtight container to prepare a powder.
<1-2> Preparation of tablets
100 mg of the conjugate of the present invention
100 mg of milk
2 mg of magnesium stearate
After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
≪ 1-3 > Preparation of capsules
100 mg of the conjugate of the present invention
100 mg of milk
2 mg of magnesium stearate
After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.
<1-4> Preparation of Injection Solution
10 [mu] g / ml of the conjugate of the present invention
Until dilute hydrochloric acid BP pH 3.5
Sodium chloride BP injected up to 1 ml
The conjugate according to the invention was dissolved in a suitable volume of injected sodium chloride BP and the pH of the resulting solution was adjusted to pH 3.5 using dilute hydrochloric acid BP and the volume was adjusted using injectable sodium chloride BP and mixed well . The solution was filled in 5 ml type I ampoule made of transparent glass, sealed in the upper lattice of the air by dissolving the glass, and sterilized by autoclave at 120 캜 for 15 minutes or longer to prepare an injection solution.
Claims (14)
Hydrophobic diosgenin;
, , , And Or a pharmaceutically acceptable salt thereof, is formed by self-assembly,
Characterized by having a particle size of less than 200 nm and a negative surface charge.
Nanoparticles.
Wherein the conjugate is any one of the following formulas (1a) to (1d):
[Formula 1a]
[Chemical Formula 1b]
(In the above formula (1b), n is an integer of 0-2)
[Chemical Formula 1c]
≪ RTI ID = 0.0 &
.
Adding carbonochloridic acid to diosgenin to obtain compound 4 (step 1);
Adding Compound 5 to Compound 4 to obtain Compound 6 (Step 2); And
Adding methotrexate (MTX) to compound 6 to obtain compound 1a (step 3);
Wherein the anticancer agent represented by the following formula (1a) and diosgenin are linked by a linker.
[Reaction Scheme 1]
Adding carbonochloridic acid to diosgenin to obtain compound 4 (step 1);
Adding compound 7 to compound 4 to obtain compound 8 (step 2);
Adding methotrexate (MTX) to compound 8 to obtain compound 9 (step 3); And
Removal of the protecting group (Boc, tert-Butyloxycarbonyl) from compound 9 to obtain compound 1b (step 4);
, Wherein the anticancer agent is represented by the following formula (1b) and Diosgenin is conjugated with a linker:
[Reaction Scheme 2]
(In the above Reaction Scheme 2, n is an integer of 0-2).
Adding succinic anhydride to diosgenin to obtain compound 10 (step 1);
Adding n-hydroxysuccinimide to compound 10 to obtain compound 11 (step 2); And
Adding cytarabine (Cyt) to compound 11 to obtain compound 1d (step 3);
Wherein the anticancer agent represented by the following formula (1d) and diosgenin are linked by a linker.
[Reaction Scheme 4]
Wherein the cancer is one selected from the group consisting of breast cancer, liver cancer, acute leukemia, chronic leukemia, lymphoma, colon cancer, rectal cancer, prostate cancer, and stomach cancer.
Wherein the nanoparticles inhibit the resistance of the anticancer agent.
Wherein the nanoparticles increase the permeability of the cancer cell membrane.
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CN111298130A (en) * | 2019-12-21 | 2020-06-19 | 河南工业大学 | Diosgenin-cytarabine liposome without cholesterol and preparation method thereof |
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