Doxorubicin-squalene micromolecular prodrug as well as preparation method and application thereof

Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and relates to construction of a doxorubicin-squalene prodrug and a self-assembled nanoparticle thereof, and application of the doxorubicin-squalene prodrug and the self-assembled nanoparticle thereof in a drug delivery system. The doxorubicin-squalene micromolecular prodrug disclosed by the invention is prepared by connecting doxorubicin (DOX) and squalene (SQ) through a hypoxia sensitive group (Ar-N = N-Ar). The structure of the compound is shown as a formula I. The doxorubicin-squalene micromolecule prodrug disclosed by the invention is obtained by connecting squalene with a hypoxia sensitive function key and connecting the squalene with doxorubicin by adopting a squalene acylation technology. The obtained doxorubicin-squalene micromolecule prodrug has a self-assembly property and can be self-assembled to form a uniform nano system. The curative effect of adriamycin can be improved, and the toxicity can be reduced. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and relates to the construction of doxorubicin-squalene prodrug and self-assembled nanoparticles thereof, and the application of the prodrug in a drug delivery system. Background Art

[0002] Doxorubicin is one of the main anticancer drugs in clinical oncology, mainly used for acute lymphocytic leukemia, acute myeloid leukemia, Wilms tumor, neuroblastoma, soft tissue and bone sarcoma, breast cancer, ovarian cancer, bladder transitional cell carcinoma, thyroid cancer, gastric cancer, and Hodgkin's disease. It can inhibit the synthesis of RNA and DNA, and is a cycle non-specific drug that has a killing effect on tumor cells in various growth cycles.

[0003] Squalene (SQ) is a terpene compound widely distributed in nature. It is a polyunsaturated hydrocarbon produced in the metabolic process of cholesterol synthesis in the human body. Squalene has a significant dynamic folding conformation, which can form a stacking structure and stabilize the nanoparticle structure.

[0004] The side effects of doxorubicin are mainly dose-limited irreversible cardiotoxicity and bone marrow suppression. Although nanotechnology has greatly improved the cardiotoxicity of doxorubicin, these new nanoformulations also have some defects, including: carrier-related toxicity, low drug loading (generally less than 10%), poor stability, etc. Therefore, how to design an efficient and low-toxic drug delivery system for the delivery of doxorubicin remains a hot topic of research.

[0005] There is no related report on doxorubicin-squalene small molecule prodrug in the prior art. Summary of the invention

[0006] In view of the above problems, we have designed a series of doxorubicin-squalene small molecule prodrugs. Doxorubicin and squalene are connected together by hypoxia-sensitive chemical linkers. The hypoxia-sensitive chemical linkers can quickly break in the hypoxic environment of tumor cells, release doxorubicin in a targeted manner, and thus reduce the toxic side effects on normal cells. At the same time, these small molecule hydrophobic prodrugs can self-assemble to form uniform prodrug nanoparticles. In these small molecule prodrug nanosystems, since the prodrug itself serves as both a carrier material and an encapsulated drug, its encapsulation rate is greater than 90% and the drug loading exceeds 75%. Based on this, the present invention designs and constructs hypoxia-sensitive doxorubicin-squalene small molecule prodrug nanoparticles, and performs PEG modification on them. After injection, they are given long circulation characteristics in the blood and applied to anti-tumor research.

[0007] The purpose of the present invention is to design and synthesize hypoxia-sensitive doxorubicin-squalene small molecule prodrugs and prepare prodrug self-assembly nano drug delivery systems. Compared with doxorubicin, the doxorubicin-squalene of the present invention has better anti-tumor effects and lower drug toxicity.

[0008] The doxorubicin-squalene small molecule prodrug of the present invention is prepared by connecting doxorubicin (DOX) and squalene (SQ) via an oxygen-sensitive group (Ar-N=N-Ar).

[0009] The structure of the doxorubicin-squalene small molecule prodrug is shown in Formula I:

[0010]

[0011] Wherein, Ar is a substituted or unsubstituted phenyl group, and the substituent is a halogen, a C1-C6 alkyl group, or a C1-C6 alkoxy group.

[0012] The present invention preferably has the following structure of doxorubicin-squalene small molecule prodrug:

[0013]

[0014] The doxorubicin-squalene small molecule prodrug of the present invention is prepared by the following method:

[0015]

[0016] wherein Ar is as described above.

[0017] The doxorubicin-squalene small molecule prodrug prepared by the present invention can be self-assembled into nanoparticles, and the small molecule prodrug nanoparticles can be non-PEGylated small molecule prodrug nanoparticles or PEG-modified small molecule prodrug nanoparticles.

[0018] (1) Preparation method of non-PEGylated small molecule prodrug self-assembled nanoparticles: a certain amount of prodrug is dissolved in an appropriate amount of DMSO, and the DMSO solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles.

[0019] (2) Preparation of PEG-modified small molecule prodrug self-assembled nanoparticles: a certain amount of DSPE-PEG 2k The prodrug is dissolved in an appropriate amount of DMSO, and the DMSO solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles.

[0020] The doxorubicin-squalene small molecule prodrug of the present invention adopts the "squalene acylation" technology to connect squalene with hypoxia-sensitive functional bonds and then connect it with doxorubicin. The obtained doxorubicin-squalene small molecule prodrug has a self-assembly property and can self-assemble to form a uniform nanosystem. The advantages of this nano drug delivery system are: (1) It adopts a one-step nanoprecipitation method, with a simple preparation process and easy industrialization; (2) The particle size is small and uniform (~100nm), which is conducive to the enrichment of nanoparticles in the tumor site through the EPR effect; (3) The ultra-high drug loading is conducive to reducing adverse reactions caused by excipients and biomaterials; (4) Through the sensitivity of the linking arm to the intracellular environment of the tumor site, the specific release of doxorubicin in the tumor cells is achieved, which improves the efficacy while reducing toxic side effects.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. A new type of doxorubicin-squalene small molecule prodrug was designed and synthesized; 2. Through the design of oxygen-deficient chemical linker arms, doxorubicin can be released inside tumor cells; 3. Uniform small molecule prodrug self-assembled nanoparticles were prepared, and the preparation method is simple and easy, with good stability and high drug loading; 4. PEG-modified small molecule prodrug self-assembled nanoparticles can effectively prolong the circulation time of the drug in the blood; 5. The prepared small molecule prodrug self-assembled nanoparticles have good anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the MS image of doxorubicin-squalene small molecule prodrug;

[0024] Figure 2 A small molecule prodrug of doxorubicin-squalene 1 H NMR spectra;

[0025] Figure 3 DOX-PRO NP S Properties diagram;

[0026] A: Particle size distribution curve; B: Transmission electron microscopy image of nanoparticles; C: Changes in particle size and PDI of nanoparticles in a 4°C environment;

[0027] Figure 4 DOX-PRO NP S Drug release under different concentrations of Na2S2O4;

[0028] Figure 5 is the survival rate of 4T1 cells;

[0029] Figure 6 The fluorescence quantitative results of DOX uptake by 4T1 cells under normoxic and hypoxic conditions;

[0030] *p<0.05; **p<0.01;

[0031] Figure 7 Blood concentration-time curves of DOX (A) and DOX-PRO NPs (B);

[0032] Figure 8 For antitumor activity;

[0033] A: Tumor change curve of tumor-bearing mice during treatment; B: Body weight change; (*p<0.05, n=5)

[0034] Fig. 9 These are various liver and kidney function indicators for mice. DETAILED DESCRIPTION

[0035] Example 1 Preparation of doxorubicin-squalene small molecule prodrug

[0036]

[0037] At 0°C, compound II (10 mmol), imidazole (15 mmol) in THF, and TBDMSCl (12 mmol in THF) were added to the mixed solution. The mixture was stirred at room temperature for 6 hours. THF was removed under reduced pressure, and the organic phase was evaporated after DCM extraction. The synthesized compound III can be used directly without purification. At room temperature, under argon protection, compound III (5 mmol), compound IV (5 mmol), EDCI (6 mmol), DMAP (catalysis) and anhydrous DCM (10 mL) were stirred overnight. The reaction solution was washed with dilute hydrochloric acid (10 mL), and the organic layer was dried over anhydrous sodium sulfate. After removing the organic solvent under reduced pressure, the residual organic matter was purified by column chromatography (PE: AcOEt = 10: 1) to obtain compound V. At room temperature, a THF solution of TBAF was added dropwise to the reaction solution. After 2 hours, it was confirmed that the TBDMS protection was successfully removed. After removing THF under reduced pressure, compound VI was obtained without other side reactions. In an ice bath, anhydrous DCM acyl chloride solution was added dropwise to the solution consisting of VI and TEA and stirred at 0°C for 30 minutes. Then, the produced salt was washed with water, and the organic phase was evaporated. The intermediate VII can be purified by column chromatography with a mobile phase of PE:AcOEt=5:1. At room temperature, DOX hydrochloride, VII, and TEA were stirred in a DMF solution overnight and diluted with DCM. The reaction solution was transferred to a silica gel chromatography, and the eluent of DCM:MeOH=10:1 was collected. After removing the organic phase, prodrug I (DOX-PRO) was obtained. The product MS is shown in Figure 1 , 1 H NMR Figure 2 .

[0038] 1H NMR(DMSO-d6,400MHz)13.97(1H,s),13.22(1H,s),10.20(1H,s),7.81-7.76(8H,m),7.53(1H,d,J=5.20Hz),7.47(2H,d,J= 7.88Hz),5.42(1H,s),5.22(1H,s),5.17-4.99(8H,m),4.9(1H,bro.s),4.85(1H,t,J=5.76Hz),4.73(1H,d,J=5.24Hz),4.5 9(2H,d,J=5.40Hz),4.18(1H,d,J=6.48Hz),3.93(3H,s),3.75(1H,bro.s),3.48(1H,bro.s),3.00-2.89(2H,m),2.43(2H,t ,J=7.08Hz),2.27(2H,t,J=6.68Hz),2.01-1.89(18H,m),1.60(8H,s),1.52(12H,s),1.34(3H,d,J=5.92Hz).ESI-MS:[M+H] + =1179.7,[M+H] + =1201.6.

[0039] Example 2 Preparation of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles (DOX-PRO NPS):

[0040] Preparation of DOX-PRO NPs by one-step precipitation method S , weigh a certain amount of doxorubicin-squalene small molecule prodrug (DOX-PRO), prepare a DOX-PRO solution with a concentration of 4 mg / mL using DMSO, and take 500 μL. 2K DSPE-PEG was prepared with DMSO at a concentration of 2 mg / mL. 2K Solution, take 200μL, mix 500μL DOX-PRO solution and 200μL DSPE-PEG 2K The solution was thoroughly mixed and slowly and uniformly added to 2 mL of deionized water under 800 rpm magnetic stirring. Stirring was performed for 4 h in the dark to disperse the solution. After 4 h, the nanoparticles were taken out and dialyzed in a dialysis bag with a molecular weight cutoff of 3500 to remove DMSO. The dialysis was performed for 24 h using distilled water as the dialysis medium. The nanoparticles obtained after dialysis were collected in a glass bottle and concentrated by rotary evaporator at 37 °C to obtain DOX-PRO NPs with a concentration of 2 mg / mL (DOX-PRO concentration 2 mg / mL). S Solution.

[0041] DOX-PRO NP prepared with a drug-excipient ratio of 5:1S The solution was detected by a particle size analyzer to have a particle size of about 60 nm, and the particle size distribution curve had a good peak shape. DOX-PRO NPs could be observed using a transmission electron microscope (TEM). S The shape is spherical ( Figure 3 A). Encapsulation efficiency can reach 94.033±4.406%, drug loading is 78.333±3.625%, and particle size distribution is Figure 3 B. PDI value Figure 3 As shown in C.

[0042] Example 3 Release of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles

[0043] Determination of DOX-PRO NPs S Release behavior in different concentrations of Na2S2O4 release medium. 0mM, 10mM, 20mM, and 40mM Na2S2O4 solutions were prepared as release medium. S 1 mL of solution (DOX PRO concentration 1 mg / mL) was placed in a dialysis bag, which was then placed in prepared Na2S2O4 release media of different concentrations. The entire system was placed in a 37°C constant temperature shaker. At the set time points including 2h, 4h, 6h, 10h, and 24h, 500 μL of samples were taken (500 μL of the corresponding release medium needed to be added to the release system for each 500 μL sample taken out), followed by ultrasonication for 10 min, and the concentration of DOX was determined using HPLC.

[0044] By using Na2S2O4 solution as a release medium to simulate the hypoxic environment in vitro, DOX-PRO NP S The release rate increases with the increase of Na2S2O4 concentration. When the concentration is 40mM, the release rate is the fastest, and about 80% is released in 24h, which shows that the nanoparticles have good drug release performance in a low oxygen environment. Figure 4 )

[0045] Example 4 In vitro antitumor activity of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles

[0046] The 4T1 cells with good growth status were digested, and then the cells were added to the 96-well plate and cultured for 24 hours. DOX and DOX-PRO NP were prepared in blank culture medium. S There were two groups of drugs with a concentration gradient of DOX (0.5μM, 1μM, 2μM, 4μM, 5μM). After 24h of cell culture, the drug-containing culture medium was added. After 72h of culture, MTT was added to each well and culture was continued for 4h. After 4h, the absorbance value of each well was measured at 570nm using a multifunctional microplate reader to calculate the cell survival rate.

[0047] The results showed that DOX and DOX-PRO NP S After 72 hours of administration, DOX-PRO NP S The cytotoxicity is slightly weaker than that of DOX itself. The structural change of DOX leads to a slight decrease in in vitro activity and a decrease in cytotoxicity, which means that the prodrug may reduce the toxic side effects of DOX. Due to the different in vitro and in vivo environments, in vitro experiments cannot reflect the characteristics of targeted drug release and long circulation of nanoformulations in prodrug design, and further pharmacodynamic experiments are needed. ( Figure 5 )

[0048] Example 5 Cellular Uptake of Doxorubicin-Squalene Small Molecule Prodrug Self-Assembled Nanoparticles

[0049] DOX-PRO is composed of squalene and DOX connected by hypoxia-sensitive bonds. The normoxic and hypoxic environments were set up to investigate the effects of DOX, DOX-PRO, and DOX-PRO NP. S DOX release under different conditions. 4T1 was inoculated in a 24-well plate, with 5*10 4 cells, cultured for 24 h, and then added with DOX, DOX PRO, and DOX PRO NP S 500 μL of culture medium (DOX concentration was 2 μM), and PBS was used as the control group. The cells were cultured in a normoxic environment or anoxic environment for 2 h and 8 h respectively (normoxic environment: incubator T = 37 ° C CO2 = 5.0%; anoxic environment: T = 37 ° C, CO2 = 5.0%, O2 = 2.5%). After 8 h, the culture medium containing the drug was discarded and washed with PBS, and then fixed with tissue fixative for 30 min, and finally stained with DAPI for 8-10 min. The cells were photographed with a fluorescent inverted microscope and the fluorescence was quantified using image J software. The results are shown in Figure 6 .

[0050] The results showed that DOX, DOX-PRO, and DOX-PRO NP S The fluorescence intensity of DOX at 8h of incubation of the three groups of drugs was higher than that at 2h, indicating that the uptake of DOX by 4T1 cells was time-dependent. According to the fluorescence quantitative results, there was no difference in the uptake of DOX by 4T1 cells in normoxic and hypoxic environments in the 2h and 8h free DOX groups, while the fluorescence intensity of DOX in hypoxic environments was higher in the DOXPRO and DOX-PRO NPS groups at 8h than in normoxic environments, indicating that hypoxic bonds are more likely to break and release DOX in hypoxic environments, increasing the uptake of cells. S The DOX uptake in the group was lower than that in the free DOX and DOX-PRO groups, which may be because DOX-PRO entered the cells through endocytosis after being made into nanoformulations, resulting in a certain time difference.

[0051] Example 6 In vivo pharmacokinetics of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles

[0052] The rats were randomly divided into 2 groups, 6 in each group, and injected with DOX solution, DOX-PRO NP and S Solution, the dosage is equivalent to DOX content (5mg / kg), 15min, 30min, 45min, 2h, 8h, 12h, 24h after administration, 200μL of blood was taken from the jugular vein of the rat, centrifuged at 3500rpm for 15min, and then the plasma was taken out and stored. Take 50μL of rat plasma, add 10μL of internal standard solution acetaminophen, 240μL of acetonitrile to precipitate protein, vortex for 3min, centrifuge at 12000rpm for 15min, take the supernatant, blow dry with nitrogen at 37℃, add distilled water to dissolve, centrifuge at 12000rpm for 5min, take 10μL of supernatant solution for LC-MS / MS analysis, and use DAS2.0 software to calculate the pharmacokinetic parameters of each group of drugs.

[0053] from Figure 7 It can be seen that the blood concentration of the free DOX group decreased rapidly 45 minutes after administration, and stabilized at a low value after 2 hours. S The blood drug concentration of the group decreased slowly. As the metabolism time prolonged, the DOX concentration in rats was higher than that in the free DOX group, indicating that DOX-PRO NP S The elimination in the body is slower, which can prolong the circulation time of DOX in the body (Table 1).

[0054] Table 1. DOX and DOX-PRO NPs S Pharmacokinetic parameters of

[0055]

[0056] Example 7 In vivo antitumor activity of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles

[0057] After digesting the 4T1 cells that were growing well, they were resuspended in PBS and placed on ice for later use. 100 μL of the cell suspension was injected subcutaneously into the right armpit of the mouse. The tumor volume was measured 7 days later. The tumor grew to 80-100 mm 3 The tumor-bearing mouse model was successfully established.

[0058] Experimental groups: saline group, DOX group, DOX-PRO NP S Group.

[0059] Dosage regimen:

[0060] (1) Normal saline group: Normal saline was injected into the tail vein once every 2 days for 4 doses.

[0061] (2) DOX group: 5 mg / kg (in terms of DOX), injected into the tail vein once every 2 days, for 4 doses.

[0062] (3)DOX-PRO NP S Group: 5 mg / kg (in terms of DOX), injected into the tail vein once every 2 days, for 4 doses.

[0063] Results determination method: Tumor size was measured every other day, V = 0.5 × a × b^2 (V tumor volume, a tumor long diameter, b tumor short diameter)

[0064] The results showed that the tumor growth rate in the saline group was the fastest during the treatment period, and the tumor volume increased nearly 7 times on the 12th day. The growth rate in the DOX group was slower than that in the saline group, but the growth rate was higher than that in the DOX-PRO NPs group. Figure 8 A). There was no significant change in the body weight of mice in the drug group and the saline group ( Figure 8 B).

[0065] Example 8 Safety Experiment of Doxorubicin-Squalene Small Molecule Prodrug Self-Assembled Nanoparticles

[0066] Blood was collected from the eye sockets of mice and centrifuged at 3000 rpm for 15 min. The upper serum was taken and tested with aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CREA) detection kits.

[0067] The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA) in the mouse serum were not significantly abnormal compared with those in the normal saline group, indicating that the two preparations had good biosafety during the treatment of mice ( Fig. 9 ).

Claims

1. Adriamycin-squalene small molecule prodrug shown in formula I: in, Ar is a substituted or unsubstituted phenyl group, and the substituent is a halogen, a C1-C6 alkyl group, or a C1-C6 alkoxy group.

2. The doxorubicin-squalene small molecule prodrug of formula I according to claim 1:

3. The preparation method of the doxorubicin-squalene small molecule prodrug of formula I according to claim 2:

4. The self-assembled nanoparticles of the doxorubicin-squalene small molecule prodrug of formula I according to claim 1 or 2, characterized in that: The small molecule prodrug nanoparticles are non-PEGylated small molecule prodrug nanoparticles and PEG-modified small molecule prodrug nanoparticles.

5. The method for preparing the self-assembled nanoparticles of the doxorubicin-squalene small molecule prodrug of formula I as claimed in claim 4, characterized in that: (1) Preparation method of non-PEGylated small molecule prodrug self-assembled nanoparticles: a certain amount of prodrug is dissolved in an appropriate amount of DMSO, and the DMSO solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; (2) Preparation method of PEG-modified small molecule prodrug self-assembled nanoparticles: a certain amount of PEG derivative and prodrug are dissolved in an appropriate amount of DMSO, and the DMSO solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles.

6. The preparation method according to claim 5, characterized in that: In the PEG-modified small molecule prodrug self-assembled nanoparticles, the mass ratio of doxorubicin-squalene small molecule prodrug to PEG derivative is 4-6:

1.

7. Use of the doxorubicin-squalene small molecule prodrug of formula I according to claim 1 or 2 or the self-assembled nanoparticles according to claim 4 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor is breast cancer.

9. Use of the doxorubicin-squalene small molecule prodrug of formula I as described in claim 1 or 2 or the self-assembled nanoparticles as described in claim 4 in the preparation of drugs with improved efficacy and reduced toxicity.

10. Use of the doxorubicin-squalene small molecule prodrug of formula I according to claim 1 or 2 or the self-assembled nanoparticles according to claim 4 in the preparation of injections.

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