Doxorubicin-squalene small molecule prodrugs, methods of making and using the same

Abstract

The application belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to construction of a doxorubicin-squalene prodrug and self-assembled nanoparticles thereof, and application of the doxorubicin-squalene prodrug in a drug delivery system. The doxorubicin-squalene small molecule prodrug is prepared by connecting doxorubicin (DOX) and squalene (SQ) through an oxygen deficiency sensitive group (Ar-N=N-Ar). The structure is shown as formula I. The doxorubicin-squalene small molecule prodrug of the application adopts a'squalene acylation' technology, connects squalene with an oxygen deficiency sensitive functional bond, and connects with doxorubicin to obtain the doxorubicin-squalene small molecule prodrug. The obtained doxorubicin-squalene small molecule prodrug has self-assembly properties and can self-assemble to form a uniform nano system. Not only the efficacy of doxorubicin can be improved, but also the toxicity can be reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to construction of doxorubicin-squalene prodrugs and self-assembled nanoparticles thereof, and application of the same in drug delivery systems. BACKGROUND

[0002] Doxorubicin is one of the main anticancer drugs in clinical oncology, and is mainly used for acute lymphoblastic leukemia, acute myeloid leukemia, nephroblastoma, 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, which can kill tumor cells in various growth cycles.

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

[0004] The side effects of doxorubicin are mainly dose-limiting irreversible cardiotoxicity and bone marrow suppression. Although nanotechnology greatly improves the cardiotoxicity of doxorubicin, these new nanofabrications 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 is still a research hotspot.

[0005] There is no related report on doxorubicin-squalene small molecule prodrugs in the prior art. SUMMARY

[0006] In view of the above problems, we designed a series of doxorubicin-squalene small molecule prodrugs, in which doxorubicin and squalene are connected by an oxygen-sensitive chemical linker. The oxygen-sensitive chemical linker can be quickly broken in the hypoxic environment of tumor cells, and doxorubicin is released in a targeted manner, thereby reducing the toxic side effects on normal cells. At the same time, these small molecule hydrophobic prodrugs can self-assemble into uniform prodrug nanoparticles. In these small molecule prodrug nanosystems, the prodrug itself acts as both a carrier material and a loaded drug, with an encapsulation efficiency greater than 90% and a drug loading capacity exceeding 75%. Based on this, we designed and constructed an oxygen-sensitive doxorubicin-squalene small molecule prodrug nanoparticle, and performed PEG modification on it. After injection, the nanoparticle has long circulation characteristics in the blood, and can be applied to anti-tumor research.

[0007] The present application aims to design and synthesize an oxygen-sensitive doxorubicin-squalene small molecule prodrug, and prepare a prodrug self-assembled nanomedicine delivery system, which has better antitumor effect and lower drug toxicity than doxorubicin.

[0008] The doxorubicin-squalene small molecule prodrug of the present application is prepared by connecting doxorubicin (DOX) and squalene (SQ) through 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, and the substituent group is halogen, C1-C6 alkyl or C1-C6 alkoxy.

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

[0013]

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

[0015]

[0016] Wherein, Ar is as previously described.

[0017] The doxorubicin-squalene small molecule prodrug prepared by the present application 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 to water under stirring, and the prodrug spontaneously forms uniform nanoparticles.

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

[0020] The doxorubicin-squalene small molecule prodrug of the application adopts a ''squalene acylation'' technology, links squalene with an oxygen deficiency sensitive functional group, and is connected with doxorubicin to obtain. The obtained doxorubicin-squalene small molecule prodrug has a self-assembly property and can self-assemble to form a uniform nano system. The nano drug delivery system has the following advantages: (1) a one-step nano precipitation method is adopted, the preparation process is simple and easy to industrialize; (2) the particle size is small and uniform (about 100 nm), which is beneficial to the enrichment of the nano particles in the tumor site through the EPR effect; (3) the drug loading capacity is high, which is beneficial to reducing the adverse reactions caused by auxiliary materials and biological materials; (4) through the sensitivity of the linking arm to the intracellular environment of the tumor site, the specific drug release of doxorubicin in the tumor cells is realized, which improves the curative effect and reduces the toxic side effects.

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

[0022] 1. A novel doxorubicin-squalene small molecule prodrug is designed and synthesized; 2. Through the design of an oxygen deficiency chemical linking arm, doxorubicin can be released in tumor cells; 3. Uniform small molecule prodrug self-assembled nanoparticles are prepared, the preparation method is simple and easy to operate, the stability is good, and the drug loading capacity is high; 4. The 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 effect. BRIEF DESCRIPTION OF DRAWINGS

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

[0024] Figure 2 is the 1 H NMR graph of the doxorubicin-squalene small molecule prodrug;

[0025] Figure 3 is the property graph of DOX-PRO NP S ;

[0026] A: Particle size distribution curve; B: Transmission electron microscope graph of the nanoparticles; C: Change of the particle size and PDI of the nanoparticles in a 4℃ environment;

[0027] Figure 4 is the drug release condition of DOX-PRO NP S under different concentrations of Na2S2O4;

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

[0029] Figure 6 is the fluorescence quantitative result of the 4T1 cell uptake of DOX in a normal oxygen and an oxygen deficiency environment;

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

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

[0032] Figure 8 for anti-tumor activity;

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

[0034] Figure 9 for various liver and kidney function indicators of mice. DETAILED DESCRIPTION

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

[0036]

[0037] Compound II (10 mmol), imidazole (15 mmol) in THF, TBDMSCl (12 mmol in THF) were added to the mixed solution at 0°C. The mixture was stirred at room temperature for 6 hours. THF was removed under reduced pressure, and the organic phase was removed by evaporation after DCM extraction. The synthesized compound III can be used directly without purification. Compound III (5 mmol), compound IV (5 mmol), EDCI (6 mmol), DMAP (catalyst) and anhydrous DCM (10 mL) were stirred overnight at room temperature under argon protection. The reaction solution was washed with dilute hydrochloric acid (10 mL), and the organic layer was dried with 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. A THF solution of TBAF was added dropwise to the reaction solution at room temperature. 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, an anhydrous DCM acyl chloride solution was added dropwise to a solution composed of VI and TEA, and stirred at 0°C for 30 minutes. Then, the resulting salt was washed with water, and the organic phase was evaporated. The intermediate VII can be purified by column chromatography with PE: AcOEt = 5:1 as the mobile phase. DOX hydrochloride, VII, TEA in DMF solution were stirred overnight at room temperature, and diluted with DCM. The reaction solution was transferred to silica gel chromatography, and the eluent of DCM: MeOH = 10:1 was collected to obtain the prodrug I (DOX-PRO) after removing the organic phase. The product MS is shown in Figure 1 、 1 H NMR is shown in 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] DOX-PRO NP was prepared by a 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. Weigh a certain amount of DSPE-PEG. 2K DSPE-PEG was prepared with DMSO at a concentration of 2 mg / mL. 2K Take 200 μL of the solution and add 500 μL of DOX-PRO solution and 200 μL of DSPE-PEG. 2K The solution was thoroughly mixed and slowly added dropwise to 2 mL of deionized water under magnetic stirring at 800 rpm. The mixture was stirred thoroughly in the dark for 4 hours to disperse the nanoparticles. After 4 hours, the nanoparticles were removed and placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis to remove DMSO. Distilled water was used as the dialysis medium, and dialysis was performed for 24 hours. The obtained nanoparticles were collected in a glass bottle and concentrated using a rotary evaporator at 37°C to obtain DOX-PRO NP at a concentration of 2 mg / mL (DOX-PRO concentration 2 mg / mL). S Solution.

[0041] DOX-PRO NP was prepared with a drug-to-excipient ratio of 5:1.S The particle size of the solution was about 60 nm, and the particle size distribution curve was well shaped. The shape of DOX-PRO NP S was spherical Figure 3 (A) as observed by transmission electron microscopy (TEM). The encapsulation efficiency was 94.033 ± 4.406%, the drug loading was 78.333 ± 3.625%, and the particle size distribution was as shown in Figure 3 B, and the PDI value was as shown in Figure 3 C.

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

[0043] The release behavior of DOX-PRO NP S in different concentrations of Na2S2O4 release medium was determined. Na2S2O4 solutions with different concentrations of 0 mM, 10 mM, 20 mM, and 40 mM were prepared as release media. 1 mL of DOX-PRO NP S solution (DOX-PRO concentration 1 mg / mL) was loaded into a dialysis bag, and the dialysis bag was placed in the prepared Na2S2O4 release medium with different concentrations. The entire system was placed in a 37°C constant temperature shaker. At the set time points including 2 h, 4 h, 6 h, 10 h, and 24 h, 500 μL of sample was taken (500 μL of sample was taken, and 500 μL of corresponding release medium was added to the release system), and then ultrasonic treatment was performed for 10 min. The concentration of DOX was determined by HPLC.

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

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

[0046] Well-grown 4T1 cells were digested, and then the cells were added to a 96-well plate and cultured for 24 h. DOX and DOX-PRO NP S were prepared with blank medium, and the concentration gradient was DOX (0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM). After 24 h of cell culture, the medium containing the drugs was added, and after 72 h of culture, MTT was added to each well and cultured for another 4 h. After 4 h, the absorbance value of each well was determined at 570 nm by a multifunctional enzyme label instrument, and the cell survival rate was calculated.

[0047] Results show that: DOX and DOX-PRO NP S After 72h of administration, under different concentrations, DOX-PRO NP S The cytotoxicity is slightly weaker than that of DOX itself, and the structural changes of DOX lead to a slight decrease in in vitro activity and cytotoxicity, which means that the prodrug may reduce the toxic side effects of DOX. Due to the difference between in vivo and in vitro environments, the characteristics of targeted drug release and long circulation of nano-preparation in prodrug design cannot be reflected in in vitro experiments, and further pharmacodynamic experiments are needed. Figure 5

[0048] Example 5 Cell uptake of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles

[0049] DOX-PRO is connected by squalene and DOX through a hypoxia-sensitive bond, and the release of DOX, DOX-PRO, DOX-PRO NP S Under different environments, 4T1 was inoculated in a 24-well plate, 5*10 4 cells per well, and cultured for 24h. After 24h, 500μL of culture medium containing DOX, DOX PRO, DOX PRO NP S (DOX concentration 2μM) was added, and PBS was set as the control group. In normoxic or hypoxic environment, incubate for 2h and 8h (normoxic environment: incubator T = 37℃ CO2 = 5.0%; hypoxic environment: T = 37℃, CO2 = 5.0%, O2 = 2.5%), discard the drug-containing culture medium and wash with PBS, then add tissue fixative and fix for 30min, finally dye with DAPI for 8-10min, take pictures with fluorescence inverted microscope and quantify fluorescence with image J software, results are shown in Figure 6 .

[0050] Results show that, in either normoxic or hypoxic environment, DOX, DOX-PRO, DOX-PRO NP S The fluorescence intensity of DOX is higher at 8h than at 2h, indicating that the uptake of DOX by 4T1 cells is time-dependent. According to the fluorescence quantification results, there is no difference in DOX uptake by 4T1 cells between normoxic and hypoxic environments at 2h and 8h for the free DOX group, while the DOX fluorescence intensity in the hypoxic environment is higher than that in the normoxic environment at 8h for the DOX PRO and DOX-PRO NP S groups, indicating that the hypoxic bond is more likely to break in the hypoxic environment, releasing more DOX and increasing cell uptake. The DOX uptake of the DOX-PRO NP group is lower than that of the free DOX and DOX-PRO groups, which may be because DOX-PRO is made into a nano-preparation and enters the cell through endocytosis, resulting in a certain time difference.​

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

[0052] Rats were randomly divided into two groups of six each, and were injected intravenously with DOX solution and DOX-PRO NP, respectively. S The solution was administered at an equivalent DOX content (5 mg / kg). Blood samples of 200 μL were collected from the jugular vein of rats at 15 min, 30 min, 45 min, 2 h, 8 h, 12 h, and 24 h post-administration. The plasma was collected after centrifugation at 3500 rpm for 15 min and stored. 50 μL of rat plasma was added to 10 μL of acetaminophen (internal standard), and 240 μL of acetonitrile to precipitate proteins. The mixture was vortexed for 3 min, centrifuged at 12000 rpm for 15 min, and the supernatant was collected. The supernatant was dried under nitrogen at 37°C, reconstituted with distilled water, centrifuged at 12000 rpm for 5 min, and 10 μL of the supernatant was injected for LC-MS / MS analysis. Pharmacokinetic parameters for each group were calculated using DAS2.0 software.

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

[0054] Table 1. DOX and DOX-PRO NP S pharmacokinetic parameters

[0055]

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

[0057] Healthy 4T1 cells were digested and resuspended in PBS. The cell suspension was kept on ice for later use. 100 μL of the cell suspension was subcutaneously injected into the right axilla of mice. The tumor volume was measured 7 days later, and the tumor had grown to 80-100 mm. 3 At that time, the tumor-bearing mouse model was successfully established.

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

[0059] Dosage regimen:

[0060] (1) Saline group: Saline, 2 days once tail vein injection, 4 times of administration.

[0061] (2) DOX group: 5 mg / kg (calculated as DOX), 2 days once tail vein injection, 4 times of administration.

[0062] (3) DOX-PRO NP S group: 5 mg / kg (calculated as DOX), 2 days once tail vein injection, 4 times of administration.

[0063] Result determination method: Measure the tumor size every other day, V = 0.5 x a x b^2. (V tumor volume, a tumor long diameter, b tumor short diameter)

[0064] The results show that the tumor growth rate of the saline group is the fastest during the treatment, and the tumor volume grows nearly 7 times on the 12th day. The growth rate of the DOX group is slower than that of the saline group, but the growth rate is higher than that of the DOX-PRO NPs Figure 8 A). The body weight of the mice in the administration groups and the saline group did not change significantly Figure 8 B).

[0065] Example 8 Safety experiment of doxorubicin-squalene small molecule prodrug self-assembled nanoparticles

[0066] Blood was collected from the mouse orbit, 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 the saline group, indicating that the two preparations had good biological safety during the treatment of mice Figure 9 ).

Claims

1. Use of self-assembled nanoparticles of doxorubicin-squalene small molecule prodrug in the preparation of an anti-breast cancer medicament, characterized in that, The doxorubicin-squalene small molecule prodrug is shown as formula I: ； The self-assembled nanoparticles of the doxorubicin-squalene small molecule prodrug are prepared by the following method: A certain amount of PEG derivative and prodrug are dissolved in a proper amount of DMSO, and the DMSO solution is slowly added into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; The mass ratio of the doxorubicin-squalene small molecule prodrug to the PEG derivative is 4-6:

1.

2. Use of self-assembled nanoparticles of doxorubicin-squalene small molecule prodrug in the preparation of a medicament for improving efficacy and reducing toxicity, characterized in that, The doxorubicin-squalene small molecule prodrug is shown as formula I: ； The self-assembled nanoparticles of the doxorubicin-squalene small molecule prodrug are prepared by the following method: A certain amount of PEG derivative and prodrug are dissolved in a proper amount of DMSO, and the DMSO solution is slowly added into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; The mass ratio of the doxorubicin-squalene small molecule prodrug to the PEG derivative is 4-6:

1.

3. Use according to claim 1 or 2, characterized in that, The preparation method of the doxorubicin-squalene small molecule prodrug is as follows: 。

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