Preparation and application of weakly alkaline dexamethasone derivatives and their preparations

Abstract

The invention discloses the preparation and application of dexamethasone weak base derivatives and preparations thereof, and belongs to the technical field of liposome medicine. The dexamethasone weak base derivatives of the invention are prepared by reacting dexamethasone and a weak base intermediate via ‑COO‑, ‑OOC‑(CH2) n1 ‑S‑(CH2) n2 ‑CO‑ or ‑OOC‑(CH2) n1 ‑S‑S‑(CH2) n2 ‑CO‑connected, and weakly basic intermediates include tetrahydropyrrolyl, piperidinyl, 4‑(1‑piperidinyl)piperidinyl, N‑methylpiperazinyl, morpholinyl or other tertiary amine structures. Weakly basic derivatives of dexamethasone can be prepared as single-loaded liposomes or double-loaded liposomes for combined use with other drugs. The liposome preparation has the characteristics of high encapsulation rate, high drug loading, high stability, etc. Through the modification of PEGylated phospholipids, the preparation can also effectively avoid the uptake of the reticuloendothelial system, thereby achieving the effect of long circulation in the blood.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome medicines and relates to the preparation and application of dexamethasone weakly alkaline derivatives and preparations thereof. Background Art

[0002] Dexamethasone (DEX) is a white crystalline powder, odorless, slightly bitter, and belongs to the class of glucocorticoid drugs. Because dexamethasone can inhibit the synthesis and release of inflammatory chemical mediators and inhibit the aggregation of inflammatory cells at the site of inflammation, the drug is widely used in allergic and autoimmune inflammatory diseases to reduce and prevent tissue responses to inflammation. In addition, dexamethasone can also act as a chemotherapeutic drug, which can reduce the number of cancer cells and inhibit the growth of cancer cells. Studies have shown that DEX can normalize the tumor microenvironment, improve the delivery efficiency of nanoformulations, and reduce the expression of collagen and fibroblasts in the tumor microenvironment to reduce the tumor interstitial fluid pressure (IFP), further promoting drug accumulation. In addition to improving the anti-tumor effect, DEX can also inhibit the synthesis and release of prostaglandins and serotonin in the central and peripheral systems. The reduction of prostaglandins and serotonin can change the permeability of the blood-brain barrier to proteins through central effects, thereby alleviating symptoms of nausea and vomiting.

[0003] Liposomes are considered to be a powerful drug delivery system due to their structural versatility, biocompatibility, biodegradability, non-toxicity and non-immunogenicity. As drug carriers, liposomes can not only passively accumulate in cancer tissues through high permeability and high retention (EPR), but also can be specifically modified to enhance targeting, improve drug therapeutic effects and reduce toxic side effects. For example, long-circulating liposomes are prepared by modifying the liposome surface with hydrophilic polymers such as polyethylene glycol (PEG). The modified liposome surface will form a hydration film, which can effectively avoid uptake by the reticuloendothelial system, thereby achieving the effect of long circulation in the blood.

[0004] The liposomes prepared by the active drug loading method have less drug leakage, high drug loading and encapsulation efficiency, and high stability. At present, most of the liposome preparations on the market are prepared by the active drug loading method. Active drug loading is mainly divided into pH gradient method, amine (ammonium) ion gradient method, calcium acetate gradient method and ion carrier gradient method. Dexamethasone is difficult to be directly encapsulated in liposomes. To solve this problem, dexamethasone can be subjected to weak alkaline structural modification, and the amine (ammonium) ion gradient method is used to stably bind the drug to the capture agent in the inner water phase, so that the drug is stably encapsulated in the liposome. Summary of the invention

[0005] The first object of the present invention is to provide a weakly basic dexamethasone derivative.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned weakly basic dexamethasone derivative.

[0007] The third purpose of the present invention is to prepare single-carrier liposomes by weakly alkaline modification of dexamethasone, which can be used to prepare drugs for treating inflammatory diseases and tumor diseases.

[0008] The fourth object of the present invention is to modify dexamethasone to be weakly alkaline and then prepare co-loaded liposomes with other drugs. The combined use of dexamethasone weakly alkaline derivatives and different types of drugs can not only achieve the purpose of synergistic enhancement and reduced toxic side effects, but also can be used to treat different types of diseases.

[0009] The technical solution of the present invention is as follows:

[0010] The invention provides a weakly basic derivative of dexamethasone, wherein the weakly basic derivative of dexamethasone is prepared by reacting dexamethasone with a weakly basic intermediate via -COO-, -OOC-(CH2) n1 -S-(CH2) n2 -CO- or -OOC-(CH2) n1 -SS-(CH2) n2 -CO- connected, the former can release the drug through esterase cleavage in the body, and the latter two can release the drug through oxidation and reduction responsiveness. The general structural formula is as follows:

[0011]

[0012] in:

[0013] R1 is -COO-, -OOC-(CH2)n1-S-(CH2)n2-CO- or -OOC-(CH2)n1-SS-(CH2)n2-CO-;

[0014] R2 is tetrahydropyrrolyl, piperidinyl, 4-(1-piperidinyl)piperidinyl, N-methylpiperazinyl, morpholinyl or other tertiary amine structures.

[0015] n1 and n2 in R1 or R2 are both the number of repeating units, and are optionally integers of 1-5.

[0016] The dexamethasone weakly basic derivative is specifically selected from the following compounds:

[0017]

[0018] The preparation method of the dexamethasone weakly basic derivative (DPD) provided by the present invention specifically comprises the following steps:

[0019] Dexamethasone and 4-piperidinylpiperidinylcarbonyl chloride are subjected to esterification reaction under the catalysis of DMAP, and the whole reaction is carried out under the protection of N2, and finally the sample is separated and purified to obtain the product;

[0020] Wherein, in terms of molar ratio, the ratio of dexamethasone to 4-piperidinylpiperidinylcarbonyl chloride and DMAP is: 1:(0.95-1):(2-3).

[0021] The preparation method of the weakly basic dexamethasone derivative (DPD-2) or (DPD-3) provided by the present invention specifically comprises the following steps:

[0022] 4-(1-piperidinyl)piperidine and thiohydroxyacetic anhydride or 1,4,5-Oxadithiepane-2,7-dione are mixed in dimethylformamide (DMF) for reaction, 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) are added and stirred, dexamethasone is added and stirred until the reaction is completed, the solvent is evaporated, and separation and purification are performed to obtain the product;

[0023] Among them, in molar ratio, the ratio of 4-(1-piperidinyl)piperidine to thioglycolic anhydride or 1,4,5-Oxadithiepane-2,7-dione is 1:(1~1.1), and the ratio of dexamethasone to 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) is 1:(2.5~3):(2.5~3).

[0024] The present invention provides a single-loaded liposome containing the above-mentioned weakly basic dexamethasone derivative and a co-loaded liposome used in combination with other drugs. The liposome comprises the following components: the weakly basic dexamethasone derivative or the weakly basic dexamethasone derivative and other drugs, phospholipids, cholesterol, PEGylated phospholipids, ammonium salt solution and buffer.

[0025] The other drugs described in the present invention include anti-inflammatory drugs, anti-tumor drugs, anti-allergic drugs, antibacterial drugs and anesthetic drugs, etc., without special limitation.

[0026] Furthermore, the other drugs are preferably anti-tumor drugs, including one or more of bortezomib, toremifene, paclitaxel, docetaxel, irinotecan, gemcitabine, and topotecan;

[0027] Furthermore, the other drug is preferably irinotecan.

[0028] Furthermore, the phospholipids are one or more of natural phospholipids or synthetic phospholipids; the natural phospholipids are selected from egg yolk lecithin, soybean phospholipids and hydrogenated soybean phospholipids; the synthetic phospholipids are selected from dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), etc.;

[0029] Furthermore, the phospholipid is preferably distearoylphosphatidylcholine (DSPC).

[0030] Furthermore, the PEGylated phospholipid is DSPE-MPEG 1000 、DSPE-MPEG 2000 or DSPE-MPEG 5000 One or more of; preferably DSPE-MPEG 2000 ;

[0031] Furthermore, in the liposome, the mass ratio of drug to phospholipid is 1:(2-20); the mass ratio of phospholipid, cholesterol and PEGylated phospholipid is (2-10):1:(0.02-0.20);

[0032] Furthermore, the mass ratio of the drug to the phospholipid is 1:(2-12), and the mass ratio of the phospholipid, cholesterol, and PEGylated phospholipid is (2-5):1:(0.05-0.15).

[0033] Furthermore, the ammonium salt solution is selected from one or more of ammonium chloride, ammonium sulfate, ammonium acetate and sucrose octasulfate triethylamine; preferably sucrose octasulfate triethylamine solution.

[0034] Furthermore, the concentration of the sucrose octasulfate triethylamine solution is 0.2-0.9M, preferably 0.4-0.8M, calculated as ammonium salt.

[0035] The present invention provides a method for preparing a liposome containing a weakly alkaline derivative of dexamethasone or a liposome containing dexamethasone and other drugs, and the specific steps are as follows:

[0036] (1) Preparation of blank liposomes: Use phospholipids, cholesterol and PEGylated phospholipids to prepare blank liposomes by thin film dispersion method or ethanol injection method, and the concentration of sucrose octasulfate triethylammonium is 0.4-0.8 M. The particle size is reduced and uniformed by liposome extrusion equipment to obtain blank liposomes.

[0037] (2) Preparation of gradient blank liposomes: Use agarose gel columns to replace the extraliposome aqueous phase with buffer salts to establish a transmembrane gradient and form gradient blank liposomes.

[0038] (3) Drug loading: Add dexamethasone weak base derivatives or dexamethasone and other drugs to the gradient blank liposomes, stir in a water bath, and immediately put in an ice water bath after taking out to obtain dexamethasone weak base derivative liposomes or dexamethasone and other drug co-loaded liposomes.

[0039] Furthermore, the concentration of sucrose octasulfate triethylammonium in step (1) is 0.4-0.8 M; and the particle size of the blank liposome is 80-150 nm.

[0040] Furthermore, in step (2), the buffer salt is selected from one or more of physiological saline (NaCl), hydroxyethylpiperazineethanesulfonic acid (HEPES), tromethamine, phosphate, glucose and other injection-acceptable buffer salts, and its pH value is 5.0 to 7.0, and the pH value is preferably 6.5.

[0041] Furthermore, the buffer salt in step (2) is preferably a combination of hydroxyethylpiperazineethanesulfonic acid (HEPES) and normal saline (NaCl), wherein the concentration of HEPES is 18 mM and the concentration of NaCl is 50 mM.

[0042] Furthermore, in step (3), the water bath temperature is 50-75° C., the stirring time is 10-100 min, and the ice water bath time is 15 min.

[0043] Compared with the existing experimental technology, the present invention has the following beneficial effects:

[0044] ① The present invention adopts an active drug loading method to prepare the dexamethasone weakly alkaline derivative liposome, which can make the dexamethasone weakly alkaline derivative form a gel-state complex in the internal water phase, effectively ensuring the stability of the preparation. The obtained preparation has a uniform particle size, an encapsulation rate of more than 98%, and good controllability and reproducibility.

[0045] ② The single-loaded or co-loaded liposomes of the present invention can effectively avoid uptake by the reticuloendothelial system, thereby achieving the effect of long circulation in the blood.

[0046] ③ The single-loaded or co-loaded liposome of the present invention can inhibit the production of inflammatory factors and improve inflammatory reactions. In addition, the combined use of dexamethasone weakly alkaline derivatives and other drugs can not only achieve the purpose of synergistic enhancement, but also be used to treat different types of diseases.

[0047] ④ The single-loaded or co-loaded liposomes of the present invention, dexamethasone can reduce the side effects of nausea and vomiting of other drugs by inhibiting the synthesis and secretion of prostaglandins and serotonin in the central and peripheral systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1The ester-linked dexamethasone and the weakly basic ligand (DPD) of Example 1 of the present invention are 1 H-NMR spectrum.

[0049] Figure 2 The dexamethasone and the weakly basic ligand (DPD-2) connected by the thioether bond of Example 2 of the present invention 1 H-NMR spectrum.

[0050] Figure 3 This is a transmission electron microscopy result image of Example 12 of the present invention.

[0051] Figure 4 The in vitro release result pictures of Example 18 of the present invention are shown. Among them, Figures A and B are the cumulative release curves of DEX released from DPD-2 and DPD-3 in release media with different concentrations of H2O2 and DTT after the prodrug is released from the liposomes; Figures C and D are the residual cumulative release curves of DPD-2 or DPD-3 after degradation in release media with different concentrations of H2O2 and DTT after release from the liposomes.

[0052] Figure 5 These are pictures of the stability investigation of co-loaded liposomes in Example 19 of the present invention, wherein Figure A shows the encapsulation efficiency, and Figure B shows the changes in particle size and PDI.

[0053] Figure 6 This is a pharmacodynamic diagram of the co-loaded preparation in Example 20 of the present invention, wherein Figure A is a tumor volume change curve, and Figure B is a mouse body weight change curve.

[0054] Figure 7 This is a graph of the cytokine content of the co-loaded preparation in Example 21 of the present invention in the tumor cell model, wherein A is interleukin-6 (IL-6), Figure B is tumor necrosis factor α (TNF-α), Figure C is vascular endothelial growth factor (VEGF), and Figure D is prostaglandin E2 (PGE2).

[0055] Figure 8 This is an immunofluorescence image of the tumor tissue of the co-loaded preparation in Example 22 of the present invention in the tumor cell model, wherein A is the platelet-endothelial cell adhesion molecule (CD31), Figure B is α-smooth muscle actin (α-SMA), and Figure C is recombinant human collagen I (Collagen-I). DETAILED DESCRIPTION

[0056] The present invention is described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Some non-essential improvements and adjustments made according to the contents of the present invention all belong to the protection scope of the present invention.

[0057] Example 1: Synthesis of ester-linked dexamethasone and weakly basic ligand (DPD)

[0058] Dexamethasone (2.97 g, 7.7 mmol) and 4-piperidinylpiperidinylcarbonyl chloride (1.74 g, 7.54 mmol) were dissolved in dichloromethane, and 1.6 mL of triethylamine was added. Then, a dichloromethane solution of 4-dimethylaminopyridine (DMAP) (2.09 g, 17.00 mmol) was slowly added dropwise under an ice bath, and stirred at room temperature for 10 h under drying tube drying. After the reaction was completed, a white solid powder was obtained by column chromatography separation and purification, and the yield was 92.8%. The structure of DPD in Example 1 was determined by hydrogen nuclear magnetic resonance spectroscopy, and the solvent was deuterated chloroform. The results are as follows: Figure 1 , the spectrum results are as follows:

[0059] 1HNMR(400MHz,Chloroform-d)δ7.20(d,J=10.1Hz,1H),6.33(dd,J=10.1,1.9Hz,1H),6.11(t,J=1.8Hz,1H), 4.91–4.76(m,2H),4.39–4.32(m,1H),4.20(s,1H),4.12(q,J=7.1Hz,1H),3.10(ddd,J=11.1,7.3,3.9Hz,1H) ,2.91–2.73(m,2H),2.64–2.31(m,10H),2.17(td,J=11.7,7.9Hz,1H),2.04(s,2H),1.96–1.70(m,6H),1.60( d,J=13.3Hz,3H),1.54(s,3H),1.46(d,J=5.6Hz,2H),1.29–1.19(m,3H),1.06(s,3H),0.91(d,J=7.3Hz,3H).

[0060] Example 2: Synthesis of Dexamethasone and Weakly Basic Ligand Connected by Thioether Bond (DPD-2)

[0061] Dissolve thiohydroxyacetic anhydride (2.0g, 15.13mmol) in 25mL DMF, then add dropwise 25mL DMF solution containing 4-(1-piperidinyl)piperidine (2.54g, 15.13mmol). After a period of reaction, add HOBT (3.07g, 22.70mmol) and EDCI (4.35g, 22.70mmol), stir for 1h, then add dexamethasone (2.97g, 7.7mmol), the solution changes from light yellow to dark red, and react overnight for 24h. After the reaction is completed, evaporate the reaction solution under reduced pressure at 75°C. Then wash the reaction solution once with 0.5N hydrochloric acid, twice with saturated sodium carbonate solution, and once with saturated brine, and finally dry with anhydrous sodium sulfate, filter, evaporate to dryness under reduced pressure, and separate and purify to obtain a white solid powder with a yield of 83.3%. The structure of DPD-2 in Example 2 was determined by hydrogen nuclear magnetic resonance spectroscopy, and deuterated DMSO was used as the solvent. Figure 2 , the spectrum results are as follows:

[0062] 1H NMR(400MHz,DMSO-d6)δ7.30(d,J=10.1Hz,1H),6.23(dd,J=10.1,1.9Hz,1H),6.01(t,J=1 .6Hz,1H),5.40(dt,J=4.4,1.8Hz,1H),5.18(s,1H),5.10(d,J=17.6Hz,1H),4.84(d,J=17 .6Hz,1H),4.35(dd,J=12.4,3.5Hz,1H),4.15(tt,J=6.8,2.2Hz,1H),3.88(d,J=13.5Hz,1 H),3.73–3.57(m,2H),3.51(d,J=1.7Hz,2H),2.99(t,J=12.6Hz,1H),2.88(ddd,J=11.3,7 .2,4.2Hz,1H),2.69–2.58(m,1H),2.55(dd,J=12.6,2.6Hz,1H),2.43(t,J=5.3Hz,5H),2. 34(td,J=13.0,12.6,5.2Hz,2H),2.23–2.07(m,2H),1.78(dd,J=12.2,5.7Hz,1H),1.74–1 .66(m,2H),1.66–1.54(m,2H),1.48(d,J=9.4Hz,8H),1.36(p,J=6.0,5.5Hz,3H),1.24(q, J=5.9,5.2Hz,1H),1.08(ddd,J=12.1,8.1,4.1Hz,1H),0.89(s,3H),0.80(d,J=7.2Hz,3H).

[0063] Example 3: Synthesis of disulfide-linked dexamethasone and weakly basic ligand (DPD-3)

[0064] Dissolve 1,4,5-Oxadithiepane-2,7-dione (2.48 g, 15.13 mmol) in 25 mL DMF, then add dropwise 25 mL DMF solution of 4-(1-piperidinyl)piperidine (2.54 g, 15.13 mmol). After a period of reaction, add HOBT (3.07 g, 22.70 mmol) and EDCl (4.35 g, 22.70 mmol), stir for 1 hour, then add dexamethasone (2.97 g, 7.7 mmol), the solution changes from light yellow to dark red, and react overnight for 18 hours. After the reaction is completed, evaporate the reaction solution under reduced pressure at 75 ° C. Then wash the reaction solution once with 0.5N hydrochloric acid, twice with saturated sodium carbonate solution, and once with saturated brine, and finally dry with anhydrous sodium sulfate, filter, evaporate to dryness, and separate and purify to obtain a white solid powder (yield 81.5%).

[0065] Example 4: Preparation of single liposomes containing weakly basic dexamethasone derivatives

[0066] Weigh 105.0 mg DSPC, 35.0 mg cholesterol, 2.8 mg DSPE-MPEG 2000 , namely (DSPC, cholesterol, DSPE-MPEG 2000 The mass ratio of phospholipids was 3:1:0.08), dissolved with 1.5 mL of ethanol, and the dissolved phospholipids were uniformly injected into the preheated 5 mL of 0.6 mol / L TEA-SOS solution with a syringe, and stirred at 65 ° C for 30 min to obtain a liposome suspension. The particle size was controlled by a liposome extruder, and extruded three times with 200 nm and 100 nm PC membranes at 65 ° C and 0.4 MPa nitrogen conditions, and then quickly cooled in an ice water bath (0-2 ° C) after extrusion, and placed at this temperature for 15 min to obtain the final blank liposomes.

[0067] Sepharose CL-4B was loaded into a chromatography column, and a 18mM HEPES+50mM NaCl solution with a pH of 6.5 was prepared, and the agarose gel column was pre-equilibrated with the solution. The blank liposomes prepared above were loaded onto the agarose gel column, and slowly eluted with the above solution, and the external aqueous phase was replaced with a 18mM HEPES+50mM NaCl solution with a pH of 6.5. In this step, the liposomes were diluted by about 1 times, and gradient blank liposomes were obtained.

[0068] Weigh 12.17 mg of DPD and mix it with the prepared gradient blank liposomes, incubate with stirring at 65°C for 40 min, quickly place it in an ice water bath to cool for 15 min, take it out and let it cool to room temperature to obtain single-loaded liposomes.

[0069] Example 5

[0070] The 12.17 mg DPD in Example 4 was replaced by 14.00 mg DPD-2, and the other parameters were the same as those in Example 4.

[0071] Example 6

[0072] The 12.17 mg DPD in Example 4 was replaced by 14.66 mg DPD-3, and the other parameters were the same as those in Example 4.

[0073] The particle size, PDI, and encapsulation efficiency of the weakly basic dexamethasone derivative single-loaded liposomes prepared in Examples 4 to 6 were tested, and the results are listed in Table 1.

[0074] Table 1: Particle size, PDI, and encapsulation efficiency of Examples 4-6

[0075] drug Particle size PDI Encapsulation efficiency Example 4 DPD 108.10±0.67 0.048±0.03 98.12％ Example 5 DPD-2 129.13±0.64 0.062±0.01 97.98％ Example 6 DPD-3 131.45±0.76 0.062±0.01 95.83％

[0076] Example 7

[0077] DSPC, cholesterol, DSPE-MPEG in Example 5 2000 The dosage is replaced by 105.0mg DSPC, 52.5mg cholesterol, 2.8mg DSPE-MPEG 2000 , namely (DSPC, cholesterol, DSPE-MPEG 2000 The mass ratio of is 2:1:0.05), and other parameters are the same as those in Example 5.

[0078] Example 8

[0079] DSPC, cholesterol, DSPE-MPEG in Example 5 2000 The dosage was replaced by 105.0mg DSPC, 26.3mg cholesterol, 2.9mg DSPE-MPEG 2000 , namely (DSPC, cholesterol, DSPE-MPEG 2000 The mass ratio of is 4:1:0.11), and other parameters are the same as those in Example 5.

[0080] Example 9

[0081] DSPC, cholesterol, DSPE-MPEG in Example 5 2000 The dosage is replaced by 105.0mg DSPC, 21mg cholesterol, 2.8mg DSPE-MPEG 2000 , namely (DSPC, cholesterol, DSPE-MPEG 2000 The mass ratio of is 5:1:0.13), and other parameters are the same as those in Example 5.

[0082] The particle size, PDI, and encapsulation efficiency of the weakly basic dexamethasone derivative single-loaded liposomes prepared in Examples 5 and 7 to 9 were tested. The results are listed in Table 2.

[0083] Table 2: Particle size, PDI, and encapsulation efficiency of Examples 5, 7 to 9

[0084] Phospholipid ratio Particle size PDI Encapsulation efficiency Example 72: 1: 0.05 121.42±0.71 0.047±0.01 84.51％ Example 53: 1: 0.08 129.13±0.64 0.062±0.01 97.98％ Example 84: 1: 0.11 135.71±0.57 0.061±0.04 80.53％ Example 95:1:0.13 136.19±2.26 0.214±0.01 40.04％

[0085] As shown in Table 2, as the cholesterol ratio decreases, the encapsulation efficiency of the liposomes increases first and then decreases. In addition, when the cholesterol ratio is low, the particle size of the liposomes increases significantly and the distribution is uneven. 2000 When the mass ratio is 3:1:0.08, the particle size and PDI of the liposomes are small and uniform, and the encapsulation efficiency reaches the highest.

[0086] Example 10

[0087] The concentration of the aqueous phase solution of sucrose octasulfate-triethylamine (SOS-TEA) in Example 4 was replaced with 0.4 mol / L, and the other parameters were the same as those in Example 4.

[0088] Embodiment 11

[0089] The concentration of the aqueous phase solution of sucrose octasulfate-triethylamine (SOS-TEA) in Example 4 was replaced with 0.8 mol / L, and the other parameters were the same as those in Example 4.

[0090] The particle size, PDI, and encapsulation efficiency of the weakly alkaline derivative of dexamethasone single liposome prepared in Example 4 and Examples 10 to 11 were tested. The results are listed in Table 3.

[0091] Table 3: Particle size, PDI, and encapsulation efficiency of Examples 4, 10, and 11

[0092] SOS-TEA concentration Particle size PDI Encapsulation efficiency Example 100.4 110.31±0.36 0.073±0.01 98.01％ Example 40.6 118.23±0.95 0.061±0.02 98.76％ Example 110.8 116.78±0.43 0.078±0.02 98.44％

[0093] It can be seen from Table 3 that when the concentration of the aqueous solution in SOS-TEA is 0.4-0.8 mol / L, the encapsulation efficiency of the dexamethasone weakly alkaline derivative liposome tends to be stable.

[0094] Example 12: Preparation of Dexamethasone Weakly Basic Derivative Co-loaded Liposomes

[0095] Weigh 70.0 mg DSPC, 23.3 mg cholesterol, 1.2 mg DSPE-MPEG 2000 , namely (DSPC, cholesterol, DSPE-MPEG 2000The mass ratio of phospholipids was 3:1:0.05), dissolved with 1.5 mL of ethanol, and the dissolved phospholipids were uniformly injected into the preheated 5 mL of 0.6 mol / L TEA-SOS solution with a syringe, and stirred at 65 ° C for 30 min to obtain a liposome suspension. The particle size was controlled by a liposome extruder, and extruded 5 times with a 100 nm PC membrane at 65 ° C and 0.4 MPa nitrogen. After extrusion, it was quickly cooled in an ice water bath (0-2 ° C), and placed at this temperature for 15 min to obtain the final blank liposome.

[0096] Sepharose CL-4B was loaded into a chromatography column, and a 18mM HEPES+50mM NaCl solution with a pH of 6.5 was prepared, and the agarose gel column was pre-equilibrated with the solution. The blank liposomes prepared above were loaded onto the agarose gel column, and slowly eluted with the above solution, and the external aqueous phase was replaced with a 18mM HEPES+50mM NaCl solution with a pH of 6.5. In this step, the liposomes were diluted by about 1 times, and gradient blank liposomes were obtained.

[0097] DPD and irinotecan (IRI) were weighed and mixed with the prepared gradient blank liposomes in a mass ratio of 1:1, with a drug-lipid ratio of 1:3, and drug loading was performed at 70°C, and stirred and incubated for 40 minutes. The liposomes were quickly placed in an ice water bath to cool for 15 minutes, and then taken out and allowed to cool to room temperature to obtain co-loaded liposomes.

[0098] Embodiment 13

[0099] The preparation of blank liposomes was the same as in Example 12. DPD and irinotecan (IRI) were weighed at a mass ratio of 2:1 and mixed with the prepared gradient blank liposomes. The drug-lipid ratio was 1:3, and the drug was loaded at 70°C and incubated with stirring for 40 minutes. The liposomes were quickly placed in an ice water bath to cool for 15 minutes, taken out and allowed to cool to room temperature to obtain co-loaded liposomes.

[0100] Embodiment 14

[0101] The preparation of blank liposomes was the same as in Example 13. DPD and irinotecan (IRI) were weighed at a mass ratio of 2:1 and mixed with the prepared gradient blank liposomes. The drug-lipid ratio was 1:2. The drug was loaded at 70°C and incubated with stirring for 40 minutes. The mixture was quickly placed in an ice water bath to cool for 15 minutes, taken out and allowed to cool to room temperature to obtain co-loaded liposomes.

[0102] Embodiment 15

[0103] The preparation of blank liposomes was the same as in Example 13. DPD and irinotecan (IRI) were weighed at a mass ratio of 2:1 and mixed with the prepared gradient blank liposomes. The drug-lipid ratio was 1:8, and the drug was loaded at 70°C and incubated with stirring for 40 minutes. The liposomes were quickly placed in an ice water bath to cool for 15 minutes, taken out and allowed to cool to room temperature to obtain co-loaded liposomes.

[0104] Example 16

[0105] The preparation of blank liposomes was the same as in Example 13. DPD and irinotecan (IRI) were weighed at a mass ratio of 2:1 and mixed with the prepared gradient blank liposomes. The drug-lipid ratio was 1:12, and the drug was loaded at 70°C and incubated with stirring for 40 minutes. The liposomes were quickly placed in an ice water bath to cool for 15 minutes, taken out and allowed to cool to room temperature to obtain co-loaded liposomes.

[0106] The particle size, PDI, and encapsulation efficiency of the dexamethasone weakly alkaline derivative co-loaded liposomes prepared in Examples 12 to 16 were tested. The results are listed in Table 4.

[0107] Table 4: Particle size, PDI, and encapsulation efficiency of Examples 12 to 16

[0108]

[0109] Example 17: The co-loaded liposomes in Example 12 were measured by transmission electron microscopy

[0110] The transmission electron microscopy results of Example 17 are shown in Figure 3 The results showed that the liposomes were spherical in shape and uniform in size.

[0111] Example 18: In vitro release

[0112] PBS buffer (pH=7.4) containing appropriate amounts of ammonium chloride and SDS was used as the release medium, and a certain concentration of hydrogen peroxide (H2O2) and dithiothreitol (DTT) were added to the release medium, respectively, to investigate the in vitro release of weakly alkaline derivative liposomes under different oxidation or reduction conditions and the degradation of weakly alkaline derivatives of dexamethasone. The single-loaded liposomes (0.5 mL) prepared in implementations 6 and 8 were added to dialysis bags (8-14 kDa) in triplicate. The dialysis bag was then immersed in a centrifuge tube containing 40 mL of the release medium, and the in vitro release was investigated in a 37°C constant temperature oscillator. Samples were taken at set time points, and the concentrations of the released weakly alkaline derivatives of dexamethasone and dexamethasone were determined by high performance liquid chromatography. Finally, the cumulative release rate R was calculated according to the following formula n And draw the curve.

[0113]

[0114] Where V0 represents the volume of release medium (mL), C n represents the concentration at the nth sampling (mg / mL), V represents the sampling and supplement volume (mL), and M represents the total amount of drug (mg).

[0115] When the weak base derivative of dexamethasone is formed by connecting dexamethasone to a weak base intermediate via a sulfur bond, dexamethasone can be released by breaking the bond through oxidation and reduction responsiveness. Figure 4 As shown in the figure, compared with the release medium containing 0.1mM H2O2 or 0.1mM DTT, both DPD-2 and DPD-3 were degraded more in the release medium containing 10mM H2O2 or 10mM DTT, and more DEX was released. This is because the oxidation response mechanism of thioether bonds and disulfide bonds is that the sulfur atom can be oxidized into hydrophilic sulfoxide or sulfone, thereby promoting the hydrolysis of adjacent ester bonds and the release of drugs. Although both DPD-2 and DPD-3 show oxidation responsiveness, the oxidation responsiveness of the thioether bond-linked derivatives is greater than that of the disulfide bond-linked derivatives because the chemical valence of the thioether is lower than that of the disulfide bond, so it is more easily oxidized. In addition, under the reduction reaction conditions of DTT, the thioether bond is prone to sulfurization, while the disulfide bond will be degraded into a hydrophilic thiol group, thereby promoting the hydrolysis of adjacent ester bonds and drug release. In Figures C and D, the remaining cumulative release curve of the weakly alkaline dexamethasone derivatives showed a downward trend after 36 h or 48 h. This may be because the release rate of the weakly alkaline dexamethasone derivatives by liposomes is lower than its degradation rate, and more derivatives are consumed to release DEX.

[0116] Example 19: Stability Test

[0117] The three batches of co-loaded liposomes prepared by the preparation method of Example 12 were packed into vials, filled with nitrogen and sealed with caps. The liposome preparations were then stored at 4±2°C in the dark, and samples were taken at 0d, 7d, 14d, 21d and 28d to measure the particle size and encapsulation efficiency of each sample. The results are shown in Figure 5 .

[0118] During the storage period of 1 month, the particle size of the co-loaded liposomes remained at about 110 nm, the PDI was less than 0.1, and the encapsulation efficiencies of IRI and DPD were both above 96%, demonstrating that the co-loaded liposomes had good storage stability.

[0119] Example 20: Pharmacodynamics Experiment

[0120] Establishment of animal model: 4T1 cells in the logarithmic growth phase were digested with trypsin, the cells were collected, and the bottom cell sediment was redispersed with an appropriate amount of sterile PBS solution to obtain a cell suspension. 200 μL of the cell suspension was subcutaneously inoculated into the axilla of the right forelimb of female Balb / c mice, with about 1×106 cells inoculated per mouse to establish a tumor-bearing mouse animal model.

[0121] Efficacy experiment: When the tumor volume reaches 200mm 3At the time of the experiment, the tumor-bearing mice were randomly divided into 8 groups, 5 mice in each group, and the groups were as follows: blank control group (normal saline group); co-loading preparation group (Example 12, IRI:DPD=1:1); irinotecan liposome and dexamethasone prodrug liposome physical mixture-1 (IRI and DPD mass ratio is 1:1) group; irinotecan liposome group; dexamethasone prodrug (DPD) liposome group and dexamethasone group. Each group was injected with drugs through the tail vein, once every two days, for a total of 14 days, and the total dosage of each group was 5 mg / kg. The tumor volume and body weight changes of mice were recorded regularly.

[0122] The results are as follows Figure 6 Compared with single-loaded liposomes, co-loaded liposomes can have a synergistic anti-tumor effect. At the same time, DPD can improve the tumor microenvironment and better inhibit tumor growth. At the same time, there is no significant change in the weight of mice.

[0123] Example 21: Cytokine Detection

[0124] Cytokine detection: In order to verify whether the co-loaded preparation can effectively improve the tumor microenvironment, regulate the production of inflammatory factors, improve inflammatory response, reduce angiogenesis, and reduce the side effects of nausea and vomiting, the concentration levels of cytokines IL-6, TNF-α, VEGF, and PGE2 in the supernatant of tumor tissue after treatment were determined by enzyme-linked immunosorbent assay (ELISA). Take the tumor tissue of each group, add the homogenate medium (0.9% saline) according to the ratio of weight (mg): volume (μL) = 1:9, grind, centrifuge at 3000rpm for 10min, and take the supernatant to determine the concentration level of cytokines in tumor tissue.

[0125] The results are as follows Figure 7 As shown. Both the DPD single-loaded liposome and the co-loaded preparation group (Example 12, IRI:DPD=1:1) can reduce the content of IL-6 in tumor tissue (P<0.01), and have an anti-inflammatory effect, but the co-loaded preparation group has a more obvious effect; the physical mixture group has a weak effect, which may be because DPD and IRI fail to reach the tumor site at the same time and cannot act at the same time. VEGF is the main factor affecting angiogenesis. Compared with the use of DPD and IRI alone, the co-loaded preparation group can significantly reduce the level of VEGF. Blocking VEGF production can promote the normalization of blood vessels in the tumor site. Normalized blood vessels can alleviate the hypoxic environment inside the tumor and promote drug penetration. From the content of PGE2, it can be seen that the PGE2 content in the IRI group is higher, slightly higher than that in the control group. The co-loaded preparation group can significantly reduce PGE2 expression (P<0.05), thereby alleviating the side effects of nausea and vomiting.

[0126] Example 22: Immunofluorescence examination

[0127] Immunofluorescence examination: Overproliferation of fibroblasts, dense matrix collagen and tumor vascular permeability are the characteristics of tumor stroma. As a marker of vascular endothelial cells, CD31 can be used to monitor changes in vascular morphology and number. Highly disordered, tortuous, uneven thickness, and excessively branched blood vessels are the main reasons for increasing tumor interstitial fluid pressure and hindering drug delivery. Fibroblasts cover between vascular endothelial cells and are an important part of the tumor tissue microenvironment. They can be labeled by α-SMA. Collagen-I can be used to label collagen in the tumor microenvironment. Collagen plays an important role in limiting the diffusion of macromolecules. In summary, the tumors of mice in each group after administration were sliced, labeled, stained, photographed with a microscope, quantified with imageJ software, and the fluorescence intensity was calculated.

[0128] The results are as follows Figure 8 As shown, compared with the control group, the co-loading preparation group (Example 12, IRI:DPD=1:1) can significantly reduce the expression of α-SMA, Collagen-Ⅰ, and CD31 in the tumor microenvironment. CD31 can be used to monitor changes in vascular morphology and quantity. From the results, it can be seen that under the action of DPD / DEX, the disorder of blood vessels is improved, and the blood vessels in the tumor tend to be normal. The normalization of blood vessels can improve the blood perfusion of the tumor site. α-SMA and Collagen-Ⅰ are the main components of the tumor matrix. The reduction in the expression of α-SMA and Collagen-Ⅰ indicates that the dense matrix components outside the tumor cells are affected, which can enable more drugs to be delivered to the tumor cells to exert their efficacy.

Claims

1. A weakly basic dexamethasone derivative or a pharmaceutically acceptable salt thereof, characterized in that: Its general structure is as follows: in; R1 is -COO-, -OOC-(CH2)n1-S-(CH2)n2-CO- or -OOC-(CH2)n1-SS-(CH2)n2-CO-; R2 is 4-(1-piperidinyl)piperidinyl; n1 and n2 in R1 are both the number of repeating units, and are optionally integers of 1-5.

2. The weakly basic dexamethasone derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Specifically selected from the following compounds:

3. The method for preparing the weakly basic dexamethasone derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The preparation method of DPD specifically comprises the following steps: dexamethasone and 4-piperidinylpiperidinyl chloride are subjected to an esterification reaction under the catalytic action of DMAP, the reaction is carried out under N2 protection throughout the process, and finally the sample is separated and purified to obtain DPD; The specific preparation method of DPD-2 or DPD-3 comprises the following steps: mixing 4-(1-piperidinyl)piperidine with thiohydroxyacetic anhydride or 1,4,5-Oxadithiepane-2,7-dione in dimethylformamide for reaction, then adding 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and stirring, then adding dexamethasone and stirring, and after the reaction is completed, evaporating the solvent, separating and purifying to obtain the product.

4. The preparation method according to claim 3, characterized in that: In the preparation method of DPD, the molar ratio of dexamethasone to 4-piperidinylpiperidinylcarbonyl chloride and DMAP is: 1: (0.95-1): (2-3); In the preparation method of DPD-2 or DPD-3, in molar ratio, the ratio of 4-(1-piperidinyl)piperidine to thioglycolic anhydride or 1,4,5-Oxadithiepane-2,7-dione is 1:(1-1.1), and the ratio of dexamethasone to 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(2.5-3):(2.5-3).

5. A liposome, characterized in that: The invention relates to a liposome containing a weakly basic dexamethasone derivative or a liposome containing dexamethasone and other drugs as described in any one of claims 1 to 2; the liposome is composed of the following components: a weakly basic dexamethasone derivative or a weakly basic dexamethasone derivative and other drugs, phospholipids, cholesterol, PEGylated phospholipids, an ammonium salt solution and a buffer solution; wherein the mass ratio of the weakly basic dexamethasone derivative or the dexamethasone and other drugs to the phospholipids is 1:(2-20), and the mass ratio of the phospholipids, cholesterol and PEGylated phospholipids is (2-10):1:(0.02-0.20).

6. The liposome according to claim 5, wherein The phospholipids are one or more of natural phospholipids and synthetic phospholipids; the ammonium salt solution is one or more of ammonium chloride, ammonium sulfate, ammonium acetate, and sucrose octasulfate triethylamine; the buffer solution is selected from one or more of physiological saline, hydroxyethylpiperazineethanesulfonic acid, tromethamine, phosphate, glucose, and other injection-acceptable buffer solutions; the other drugs are selected from anti-inflammatory drugs, anti-tumor drugs, anti-allergic drugs, antibacterial drugs, and anesthetic drugs.

7. The liposome according to claim 6, wherein The natural phospholipid is selected from egg yolk lecithin, soybean lecithin and hydrogenated soybean lecithin; the synthetic phospholipid is selected from dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylcholine and distearoylphosphatidylcholine ethanolamine; the ammonium salt solution is selected from sucrose octasulfate triethylamine solution; and the pH value of the buffer solution is 5.0-7.

0.

8. The liposome according to claim 6, wherein The concentration of the ammonium salt solution is 0.2-0.9M, calculated as ammonium salt.

9. The method for preparing liposomes according to any one of claims 5 to 8, characterized in that: The following steps are involved: (1) Preparation of blank liposomes: using the prescribed amount of phospholipids, cholesterol and PEGylated phospholipids, a blank liposome initial product is prepared by a thin film dispersion method or an ethanol injection method, and the concentration of sucrose octasulfate triethylammonium is 0.4 to 0.8 M; the particle size is reduced and uniformed by a liposome extrusion device to obtain blank liposomes with a particle size of 80 to 150 nm; (2) Preparation of gradient blank liposomes: using an agarose gel column to replace the extraliposome aqueous phase with a buffer salt, establishing a transmembrane gradient, and forming gradient blank liposomes; (3) Drug loading: Add dexamethasone weak base derivatives or dexamethasone and other drugs to gradient blank liposomes, stir in a water bath at 50-75°C, and immediately place in an ice water bath to obtain dexamethasone weak base derivative single-loaded liposomes or dexamethasone and other drugs co-loaded liposomes.

10. Use of the weakly basic dexamethasone derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, and the liposome according to claim 5 in preparing a drug for treating inflammatory diseases and tumor diseases; and / or preparing a combined drug; and / or preparing a drug delivery system.

Images