CN115252554B - Preparation, composition and application of novel 4 (3H) -quinazolinone analogue/cyclic dinucleotide cGAMP co-carrier liposome in antitumor drugs - Google Patents
Preparation, composition and application of novel 4 (3H) -quinazolinone analogue/cyclic dinucleotide cGAMP co-carrier liposome in antitumor drugs Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, reports a 4 (3H) -quinazolinone analogue/cyclic dinucleotide cGAMP co-carried liposome compound, invents a folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carried liposome and a preparation method of an anti-PD-L1 antibody targeted 4 (3H) -quinazolinone analogue/cGAMP co-carried liposome compound, and an anti-tumor activity research, wherein research results show that the co-carried liposome compound medicament has a synergistic anti-tumor effect, and the targeted co-carried liposome compound has significantly improved anti-tumor activity compared with a non-targeted medicament. Therefore, the 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome compound has good application prospect in the aspect of antitumor drugs.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a 4 (3H) -quinazolinone analogue single-carrier liposome, a 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome, a folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome, and an anti-PD-L1 antibody targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome compound composition, a preparation method and application thereof in medicines such as autoimmune diseases, allergies, inflammations, bone diseases, diabetes mellitus, bone mineralization and soft tissue calcification diseases, viruses, calcium pyrophosphate dihydrate deposition diseases, malignant tumors and the like.
Background
In recent years, the field of tumor immunotherapy has been unprecedented, and has a milestone significance in the history of tumor fighting in human beings. Tumor immunotherapy aims at activating the immune system of the tumor to resist tumor cells, and has the characteristics of safety, remarkable effect, relapse prevention and the like. Immune checkpoint inhibitors are an important branch of tumor immunotherapy that activate the adaptive regulation of T cell killing tumor cells by disrupting the pathway of immune co-suppression signaling. With intensive clinical research, immune checkpoint inhibitors are found to have certain limitations and drug resistance, and have low response values to non-inflammatory cancers. Scientists have therefore proposed a broader strategy for immunotherapy to increase tumor immunogenicity by activating the innate immune system, resulting in tumor immunotherapy.
The cGAS-STING-TBK1-IRF3 signaling pathway is an important component of the innate immune system. When dsDNA of tumor cells enters normal cells, it is rapidly recognized and bound by cyclic Guanosine Monophosphate (GMP) -Adenosine Monophosphate (AMP) synthase (cGAS), and the complex of dsDNA-cGAS catalyzes the production of cyclic dinucleotides 2',3' -cGAMP (cGAMP) from Guanosine Triphosphate (GTP) and Adenosine Triphosphate (ATP). The resulting cGAMP activates interferon gene Stimulators (STING) causing downstream pathways to produce type I interferons and various cytokines, which in turn activate CD8 positive T cells to clear tumor cells. The cGAMP is used as a secondary signal molecule, and the exogenous addition can improve the immunity of the organism and increase the anti-tumor effect.
The 4 (3H) -quinazolinone compound is a nitrogen-containing heterocyclic compound with wide pharmacological activity, and has good activity in the aspects of resisting cancer, tumor, hypertension, microorganism and the like. The natural products contain quinazolinone structures such as echinozolone, chrysogenine, poncirin (glycosamine), rutaecarpine (rutaecarpine) and febridine (febrifugine) in a large number of natural products. Therefore, molecules with 4 (3H) -quinazolinone as a framework are often used for anti-tumor activity research.
The liposome is a miniature vesicle with a bilayer, can load hydrophilic and hydrophobic drugs, namely hydrophilic molecules are loaded in the inner cavity of the liposome, and hydrophobic molecules are embedded in the lipid bilayer, and the liposome has the characteristics of good biocompatibility, slow release of the drugs and reduction of drug toxicity. The liposome belongs to a nano carrier, can effectively encapsulate various medicines, improves the stability of the medicines, prolongs the half life in vivo and improves the medicine effect.
In recent years, the research of targeting liposome has been greatly broken through in the aspect of anti-tumor, wherein the research of targeting liposome by folic acid is most mature. The research shows that the folic acid receptor is over-expressed in various tumor cells, so that the research utilizes the advantages of high chemical stability, high tissue compatibility, high penetrating power, short target time, high affinity with the receptor, almost no immunogenicity, low price, easy obtainment and the like of folic acid small molecules, and prepares folic acid targeted liposome for the synergistic anti-tumor research of the double-carrier drugs.
When a tumor occurs, antigens on the surface of the cell membrane are easily recognized by immune cells, thereby causing the immune system to attack tumor tissues. However, tumor cells modify their surface antigens, recruit suppressive immune cells or molecules, and alter the microenvironment surrounding tumor tissues to evade the monitoring, recognition and attack of the immune system of the body to continue dividing and growing, for example, tumor cells overexpress programmed death ligand-1 (PD-L1) to bind to programmed death receptor-1 (PD-1) on the surface of T cells, which will shut down the T cell immune response and evade the clearance of the immune system. The research utilizes the characteristics that the PD-L1 antibody has tumor tissue specificity, improves the enrichment amount of the drug at the tumor part, reduces toxic and side effects and improves bioavailability, and the PD-L1 antibody itself has the characteristic of improving the response of T cells to tumor cells so as to kill the tumor cells, so that the anti-PD-L1 targeted double-carrier liposome compound is prepared for anti-tumor research.
The study reports 4 (3H) -quinazolinone analogue single-carrier liposome, 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome, folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome and anti-PD-L1 targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome compound, and anti-tumor activity study is carried out by using the compound, thus providing innovative ideas and basis for immune anti-tumor drug development.
Disclosure of Invention
The invention provides a 4 (3H) -quinazolinone analogue single-carrier liposome, a 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome, a folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome and an anti-PD-L1 targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome compound, a preparation method and application thereof in antitumor drugs. Research shows that the co-carried liposome composite medicine has synergistic antitumor effect, and the targeting co-carried liposome composite has obviously improved antitumor activity compared with non-targeting medicine.
The specific invention comprises the following steps:
1. a composition and preparation method of 4 (3H) -quinazolinone analogue single carrier liposome comprises liposome and 4 (3H) -quinazolinone analogue. Wherein the lipophilic 4 (3H) -quinazolinone analog is entrapped in the hydrophobic region of the liposome core.
The liposome in the 4 (3H) -quinazolinone analog single-carrier liposome is prepared from soybean phospholipid HSPC, cholesterol CHOL, dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG) 2000 ) Composition is prepared.
The preparation method of the 4 (3H) -quinazolinone analogue single liposome preparation comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL and DPPE-PEG 2000 And 4 (3H) -quinazolinone analog are added into dichloromethane, mixed evenly, and the solvent is suspended by a rotary evaporator until the mixture forms a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, extruding the hydrated liposome in a liposome extruder to pass through a 100nm polycarbonate microporous filter membrane to form uniform single-chamber liposome, and then dialyzing to remove free ammonium sulfate;
step three: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
2. A composition of 4 (3H) -quinazolinone analog/cGAMP co-carrier liposome complex and its preparation method are provided, which comprises liposome, 4 (3H) -quinazolinone analog, and cGAMP. The method is characterized in that the hydrophobic 4 (3H) -quinazolinone analogue in the co-carrier liposome is wrapped in a hydrophobic region of the liposome, and the hydrophilic cGAMP is wrapped in a hydrophilic region in the center of the liposome. The liposome of the 4 (3H) -quinazolinone analog/cGAMP co-carried liposome preparation is prepared from soybean phospholipidHSPC, cholesterol CHOL, dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG) 2000 ) Composition is prepared.
The preparation method of the 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome complex comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL, DPPE-PEG 2000 And 4 (3H) -quinazolinone analog are added into dichloromethane, mixed evenly, and the solvent is suspended by a rotary evaporator until the mixture forms a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, extruding the hydrated liposome in a liposome extruder to pass through a 100nm polycarbonate microporous filter membrane to form uniform single-chamber liposome, and then dialyzing to remove free ammonium sulfate;
step three: adding cGAMP into the dialyzed liposome solution, slightly stirring and incubating for 1h in a magnetic stirrer with an oil bath temperature of 60 ℃, and dialyzing the incubated liposome to remove free cGAMP;
step four: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
3. A folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome complex composition and a preparation method thereof are provided, wherein the folic acid targeted liposome composition comprises folic acid targeted liposome, 4 (3H) -quinazolinone analogue and cGAMP. The folic acid targeting co-carrier liposome is characterized in that a hydrophobic 4 (3H) -quinazolinone analogue in the folic acid targeting co-carrier liposome is wrapped in a hydrophobic area of the liposome, and a hydrophilic cGAMP is wrapped in a hydrophilic area in the center of the liposome.
The liposome of the folic acid targeted 4 (3H) -quinazolinone analog/cGAMP co-carried liposome preparation is prepared from soybean phospholipid HSPC, cholesterol CHOL, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG) 2000 ) Dipalmitoyl phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG) 2000 FA) composition.
The preparation method of the folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome complex comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL, DSPE-PEG 2000 、DSPE-PEG 2000 -FA and 4 (3H) -quinazolinone analogs were added to dichloromethane, mixed well, and the solvent was suspended using a rotary evaporator until the mixture formed a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, extruding the hydrated liposome in a liposome extruder to pass through a 100nm polycarbonate microporous filter membrane to form uniform single-chamber liposome, and then dialyzing to remove free ammonium sulfate;
step three: adding cGAMP into the dialyzed liposome solution, slightly stirring and incubating for 1h in a magnetic stirrer with an oil bath temperature of 60 ℃, and dialyzing the incubated liposome to remove free cGAMP;
step four: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
4. An anti-PD-L1 antibody targeted 4 (3H) -quinazolinone analogue/cGAMP co-carried liposome complex composition and a preparation method thereof, wherein the anti-PD-L1 targeted liposome comprises an anti-PD-L1 targeted liposome, a 4 (3H) -quinazolinone analogue and a cGAMP. The anti-PD-L1 targeting co-carrier liposome is characterized in that the hydrophobic 4 (3H) -quinazolinone analogue in the anti-PD-L1 targeting co-carrier liposome is wrapped in a hydrophobic region of the liposome, and the hydrophilic cGAMP is wrapped in a hydrophilic region in the center of the liposome.
The liposome of the anti-PD-L1 antibody targeted 4 (3H) -quinazolinone analogue/cGAMP co-carried liposome preparation is prepared from soybean phospholipid HSPC, cholesterol CHOL, dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG) 2000 ) Dipalmitoyl phosphoethanolamine-polyethylene glycol-maleimide (DPPE-PEG) 2000 -MAL).
The preparation method of the anti-PD-L1 targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome preparation comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL, DPPE-PEG 2000 、DPPE-PEG 2000 -MAL and 4 (3H) -quinazolinone analogs are added to methylene chloride,uniformly mixing, and suspending the solvent by using a rotary evaporator until the mixture forms a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, extruding the hydrated liposome in a liposome extruder to pass through a 100nm polycarbonate microporous filter membrane to form uniform single-chamber liposome, and then dialyzing to remove free ammonium sulfate;
step three: adding cGAMP into the dialyzed liposome solution, and incubating for 1h with gentle stirring in a magnetic stirrer with an oil bath temperature of 60 ℃;
step four: adding thiolated anti-PD-L1 and a reducing agent sodium dithionite solution into the incubated liposome of the cGAMP, incubating overnight at room temperature in a dark place, and dialyzing the incubated liposome;
step five: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
The encapsulation efficiency of the four liposomes of the invention was measured and found to be 90% or more for both the 4 (3H) -quinazolinone analog and the cGAMP.
5. The research result of the 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome prepared by the invention on a mouse tumor model shows that the anti-tumor effect of the co-carrier liposome compound is better than that of a single carrier liposome, which indicates that the co-carrier liposome has good synergistic effect in anti-tumor aspect.
6. The research result of the folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carried liposome prepared by the invention on a mouse tumor model shows that the folic acid targeted co-carried liposome has better anti-tumor effect than that of a non-targeted co-carried liposome, which indicates that the folic acid targeted liposome is beneficial to the aggregation of medicines in tumor microenvironment to increase the medicine effect.
7. The research result of the anti-PD-L1 antibody targeted 4 (3H) -quinazolinone analog/cGAMP co-carried liposome on a mouse tumor model shows that the anti-PD-L1 targeted co-carried liposome has better anti-tumor effect than non-targeted co-carried liposome and better folic acid targeted co-carried liposome. The anti-PD-L1 targeted liposome not only increases the drug concentration of the tumor microenvironment, but also plays an anti-tumor role.
Detailed Description
The following is a detailed description of the present invention by way of examples. In the present invention, the following examples are given for better illustration of the present invention and are not intended to limit the scope of the present invention.
Example 1.4 preparation of (3H) -quinazolinone analog 1, 4 (3H) -quinazolinone analog 2 and characterization of 4 (3H) -quinazolinone analog 1,2- [ (5-methoxy- (imidazo [4,5-B ] pyridine) -2-thio) methyl ] -7-fluoro-quinazolin-4 (3H) -one preparation method:
methyl 2-amino-4-fluorobenzoate (338 mg,2 mmol) and chloroacetonitrile (127. Mu.L, 2 mmol) were added to 4M HCl/dioxane (5 mL) and reacted overnight at 97 ℃. After working up, the reaction mixture was cooled to room temperature, 20mL of saturated sodium bicarbonate solution was slowly added dropwise under ice bath, solids were precipitated, filtered off with suction, and the filter cake was washed successively with water, ethanol, diethyl ether, respectively, to give 2- (chloromethyl) -7-fluoroquinazolin-4 (3H) -one (239 mg, 56%) as a brown solid. 1 H NMR(400MHz,DMSO)δ12.69(s,1H),8.19(t,J＝8.2Hz,1H),7.48(d,J＝8.4Hz,1H),7.41(t,J＝8.7,1H),4.53(s,2H). 13 C NMR(101MHz,DMSO)δ167.49,164.99,161.27,154.36,150.99,129.53,129.42,118.76,116.37,116.14,113.08,112.86,43.51.
5-methoxy-2-mercaptoimidazo [4,5-b]Pyridine (91 mg,0.5 mmol), 2- (chloromethyl) -7-fluoroquinazolin-4 (3H) -one (106 mg,0.5 mmol) and sodium hydroxide (100 mg,2.5 mmol) were dissolved in methanol (7 mL) and stirred at room temperature overnight. The work-up reaction was carried out and the organic solvent methanol was removed under reduced pressure to give a crude product which was purified using a 300-400 mesh silica gel column to give the final product, analog 1 (122 mg, 68%). 1 H NMR(400MHz,DMSO)δ13.22(s,1H),12.69(s,1H),8.22–8.10(m,1H),7.83(s,1H),7.48-7.36(m,2H),6.63(d,J＝8.6Hz,1H),4.51(s,2H),3.86(s,3H). 13 C NMR(101MHz,DMSO)δ167.35,164.84,162.59,161.10,154.45,150.11,149.98,147.43,144.67,129.54,129.43,126.20,121.88,118.44,116.05,115.82,112.21,111.99,109.60,54.52,36.00.ESI-MS m/z calcd for C 16 H 12 FN 5 O 2 S + 358.0768,found 358.0762[M+H] + .
A process for the preparation of the 4 (3H) -quinazolinone analog 2,2- [ (4-methyl- (thiazolo [4,5-B ] pyridine) -2-thio) methyl ] -7-fluoro-quinazolin-4 (3H) -one:
4-methyl-2-mercaptobenzothiazole (91 mg,0.5 mmol), 2- (chloromethyl) -7-fluoroquinazolin-4 (3H) -one (106 mg,0.5 mmol) and sodium hydroxide (100 mg,2.5 mmol) were dissolved in methanol (7 mL) and stirred overnight at ambient temperature. The work-up reaction was carried out and the organic solvent methanol was removed under reduced pressure to give a crude product which was purified by a 300-400 mesh silica gel column to give analog 2 (100 mg, 56%) as a white solid as the final product. 1 H NMR(400MHz,DMSO)δ12.66(s,1H),8.17(dd,J＝8.7,2.4Hz,1H),7.86–7.76(m,1H),7.38(ddd,J＝11.2,9.5,2.5Hz,2H),7.29–7.20(m,2H),4.62(s,2H),2.58(s,3H). 13 C NMR(101MHz,DMSO)δ167.49,164.99,164.22,161.02,156.09,151.93,149.73,149.60,135.16,131.33,129.72,129.61,127.36,125.28,119.64,118.14,116.19,115.96,111.87,111.65,35.95,18.23.ESI-MS m/z calcd for C 17 H 12 FN 3 OS 2 + 358.0479,found 358.0488[M+H] + .
Example 2.4 preparation of (3H) -quinazolinone analog Single Apolipoplasts
450mg of soybean phospholipid HSPC, 150mg of cholesterol CHOL and 120mg of DPPE-PEG 2000 And 5mg of 4 (3H) -quinazolinone analogue is added into 50mL of dichloromethane, then transferred into a 1L eggplant-shaped bottle, dried in a vacuum spin mode to form a film, and the film is continuously distilled for more than 3 hours after the film is formed, so that residual trace organic solvent dichloromethane is removed. And (3) adding 60mL of 250mM ammonium sulfate solution for hydration (the hydration temperature is 60 ℃), and allowing the hydrated liposome to pass through a 100nm polycarbonate microporous filter membrane back and forth under the extrusion of a liposome extruder to form uniform single-chamber liposome. Loading the homogenized liposome into 3500Da dialysis bag, dialyzing to remove free ammonium sulfate (dialysis buffer 500mL of 5% glucose solution, dialyzing for three times, changing dialysate every 6 hr), adding trehalose (mass 10% of liposome volume), and lyophilizing to obtain lip/4 (3H) -quinazolinoneAnd (3) preserving the lyophilized powder of the pinone analogue at-20 ℃.
Encapsulation efficiency determination of 4 (3H) -quinazolinone analog single-carrier liposome.
Taking a certain amount of 4 (3H) -quinazolinone analogue single-carrier liposome freeze-dried powder, adding 700 mu L of demulsifier (methanol: isopropanol=7:3, V/V) and 300 mu L of water, shaking and mixing uniformly, centrifuging at 10000rpm for 1min, taking supernatant, running HPLC, and measuring the drug concentration by a concentration-peak area standard curve, thereby calculating the encapsulation rate of cGAMP to be 95.6%.
Example 3 preparation of cgamp single carrier liposomes.
Cyclic dinucleotides cGAMP are synthesized catalytically by cyclic cGMP-AMP dinucleotide synthetases (cGAS) under activated conditions following DNA binding according to literature methods. The purity is above 98%. (Pingwei Li, et al, immunity,2013,39 (6), 1019-1031).
450mg of soybean phospholipid HSPC, 150mg of cholesterol CHOL and 120mg of DPPE-PEG 2000 Adding into 50mL of dichloromethane, transferring into a 1L eggplant-shaped bottle, spin-drying under vacuum to form a film, and continuously spin-evaporating for more than 3 hours after the film is formed to remove residual trace organic solvent dichloromethane. And (3) adding 60mL of 250mM ammonium sulfate solution for hydration (the hydration temperature is 60 ℃), and allowing the hydrated liposome to pass through a 100nm polycarbonate microporous filter membrane back and forth under the extrusion of a liposome extruder to form uniform single-chamber liposome. The homogenized liposomes were placed in 3500Da dialysis bags and dialyzed thoroughly to remove free ammonium sulfate (dialysis buffer 500mL of 5% dextrose solution, dialyzed three times, dialysate changed every 6 hours). 90mg of cGAMP is added into a liposome solution, the liposome is incubated for 2 hours with slight stirring in a magnetic stirrer with an oil bath temperature of 70 ℃, the incubated liposome is placed into a 3500Da dialysis bag for complete dialysis to remove free cGAMP (5% glucose solution with a dialysis buffer of 500mL is dialyzed for three times, and the dialysis solution is replaced every 6 hours), trehalose (the mass is 10% of the volume of the liposome) is added into the dialyzed liposome for freeze drying, and lip/cGAMP freeze-dried powder is obtained and stored at-20 ℃.
The encapsulation efficiency of the cGAMP single-carrier liposome is measured by taking a certain amount of lip/cGAMP single-carrier liposome freeze-dried powder, adding 700 mu L of demulsifier (methanol: isopropanol=7:3, V/V) and 300 mu L of water, shaking and mixing uniformly, centrifuging at 10000rpm for 1min, taking the supernatant, running HPLC, and measuring the drug concentration by a concentration-peak area standard curve, thereby calculating the encapsulation efficiency of the cGAMP to be 90.6%.
Example 4.4 preparation of (3H) -quinazolinone analog 1/cGAMP co-carried liposomes
450mg of soybean phospholipid HSPC, 150mg of cholesterol CHOL and 120mg of DPPE-PEG 2000 Adding 5mg of 4 (3H) -quinazolinone analogue 1 into 50mL of dichloromethane, transferring into a 1L eggplant-shaped bottle, performing vacuum spin drying to form a film, and continuously performing spin evaporation for more than 3 hours after the film is formed to remove residual trace organic solvent dichloromethane. And adding 60mL of 250mM ammonium sulfate solution for hydration (the hydration temperature is 60 ℃), and allowing the hydrated liposome to pass through a 100nm polycarbonate microporous filter membrane for 21 times under the extrusion of a liposome extruder to form uniform single-chamber liposome. The homogenized liposomes were placed in 3500Da dialysis bags and dialyzed thoroughly against free small molecule inhibitors and ammonium sulfate (dialysis buffer 500mL of 5% dextrose solution, dialyzed three times, dialysate changed every 6 hours). 55mg of cGAMP is added into a liposome solution, the mixture is incubated for 2 hours with slight stirring in a magnetic stirrer with an oil bath temperature of 70 ℃, the incubated liposome is filled into 3500Da dialysis bags, free cGAMP is fully dialyzed and removed (dialysis buffer is 500mL of 5% glucose solution and is dialyzed for three times, dialysis liquid is replaced every 6 hours), trehalose (with a mass of 10% of the volume of the liposome) is added into the dialyzed liposome, and freeze-drying is carried out, so that lip/cGAMP-1 co-carrier liposome freeze-dried powder is obtained, and the liposome is preserved at the temperature of minus 20 ℃.
Encapsulation efficiency of co-carrier liposomes: a certain amount of lip/cGAMP-1 co-carrier liposome freeze-dried powder was taken, 700 μl of demulsifier (methanol: isopropanol=7:3, V/V) and 300 μl of water were added, shaking and mixing were carried out, centrifugation was carried out at 10000rpm for 1min, the supernatant was taken out of HPLC, and the drug concentration was determined by a concentration-peak area standard curve, and further the encapsulation efficiency of 4 (3H) -quinazolinone analogue 1 was calculated to be 97% and cGAMP encapsulation efficiency was calculated to be 90.2%.
Example 5 preparation of folate-targeted 4 (3H) -quinazolinone analog 1/cGAMP co-loaded liposomes
450mg of soybean phospholipid HSPC, 150mg of cholesterol CHOL and 120mg of DSPE-PEG 2000 、30mg DSPE-PEG 2000 FA and 5mg of 4 (3H) -quinazolinone analog 1 are added into 50mL of dichloromethane, transferred into a 1L eggplant-shaped bottle, dried in a vacuum spin mode to form a film, and the film is continuously distilled for more than 3 hours after the film is formed, and residual trace organic solvent dichloromethane is removed. And (3) adding 60mL of 250mM ammonium sulfate solution for hydration (the hydration temperature is 60 ℃), and allowing the hydrated liposome to pass through a 100nm polycarbonate microporous filter membrane back and forth under the extrusion of a liposome extruder to form uniform single-chamber liposome. The homogenized liposomes were placed in 3500Da dialysis bags and dialyzed thoroughly against free small molecule inhibitors and ammonium sulfate (dialysis buffer 500mL of 5% dextrose solution, dialyzed three times, dialysate changed every 6 hours). 55mg of cGAMP is added into a liposome solution, the liposome is incubated for 2 hours with slight stirring in a magnetic stirrer with an oil bath temperature of 70 ℃, the incubated liposome is filled into 3500Da dialysis bags, free cGAMP is fully dialyzed and removed (dialysis buffer is 500mL of 5% glucose solution and is dialyzed for three times, dialysis liquid is replaced every 6 hours), trehalose (with a mass of 10% of the volume of the liposome) is added into the dialyzed liposome, and freeze-drying is carried out, so that lip/cGAMP-1-FA co-carrier liposome freeze-dried powder is obtained, and the liposome is preserved at the temperature of minus 20 ℃.
The encapsulation efficiency of the lip/cGAMP-1-FA co-carrier liposome is measured by taking a certain amount of lip/cGAMP-1-FA co-carrier liposome freeze-dried powder, adding 700 mu L of demulsifier (methanol: isopropanol=7:3, V/V) and 300 mu L of water, shaking and mixing uniformly, centrifuging at 10000rpm for 1min, taking supernatant, running HPLC, and measuring the drug concentration by a concentration-peak area standard curve, thereby calculating that the encapsulation efficiency of the 4 (3H) -quinazolinone analogue 1 is 95.7%, and the encapsulation efficiency of the cGAMP is 92.3%.
Example 6 preparation of anti-PD-L1 targeted 4 (3H) -quinazolinone analog 1/cGAMP co-carried liposomes
450mg of soybean phospholipid HSPC, 150mg of cholesterol CHOL and 120mg of DPPE-PEG 2000 、30mg DPPE-PEG 2000 MAL and 5mg of 4 (3H) -quinazolinone analog 1 are added into 50mL of dichloromethane, transferred into a 1L eggplant-shaped bottle, dried in a vacuum spin mode to form a film, and the film is continuously spin-evaporated for more than 3 hours after the film is formed, and residual trace organic solvent dichloromethane is removed. 60mL of 250mM ammonium sulfate solution is added for hydration (the hydration temperature is 60 ℃), and the hydrated liposome is extruded out of the liposomeThe liposome after homogenization is filled into 3500Da dialysis bags to fully dialyze and remove free small molecular inhibitor and ammonium sulfate (dialysis buffer is 500mL of 5% glucose solution, dialyzed three times, and dialysate is changed every 6 hours). 55mg of cGAMP was added to the liposome solution, incubated with gentle stirring in a magnetic stirrer at an oil bath temperature of 70℃for 2h, after incubation, the thiolated anti-PD-L1 (total mass of phospholipids: anti-PD-L1 mass = 1mg: 20. Mu.g) and a reducing agent sodium dithionite solution (final concentration of 1 mM) were added and incubated overnight at room temperature protected from light; the antibody-linked liposome is put into a 30kD dialysis bag for fully dialyzing to remove free anti-PD-L1 (the molecular weight is about 15 kD) and cGAMP (5% glucose solution with a dialysis buffer of 500mL, dialyzing for three times, and changing the dialyzate every 6 hours), and the dialyzate-finished liposome is added with trehalose (the mass is 10% of the volume of the liposome) for freeze drying, and the lip/cGAMP-1-anti-PD-L1 co-carried liposome lyophilized powder is preserved at the temperature of minus 20 ℃.
The encapsulation efficiency of the lip/cGAMP-1-anti-PD-L1 co-carrier liposome is measured by taking a certain amount of lip/cGAMP-1-anti-PD-L1 co-carrier liposome freeze-dried powder, adding 700 mu L of demulsifier (methanol: isopropanol=7:3, V/V) and 300 mu L of water, shaking and mixing uniformly, centrifuging at 10000rpm for 1min, taking supernatant, running HPLC, and measuring the drug concentration by a concentration-peak area standard curve, and further calculating that the encapsulation efficiency of the 4 (3H) -quinazolinone analogue 1 is 94.1% and the encapsulation efficiency of the cGAMP is 91%.
The lip/cGAMP-1-anti-PD-L1 co-carried liposome protein connection rate is determined by taking 300 mu L of liposome solution, adding 0.4mL of methanol, and vortex oscillating for 30s; 0.2mL of methylene chloride was added and vortexed for 30s; 0.1mL dd H was added 2 O, vortex oscillation for 30s;9000g of the mixture was centrifuged for 1min, and the upper layer was removed to obtain an organic methylene chloride layer. Adding 0.3mL of methanol, and carrying out vortex oscillation for 30s;9000g was centrifuged for 2min and the supernatant carefully removed to give a white pellet. N (N) 2 The residual solvent was blown dry and 200. Mu.L HEPES (20mM HEPES,140mM NaCl,2%SDS) was added to solubilize the protein. Then, according to the test method of the BCA kit, the protein ligation rate of the lip/cGAMP-1-anti-PD-L1 co-carrier liposome was determined to be 95%.
Measurement of Lip/cGAMP-1-anti-PD-L1 Co-apolipoprotein connection Density 25. Mu.L of Lip/cGAMP-1-anti-PD-L1 co-apolipoprotein solution was taken, added to 3.975mL of dichloromethane followed by 4mL of FeCl 3 Color reagent (color reagent: 2.7g ferric trichloride+3 g ammonium thiocyanate+100 mL water), shaking and mixing; centrifugation at 4000rpm for 10min, careful removal of the upper layer, removal of the lower layer solution and measurement of 485nm UV absorption. And drawing a standard curve through phospholipid concentration-absorbance, calculating the phospholipid quality, and obtaining the lip/cGAMP-1-anti-PD-L1 co-carried liposome protein connection density of 19.2 mug/mg according to the protein connection rate. Example 7 preparation of anti-PD-L1 antibody-targeted 4 (3H) -quinazolinone analog 2/cGAMP co-carried liposomes
450mg of soybean phospholipid HSPC, 150mg of cholesterol CHOL and 120mg of DPPE-PEG 2000 、30mg DPPE-PEG 2000 MAL and 5mg of 4 (3H) -quinazolinone analog 2 are added into 50mL of dichloromethane together, transferred into a 1L eggplant-shaped bottle, dried in a vacuum spin mode to form a film, and the film is continuously spin-evaporated for more than 3 hours after the film is formed, and residual trace organic solvent dichloromethane is removed. And (3) adding 60mL of 250mM ammonium sulfate solution for hydration (the hydration temperature is 60 ℃), allowing the hydrated liposome to pass through a 100nm polycarbonate microporous membrane back and forth under the extrusion of a liposome extruder to form uniform single-chamber liposome, and filling the uniform liposome into a 3500Da dialysis bag for fully dialyzing to remove free small molecule inhibitor and ammonium sulfate (5% glucose solution with a dialysis buffer of 500mL, dialyzing for three times, and changing dialysate every 6 hours). 55mg of cGAMP was added to the liposome solution, incubated with gentle stirring in a magnetic stirrer at an oil bath temperature of 70℃for 2h, after incubation, the thiolated anti-PD-L1 (total mass of phospholipids: anti-PD-L1 mass = 1mg: 20. Mu.g) and a reducing agent sodium dithionite solution (final concentration of 1 mM) were added and incubated overnight at room temperature protected from light; the antibody-linked liposome is put into a 30kD dialysis bag for fully dialyzing to remove free anti-PD-L1 (the molecular weight is about 15 kD) and cGAMP (5% glucose solution with a dialysis buffer of 500mL, dialyzing for three times, and changing the dialyzate every 6 hours), and the dialyzate-finished liposome is added with trehalose (the mass is 10% of the volume of the liposome) for freeze drying, and the lip/cGAMP-2-anti-PD-L1 co-carried liposome lyophilized powder is preserved at the temperature of minus 20 ℃.
The encapsulation efficiency of the lip/cGAMP-2-anti-PD-L1 co-carrier liposome is measured by taking a certain amount of lip/cGAMP-2-anti-PD-L1 co-carrier liposome freeze-dried powder, adding 700 mu L of demulsifier (methanol: isopropanol=7:3, V/V) and 300 mu L of water, shaking and mixing uniformly, centrifuging at 10000rpm for 1min, taking supernatant, running HPLC, and measuring the drug concentration by a concentration-peak area standard curve, and further calculating that the encapsulation efficiency of the 4 (3H) -quinazolinone analogue 2 is 95% and the encapsulation efficiency of the cGAMP is 91%.
The link rate of lip/cGAMP-2-anti-PD-L1 co-carried liposome protein is determined by taking 300 mu L of liposome solution, adding 0.4mL of methanol, and vortex oscillating for 30s; 0.2mL of methylene chloride was added and vortexed for 30s; 0.1mL dd H was added 2 O, vortex oscillation for 30s;9000g of the mixture was centrifuged for 1min, and the upper layer was removed to obtain an organic methylene chloride layer. Adding 0.3mL of methanol, and carrying out vortex oscillation for 30s;9000g was centrifuged for 2min and the supernatant carefully removed to give a white pellet. N (N) 2 The residual solvent was blown dry and 200. Mu.L HEPES (20mM HEPES,140mM NaCl,2%SDS) was added to solubilize the protein. Then, according to the test method of the BCA kit, the protein ligation rate of the lip/cGAMP-2-anti-PD-L1 co-carrier liposome was determined to be 93%.
Measurement of Lip/cGAMP-2-anti-PD-L1 Co-apolipoprotein connection Density 25. Mu.L of Lip/cGAMP-2-anti-PD-L1 co-apolipoprotein solution was taken, added to 3.975mL of dichloromethane followed by 4mL of FeCl 3 Color reagent (color reagent: 2.7g ferric trichloride+3 g ammonium thiocyanate+100 mL water), shaking and mixing; centrifugation at 4000rpm for 10min, careful removal of the upper layer, removal of the lower layer solution and measurement of 485nm UV absorption. And drawing a standard curve through phospholipid concentration-absorbance, calculating the phospholipid quality, and obtaining the lip/cGAMP-2-anti-PD-L1 co-carried liposome protein connection density of 19 mug/mg according to the protein connection rate.
Example 7 detection of the antitumor Effect of Co-carried Liposome Complex on tumor-bearing mice Using tumor-bearing murine model
Animals: BALB/C common mice, C57BL/6 common mice, male, body weight 20-22g,7-8 weeks old, SPF grade, purchased from Shanghai Laike laboratory animal Limited.
Feeding conditions: all mice were free to feed and drink water and were kept at room temperature (23.+ -. 2). Degree.C. The feed and water are subjected to high-pressure sterilization treatment, and all experimental feeding processes are SPF level.
Dose setting:
(1) All 4 (3H) -quinazolinone analogs/cyclic nucleotide cGAMP co-liposome complexes, 4 (3H) -quinazolinone analogs (0.5 mg/kg)/cyclic nucleotide cGAMP (5 mg/kg)
(2) All 4 (3H) -quinazolinone analogs Single apoliposomal complexes, 4 (3H) -quinazolinone analogs (0.5 mg/kg)
(3) All cGAMP single liposome complexes, cGAMP (5 mg/kg)
Test control
Negative control: PBS solution
Positive control: cGAMP single carrier liposome, cGAMP dosage 5mg/kg
Administration method
Route of administration: intraperitoneal injection administration
Number of administrations: 1 time per day for 21 consecutive days
Number of animals per group: 10 pieces of
Tumor cell line
The mouse colorectal cancer cell line CT26 and the mouse breast cancer cell line 4T1 are all purchased from a cell bank of China academy of sciences.
Main step of the test
Establishment and intervention of tumor model mice
Cell culture, passage, collection of cells at the logarithmic phase of cells, and concentration of (1.0X10) 7 ) Per ml of cell suspension, mice were injected with 0.2ml of cell suspension (cell number 2.0X10 6 One/one), the tumor formation was successful about 8 days, and the tumor formation was randomly divided into 8 groups. The first group is a negative control group; the second group is a group of cGAMP single-carrier liposomes (5 mg/kg cGAMP); the third group was the 4 (3H) -quinazolinone analog-1 single liposome group (0.5 mg/kg); the fourth group was 4 (3H) -quinazolinone analog-2 single liposome group (0.5 mg/kg); the fifth group was co-carried liposomes (cGAMP 5mg/kg/4 (3H) -quinazolinone analog-1, 0.5 mg/kg); the sixth group is folic acid targeting co-carrier liposome group cGAMP 5mg/kg/4 (3H) -quinazolinone analogue-1, 0.5 mg/kg); the seventh group is anti-PD-L1 antibody targeting co-Apoliposomal group cGAMP 5mg/kg/4 (3H) -quinazolinone analogue-1, 0.5 mg/kg); the eighth group was anti-PD-L1 antibody targeting co-carrier liposome group cGAMP 5mg/kg/4 (3H) -quinazolinone analog-2, 0.5 mg/kg.
The administration was carried out 1 time per day for 21 days. After 21 days, mice were sacrificed and weighed for tumor weight, and tumor inhibition rate was calculated.
Statistical analysis
Data are expressed as x±s, treated with SPSS10.0 software, and the significance of each group of tumor weight differences was compared using one-way ANOVA test with a significance level a=0.05.
Experimental results
The mice are inoculated with tumor cells subcutaneously for 8 days to prepare a successful subcutaneous transplantation tumor model, and the novel 4 (3H) -quinazolinone analog/cGAMP complex co-carrier liposome can obviously inhibit tumor growth, and the tumor weight after 21 days of administration is obviously lower than that of a negative control group. The tumors were weighed and analyzed for significance, and the tumor mass of the mice in the 5-group administration group was significantly reduced compared to the negative control group, wherein the tumor mass of the mice in the co-loaded liposome administration group was smaller than that of the single-loaded liposome administration group, indicating that the combination of the 4 (3H) -quinazolinone analog and the cGAMP has a synergistic effect in anti-tumor aspect. The tumor mass of the mice of the targeted co-carried liposome group is smaller than that of the mice of the non-targeted group, and obvious differences exist, which indicates that the targeted liposome can improve the tumor inhibiting effect.
The specific results are shown in the following Table
TABLE 1 inhibition of BalB/C murine colorectal cancer cell CT26 subcutaneous transplantation tumor by novel 4 (3H) -quinazolinone analog/cyclic dinucleotide cGAMP co-carrier liposomes (n=10, mean.+ -. SD)
Note that: * P <0.05vs negative control; * P <0.01vs negative control group.
TABLE 2 inhibition of BalB/C murine breast cancer 4T1 subcutaneous transplantation tumor by novel 4 (3H) -quinazolinone analog/cyclic dinucleotide cGAMP co-carrier liposomes (n=10, mean.+ -. SD)
Note that: * P <0.05vs negative control; * P <0.01vs negative control group.
Claims (7)
1. A4 (3H) -quinazolinone analog/cGAMP co-carried liposome complex is characterized in that the 4 (3H) -quinazolinone analog/cGAMP co-carried liposome complex consists of liposome, 4 (3H) -quinazolinone analog and cyclic dinucleotide cGAMP, wherein the liposome consists of soybean phospholipid HSPC, cholesterol CHOL, dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000DPPE-PEG 2000 Composition, the 4 (3H) -quinazolinone analog is 2- [ (5-methoxy- (imidazo [4, 5-B)]Pyridine) -2-thio) methyl]-7-fluoro-quinazolin-4 (3H) -one or 2- [ (4-methyl- (thiazolo [4, 5-B)]Pyridine) -2-thio) methyl]-7-fluoro-quinazolin-4 (3H) -one;
the preparation method of the 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome complex comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL, DPPE-PEG 2000 And 4 (3H) -quinazolinone analog are added into dichloromethane, mixed evenly, and the solvent is suspended by a rotary evaporator until the mixture forms a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, extruding the hydrated liposome in a liposome extruder to pass through a 100nm polycarbonate microporous filter membrane to form uniform single-chamber liposome, and then dialyzing to remove free ammonium sulfate;
step three: adding cyclic dinucleotide cGAMP into the dialyzed liposome solution, slightly stirring and incubating for 1h in a magnetic stirrer with an oil bath temperature of 60 ℃, and dialyzing the incubated liposome to remove free cGAMP;
step four: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
2. A folic acid targeted 4 (3H) -quinazolinone analog/cGAMP co-carried liposome complex is characterized in that the folic acid targeted 4 (3H) -quinazolinone analog/cGAMP co-carried liposome complex consists of folic acid targeted liposome, 4 (3H) -quinazolinone analog and cyclic dinucleotide cGAMP, and the liposome consists of soybean phospholipid HSPC, cholesterol CHOL, distearoyl phosphatidylethanolamine-polyethylene glycol 2000DSPE-PEG 2000 Dipalmitoyl phosphatidylethanolamine-polyethylene glycol-folic acid DSPE-PEG 2000 -FA composition, said 4 (3H) -quinazolinone analog being 2- [ (5-methoxy- (imidazo [4, 5-B)]Pyridine) -2-thio) methyl]-7-fluoro-quinazolin-4 (3H) -one or 2- [ (4-methyl- (thiazolo [4, 5-B)]Pyridine) -2-thio) methyl]-7-fluoro-quinazolin-4 (3H) -one; the preparation method of the folic acid targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome complex comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL, DSPE-PEG 2000 、DSPE-PEG 2000 -FA and 4 (3H) -quinazolinone analogs were added to dichloromethane, mixed well, and the solvent was suspended using a rotary evaporator until the mixture formed a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, extruding the hydrated liposome in a liposome extruder to pass through a 100nm polycarbonate microporous filter membrane to form uniform single-chamber liposome, and then dialyzing to remove free ammonium sulfate;
step three: adding cGAMP into the dialyzed liposome solution, slightly stirring and incubating for 1h in a magnetic stirrer with an oil bath temperature of 60 ℃, and dialyzing the incubated liposome to remove free cGAMP;
step four: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
3. An anti-PD-L1 antibody targeted 4 (3H) -quinazolinone analog/cGAMP co-carrier liposome complex, which is characterized in that the anti-PD-L1 targeted 4 (3H) -quinazolinone analog/cGAMP co-carrier liposome complexThe body complex consists of an anti-PD-L1 antibody targeting liposome, a 4 (3H) -quinazolinone analogue and cGAMP, wherein the liposome consists of soybean phospholipid HSPC, cholesterol CHOL, dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000DPPE-PEG 2000 Dipalmitoyl phosphatidylethanolamine-polyethylene glycol-maleimide DPPE-PEG 2000 -MAL composition, said 4 (3H) -quinazolinone analog being 2- [ (5-methoxy- (imidazo [4, 5-B)]Pyridine) -2-thio) methyl]-7-fluoro-quinazolin-4 (3H) -one or 2- [ (4-methyl- (thiazolo [4, 5-B)]Pyridine) -2-thio) methyl]-7-fluoro-quinazolin-4 (3H) -one;
the preparation method of the anti-PD-L1 targeted 4 (3H) -quinazolinone analogue/cGAMP co-carrier liposome complex comprises the following steps:
step one: soybean phospholipid, cholesterol CHOL, DPPE-PEG 2000 、DPPE-PEG 2000 -MAL and 4 (3H) -quinazolinone analogs are added to dichloromethane, mixed well, and the solvent is suspended using a rotary evaporator until the mixture forms a uniform film;
step two: adding 250mM ammonium sulfate solution for hydration, allowing the hydrated liposome to pass through 100nm polycarbonate microporous membrane under extrusion of liposome extruder to form uniform single-chamber liposome, and dialyzing to remove free ammonium sulfate;
step three: adding cGAMP into the dialyzed liposome solution, and incubating for 1h with gentle stirring in a magnetic stirrer with an oil bath temperature of 60 ℃;
step four: adding thiolated anti-PD-L1 and a reducer sodium dithionite solution into the incubated liposome containing the cGAMP, incubating overnight at room temperature in a dark place, and dialyzing the incubated liposome;
step five: adding 5-10% of freeze-drying protective agent which is mannitol, sucrose, trehalose or maltodextrin to obtain freeze-dried powder.
4. Use of the 4 (3H) -quinazolinone analog/cGAMP co-loaded liposome complex of claim 1 in the preparation of an antitumor drug.
5. Use of the folate-targeted 4 (3H) -quinazolinone analog/cGAMP co-loaded liposome complex of claim 2 in the preparation of an antitumor drug.
6. Use of the anti-PD-L1 antibody-targeted 4 (3H) -quinazolinone analog/cGAMP co-loaded liposome complex of claim 3 in the preparation of an anti-tumor medicament.
7. A medicament comprising a 4 (3H) -quinazolinone analog/cGAMP co-encapsulation liposome complex of claim 1 or a folic acid-targeted 4 (3H) -quinazolinone analog/cGAMP co-encapsulation liposome complex of claim 2 or an anti-PD-L1 antibody-targeted 4 (3H) -quinazolinone analog/cGAMP co-encapsulation liposome complex of claim 3.
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| PCT/CN2022/089281 WO2022233255A1 (en) | 2021-05-01 | 2022-04-26 | Preparation and composition of novel 4(3h)-quinazolinone analogue/cyclic dinucleotide cgamp co-loaded liposome and application thereof in antitumor drugs |
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