Composition and application thereof in preparation of antitumor drugs
By preparing nano-scale pharmaceutical compositions, including comparitin A4 and albumin, the problem of the difficulty of comparitin A4 phosphate molecules to penetrate tumor tissue and toxicity to vascular endothelial cytosine, the tumor tissue penetration and immune cell activation are achieved, and the tumor treatment effect is improved.
Patent Information
- Application Number
- CN202311830504.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-07-08
AI Technical Summary
In the prior art, the conpritine A4 phosphate molecule has poor water solubility, is difficult to effectively penetrate tumor tissue, and is toxic to vascular endothelial cells, resulting in insignificant tumor treatment effect and cardiomyotoxicity, and pMMR-type solid tumors have serious resistance to immunotherapy.
A nano-scale pharmaceutical composition system is used, including a therapeutically effective amount of conpritine A4 compound and albumin, and is prepared into a nano-formula with an average particle size of 25 nm-400 nm for intravenous injection, which enhances tumor tissue penetration ability and activates anti-tumor activity of immune cells, and reduces damage to vascular endothelial cells.
Nano preparations can effectively penetrate tumor tissue, increase T cell infiltration, activate immune cell killing activity, have good tumor targeting and biosafety, significantly improve tumor treatment effect, and reduce vascular endothelial cytotoxicity.
Smart Images
Figure CN120267629A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medicine and relates to a nano-system of a pharmaceutical composition for anti-tumor immunotherapy. Specifically, the pharmaceutical composition promotes the killing and tissue infiltration of tumor cells by T cells by enhancing the expression of co-stimulatory signal molecules and chemokines in tumor cells, and overcomes the drug resistance of tumors to immunotherapy. Background Art
[0002] The incidence and mortality of cancer worldwide are still increasing rapidly. Immunotherapy regimens led by immune checkpoint inhibitors have made breakthrough progress in patients with solid tumors with deficient DNA mismatch repair (dMMR) and have been approved as first-line treatment for advanced metastatic patients. However, the vast majority of solid tumor patients have a tumor phenotype with perfect DNA mismatch repair (pMMR), have a low response to immunotherapy, and have a relatively serious drug resistance phenomenon. Therefore, there is an urgent clinical need for strategies that can overcome the drug resistance of pMMR solid tumor patients to immunotherapy.
[0003] Combretastatin is a natural product isolated from Combretum caffrum. The main structure of this class of compounds contains two benzene rings connected by a carbon bridge with a cis double bond. Among the multiple combretastatin compounds initially isolated, combretastatin A4 has the highest killing intensity against tumor cells. In its structure, the 3, 4, and 5 positions of one benzene ring are substituted by methoxy groups, and the 3rd and 4th positions of the other benzene ring are substituted by a hydroxyl group and a methoxy group, respectively. However, the water solubility of combretastatin A4 is poor. Researchers modified a phosphate group on the hydroxyl group substituted at the 3rd position of its benzene ring to obtain a hydrophilic combretastatin A4 phosphate molecule.
[0004] Molecules administered by systemic injection often need to cross the vascular barrier to reach the tumor tissue. However, water-soluble molecules have poor ability to cross cell membranes. Most of the drugs of combretastatin A4 phosphate molecule are blocked in endothelial cells after intravenous injection and cause serious toxicity to vascular endothelial cells. Clinical trial results show that combretastatin A4 phosphate molecule has not achieved significant efficacy in the field of tumor treatment and damages vascular endothelial cells, with strong cardiotoxicity. Summary of the Invention
[0005] In order to achieve the object of the present invention, that is, to address the current situation and problems of drug resistance of pMMR solid tumors to immunotherapy, the present invention provides a nano-system of a pharmaceutical composition for anti-tumor immunotherapy. The pharmaceutical composition specifically includes: a therapeutically effective amount of combretastatin A4 compound, a therapeutically effective amount of one or more immune checkpoint inhibitors, and albumin.
[0006] Among them, the drug composition nano-system can effectively penetrate the cell barrier to reach the tumor tissue, and will not damage the vascular endothelial cells. It can not only increase the number of T cells infiltrating in the solid tumor, but also effectively activate the anti-tumor killing activity of immune cells, and at the same time has excellent tumor targeting and biological safety.
[0007] Further, the drug composition nano-system is characterized in that the average particle size of the nano-preparation is 25 nm - 400 nm, preferably 100 nm - 250 nm.
[0008] Further, the drug composition nano-system is characterized in that it contains the compound Combretastatin A4.
[0009] Further, the drug composition nano-system is characterized in that the albumin includes but is not limited to serum albumin, ovalbumin, lactalbumin, myoalbumin, wheat albumin, soybean albumin and other types of albumin, or combinations thereof, preferably human serum albumin.
[0010] Further, in the drug composition nano-system, the molar ratio of albumin to the compound Combretastatin A4 is 1 to 1000, preferably the molar ratio is 10.
[0011] Further, the composition further comprises one or more excipients that have been applied in clinical trials or are already on the market.
[0012] Further, when the drug composition nano-system is applied, it can be made into an intravenous injection preparation or a freeze-dried powder injection.
[0013] Further, the drug composition nano-system can be used to treat a variety of cancers including but not limited to colon cancer, triple-negative breast cancer, malignant melanoma, non-small cell lung cancer, liver cancer, renal cell carcinoma, prostate cancer, ovarian cancer or gastric cancer.
[0014] The present invention has the following beneficial effects:
[0015] The drug composition nano-system of the present invention can effectively cross the vascular barrier during tumor treatment, reducing damage to vascular endothelial cells; it can effectively promote the secretion of co-stimulatory molecule CD80 and chemokine CXCL10 in tumor tissues, and significantly increase the infiltration of CD8 + T lymphocyte numbers.
[0016] The drug composition nano-system of the present invention has good tumor cell targeting, can specifically accumulate in tumor cells rather than endothelial cells, reducing the toxic and side effects on blood vessels.
[0017] The nanosystem of the pharmaceutical composition described in the present invention has a synergistic therapeutic effect, making its anti-tumor immune pharmacodynamic effect superior to the nanosystem formed by a single component.
[0018] The nanosystem of the pharmaceutical composition described in the present invention can activate the tumor immune system, has a good synergistic effect on tumor immunotherapy, and has great clinical research significance for malignant tumors lacking effective therapeutic drugs, especially pMMR solid tumors, providing a new idea for sensitizing existing immunotherapy in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 . Penetration pictures of HUVEC cell spheres of different nano-compositions.
[0021] Figure 2 . Statistical results of the penetration distance of HUVEC cell spheres of different nano-compositions.
[0022] Figure 3 . In vivo vascular endothelial cell penetration pictures of different nano-compositions.
[0023] Figure 4 . Statistical results of the penetration distance of in vivo vascular endothelial cells of different nano-compositions.
[0024] Figure 5 . Detection results of the toxicity of different nano-compositions to vascular endothelial cells.
[0025] Figure 6 . Detection results of the toxicity of different nano-compositions to vascular endothelial cells.
[0026] Figure 7 . Infiltration of T cells in tumor tissues by different nano-combinations of compositions.
[0027] Figure 8 . Anti-tumor pharmacodynamic effect of nano-composition combined with anti-CTLA-4 antibody. DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only described by way of example and do not mean any limitation to the present invention. Those skilled in the art can also make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application. It should be understood that the present invention intends to cover the variations and modifications included in the appended claims.
[0030] Example 1: Preparation schemes of nano-composition A-1 and nano-composition A-2
[0031] Measure an appropriate amount of the stock albumin solution, dilute the albumin solution (50 mg / mL) with double-distilled water and place it in an ultrasonic vial. Thoroughly premix it with a 1 mL pipette. During the water bath heating process, add 5 mg of combretastatin A4 dropwise and mix well. Place the obtained preparation in an ice-water bath and use a cell ultrasonic disruptor to ultrasonicate for 3 cycles (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, cool the preparation in a 4°C ice-water bath for 1 - 2 minutes. Finally, centrifuge the preparation at 3500 rpm for 3 min. After removing the supernatant, the prepared nano-composition A-1 is obtained.
[0032] Measure an appropriate amount of lecithin and 5 mg of combretastatin A4, dissolve them in chloroform and place them in a round-bottomed flask. After vacuum distillation, wash the inner wall of the flask with 1 mL of double-distilled water to elute the drug-lecithin mixture. Place the obtained preparation in an ice-water bath and use a cell ultrasonic disruptor to ultrasonicate for 3 cycles (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, cool the preparation in a 4°C ice-water bath for 1 - 2 minutes. Finally, centrifuge the sample at 3500 r.p.m. for 3 min. After removing the supernatant, the prepared nano-composition A-2 is obtained.
[0033] Example 2: Preparation protocols for nano-composition B-1 and nano-composition B-2
[0034] Measure an appropriate amount of the stock albumin solution, dilute the albumin solution (50 mg / mL) with double-distilled water and place it in an ultrasonic vial. Thoroughly premix it with a 1 mL pipette. During the water bath heating process, add 0.5 mg of combretastatin A4 dropwise and mix well. Place the obtained preparation in an ice-water bath and use a cell ultrasonic disruptor to ultrasonicate for 3 cycles (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, cool the preparation in a 4°C ice-water bath for 1 - 2 minutes. Finally, centrifuge the preparation at 3500 rpm for 3 min. After removing the supernatant, the prepared nano-composition B-1 is obtained.
[0035] Measure an appropriate amount of lecithin and 0.5 mg of combretastatin A4, dissolve them in chloroform and place them in a round-bottomed flask. After vacuum distillation, wash the inner wall of the flask with 1 mL of double-distilled water to elute the drug-lecithin mixture. Place the obtained preparation in an ice-water bath and use a cell ultrasonic disruptor to ultrasonicate for 3 cycles (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, cool the preparation in a 4°C ice-water bath for 1 - 2 minutes. Finally, centrifuge the sample at 3500 r.p.m. for 3 min. After removing the supernatant, the prepared nano-composition B-2 is obtained.
[0036] Example 3: Preparation protocols for nano-composition C-1 and nano-composition C-2
[0037] Measure an appropriate amount of the stock albumin solution, dilute the albumin solution (50 mg / mL) with double-distilled water and place it in an ultrasonic vial. Use a 1 mL pipette to premix it thoroughly. During the water bath heating process, add 0.25 mg of combretastatin A4 dropwise and mix well. Place the above-prepared preparation in an ice-water bath, and use a cell ultrasonic disruptor to perform 3 cycles of ultrasonication (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, place the preparation in a 4°C ice-water bath to cool for 1 - 2 minutes. Finally, centrifuge the preparation at 3500 rpm for 3 min. After removing the supernatant, the prepared nano-composition C-1 is obtained.
[0038] Measure an appropriate amount of lecithin and 0.25 mg of combretastatin A4, dissolve them with chloroform and place them in a round-bottom flask. After vacuum distillation, use 1 mL of double-distilled water to wash the drug and lecithin mixture on the inner wall of the flask. Place the above-prepared preparation in an ice-water bath, and use a cell ultrasonic disruptor to perform 3 cycles of ultrasonication (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, place the preparation in a 4°C ice-water bath to cool for 1 - 2 minutes. Finally, centrifuge the sample at 3500 r.p.m. for 3 min. After removing the supernatant, the prepared nano-composition C-2 is obtained.
[0039] Example 4: Preparation protocols for nano-compositions D-1 and D-2
[0040] Measure an appropriate amount of the stock albumin solution, dilute the albumin solution (50 mg / mL) with double-distilled water and place it in an ultrasonic vial. Use a 1 mL pipette to premix it thoroughly. During the water bath heating process, add 0.1 mg of combretastatin A4 dropwise and mix well. Place the above-prepared preparation in an ice-water bath, and use a cell ultrasonic disruptor to perform 3 cycles of ultrasonication (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, place the preparation in a 4°C ice-water bath to cool for 1 - 2 minutes. Finally, centrifuge the preparation at 3500 rpm for 3 min. After removing the supernatant, the prepared nano-composition C-1 is obtained.
[0041] Measure an appropriate amount of lecithin and 0.1 mg of combretastatin A4, dissolve them with chloroform and place them in a round-bottom flask. After vacuum distillation, use 1 mL of double-distilled water to wash the drug and lecithin mixture on the inner wall of the flask. Place the above-prepared preparation in an ice-water bath, and use a cell ultrasonic disruptor to perform 3 cycles of ultrasonication (ultrasonic power 300 W), 5 minutes for each cycle. After each ultrasonication, place the preparation in a 4°C ice-water bath to cool for 1 - 2 minutes. Finally, centrifuge the sample at 3500 r.p.m. for 3 min. After removing the supernatant, the prepared nano-composition C-2 is obtained.
[0042] Example 5: In vitro detection of the ability of nano-compositions A-D to penetrate vascular endothelial cells
[0043] The vascular endothelial cells HUVEC were cultured in a low-attachment 96-well plate to form cell spheres. After staining the nano-composition with the fluorescent dye DiO and adding it to the cell spheres, after co-culturing for 24 h, a fluorescence microscope was used to detect the fluorescence intensity and penetration depth in different cell spheres to judge the ability of the nano-composition to penetrate vascular endothelial cells, as shown in Figure 1 and 2 . The experimental results showed that the nano-compositions A1, B1, C1, and D1 could effectively penetrate vascular endothelial cells, while the nano-compositions A2, B2, C2, and D2 could not effectively penetrate vascular endothelial cells. This indicated that the nano-compositions of albumin and combretastatin A4 could effectively penetrate vascular endothelial cells, while the compositions prepared with nano-lecithin did not possess this ability.
[0044] Example 6: In vivo detection of the ability of nano-compositions A-D to penetrate vascular endothelial cells
[0045] Female Balb / c mice weighing about 18 - 22 g were selected to establish a subcutaneous tumor-bearing model of ectopic colon cancer (CT26), and the nano-composition stained with the fluorescent dye DiO was used to trace its distribution in tumor-bearing mice. One week after tumor-bearing in mice, the nano-compositions A-D loaded with DiO (0.1 mL / 10 g) were injected via the tail vein. 12 h after administration, the tumor-bearing mice were anesthetized with 2% sodium pentobarbital solution and then sacrificed. The vascular endothelial cells of the tumor tissue were stained and labeled with a fluorescently labeled anti-CD31 antibody, and a laser confocal microscope was used to measure the diffusion distance of the nanoparticles near the endothelial cells, as shown in Figure 3 and 4 . The experimental results showed that the nano-compositions A1, B1, C1, and D1 could effectively penetrate the vascular endothelial cells in the tumor tissue of mice, while the nano-compositions A2, B2, C2, and D2 could not effectively penetrate the blood vessels. This further indicated that the nano-compositions of albumin and combretastatin A4 could effectively penetrate the vascular endothelial cells in vivo, while the nano-compositions prepared with lecithin did not possess this ability.
[0046] Example 7: In vitro detection of the toxicity of nano-compositions A-D to vascular endothelial cells
[0047] The vascular endothelial cells HUVEC were seeded in a 96-well plate at a density of 1×10 4 / mL. After the cells adhered to the wall (about 24 h), the compositions A-D and the combretastatin A4 phosphate molecule corresponding to the dose of combretastatin A4 were added. After co-culturing with the drugs for 24 h, 10 μL of CCK8 solution was added to each well and incubated at 37 °C for 0.5 h, and the absorbance value was measured at a wavelength of 450 nm. Using the untreated cells as a reference, the cell survival rate was calculated, as shown in Figure 5。The experimental results showed that Combretastatin A4 phosphate molecules and nano-combinations A2, B2, C2, and D2 at equal doses had obvious toxicity to endothelial cells, while nano-combinations A1, B1, C1, and D1 had no obvious toxic side effects on HUVEC cells. This indicates that the nano-combination of albumin and Combretastatin A4 can effectively penetrate vascular endothelial cells without damaging them, while the nano-combinations prepared from Combretastatin A4 phosphate molecules and lecithin do not have this ability.
[0048] Example 8: In vivo detection of the toxicity of nano-combinations A-D to vascular endothelial cells
[0049] Female Balb / c mice weighing about 18 - 22 g were selected to establish a subcutaneous tumor-bearing model of ectopic colon cancer (CT26). One week after tumor-bearing in the mice, Combinations A1 and A2 (20 mg / kg of Combretastatin A4) and Combretastatin A4 phosphate molecules corresponding to the dose of Combretastatin A4 were injected via the tail vein. Five days after administration, the tumor-bearing mice were anesthetized with 2% sodium pentobarbital solution and then sacrificed. The vascular endothelial cells of the tumor tissue were stained and labeled with a fluorescently labeled anti-CD31 antibody, and after photographing with a laser confocal microscope, quantification was performed, as shown in Figure 6 。The experimental results showed that, compared with the normal saline (control) group, Combretastatin A4 phosphate molecules and nano-combination A2 at equal doses had obvious toxicity to endothelial cells in the tumor tissue, while nano-combination A1 had no obvious vascular toxicity. This indicates that the nano-combination of albumin and Combretastatin A4 can effectively penetrate vascular endothelial cells without damaging them, while the nano-combinations prepared from Combretastatin A4 phosphate molecules and lecithin do not have this ability.
[0050] Example 9: Infiltration of T cells in tumor tissue by nano-combinations
[0051] Balb / c mice weighing about 18 - 22 g were selected. A lung metastasis model was constructed by injecting CT26 colon cancer cells via the tail vein, and frozen sections of the tumor tissue were made and fluorescently stained to detect the infiltration of T cells in the tumor. One week after tumor inoculation in the mice, they were randomly divided into three groups: a 10 mL / kg normal saline (control) group; 20 mg / kg nano-combinations A1 and A2. Seven days after administration, the tumor-bearing mice were anesthetized with 2% sodium pentobarbital solution and then sacrificed, and the tumor tissue of the tumor-bearing mice was isolated. The tumor tissue was made into 8-μm frozen section samples, the cell nuclei were labeled with DAPI, the infiltration of T cells was detected with an anti-CD8 antibody, and photographs were taken with a laser confocal microscope, as shown in Figure 7As shown. The results showed that the number of T cells in the lung metastases after treatment with composition A1 was significantly increased, while that with nano - composition A2 did not increase. This indicates that the nano - composition of albumin and combretastatin A4 can effectively enhance the infiltration of T cells in tumors, while the nano - composition prepared with lecithin does not have this ability.
[0052] Example 10: Antitumor efficacy of nano - composition combined with anti - CTLA - 4 antibody
[0053] Balb / c mice weighing about 18 - 22 g were selected and a lung metastasis model was established by injecting colon cancer CT26 cells via the tail vein. One week after tumor inoculation in the mice, they were randomly divided into six groups: ① 10 mL / kg normal saline (control) group; ②③ 20 mg / kg nano - composition A1 and A2; ④ 10 μg / mouse anti - CTLA - 4 antibody group; ⑤⑥ 20 mg / kg nano - composition A1 and A2 combined with anti - CTLA - 4 antibody group. The nano - compositions were administered on days 0, 3, and 6, and the anti - CTLA - 4 antibody was administered on days 1, 4, and 7. Two weeks after the last administration, the tumor - bearing mice were anesthetized with 2% sodium pentobarbital solution and sacrificed, and the lung tissues of the tumor - bearing mice were isolated. After picric acid staining, the number of lung metastases in each group was calculated. See Figure 8 . The results showed that compared with the single - treatment group, the number of lung metastases after treatment with nano - composition A1 combined with anti - CTLA - 4 antibody was significantly reduced, while that with nano - composition A2 was not reduced. This indicates that the nano - composition of albumin and combretastatin A4 can effectively enhance the intratumoral distribution of combretastatin A4 and produce a strong antitumor effect, while the nano - composition prepared with lecithin does not have this ability.
Claims
1. A nano-composition for overcoming pMMR solid tumors, characterized in that, (1) The active ingredients in the composition are Combretastatin A1 and Combretastatin A4 compounds; (2) The main carrier components in the composition are one or more combinations of serum albumin, ovalbumin, lactalbumin, myoalbumin, wheat albumin, soybean albumin, and other types of albumin; (3) Other excipients in the composition are one or more combinations of phospholipids, PLGA, PLA, etc.; (4) The average particle size of the composition preparation is 25 nm - 400 nm.
2. Method for preparing a nano-composition, characterized in that, It includes the following steps: (1) Dilute the albumin solution with double-distilled water to different concentrations; (2) Thoroughly mix Combretastatin A1 and Combretastatin A4 molecules at different concentrations with the albumin solution in step (1); (3) Ultrasonicate the mixture in step (2) at different powers using a cell ultrasonic disruptor, and after centrifugation, obtain the albumin nanocomposite containing Combretastatin A1 and Combretastatin A4.
3. The nano-composition according to claim 1, characterized in that, The anti-tumor drugs mentioned are Combretastatin A1, Combretastatin A4, etc.
4. The nano-composition according to claim 1, wherein, The composition carrier mentioned is serum albumin, ovalbumin, lactalbumin, myoalbumin, wheat albumin, soybean albumin, and other types of albumin, etc.
5. The nano-composition according to claim 1, characterized in that, Other excipients of the composition are phospholipids, PLGA, PLA, etc.
6. The nano-composition according to claim 1, wherein The average particle size of the composition preparation mentioned is 25nm - 400 nm.
7. The nano-composition according to claim 2, characterized in that, The albumin concentration is diluted with double-distilled water to 10 - 100 mg / mL.
8. The nano-composition according to claim 2, characterized in that, The dosages of Combretastatin A1 and Combretastatin A4 molecules added are 5 mg, 0.5 mg, 0.25 mg, 0.1 mg, etc.
9. The nano-composition according to claim 2, wherein The ultrasonic powers for preparing the nanocomposite mentioned are 80W, 100W, 200W, and 300W.