Preparation method and application of self-assembled conjugate of non-steroidal anti-inflammatory drug and irinotecan

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of a self-assembled conjugate of a non-steroidal anti-inflammatory drug and irinotecan. Water-insoluble non-steroidal anti-inflammatory drugs (ibuprofen, oxaprozin, naproxen, indometacin and the like) are introduced into a water-soluble anti-tumor drug irinotecan through esterification reaction and are self-assembled into nano particles in an aqueous solution, and then the self-assembled nano conjugate with an anti-tumor effect is formed. The inhibition activity of the nano particles prepared by the method on tumor cells is far better than that of irinotecan and a corresponding physical mixture of a non-steroidal anti-inflammatory drug and irinotecan, cancer cell proliferation can be accurately blocked by synergistically inhibiting protein expression of COX-2 and Topo I, particularly, the inhibition effect on Topo I is better, the anti-tumor treatment effect is greatly improved, and the nano particles have good application prospects. And a more efficient scheme is provided for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a preparation method and application of a self-assembled conjugate of a non-steroidal anti-inflammatory drug and irinotecan. Background Art

[0002] Cancer is a major global public health issue in the 21st century and has long been one of the leading causes of death worldwide. Chemotherapy, which kills rapidly dividing cells by interfering with cell proliferation through cytotoxic compounds, remains an effective treatment for many cancers due to its systemic therapeutic effects. However, due to the lack of tumor selectivity of cytotoxic drugs, systemic administration often affects both cancer cells and healthy organs, leading to serious side effects, treatment interruptions, and limited efficacy.

[0003] In recent years, nanoparticle drug delivery systems have emerged as an important strategy to overcome the shortcomings of traditional chemotherapy, demonstrating numerous advantages, including increased drug solubility, enhanced tumor targeting, improved pharmacokinetic properties, and optimized drug release patterns. Nanoparticle drug delivery systems can load anticancer drugs into nanoparticles, such as liposomes, polymers, or inorganic materials, through chemical conjugation or physical encapsulation, and then deliver them to solid tumors. Compared to free drugs, these nanocarriers can significantly enhance antitumor efficacy and mitigate toxic side effects on normal tissues. However, most nanocarriers lack inherent therapeutic activity and possess low drug loading capacity. Furthermore, some carriers may cause severe inflammatory reactions, such as nephrotoxicity and hepatotoxicity, during degradation, metabolism, and excretion. Studies have shown that amphiphilic drug-drug conjugates can self-assemble into nanoparticles in aqueous environments without the need for a carrier. This strategy circumvents the issue of carrier toxicity while offering the advantages of nanoscale size and a fixed drug-drug ratio.

[0004] Colorectal cancer is one of the most common malignant tumors in the world, and its morbidity and mortality rates are among the highest among all cancer types. Faced with the increasingly severe morbidity, the medical community urgently needs to find highly effective and low-toxic treatment options. Although surgery is the main clinical treatment, patients in the middle and late stages often require combined radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, these therapies currently still have limitations such as bone marrow suppression, radiation damage, and heart, liver, and kidney dysfunction. Among them, the chemotherapy regimen of irinotecan combined with 5-fluorouracil, folinic acid, or oxaliplatin remains an important treatment option, but drug resistance and adverse reactions still seriously restrict its clinical efficacy.

[0005] Inflammation is the body's immune response to infection or tissue damage. Its continued presence can indirectly promote the proliferation advantage of mutant cells, thereby inducing tumors. Studies have shown that inflammatory bowel disease of the large intestine is one of the main risk factors for colorectal cancer. A large number of preclinical and epidemiological studies have confirmed that the combination of nonsteroidal anti-inflammatory drugs (NSAIDs) with chemotherapy or immunotherapy can significantly improve the therapeutic effect of tumors and reduce adverse reactions. Therefore, if a combination regimen of NSAIDs and irinotecan can be developed, it is expected to improve the treatment effect of colorectal cancer. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned existing technologies, water-insoluble non-steroidal anti-inflammatory drugs (ibuprofen, oxaprozin, naproxen, indomethacin, etc.) are introduced into the water-soluble anti-tumor drug irinotecan through an esterification reaction, and self-assembled into nanoparticles in an aqueous solution, thereby forming self-assembled nanoconjugates with anti-tumor effects, which are then used to prepare anti-tumor nanomedicines to improve the therapeutic effect of tumors.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a self-assembled conjugate of a non-steroidal anti-inflammatory drug and irinotecan, wherein the self-assembled conjugate includes compounds represented by formulas 6-9, which are self-assembled conjugates of oxaprozin, ibuprofen, naproxen, indomethacin and irinotecan, respectively:

[0009]

[0010] Preferably, the self-assembling conjugate is a compound represented by Formula 7, which is a self-assembling conjugate of ibuprofen and irinotecan:

[0011]

[0012] The second aspect of the present invention provides a method for preparing the self-assembled conjugate of the non-steroidal anti-inflammatory drug and irinotecan described in the first aspect, specifically comprising: dissolving the non-steroidal anti-inflammatory drugs oxaprozin, ibuprofen, naproxen, and indomethacin in anhydrous dichloromethane, then adding dicyclohexylcarbodiimide, and then adding irinotecan and 4-dimethylaminopyridine after the reaction. After the reaction is complete, filtering to remove the white solid, and then purifying the resulting crude product by silica gel column chromatography to obtain the target conjugate 6-9.

[0013] The structural formulas of the nonsteroidal anti-inflammatory drugs oxaprozin, ibuprofen, naproxen, and indomethacin are shown in Formulas 1-4, respectively, and the structural formula of irinotecan is shown in Formula 5:

[0014]

[0015] Among them, compounds 1-4 are non-steroidal anti-inflammatory drugs, namely oxaprozin, ibuprofen, naproxen and indomethacin, which exert anti-inflammatory effects by inhibiting prostaglandin cyclooxygenase (COX); compound 5 is the anti-tumor drug irinotecan, a semi-synthetic water-soluble camptothecin derivative, approved by the FDA for the treatment of various solid tumors. It can act on DNA topoisomerase I, prevent DNA replication and cause DNA breakage.

[0016] Preferably, the method further comprises the step of preparing the self-assembled conjugate into nanoparticles: dissolving conjugates 6-9 in DMSO respectively, stirring, adding the solution to water, continuing to stir and mix, and then dialyzing the resulting solution with water for 20-30 hours. After dialysis, the target nanoparticles are obtained by drying.

[0017] Preferably, after adding dicyclohexylcarbodiimide, the reaction is stirred at 0° C. for 8-15 minutes.

[0018] Preferably, after adding irinotecan and 4-dimethylaminopyridine, stirring is carried out at room temperature for 4-10 hours.

[0019] Preferably, the molar ratio of the nonsteroidal anti-inflammatory drug to irinotecan, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1:1:2-3:2-3.

[0020] More preferably, the molecular weight cut-off of the dialysis is 900-1500 g / mol.

[0021] More preferably, the dialysis time is 23-25 ​​hours, and the dialysis water is replaced at 1, 2, 3, 5, 7, 10, 14, and 20 hours, respectively.

[0022] More preferably, the concentration of the conjugate in DMSO is 7-15 mg / 3-8 mL, and the volume ratio of DMSO to water is 1:2.

[0023] More preferably, the conjugate is dissolved in DMSO and stirred at room temperature for 3-10 minutes; the conjugate solution is added to water and stirred for 20-60 minutes to mix.

[0024] The third aspect of the present invention provides use of the self-assembled conjugate of the nonsteroidal anti-inflammatory drug and irinotecan described in the first aspect in the preparation of an anticancer drug.

[0025] Preferably, the anticancer drugs include but are not limited to anti-colorectal cancer drugs.

[0026] The fourth aspect of the present invention provides use of the self-assembled conjugate of the nonsteroidal anti-inflammatory drug described in the first aspect and irinotecan in the preparation of a drug for inhibiting tumor cell proliferation.

[0027] Preferably, the tumor cells include but are not limited to colorectal cancer cells HT-29 and HCT-116.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention discloses a method for preparing a self-assembled conjugate of a nonsteroidal anti-inflammatory drug (NSAID) and irinotecan. Water-insoluble NSAIDs (ibuprofen, oxaprozin, naproxen, indomethacin, etc.) are introduced into the water-soluble anti-tumor drug irinotecan through an esterification reaction. The conjugate self-assembles into nanoparticles in an aqueous solution, thereby forming a self-assembled nanoconjugate with anti-tumor activity. The nanoparticles prepared using the present method have tumor cell inhibitory activity far exceeding that of irinotecan and the corresponding physical mixtures of NSAIDs and irinotecan. They can synergistically inhibit the expression of COX-2 and Topo I proteins, precisely blocking cancer cell proliferation (particularly with a superior inhibitory effect on Topo I), significantly improving the anti-tumor therapeutic effect and providing a more efficient solution for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the self-assembly of the conjugate of nonsteroidal anti-inflammatory drugs and irinotecan into nanoparticles;

[0031] Figure 2 Dynamic light scattering and transmission electron microscopy characterization images of nanoparticle 7;

[0032] Figure 3 The inhibitory effect of nanoparticle 7 on COX-2 and Topo I proteins in HT-29 cells;

[0033] Figure 4 This is a photo of a solid tumor after nanoparticle 7 was used to treat HT-29 nude mouse xenograft tumors;

[0034] Figure 5 The effect of nanoparticle 7 on the growth volume change of HT-29 nude mouse xenograft tumor;

[0035] Figure 6 The inhibitory effect of nanoparticle 7 on the growth of HT-29 nude mouse xenograft tumors;

[0036] Figure 7 This is the effect of nanoparticle 7 on the body weight of nude mice bearing HT-29 xenograft tumors. DETAILED DESCRIPTION

[0037] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] The present invention provides a method for preparing a self-assembled conjugate of a non-steroidal anti-inflammatory drug and irinotecan, wherein a water-insoluble non-steroidal anti-inflammatory drug (ibuprofen, oxaprozin, naproxen, indomethacin, etc.) is introduced into a water-soluble anti-tumor drug irinotecan through an esterification reaction, and the drug self-assembles into nanoparticles in an aqueous solution, thereby forming a self-assembled nanoconjugate with anti-tumor effect ( Figure 1 As shown in the following formula, conjugates 6, 7, 8, and 9 are conjugates of irinotecan with ibuprofen, oxaprozin, naproxen, and indomethacin, respectively.

[0040]

[0041] Furthermore, conjugate 6-9 was dissolved in DMSO and stirred at room temperature. The solution was then slowly added dropwise to stirred deionized water and stirred for 20-60 minutes. The resulting solution was then dialyzed against deionized water for 20-30 hours, with the dialyzed water replaced during the period. The dialyzed aqueous solution was freeze-dried to obtain the target nanoparticles 6-9, which were then used to prepare anti-tumor nanomedicines, yielding the following research results:

[0042] (1) In vitro antitumor activity

[0043] The MTT assay was used to evaluate the inhibitory effect of nanoparticles (6-9) on the proliferation of human colorectal cancer cells HT-29, HCT-116 and normal liver cells LO2. The study found that almost all nanoparticles were more cytotoxic than irinotecan and NSAID / irinotecan mixtures on the tested cells. For HT-29 cells, all nanoparticles (IC 50 =1.15~5.82μM) had a better proliferation inhibitory effect than irinotecan (IC 50 =6.45 μM). Among them, nanoparticle 7 showed the strongest activity (IC 50 =1.15 μM), which is 4.5 times more potent than the ibuprofen / irinotecan mixture. Except for nanoparticle 7, the antitumor activity of the remaining nanoparticles was significantly better than that of the corresponding physical mixture. For HCT-116 cells, all nanoparticles (IC 50=6.56~16.62μM) were higher than irinotecan (IC 50 =28.62μM), and all of them were better than their physical mixtures. In LO2 normal cells, except for nanoparticle 8, the IC 50 The values ​​(18.78-30.46 μM) were higher than those of irinotecan (IC 50 =19.80 μM). In summary, all nanoparticles (especially nanoparticle 7) showed the strongest cytotoxicity against HT-29 cells, but had weaker cytotoxicity against LO2 cells.

[0044] (2) Nanoparticle 7 inhibits the expression of COX-2 and Topo I proteins in HT-29 cells

[0045] Nanoparticle 7 significantly inhibited COX-2 protein expression, with an effect comparable to that of the ibuprofen / irinotecan mixture. Furthermore, compared with the control group, nanoparticle 7, irinotecan, and the ibuprofen / irinotecan mixture all significantly inhibited Topo I expression, but nanoparticle 7 had a more potent inhibitory effect on Topo I, significantly surpassing both irinotecan and the ibuprofen / irinotecan mixture. These results suggest that nanoparticle 7 can block HT-29 cell proliferation by synergistically inhibiting COX-2 and Topo I protein expression.

[0046] (3) In vivo antitumor activity

[0047] The tumor volume of mice treated with nanoparticle 7 was significantly smaller than that of mice treated with irinotecan or ibuprofen / irinotecan mixture, indicating that nanoparticle 7 has stronger anti-tumor ability in vivo. The efficacy of the nanoparticle 7 treatment group was significantly better than that of the irinotecan and ibuprofen / irinotecan mixture treatment groups, but there was no significant difference in efficacy between the latter two groups. These observations are consistent with the results of in vitro evaluation. The results showed that the tumor inhibition rate of the nanoparticle 7 treatment group was 55.3%, which was significantly higher than that of the irinotecan group (38.6%) and the ibuprofen / irinotecan mixture group (41.2%). These results further demonstrate that the therapeutic effect of nanoparticle 7 is better than that of irinotecan ibuprofen / irinotecan mixture. In addition, under the current treatment regimen, nanoparticle 7 treatment was well tolerated and did not cause significant weight loss in mice.

[0048] In order to comprehensively and clearly present the technical solutions and significant advantages of the present invention, the present invention is described in detail below in conjunction with specific embodiments.

[0049] Example 1: Preparation of Conjugate 6

[0050] 0.5 mmol of oxaprozin (Compound 1) was dissolved in anhydrous dichloromethane (10 mL), followed by the addition of dicyclohexylcarbodiimide (DCC, 1.0 mmol) and stirring at 0°C for 10 minutes. Irinotecan (0.5 mmol; Compound 5) and 4-dimethylaminopyridine (DMAP, 1.0 mmol) were then added. The reaction mixture was stirred at room temperature for 6 hours, after which the white solid (dicyclohexylurea) was removed by filtration. The crude product was purified by silica gel column chromatography to obtain the target conjugate 6 (Compound 6) in a 54.5% yield.

[0051]

[0052] The H-NMR and C-NMR spectra of the obtained conjugate 6 are as follows: 1 H NMR (600MHz, CDCl3) δ8.14(d,J=9.2Hz,1H),7.85(d,J=2.4Hz,1H),7.56–7.53(m,3H),7.49–7.47(m,2H),7.28–7.26(m ,2H),7.25–7.23(m,3H),7.18(s,1H),5.70(d,J=17.0Hz,1H),5.42(d,J=17.0Hz,1H),5.23–5.12(m,2H),4.58(d,J=11. 5Hz,1H),4.51(d,J=11.9Hz,1H),3.56(s,2H),3.34(s,1H),3.23–3.12(m,8H),2.96(s,1H),2.78(s,2H),2.51(s,1H), 2.41(s,2H),2.35–2.28(m,2H),2.19–2.13(m,1H),2.02–1.91(m,6H),1.39(t,J=7.2Hz,3H),0.97(t,J=7.5Hz,3H)ppm. 13C NMR (150MHz, CDCl3) δ170.98,167.50,161.27,157.39,152.95,151.61,150.08,147.26,146.88,1 45.76,145.59,145.40,135.15,132.38,131.89,128.87,128.61,128.51,128.48,128.04,127.88 ,127.57,127.21,126.45,125.65,120.18,114.75,95.94,76.28,67.22,63.77,50.72,49.34,43. 44,43.03,31.92,31.59,30.94,30.21,29.77,26.56,25.69,23.23,22.83,22.50,14.10,7.64ppm.

[0053] The high-resolution mass spectrometry results of the obtained conjugate 6 are as follows: HR-MS (m / z) (ESI): [M+H] + :862.3771.

[0054] Example 2: Preparation of Conjugate 7

[0055] 0.5 mmol of ibuprofen (Compound 2) was dissolved in anhydrous dichloromethane (10 mL), followed by the addition of dicyclohexylcarbodiimide (DCC, 1.0 mmol) and stirring at 0°C for 10 minutes. Irinotecan (0.5 mmol) and 4-dimethylaminopyridine (DMAP, 1.0 mmol) were then added. The reaction mixture was stirred at room temperature for 6 hours, after which the white solid (dicyclohexylurea) was removed by filtration. The crude product was purified by silica gel column chromatography to obtain the target conjugate 7 (Compound 7) in a 53.2% yield.

[0056]

[0057] The H-NMR and C-NMR spectra of the obtained conjugate 7 are as follows: 1H NMR (600MHz, CDCl3) δ8.22(d,J=9.1Hz,1H),7.86(d,J=2.4Hz,1H),7.61(dd,J=9.1,2.3Hz,1H),7.18(d,J=8.0Hz,2H),7.10(d,J=8.1Hz,2H),6.85 (s,1H),5.66(d,J=17.0Hz,1H),5.40(d,J=17.0Hz,1H),5.21(d,J=1.9Hz,2H),4.60(d,J=11.9Hz,1H),4.52(d,J=12.0Hz,1H),3.90(q,J=7.2Hz,1 H),3.57(s,2H),3.35(s,1H),3.18–3.13(m,3H),2.97(t,J=11.9Hz,1H), 2.87–2.76(m,2H),2.53(s,1H),2.45–2.33(m,5H),2.26–2.22(m,1H),2. 11–2.08(m,1H),2.02–1.86(m,6H),1.76–1.72(m,1H),1.50(d,J=7.2Hz, 3H),1.40(t,J=7.7Hz,3H),0.90(t,J=7.5Hz,3H),0.79–0.78(m,6H)ppm. 13 C NMR (150MHz, CDCl3) δ173.66,167.76,157.40,152.97,151.77,150.04,147.38,146.60, 145.70,145.26,140.72,136.78,131.89,129.66,127.53,127.36,127.17,125.54,120.0 8,114.83,96.18,75.90,67.25,63.76,49.29,45.20,44.54,43.43,43.04,31.84,30.16, 29.76,29.43,26.59,25.70,23.22,22.83,22.52,22.42,22.39,18.32,14.12,7.54ppm..

[0058] The high-resolution mass spectrometry results of the obtained conjugate 7 are as follows: HR-MS (m / z) (ESI): [M+H] + :775.4032.

[0059] Example 3: Preparation of Conjugate 8

[0060] 0.5 mmol of naproxen (Compound 3) was dissolved in anhydrous dichloromethane (10 mL), followed by the addition of dicyclohexylcarbodiimide (DCC, 1.0 mmol) and stirring at 0°C for 10 minutes. Irinotecan (0.5 mmol) and 4-dimethylaminopyridine (DMAP, 1.0 mmol) were then added. The reaction mixture was stirred at room temperature for 6 hours, after which the white solid (dicyclohexylurea) was removed by filtration. The crude product was purified by silica gel column chromatography to obtain the target conjugate 8 (Compound 8) in a 57.8% yield.

[0061]

[0062] The H-NMR and C-NMR spectra of the obtained conjugate 8 are as follows: 1 H NMR (600MHz, CDCl3) δ7.93(d,J=9.1Hz,1H),7.83(d,J=1.9Hz,1H),7.61(d,J=8.5H z,1H),7.58(dd,J=9.1,2.4Hz,1H),7.53(s,1H),7.51(d,J=8.9Hz,1H),7.32(dd,J =8.4,1.6Hz,1H),6.87(dd,J=8.9,2.5Hz,1H),6.78(d,J=2.3Hz,1H),6.56(s,1H), 5.67(d,J=17.0Hz,1H),5.42(d,J=17.0Hz,1H),5.13(d,J=2.6Hz,2H),4.59(d,J=1 2.5Hz,1H),4.51(d,J=12.7Hz,1H),4.02(q,J=7.1Hz,1H),3.66(s,3H),3.34(t,J= 10.5Hz,1H),3.26–3.06(m,6H),2.97(d,J=12.2Hz,1H),2.50(d,J=10.2Hz,1H),2. 34–2.27(m,2H),2.21–2.08(m,4H),2.06–2.01(m,2H),1.93–1.87(m,2H),1.74–1. 62(m,2H),1.52(d,J=7.1Hz,3H),1.39(t,J=7.5Hz,3H),0.86(t,J=7.5Hz,3H)ppm. 13C NMR (150MHz, CDCl3) δ173.54,167.84,157.39,157.29,152.98,151.30,150.00,147.07,146.30,1 45.02,144.90,134.53,133.89,131.89,129.04,128.87,127.61,127.35,126.93,126.40,125.86 ,125.38,120.58,118.89,114.72,105.41,96.08,75.79,67.46,63.67,55.20,50.19,49.19,44.8 3,43.46,43.06,31.71,30.20,29.75,26.66,25.78,23.15,22.97,22.57,18.50,14.02,7.52ppm.

[0063] The high-resolution mass spectrometry results of the obtained conjugate 8 are as follows: HR-MS (m / z) (ESI): [M+H] + :799.3659.

[0064] Example 4: Preparation of Conjugate 9

[0065] 0.5 mmol of indomethacin (Compound 4) was dissolved in anhydrous dichloromethane (10 mL), followed by the addition of dicyclohexylcarbodiimide (DCC, 1.0 mmol) and stirring at 0°C for 10 minutes. Irinotecan (0.5 mmol) and 4-dimethylaminopyridine (DMAP, 1.0 mmol) were then added. The reaction mixture was stirred at room temperature for 6 hours, after which the white solid (dicyclohexylurea) was removed by filtration. The crude product was purified by silica gel column chromatography to obtain the target conjugate 9 (Compound 9) in a 49.2% yield.

[0066]

[0067] The H-NMR and C-NMR spectra of the obtained conjugate 9 are as follows: 1H NMR (600MHz, CDCl3) δ8.11(d,J=9.1Hz,1H),7.83(d,J=2.1Hz,1H),7.53(d, J=8.9Hz,1H),7.50(d,J=8.3Hz,2H),7.31(d,J=8.0Hz,2H),6.95(s,1H),6.9 1(d,J=2.4Hz,1H),6.68(d,J=9.0Hz,1H),6.49(dd,J=9.0,2.4Hz,1H),5.65( d,J=17.1Hz,1H),5.40(d,J=17.1Hz,1H),5.19(d,J=3.4Hz,2H),4.58(d,J=1 2.7Hz,1H),4.51(d,J=12.0Hz,1H),3.89(d,J=16.0Hz,1H),3.79(d,J=16.1H z,1H),3.71(s,3H),3.59–3.48(m,1H),3.33(s,1H),3.16–3.12(m,3H),2.98 –2.72(m,3H),2.55–2.50(m,1H),2.45(s,3H),2.39–2.27(m,4H),2.21–2.12 (m,2H),2.08–1.92(m,6H),1.39(t,J=7.7Hz,3H),0.94(t,J=7.5Hz,3H)ppm. 13 C NMR (150MHz, CDCl3) δ169.86,168.25,167.50,157.32,156.04,152.93,151.51,150.08,147.19,146.84 ,145.43,145.36,139.26,136.15,133.66,131.90,131.24,130.73,130.38,129.01,127.51,127.18,12 5.62,120.27,114.93,114.67,112.18,111.45,100.71,95.59,76.33,67.31,63.77,55.62,53.56,49.3 2,43.45,43.06,31.82,31.59,30.00,29.78,26.61,25.70,23.24,22.88,22.54,14.14,13.45,7.62ppm.

[0068] The high-resolution mass spectrometry results of the obtained conjugate 9 are as follows: HR-MS (m / z) (ESI): [M+H] + :926.3495.

[0069] Example 5: Preparation of Nanoparticles 6-9

[0070] Dissolve 10 mg of conjugate 6-9 in 4 mL of DMSO and stir at room temperature for 5 minutes. Subsequently, slowly add the solution dropwise to 8 mL of stirred deionized water and continue stirring for 30 minutes. The resulting solution is then dialyzed with deionized water (molecular weight cut-off = 1000 g / mol) for 24 hours, and the dialyzed water is replaced at 1, 2, 3, 5, 7, 10, 14, and 20 hours. The dialyzed aqueous solution is then freeze-dried to obtain the target nanoparticles 6-9. At the same time, the particle size and morphology of the nanoparticles are characterized by dynamic light scattering and transmission electron microscopy, respectively. Among them, the particle size and morphology of nanoparticle 7 are as follows: Figure 2 Dynamic light scattering (DLS) measurements indicate that nanoparticle 7 has an average hydrodynamic diameter of approximately 106.0 nm and a polydispersity index of 0.084. Transmission electron microscopy (TEM) images reveal that the average size of nanoparticle 7 is approximately 73.5 nm, which is smaller than the DLS measurement due to nanoparticle shrinkage during TEM sample preparation.

[0071] Example 6: In vitro cytotoxic activity test of nanoparticles 6-9

[0072] The anticancer activity and toxicity of nanoparticles 6-9 were investigated using human colon cancer cells (HT-29 and HCT-116) and normal human hepatocytes (LO2). The experimental design was as follows: untreated cells served as negative controls, while irinotecan, an oxaprozin / irinotecan mixture, an ibuprofen / irinotecan mixture, a naproxen / irinotecan mixture, and an indomethacin / irinotecan mixture served as positive controls. Nanoparticles 6-9 were first prepared in saline to a 2 mmol / L stock solution and subsequently diluted to a working concentration range of 1.25, 2.5, 5, 10, 20, 40, and 80 μmol / L using a serial dilution method in culture medium (McCoy's 5A supplemented with 10% FBS). Simultaneously, the mixture and irinotecan were treated using the same method.

[0073] Cells in logarithmic growth phase were plated at 5×10 4Cells were seeded at a density of 100 cells / mL in a 96-well plate and allowed to adhere for 12 hours under standard culture conditions. After adhesion was completed, 20 μL of compound solution of different concentrations was added to each well and the cells were placed in a 37°C incubator for further culture for 72 hours. Subsequently, 5 mg / mL of MTT reagent was added to each well and incubated for another 4 hours to form formazan crystals. 150 μL of DMSO was then added to each well to dissolve the crystals, and the absorbance was measured using a microplate reader at dual wavelengths (main wavelength 570 nm, reference wavelength 630 nm). Finally, a dose-response curve was generated based on the data from three repeated experiments, and nonlinear regression analysis was performed using SPSS software to calculate the half-maximal inhibitory concentration (IC 50 )value.

[0074] The experimental results are shown in Table 1. All nanoparticles (especially nanoparticle 7) showed the strongest cytotoxicity to HT-29 cells, but had weak cytotoxicity to LO2 cells.

[0075] Table 1 Cytotoxic effects of nanoparticles (6-9), NSAIDs and irinotecan mixtures, and irinotecan on HT-29, HCT-116, and LO2 cells

[0076]

[0077]

[0078] Example 7: Test of Nanoparticle 7 Inhibiting the Expression of COX-2 and Topo I Protein in HT-29 Cells

[0079] HT-29 cells were treated with 20 μM nanoparticle 7 at 37°C for 24 hours. Untreated cells were used as negative controls, and cells treated with 20 μM irinotecan or a mixture of ibuprofen and irinotecan were used as positive controls. After treatment, the cells were collected and precipitated by centrifugation. The precipitate was then washed twice with phosphate-buffered saline (PBS) at 4°C and resuspended in lysis buffer (dissolved). The cells were lysed on ice for 30 minutes and the clear supernatant was obtained by centrifugation (20,000 g, 4°C, 10 minutes). The protein concentration was then determined using the bicinchoninic acid (BCA) method, and equal amounts of protein samples were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation. After electrophoresis, the proteins were transferred to PVDF Hybond-P membranes (GE Healthcare) using standard methods. The membrane was then blocked with 5% skim milk in TBST buffer for 1 hour at room temperature, followed by incubation with a primary antibody (mouse anti-human Actin monoclonal antibody, Abcam) overnight at 4°C. The following day, the membrane was incubated with a peroxidase-conjugated secondary antibody (HRP-IgG, Abcam) for 2 hours at room temperature. After four TBST washes (20 minutes each), the membrane was developed using a chemiluminescent detection reagent (Thermo Fisher Scientific), and protein bands were recorded on X-ray film using standard darkroom development and fixing procedures.

[0080] The experimental results are shown in Figure 3 , it can be seen that nanoparticle 7 can block the proliferation of HT-29 cells by synergistically inhibiting the expression of COX-2 and Topo I proteins.

[0081] Example 8: In vivo anti-tumor activity test of nanoparticle 7

[0082] (1) Experimental method: The in vivo antitumor activity of nanoparticle 7 was evaluated using the HT-29 xenograft tumor model.

[0083] (2) Subjects: BALB / c nude mice, provided by Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. Laboratory Animal Production License: SCXK (Zhejiang) 2019-0004. Laboratory Animal Use License: SYXK (Suzhou) 2012-0004. Age: 6 weeks, weight: 22-25 g, sex: male, number of animals: 3 per group, totaling 12.

[0084] (3) Drugs and reagents: Nanoparticles 7 were dissolved in physiological saline, and irinotecan and ibuprofen / irinotecan mixture were dissolved in DMSO.

[0085] (4) Groups and dosing regimens are as follows:

[0086] Group 1: normal saline (blank control), once every 3 days for 3 weeks;

[0087] Group 2: ibuprofen / irinotecan mixture (2.7 + 7.6 mg / kg), once every 3 days for 3 weeks;

[0088] Group 3: irinotecan (7.6 mg / kg), once every 3 days for 3 weeks;

[0089] Group 4: nanoparticle 7 (10 mg / kg, equimolar to Ir), once every 3 days for 3 weeks;

[0090] The cultured human lung cancer HT-29 cell suspension was collected at a concentration of 5×10 6 Each nude mouse was inoculated with 0.1 mL of the solution subcutaneously on its back. The diameter of the transplanted tumor was measured with a vernier caliper every day. After 19 days of inoculation, the tumor grew to 100 mm. 3 At 4 hr, the animals were randomly divided into groups of 3. Simultaneously, each group of nude mice began receiving drug treatment (see Groups and Dosage Schedule for dosing). Tumor diameter was measured to dynamically observe the anti-tumor efficacy of the test samples. Following the experiment, the nude mice were sacrificed, and the tumor masses were surgically removed and weighed.

[0091] The formula for calculating tumor volume (TV) is: TV = 1 / 2ab 2 , where a and b represent the length and width respectively.

[0092] The relative tumor volume (RTV) was calculated based on the measurement results. The calculation formula is: RTV = V t / V0, where V0 is the tumor volume measured at the time of cage administration (i.e., d0), V t is the tumor volume at each measurement.

[0093] Evaluation index of antitumor activity: relative tumor proliferation rate T / C (%), calculated as follows:

[0094]

[0095] Among them, T RTV :RTV in treatment group; C RTV : Model group RTV.

[0096] Evaluation index of antitumor activity: tumor growth inhibition rate (%), calculated as follows:

[0097]

[0098] (5) Experimental results: The in vivo anti-tumor activity test results of nanoparticles 7 are as follows Figure 4 、 5 , 6, and 7.

[0099] like Figure 4 and 5 As shown in Figure 3, the tumor volume of mice treated with nanoparticle 7 was significantly smaller than that of mice treated with irinotecan or ibuprofen / irinotecan mixture, indicating that nanoparticle 7 has stronger anti-tumor ability in vivo. Figure 6 As shown in Figure 2, the tumor inhibition rate of the nanoparticle 7 treatment group was 55.3%, which was significantly higher than that of the irinotecan group (38.6%) and the ibuprofen / irinotecan mixture group (41.2%). Figure 7 As shown, there was no significant weight loss in mice after treatment, indicating that nanoparticle 7 treatment was well tolerated.

[0100] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A self-assembled conjugate of a nonsteroidal anti-inflammatory drug and irinotecan, characterized in that: The self-assembling conjugates include compounds shown in Formulas 6-9, which are self-assembling conjugates of oxaprozin, ibuprofen, naproxen, indomethacin and irinotecan:

2. The self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 1, characterized in that: The self-assembling conjugate is a compound shown in Formula 7, which is a self-assembling conjugate of ibuprofen and irinotecan:

3. The method for preparing the self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 1, characterized in that: The non-steroidal anti-inflammatory drugs oxaprozin, ibuprofen, naproxen, and indomethacin were dissolved in anhydrous dichloromethane, followed by the addition of dicyclohexylcarbodiimide. After the reaction, irinotecan and 4-dimethylaminopyridine were added. After the reaction was complete, the white solid was removed by filtration, and the resulting crude product was purified by silica gel column chromatography to obtain the target conjugate 6-9.

4. The method for preparing the self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 3, characterized in that: The method also includes the step of preparing the self-assembled conjugate into nanoparticles: dissolving conjugates 6-9 in DMSO respectively, stirring, adding the solution to water, continuing to stir and mix, and then dialyzing the resulting solution with water for 20-30 hours. After dialysis, the target nanoparticles are obtained by drying.

5. The method for preparing the self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 3, characterized in that: After adding dicyclohexylcarbodiimide, the reaction was stirred at 0°C for 8-15 minutes.

6. The method for preparing the self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 3, characterized in that: After adding irinotecan and 4-dimethylaminopyridine, the mixture was stirred at room temperature for 4-10 hours.

7. The method for preparing the self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 3, characterized in that: The molar ratio of the nonsteroidal anti-inflammatory drug to irinotecan, dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1:1:2-3:2-3.

8. The method for preparing the self-assembled conjugate of nonsteroidal anti-inflammatory drugs and irinotecan according to claim 4, characterized in that: The molecular weight cut-off of the dialysis is 900-1500 g / mol.

9. Use of the self-assembled conjugate of the nonsteroidal anti-inflammatory drug and irinotecan according to claim 1 in the preparation of anticancer drugs.

10. Use of the self-assembled conjugate of the nonsteroidal anti-inflammatory drug and irinotecan according to claim 1 in the preparation of a drug for inhibiting tumor cell proliferation.

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