Imiquimod prodrugs, methods of making and using the same

Abstract

The application discloses an imiquimod prodrug and a preparation method and application thereof, and the structural formula of the imiquimod prodrug is shown in the following formula: the preparation method of the imiquimod prodrug comprises the following steps: step one, dissolving excessive imiquimod in anhydrous dioxane under a protective atmosphere, and dropping propargyl chloroformate dissolved in anhydrous dioxane under ice bath to obtain a mixture; step two, stirring the mixture obtained in the step one at 45-55 DEG C overnight; step three, filtering the product obtained in the step two, recovering excessive imiquimod, and purifying the filtrate after concentration and drying under reduced pressure to obtain the imiquimod prodrug. By using propargyloxy carbonyl to protect the amino group of the IMQ drug, the toxicity of the IMQ drug to cells is significantly reduced, and the systemic inflammatory response in mice is reduced; by using propargyloxy carbonyl to protect the amino group of the DOX drug, the toxicity of the DOX drug to cells is significantly reduced, and the cardiotoxicity in mice is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an imiquimod prodrug, its preparation method, and its application. Background Technology

[0002] Due to the cumbersome and costly process of producing personalized cancer cell vaccines in vitro, immunogenic cell death (ICD) has emerged as one of the most attractive strategies for cancer immunotherapy. This approach can activate the immune response in the tumor microenvironment through damage-associated molecular patterns (DAMPs) and specific tumor-associated antigens (TAAs).

[0003] Currently, several strategies exist for in-situ tumor vaccine preparation using ICDs and delivery of immune adjuvants. For example, some systems utilize various nanomaterials to prepare in-situ tumor vaccines, and some have even undergone clinical trials. However, significant challenges exist in the preclinical and clinical translational research of nanomaterials, as a common but crucial requirement is ensuring the safety of nanomaterials for patients. Some systems also utilize photodynamic therapy (PDT) or photothermal therapy (PTT) combined with immunomodulators for tumor immunotherapy, but their clinical translation is limited by thermal damage to normal tissues, as well as the short lifespan and short diffusion distance of photosensitizers. Radiotherapy (RT), while used to induce anti-tumor immunity, also faces the risk of significant irreversible and severe damage to normal tissues. Chemotherapy plays a dominant role in clinical trials by inducing potent immune responses through ICDs. However, the random biodistribution of systemic administration leads to significant toxic side effects on normal tissues, a major factor limiting its development. Furthermore, the use of attractive Toll-like receptor (TLR) agonists can activate TLR signaling to initiate innate immune responses, potentially enhancing the intensity of anti-tumor responses. However, agonists often cause severe systemic inflammation and chronic disease due to systemic diffusion or leakage after encapsulation. Therefore, developing effective, highly targeted, and biosafe in situ vaccine strategies is both urgent and challenging.

[0004] Bioorthogonal chemistry, capable of facilitating non-natural chemical transformations in complex biological environments, is a powerful tool for understanding and inquiring about biological processes. In recent years, transition metal catalysts (TMCs), including Pd, Au, Ru, Ir, and Cu, have emerged as outstanding candidates for bioorthogonal catalysts. Various metal complexes, metal nanoparticles, artificial enzymes, and removable devices have been reported to achieve excellent catalytic activity and therapeutic effects in both in vivo and in vitro environments. The development of different types of functional catalysts has enabled the in-situ synthesis of prodrugs in vivo, and the improved safety of prodrugs reduces the off-target toxicity of chemotherapeutic drugs. However, exploration of bioorthogonal catalysis in emerging immunotherapies is limited. Current reports have explored the use of nanomaterials or antibodies, but nanomaterials suffer from complex preparation and difficulties in controlling safety, stability, and targeting in vivo. Meanwhile, antibodies are limited in clinical application due to high cost and low efficacy. Summary of the Invention

[0005] Purpose of the Invention: In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide an imiquimod prodrug that enhances the immune stimulation effect. Another purpose of this invention is to provide a method for preparing an imiquimod prodrug. A further purpose of this invention is to provide a combination drug composition with tumor immunotherapy effects. Yet another purpose of this invention is to provide the application of an imiquimod prodrug or combination drug composition in tumor immunotherapy using orthogonal catalytic microneedles.

[0006] Technical solution: To achieve the above objectives, in a first aspect, the present invention provides an imiquimod prodrug, the structural formula of which is:

[0007]

[0008] Secondly, the present invention provides a method for preparing an imiquimod prodrug, comprising the following steps:

[0009] Step 1: Dissolve excess imiquimod in anhydrous dioxane under a protective atmosphere, and add propargyl chloroformic acid dissolved in anhydrous dioxane dropwise under an ice bath to obtain a mixture;

[0010] Step 2: Stir the mixture obtained in Step 1 at 45-55°C overnight;

[0011] Step 3: Filter the product obtained in step 2, recover the excess imiquimod, and purify the filtrate by column chromatography to obtain the imiquimod prodrug.

[0012] Further, in step one, the mass-to-volume ratio of imiquimod, anhydrous dioxane, and propargyl chloroformic acid is 300 mg: 40 mL: 0.2–0.3 mL. The protective atmosphere is nitrogen. Preferably, the mass-to-volume ratio of imiquimod, anhydrous dioxane, and propargyl chloroformic acid is 300 mg: 40 mL: 0.232 mL.

[0013] Further, in step three, the eluent for purification is a mixed solution of methanol and dichloromethane with a volume ratio of 1:9 to 10. Preferably, the volume ratio of methanol to dichloromethane is 1:10.

[0014] Thirdly, the present invention provides a combination drug composition with tumor immunomodulatory effects, the structural formula of which is as follows: The combination drug composition comprises imiquimod prodrug and doxorubicin prodrug or their solvates, polymorphs, isomers, or pharmaceutically acceptable salts, wherein the mass ratio of imiquimod prodrug to doxorubicin prodrug is 1-2:5, the mass ratio is 1:5 in mouse experiments, and the concentration ratio is 2:5 in cell experiments. The imiquimod prodrug is the above-mentioned imiquimod prodrug or its solvates, polymorphs, isomers, or pharmaceutically acceptable salts. The structural formula of the doxorubicin prodrug is:

[0015]

[0016] Furthermore, the preparation method of the doxorubicin prodrug includes the following steps:

[0017] S1. Dissolve doxorubicin and N-(propyneoxycarbonyl)succinimide in anhydrous DMF;

[0018] S2. Add N,N-diisopropylethylamine dissolved in DMF dropwise to the solution obtained in S1, and stir at room temperature for 4 to 5 hours;

[0019] S3. Ethyl acetate was added to the solution obtained in S2, and the solution was washed with HCl, saturated sodium bicarbonate, and saturated saline solution, respectively. The organic phase was collected, dried with anhydrous sodium sulfate, and the product was purified by column chromatography to obtain the doxorubicin prodrug.

[0020] Furthermore, in S1, the mass-to-volume ratio of doxorubicin, N-(propyneoxycarbonyl)succinimide, and anhydrous DMF is 464 mg: 158 mg: 5–10 mL.

[0021] Further, in step S3, the eluent for purification is a mixed solution of methanol and dichloromethane with a volume ratio of 1:9 to 10. Preferably, the volume ratio of methanol to dichloromethane is 1:10.

[0022] Fourthly, the present invention provides the use of the above-mentioned imiquimod prodrug or the above-mentioned combination drug composition in the preparation of tumor immunotherapy drugs using orthogonal catalytic microneedles.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0024] 1. The imiquimod prodrug P-IMQ involved in this invention, compared with the imiquimod original drug IMQ, significantly reduces its cytotoxicity by using propyne-oxycarbonyl to protect the amino group of the IMQ drug, and also reduces the systemic inflammatory response in mice.

[0025] 2. The doxorubicin prodrug P-DOX and doxorubicin technical drug DOX involved in this invention significantly reduce the cytotoxicity of DOX by protecting the amino group of DOX drug with propoxycarbonyl, and also reduce the cardiotoxicity in mice.

[0026] 3. The combined drug composition of P-IMQ and P-DOX involved in this invention, when combined with bioorthogonal catalytic microneedles for treatment, can effectively inhibit tumor growth in tumor models while inducing ICD, thereby inhibiting the growth of distant tumors and lung metastasis by inducing a tumor immune response.

[0027] 4. The preparation method of doxorubicin prodrug provided by the present invention is simple to operate, and the obtained doxorubicin prodrug has high chemical purity and high yield, with a yield of over 80%.

[0028] 5. The preparation method of the imiquimod prodrug provided by the present invention is simple to operate and can obtain imiquimod prodrug with high chemical purity in one step. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of in-situ bioorthogonal catalysis mediated by microneedle patches of prodrugs;

[0030] Figure 2 These are transmission electron microscope (TEM) images of TiO2 nanosheets (TNS) and Pd-deposited TiO2 nanosheets (Pd-TNS) of the present invention; (a) TEM image of TiO2 nanosheets, scale bar 100 nm, (b) TEM image of Pd-TNS, scale bar 100 nm, (cf) scanning TEM / energy elemental map of Pd-TNS, scale bar 20 nm.

[0031] Figure 3 The structure of the Pd-TNS loaded microneedle array patch (PT-MN) of the present invention is characterized as follows: (a) appearance of the Pd-TNS loaded microneedle array patch (PT-MN), (b) scanning electron microscope (SEM) image of PT-MN, scale bar 200 μm, (c) SEM image of a longitudinal section of a microneedle, scale bar 200 μm, and (d) SEM image of the distribution of Pd-TNS in the matrix of a longitudinal section of a microneedle, scale bar 4 μm.

[0032] Figure 4 This is a diagram illustrating the mechanical behavior of microneedles;

[0033] Figure 5 This is a schematic diagram of decageing non-fluorescent AC-Rho 110 or P-Rho 110 into fluorescent Rho 110;

[0034] Figure 6 These are confocal laser microscope images of fluorescence changes in B16-F10 cells mediated by PT-MN uncoiling of P-Rho 110. (a) Fluorescence changes in B16-F10 cells treated with a combination of P-Rho 110 and PT-MN at different time points; (b) Quantitative fluorescence plot of (a); (c) Fluorescence changes in B16-F10 cells treated with P-Rho 110 alone at different time points; (d) Quantitative fluorescence plot of (c); (e) Fluorescence changes in B16-F10 cells treated with PT-MN alone at different time points; (f) Quantitative fluorescence plot of (e).

[0035] Figure 7 This is a flow cytometry analysis of the fluorescence changes of P-Rho 110 mediated by PT-MN in B16-F10 cells. (a) Fluorescence changes in B16-F10 cells treated with a combination of P-Rho 110 and PT-MN at different time points, (b) Fluorescence changes in B16-F10 cells treated with P-Rho 110 alone at different time points, (c) Fluorescence changes in B16-F10 cells treated with PT-MN alone at different time points, and (d) Quantitative analysis line graphs of (ac).

[0036] Figure 8 The images show the liquid phase analysis of P-DOX and P-IMQ release mediated by Pd-TNS. (a) Liquid phase waterfall plot of P-DOX and Pd-TNS suspension in PBS over time, (b) Line plot of quantitative analysis of (a), (c) Liquid phase waterfall plot of P-IMQ and Pd-TNS suspension in PBS over time, and (d) Line plot of quantitative analysis of (c).

[0037] Figure 9 The survival rates of B16-F10 and 4T1 cells treated with DOX, P-DOX and P-DOX / PT-MN combination at different drug concentrations are shown in (a) survival curve of B16-F10 cells and (b) survival curve of 4T1 cells.

[0038] Figure 10 This is a schematic diagram of cell apoptosis detected by flow cytometry using the Annexin V-FITC / PI double staining method.

[0039] Figure 11These are confocal microscopy images of B16-F10 cells stained with Calcein-AM and PI 24 hours after treatment with normal culture, PT-MN, P-DOX, DOX, and P-DOX / PT-MN combination, respectively.

[0040] Figure 12 These are confocal microscopy images of B16-F10 cells treated with normal culture, PT-MN, P-DOX, DOX, and P-DOX / PT-MN combination for 24 hours after immunofluorescence staining with CRT antibody and HMGB1 antibody.

[0041] Figure 13 The analysis included flow cytometry analysis of B16-F10 cells treated with normal culture, PT-MN, P-DOX, DOX, and P-DOX / PT-MN combination for 24 hours, respectively, using CRT antibody for immunofluorescence staining, and analysis of intracellular and extracellular ATP using the kit. (a) Flow cytometry analysis using CRT antibody for immunofluorescence staining, (b) Quantification of fluorescence intensity of (a), (c) Determination of extracellular ATP content using the kit, and (d) Determination of intracellular ATP content using the kit.

[0042] Figure 14 The survival rates of B16-F10 cells treated with different drugs and concentrations are as follows: (a) survival rate of B16-F10 cells treated with IMQ for 48 hours, (b) survival rate of B16-F10 cells treated with P-IMQ for 48 hours, and (c) survival rate of B16-F10 cells treated with DOX / IMQ and P-DOX / P-IMQ for 48 hours.

[0043] Figure 15 This involves using a Transwell apparatus to measure the process of co-culturing B16-F10 cells with dendritic cells.

[0044] Figure 16 These are CD80 and CD86 indicators of dendritic cells after different culture methods;

[0045] Figure 17 The values ​​are the cytokine content in culture media with different culture methods: (a) IL-6 content in cell supernatant, (b) IL-12 content in cell supernatant, and (c) TNF-α content in cell supernatant.

[0046] Figure 18 This is a schematic diagram of the skin insertion capability of PT-MN patch. (a) Photograph of PT-MN patch inserted into the skin, (b) Photograph of the skin after PT-MN patch removal, (c) Photograph of the skin 2 hours after PT-MN patch removal;

[0047] Figure 19This document presents the process and results of PT-MN-mediated in vivo activation of combined prodrugs for anticancer research. (a) Schematic diagram of PT-MN-mediated combined prodrug activation for melanoma treatment; (b) Body weight change curves of mice in each group; (c) Tumor growth kinetics of mice in each group; (d) Mean tumor mass of each group.

[0048] Figure 20 This is a diagram analyzing the cell death mechanisms in tumor tissues after different treatments.

[0049] Figure 21 This is an immunofluorescence staining analysis of CRT and HMGB1 in tumor tissues after different treatments;

[0050] Figure 22 This is an analysis of CD4 and CD8 immunofluorescence staining in tumor tissues after different treatments;

[0051] Figure 23 This is a schematic diagram showing the changes in immune factor indicators in mouse serum after different treatments using enzyme-linked immunosorbent assay (ELISA). (a) Changes in serum IL-6, (b) Changes in serum THF-α, and (c) Changes in serum IFN-γ.

[0052] Figure 24 The diagram shows the process and results of using combined treatment methods to treat bilateral tumor models; (a) Schematic diagram of PT-MN-mediated combined prodrug activation for the treatment of bilateral tumors; (b) Curves of body weight change in mice in each group, where group A is the PBS injection group, group B is the PT-MN group, group C is the DOX injection group, group D is the DOX and IMQ injection group, group E is the P-DOX and P-IMQ injection group, and group F is the P-DOX and P-IMQ combined PT-MN treatment group; (c) Primary tumor growth kinetics in each group of mice; (d) Distal tumor growth kinetics in each group of mice.

[0053] Figure 25 The analysis included cell death mechanisms and T cell infiltration in distal tumor tissues after different treatments; (a) distal tumor sections were stained with hematoxylin and eosin to observe pathological features, and (b) distal tumor sections were treated with CD4 and CD8 antibodies for immunofluorescence staining.

[0054] Figure 26 The flow cytometry analysis of dendritic cell maturity in distal tumor tissues after different treatments; (a) CD80 and CD86 indices of dendritic cells in distal tumor tissues after different treatments, (b) quantitative analysis of (a).

[0055] Figure 27The flow cytometry detection of regulatory T cells in distal tumor tissues after different treatments; (a) the proportion of Foxp3-positive cells among CD4-positive T cells in distal tumor tissues after different treatments, and (b) quantitative analysis of (a).

[0056] Figure 28 The flow cytometry analysis of dendritic cell maturity in lymph node tissues after different treatments; (a) CD80 and CD86 indices of dendritic cells in lymph node tissues after different treatments, (b) quantitative analysis of (a).

[0057] Figure 29 The proportions of CD4-positive and CD8-positive T cells in spleen tissue after different treatments were detected by flow cytometry; (a) the proportions of CD4-positive and CD8-positive T cells in spleen tissue after different treatments, and (b) the quantitative analysis of (a). Detailed Implementation

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer. The PDMS silicon mold was purchased from Taizhou Microchip Medical Technology Co., Ltd.

[0059] Example 1

[0060] A method for preparing an imiquimod prodrug having general formula (1) includes the following steps:

[0061] Imiquimod (300 mg, 1.25 mmol) was dissolved in 40 mL of anhydrous dioxane under nitrogen protection. Proprynnechloroformic acid (232 μL, 2.5 mmol) dissolved in 5 mL of anhydrous dioxane was added dropwise under ice bath conditions. The mixture was then stirred overnight at 50 °C. A portion of the imiquimod feedstock was recovered by filtration. The filtrate was concentrated and dried under reduced pressure, and the product was purified by column chromatography using methanol:dichloromethane = 1:10 as the eluent. The resulting imiquimod prodrug (compound 1) was a white solid (90 mg, yield 22%). 1 H NMR (400MHz, CDCl3) δ8.26-8.12(m,1H),7.98-7.89(m,1H),7.81(s,1H),7.73-7.60(m,1H),7.52-7.48(m,1H),4.89(d,J=2 .4Hz, 1H), 4.78 (d, J = 2.4Hz, 1H), 4.33 (dd, J = 17.6, 7.2Hz, 2H), 2.38 (t, J = 2.4Hz, 1H), 2.36-2.31 (m, 1H), 1.06-1.02 (m, 1H).

[0062]

[0063] Example 2

[0064] A method for preparing an doxorubicin prodrug having general formula (2) includes the following steps:

[0065] Doxorubicin (464 mg, 0.8 mmol) and N-(propyneoxycarbonyl)succinimide (158 mg, 0.8 mmol) were dissolved in 5 mL of anhydrous DMF. Then, N,N-diisopropylethylamine (210 μL, 1.2 mmol) dissolved in 2 mL of DMF was added dropwise to the solution, and the mixture was stirred at room temperature for 4 hours. 20 mL of ethyl acetate was added to the solution, and the mixture was washed twice with 1N HCl, once with saturated sodium bicarbonate, and once with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and purified by column chromatography using methanol:dichloromethane (1:10) as the eluent. The collected organic phase was concentrated and dried under reduced pressure to obtain compound 2 as a red solid (423 mg, 84% yield). 1 H NMR (400MHz, DMSO-d6) δ13.89(s,1H),13.12(s,1H),7.79-7.71(m,2H),7.49(d,J=8.4Hz,1H),7.01(d,J=8.0Hz,1H), 5.36(s,1H),5.19(d,J=3.6Hz,1H),4.88-4.82(m,2H),4.74(d,J=6.0Hz,1H),4.59(s,2H),4.53(d,J=2.4Hz,2H),4.17 (d,J=6.8Hz,1H),3.92(s,3H),3.74-3.67(m,1H),3.45(s,1H),2.93(d,J=18.4Hz,1H),2.78(d,J=18.0Hz,1H),2.20(d ,J=14.8Hz,1H),2.06-2.01(m,1H),1.85(td,J=13.2,4.0Hz,1H),1.47(dd,J=12.8,4.4Hz,1H),1.13(d,J=6.4Hz,3H).

[0066]

[0067] Example 3

[0068] The imiquimod prodrug of general formula (1) obtained in Example 1 and the doxorubicin prodrug of general formula (2) obtained in Example 2 can be used as tumor immunotherapy drugs in orthogonal catalytic microneedles. The specific steps are as follows:

[0069] like Figure 1As shown in the figure, a 15×15 microneedle array of swellable materials with catalyst supported on polyethylene was prepared using a PDMS mold. The actual product is shown in the figure. Figure 3 As shown in ab. The microneedles are formed from a polyvinyl alcohol (PVA) matrix containing palladium nanoparticles (Pd-TNS) supported on TiO2 nanosheets dispersed therein. The combined prodrug, after systemic administration, is activated by the Pd-TNS in the microneedles into a therapeutic agent or immune agonist. The combined therapy induces immunogenic cell death (ICD), promotes dendritic cell maturation, stimulates T cell increase, and can significantly inhibit the growth of primary tumors, distant tumors, and lung metastases. The catalyst loaded in the microneedles is... Figure 2 The image shows TiO2 nanosheets with Pd nanoparticle deposition, and the catalyst Pd-TNS is approximately 200 nm in size. The catalyst is uniformly distributed within the microneedles as shown. Figure 3 The SEM image of cd is shown. Figure 4 Microneedles have sufficient mechanical properties to penetrate the skin, and the yield strength of PT-MN is 0.85 N / needle.

[0070] Example 4

[0071] The catalyst or bioorthogonal catalytic microparticle in Example 3 has the effect of catalyzing the in vitro release of imiquimod prodrug and mycin prodrug. The specific steps are as follows:

[0072] (1)Use Figure 5 The probe shown is used to test the catalytic release of microneedles in B16-F10 cells, Ex = 485 nm, Em = 520 nm. The results show that the microneedles can catalyze the uncoiling of the non-fluorescent probe P-Rho 110 into the fluorescent Rho 110 in B16-F10 cell culture medium, and this can be observed in cells using confocal microscopy (…). Figure 6 (Scale bar 50 μm) or flow cytometry ( Figure 7 (2) A catalyst of 1 mg / mL Pd-TNS in PBS solution can catalyze a prodrug of 100 μM P-DOX ( ) Figure 8 a) or P-IMQ ( Figure 8 c) Released as drug DOX or IMQ, and both the prodrug reduction rate and the drug release rate are close to 80%. Figure 8 (b, 8d). (3) The 48-hour cytotoxicity of the PT-MN-catalyzed prodrug P-DOX was determined using the CCK8 assay. Figure 9 As shown, data points represent mean ± standard deviation (n = 3), and the maximum half-maximal inhibitory concentration (IC50) of P-DOX combined with PT-MN for B16-F10 cells. 50The value was 0.16±0.04 μM, much smaller than that of P-DOX (1.99±0.21 μM), and comparable to that of DOX (0.16±0.04 μM). Figure 9 a). Similarly, in 4T1 cells, the maximum half-maximal inhibitory concentration (IC50) of cells treated with the combined P-DOX and PT-MN was [data missing]. 50 The value was 1.5 ± 0.3 μM, much smaller than that of P-DOX (9.7 ± 0.6 μM) and similar to that of DOX (1.1 ± 0.2 μM). Figure 9 a). The above results all demonstrate that both catalysts and microneedles can catalyze the release of prodrugs in an in vitro environment.

[0073] Example 5

[0074] The bioorthogonal catalytic microneedles in Example 3 can catalyze the release of P-DOX in vitro, thereby inhibiting B16-F10 cells and inducing immunogenic cell death (ICD). The specific steps are as follows:

[0075] (1) B16-F10 cells were treated with P-DOX and PT-MN for 24 hours. After treatment, the cells were stained for apoptosis using Annexin V-FITC / PI kit. Flow cytometry showed that the treated cells exhibited significant apoptosis. Figure 10 (2) Live and dead cell staining was performed using Calcein-AM / PI, and confocal microscopy images showed that the treated cells exhibited significant cell death. Figure 11 (Scale bar 50 μm) Calcein-AM penetrates the cell membrane of living cells. After hydrolysis by esterase, it emits green fluorescence intracellularly. PI staining shows nucleic acids of necrotic and apoptotic cells with damaged membranes. (3) Immunofluorescence staining of treated cells with CRT and HMGB1 antibodies showed that the treated cells had obvious membrane exposure of CRT protein and translocation of HMGB1 into the cytoplasm. Figure 12 (Scale bar 50 μm), flow cytometry results also showed that the treated cells had significant membrane exposure of CRT protein ( Figure 13 (4) The ATP content of the treated cell supernatant was measured, and the results showed a significant decrease in intracellular ATP content. Figure 13 d) and a significant increase in extracellular ATP content ( Figure 13 c).

[0076] Comparative Example 1

[0077] The remaining steps of this comparative example are the same as those in Example 5, except that: only orthogonal catalytic microneedles PT-MNs are introduced; treatment without introducing PT-MNs is performed directly with doxorubicin prodrug; and doxorubicin is used directly. In the results of the comparative examples, only the group using doxorubicin directly showed an effect that was close to that of the experimental group; the other comparative examples showed no effect.

[0078] Example 6

[0079] The imiquimod prodrug in Example 2 showed very low toxicity to B16-F10 cells, maintaining a survival rate of 90% at 12.5 μM. Figure 14 b), while imiquimod itself begins to show significant cytotoxicity after 2.5 μM ( Figure 14 a) Data points represent mean ± standard deviation (n = 3). The 48-hour maximum half-maximal inhibitory concentration (IC50) of doxorubicin prodrug combined with imiquimod prodrug for B16-F10 was determined. 50 The value was 0.13±0.02μM, which is much lower than the 2.0±0.3μM value of the combination of doxorubicin and imiquimod. Figure 14 c). This demonstrates the safety of the combined use of imiquimod prodrug and doxorubicin prodrug for cells.

[0080] Example 7

[0081] Doxorubicin prodrug and imiquimod prodrug were used in combination at a concentration ratio of 5:2 in the co-culture of B16-F10 cells and bone marrow-derived dendritic cells. For specific implementation details, see [link to implementation details]. Figure 15 The combined drug was incubated with B16-F10 cells for 24 hours in the presence of PT-MN, followed by co-culturing with dendritic cells for 24 hours. The results are as follows:

[0082] (1) Flow cytometry was used to detect the maturity of dendritic cells. Figure 16 The results showed that the CD80 and CD86 levels of dendritic cells in the treatment group increased significantly, indicating that the dendritic cells had a significant maturation trend. (2) The cytokine content in the cell supernatant after treatment was determined by enzyme-linked immunosorbent assay (ELISA). Figure 17 It can be seen that: IL-6 ( Figure 17 a) IL-12 Figure 17 b) and TNF-α Figure 17 The levels of c) were significantly increased. These results demonstrate that the combined use of doxorubicin prodrug and imiquimod prodrug, catalyzed by PT-MN, can induce the maturation of co-cultured dendritic cells and the release of various cytokines.

[0083] Comparative Example 2

[0084] The remaining steps of this comparative example are the same as those in Example 7, except that: only orthogonal catalytic microneedles PT-MNs are introduced; treatment without introducing PT-MNs is performed directly using doxorubicin prodrug and imiquimod prodrug in combination; and doxorubicin and imiquimod are performed directly in combination. In the results of the comparative examples, the group using doxorubicin prodrug and imiquimod prodrug in combination showed a weak effect, the group using doxorubicin and imiquimod in combination showed a significant effect that was close to that of the experimental group, and the other comparative examples showed no significant effect.

[0085] Example 8

[0086] PT-MNs were applied to the skin surface of mice for treatment. Figure 18 a), removed one day later ( Figure 18 b) The skin returns to its original state within 2 hours. Figure 18 c) Demonstrating the safety of PT-MN application during treatment. A PT-MN patch was inserted into the skin, and micropores were observed after removing the microneedles at the end of each treatment. The micropores were temporary and gradually recovered within 2 hours after PT-MN removal.

[0087] Example 9

[0088] The combination of doxorubicin prodrug and imiquimod prodrug, along with orthogonal catalytic microneedles (PT-MNs), was used to treat melanoma in mice. The specific procedure is as follows:

[0089] like Figure 19 As shown in Figure a, B16-F10 cells were injected subcutaneously into C57BL / 6J mice to construct a tumor-bearing mouse model. Five days later, mice were divided into groups of five according to standard methods. The treatment group (group F) received intravenous injections of doxorubicin prodrug (100 mg / kg) and imiquimod prodrug (20 mg / kg) into the peritoneal cavity. PT-MNs were cut to the appropriate size for the tumor and applied to it. The microneedle patch was removed 24 hours later. Treatment was repeated three times, every three days, with tumor size and mouse weight monitored during this period. On day 12, mouse serum was collected for cytokine detection, and tumors were collected for HE staining and immunohistochemical experiments.

[0090] Comparative Example 3

[0091] The remaining steps of this comparative example are the same as those in Example 9, except that: A: no treatment group; B: only orthogonal catalytic microneedles PT-MNs are introduced; C: no PT-MNs are introduced for treatment, and doxorubicin is used directly; D: doxorubicin and imiquimod are used in combination directly; E: doxorubicin prodrug and imiquimod prodrug are used in combination directly.

[0092] The results are as follows:

[0093] (1) As Figure 19As shown in b, group A was the PBS injection group, group B was the PT-MN group, group C was the DOX injection group, group D was the DOX and IMQ injection group, group E was the P-DOX and P-IMQ injection group, and group F was the P-DOX and P-IMQ combined with PT-MN treatment group. Data points represent mean ± standard deviation (n = 5). The body weight of mice in all experimental groups did not decrease, indicating that the combined drug treatment was biosafety-free in mice.

[0094] (2) By Figure 19 As shown in c and 19d, tumor growth in the treatment group mice was significantly inhibited. Data points represent mean ± standard deviation (n = 5). Tumor sections were stained with hematoxylin-eosin to observe pathological features and stained with TUNEL to detect apoptosis. Figure 20 As can be seen, significant apoptosis and necrosis of tumors were observed in the treatment group. Scale bar: 100μm. Groups C, D, and E also showed some tumor-suppressive effects, but these were far less than those in the experimental group.

[0095] (3) By Figure 21 As shown, tumor sections were treated with CRT and HMGB1 antibodies and then stained with immunofluorescence. The tumors in the treatment group showed immunogenic cell death and a significant increase in CRT and HMGB1 positivity. Scale bar 50 μm.

[0096] (4) By Figure 22 As shown, tumor sections were treated with CD4 and CD8 antibodies for immunofluorescence staining. The treated group showed a significant increase in CD4 and CD8 positivity, indicating significant T cell infiltration. Scale bar: 50 μm.

[0097] (5) By Figure 23 As shown, the serum levels of IL-6, TNF-α, and IFN-γ in the treatment group mice were significantly increased, while the levels in groups D and E showed some upward trend, but were far less than those in the experimental group. Data points represent mean ± standard deviation (n = 3).

[0098] The above results all indicate that the combined use of doxorubicin prodrug and imiquimod prodrug, under the catalysis of PT-MNs, can significantly inhibit the growth of mouse melanoma, induce immunogenic cell death, and cause immune cell infiltration and an increase in inflammatory cytokines.

[0099] Example 10

[0100] The combination of doxorubicin prodrug and imiquimod prodrug, along with orthogonal catalytic microneedles (PT-MNs), was used to treat a bilateral melanoma model in mice. The specific procedure is as follows:

[0101] like Figure 24As shown in Figure a, B16-F10 cells were injected subcutaneously into C57BL / 6J mice to construct in situ tumors. Three days later, B16-F10 cells were injected subcutaneously into the other side of the mouse to construct distal tumors. Four days later, mice were divided into groups of five according to standard methods. The treatment group (Group F) received intravenous injections of doxorubicin prodrug (100 mg / kg) and imiquimod prodrug (20 mg / kg) into the peritoneal cavity of the mice. PT-MNs were cut to the appropriate size for the tumor and attached to the in situ tumor. The microneedle patch was removed after 24 hours. Treatment was repeated three times, once every three days, during which the size of the two tumors and the weight of the mice were measured. At day 12, distal tumors were collected for HE staining and immunohistochemistry experiments. Distal tumors, lymph nodes, and spleens were collected from the mice for flow cytometry analysis. Data points are expressed as mean ± standard deviation (n = 5).

[0102] Comparative Example 4

[0103] The remaining steps of this comparative example are the same as those in Example 10, except that: A: no treatment group; B: only orthogonal catalytic microneedles PT-MNs are introduced; C: no PT-MNs are introduced for treatment, and doxorubicin is used directly; D: doxorubicin and imiquimod are used in combination directly; E: doxorubicin prodrug and imiquimod prodrug are used in combination directly.

[0104] The results are as follows:

[0105] (1) As Figure 24 As shown in b, the body weight of mice in all experimental groups did not decrease, indicating that the combined drug treatment was biosafety-free for mice. (2) Orthotopic tumors in the treatment group mice ( Figure 24 c) and distal tumors ( Figure 24 d) Growth was significantly inhibited, and HE staining ( Figure 25 (a, scale bar 100μm) Obvious necrosis of distal tumors was observed in the treatment group. Groups C, D, and E also showed some tumor suppression effects, but were far less than the experimental group. (3) From Figure 25 As shown in b, the distal tumors in the treatment group showed significant T-cell infiltration and a significant increase in CD4 and CD8 positivity, scale bar 50 μm. (4) From Figure 26 As shown, the dendritic cells in the distal tumors of the treatment group showed a significant increase in CD80 and CD86 positivity, indicating an increased proportion of mature dendritic cells. Figure 26 b). Group D showed a certain increasing trend, but it was far less than the experimental group. (5) From Figure 27 As shown, the proportion of CD4 and Foxp3 positive cells in the distal tumors of the treatment group decreased significantly. Cells that are positive for both CD4 and Foxp3 are regulatory T cells, demonstrating a decrease in the proportion of helper T cells. Figure 27 b). Groups C, D, and E also showed a certain downward trend, but it was far less than that of the experimental group. (6) From Figure 28As shown, the dendritic cells in the lymph nodes of the treatment group showed a significant increase in CD80 and CD86 positivity. Cells that were simultaneously positive for both CD80 and CD86 were mature dendritic cells, demonstrating an increased proportion of mature dendritic cells. Figure 28 b). Groups C, D, and E showed a certain increasing trend, but it was far less than that of the experimental group. (7) From Figure 29 As shown, the proportion of CD3 and CD8 positive cells in the spleen of the treatment group was significantly increased, demonstrating an increase in the proportion of cytotoxic T cells. Figure 29 (b) Groups D and E showed a certain increasing trend, but it was far less than that of the experimental group.

[0106] The above results all indicate that the combined use of doxorubicin prodrug and imiquimod prodrug, under the catalysis of PT-MNs, can significantly inhibit the growth of melanoma in mice, while inducing a tumor immune response and inhibiting the growth of distal tumors.

Claims

1. An imiquimod prodrug, characterized in that: The structural formula of the imiquimod prodrug is: 。 2. The method for preparing an imiquimod prodrug according to claim 1, characterized in that, Includes the following steps: Step 1: Dissolve excess imiquimod in anhydrous dioxane under a protective atmosphere, and add propargyl chloroformic acid dissolved in anhydrous dioxane dropwise under an ice bath to obtain a mixture; Step 2: Stir the mixture obtained in Step 1 at 45~55℃ overnight; Step 3: Filter the product obtained in step 2, recover the excess imiquimod, and purify the filtrate by column chromatography to obtain the imiquimod prodrug.

3. The method for preparing an imiquimod prodrug according to claim 2, characterized in that: In step one, the mass-to-volume ratio of imiquimod, anhydrous dioxane, and propargyl chloroformic acid is 300 mg: 40 mL: 0.2~0.3 mL.

4. The method for preparing an imiquimod prodrug according to claim 2, characterized in that: In step one, the protective atmosphere is nitrogen or argon.

5. The method for preparing an imiquimod prodrug according to claim 2, characterized in that: In step three, the eluent for purification is a mixed solution of methanol and dichloromethane with a volume ratio of 1:9~10.

6. A combination pharmaceutical composition with tumor immunomodulatory effects, characterized in that, Its structural formula is as follows: The combined pharmaceutical composition comprises imiquimod prodrug and doxorubicin prodrug or a pharmaceutically acceptable salt thereof, wherein the mass ratio of imiquimod prodrug to doxorubicin prodrug is 1-2:5, wherein the imiquimod prodrug is the imiquimod prodrug of claim 1 or a pharmaceutically acceptable salt thereof, and the structural formula of the doxorubicin prodrug is: 。 7. A combination pharmaceutical composition with tumor immunomodulatory effects according to claim 6, characterized in that: The preparation method of the doxorubicin prodrug includes the following steps: S1. Dissolve doxorubicin and N-(propyneoxycarbonyl)succinimide in anhydrous DMF; S2. Add N,N-diisopropylethylamine dissolved in DMF dropwise to the solution obtained in S1, and stir at room temperature for 4-5 hours; S3. Ethyl acetate was added to the solution obtained in S2, and the solution was washed with HCl, saturated sodium bicarbonate, and saturated saline solution, respectively. The organic phase was collected, dried with anhydrous sodium sulfate, and the product was purified by column chromatography to obtain the doxorubicin prodrug.

8. The combination pharmaceutical composition with tumor immunomodulatory effects according to claim 7, characterized in that: In S1, the mass-to-volume ratio of doxorubicin, N-(propyneoxycarbonyl)succinimide, and anhydrous DMF is 464 mg: 158 mg: 5~10 mL.

9. A combination pharmaceutical composition with tumor immunomodulatory effects according to claim 7, characterized in that: In step S3, the eluent for purification is a mixed solution of methanol and dichloromethane with a volume ratio of 1:9~10.

10. The use of the imiquimod prodrug of claim 1 or the combination pharmaceutical composition of claim 6 in the preparation of an immuno-oncology drug.

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