Co-assembled nanoparticles of fatty acid coupled prodrug compound containing STING agonist and IDO1 inhibitor, and preparation and application thereof

Abstract

The invention belongs to the field of biomedical technologies and pharmaceutical preparations, and particularly discloses co-assembled nanoparticles of a fatty acid coupled prodrug compound containing an STING agonist and an IDO1 inhibitor as well as preparation and application of the co-assembled nanoparticles of the fatty acid coupled prodrug compound containing the STING agonist and the IDO1 inhibitor. According to the invention, a STING agonist MSA-2 and an IDO1 inhibitor NLG919 are respectively subjected to prodrug chemical modification, so that the STING agonist MSA-2 and the IDO1 inhibitor NLG919 are assembled together to form nanoparticles. The nano preparation co-loaded with the two drugs can activate an STING pathway and start anti-tumor immune response through a single drug delivery system, and can effectively block an IDO1 negative feedback immunosuppression pathway induced by STING activation at the same time. The invention has the beneficial effects that through a synergistic effect mechanism, the anti-tumor immune curative effect of the drug is obviously enhanced, the drug resistance of STING agonist single-drug treatment can be effectively overcome, the whole-body toxic and side effects of the drug are reduced, and a novel strategy with great potential is provided for clinical tumor immune treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedical technology and pharmaceutical formulation, specifically to the co-assembled nanoparticles containing a fatty acid-coupled prodrug compound comprising a STING agonist and an IDO1 inhibitor, their preparation, and their application. Background Technology

[0002] Tumor immunotherapy, particularly immune checkpoint inhibitors, has made groundbreaking progress in the treatment of various cancers. However, a large number of patients cannot benefit from it due to the lack of immune cell infiltration in their tumor microenvironment (TME) (i.e., "cold tumors"). Interferon gene-stimulating protein (STING) agonists can activate the innate immune response and induce the production of various inflammatory cytokines such as type I interferon, thereby transforming "cold tumors" into "hot tumors," demonstrating great potential in the field of tumor immunotherapy.

[0003] Therefore, STING agonist therapy is a promising new strategy for tumor immunotherapy. Despite significant preclinical studies, the monotherapy efficacy of STING agonists in clinical trials has been unsatisfactory, and combination therapy with first-line immune checkpoint therapies has failed to demonstrate significant advantages, severely hindering the clinical translation of STING agonists. Recent studies have shown that activation of the STING pathway, while inducing anti-tumor immunity, may also trigger negative feedback regulatory mechanisms, leading to immunosuppression. For example, interferon-γ (IFN-γ) produced after STING activation can sustainably upregulate the expression of the immune metabolic checkpoint enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1, by depleting tryptophan and producing the immunosuppressive metabolite kynurenine, can inhibit the function of effector T cells and promote the proliferation of regulatory T cells (Tregs), thereby creating an immunosuppressive tumor microenvironment, ultimately leading to tumor immune escape and treatment failure.

[0004] Currently, there is no effective strategy to achieve specific activation of the STING pathway and simultaneous inhibition of the IDO1 pathway using a single formulation. Therefore, overcoming the immunosuppression caused by the activation of the STING agonist's own negative feedback pathway is a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This invention provides a co-loaded nanoparticle for tumor immunotherapy, and also provides a method for synthesizing and preparing a STING agonist fatty acid-coupled prodrug compound and an IDO1 inhibitor fatty acid-coupled prodrug compound, as well as a method for co-assembling the two drugs and their application in tumor immunotherapy.

[0006] The purpose of this invention is to solve the problems mentioned in the background art above, and to provide a novel pharmaceutical formulation and its application that can synergistically activate the STING pathway and inhibit the IDO1 pathway, thereby effectively overcoming STING agonist treatment resistance.

[0007] This invention constructs two fatty acid-coupled prodrug compounds by introducing long-chain unsaturated fatty acids (eicosapentaenoic acid, docosahexaenoic acid, etc.) into the STING agonist MSA-2 molecule and the IDO1 inhibitor NLG919 molecule. The two prodrug compounds are then combined in a reasonable ratio to form co-loaded nanoparticles. These co-assembled nanoparticles can effectively activate the body's innate immune response, while relieving STING agonist-induced immune tolerance and enhancing tumor immunotherapy.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A co-assembled nanoparticle comprising a fatty acid-coupled prodrug compound of a STING agonist and an IDO1 inhibitor, comprising at least one fatty acid-coupled prodrug compound I of a STING agonist and at least one fatty acid-coupled prodrug compound II of an IDO1 inhibitor, and a polyethylene glycol-modified lipid molecule; wherein compound I and compound II each independently have the structure shown in the following formula:

[0010] MLR;

[0011] M comes from a STING agonist or an IDO1 inhibitor; L is a chemical bond or linker segment.

[0012] R comes from long-chain unsaturated fatty acids from C4 to C30.

[0013] Preferably, the lipid molecule is a polyethylene glycol-coupled lipid molecule. More preferably, the polyethylene glycol-coupled lipid molecule is distearylphosphatidylethanolamine-polyethylene glycol, such as distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG). 2000 ).

[0014] Preferably, the STING agonist is MSA-2; and the IDO1 inhibitor is NLG919.

[0015] Preferably, the molar ratio of the STING agonist prodrug compound to the lipid molecule is 50-100:1; the molar ratio of the IDO1 inhibitor prodrug compound to the lipid molecule is 10-30:1. More preferably, the molar ratio of the STING agonist prodrug compound to the lipid molecule is 60-90:1; the molar ratio of the IDO1 inhibitor prodrug compound to the lipid molecule is 15-30:1. Even more preferably, the molar ratio of the STING agonist prodrug compound to the lipid molecule is 70-80:1; the molar ratio of the IDO1 inhibitor prodrug compound to the lipid molecule is 20-25:1. Specifically preferably, the molar ratio of the STING agonist prodrug compound, the IDO1 inhibitor prodrug compound, and the lipid molecule (polyethylene glycol-coupled lipid molecule) is 74:22:1.

[0016] Preferably, the connecting segment is -O(CH2). n1 -SS-(CH2) n2 0-, n1 and n2 are each independently selected from integers from 2 to 10. More preferably, n1 and n2 are each independently selected from integers from 2 to 8. Even further, n1 and n2 are each independently selected from 2, 3, 4, and 5; specifically, n1 and n2 are 2 or 3.

[0017] Preferably, R is a C4-C30 long-chain unsaturated alkyl group. Further, R is derived from a C10-C30 long-chain unsaturated fatty acid; R is a C10-C30 long-chain unsaturated alkyl group containing 3-15 carbon-carbon double bonds. More preferably, the long-chain unsaturated fatty acid is selected from one or more of oleic acid (OA), linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

[0018] Preferably, compounds I and II have the structures shown in formula (I) and formula (II), respectively:

[0019] MSA-2-L1-R (I);

[0020] NLG919-L2-R (II);

[0021] Where L1 is -O(CH2) n1 -SS-(CH2) n2 O-, n1 and n2 are independent integers from 2 to 5; L2 is a chemical bond.

[0022] Preferably, the average particle size of the co-assembled nanoparticles is 50-500 nm; more preferably, the average particle size of the co-assembled nanoparticles is 50-200 nm.

[0023] Preferably, the preparation methods of compound I and compound II are as follows:

[0024] The STING agonist or IDO1 inhibitor reacts with the precursor compound corresponding to L to obtain an intermediate, which is further reacted with the fatty acid corresponding to R to obtain the MLR;

[0025] Alternatively, when R is a chemical bond, the STING agonist or IDO1 inhibitor reacts directly with the fatty acid corresponding to R to obtain the MLR.

[0026] Taking MSA-2 as an example, the structure of its corresponding TING agonist fatty acid-coupled prodrug compound I is as follows: Figure 1 As shown in the figure, the preparation method of MSA-2-SS-DHA includes:

[0027] Step (1): Dissolve compound MSA-2 in an organic solvent (e.g., anhydrous dichloromethane); add 2,2'-dithiodiethanol, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the solution in sequence, and stir the resulting mixture at 40-50°C (e.g., 45°C) until the reaction is complete to obtain the corresponding intermediate product MSA-2-SS-OH, the structure of which is shown below. Figure 1 ;

[0028] Step (2): Dissolve MSA-2-SS-OH in an organic solvent (anhydrous dichloromethane), add docosahexaenoic acid, 4-dimethylaminopyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and stir the resulting mixture at 40-50°C (e.g., 45°C) until the reaction is complete, to obtain the following... Figure 1 The product shown is MSA-2 coupled with a docosahexaenoic acid prodrug compound (MSA-2-SS-DHA).

[0029] Preferably, the molar ratio of the compound MSA-2, 2,2'-dithiodiethanol, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is (0.5~0.8):1:(1~2):(1~2). The molar ratio of MSA-2-SS-OH, docosahexaenoic acid, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is (1~1.5):1:(1~2):(1~2).

[0030] When the IDO1 inhibitor is NLG919, its preparation method can refer to step (2) of the preparation of MSA-2-SS-DHA, and also refer to Figure 4 The structure shown in the NLG919-conjugated docosahexaenoic acid prodrug compound (NLG919-DHA) was obtained.

[0031] A method for preparing co-assembled nanoparticles containing a STING agonist and an IDO1 inhibitor fatty acid-coupled prodrug compound as described in any of the above technical solutions includes: dissolving a STING agonist fatty acid-coupled prodrug compound I and an IDO1 inhibitor fatty acid-coupled prodrug compound II in an organic solvent (such as DMSO), adding polyethylene glycol-modified lipid molecules and dissolving them completely, injecting the solution into water, and obtaining uniformly dispersed co-assembled nanoparticles.

[0032] This invention provides a novel drug combination strategy that co-assembles a STING agonist prodrug and an IDO1 inhibitor prodrug into nanoparticles. The STING agonist is the non-nucleotide small molecule agonist MSA-2, and the IDO1 inhibitor is NLG919.

[0033] This invention employs a prodrug modification strategy, linking MSA-2 to a long-chain unsaturated fatty acid—docosahexaenoic acid (DHA)—via a disulfide bond that can be cleaved under reducing conditions in the tumor microenvironment, forming the MSA-2 prodrug (MSA-SS-DHA); simultaneously, linking NLG919 to DHA via an ester bond, forming the NLG919 prodrug (NLG919-DHA). This prodrug design not only solves the problems of poor water solubility and difficulty in co-formulation of MSA-2 and NLG919, but also endows the nano-formulation with stimulus-responsive release properties.

[0034] This invention also provides a method for preparing the drug co-assemblies. The method includes dissolving MSA-2 and NLG919 prodrugs in an organic solvent (such as DMSO), followed by mixing with an aqueous phase under ultrasonic or stirring conditions. The two unsaturated fatty acid-coupled prodrug molecules then self-assemble to form nanoparticles. To increase the stability of the nanoparticles and prolong their circulation time in vivo, polyethylene glycol-modified lipid molecules, such as distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG), can be selectively added. 2000 ).

[0035] In some implementations, the molar ratio of the STING agonist prodrug compound, the IDO1 inhibitor prodrug compound, and the polyethylene glycol lipid molecule is 74:22:1.

[0036] This invention provides the use of the aforementioned drug as a prodrug in the preparation of pharmaceutical formulations. The pharmaceutical formulation may be, but is not limited to, an antitumor drug.

[0037] In some embodiments, the cancer is selected from breast cancer, melanoma, colon cancer, lung cancer, pancreatic cancer, kidney cancer, head and neck cancer, lymphoma, skin cancer, urothelial carcinoma, gastric cancer, or hepatocellular carcinoma. In particular, the pharmaceutical compositions of the present invention have excellent efficacy in treating tumors that are insensitive to monotherapy with STING agonists or have developed acquired resistance.

[0038] The present invention demonstrates that the drug precursors formed by coupling with unsaturated fatty acids can be used for a variety of purposes due to the increased flexibility of the drug molecules, including but not limited to: being encapsulated in liposomes to prepare drug-loaded liposome drugs.

[0039] Furthermore, according to the above-mentioned method for preparing the pharmaceutical formulation: DSPE-PEG 2000 MSA-2 and NLG919 prodrug conjugates are dissolved in organic solvents, mixed, and then injected into an aqueous phase. They can self-assemble to form co-assembled nanoparticle prodrugs containing both drug components. Specifically, uniformly dispersed co-assembled nanoparticles are obtained using organic solvent evaporation or thin-film dispersion methods. This method can increase the water solubility of the drug, enabling intravenous injection, improving drug bioavailability, and prolonging the blood circulation half-life.

[0040] Characterization data of the co-assembled nanoparticles show that the co-assembled nanoparticles containing the STING agonist prodrug compound and the IDO1 inhibitor prodrug compound have good particle size and polydispersity index (PDI) stability.

[0041] In vivo animal studies showed that in STING-sensitive animal models, the co-assembled nanoparticles exhibited more sustained tumor growth inhibition and better biocompatibility compared to single STING agonist prodrug compounds or single IDO1 inhibitor prodrug compounds. In STING-resistant animal models, the co-assembled nanoparticles containing both STING agonist and IDO1 inhibitor prodrug compounds significantly inhibited STING-resistant tumor growth and prolonged the survival of tumor-bearing animals.

[0042] The gain effect of this invention is as follows:

[0043] (1) In this invention, STING agonist and IDO1 inhibitor are prodrug modified and co-delivered via a single nanoplatform. Activation of STING is responsible for activating anti-tumor immunity and recruiting immune cells to the tumor, while IDO1 inhibitor is responsible for relieving the negative feedback immunosuppression induced by STING agonist monotherapy, thereby greatly enhancing CD8 activation. + T-cell-mediated anti-tumor immunity achieves synergistic therapeutic effects and can effectively reverse the resistance of solid tumors to STING agonists.

[0044] (2) The present invention can significantly improve the pharmacokinetic behavior of two small molecule immunomodulatory drugs with different functions by nano-co-delivery. Through the enhanced permeability and retention (EPR) effect of nano-sized drugs, the drugs are enriched in tumor tissue and tumor draining lymph nodes, thereby improving the utilization efficiency of immunomodulators.

[0045] (3) This invention effectively reduces the non-specific inflammatory response and toxic side effects caused by free drugs (especially STING agonists) in the systemic circulation through prodrug design and nanocarrier encapsulation, thereby improving the safety of treatment. Attached Figure Description

[0046] Figure 1 This is the synthetic route for the MSA-2-docosahexaenoic acid coupling prodrug compound in Example 1.

[0047] Figure 2 The image shows the 1H NMR spectrum of the MSA-2-docosahexaenoic acid coupling prodrug intermediate product in Example 1.

[0048] Figure 3 The image shows the 1H NMR spectrum of the MSA-2-docosahexaenoic acid-coupled prodrug compound in Example 1.

[0049] Figure 4 This is the synthetic route for the NLG919-docosahexaenoic acid coupling prodrug compound in Example 2.

[0050] Figure 5 The image shows the 1H NMR spectrum of the NLG919-docosahexaenoic acid prodrug compound in Example 2.

[0051] Figure 6 The image shows a transmission electron microscope (TEM) image and particle size distribution of the fatty acid-coupled co-assembled nanoparticles in Example 3.

[0052] Figure 7 This is a diagram showing the in vitro stability analysis of the fatty acid-coupled co-assembled nanoparticles in Example 4.

[0053] Figure 8 AB represents the tumor growth curves and survival status of the STING agonist-sensitive MC38 tumor model after drug treatment in Example 5.

[0054] Figure 9 AB represents the tumor growth curves and survival status of the STING agonist-resistant MC38 / R tumor model after drug treatment in Example 5.

[0055] Figure 10AD is an in vivo immune activation analysis diagram of the fatty acid-coupled co-assembled nanoparticles in Example 6. Detailed Implementation

[0056] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0058] DSPE-PEG (molecular weight 2000) (CAS: 147867-65-0) was purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0059] The present invention will be further described below with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0060] Example 1: Synthesis of MSA-2-conjugated docosahexaenoic acid prodrug compound (MSA-2-SS-DHA)

[0061] The synthetic route of MSA-2 via disulfide coupling with docosahexaenoic acid (DHA) is as follows: Figure 1 As shown, it includes the following steps:

[0062] Step 1: In a dry reaction flask, compound MSA-2 (200 mg, 0.68 mmol) was dissolved in anhydrous dichloromethane (5 mL). Then, 2,2'-dithiodiethanol (157.2 mg, 1.0 mmol), 4-dimethylaminopyridine (DMAP) (166.2 mg, 1.4 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (211 mg, 1.4 mmol) were added sequentially to the solution. The resulting mixture was stirred at 45°C for 4 hours. After the reaction was complete, the solvent was removed by vacuum distillation to obtain a crude residue. This residue was redissolved in dichloromethane and washed sequentially with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. The separated organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated again under reduced pressure to obtain the crude product. Finally, the crude product was purified by rapid column chromatography using silica gel as the stationary phase (eluent: dichloromethane / methanol v / v = 20:1) to obtain the target product MSA-2-SS-OH, a pink solid with a mass of 212 mg and a yield of 72.4%. 1 The chemical structure of the product was analyzed and confirmed by 1H NMR. 1 H NMR spectra and peak assignments, such as Figure 2 As shown.

[0063] Step 2: The product from step (1) (300 mg, 0.70 mmol) was dissolved in anhydrous dichloromethane (2 mL). Then, docosahexaenoic acid (DHA) (190.8 mg, 0.58 mmol), 4-dimethylaminopyridine (DMAP) (106.2 mg, 0.87 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (135 mg, 0.87 mmol) were added sequentially to the solution. The reaction mixture was stirred at 45°C for 4 hours. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was redissolved in dichloromethane and washed sequentially with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent: dichloromethane / methanol, volume ratio = 15:1) to obtain the target product. The final product was an orange solid, with a compound yield of 300 mg and a yield of 68%. 1 The chemical structure of the product was analyzed and confirmed by 1H NMR. 1 H NMR spectra and peak assignments, such as Figure 3 As shown.

[0064] Example 2: Synthesis of NLG919-conjugated docosahexaenoic acid prodrug compound (NLG919-DHA)

[0065] The synthetic route of NLG919 via ester coupling with docosahexaenoic acid is as follows: Figure 4 As shown, the steps are as follows:

[0066] NLG919 (160 mg, 0.57 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (5 mL). DHA (223.2 mg, 0.70 mmol), 4-dimethylaminopyridine (DMAP, 104 mg, 0.85 mmol, 1.5 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (132 mg, 0.85 mmol) were added sequentially to this solution. The reaction mixture was stirred overnight at 45°C. After the reaction was complete, the solvent was removed under reduced pressure. The residue was redissolved in dichloromethane and washed sequentially with 5% citric acid aqueous solution, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (eluent: dichloromethane / methanol = 10:1), yielding the final product as an orange solid with a yield of 188 mg, representing a yield of 61.7%. 1 The chemical structure of the product was analyzed and confirmed by 1H NMR. 1 H NMR spectra and peak assignments, such as Figure 5 As shown.

[0067] Example 3: Preparation and characterization of co-assembled nanoparticles with immunomodulatory functions (iBINP)

[0068] 1.5 mg of the MSA-2-coupled docosahexaenoic acid prodrug compound and 0.4 mg of the NLG919-coupled docosahexaenoic acid prodrug compound synthesized in Examples 1-2 were dissolved in 0.1 mL of dimethyl sulfoxide (DMSO). After complete dissolution, 0.19 mg of DSPE-PEG was added. 2000 (MSA-2-SS-DHA, NLG919-DHA and DSPE-PEG 2000 The molar ratio is 74:22:1). Under vigorous stirring or sonication conditions, the mixture contains two prodrugs and DSPE-PEG. 2000The DMSO solution was rapidly injected into 0.9 mL of deionized water, and the mixture immediately became a clear or translucent suspension. The residual dimethyl sulfoxide was removed by dialysis (molecular weight cutoff 3500) to obtain uniformly dispersed co-assembled nanoparticles containing the STING agonist and the IDO1 inhibitor (denoted as iBINP).

[0069] like Figure 6 As shown, transmission electron microscopy clearly reveals the regular morphology, uniform particle size, and high dispersibility. Dynamic light scattering particle size analysis determined the iBINP particle size to be 110.03 ± 7.79 nm, and the particle size distribution exhibited a single peak, indicating that the co-assembled nanoparticles had a uniform particle size distribution.

[0070] Example 4: In vitro stability test of co-assembled nanoparticles (iBINP) containing STING agonist prodrug and IDO1 inhibitor prodrug

[0071] The iBINPs prepared in Example 3 were dispersed in two systems: ultrapure water (DI Water) and PBS containing 10% fetal bovine serum. The particle size and PDI were measured using a dynamic light scattering instrument. Three parallel samples were measured each time. The solid line represents the particle size, and the dashed line represents the PDI.

[0072] The results are as follows Figure 7 As shown in AB, the nano-co-assembled particles remained stable for at least 7 days, with no significant changes in particle size and PDI observed.

[0073] Example 5: In vivo antitumor effects of iBINP in STING agonist-sensitive and drug-resistant tumor models

[0074] Subcutaneous tumor models were established in wild-type MC38 mice sensitive to STING agonists and STING agonist-resistant MC38 / R mice. Tumors were allowed to grow to 100 mm. 3 Mice were then randomly assigned to three groups: a saline group, a STING agonist prodrug nanoparticle group (STING-NP), an IDO1 inhibitor prodrug nanoparticle group (IDO1-NP), a free drug combination of MSA-2 and NLG919 (FDC), and an iBINP group. All drugs were administered via tail vein injection. (The nanoparticles in the STING-NP and IDO1-NP groups were prepared using a method similar to that in Example 3; the FDC group consisted of a simple mixture of free MSA-2 and NLG919.) The MSA-2 dose was 30 mg / kg in each group, and the NLG919 dose was 10 mg / kg. The drugs were administered three times, with a two-day interval between doses.

[0075] The results are as follows Figure 8As shown in Figures AB, when treating wild-type MC38 subcutaneous tumors with the drug, there was no statistically significant difference in tumor growth between the STING-NP monotherapy group and the iBINP nanoassembly group combined with IDO1-NP, as the tumor is sensitive to the STING agonist. The combination of the two drugs did not demonstrate any advantage; however, the combination therapy group showed a significant advantage in prolonging the survival of mice compared to other control groups. These results suggest that iBINP treatment can inhibit tumor growth in mice in the long term and prolong the survival of tumor-bearing mice.

[0076] The results are as follows Figure 9 As shown in AB, STING-NP monotherapy was almost ineffective against drug-resistant MC38 / R tumors. However, the iBINP treatment group showed a strong tumor-suppressive effect and significantly prolonged the survival of mice. These results strongly demonstrate that the nanocomplex of the present invention can effectively overcome STING agonist acquired resistance mediated by the IDO1 pathway in solid tumors.

[0077] Example 6: The remodeling effect of iBINP on the tumor immune microenvironment

[0078] Similar to Example 5, C57BL / 6 mice were used as experimental subjects, and MC38 cells were inoculated to establish a subcutaneous colorectal cancer tumor model. When the tumor volume reached approximately 100 mm... 3 Each group was administered 1) saline solution, 2) single STING agonist prodrug nanoparticles (STING-NP), 3) single IDO1 inhibitor prodrug nanoparticles, 4) a combination of free MSA-2 and NLG919 (FDC group), and 5) co-assembled nanoparticles (iBINP). The MSA-2 dose was 30 mg / kg and the NLG919 dose was 10 mg / kg in each group, administered three times, with a two-day interval between doses.

[0079] On the second day after the end of the drug treatment cycle, tumor tissue was taken for flow cytometry analysis. The results were as follows: Figure 10 AD results showed that, compared with the free drug combination group, iBINP treatment significantly increased intratumoral CD8 levels. + T cell infiltration reduced the proportion of intratumoral regulatory T cells (Tregs); compared with the STING-NP monotherapy group, the iBINP treatment group showed a higher proportion of CD8+ cells in tumor tissue. + Although the proportion of T cells did not increase significantly, the proportion of Tregs with immunosuppressive functions decreased significantly, thereby reducing the CD8+ level. + The / Treg ratio increased significantly, creating a tumor immune microenvironment conducive to anti-tumor immunity.

[0080] The above data indicate that the co-assembled prodrug nanoparticles (iBINP) have a good in vivo immune activation effect, which can effectively activate innate and adaptive immunity, and overcome immune tolerance caused by STING pathway activation, thereby promoting the body's anti-tumor immune effect.

Claims

1. A co-assembled nanoparticle comprising a STING agonist and an IDOl inhibitor and a fatty acid conjugated prodrug compound, characterized in that, A fatty acid conjugated prodrug compound I comprising at least one STING agonist and a fatty acid conjugated prodrug compound II comprising at least one IDO1 inhibitor, and a pegylated lipid molecule; the compound I and the compound II each independently having a structure represented by the following formula: M-L-R; M is from a STING agonist or an IDO1 inhibitor; L is a chemical bond or a linking segment; R is from a long chain unsaturated fatty acid with a length of C10-C30.

2. The co-assembled nanoparticle comprising a fatty acid-coupled prodrug compound of a STING agonist and an IDO1 inhibitor according to claim 1, characterized in that, The lipid molecule is distearoylphosphatidylethanolamine-polyethylene glycol.

3. The co-assembled nanoparticle comprising a fatty acid-coupled prodrug compound of a STING agonist and an IDO1 inhibitor of claim 1, wherein, The STING agonist is MSA-2; the IDO1 inhibitor is NLG919.

4. The co-assembled nanoparticle comprising a fatty acid-coupled prodrug compound of a STING agonist and an IDO1 inhibitor according to claim 3, characterized in that, The molar ratio of the STING agonist prodrug compound to the lipid molecule is 50-100:1; the molar ratio of the IDO1 inhibitor prodrug compound to the lipid molecule is 10-30:

1.

5. The co-assembled nanoparticle comprising a fatty acid-coupled prodrug compound of a STING agonist and an IDO1 inhibitor of claim 1, wherein, said connecting segment is -O(CH2) n1 -S-S-(CH2) n2 O-, n1 and n2 are each independently an integer from 2 to 10; and R is a long chain unsaturated alkanoyl group having from 10 to 30 carbon atoms, containing from 3 to 15 carbon-carbon double bonds.

6. The co-assembled nanoparticle comprising a fatty acid-coupled prodrug compound of a STING agonist and an IDO1 inhibitor of claim 1, wherein, The compound I and the compound II each independently have a structure represented by the following formula (I) and formula (II): MSA-2-L1-R; NLG919-L2-R; wherein L1is -0(CH2) n1 -S-S-(CH2) n2 O-, n1and n2are each independently an integer from 2 to 5; L2is a bond; and said R is from one or more of oleic acid, linolenic acid, a-linolenic acid, g-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid.

7. The co-assembled nanoparticle comprising a fatty acid-conjugated prodrug compound of a STING agonist and an IDO1 inhibitor according to any one of claims 1 to 6, characterized in that, The preparation method of the compound I and the compound II is as follows: The STING agonist or the IDO1 inhibitor reacts with a precursor compound corresponding to L to obtain an intermediate, and the intermediate further reacts with a fatty acid corresponding to R to obtain the M-L-R; Or when R is a chemical bond, the STING agonist or the IDO1 inhibitor directly reacts with a fatty acid corresponding to R to obtain the M-L-R.

8. A method of preparing a co-assembled nanoparticle comprising a fatty acid- conjugated prodrug compound of a STING agonist and an IDO1 inhibitor of any one of claims 1-6, comprising: The fatty acid conjugated prodrug compound I of the STING agonist and the fatty acid conjugated prodrug compound II of the IDO1 inhibitor are dissolved in an organic solvent, a pegylated lipid molecule is added and dissolved sufficiently, and the dissolved solution is injected into water to obtain uniformly dispersed co-assembled nanoparticles.

9. Use of the co-assembled nanoparticles comprising the fatty acid conjugated prodrug compound of the STING agonist and the IDO1 inhibitor according to any one of claims 1-6 in the preparation of a medicament for treating cancer.

10. Use according to claim 9, characterized in that, The cancer is selected from one or more of breast cancer, melanoma, colon cancer, lung cancer, pancreatic cancer, renal cancer, head and neck cancer, lymphoma, skin cancer, urothelial cancer, gastric cancer, or hepatocellular carcinoma.

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