Pharmaceutical composition as well as preparation and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a pharmaceutical composition as well as preparation and application thereof. The invention provides a pharmaceutical composition. The pharmaceutical composition comprises a PCSK9 inhibitor and an STING agonist. According to the pharmaceutical composition, by virtue of synergism of biological functions and chemical properties of the PCSK9 inhibitor and the STING agonist, a cGAS-STING signal channel is powerfully activated, a preparation process is simplified, namely, rapid and large-scale preparation can be realized by adopting a microfluidic method, industrial production and amplification are easy, and the pharmaceutical composition has huge potential in clinical tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a pharmaceutical composition and its preparation and application. Background Art

[0002] Tumor immunotherapy is a breakthrough treatment strategy in cancer treatment. It can stimulate the immune system to enable cancer patients to obtain a lasting protective anti-tumor response. This treatment strategy has made rapid progress in the past 30 years. It currently shows significant and lasting clinical benefits in some refractory tumors and is widely used in the treatment of various types of cancer. However, due to the complexity and heterogeneity of the tumor immune microenvironment, in actual clinical practice, there are still a large number of solid tumor patients who are unable to produce sufficient anti-tumor immune response to tumor immunotherapy, resulting in immunotherapy failing to show satisfactory results in certain clinical settings. Therefore, how to improve the tumor microenvironment and enhance the tumor immune response has become an important issue that needs to be solved urgently.

[0003] Infiltrating T cells at the tumor site are key factors in mediating effective anti-tumor immune responses, however, tumors can support immune evasion by establishing an immunosuppressive environment. The tumor microenvironment is highly heterogeneous in different tumors. According to the type, number, and location of immune cells at the tumor site, the tumor immune microenvironment can be roughly divided into two categories: hot tumors and cold tumors. Among them, hot tumors have higher cytotoxic T lymphocyte infiltration and respond well to immunotherapy, but unfortunately, in more cases, malignant tumors are cold tumors that are insensitive to immunotherapy and lead to poor patient prognosis and shortened overall survival. Therefore, finding a strategy that can effectively activate the immune response of cold tumors can effectively solve the problem of poor immune response at the tumor site.

[0004] An important feature of hot tumors is the high expression of type I interferon (IFN) and IFN-induced genes. It has been found that IFN and CD8 + There is a positive correlation between T cells and related cell markers. IFN is crucial in the early stages of the initiation of anti-tumor immune responses, and mice lacking interferon receptors cannot induce tumor-specific CD8 + Proliferation and differentiation of T cells. IFN is mainly secreted by innate immune cells, dendritic cells (DC). DC cells are important antigen presenting cells in anti-immune responses and are also the first core immune cells to respond to tumors and regulate immune responses. In the early stages of tumorigenesis, DC cells first infiltrate the tumor site and appear in the tumor-draining lymph nodes. The lack of IFN signals directly affects CD8 +Proliferation, recruitment and activation of T cells. Therefore, IFN plays a crucial bridging role in anti-tumor immunity. The ability of tumor-infiltrating dendritic cells to secrete IFN is a key factor affecting tumor T cell infiltration. Therefore, finding a suitable strategy to increase the expression of IFN in tumor-infiltrating dendritic cells is expected to reshape the tumor microenvironment and make it possible to transform cold tumors with inert immune response into hot tumors with immune activation.

[0005] The cGAS-STING signaling pathway is the core signaling pathway that regulates the secretion of IFN by dendritic cells. Dendritic cells sense exogenous DNA and initiate innate immune responses through this signaling pathway. However, due to the important immunoregulatory function of this pathway, the activation of this signaling pathway is also strictly regulated by the body. DNA is an important pathogen-associated molecular pattern of host cells during microbial or viral infection. When it is captured by dendritic cells, it can be bound by cGAS in the cytoplasm and activate the enzymatic activity of cGAS, and synthesize 2′3′-cGAMP with ATP and GTP as substrates; then 2′3′-cGAMP binds to STING located in the endoplasmic reticulum, and after sensing the signal, STING initiates vesicle transport from the endoplasmic reticulum to the Golgi apparatus; after reaching the Golgi apparatus, STING further recruits and activates phosphorylated TBK1 and forms protein dimers; protein dimers jointly recruit IRF3 and phosphorylate and dimerize IRF3; phosphorylated and dimerized IRF3 exerts transcription factor activity and eventually enters the cell nucleus to initiate the expression of type I interferon and other immune regulatory factors. In order to avoid uncontrolled over-activation of the cGAS-STING signaling pathway, the body has adopted a variety of methods to limit the signal transduction of the pathway, resulting in the activation of the cGAS-STING signaling pathway involving multiple biological processes. The key links include not only the activation of the signal receptor STING, but also the trans-organelle transport of STING and the formation of the STING-TBK1 complex. The lack of regulation of any key link will affect the agonist efficiency of the cGAS-STING signaling pathway. The complex regulatory process of the cGAS-STING signaling pathway not only makes the development of STING agonists challenging, but also provides opportunities for the development of more efficient STING agonist strategies.

[0006] The current cGAS-STING signaling pathway agonist strategy is mainly based on single-target agonists developed based on the signal receptor STING protein. By giving the signal receptor STING pathway initiation signal, downstream signal transduction is initiated. A series of STING agonists developed based on this signal pathway have shown great application potential in tumor immunotherapy. According to the structural characteristics, they can be divided into cyclic dinucleotides (CDN) STING agonists and metal ion STING agonists. Among them, manganese ions are the STING agonists with the most clinical application potential discovered in recent years. They can activate cGAS and then activate STING to finally initiate signal transduction. Compared with other organic small molecule STING agonists, manganese ions not only have better physical and chemical stability and lower use costs, but are also the only dietary supplement approved by the FDA for clinical use. Therefore, the use of manganese ions for tumor immunotherapy through systemic administration has great clinical application prospects. However, compared with organic small molecule STING agonists, manganese ions have problems such as unstable pharmacokinetics and poor distribution in tumor tissues. Therefore, how to improve the pharmacokinetics and tumor accumulation of manganese ions and thus enhance the agonist efficiency of the cGAS-STING signaling pathway has become an urgent problem that needs to be solved.

[0007] At present, a large number of studies have attempted to solve the above-mentioned obstacles to the clinical application of manganese ions. Among them, fixing free manganese ions by mineralization and using nanoliposomes to load and improve the physical properties of precipitation is a very promising solution strategy. The current relatively successful representative solutions include: inorganic phosphate-manganese mineralized core nanoliposomes using the traditional microemulsion preparation process, and cyclic dinucleotides (CDN)-manganese ore core nanoliposomes using the traditional film dispersion preparation process. Although the former study solves the problem of manganese ion delivery and makes efficient systemic administration of metal ions possible, the efficiency of STING agonists based on manganese ions still has a lot of room for improvement. Although the latter uses biologically active CDN to mineralize manganese ions and prepare nanoparticles based on the former, CDN and manganese ions are agonized for the same target, and the agonist efficiency is still limited by the trans-organelle transport and TBK1 recruitment process after STING activation. In addition, both solutions have the problem that the preparation process is complicated and not conducive to industrial scale-up and further clinical transformation.

[0008] Although existing research has brought hope for solving key problems, existing solutions still cannot break through the limitations of traditional single-target STING stimulant strategies and traditional nanoliposome preparation processes and delivery system delivery efficiency, resulting in the cGAS-STING stimulant efficiency of manganese ions being firmly limited by the STING protein signal perception ability, while the downstream effector link that affects the signal transduction efficiency more critically: STING's trans-organelle transport process is completely lacking in regulation; the complex nanoliposome preparation process also leads to the liposomes that have shown great clinical application potential in preclinical studies being abandoned due to the inability to industrial standardized production. Therefore, as research in this field gradually deepens, there is an urgent need to provide more efficient STING stimulant strategies and new nanoliposome preparation processes. Summary of the invention

[0009] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, one object of the present invention is to provide a pharmaceutical composition and its preparation and use. The pharmaceutical composition provided by the present invention includes a PCSK9 inhibitor and a STING agonist, and utilizes the synergy of the biological functions and chemical properties of the two to achieve the biological purpose of simultaneously regulating STING activation and STING trans-organelle transport, and ultimately strongly activating the cGAS-STING signaling pathway. At the same time, a microfluidics-based preparation process has been developed to further solve the subsequent industrial amplification problem, which has great potential.

[0010] To this end, the present invention provides a pharmaceutical composition in a first aspect, the pharmaceutical composition comprising a PCSK9 inhibitor and a STING agonist;

[0011] The STING agonist comprises at least one selected from manganese ions and cyclic dinucleotides.

[0012] The inventors found that PCSK9 inhibitors that can inhibit the expression and function of PCSK9 protein can not only be used to treat hyperlipidemia and cardiovascular diseases, but also have unexpected advantages in tumor immunotherapy, that is, they can affect the activation of the STING pathway in a way that is independent of STING agonists. By combining PCSK9 inhibitors with STING agonists, it was found that the two synergistically and effectively activated the STING signaling pathway and the release of type I interferon, while the use of PCSK9 inhibitors or STING agonists alone lacked this effect.

[0013] According to an embodiment of the present invention, the cyclic dinucleotide includes at least one selected from cGAMP and c-di-AMP.

[0014] According to an embodiment of the present invention, the pharmaceutical composition further comprises a drug delivery carrier.

[0015] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0016] According to an embodiment of the present invention, the drug delivery carrier has a phospholipid shell modified with polysaccharides, and the phospholipid shell encapsulates the PCSK9 inhibitor and the STING agonist. By modifying the surface of the phospholipid shell with polysaccharides, such as modifying the natural polysaccharide hyaluronic acid with CD44 targeting and tumor tissue targeting functions, the drug composition is further endowed with CD44 targeting and tumor targeting accumulation capabilities.

[0017] According to an embodiment of the present invention, the PCSK9 inhibitor includes at least one selected from siRNA targeting PCSK9, antibodies that specifically bind to PCSK9 protein, and small molecule compounds that inhibit PCSK9 secretion by inhibiting vesicle transport from the endoplasmic reticulum to the Golgi apparatus.

[0018] According to an embodiment of the present invention, the small molecule compound includes at least one selected from AL-1576 and CVI-LM001.

[0019] According to an embodiment of the present invention, the PCSK9 inhibitor is selected from siRNA targeting PCSK9.

[0020] According to an embodiment of the present invention, the phospholipid shell includes at least one selected from cationic lipids, polyethylene glycol lipids, and auxiliary lipids.

[0021] According to an embodiment of the present invention, the cationic lipid includes at least one selected from Dlin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP, DOTAP, DOTMA, and DC-Chol.

[0022] According to an embodiment of the present invention, the polyethylene glycol lipid includes at least one selected from DMG-PEG, DSPE-PEG, DOPE-PEG, and DSG-PEG.

[0023] According to an embodiment of the present invention, the helper lipid includes at least one selected from DOPE, DSPC, DPPC, DMPC, DOPC, DSPE, and ALC-0159.

[0024] According to an embodiment of the present invention, the molar ratio of the cationic lipid, the polyethylene glycol lipid, and the auxiliary lipid is (1-1): (1-10): (1-50).

[0025] According to an embodiment of the present invention, the phospholipid shell comprises DOTAP, DSPE-PEG, DOPE.

[0026] According to an embodiment of the present invention, the polysaccharide includes at least one selected from hyaluronic acid, sodium alginate, dextran and derivatives thereof.

[0027] According to an embodiment of the present invention, the polysaccharide is selected from hyaluronic acid.

[0028] The second aspect of the present invention provides a method for preparing the pharmaceutical composition according to the first aspect, comprising:

[0029] (1) preparing a PCSK9 inhibitor solution and a STING agonist solution respectively, and adding the STING agonist solution to the PCSK9 inhibitor solution to obtain an aqueous phase component;

[0030] (2) preparing a phospholipid shell that is not modified with polysaccharide to obtain an alcohol phase component;

[0031] (3) Mixing the aqueous phase component and the alcohol phase component, and adding the polysaccharide solution thereto to obtain the pharmaceutical composition.

[0032] The present invention makes full use of the chemical characteristics of the chelation of PCSK9 inhibitors and STING agonists, abandons the traditional microemulsion and film dispersion-based liposome preparation process, and develops a preparation strategy suitable for industrial scale-up production that is significantly superior to existing solutions in terms of stability and controllability. This preparation method has unique advantages in the preparation of functional drug carriers, greatly simplifies the preparation process, is conducive to industrial production scale-up, and also uses safer production excipients, which is conducive to the clinical application transformation of the nucleic acid drug delivery system.

[0033] According to an embodiment of the present invention, in step (1), the concentration of the PCSK9 inhibitor solution is 1-999 μg / ml, and the concentration of the STING agonist solution is 1-999 mmol / ml.

[0034] According to an embodiment of the present invention, the step (3) further comprises:

[0035] The aqueous phase component and the alcohol phase component are mixed through a microfluidic chip.

[0036] According to an embodiment of the present invention, the flow rate during the mixing process through the microfluidic chip is 5-20 ml / min.

[0037] According to an embodiment of the present invention, the volume ratio of the water phase component to the alcohol phase component is (1:1)-(1:10).

[0038] According to an embodiment of the present invention, the preparation method further comprises:

[0039] The obtained pharmaceutical composition is dialyzed and filtered.

[0040] The third aspect of the present invention provides use of the pharmaceutical composition described in the first aspect in preparing a drug for treating tumors, preventing tumor recurrence or metastasis.

[0041] According to an embodiment of the present invention, the tumor includes at least one selected from colon cancer, breast cancer, melanoma, and lung cancer.

[0042] The fourth aspect of the present invention provides a vaccine, which comprises an antigen and the pharmaceutical composition described in the first aspect.

[0043] According to an embodiment of the present invention, the antigen comprises a tumor antigen or a pathogenic bacteria antigen.

[0044] The fifth aspect of the present invention provides use of the vaccine described in the fourth aspect in preventing or treating tumors or infectious diseases.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The cGAS-STING signaling pathway agonist strategy is more efficient: The present invention provides a cGAS-STING agonist strategy based on biological synergy, which utilizes the synergy of the biological functions of PCSK9 inhibitors and STING agonists, breaking through the limitations of traditional single-target STING agonist development strategies on the immunotherapy potential of the cGAS-STING signaling pathway, establishing a biological basis for the discovery of more efficient STING agonist strategies in the future, and also establishing a new platform for nanoparticle delivery systems for systemic cancer treatment;

[0047] (2) The preparation process is simple and easy to scale up for industrial production: This preparation method makes full use of the chemical properties of PCSK9 inhibitors and STING agonists in alkaline environment chelation / acidic environment dissociation, greatly simplifies the preparation process, and not only achieves the goal of rapid, controllable and standardized preparation of pharmaceutical compositions loaded with PCSK9 inhibitors and STING agonists mineralized cores, but also provides convenience for further optimizing the surface properties of pharmaceutical compositions. The microfluidic process used in the present invention is significantly superior to traditional microemulsion and thin film dispersion methods in terms of stability and controllability, which is conducive to the application of the process to large-scale industrial production, and ultimately achieves the clinical goal of initiating a strong anti-tumor immune response by systemic intravenous injection and delivery of active components.

[0048] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0050] Figure 1 The flowchart of preparing the pharmaceutical composition based on the microfluidic method in Example 1 of the present invention is shown;

[0051] Figure 2 Figure 2 shows the characterization and in vitro efficacy verification of the pharmaceutical composition in Example 2 of the present invention. Figure A shows the siPCSK9-Mn prepared by microfluidics. 2+ HLNP, Mn 2+ Particle size comparison of HLNP, Blank HLNP and siPCSK9 HLNP; Figure B shows the particle size of siPCSK9-Mn 2+ Acid response release of HLNPs in an acidic environment simulating endosomes (pH = 4.5) versus physiological pH (pH = 7.4); Figure C shows the release of Blank HLNPs, scrambled siRNA (labeled as Ctrl-siRNA) and PCSK9-siRNA in response to Mn 2+ Comparison of the activity of synergistically inducing BMDC to secrete IFN-β; Figure D shows the activity of siPCSK9-Mn 2+ Quantification of siRNA in HLNPs; Panel E shows the quantification of Blank HLNPs, siPCSK9 HLNPs, Mn 2+ HLNPs and siPCSK9-Mn 2+ Comparison of HLNP-induced BMDC secretion of IFN-β; Figure F shows the detection of Ctrl-siRNA-Mn by Western blot 2+ HLNP, siPCSK9 HLNP, Mn 2+ HLNPs and siPCSK9-Mn 2+ Figure 1 shows the results of HLNP-induced phosphorylation of TBK1 and STING in BMDC cells; Figure G shows the flow cytometry detection of Ctrl-siRNA-Mn 2+ HLNP, Mn 2+ HLNPs, siPCSK9 HLNPs, and siPCSK9-Mn 2+ The result of HLNP-induced CD86-positive cell population in BMDC cells; Figure H is the result obtained by quantitative analysis of Figure G; Figures I and J respectively show the synergistic effect of c-di-AMP and cGAMP when used in combination with PCSK9 siRNA;

[0052] Figure 3 It shows that siPCSK9-Mn in Example 2 of the present invention 2+The therapeutic effect of HLNP in MC38 subcutaneous tumor model. Figure A shows the animal modeling and specific dosing regimen; Figures B, EH show the results of untreated, siPCSK9HLNP, Mn 2+ HLNPs and siPCSK9-Mn 2+ Tumor size change curves of mice treated with HLNP preparations; Figure C shows the tumor size change curves of mice treated with siPCSK9 HLNPs, Mn 2+ HLNPs and siPCSK9-Mn 2+ Figure D shows the body weight change curves of mice without treatment, mice administered with siPCSK9 HLNP, and mice treated with Mn 2+ HLNPs and siPCSK9-Mn 2+ Figure I shows the survival rate of mice without treatment, mice administered siPCSK9 HLNP, Mn 2+ HLNPs and siPCSK9-Mn 2+ Representative images of tumors in mice treated with HLNPs on day 15 of treatment; Figures J and K show flow cytometry analysis of tumors in mice without treatment, treated with siPCSK9 HLNPs, and Mn 2+ HLNPs and siPCSK9-Mn 2+ HLNP preparations induce CD8 cell populations in mouse tumor tissues; Figure L shows the administration of Mn 2+ HLNP, siPCSK9HLNP, siPCSK9-Mn 2+ Tumor accumulation within 72 h after HLNP preparation;

[0053] Figure 4 It shows that siPCSK9-Mn in Example 2 of the present invention 2+ The therapeutic effect of HLNP in 4T1 orthotopic tumor model. Figure A shows the untreated, siPCSK9 HLNP, Mn 2+ HLNPs and siPCSK9-Mn 2+ In vivo imaging of tumor sites in mice treated with HLNP formulations; Figure B shows tumors in mice without treatment, treated with siPCSK9 HLNPs, and Mn 2+ HLNPs and siPCSK9-Mn 2+ Figure 2 shows the fluorescence signal intensity of tumor cells in the lesion sites of mice treated with HLNP preparations; Figure C shows the fluorescence signal intensity of tumor cells in the lesion sites of mice treated with siPCSK9 HLNPs, Mn 2+ HLNPs and siPCSK9-Mn 2+ Body weight change curve of mice treated with HLNP preparation. DETAILED DESCRIPTION

[0054] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0055] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0056] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0057] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0058] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0059] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0060] Definitions and explanations of terms

[0061] The term "PCSK9 inhibitor" is understood to mean an agent capable of inhibiting the synthesis of PCSK9 during its transcription and post-transcriptional modification, as well as during its intracellular vesicular transport and protein secretion after translation.

[0062] The term "cGAMP" is cyclic GMP-AMP, which is a diffusible cyclic dimeric nucleotide.

[0063] The term "c-di-AMP" is cyclic dimer adenosine monophosphate.

[0064] The term "AL-1576" is a SNO-CoA reductase 2 inhibitor, which increases the inactivation of PCSK9 caused by nitric oxide by inhibiting the activity of SNO-CoA reductase 2.

[0065] The term "CVI-LM001" refers to an oral small molecule PCSK9 synthesis inhibitor developed by CVI Pharmaceuticals.

[0066] The term "pharmaceutically acceptable" indicates that the pharmaceutical composition can be administered to a subject without producing adverse physiological reactions that prevent the administration of the pharmaceutical composition. For example, "pharmaceutically acceptable excipients" refer to excipients useful in preparing generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of these excipients or diluents include, but are not limited to, water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, liposomes and non-aqueous vehicles, such as fixed oils, may also be used.

[0067] The term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and agents for delayed absorption, etc. that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition. Of course, pharmaceutically acceptable carriers may also include trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, to extend the shelf life or efficacy of the antibody.

[0068] According to an embodiment of the present invention, a first aspect of the present invention provides a pharmaceutical composition, comprising a PCSK9 inhibitor and a STING agonist;

[0069] The STING agonist comprises at least one selected from manganese ions and cyclic dinucleotides.

[0070] PCSK9 (proprotein convertase subtilisin / kexin9) is a secreted protein that plays an important role in regulating cholesterol metabolism. It can inhibit the liver's absorption and metabolism of cholesterol by binding to the LDLR receptor in the liver to mediate LDLR degradation. A series of antibodies and siRNA drugs developed with PCSK9 as a therapeutic target have been shown to have therapeutic effects in hyperlipidemia and cardiovascular diseases. However, inhibiting PCSK9 protein expression and function can also bring unexpected effects in tumor immunotherapy, and can affect STING pathway activation in a way that is independent of STING agonists [3,4][2,3]. Based on this, the inventors combined PCSK9 inhibitors (such as siRNA that specifically binds to PCSK9) with STING agonists (such as manganese ions) and found that the two synergistically activated the STING signaling pathway and the release of type I interferon in a component-dependent manner, while the combination of scrambled siRNA and manganese ions as a control lacked this effect.

[0071] In addition, due to the influence of the structure and function of different types of PCSK9 inhibitors and / or STING agonists, as well as the differences in the responses of different cell lines and animals / human bodies to PCSK9 inhibitors / STING agonists, the content and ratio of PCSK9 inhibitors and STING agonists cannot be accurately defined. In theory, any drug content, ratio and combination that can achieve the goal of inhibiting PCSK9 expression in vivo and the goal of stimulating the cGAS-STING signaling pathway are within the protection scope of the present invention.

[0072] According to an embodiment of the present invention, the pharmaceutical composition further comprises a drug delivery carrier. The drug combination of the present invention is not limited by the drug delivery carrier, and any drug carrier that can use the drug combination systemically through intravenous administration can exert the effect of tumor treatment and is within the protection scope of the present invention.

[0073] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0074] According to an embodiment of the present invention, the drug delivery carrier has a phospholipid shell modified with polysaccharide, and the phospholipid shell encapsulates the PCSK9 inhibitor and the STING agonist.

[0075] According to an embodiment of the present invention, the PCSK9 inhibitor includes at least one selected from siRNA targeting PCSK9, an antibody specifically binding to the PCSK9 protein, and a small molecule compound.

[0076] According to a specific embodiment of the present invention, the antibody can be Alirocumab, Evolocumab, SHR-1209, AK102, IBI306, etc. The siRNA can be the marketed Inclisiran and the siRNA with similar structure and function used in the present invention. The small molecule compound can be AL-15761, CVI-LM00 and other structural and functional analogs.

[0077] The PCSK9 inhibitors used in the present invention are not limited to the above preparations. Other substances that can inhibit the expression and function of PCSK9 protein, including antibodies, siRNA and small molecule compounds with similar biological functions, can also be used in the present invention.

[0078] According to an embodiment of the present invention, the PCSK9 inhibitor is selected from siRNA targeting PCSK9. siRNA and STING agonist have the chemical properties of chelation in alkaline environment / dissociation in acidic environment, which provides a strong basis for the efficient preparation of pharmaceutical compositions.

[0079] According to an embodiment of the present invention, the phospholipid shell includes at least one selected from cationic lipids, polyethylene glycol lipids, and auxiliary lipids. The components of the phospholipid shell of the present invention include but are not limited to cationic lipids, polyethylene glycol lipids, and auxiliary lipids. For example, steroid lipids can be additionally added according to the chemical properties of the phospholipids.

[0080] According to an embodiment of the present invention, the cationic lipid includes at least one selected from Dlin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP, DOTAP, DOTMA, and DC-Chol.

[0081] According to an embodiment of the present invention, the polyethylene glycol lipid includes at least one selected from DMG-PEG, DSPE-PEG, DOPE-PEG, and DSG-PEG.

[0082] According to an embodiment of the present invention, the auxiliary lipid includes at least one selected from DOPE, DSPC, DPPC, DMPC, DOPC, DSPE, and ALC-0159. The selection of cationic lipids, polyethylene glycol lipids, and auxiliary lipids in the components of the phospholipid shell includes but is not limited to the above specific selections, and other lipids with similar functions can be used in the preparation of the phospholipid shell of the present invention.

[0083] According to an embodiment of the present invention, the molar ratio of the cationic lipid, the polyethylene glycol lipid, and the auxiliary lipid is (1-10): (1-10): (1-50).

[0084] According to an embodiment of the present invention, the phospholipid shell includes DOTAP, DSPE-PEG, and DOPE.

[0085] According to an embodiment of the present invention, the polysaccharide includes at least one selected from hyaluronic acid, sodium alginate, dextran and derivatives thereof.

[0086] According to an embodiment of the present invention, the polysaccharide is selected from hyaluronic acid.

[0087] According to an embodiment of the present invention, the second aspect of the present invention provides a method for preparing the pharmaceutical composition according to the first aspect, comprising:

[0088] (1) preparing a PCSK9 inhibitor solution and a STING agonist solution respectively, and adding the STING agonist solution to the PCSK9 inhibitor solution to obtain an aqueous phase component;

[0089] (2) preparing a phospholipid shell that is not modified with polysaccharide to obtain an alcohol phase component;

[0090] (3) Mixing the aqueous phase component and the alcohol phase component, and adding the polysaccharide solution thereto to obtain the pharmaceutical composition.

[0091] The present invention makes full use of the chemical characteristics of the chelation of PCSK9 inhibitors and STING agonists, and uses microfluidics to achieve rapid, large-scale and stable production of the pharmaceutical composition provided by the present invention. At the same time, the controllability of the method is significantly better than that of the prior art.

[0092] According to an embodiment of the present invention, in step (1), the concentration of the PCSK9 inhibitor solution is 1-999 μg / ml, and the concentration of the STING agonist solution is 1-999 mmol / ml.

[0093] According to an embodiment of the present invention, step (3) further comprises:

[0094] The aqueous phase component and the alcohol phase component are mixed through a microfluidic chip.

[0095] According to an embodiment of the present invention, the flow rate during the mixing process through the microfluidic chip is 5-20 ml / min.

[0096] According to an embodiment of the present invention, the volume ratio of the water phase component to the alcohol phase component is (1:1)-(1:10).

[0097] According to an embodiment of the present invention, the preparation method further comprises:

[0098] The obtained pharmaceutical composition is dialyzed and filtered to meet the requirements of subsequent cell treatment or animal administration.

[0099] According to an embodiment of the present invention, the third aspect of the present invention provides use of the pharmaceutical composition described in the first aspect in preparing a drug, wherein the drug is used to treat tumors, prevent tumor recurrence or metastasis.

[0100] According to an embodiment of the present invention, the tumor includes at least one selected from colon cancer, breast cancer, melanoma, and lung cancer.

[0101] According to an embodiment of the present invention, a fourth aspect of the present invention provides a vaccine, comprising an antigen and the pharmaceutical composition described in the first aspect.

[0102] According to an embodiment of the present invention, the antigen includes a tumor antigen or a pathogenic bacteria antigen.

[0103] According to an embodiment of the present invention, the fifth aspect of the present invention provides use of the vaccine described in the fourth aspect in preventing or treating tumors or infectious diseases.

[0104] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0105] Example 1 Preparation of a pharmaceutical composition having a drug delivery carrier

[0106] For detailed process, see Figure 1 .

[0107] (1) Preparation of microfluidic aqueous phase components:

[0108] To the PCSK9-siRNA with a final concentration of 200-1000 μg / ml, a manganese ion solution with a final concentration of 20-60 mmol / L is added dropwise under vortexing conditions. The solution obtained in this step is used as the aqueous flow channel working fluid in the later microfluidic preparation process;

[0109] Among them, the “final concentration” is based on the volume of the prepared microfluidic aqueous phase component;

[0110] (2) Preparation of microfluidic alcohol phase components:

[0111] The phospholipid shell components are selected as DOTAP, DOPE and DSPE-PEG2K, with concentrations of 42.5 mg / ml, 42.5 mg / ml and 15 mg / ml respectively, and dissolved in ethanol. The solution obtained in this step is used as the organic phase flow channel working fluid in the later microfluidic preparation process;

[0112] (3) Microfluidic preparation of pharmaceutical compositions:

[0113] The obtained aqueous phase component and alcohol phase component were loaded into a microfluidic chip at a volume ratio of 3:1, and the flow rate was set to 8 ml / min to prepare a pharmaceutical composition loaded with siRNA and manganese ions;

[0114] Under vortex shaking conditions, hyaluronic acid (HA) with a final concentration of 2 mg / ml was added dropwise to the solution containing lipid nanoparticles to obtain a pharmaceutical composition with surface modified hyaluronic acid (expressed as PCSK9-siRNA-Mn 2+ HLNPs or siPCSK9-Mn 2+ HLNP);

[0115] Here, the "final concentration" is based on the volume of the prepared pharmaceutical composition.

[0116] To meet the needs of cell treatment or animal administration, the preparation method of the pharmaceutical composition may further include:

[0117] (4) Dialysis and filtration:

[0118] The HLNPs prepared in step (3) were transferred to a dialysis tube for dialysis. The dialysis external solution was 2 L of 0.9% NaCl (pH = 8). The dialysis was performed overnight at 4°C. Before use, the HLNPs were sterile filtered with a 220 nm filter membrane, and then used for subsequent cell treatment or animal administration.

[0119] Example 2 Effect Verification

[0120] The preparation method in Example 1 was used to prepare a pharmaceutical composition loaded with scrambled siRNA and manganese ions (Ctrl-siRNA-Mn 2+ HLNPs), blank HLNPs, PCSK9-siRNA-loaded HLNPs (siPCSK9HLNPs), scrambled siRNA-loaded HLNPs (Ctrl-siRNAHLNPs), and Mn 2+ The pharmaceutical composition (Mn 2+ HLNP) was used for control experiments. PCSK9-siRNA includes a sense strand (PCS-A2-S) and an antisense strand (PCS-A2-AS), and scrambled siRNA also includes a sense strand (Ctrl-S) and an antisense strand (Ctrl-AS). The corresponding sequences are shown in Table 1.

[0121] Table 1 siRNA sequence list

[0122]

[0123] Among them, stability modification of siRNA can not only improve its delivery efficiency in vivo, but also reduce the immunogenicity of siRNA. In addition, 2'OMe represents that the hydroxyl group at the 2' position of ribose is replaced by a methoxy group, the lowercase base is the site of methoxy chemical modification, dT*dT represents that there are two deoxyribonucleic acids dTdT hanging in a single strand at the 3' end of each chain, and * represents the phosphorothioate modification between the two deoxyribonucleic acids.

[0124] (1) Characterization of HLNPs

[0125] The prepared HLNPs were characterized, and the ability of different HLNPs at the same dose to activate the cGAS-STING signaling pathway of BMDCs, as well as the ability of PCSK9-siRNA and manganese ions to activate BMDC maturation in a component-dependent manner were tested. Figure 2 shown.

[0126] The experimental results show that the microfluidic process can continuously, stably and controllably prepare HLNPs. 2+ HLNP and Mn 2+ HLNP, the particle size is between 80-100nm (see Figure 2 Figure A), and siPCSK9-Mn 2+ HLNPs can achieve acid-responsive release in an acidic environment (pH = 4.5) simulating endosomes compared to physiological pH (pH = 7.4) (see Figure 2 (Fig. B).

[0127] By determination, PCSK9-siRNA-Mn 2+ HLNP, Ctrl-siRNA-Mn 2+ HLNP, Mn 2+ Mn of HLNP 2+ The loading capacity is 1.8-2mM. The principle and method of manganese ion determination and the preparation method of related reagents refer to the "National Standard of the People's Republic of China - Determination of Manganese in Water Quality - Potassium Periodate Spectrophotometer Method - GB11906-89". Specifically, the method for detecting the manganese ion encapsulation rate of the preparation is: take PCSK9-siRNA-Mn 2+ HLNP, Ctrl-siRNA-Mn 2+ HLNP, Mn 2+ HLNP 5μl, then add 0.5% TritonX-10038μl for lysis, then detect according to the method described in GB11906-89, the total detection system is proportionally reduced to 200μl, and finally detected in a 96-well plate, and the concentration is calculated using the manganese ion standard curve determined by the same method.

[0128] Then the PCSK9-siRNA-Mn prepared in Example 1 2+ The HLNP preparation was diluted 2 times, and the siRNA concentration was adjusted from 200 μg / ml to 100 μg / ml. Then 1 μl of the preparation was added with 19 μl of 0.5% TritonX-100 for lysis, and then 4 μl of 6X Loading Buffer was added for agarose gel electrophoresis. Finally, the siRNA standard of 100 pg was compared for quantification. The results showed that PCSK9-siRNA-Mn 2+ The siRNA encapsulation efficiency of HLNPs was 98.46% (see Figure 2 (Fig. D).

[0129] In addition, compared with Ctrl-siRNA-Mn 2+ For HLNP, PCSK9-siRNA and Mn 2+ Combined use can significantly activate BMDC to secrete type I interferon, and this effect is sequence-dependent (see Figure 2 Figure C). PCSK9-siRNA and Mn 2+ The STING activation effect of PCSK9-siRNA-Mn 2+ The secretion of type I interferon by BMDCs treated with HLNPs was much higher than that of siPCSK9 HLNPs and Mn 2+ HLNP (see Figure 2 Figure E), indicating that inhibition of PCSK9 expression is associated with Mn 2+ The STING agonism of PCSK9 has a strong biological synergy. Western blot (WB) analysis of TBK1 and STING phosphorylation also increased significantly when PCSK9 expression was reduced, indicating that the STING signaling pathway was strongly activated. This result is consistent with the results of Figure E (see Figure 2 F), flow cytometry analysis of the number of CD86-positive cells in BMDC cells (see Figure 2 The same trend was also shown in Figure GH (Fig. 3B), and the CD86-positive cell population, a mature marker of BMDC cells, was also found in the siPCSK9-Mn 2+ HLNPs resulted in a component-dependent increase, further demonstrating that the inhibition of PCSK9 expression was associated with Mn 2+The STING agonism has potential biological synergy. Using the reporter cell THP-1ISG that can specifically characterize the activation of the STING signaling pathway, we analyzed the synergistic effects of two other STING agonists, c-di-AMP and cGAMP, with PCSK9 siRNA. The results showed that the combination of these two STING agonists and siPCSK9 can also achieve strong activation of the cGAS-STING signaling pathway (see Figure 2 (Fig. 1J).

[0130] (2) siPCSK9-Mn 2+ The therapeutic effect of HLNP in MC38 subcutaneous tumor model

[0131] siPCSK9-Mn was validated in vitro 2+ After HLNP induced a strong biological synergy of STING activation in BMDC cells, siPCSK9-Mn was verified in vivo. 2+ The therapeutic effect of HLNP on MC38 subcutaneous tumor model, the results are as follows Figure 3 shown.

[0132] In the in vivo experiment, the effects of siPCSK9 HLNP and siPCSK9-Mn on the 2+ HLNP and Mn 2+ The therapeutic effect of HLNP preparation on MC38 colon cancer subcutaneous mouse model. Figure 3 As shown in Figure A, siPCSK9 (100 μg / dose), Mn 2+ MC38 tumor-bearing mice were treated with the drugs (60 μg / dose) separately or in combination, with tail vein injection every three days for three times. During the period, the tumor size and body weight changes of each group were detected and monitored for 40 days.

[0133] The experimental results showed that the siPCSK9 HLNP and Mn 2+ Compared with HLNP, administration of siPCSK9-Mn 2+ HLNP has obvious therapeutic advantages, which can significantly inhibit tumor growth and significantly improve animal survival 25 days after stopping administration (see Figure 3 BI Figure), and the body weight did not decrease by more than 10%, indicating that siPCSK9-Mn 2+ HLNPs can activate tumor immunity and have good safety (see Figure 3 In addition, the immune response of tumor tissues showed that siPCSK9-Mn 2+ Compared with the nanoformulations loaded with single components, the infiltration of CD8 cells in tumor tissues was also significantly increased (see Figure 3 Meanwhile, the tumor tissue experiment of nanoformulation also showed that the PCSK9-siRNA-Mn prepared in Example 1 2+ After HLNPs were fluorescently labeled, tumor accumulation of the agent within 72 hours could be observed in tumor tissue (see Figure 3 (Fig. L).

[0134] (3) siPCSK9-Mn 2+ The therapeutic effect of HLNP in 4T1 orthotopic tumor model

[0135] Next, siPCSK9-Mn was tested in an orthotopic mouse model of breast cancer. 2+ The therapeutic effect of HLNP was determined using the same dosing regimen as described above for the MC38 colon cancer tumor model. In vivo imaging of the tumor site was performed over a two-week period (see Figure 4 Figure A) found that siPCSK9-Mn 2+ Compared with other single-component nanoformulations, HLNP also has obvious therapeutic advantages in the treatment of breast cancer without seriously affecting the weight of animals. The fluorescence signal of tumor cells in the lesion site is significantly reduced (see Figure 4 (Figure BC).

[0136] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0137] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a PCSK9 inhibitor and a STING agonist; The STING agonist comprises at least one selected from manganese ions and cyclic dinucleotides; Optionally, the cyclic dinucleotide comprises at least one selected from cGAMP and c-di-AMP.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises a drug delivery vehicle; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

3. The pharmaceutical composition according to claim 2, characterized in that The drug delivery carrier has a phospholipid shell modified with polysaccharide, and the phospholipid shell encapsulates the PCSK9 inhibitor and the STING agonist.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The PCSK9 inhibitor comprises at least one selected from siRNA targeting PCSK9, antibodies specifically binding to PCSK9 protein, and small molecule compounds inhibiting PCSK9 secretion by inhibiting vesicle transport from the endoplasmic reticulum to the Golgi apparatus; Optionally, the small molecule compound includes at least one selected from AL-1576 and CVI-LM001; Optionally, the PCSK9 inhibitor is selected from siRNA targeting PCSK9.

5. The pharmaceutical composition according to claim 3, characterized in that The phospholipid shell comprises at least one selected from cationic lipids, polyethylene glycol lipids, and auxiliary lipids; Optionally, the cationic lipid comprises at least one selected from Dlin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, DODMA, DODAP, DOTAP, DOTMA, DC-Chol; Optionally, the polyethylene glycol lipid comprises at least one selected from DMG-PEG, DSPE-PEG, DOPE-PEG, and DSG-PEG; Optionally, the helper lipid comprises at least one selected from DOPE, DSPC, DPPC, DMPC, DOPC, DSPE, ALC-0159; Optionally, the molar ratio of the cationic lipid, the polyethylene glycol lipid, and the helper lipid is (1-10): (1-10): (1-50); Optionally, the phospholipid shell comprises DOTAP, DSPE-PEG, DOPE.

6. The pharmaceutical composition according to claim 3, characterized in that The polysaccharide comprises at least one selected from hyaluronic acid, sodium alginate, dextran and derivatives thereof; Optionally, the polysaccharide is selected from hyaluronic acid.

7. A method for preparing the pharmaceutical composition according to any one of claims 3 to 6, characterized in that: include: (1) preparing a PCSK9 inhibitor solution and a STING agonist solution respectively, and adding the STING agonist solution to the PCSK9 inhibitor solution to obtain an aqueous phase component; (2) preparing a phospholipid shell that is not modified with polysaccharide to obtain an alcohol phase component; (3) Mixing the aqueous phase component and the alcohol phase component, and adding the polysaccharide solution thereto to obtain the pharmaceutical composition.

8. The preparation method according to claim 7, characterized in that: In step (1), the concentration of the PCSK9 inhibitor solution is 1-999 μg / ml, and the concentration of the STING agonist solution is 1-999 mmol / ml.

9. The preparation method according to claim 7, characterized in that: The step (3) further comprises: Mixing the aqueous phase component and the alcohol phase component through a microfluidic chip; Optionally, the flow rate during the mixing process through the microfluidic chip is 5-20 ml / min; Optionally, the volume ratio of the aqueous phase component to the alcohol phase component is (1:1)-(1:10).

10. The preparation method according to any one of claims 7 to 9, characterized in that: The preparation method further comprises: The obtained pharmaceutical composition is dialyzed and filtered.

11. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a drug, characterized in that: The drug is used to treat tumors, prevent tumor recurrence or metastasis; Optionally, the tumor comprises at least one selected from colon cancer, breast cancer, melanoma, and lung cancer.

12. A vaccine, characterized in that The vaccine comprises an antigen and the pharmaceutical composition according to any one of claims 1 to 6.

13. The vaccine according to claim 12, characterized in that The antigens include tumor antigens or pathogenic bacteria antigens.

14. Use of the vaccine according to claim 12 or 13 in preventing or treating tumors or infectious diseases.

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