Rapamycin lipid polymer hybrid nanoparticles

Abstract

The present application relates to a nanoparticle comprising a lipid, a polymer and an immunosuppressive agent, wherein the lipid comprises an anionic lipid and / or a PEGylated lipid. Furthermore, the invention also relates to a preparation method of the nanoparticles, a pharmaceutical composition containing the nanoparticles, and application of the nanoparticles and / or the pharmaceutical composition in inducing immune tolerance.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a lipid polymer nanoparticle encapsulating an immunosuppressant, a preparation method thereof, and related applications. Background Art

[0002] Adaptive immune response refers to the whole process of the body's antigen-specific T / B lymphocytes activating, proliferating, and differentiating into effector cells after receiving antigen stimulation, producing a series of biological effects. It includes the process of establishing an adaptive response after the first antigen stimulation, as well as the rapid activation and clearance process after subsequent antigen exposure. Specifically, T and B lymphocytes are activated after recognizing antigens presented by antigen-presenting cells (APCs). After activation, lymphocytes expand and differentiate into effector cells, and mediate the elimination of antigens by inducing humoral or cytotoxic reactions. After the antigen is cleared, the corresponding activated cells decrease, and memory T and B lymphocytes are produced, which rapidly proliferate and play a role during the next activation.

[0003] Specific immune tolerance is generally caused by interference with antigen presentation signals and T or B activation signals during the adaptive immune response, which results in tolerance. For example, APC cells, mainly DC cells, are induced to form tolerant APCs, which in turn induce T cells to transform into specifically tolerant Treg cells, which in turn become tolerant to antigens. Specific immune tolerance technology is of great significance for the treatment of autoimmune diseases and repeated administration of antigenic protein drugs and viral drugs.

[0004] Due to the generation of specific tolerance, the recognition of specific antigens and the interference of immune function need to be carried out simultaneously. Nanoparticles can carry one or more immunogens and immunosuppressants at the same time, and the cargo can be loaded inside or modified on its surface. After the immunogen or immunosuppressant is encapsulated by nanoparticles, it can be protected to a certain extent. In addition, nanoparticles can be provided with targeting functions by adjusting the physicochemical properties (charge, size, etc.) or surface ligand modification, so as to deliver the immunogen or immunosuppressant to immune cells in a targeted manner, which is conducive to reducing the side effects caused by off-target effects and systemic immunosuppression. In view of the above advantages, nanoparticles have become the most promising technology for inducing specific immune tolerance. For example, nanoparticles that synchronously target specific antigen receptors and CD22 on the surface of B cells can induce CD22 and BCR receptor binding, thereby depleting the specific B cell pool, thereby inhibiting the immune response. Nanoparticles loaded with MHC-I type antigen peptide molecules on the surface are used to present antigens to T cells in the absence of stimulation signals, thereby inducing tolerance. Selecta uses PEG-PLA polymer nanoparticles loaded with the immunosuppressant rapamycin and the immunogen for simultaneous injection. It can deliver rapamycin to APC cells to produce APC cells that are tolerant to the corresponding immunogen, thereby inducing Tregs cells, inhibiting the activation and proliferation of B cells and thus inhibiting the production of corresponding antibodies.

[0005] By comparing the above methods, we can see that the method of inducing tolerance by targeting T and B cells has the disadvantages of high targeting requirements and complex screening of targeting ligands. From Selecta's research, APC has a strong phagocytic ability. After the antigen protein and nanoparticle system are administered, they are easily phagocytosed by APC cells. Therefore, the targeting requirements for nanoparticles are relatively low, and the antigen protein and nanoparticles can be administered independently and synchronously. These characteristics mean that immunomodulatory nanoparticles based on targeting APC have great advantages in inducing specific tolerance to therapeutic proteins and AAV gene drugs and inhibiting the production of immune antibodies.

[0006] However, the application results of Selecta's existing immune nanoparticles in inhibiting the immune response of AAV drugs show that the effect is poor when the viral load is high or there are more pre-existing antibodies, and the dosage needs to be further increased, indicating that the efficiency of the nanoparticles still needs to be improved. Summary of the invention

[0007] In a first aspect, the present invention provides a nanoparticle comprising a lipid, a hydrophobic polymer and an immunosuppressant. The lipid comprises an anionic lipid and / or a PEGylated lipid. When the nanoparticle and the immunogen are administered to a subject simultaneously, the subject can be induced to produce tolerance to the immunogen. The simultaneous administration means that the time interval between the administration of the nanoparticle and the immunogen to the subject does not exceed 24 hours.

[0008] In certain embodiments, the immunogen is selected from the group consisting of a self-antigen, a foreign antigen, and an immunogenic therapeutic drug.

[0009] In a preferred embodiment, anionic lipids and / or PEGylated lipids are distributed on the surface of the nanoparticles.

[0010] In certain embodiments, the lipid is selected from the group consisting of a natural lipid and a semi-synthetic lipid.

[0011] In certain embodiments, the lipid is selected from natural phospholipids, semisynthetic phospholipids, anionic phospholipid lipids and phospholipid lipids modified by polyethylene glycol (PEG). Particularly preferably, the lipid comprises phospholipid lipids modified by polyethylene glycol and anionic phospholipid lipids, wherein the anionic phospholipid lipid is particularly preferably a compound with phosphatidylserine (PS) as a backbone.

[0012] In a preferred embodiment, the anionic lipid is a compound represented by Formula 1. The compound represented by Formula 1 is a compound with phosphatidylserine as a skeleton.

[0013]

[0014] Wherein, R1 and R2 are respectively selected from (Cn)-hydrocarbon groups, wherein n is any integer between 13 and 17, more specifically, n is selected from 13, 14, 15, 16 or 17. In certain embodiments, the hydrocarbon group contains or does not contain a branch. In certain embodiments, the hydrocarbon group is selected from an unsaturated hydrocarbon group or a saturated hydrocarbon group. The saturated hydrocarbon group refers to a group from a hydrocarbon compound containing only a carbon-carbon single bond. The unsaturated hydrocarbon group refers to a group from a hydrocarbon compound containing a carbon-carbon double bond and / or a carbon-carbon triple bond, and the unsaturated hydrocarbon group may or may not contain a carbon-carbon single bond. The "n" in the "Cn" refers to the number of carbons on the main chain of the hydrocarbon group.

[0015] In a preferred embodiment, in Formula 1, R1 and R2 are both C17 saturated hydrocarbon groups without branches, that is, the anionic lipid is distearoylphosphatidylserine (DSPS).

[0016] In a preferred embodiment, the immunosuppressant is an mTOR inhibitor, preferably rapamycin and / or its lipophilic derivatives.

[0017] In a preferred embodiment, the hydrophobic polymer is biodegradable and / or bioerodible. The polymer is selected from polyesters and / or copolymers of polyesters. The polyester is selected from polylactic acid and polyglycolic acid. More preferably, the polymer is polylactic acid (PLA).

[0018] The PLA of the present invention is characterized in that the molecular weight is about 10,000 to about 100,000D, preferably about 20,000 to about 60,000D.

[0019] The PLA of the present invention is characterized in that its terminal group can be an ester group, a carboxyl group or a hydroxyl group, preferably a carboxyl group.

[0020] The PEGylated lipid of the present invention is preferably a phospholipid modified with polyethylene glycol, more preferably PEGylated distearoylphosphatidylethanolamine (DSPE-PEG).

[0021] The DSPE-PEG of the present invention is characterized in that the molecular weight of the PEG is about 2000-about 10000 D, preferably about 5000 D. The DSPE-PEG of the present invention is characterized in that the terminal group of the PEG is methoxy, hydroxyl or carboxyl, etc., preferably methoxy.

[0022] In one embodiment, the nanoparticles comprise the lipid, the hydrophobic polymer and the immunosuppressant, the lipid comprises the compound represented by Formula 1 and DSPE-PEG, the hydrophobic polymer is polylactic acid (PLA), and the immunosuppressant comprises rapamycin and / or its lipophilic derivatives.

[0023] In a specific embodiment, the compound represented by Formula 1 is DSPS.

[0024] In a specific embodiment, the mass fraction of the polylactic acid in the polymer and the lipid is between about 60wt% and about 99wt%, preferably between about 80wt% and about 99wt%, more preferably between about 90wt% and about 99wt%, still more preferably between about 92.5wt% and about 98.5wt%, and more preferably about 94.0wt% to about 98.5wt%.

[0025] In a specific embodiment, the mass fraction of the DSPE-PEG in the polymer and the lipid is between about 0.5wt% and about 40wt%, preferably between about 0.5wt% and about 20wt%, more preferably between about 0.5wt% and about 10wt%, still more preferably between about 0.5wt% and about 5wt%, and more preferably about 0.5wt% to about 3.0wt%.

[0026] In a specific embodiment, the mass fraction of the DSPS in the polymer and the lipid is between about 1wt% and about 40wt%, preferably between about 1wt% and about 20wt%, more preferably between about 1wt% and about 10wt%, still more preferably between about 1wt% and about 5wt%, and more preferably about 1-3wt%.

[0027] In a specific embodiment, the mass fraction of the rapamycin or its derivative in the nanoparticles is between about 1wt% and about 40wt%, preferably between about 1wt% and about 20wt%, more preferably between about 1wt% and about 10wt%, and even more preferably between about 5wt% and about 10wt%.

[0028] The nanoparticles of the present invention may have an average particle size of about 50 to about 500 nm, preferably an average particle size of about 100 nm to about 300 nm.

[0029] In a second aspect, the present application relates to a method for preparing the nanoparticles.

[0030] In certain embodiments, the nanoparticles are prepared by an emulsification solvent evaporation method. The emulsification solvent evaporation method comprises the following steps: (1) dissolving the immunosuppressant, the PEGylated lipid, the anionic lipid, and the hydrophobic polymer in an organic solvent that is immiscible with water; (2) adding the organic solvent solution obtained in step (1) to an aqueous solution containing a surfactant, and obtaining an emulsion by ultrasonic or homogenization emulsification; (3) redispersing the emulsion in an aqueous solution containing a surfactant, and removing the organic solvent by evaporation or ultrafiltration to obtain nanoparticles. The emulsification refers to the operation of dispersing one liquid into another liquid that is immiscible with the liquid to form an emulsion.

[0031] In certain embodiments, the nanoparticles are prepared by a nanoprecipitation method. The nanoprecipitation method comprises the following steps: (1) dissolving the immunosuppressant, the PEGylated lipid, the anionic lipid, and the hydrophobic polymer in a water-miscible organic solvent, and mixing the solution with an aqueous solution containing or not containing a surfactant through a microfluidic chip; (2) dispersing the mixed solution into an aqueous solution containing / not containing a surfactant, and removing the organic solvent by volatilization or ultrafiltration to obtain nanoparticles.

[0032] In a third aspect, the present application relates to a pharmaceutical composition comprising any of the aforementioned nanoparticles and / or a pharmaceutically acceptable carrier.

[0033] In certain embodiments, the pharmaceutical composition further comprises a drug not contained in the nanoparticles, preferably an immunosuppressant. The drug not contained in the nanoparticles may be the same as or different from the drug contained in the nanoparticles.

[0034] In certain embodiments, the immunosuppressant is selected from one or more of the following groups: glucocorticoids, cytokine synthesis inhibitors, antimetabolites, alkylating agents, B cell-targeted antibodies, TNF-α inhibitors, NF-κB inhibitors and other immunosuppressants, such as CTLA4 protein and / or its analogs, competitive inhibitors of CD28 / B7 pathway, etc.

[0035] In a preferred embodiment, the immunosuppressant is CTLA4 protein and / or its analogs.

[0036] In a fourth aspect, the present application also relates to any of the aforementioned nanoparticles in combination with a drug. In a preferred embodiment, the drug in combination is an immunosuppressant.

[0037] In certain embodiments, the co-administered drug may be the same as or different from the drug contained in the nanoparticles.

[0038] In certain embodiments, the immunosuppressant is selected from one or more of the following groups: glucocorticoids, cytokine synthesis inhibitors, antimetabolites, alkylating agents, B cell-targeted antibodies, TNF-α inhibitors, NF-κB inhibitors and other immunosuppressants, such as CTLA4 protein and / or its analogs, competitive inhibitors of CD28 / B7 pathway, etc.

[0039] In a preferred embodiment, the immunosuppressant is CTLA4 protein and / or its analogs.

[0040] In a fifth aspect, the present application also relates to the use of any of the aforementioned nanoparticles and / or any of the aforementioned pharmaceutical compositions in the prevention and / or treatment of autoimmune diseases, and / or in inhibiting the immune response of a subject to a therapeutic drug.

[0041] In certain embodiments, the therapeutic drug is selected from one or more of the following groups: protein drugs, viral drugs, etc.

[0042] In certain embodiments, the therapeutic agent is an adeno-associated virus (AAV).

[0043] In certain embodiments, the therapeutic agent is uricase.

[0044] In a sixth aspect, the present application also relates to a method for inducing immune tolerance using the nanoparticles, which comprises administering the nanoparticles and / or the pharmaceutical composition and the therapeutic drug to a subject.

[0045] In a preferred embodiment, the time interval between the administration of the nanoparticles and / or the pharmaceutical composition and the administration of the therapeutic drug to the subject is no more than 24 hours.

[0046] According to those skilled in the art, anionic lipids phosphatidylglycerol (PG) and phosphatidylserine (PS) have their unique roles in the immune system. On the one hand, compounds containing phosphatidylglycerol (PG) will bind to complement C1q in the blood after entering the blood circulation, and through the action of C1q binding to scavenger receptors such as class F scavenger receptors (SR-F1), the compounds are selectively absorbed by APCs expressing the above receptors in the liver and spleen. On the other hand, when cells undergo apoptosis, the proportion of negatively charged phosphatidylserine (PS) on the cell surface will increase, and through PS binding to APC cell surface receptors such as stable protein-2, TIM-4 and CD300f, apoptotic cells are induced to be engulfed by APC cells without immune response, indicating that the compounds containing PS have advantages in targeting APC cells and inducing or helping immune tolerance. The inventors thus applied PS and PG to the preparation of nanoparticle prescriptions for inducing immune tolerance, and compared the effects of inducing immune tolerance by different prescriptions, and determined that nanoparticles containing PS have good ability to induce immune tolerance in immune models.

[0047] The beneficial effects of the present invention are as follows: the present invention uses a nanoprecipitation method or an emulsified solvent volatilization method to prepare a nanoparticle containing an immunosuppressant, a PEGylated lipid, an anionic phospholipid and a hydrophobic polymer. When the nanoparticles and the immunogen are administered to a subject simultaneously, the negatively charged phospholipids and PEG molecules distributed on the surface of the nanoparticles enhance the ratio of the nanoparticles and the immunosuppressants carried by the nanoparticles to reach the APC cells, thereby enhancing the immune tolerance effect against the antigens that arrive synchronously. In view of the fact that the lipid component of the nanoparticles has the potential to enhance the drug contained in the nanoparticles to reach the APC, the inventors screened the ratio of different components in the nanoparticles, investigated the effects of nanoparticles of different prescriptions on the ability to induce immune tolerance in vivo, and then found a nanoparticle prescription with stronger induction of immune tolerance.

[0048] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0050] Figure 1These are the in vivo screening results of nanoparticles with different formulations in Example 6 for inducing AAV8 capsid immune tolerance.

[0051] Figure 2 These are the in vivo screening results of nanoparticles with different formulations in Example 7 to induce AAV8 capsid immune tolerance.

[0052] Figure 3 These are the in vivo screening results of nanoparticles with different formulations in Example 8 that induce AAV8 capsid immune tolerance.

[0053] Figure 4 These are the in vivo screening results of nanoparticles with different formulations in Example 9 to induce AAV8 capsid immune tolerance.

[0054] Figure 5 These are the in vivo screening results of nanoparticles with different formulations in Example 10 to induce AAV8 capsid immune tolerance.

[0055] Figure 6 The nanoparticles with different prescriptions in Example 11 inhibit the production of uricase antibodies.

[0056] Figure 7 The nanoparticles combined with CTLA-4 protein in Example 12 inhibit the production of AAV antibodies. DETAILED DESCRIPTION

[0057] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0058] Definition of terms

[0059] Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Generally, the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and proteins and nucleic acids described herein are concepts recognized by those skilled in the art.

[0060] In the present application, the term "lipid" generally refers to lipid compounds. Specifically, the lipid refers to fatty acids and esters and derivatives thereof formed by the action of fatty acids and alcohol compounds. In the examples of the present application, the lipid is generally an amphipathic lipid, and the amphipathicity means that the lipid has both hydrophilicity and hydrophobicity, and correspondingly, the lipid has both a hydrophilic part and a hydrophobic part in structure. It should be understood by those skilled in the art that the hydrophobic part of the amphipathic lipid should contain a hydrophobic group, and the hydrophilic part of the amphipathic lipid should contain a hydrophilic group.

[0061] In the present application, the term "anionic lipid" generally refers to a lipid that is electronegative under the condition of pH = 7.4. According to those skilled in the art, "anionic lipid" and "negatively charged lipid" can be used interchangeably. In certain embodiments, the anionic lipid can be a lipid whose hydrophilic group is electronegative under the condition of pH = 7.4. The hydrophilic group of the anionic lipid can be selected from PS (phosphatidylserine), PG (phosphatidylglycerol) and PA (phosphatidic acid).

[0062] In the present application, the term "PEGylated lipid" generally refers to a special lipid prepared by attaching a polyethylene glycol (PEG) chain to a lipid molecule. PEGylated lipids are widely used in the fields of biomedicine and drug delivery. In the present application, the chain length of the PEG refers to the length of the PEG main chain, and the main chain specifically refers to the part that does not include the branched structure in the PEG. It will be appreciated by those skilled in the art that for linear polymers that do not include branched structures, the molecular weight of the PEG can qualitatively describe the chain length, that is, the same or different molecular weights can be used to represent that the chain lengths are the same or different, and the degree of differentiation of the two is the same.

[0063] In the present application, the term "nanoparticle" generally refers to particles with a diameter between 1-1000 nm. In the field of biomedicine, the nanoparticles are generally used for drug delivery, bioimaging, diagnosis and treatment.

[0064] In this application, the term "immunosuppressant" generally refers to a substance that has an immunosuppressive effect on the immune system. Examples of immunosuppressants may include, but are not limited to, glucocorticoids, mTOR inhibitors, antimetabolites, alkylating agents, B cell targeted drugs, TNF-α inhibitors, and NF-κB inhibitors. In certain embodiments, the immunosuppressant may be rapamycin and / or its lipophilic derivatives.

[0065] In the present application, the term "antigen" refers to a compound that can be recognized and bound by T / B lymphocyte surface antigen recognition receptors (TCR / BCR) in vivo and in vitro. Further, the antigen can cause T / B lymphocyte activation, proliferation and differentiation to produce antigen-specific effector T cells and / or antibodies, and these products can also be combined with the antigen in vivo and in vitro. "Immunogen" refers to an antigen that can trigger an immune response, preferably an antigen that triggers an active immune response. "Antigen" and "immunogen" can be proteins, polypeptides, polysaccharides, lipids or nucleic acids. "Antigen specificity" refers to the ability of a subject to recognize a single antigen or a molecule (such as an epitope or mimotope) that is extremely similar to the antigen and to produce an immune response against it. "Anti-responsiveness", "tolerance" or "antigen-specific tolerance" refers to the insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen-specific and persists after cessation of exposure to the immunogen. The therapies described herein for preventing and / or treating autoimmune diseases and suppressing the immune response of subjects to therapeutic drugs are antigen-specific.

[0066] In the present application, "CTLA4" or "CTLA-4" is cytotoxic T lymphocyte-associated protein 4, also known as CD152 (differentiation cluster 152), which is a leukocyte differentiation antigen and a transmembrane receptor on T cells. It shares the B7 molecule ligand with CD28, and CTLA-4 induces T cell anergy after binding to the B7 molecule, and participates in the negative regulation of the immune response. The term "CTLA-4 protein" refers to a fusion protein consisting of the Fc domain of human immunoglobulin IgG and the extracellular domain of CTLA-4 protein. The fusion protein has a high binding affinity for CD80 and CD86. The CTLA-4 protein can block CD28-mediated T cell co-stimulatory signals, thereby inducing immunosuppression and tolerance. Exemplary CTLA-4 proteins may include, but are not limited to, Abatacept, Belatacept and biosimilars thereof.

[0067] In the present application, the term "drug" or "pharmaceutical composition" generally refers to a compound or composition that can induce a desired therapeutic effect when properly administered to a patient. Preferably, the pharmaceutical composition comprises the drug (e.g., lipopolymer nanoparticles) of the present application or its variant, prodrug or other biologically active effective form, and a carrier, such as a diluent, a pharmaceutical excipient, a buffer, a preservative and a tension regulator.

[0068] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering a therapeutic agent, such as a nanoparticle. A pharmaceutically acceptable carrier refers to any drug carrier that does not itself induce the production of antibodies that are harmful to the subject receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may include liquids, such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may also be present in these carriers.

[0069] In this application, the terms "effective amount" and "therapeutically effective amount" are used interchangeably, which generally refer to an amount or dosage sufficient to produce a desired therapeutic result when a pharmaceutical composition comprising one or more nanoparticles is administered. More specifically, a therapeutically effective amount is an amount of a drug or its pharmaceutical composition sufficient to treat a specified condition, illness or disease over a period of time, and specifically refers to an amount of the drug or its pharmaceutical composition that improves, alleviates, mitigates and / or delays one or more of its symptoms. Determining the effective amount or therapeutically effective amount of a given pharmaceutical composition is entirely within the capabilities of those skilled in the art.

[0070] In this application, the term "treatment" generally refers to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include those already having the disorder as well as those susceptible to the disorder or those in whom the disorder is to be prevented.

[0071] In the present application, the term "patient" or "subject" may generally include humans, non-human primates (e.g., monkeys) or other animals, particularly mammals, such as cattle, horses, pigs, sheep, goats, dogs, cats or rodents, such as mice and rats. In addition, the subject described in the present application should be understood in a broader sense, such as cells or single-cell organisms of any origin can be used as subjects. In a particularly preferred embodiment, the patient or subject is a human.

[0072] In the present application, the term "and / or" should be understood as any one element or a combination of any several elements used for connection.

[0073] In this application, the term "comprises", "comprising" or "including" generally means including the features explicitly stated, but not excluding other elements.

[0074] In this application, the term "selected from" generally refers to the objects selected and all combinations thereof. For example, "selected from A, B and C" means all combinations including A, B and C, for example, A, B, C, A+B, A+C, B+C, or A+B+C.

[0075] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. DETAILED DESCRIPTION OF THE INVENTION

[0078] Nanoparticles

[0079] The nanoparticles provided in the present application are tiny particles with a diameter between 1-1000 nm.

[0080] In a preferred embodiment, the nanoparticle may comprise an immunosuppressant, a lipid and a polymer. The immunosuppressant may be located at any part of the nanoparticle comprising the lipid and the polymer.

[0081] Those skilled in the art will appreciate that in the nanoparticles comprising an immunosuppressant, a lipid and a polymer, the drug, the lipid and the polymer may be selected differently.

[0082] In some embodiments, the immunosuppressant can be selected from mTOR inhibitors, TGF-β signaling agents, TGF-β receptor agonists, histone deacetylase (HDAC) inhibitors, corticosteroids, mitochondrial function inhibitors, P38 inhibitors, NF-κβ inhibitors, adenosine receptor agonists, prostaglandin E2 agonists, phosphodiesterase inhibitors, proteasome inhibitors, kinase inhibitors, G-protein coupled receptor agonists, G-protein coupled receptor inhibitors, glucocorticoids, retinoids, cytokine inhibitors, interleukins, cytokine receptor inhibitors, aryl hydrocarbon receptor inhibitors, cytokine receptor agonists, peroxisome proliferator-activated receptor inhibitors, peroxisome proliferator-activated receptor agonists, histone deacetylase inhibitors, calcineurin inhibitors and phosphatase inhibitors. One or more. In some embodiments, the immunosuppressant can also be selected from IDO, vitamin D3, cyclosporine A, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, triptolide and cyclosporine A.

[0083] In a preferred embodiment, the immunosuppressant may be an mTOR inhibitor. The mTOR inhibitor may be selected from rapamycin or a derivative thereof (preferably a lipophilic derivative), rhubarb acid (chrysophanol), deforolimus (MK-8669), KU-0063794, PI-103, PP242, temsirolimus and WYE-354. In a more preferred embodiment, the immunosuppressant may be rapamycin or a lipophilic derivative thereof, specific examples of which may include but are not limited to BA9, CCL-779, RAD001, AP23573, C20-methallyl rapamycin (C20-Marap), C16-(S)-butylsulfonylamino rapamycin (C16-BSrap), C16-(S)-3-methylindorapamycin (C16-iRap), AZD8055 and BEZ235 (NVP-BEZ235).

[0084] The polymer may be a natural or non-natural (synthetic or semi-synthetic) polymer. The polymer may be a homopolymer comprising one monomer or a copolymer comprising two or more monomers. The polymer may or may not contain side chains. The polymer may or may not contain modifications.

[0085] In a preferred embodiment, the polymer is biodegradable and / or bioerodible. It should be understood by those skilled in the art that the biodegradable or bioerodible means that the polymer with this property can be gradually decomposed into small molecules that are harmless to the subject without applying excess external force. The biodegradable means that the polymer can be gradually decomposed into smaller molecules in the subject through biochemical reactions (such as the action of enzymes), and finally converted into substances that are harmless to the human body, such as carbon dioxide and water, and other small molecules that can be processed by the subject's metabolic system. The bioerodible means that the polymer gradually dissolves or erodes in the subject through hydrolysis or other chemical processes, resulting in a reduction in material mass and size. It should be noted that both the biodegradable and the bioerodible can convert high molecular polymers into small molecules, preferably small molecules that are harmless to the subject. Biodegradable is mainly converted through biochemical pathways, and bioerodible is mainly converted through chemical pathways.

[0086] Since the polymer is preferably a biodegradable and / or bioerodible polymer, the polymer may be selected from one or more of the following: polyesters, polyethers, polyester ethers, polyanhydrides, natural polymers and related copolymers. The related copolymers may be composed of monomers from different related homopolymers. Specifically, the polymer may be selected from one or more of the following: polylactic acid (PLA), polyhydroxyalkanoate (PHA), polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), polydioxanone (PDS), polytrimethylene carbonate, poly-ε-caprolactone, polycarbonate, polyurea, chitosan, alginate and collagen, preferably polylactic acid (PLA). More specifically, the lactic acid monomers constituting the polylactic acid may be L-lactic acid and / or D-lactic acid. It will be understood by those skilled in the art that the L-lactic acid and D-lactic acid refer to stereoisomers with different chirality.

[0087] It will be appreciated by those skilled in the art that the polymer is a macromolecule with a large molecular weight formed by small molecule monomers being connected by covalent bonds with each other. Wherein, in the polymer composed of the same monomer, the number of monomers contained in each polymer molecule can be the same or different, and the difference in the number of monomers leads to the molecular weight difference of the polymer molecule. It should be noted that, unless otherwise indicated, the molecular weight of the polymer molecule in the same system in the present application should be the same, and the molecular weight of the polymer molecule in different systems can be different. The system can refer to the different prescriptions of the nanoparticles described in the present application. In a specific embodiment, the polymer is polylactic acid (PLA). Specifically, the molecular weight of the polylactic acid molecule can be between about 500D-about 100kD, preferably between about 1kD-about 100kD, more preferably between about 5kD-about 80kD, and even more preferably between about 10kD-about 50kD. More specifically, the molecular weight of the polylactic acid molecule may be about 10 kD, about 15 kD, about 20 kD, about 25 kD, about 30 kD, about 35 kD, about 40 kD, about 45 kD, or about 50 kD.

[0088] Other examples of polymers suitable for use in the present invention can include, but are not limited to, polyethylene, polypropyl fumarate, polyamide, polyacetal, polyether, polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, poly-β-hydroxyalkanoate, polyorthoester, polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polyurea, polystyrene, polylysine, polylysine-PEG copolymer, polyethyleneimine, and ethyleneimine-PEG copolymer.

[0089] In certain embodiments, the lipid may be stearic acid.

[0090] In certain embodiments, the lipid may be an anionic lipid, more preferably an anionic phospholipid. In a specific embodiment, the hydrophilic group of the anionic phospholipid may be negatively charged under the subject's physiological conditions. The physiological conditions may refer to the in vivo physiological environment when the subject's body reaches acid-base balance, specifically, it may refer to a pH value in the subject's body between about 7.0 and about 7.8, preferably between about 7.2 and about 7.6, more preferably between about 7.3 and about 7.5, and even more preferably about 7.4. In certain embodiments, the pH may be measured by a pH meter. In certain embodiments, the pH may be predicted by judging whether the subject has acid-base imbalance and whether the pH value in the subject's body is within the physiological conditions. If the subject does not have acid-base imbalance, the pH value in the subject's body is within the physiological conditions.

[0091] In a further embodiment, the hydrophilic group of the anionic lipid may be negatively charged. Specifically, the negatively charged hydrophilic group may be selected from one or more of the following: phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA) and cardiolipin (CL). Since the negative charge of the anionic lipid usually comes from the hydrophilic group, the hydrophilic group may also be referred to as the skeleton of the anionic lipid.

[0092] In a preferred embodiment, the hydrophilic group of the anionic lipid may be phosphatidylserine (PS). In certain embodiments, the hydrophobic group of the anionic lipid may be a fatty acid chain. Specifically, the hydrophobic group of the anionic lipid may be an unsaturated fatty acid or a saturated fatty acid. More specifically, the hydrophobic group of the anionic lipid may be selected from one or more of the following: stearic acid, palmitic acid, oleic acid, linolenic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid.

[0093] In some embodiments, the lipid can be a polyethylene glycol (PEG) lipid. PEGylated lipid refers to a modified lipid formed by chemically connecting a PEG molecule to a lipid molecule. This modification can give the nanoparticle a variety of unique properties, such as increasing stability, extending blood circulation time, improving tumor targeting efficiency and enhancing drug efficacy. It should be noted that the introduction of PEGylated lipids in this application can play a role in promoting the relevant functions of the nanoparticles, especially the nanoparticles comprising the anionic lipids. It should be understood that the effect of PEGylated lipids on the functional regulation of the nanoparticles is broad-spectrum, rather than for a specific effect (such as helping to induce immune tolerance). Specifically, the molecular weight of the PEG can be between about 500D-about 10kD, preferably between about 1kD-about 6kD. More specifically, the molecular weight of the PEG can be about 1kD, can be about 2kD, can be about 3kD, can be about 4kD, can be about 5kD, can be about 6kD. The PEGylated lipid may also contain other modifications on the hydroxyl side of PEG, or may not contain modifications (PEG-H or PEG); for example, the hydroxyl side of the PEG forms a methyl ether (MPEG); for example, the hydrogen on the hydroxyl side of the PEG is replaced by a carboxyl group (PEG-COOH); for example, the hydrogen on the hydroxyl side of the PEG hydroxyl side is replaced by mannose (PEG-Mannose); for example, the hydrogen on the hydroxyl side of the PEG is replaced by sialic acid (PEG-Sialic acid). In the nanoparticles of different prescriptions, the polymer, the lipid, and the immunosuppressant have different ratios. Specifically, the ratio can be expressed as a mass fraction. It should be noted that the denominator of the mass fraction can be the sum of the masses of the lipid and the polymer, the mass of the drug does not participate in the calculation of the denominator of the mass fraction, and the numerator of the mass fraction can be the mass of the corresponding substance, and the mass of the drug participates in the calculation of the numerator of the mass fraction. Those skilled in the art should be able to understand that the method for calculating the mass fraction is limited to the present application, and other algorithms may exist in current or future literature. However, when the mass fraction of the corresponding substance in other algorithms is calculated using the calculation method used in the present application and falls within the protection scope of the present application, the corresponding scheme should be deemed to fall within the protection scope of the present application.

[0094] The polymer contained in the nanoparticles can be polylactic acid (PLA). The mass fraction of the polylactic acid can be between about 45wt% and about 99wt%, preferably between about 80wt% and about 99wt%, more preferably between about 90wt% and about 99wt%, still more preferably between about 92.5wt% and about 97.5wt%, and most preferably about 95wt%. More specifically, the mass fraction of the polylactic acid can be about 45wt%, about 50wt%, about 55wt%, about 60wt%, about 65wt%, can be about 70wt%, can be about 72.5wt% (e.g., about 73wt%), can be about 75wt%, can be about 77.5wt%, can be about 80wt%, can be about 82.5wt%, can be about 85wt% (e.g., about 83.8wt%), can be about 87.5wt%, can be about 90wt%, can be about About 91wt%, may be about 92wt% (for example, about 92.4wt%), may be about 93wt%, may be about 94wt% (for example, about 93.6wt%), may be about 95wt% (for example, about 95.4wt%, about 94.8wt%), may be about 96wt%, may be about 97wt% (for example, about 96.8wt%, about 97.4wt%), may be about 98wt% (for example, about 97.4wt%), may be about 99wt% (for example, about 98.7wt%).

[0095] The polymer included in the nanoparticle can be a copolymer of PEG and PLA (PEG-PLA). The PEG-PLA is usually synthesized by connecting PEG with PLA monomer or prepolymer through a suitable chemical reaction. The mass fraction of the PEG-PLA can be about 5wt%-about 50wt%, preferably about 5wt%-about 30wt%, more preferably about 10wt%-about 25wt%, and even more preferably about 25wt%. More specifically, the mass fraction of the PEG-PLA can be about 5wt%, about 10wt%, about 15wt%, about 20wt%, about 25wt%, about 30wt%, about 35wt%, about 40wt%, about 45wt%, or about 50wt%.

[0096] The lipid contained in the nanoparticle can include anionic lipids. Specifically, the hydrophilic group can be a lipid of phosphatidylserine (PS) (can also be expressed as a lipid with phosphatidylserine as a skeleton). More specifically, the anionic lipid can be distearoylphosphatidylserine (DSPS). The mass fraction of the DSPS can be between about 0-about 40wt%, preferably between about 0-about 20wt%, more preferably between about 0-about 10wt%, still more preferably between about 0-about 5wt%, most preferably about 2wt%. More specifically, the mass fraction of the DSPS can be about 0, about 0.5wt%, about 1wt%, about 1.5wt%, about 2wt%, about 2.5wt%, about 3wt%, about 3.5wt%, about 4wt%, about 4.5wt%, about 5wt%, about 10wt%, about 15wt%, about 20wt%, about 25wt%, about 30wt%, about 35wt%, about 40wt%.

[0097] The lipid contained in the nanoparticles can be an anionic lipid. Specifically, it can be stearic acid. The mass fraction of the stearic acid can be between about 1wt% and about 40wt%, preferably between about 1wt% and about 20wt%, more preferably between about 1wt% and about 10wt%, and most preferably about 10wt%.

[0098] The lipid contained in the nanoparticle can include PEGylated lipids. Specifically, it can be selected from one or more of the following: dimyristoylglycerol-polyethylene glycol (DMG-PEG) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), preferably DSPE-PEG. The mass fraction of the DSPE-PEG can be between about 0-about 40wt%, preferably between about 0-about 20wt%, more preferably between about 0-about 10wt%, even more preferably between about 0-about 5wt%, even more preferably between about 0.5wt%-about 3wt%.

[0099] The immunosuppressant contained in the nanoparticles may specifically be rapamycin and / or its lipophilic derivatives. The mass fraction of the immunosuppressant may be between about 1 and about 30 wt%, preferably between about 1 wt% and about 20 wt%, more preferably between about 1 wt% and about 10 wt%, more preferably between about 5 wt% and about 10 wt%, and most preferably about 8 wt%. More specifically, the mass fraction of the drug may be about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt%.

[0100] Those skilled in the art will appreciate that the nanoparticles comprising the rapamycin and / or its lipophilic derivative, the anionic lipid, the PEGylated lipid and the polymer may have the function of inducing specific immune tolerance. The inducing specific immune tolerance function possessed by the nanoparticles is dominated by the immunosuppressant contained therein, and is enhanced by the targeted APC delivery effect of the nanoparticles composed of the anionic lipid, the PEGylated lipid and the polymer.

[0101] Preparation method

[0102] The nanoparticles described in the present application can be prepared by an emulsification solvent evaporation method. The emulsification solvent evaporation method can include the following steps:

[0103] a) dissolving the immunosuppressant, the polymer and the lipid in an organic solvent to obtain an oil phase;

[0104] b) dissolving polyvinyl alcohol (PVA) in phosphate buffer (PB) to obtain aqueous phase 1 and aqueous phase 2, wherein the aqueous phase 1 and aqueous phase 2 are PVA-PB solutions with different concentrations;

[0105] c) adding the oil phase to the water phase 1 and performing emulsification treatment;

[0106] d) thoroughly mixing the aqueous phase 2 with the mixture obtained in step (c);

[0107] e) purifying the mixture obtained in step (d).

[0108] The organic solvent included in the emulsified solvent volatilization method can be arbitrarily selected, and can include but is not limited to other organic solvents that are immiscible with water, such as dichloromethane, toluene, chloroform, ethyl acetate, etc. The concentration of the polymer in the oil phase can be between about 10 and about 100 mg / mL, preferably between about 30 and about 70 mg / mL, and more preferably about 50 mg / mL.

[0109] The emulsification can be achieved by ultrasound, homogenization, etc.

[0110] In step (e), the purification may include removing free rapamycin or its derivatives in the system. The removal may be accomplished by ultrafiltration concentration using an ultrafiltration tube, tangential flow, high-speed centrifugation or other liquid replacement methods.

[0111] The nanoparticles described in the present application may be prepared by a nanoprecipitation method. The nanoprecipitation method may include the following steps:

[0112] a) dissolving the immunosuppressant, the polymer and the lipid in an organic solvent to obtain an oil phase;

[0113] b) dissolving polyvinyl alcohol (PVA) in phosphate buffer (PB) to obtain aqueous phase 1 and aqueous phase 2, wherein the aqueous phase 1 and aqueous phase 2 are PVA-PB solutions with different concentrations;

[0114] c) mixing the oil phase with the aqueous phase 1 and adding the mixture to the aqueous phase 2 to obtain a nanoparticle mixture;

[0115] d) purifying the mixture obtained in step (c).

[0116] The organic solvent included in the nanoprecipitation method can be arbitrarily selected, and can include but is not limited to tetrahydrofuran, ethanol, acetone, dimethyl sulfoxide, methanol and other organic solvents that are miscible with water. The concentration of the polymer in the oil phase can be between about 1 and about 30 mg / mL, preferably between about 5 and about 20 mg / mL, and more preferably between about 10 and about 15 mg / mL.

[0117] During the mixing process, the volume ratio of the oil phase, the water phase 1 and the water phase 2 may be 1:1:2.

[0118] In step (c), the mixing method may be performed in a microfluidic chip.

[0119] In addition, the nanoparticles can be prepared by spray drying or other processes. The conditions used in preparing the nanoparticles can be changed to produce particles with desired size and properties (e.g., hydrophobicity, hydrophilicity, external morphology, "stickiness", shape, etc.). The methods for preparing these synthetic nanocarriers and the conditions used (e.g., solvents, temperatures, concentrations, air flow rates, etc.) can depend on the materials to be attached to these synthetic nanocarriers and / or the composition of the polymer matrix.

[0120] After obtaining a mixture containing the nanoparticles, the content of each component in the nanoparticles and the physical properties (such as potential, particle size, drug loading, etc.), chemical properties, and biological properties (such as immunosuppressive function) can be evaluated. The potential and the particle size can be measured by a laser particle size analyzer. The drug loading can be calculated by testing the drug content in a certain mass of the nanoparticles. The biological property can specifically refer to the downstream biological function of the nanoparticles (such as the function of inducing immune tolerance). It should be understood by those skilled in the art that the nanoparticles of different prescriptions have different effects on inducing immune tolerance. The immune tolerance can be for specific drugs (such as AAV, uricase, etc.) or for other drugs, and the effect can be characterized by the relative content of anti-drug antibodies. The relative content of the anti-drug antibodies can be detected by antibody-antigen reaction, and the antibody-antigen reaction can include but is not limited to enzyme-linked immunosorbent assay (ELISA), immunoprecipitation (IP), immunofluorescence (IF), protein immunoblotting (WB) and immunohistochemistry (IHC).

[0121] More specifically, the method for evaluating the biological property may include one or more of the following steps: (1) administering the nanoparticles and the drug that induces immune tolerance to a subject; the administration time may be the same or different, and preferably the time difference between the two is controlled within 24 hours; the administration method may be selected from oral, nasal, pharyngeal, duodenal, intravenous, intramuscular, peritoneal, transsternal, subcutaneous, spinal, joint and transdermal; (2) obtaining the concentration of anti-drug antibodies in the subject's body fluid after a period of time; the body fluid may include but is not limited to blood, plasma, serum, urine, semen and sweat.

[0122] Pharmaceutical composition

[0123] The pharmaceutical composition comprising nanoparticles described in the present application can be prepared using traditional drug mixing methods. These methods include liquid-liquid mixing, wherein one or more mixtures containing one or more nanoparticles are directly mixed or mixed together via one or more containers containing diluents. The nanoparticles can also be produced or stored in powder form, so dry powder-powder mixing can be carried out, because two or more powders can be resuspended in a common medium. The method of preparation depends on the characteristics of the nanoparticles and their interactions.

[0124] The pharmaceutical composition preparation containing the nanoparticles may contain one or more of the following substances: inorganic or organic buffers (e.g., sodium or potassium salts of phosphoric acid, carbonic acid, acetic acid, citric acid), pH adjusters (e.g., hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid, acetic acid salts, amino acids or amino acid salts), antioxidants (e.g., ascorbic acid, α-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), low temperature / freeze drying stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic regulators (e.g., salt sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), defoaming agents (e.g., polydimethylsilicone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers, viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethyl cellulose) and co-solvents (e.g., glycerol, polyethylene glycol, etc.).

[0125] Drug combination

[0126] The pharmaceutical composition described in the present application can also be used in combination with other drugs. Other drugs can be selected from glucocorticoids, cytokine synthesis inhibitors, antimetabolites, alkylating agents, antibodies targeting B cells, TNF-α inhibitors, NF-κB inhibitors and other immunosuppressants, such as CTLA4 protein, competitive inhibitors of CD28 / B7 pathway, etc. The glucocorticoids can include but are not limited to hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone acetonide, dexamethasone and betamethasone; the calcineurin inhibitors can include but are not limited to cyclosporine, tacrolimus, everolimus and diltiazem. The antimetabolites may include, but are not limited to, methotrexate, 5-fluorouracil, 6-mercaptopurine, thioguanine, capecitabine, gemcitabine, cytarabine, and flutolan.

[0127] It should be understood that the pharmaceutical composition may be manufactured in any suitable manner, and the present application is in no way limited to compositions that can be produced using the methods described herein. The selection of an appropriate method may require attention to the characteristics of the specific moieties being combined.

[0128] In some embodiments, the pharmaceutical composition is manufactured under aseptic conditions or is finally sterilized. This can ensure that the resulting composition is sterile and non-infectious, thereby improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when the subject receiving the pharmaceutical composition is immunocompromised, suffering from infection and / or susceptible to infection.

[0129] The pharmaceutical composition can be administered by various routes, which may include, but are not limited to, oral, nasal, pharyngeal, duodenal, intravenous, intramuscular, peritoneal, transsternal, subcutaneous, spinal, transarticular, transdermal, or a combination of these routes. In a preferred embodiment, the pharmaceutical composition can be administered to a subject intravenously in the form of a solution. The pharmaceutical composition can be administered in an effective amount, such as an effective amount described elsewhere herein. According to the present application, the dosage of the dosage form contains different amounts of the nanoparticles. The effective amount of the nanoparticles present in the dosage form of the present application and even the immunosuppressant contained therein can be changed according to the nature of the immunosuppressant, the therapeutic benefit to be achieved, and other possible parameters. In various embodiments, a dose range study can be performed to establish the optimal therapeutically effective amount of the nanoparticles to be present in the dosage form and the therapeutically effective amount of the immunosuppressant. In various embodiments, the nanoparticles and / or immunosuppressants are present in the dosage form in an effective amount, and when administered to a subject, the nanoparticles and / or immunosuppressants effectively produce a tolerogenic immune response to these antigens. Using conventional dose range studies in subjects, it may be possible to determine the amount of immunosuppressants that are effective for inducing immune tolerance. The dosage form of the present application can be administered at a variety of frequencies. In a preferred embodiment, at least one administration of the dosage form is sufficient to produce a pharmacologically relevant response. In a plurality of more preferred embodiments, at least two administrations, at least three administrations, or at least four administrations of the dosage form are utilized to ensure a pharmacologically relevant response.

[0130] Prophylactic administration of these inventive compositions may be initiated prior to the onset of a disease, disorder or condition, or therapeutic administration may be initiated after the disease, disorder or condition is established.

[0131] In various embodiments, the nanoparticles may be administered one or more times, including but not limited to, at about 30 days, about 25 days, about 20 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, and 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or about 0 days prior to administration of a specific drug or physiological overimmunity. Additionally or alternatively, the nanoparticles may be administered to a subject after administration of a specific drug or physiological overimmunity. In various embodiments, the nanoparticles can be administered one or more times including but not limited to about 30 days, about 25 days, about 20 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, and 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or about 0 days after administration of a particular drug or physiological over-immunity is produced.

[0132] In some embodiments, a maintenance dose is administered to the subject after the initial administration has produced an induced immune tolerance response in the subject, for example, in order to maintain the induced immune tolerance effect reached after the initial dose, in order to prevent the undesirable immune response in the subject, or, in order to prevent the subject from becoming a subject in the risk of an immune response that is undesirable or undesirable. In some embodiments, the maintenance dose is the same dose as the initial dose received by the subject. In some embodiments, the maintenance dose is a dose lower than the initial dose. For example, in some embodiments, the maintenance dose is an initial dose of about 3 / 4, about 2 / 3, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 8, about 1 / 10, about 1 / 20, about 1 / 25, about 1 / 50, about 1 / 100, about 1 / 1,000, about 1 / 10,000, about 1 / 100,000, or about 1 / 1,000,000 (weight / weight).

[0133] Treating Diseases

[0134] For the purpose of immunosuppression, the compositions and methods described herein can be used to induce or enhance a tolerogenic immune response and / or inhibit, regulate, guide or redirect an undesirable immune response. These immune responses are usually caused by excessive immunity, which can be drug-induced or physiological. The physiological excessive immunity refers to an excessive immune state other than drug-induced, which can include autoimmune diseases, inflammatory diseases, allergies, organ or tissue rejection and graft-versus-host disease. The drug-induced excessive immunity can specifically be excessive immunity induced by the therapeutic drug itself or the drug carrier. The nanoparticles and the pharmaceutical composition can also be used for subjects who have undergone or are about to undergo transplantation.

[0135] Autoimmune diseases may include, but are not limited to, systemic lupus erythematosus, lupus nephritis, type 1 diabetes, systemic sclerosis, idiopathic inflammatory myopathy, Sjögren's syndrome, systemic vasculitis, rheumatoid arthritis, primary membranous nephropathy, limbic encephalitis, sclerosing myositis, antiphospholipid syndrome, autoimmune hemolytic anemia, IgG4-related disease, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, polyarteritis nodosa, chronic autoimmune hepatitis, primary biliary cirrhosis, myasthenia gravis, neuromyelitis optica spectrum disorder, multiple sclerosis, Lambert-Eaton syndrome, Hashimoto's thyroiditis, Graves' disease, and paraneoplastic cerebellar syndrome.

[0136] Therapeutic drugs may include, but are not limited to, human tumor antigens, cytokines, hormones and growth factors, recombinant enzymes and other therapeutic protein drugs, and viral drugs such as AAV, adenovirus, retrovirus and lentivirus.

[0137] This application also involves the following:

[0138] 1. A nanoparticle comprising a lipid, a hydrophobic polymer and an immunosuppressant, wherein the lipid comprises an anionic lipid and a polyethylene glycol (PEG)-ylated lipid.

[0139] 2. The nanoparticle according to item 1, wherein anionic lipids and PEGylated lipids are distributed on the surface of the nanoparticle.

[0140] 3. The nanoparticle according to item 1 or 2, wherein the anionic lipid is a compound represented by formula 1:

[0141]

[0142] Wherein, R1 and R2 are respectively selected from the hydrocarbon group of (Cn)-, wherein n represents the number of carbon atoms on the main chain, and n is selected from 13, 14, 15, 16 or 17.

[0143] 4. The nanoparticle according to item 3, wherein R1 and R2 are C17 saturated hydrocarbon groups without branches, and the anionic lipid is 1,2-dioctadecanoyl-sn-glycero-3-phospho-L-serine (DSPS).

[0144] 5. The nanoparticle according to item 1 or 2, wherein the immunosuppressant is an mTOR inhibitor, preferably rapamycin and / or its lipophilic derivatives.

[0145] 6. The nanoparticles according to item 1 or 2, wherein the hydrophobic polymer is a polyester, preferably polylactic acid (PLA).

[0146] 7. The nanoparticles according to item 6, wherein the molecular weight of the PLA is 10000-100000D, preferably 20000-60000D.

[0147] 8. The nanoparticles according to item 6, wherein the terminal group of the PLA is selected from an ester group, a carboxyl group or a hydroxyl group, preferably a carboxyl group.

[0148] 9. The nanoparticle according to item 1 or 2, wherein the PEGylated lipid is PEGylated distearoylphosphatidylethanolamine (DSPE-PEG).

[0149] 10. The nanoparticle according to item 9, wherein the molecular weight of the PEG is 2000-10000D, preferably 5000D.

[0150] 11. The nanoparticle according to item 9, wherein the terminal group of the PEG is selected from methoxy, hydroxyl or carboxyl, preferably methoxy.

[0151] 12. The nanoparticle according to any one of items 1 to 11, which, when the nanoparticle and the immunogen are administered to a subject simultaneously, can induce tolerance to the immunogen in the subject.

[0152] 13. The nanoparticle according to item 12, wherein the immunogen is selected from the group consisting of self-antigens, foreign antigens and immunogenic therapeutic drugs.

[0153] 14. The nanoparticle according to any one of items 1 to 11, wherein the anionic lipid is DSPS.

[0154] 15. The nanoparticles according to item 14, wherein the mass fraction of polylactic acid in the hydrophobic polymer and the lipid is between 60 wt% and 99 wt%, preferably between 90 wt% and 98 wt%.

[0155] 16. The nanoparticles according to item 14, wherein the mass fraction of DSPE-PEG in the hydrophobic polymer and the lipid is between 0.1 wt% and 10 wt%, preferably between 0.5 wt% and 5 wt%.

[0156] 17. The nanoparticles according to item 14, wherein the mass fraction of DSPS in the hydrophobic polymer and the lipid is between 0.1 wt% and 30 wt%, preferably 0.5 wt% and 5 wt%.

[0157] 18. The nanoparticles according to item 14, wherein the mass fraction of rapamycin or its lipophilic derivative is 1 wt%-40 wt%, preferably 1 wt%-20 wt%, more preferably 1-10 wt%.

[0158] 19. The nanoparticles according to any one of items 1 to 18, wherein the particle size of the nanoparticles is 50-500 nm, preferably 100 nm-300 nm.

[0159] 20. The nanoparticles according to any one of items 1 to 19 are prepared by an emulsification solvent evaporation method, which comprises the following steps: (1) dissolving the immunosuppressant, the PEGylated lipid, the anionic lipid, and the hydrophobic polymer in an organic solvent immiscible with water; (2) adding the organic solvent solution obtained in step (1) to an aqueous solution containing a surfactant, and obtaining an emulsion through ultrasonic or homogenization emulsification; (3) redispersing the emulsion in an aqueous solution containing a surfactant, and then removing the organic solvent to obtain nanoparticles.

[0160] 21. The nanoparticles according to any one of items 1 to 19 are prepared by a nanoprecipitation method, which comprises the following steps: (1) dissolving the immunosuppressant, the PEGylated lipid, the anionic lipid, and the hydrophobic polymer in a water-miscible organic solvent, and mixing them with an aqueous solution containing or not containing a surfactant through a microfluidic chip; (2) dispersing the mixed solution into an aqueous solution containing or not containing a surfactant, and then removing the organic solvent to obtain nanoparticles.

[0161] 22. A pharmaceutical composition comprising the nanoparticles according to any one of items 1 to 19 and / or a pharmaceutically acceptable carrier.

[0162] 23. The pharmaceutical composition according to item 22, further comprising a drug not contained in the nanoparticles, wherein the drug is preferably an immunosuppressant.

[0163] 24. According to the pharmaceutical composition of item 23, the immunosuppressant is selected from one or more of the following groups: glucocorticoids, cytokine synthesis inhibitors, antimetabolites, alkylating agents, antibodies targeting B cells, TNF-α inhibitors, NF-κB inhibitors and competitive inhibitors of CD28 / B7 pathway.

[0164] 25. A pharmaceutical composition according to item 23, wherein the immunosuppressant is cytotoxic T lymphocyte antigen 4 (CTLA-4) protein and / or its analogs.

[0165] 26. The nanoparticles described in any one of items 1 to 19 and / or the pharmaceutical composition described in any one of items 22 to 25, for use in preventing and / or treating autoimmune diseases, and / or in inhibiting an immune response of a subject to a therapeutic drug.

[0166] 27. The use according to item 26, wherein the therapeutic drug is selected from protein drugs and viral drugs.

[0167] 28. A method for inducing immune tolerance, comprising the use of the nanoparticles described in any one of items 1 to 19 and / or the pharmaceutical composition described in any one of items 22 to 25.

[0168] Without intending to be bound by any theory, the following examples are merely intended to illustrate the nanoparticle preparation method and use of the present invention, and are not intended to limit the scope of the present invention.

[0169] The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials, reagents, etc. used are all purchased from conventional reagent manufacturers unless otherwise specified.

[0170] Since the nanoparticles described in the present application contain lipids, polymers, and immunosuppressants (mainly rapamycin or its derivatives), they can be referred to as lipid-polymer hybrid nanoparticles, rapamycin nanoparticles, and other related names.

[0171] Example

[0172] Example 1 Preparation of lipid nanoparticles (LNP) by nanoprecipitation method

[0173] The prescribed amount of rapamycin, cationic lipid, DSPC, cholesterol, and DMG-PEG2k were dissolved in ethanol to obtain a lipid solution, wherein the lipid concentration was about 10 mg / mL. One volume of the ethanol solution was mixed with three volumes of a pH=4.0 50 mM citric acid solution through a microfluidic chip, then dialyzed overnight at room temperature, and then ultrafiltered and concentrated to a certain volume using an ultrafiltration tube, and then 1 / 4 volume of a 40% sucrose mother solution was added and frozen at -80°C.

[0174] Example 2: Preparation of lipid polymer nanoparticles by nanoprecipitation method

[0175] The prescribed amount of rapamycin (Rapam) or its derivatives, polymers and lipids are dissolved in acetone to obtain a lipid polymer solution, with a polymer concentration of about 10-15 mg / mL. PVA is dissolved in pH=8.0 100mM PB to obtain 2% and 0.25% PVA solutions respectively. Equal volumes of lipid polymer solution and 2% PVA solution are mixed through a microfluidic chip and flow into a solution containing 0.25% PVA with a volume of 2 times the volume of acetone solution, and then 15 times the volume of acetone solution is added. Deionized water is obtained to obtain a nanoparticle solution with a volume of about 19 times the volume of the organic phase. Stir at room temperature for 3 hours, evaporate the organic solvent, use an ultrafiltration tube to ultrafilter and concentrate the solution to remove free rapamycin, then make the volume constant to a certain volume, add 1 / 4 volume of 40% sucrose mother solution, and freeze at -80°C.

[0176] Example 3: Preparation of polymer lipid nanoparticles by emulsification solvent evaporation method

[0177] The prescribed amount of rapamycin or its derivatives, polymers and lipids were dissolved in 1 volume of dichloromethane as the oil phase, wherein the polymer concentration was about 50 mg / mL. PVA was dissolved in pH=8.0 100mM PB to obtain 5% and 0.25% PVA solutions, respectively. 3 volumes of 5% PVA solution were taken as aqueous phase 1 and 30 volumes of 0.25% PVA solution were taken as aqueous phase 2 for standby use. The oil phase was added to aqueous phase 1, and the cell sonicator was used under ice bath at 200W power for 2 minutes. The ultrasonic mode was 5S working and 5S intermittent. Then aqueous phase 2 was added to the centrifuge tube and sonicated again in the same ultrasonic mode for 2 minutes. Finally, the solution was poured into an open beaker, stirred at room temperature for 3 hours, and the organic solvent was evaporated. The free rapamycin was removed by ultrafiltration concentration and liquid exchange using an ultrafiltration tube, and then the volume was fixed to a certain volume, 1 / 4 volume of 40% sucrose mother solution was added, and it was frozen at -80°C.

[0178] Example 4: Characterization of Nanoparticles

[0179] 1. Particle size and potential

[0180] The particle size and potential of nanoparticles were measured using a laser particle size analyzer.

[0181] 2. Drug loading test

[0182] Accurately transfer about 10 mg of nanoparticles (M total), dissolve them in acetonitrile by ultrasonication for 30 min, and dilute to a 10 mL volumetric flask. Use HPLC to detect the drug content, calculate the drug concentration in the solution based on the standard curve, and calculate the total amount of drug in the nanoparticles as M drug based on the total volume and concentration. Calculate the drug loading according to the following formula:

[0183] Drug loading = M drug / M total 100%

[0184] Example 5: Screening method for inducing AAV8 capsid immune tolerance in mice

[0185] Experimental design: VGB-R04 is a gene therapy drug for hemophilia B with AAV8 as the vector. VGB-R04 can show good pharmacological activity in C57BL / 6 mice. Therefore, VGB-R04 was selected as the AAV virus antigen model, and male C57BL / 6 mice were used as the screening animals for in vivo immune tolerance. Male C57BL / 6 mice were randomly divided into groups, with 4 mice in each group. On the day of administration, VGB-R04 was first injected into the tail vein, followed by an immediate injection of immunosuppressive nanoparticles of different prescriptions; plasma was collected from all mice at different time points (before administration, 2 weeks and 4 weeks after administration) to detect anti-AAV8 antibodies (anti-AAV8 ADA).

[0186] Anti-AAV8 ADA detection method: ELISA method is used for detection. AAV8 empty shell virus is coated on the enzyme labeling plate as a capture reagent, and plasma samples diluted at different dilution multiples are added, and then biotin-labeled AAV8 empty shell virus is added as a detection reagent for co-incubation; unbound substances are removed by washing the plate, and streptavidin-labeled horseradish peroxidase is added for incubation, and then the plate is washed and the substrate is added. TMB and peroxide in the substrate solution produce a colorimetric signal that is proportional to the ADA concentration in the sample under the action of HRP.

[0187] Example 6: Screening of AAV8 capsid immune tolerance induced in mice 1

[0188] The purpose of the first round of screening is to determine a rapamycin nanoparticle formulation with better immunosuppressive effect, and to determine whether the different preparation methods of Example 2 and Example 3 have an effect on the effect of rapamycin lipid polymer nanoparticles.

[0189] (1) According to the method of Example 1, the preparation of prescriptions F1 and F2 was carried out. Taking F1 as an example, 2.5 mg of rapamycin, 6 mg of L7-526-1 (independently synthesized cationic lipid), 1.335 mg of DSPC, 2.515 mg of cholesterol, and 0.635 mg of DMG-PEG2k were dissolved in 1 mL of ethanol. The ethanol solution was mixed with 3 mL of pH = 4.0 50 mM citric acid solution through a microfluidic chip. The obtained lipid nanoparticles were dialyzed in a 20 mM Tris solution overnight and then filtered through an ultrafiltration tube ( The solution was concentrated to 0.8 mL by ultrafiltration using a 50 kDa Ultra MWCO) at 3000 r / min (about 1476×g), and then 0.2 mL of 40% sucrose mother solution was added and the solution was frozen at -80°C.

[0190] (2) F3-F5 prescription preparation was carried out according to Example 3:

[0191] Preparation of F3: 8 mg of rapamycin, 37.5 mg of polylactic acid, 12.5 mg of PLA-PEG5k, and 2.5 mg of Span40 were dissolved together in 1 mL of dichloromethane as the oil phase. PVA was dissolved in pH=8.0 100 mM PB to obtain 5% and 0.25% PVA solutions, respectively. 3 mL of 5% PVA solution was taken as aqueous phase 1, and 30 mL of 0.25% PVA solution was taken as aqueous phase 2 for standby use. The oil phase was added to aqueous phase 1, and ultrasonicated for 2 minutes at 200 W power using a cell sonicator under an ice bath. The ultrasonic mode was 5S working and 5S intermittent. Then aqueous phase 2 was added to a centrifuge tube and ultrasonicated again for 2 minutes using the same ultrasonic mode. Finally, the solution was poured into an open beaker, stirred at room temperature for 3 hours, and the organic solvent was evaporated. Use an ultrafiltration tube ( Ultrafiltration and concentration were performed at 3000 r / min (about 1476 × g) using Ultra 50kDa MWCO) to 0.8 mL, and 10 mL of 20 mM Tris was added for resuspending and ultrafiltration and concentration was performed to remove free rapamycin. The mixture was repeated once and finally concentrated to 0.8 mL. Then 0.2 mL of 40% sucrose mother solution was added and the mixture was frozen at -80°C.

[0192] Preparation of F4: 8 mg of rapamycin, 37.5 mg of polylactic acid, 5 mg of DMG-PEG2k, and 12.5 mg of DSPS were dissolved together in 1 mL of dichloromethane as the oil phase. PVA was dissolved in 100 mM PB at pH = 8.0 to obtain 5% and 0.25% PVA solutions, respectively. 3 mL of 5% PVA solution was taken as aqueous phase 1 and 30 mL of 0.25% PVA solution was taken as aqueous phase 2 for standby use. The oil phase was added to aqueous phase 1, and ultrasonicated for 2 minutes at 200 W power using a cell sonicator under an ice bath. The ultrasonic mode was 5S working and 5S intermittent. Then aqueous phase 2 was added to the centrifuge tube and ultrasonicated again for 2 minutes in the same ultrasonic mode. Finally, the solution was poured into an open beaker, stirred at room temperature for 3 hours, and the organic solvent was evaporated. Use an ultrafiltration tube ( Ultrafiltration and concentration were performed at 3000 r / min (about 1476 × g) using Ultra 50kDa MWCO) to 0.8 mL, and 10 mL of 20 mM Tris was added for resuspending and ultrafiltration and concentration was performed to remove free rapamycin. The mixture was repeated once and finally concentrated to 0.8 mL. Then 0.2 mL of 40% sucrose mother solution was added and the mixture was frozen at -80°C.

[0193] Preparation of F5: 8 mg of rapamycin, 37.5 mg of polylactic acid, 5 mg of DMG-PEG2k, and 12.5 mg of DSPG were dissolved together in 1 mL of dichloromethane as the oil phase. PVA was dissolved in 100 mM PB at pH = 8.0 to obtain 5% and 0.25% PVA solutions, respectively. 3 mL of 5% PVA solution was taken as aqueous phase 1 and 30 mL of 0.25% PVA solution was taken as aqueous phase 2 for standby use. The oil phase was added to aqueous phase 1, and ultrasonicated for 2 minutes at 200 W power using a cell sonicator under an ice bath. The ultrasonic mode was 5S working and 5S intermittent. Then aqueous phase 2 was added to the centrifuge tube and ultrasonicated again for 2 minutes in the same ultrasonic mode. Finally, the solution was poured into an open beaker, stirred at room temperature for 3 hours, and the organic solvent was evaporated. Use an ultrafiltration tube ( Ultrafiltration (50 kDa MWCO) at 3000 r / min (about 1476 × g) was concentrated to 0.8 mL, and 10 mL of 20 mM Tris was added for resuspending and ultrafiltration was performed.

[0194] (3) Preparation of Prescription F6 was carried out according to the method of Example 2:

[0195] 1.6 mg of rapamycin, 7.5 mg of PLA, and 2.5 mg of PEG-PLA were dissolved in 1 mL of acetone. PVA was dissolved in 100 mM PB at pH = 8.0 to obtain 2% and 0.25% PVA solutions, respectively. The polymer solution and 1 mL of 2% PVA solution were mixed through a microfluidic chip and flowed into a solution containing 2 mL of 0.25% PVA. Then, 15 mL of deionized water was added to obtain a nanoparticle solution with a volume of about 19 times that of the organic phase. Stir at room temperature for 3 h, evaporate the organic solvent, and filter using an ultrafiltration tube ( Ultrafiltration and concentration to 0.8 mL (Ultrasonic 50 kDa MWCO) at 3000 r / min (about 1476 × g), add 10 mL 20 mM Tris to resuspend and ultrafiltration and concentration to remove free rapamycin, repeat once, finally concentrate to 0.8 mL, then add 0.2 mL 40% sucrose mother solution and freeze at -80 °C.

[0196] (4) The nanoparticles were characterized according to Example 4. The results are shown in Table 1 below:

[0197] Table 1. Nanoparticle prescription information1

[0198]

[0199]

[0200] (5) The in vivo induction of AAV8 capsid tolerance function was tested according to the method of Example 5.

[0201] Twenty-eight C57 mice were randomly divided into seven groups. On the day of administration, 8E11 vg / kg VGB-R04 was first injected into the tail vein, and then all experimental groups except the control group were immediately injected with nanoparticles of different formulations. The dose of rapamycin was 5 mg / kg. Plasma was collected before administration and 1, 2, and 4 weeks after administration to detect anti-AAV8 antibodies (anti-AAV8 ADA).

[0202] The results are as follows Figure 1 As shown, from the results of the first round of in vivo screening, it can be seen that the Selecta formulation (F3) prepared by the emulsification method has the best tolerance induction ability. The lipid nanoparticle formulations F1 and F2 do not have the ability to induce tolerance. The formulations F4 (PLA40k / DSPE-PEG2k / DSPS) and F5 (PLA40k / DSPE-PEG2k / DSPG) with added lipid and polymer complexes have the ability to induce immune tolerance, among which the DSPS formulation is better and slightly weaker than the Selecta formulation (F3). The effect of F6 prepared by Example 2 is slightly worse than that of F3 prepared by Example 3, so the emulsification method of Example 3 is adopted for the subsequent preparation of lipid polymer nanoparticles.

[0203] Example 7: Screening of AAV8 capsid immune tolerance induced in mice 2

[0204] This round of in vivo screening mainly examined the effects of different lipids and different ratios as well as the molecular weight of PEG in PEGylated lipids on the immunosuppressive function.

[0205] (1) Different formulations were prepared and characterized according to Examples 3 and 4. The formulation composition and particle size are shown in Table 2: wherein DSPE-PEG5k-H indicates that the terminal group of DSPE-PEG5k is a carboxyl group.

[0206] Table 2. Nanoparticle prescription information,

[0207]

[0208]

[0209] (2) The in vivo induction of AAV8 capsid tolerance function was tested according to the method of Example 5.

[0210] A total of 55 male C57BL / 6 mice were randomly divided into 11 groups. On the day of administration, 8E11 vg / kg VGB-R04 was first injected into the tail vein, and then all experimental groups except the control group were immediately injected with nanoparticles of different formulations. The dose of rapamycin was 5 mg / kg; plasma was collected from all mice at different time points (before administration, 1 week, 2 weeks and 4 weeks after administration) to detect anti-AAV8 antibodies (anti-AAV8 ADA).

[0211] The results are as follows Figure 2 As shown, the results show that when 5-15% DSPS (F7, F8 and F9) is added to the Selecta formula (F15), the ability of the nanoparticles to induce immune tolerance is reduced, and the formula with 5% DSPS has the least reduction. After adding 10% stearic acid (F14), the ability of the nanoparticles to induce immune tolerance is reduced, and the reduction is small. Therefore, we speculate that although anionic lipids such as stearic acid or DSPG, DSPS have the potential to increase APC targeting, their addition amount needs to be screened and optimized. Comparing the results of F10 (PLA40k / DSPE-PEG2k / DSPS=82 / 3 / 15) and F11 (PLA40k / DSPE-PEG2k / DSPS=83.8 / 1.2 / 15), it can be seen that the proportion of DSPE-PEG is reduced and the immune tolerance of the nanoparticles is increased, indicating that the proportion of DSPE-PEG in the formula is also an important factor affecting the tolerance inducing ability of the nanoparticles.

[0212] In addition, the chain length of PEG on the surface of nanoparticles may affect the circulation and distribution behavior of nanoparticles. Therefore, the inventors used DSPE-PEG5k-H (F16) to replace PEG5k-PLA50k in Selecta prescription F15 to investigate the effect of PEG lipids on the tolerance-inducing ability of nanoparticles. The molar mass fraction of PEG5k in lipids and polymers in F16 was roughly the same as that in F15. The results are shown in Figure 2. Figure 2 As shown, the hydrophobic part of the PEG polymer used in the two is different, but under the conditions of similar PEG molar ratio and similar PEG chain length, the ability of F16 to induce immune tolerance is close to that of F15, proving that DSPE-PEG5k can replace PEG5k-PLA50k in the prescription. At the same time, the inventors speculate that the amount of PEG distributed on the surface of the nanoparticles and the chain length of PEG have a significant effect on the tolerance-inducing ability of the nanoparticles.

[0213] It should be noted that the PEGs used in the present invention are all linear polymers, i.e., without side chains. Those skilled in the art should understand that for linear polymers of the same composition without side chains, the same or different molecular weights can directly represent the same or different chain lengths, and the degree of differentiation of the two is the same, so in most cases, the two characterize roughly the same content.

[0214] Example 8: Screening of AAV8 capsid immune tolerance induced in mice 3

[0215] The purpose of this round of in vivo screening was to further optimize and screen the ratio of DSPS and DSPE-PEG lipid addition, to further investigate the PEG molecular weight of the DSPE-PEG used, and the effects of the end groups of PEGylated lipids on the immunosuppressive effects of molecules targeting APC function, such as mannose and sialic acid.

[0216] (1) Different formulations were prepared and characterized according to Examples 3 and 4. The formulation composition and particle size are shown in Table 3: wherein M represents DSPE-PEG2k-Mannose, and S represents DSPE-PEG2k-Sialic Acid.

[0217] Table 3. Nanoparticles Prescription Information

[0218]

[0219] (2) The in vivo induction of AAV8 capsid tolerance function was tested according to the method of Example 5.

[0220] A total of 52 male C57BL / 6 mice were randomly divided into 13 groups. On the day of administration, 8E11 vg / kg VGB-R04 was first injected into the tail vein, and then all experimental groups except the control group were immediately injected with nanoparticles of different formulations. The dose of rapamycin was 5 mg / kg. Plasma was collected before administration and 1, 2 and 4 weeks after administration to detect anti-AAV8 antibodies (anti-AAV8 ADA).

[0221] The results are as follows Figure 3 As shown, the comparison of F17, 18, 19, and 20 shows that in the PLA / DSPS two-component nanoparticles, when the mass of DSPS accounts for about 5% of the total mass of polymers and lipids, the nanoparticles have the best tolerance-inducing effect. F22 and F27 show that the immune tolerance ability of adding DSPS to the PLA / PEG5k-PLA50k prescription decreases. The results of F21 and F28 further show that the DSPE-PEG5k prescription is significantly better than the DSPE-PEG2k prescription, proving that the chain length of PEG in the DSPE-PEG used has a significant effect on the ability of nanoparticles to induce immune tolerance. The in vivo data of F23-25 ​​show that the prescription containing DSPE-PEG2k-mannose or sialic acid under this ratio does not significantly increase the ability to induce immune tolerance compared with Selecta's original prescription F27. The results of F18, F28, and F27 show that the nanoparticles formed by PLA / DSPS composite (F18) or PLA / DSPE-PEG5k / DSPS (F28) have a slightly stronger tolerance-inducing effect than the Selecta prescription.

[0222] Example 9: Screening of AAV8 capsid immune tolerance in mice

[0223] After studying Examples 6-8, the inventors determined that F18 (PLA40k / DSPS / Rapam=95 / 5 / 16) and F28 (PLA40k / DSPE-PEG5k / DSPS / Rapam=95.4 / 2.6 / 2 / 16) have better immunosuppressive effects. Therefore, the purpose of this round of in vivo screening is to further optimize the ratio of PLA, DSPS and DSPE-PEG addition and to investigate the effect of rapamycin loading and PLA molecular weight on the immunosuppressive effect of nanoparticles. The further optimization of the ratio of PLA, DSPS and DSPE-PEG addition is based on the prescriptions of F18 and F28, and the ratio of each component is fine-tuned to explore a better prescription for nanoparticles.

[0224] (1) Different formulations were prepared and characterized according to Examples 3 and 4. The formulation composition and particle size are shown in Table 4:

[0225] Table 4. Nanoparticles Prescription Information

[0226]

[0227] (2) The in vivo induction of AAV8 capsid tolerance function was tested according to the method of Example 5.

[0228] A total of 48 male C57BL / 6 mice were randomly divided into 12 groups. On the day of administration, 8E11 vg / kg VGB-R04 was first injected into the tail vein, and then all experimental groups except the control group were immediately injected with nanoparticles of different formulations. The dose of rapamycin was 5 mg / kg. Plasma was collected before administration and 1, 2 and 4 weeks after administration to detect anti-AAV8 antibodies (anti-AAV8 ADA).

[0229] The results are as follows Figure 4 As shown, the comparison between F31 (PLA40k / DSPE-PEG5k / DSPS=95.4 / 2.6 / 2) and F37 (PLA20k / DSPE-PEG5k / DSPS=95.4 / 2.6 / 2) shows that the effect of PLA20k prescription is worse than that of PLA50k prescription, so PLA40k will continue to be used as the nanoparticle prescription. From F29-30 and F31, F33 and F34, the reduction of drug loading significantly increases the ability of PLA50k / DSPE-PEG5k / DSPS and PLA50k / DSPS to induce immune tolerance.

[0230] Example 10: Screening of AAV8 capsid immune tolerance induced in mice 5

[0231] The purpose of this round of in vivo screening was to further optimize the ratio of DSPS and DSPE-PEG lipid addition and to investigate the effects of rapamycin derivatives and their drug loading on the immunosuppressive effect.

[0232] (1) Different formulations were prepared and characterized according to Examples 3 and 4. The formulation composition and particle size are shown in Table 5: BA9 represents Umirolimus (Cas: 851536-75-9), a derivative of rapamycin.

[0233] Table 5. Nanoparticles Prescription Information

[0234]

[0235]

[0236] (2) The in vivo induction of AAV8 capsid tolerance function was tested according to the method of Example 5.

[0237] A total of 56 male C57BL / 6 mice were randomly divided into 14 groups. On the day of administration, 4E11 vg / kg VGB-R04 was first injected into the tail vein, and then all experimental groups except the control group were immediately injected with nanoparticles of different formulations. The dose of rapamycin was 5 mg / kg. Plasma was collected before administration and 1, 2 and 4 weeks after administration to detect anti-AAV8 antibodies (anti-AAV8 ADA).

[0238] The results are as follows Figure 5 As shown, from the comparison of the effects of F40, F41 and F42, the nanoparticles containing PEGylated lipids have a stronger ability to induce immune tolerance. Compared with F42, F43 and selecta prescription F52, nanoparticles with stronger tolerance induction ability can be obtained by compounding DSPE-PEG5k or DSPE-PEG5k / DSPS mixture with PLA in a suitable ratio. From the results of F43-F46, the optimal addition ratio of DSPE-PEG5k is about 1.3%. From the results of F42 (PLA40k / DSPE-PEG5k / DSPS / Rapam=95.4 / 2.6 / 2 / 8) and F44 (PLA40k / DSPE-PEG5k / DSPS / Rapam=97.4 / 2.6 / 0 / 8), DSPS and DSPE-PEG5k must be compounded at a suitable ratio to significantly increase the tolerance induction ability of nanoparticles. Comparing F40 and F41, it can be seen from the results of F44 and F47 as well as F45 and F49 that under the PLA / DSPE-PEG5k / DSPS formulation, the ability of rapamycin derivative BA9 to induce immune tolerance is better than that of rapamycin derivative BA9. Moreover, from F47, F48, and F51, as the BA9 drug loading increases from 2.7% to 4.4% and 7.0% respectively, the tolerance induction ability of the nanoparticles does not decrease significantly like the rapamycin nanoparticle formulation, indicating that the lipophilic derivative of rapamycin has the function of further improving the efficiency of carrier use.

[0239] Example 11: Effect of administration of rapamycin lipid nanoparticles to inhibit uricase antibodies

[0240] Pegylated recombinant uricase is a powerful uric acid-lowering drug and is currently the only effective drug for treating chronic gout that is ineffective with other therapies. However, the drug is highly immunogenic and will produce anti-drug antibodies (ADA) after multiple administrations, which will lead to a decrease in the drug's ability to lower uric acid and an increase in the incidence of immune responses to infusions. In this example, Rapa immune nanoparticles are used in conjunction with the pegylated recombinant uricase drug (PUOX) being studied by the Shanghai Institute of Biology to investigate the role of immune nanoparticles in inhibiting the production of anti-PUOX antibodies and maintaining their blood drug concentrations.

[0241] (1) Different formulations were prepared and characterized according to Examples 3 and 4. The formulation composition and particle size are shown in Table 6:

[0242] Table 6. Nanoparticles Prescription Information

[0243]

[0244] (2) A total of 25 C57BL / 6 male mice were randomly divided into 5 groups, with 5 mice in each group. The group design is shown in Table 7 below. Rapaimmune nanoparticles with different prescriptions and uricase PUOX were mixed in a certain volume and injected into the tail vein of each group of mice according to the dose in Table 9. The drug was administered once every 2 weeks, for a total of 4 times. 100uL of blood was collected from each mouse before administration and 2h after each administration, and the blood was coagulated overnight at 2-8℃. 20ul of serum was obtained by centrifugation at 4000rpm and frozen at -80℃ for blood drug concentration and anti-drug antibody detection.

[0245] Table 7. Animal experiment group information

[0246] Group PUOX Rapa 1 0.5mg / kg / NA 2 0.5mg / kg F53, 2.5 mg / kg NA 3 0.5mg / kg F53, 10.0 mg / kg NA 4 0.5mg / kg F54, 2.5 mg / kg NA 5 0.5mg / kg F54, 10.0 mg / kg NA

[0247] (3) Detection method: PUOX and anti-PUOX antibodies in the blood were detected by ELISA enzyme-linked immunosorbent assay. The coated antibody used to detect blood drug concentration was anti-PUOX, with a coating concentration of 10ug / ml; the detection antibody was anti-PEG-biotin, with a final concentration of 0.25ug / ml; and the color development reagent was ST-HRP. The coated antibody used for anti-PUOX detection was PUOX protein, with a coating concentration of 2ug / ml; the detection antibody was biotin-labeled goat anti-mouse IgG (Fc) HRP antibody; the unbound substances were removed by washing the plate, and TMB was added for color development.

[0248] Table 8. Blood drug concentration data of different groups

[0249]

[0250]

[0251] Table 9. Antidrug antibody concentration data of different groups

[0252]

[0253] From Tables 8, 9 and Figure 6It can be seen that with the increase in the number of dosing, anti-drug antibodies gradually increased. Among them, the antibodies in the single-dose group G1 and the F53 low-dose group G2 increased sharply by about 16 times after the fourth dose, and the F53 high-dose group G3 and the F54 different dose groups G4 and G5 increased by 3.56, 4.98 and 4.02 times, respectively. Correspondingly, the blood drug concentration of the single-dose group G1 and the F53 low-dose group G2 decreased significantly after the third dose due to the presence of anti-drug antibodies; while the combined administration groups G3-G5 maintained a high level of overall anti-drug antibody levels due to the low overall level of anti-drug antibodies. The experimental results of rapam immune nanoparticles combined with PUOX showed that the PLA / DSPE-PEG5k / DSPS / Rapam nanoparticles formula has the ability to inhibit the production of drug antibodies, and the F54 formula containing the DSPE-PEG5k / DSPS mixture is the best.

[0254] Example 12: Rapamycin nanoparticles combined with CTLA4 inhibit the production of AAV antibodies

[0255] Experimental design: Cytotoxic T lymphocyte antigen 4 (CTLA4) is a CD28 family receptor that is mainly expressed in activated T cells; although CTLA-4 is homologous to CD28, its affinity for co-stimulatory molecules CD80 and CD86 is much higher than that of CD28. After binding to its co-stimulatory molecules, CTLA4 interferes with the production of IL-2 and interrupts the cell cycle process, thereby inhibiting the proliferation of activated T cells. The CTLA4 protein is composed of the human IgG Fc domain and the CTLA-4 extracellular domain, and has a high binding affinity for CD80 and CD86; it can block CD28-mediated T cell co-stimulatory signals, thereby inducing immunosuppression and tolerance. Therefore, this study used C57BL / 6 mice to evaluate the effects of rapamycin nanoparticles and CTLA4 protein alone or in combination on the inhibition of AAV antibody production.

[0256] (1) Rapa nanoparticles were prepared and characterized according to Examples 3 and 4. The formulation composition and particle size are shown in Table 10:

[0257]

[0258]

[0259] (2) A total of 44 C57BL / 6 mice were randomly divided into 11 groups, with 4 mice in each group, half male and half female. The group design is shown in Table 11 below. On the day of administration, nanoparticles and / or CTLA4 protein were first injected into the tail vein, followed by an immediate injection of VGB-R04. Plasma was collected from all mice at different time points (before administration, 2 weeks and 4 weeks after administration) to detect anti-AAV8 antibodies (anti-AAV8ADA). The results are shown in Figure 7 .

[0260] Table 11. Animal experiment group information

[0261]

[0262] like Figure 7 As shown, both the PLA / DSPE-PEG5k / DSPS / Rapam nanoparticle formulation and the CTLA4 protein have the ability to inhibit the production of AAV antibodies, and the antibody titer is minimal when the two are used in combination, indicating that the two have a synergistic effect. When the viral load is high, the low-dose PLA / DSPE-PEG5k / DSPS / Rapam nanoparticle formulation has a weak ability to inhibit antibody production. Therefore, under high viral loads, AAV antibody production can be inhibited by increasing the dosage of PLA / DSPE-PEG5k / DSPS / Rapam nanoparticles and combining them with CTLA.

Claims

1. A nanoparticle comprising a lipid, a hydrophobic polymer and an immunosuppressant, wherein the lipid comprises an anionic lipid and a polyethylene glycol (PEG)-ylated lipid.

2. The nanoparticle according to claim 1, wherein anionic lipids and PEGylated lipids are distributed on the surface of the nanoparticle.

3. The nanoparticle according to claim 1 or 2, wherein the anionic lipid is a compound represented by formula 1: in, R1 and R2 are each selected from a hydrocarbon group of (Cn)-, wherein n represents the number of carbon atoms in the main chain, and n is selected from 13, 14, 15, 16 or 17.

4. The nanoparticle according to claim 3, wherein R1 and R2 are C17 saturated hydrocarbon groups without branches, and the anionic lipid is 1,2-dioctadecanoyl-sn-glycero-3-phospho-L-serine (DSPS).

5. The nanoparticle according to claim 1 or 2, wherein the immunosuppressant is an mTOR inhibitor, preferably rapamycin and / or its lipophilic derivatives.

6. The nanoparticles according to claim 1 or 2, wherein the hydrophobic polymer is polyester, preferably polylactic acid (PLA).

7. The nanoparticle according to claim 1 or 2, wherein the PEGylated lipid is PEGylated distearoylphosphatidylethanolamine (DSPE-PEG).

8. The nanoparticles according to claim 4, wherein the mass fraction of DSPS in the hydrophobic polymer and the lipid is between 0.1 wt% and 30 wt%, preferably 0.5 wt% and 5 wt%.

9. A pharmaceutical composition comprising the nanoparticles according to any one of claims 1 to 8 and / or a pharmaceutically acceptable carrier.

10. Use of the nanoparticles according to any one of claims 1 to 8 and / or the pharmaceutical composition according to claim 9 in preventing and / or treating autoimmune diseases and / or in inhibiting an immune response of a subject to a therapeutic drug.

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