Nanoparticles of mycophenolate prodrugs and their therapeutic applications

Abstract

Nanoparticles containing multiple therapeutic molecules arranged in the form of spherical micelles are described, which are suitable for topical delivery and release of mycophenolic acids and are therefore effective in treating autoimmune diseases, fibrosis and / or organ rejection, particularly lung diseases. Also described are pharmaceutical compositions containing nanoparticles in a pharmaceutically acceptable solvent and methods for producing said nanoparticles.

Description

[Technical Field]

[0001] This invention relates to the fields of medicine and healthcare.

[0002] In particular, this invention relates to the delivery / formulation of active ingredients, more specifically mycophenolic acids, in nanoparticles having a micelle structure, and their use in the treatment of autoimmune diseases, fibrosis, and transplant rejection diseases, especially diseases affecting the lungs. [Background technology]

[0003] Mycophenolic acid is a 2-benzofuran immunosuppressant used to prevent organ rejection in heart, kidney, and liver transplants, and to treat autoimmune diseases. This compound is produced by Penicillium stoloniferum and acts by blocking de novo purine synthesis through reversible, non-competitive inhibition of inosine monophosphate dehydrogenase (IMPDH). Inhibition of this enzyme reduces lymphocyte proliferation and diminishes their ability to recognize and attack transplanted organs.

[0004] Mycophenolic acid (MPA) is primarily administered orally or intravenously in the form of capsules, tablets, or suspensions, mainly as its sodium salt or mofetil ester. Typical dosages are 180 mg, 360 mg, and 500 mg for tablets, 250 mg for capsules, 1 g / 5 ml for oral suspension, and 500 mg for intravenous administration.

[0005] After oral administration, the sodium salt and mofetil ester are absorbed primarily in the small intestine in the form of mycophenolic acid. In the case of mofetil ester, activation occurs in the intestines before systemic delivery by carboxylesterase (CES), which hydrolyzes the ester to mycophenolic acid. The main first-pass metabolism is observed in the liver, where mycophenolic acid is converted primarily to the inactive metabolite 7-O glucuronide by glucuronosyltransferase.

[0006] As explained by Staatz et al. ("Clinical pharmacokinetics and pharmacodynamics of mycophenolate in solid organ transplant recipients", Clin Pharmacokinet. 2007; 46(1): 13-58), the blood concentration of this metabolite is 20 to 100 times higher than that of mycophenolic acid (MPA). Some of this inactive metabolite reaches the gastrointestinal tract again via bile excretion, where it is converted back to mycophenolic acid, causing a second peak in blood concentration 6 to 12 hours later. Three other metabolites are also produced, including acyl glucuronide derivatives, which, although produced in smaller quantities, have pharmacological effects comparable to MPA. MPA is mainly excreted in the urine (87%) in the form of the inactive metabolite 7-O glucuronide.

[0007] The usual dosage of mycophenolate mofetil is 2g per day for kidney transplants, 3g per day for heart transplants, and 3g per day for liver transplants. When used off-label for lupus nephritis, the dosage is 0.5-3g per day, and for interstitial lung disease, it is 1-3g per day.

[0008] High-dose systemic therapy required to compensate for the first-pass effect and achieve therapeutic concentrations of MPA in the target organ or tissue is associated with serious side effects, primarily in the gastrointestinal and genitourinary tracts. Furthermore, the resulting significant immunosuppression can lead to opportunistic infections, fatal infections, sepsis, and hematological disorders (neutropenia or anemia).

[0009] Mycophenolic acid and mofetil ester molecules possess phenolic groups suitable for esterification reactions. Xie H. et al. have described a self-assembled structure obtained by esterifying the phenolic group of mycophenolic acid mofetil with alcohols of different chain lengths ("Target-oriented delivery of selfassembled immunosuppressant cocktails prolongs allogeneic orthotopic liver transplant survival", Journal of Controlled Release, 2020, Volume 328: 237-25). It should be noted that these nanoparticles have a morpholine ring, and its nitrogen atom is protonated at acidic pH levels in tissues and body fluids where inflammatory responses are progressing. pH-dependent protonation of nanoparticles with a morpholine structure has also been reported by Zhang et al. ("Morpholino-decorated long circulating polymeric micelles with the function of surface charge transition triggered by pH changes", Chem Commun (Camb); 2013, 49(66): 7286-8).

[0010] However, the therapeutic application of positively charged nanoparticles is limited, particularly in the field of lung diseases, mainly due to their lack of mucosal penetration (Lai K. et al., “Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues”; 2009, Adv Drug Deliv Rev., 61(2): 158-71.) and dose-dependent irritation to the airways caused by changes in the osmotic pressure of the lung fluid (Son YJ et al., in “Optimizing spray-dried porous particles for high dose delivery with a portable dry powder inhaler”; 2021, Pharmaceutics. 13(9):1528.).

[0011] Furthermore, protonation of nanoparticles can cause structural changes due to repulsion between the positive charges of the constituent monomers, potentially leading to the destruction of the nanoparticle structure (Mu Y. et al. in “Advances in pH-responsive drug delivery systems”; 2021, OpenNano and Nakayama M. et al. in “Polymeric micelles with stimuli-triggering systems for advanced cancer drug targeting”; 2014, J Drug Target., 22(7): 584-99). If not properly considered, this phenomenon can lead to uncontrolled release of the active ingredient before it reaches the target site, potentially negatively impacting therapeutic efficacy (Alsawaftah N. et al. in “pH responsive nanocarriers in cancer therapy”; 2022, Polymers., 14(5): 936).

[0012] Mycophenolic acid, its mofetil ester, and its sodium salt are poorly soluble in aqueous media (0.0355 mg / ml for mycophenolic acid, 0.043 mg / ml for mycophenolate mofetil, and 0.057 mg / ml for mycophenolate sodium salt (Source: Drugbank)), therefore the use of solubilizers is necessary.

[0013] To overcome the limitation of mycophenolic acid's low solubility, various methods have been employed to improve its water solubility, including nanoparticle systems (e.g., liposomes (Patel et. al, “Liposomal delivery of Mycophenolic Acid with Quercitin for improved breast cancer therapy in SD rats”, Front Bioeng Biotechnol. 2020 Jun 16; 8: 631), cyclodextrins (Khalafi et. al, “Investigation of the inclusion complex of beta-cyclodextrin with Mycophenolate mofetil”, Spectrochim Acta A Mol Biomol Spectrosc. 2012 May; 90: 45-9; Iqbal et. al, “Solid lipid nanoparticles of Mycophenolate mofetil: an attempt to control the release of an immunosuppressant”, Int J Nanomedicine. 2020 Aug 5; 15: 5603-5612) and polymer nanoparticles (Shirali et. al.) Several formulation strategies have been developed, including the use of “Nanoparticles delivery of Mycophenolic acid upregulates PD-L1 on dendritic cells to prolong murine allograft survival”, Am J Transplant. 2011 Dec; 11(12): 2582-92).

[0014] The above strategy involves encapsulating MPA within an excipient capable of forming nanostructures. However, this method has a major limitation: the amount of drug is reduced, mainly due to the need to use an excessive amount of excipient relative to the active ingredient. Furthermore, the above nanostructure system may exhibit a so-called "burst release" phenomenon, where the active ingredient contained within is released faster than the steady state, potentially affecting the therapeutic effect.

[0015] For example, in the treatment of autoimmune diseases, fibrosis, and organ rejection, particularly diseases affecting the lungs, the inventors have created novel nanoparticles containing multiple therapeutic molecules that contain mycophenolic acid, in order to achieve a more efficient system for delivering mycophenolic acid to target tissues or organs and to overcome the limitations of the prior art formulation strategies described above. [Overview of the Initiative]

[0016] The first aspect of the invention is a nanoparticle comprising a plurality of therapeutic molecules arranged in the form of a spherical micelle, wherein the therapeutic molecules are of general formula (I)

[0017] [ka]

[0018] (In the formula, R is a linear or branched saturated or unsaturated alkyl chain having 5 to 22 carbon atoms, R 1 (This is an OH molecule bound to a binding molecule selected as needed from the group consisting of polyethylene glycol (PEG), triethylene glycol (TEG), sugars such as hyaluronic acid, mannose, and trehalose, proteins, peptides, antibodies, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), aptamers, and combinations thereof.) It is represented as follows.

[0019] A further object of the invention is a pharmaceutical composition containing the nanoparticles described above, as defined in the claims, and the therapeutic use of the nanoparticles or the pharmaceutical composition.

Brief Description of Drawings

[0020] [Figure 1] Figure 1 is a correlation diagram obtained from the analysis of PRO-MPA-C16 nanoparticle samples at various concentrations in milli-Q water. [Figure 2] Figure 2 shows the scattering intensity (derived count rate) as a function of the concentration of PRO-MPA-C16 nanoparticles. [Figure 3] Figure 3 shows the concentration of IMPDH when cells are treated by varying the concentration and treatment time of PRO-MPA-C16 nanoparticles. [Figure 4] Figure 4 shows the concentration of IMPDH when cells are treated in the presence of sputum layers from patients with bronchiectasis / cystic fibrosis. [Figure 5] Figure 5 shows the cell viability after treating PBMC with PRO-MPA-C16 nanoparticles for 72 hours. [Figure 6] Figure 6 shows the activity of IMPDH after treating PBMC and T-Jurkat cells with PRO-MPA-C16 nanoparticles for 24 hours. [Figure 7] Figure 7 shows the pulmonary ventilation level when PRO-MPA-C16 nanoparticles are inhaled into SSc-ILD model animals.

Modes for Carrying Out the Invention

[0021] Additional features and advantages of the invention are defined in the dependent claims which form an integral part of the specification.

[0022] In this specification, the term "in the form of spherical micelles" is intended to mean a spherical structure resulting from the clustering of the above-mentioned therapeutic molecules, wherein the hydrophobic alkyl chains of the molecules are located within the spherical structure to form a so-called micelle "core", and the hydrophilic groups corresponding to the hydroxyphthalide moieties of the therapeutic molecules are oriented towards the outside of the structure to form a so-called micelle "corona".

[0023] Preferably, in the nanoparticle therapeutic molecules of this invention, R is a linear or branched saturated or unsaturated alkyl chain having 5 to 22 carbon atoms, preferably 8 to 17 carbon atoms, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 carbon atoms.

[0024] Most preferably, R is a saturated alkyl linear chain having 16 carbon atoms.

[0025] In this invention, the R in therapeutic molecules of nanoparticles 1 The base is bound to the above-mentioned binding molecule as needed.

[0026] In this invention, the term "bound" refers to the R of the therapeutic molecule. 1 This refers to the presence of an ester covalent bond between the group and the carboxyl group or derivative thereof of the bonded molecule, and a spacer may also be present.

[0027] Therefore, in one embodiment of the invention, R 1 The group is a binding molecule (for example, the R 1 It is directly bonded to hyaluronic acid by forming an ester bond between one or more carboxyl groups of the base.

[0028] The hyaluronic acid on the surface of the nanoparticles of this invention is advantageous in enabling the nanoparticles to be directed towards cells expressing the intercellular adhesion molecule CD44.

[0029] If necessary, the binding molecule, for example, a sugar such as polyethylene glycol or mannose, can be functionalized with one or more functional groups suitable for forming an ester bond, for example, one or more carboxyl groups or one or more acyl groups. Examples of functionalized binding molecules suitable for use in this invention, though not limited to these, include heterobifunctional polyethylene glycol (OH-PEG-COOH), m-PEG-acyl chloride, 5-(α-dimannopyranosyloxy)pentanoic acid (α-Man-Bu-COOH), and (((α-dimannopyranosyloxy)ethoxy)ethoxy)propionic acid (α-Man-TEG-COOH).

[0030] In another aspect, the R 1 group is attached to the binding molecule via a bifunctional spacer. In this aspect, the bifunctional spacer has a first functional group suitable for forming a covalent bond with the binding molecule and a second functional group (e.g., a carboxyl group or a derivative thereof) suitable for forming an ester bond with the R 1 group.

[0031] R 1 Bifunctional spacers suitable for use in attaching the R

[0032] group to a protein binding molecule include, but are not limited to, maleimide-PEG-acetic acid (Mal-PEG-COOH) which can react with thiol groups present in protein molecules. 1 The attachment between the binding molecule selected from messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and aptamer and the R 1 group may be achieved by using a bifunctional spacer that includes (i) a functional group capable of reacting with the amino group of, for example, an aptamer (a modified aptamer having an amino group at the 5' end), and (ii) a carboxyl group capable of reacting with the R

[0033] As will be apparent from the following detailed description, the present invention provides novel nanoparticles having a micelle structure composed of a plurality of prodrug molecules obtained by esterifying mycophenolic acid with a long-chain alcohol, which can deliver the mycophenolic acid and release it locally.

[0034] As used herein, "prodrug" refers to a pharmaceutical that does not exhibit activity until it is appropriately activated / metabolized in the body.

[0035] This invention is based on the inventor's remarkable discovery that, despite its high lipophilicity and lack of amphiphilic structure, the mycophenolic acid prodrug molecule can spontaneously cluster with each other in water to form a micelle structure (self-assembly) without the use of other substances.

[0036] The inventors also noted that the apparent solubility of the nanoparticles of this invention in water increased dramatically (at least eight times) compared to the solubility of mycophenolic acid in water.

[0037] The properties of these nanoparticles are completely unexpected based on prior art teachings. For example, Sifei Han's paper (Han S. et al, “Targeted delivery of a model immunomodulator to the lymphatic system: comparison of alkyl ester versus triglyceride mimetic lipid prodrug strategies”; 2014 J Control Release 177: 1-10) describes the use of MPA lipophilic esters to increase the distribution of lipids and lipoproteins in the lymphatic circulation.

[0038] International Publication No. 2017 / 019636 describes a lipophilic prodrug molecule of mycophenolic acid and a nanoparticle system (e.g., liposomes, micelles, etc.) for its delivery, wherein the prodrug molecule is complexed with one or more lipid components of the nanoparticle or encapsulated within one or more lipid components of the nanoparticle.

[0039] In vitro studies by the same inventors have also shown that nanoparticles possessing the above characteristics have the advantage of having an excellent ability to pass through thick mucous secretions such as bronchial mucus.

[0040] As shown in Figure 4, when human bronchial epithelial cells were treated with the nanoparticles of this invention in the presence of a layer of sputum from patients with bronchiectasis / cystic fibrosis, which is known to be particularly thick and viscous, the level of the target enzyme, inosine monophosphate dehydrogenase (IMPDH), was significantly reduced in these cells compared to mycophenolic acid used as a control.

[0041] These remarkable findings not only reveal that the nanoparticles of this invention can penetrate mucus secretions of complex densities and viscosities particularly effectively without the need for auxiliary components such as polyethylene glycol, but also clearly demonstrate that the nanoparticles simultaneously maintain their integrity and the ability to locally release the active ingredient, mycophenolic acid. Further experiments confirmed that the nanoparticles of this invention exhibit activity that suppresses the proliferation of immune cells by reducing the level of the IMPDH enzyme, and that this activity is dose-dependent (Figures 5 and 6). Based on the results of in vitro tests, the therapeutic activity of the nanoparticles of this invention was also verified in vivo using a mouse model of interstitial lung disease (SSc-ILD model), and it was shown that inhalation of the nanoparticles of this invention by SSc-ILD animals significantly improved the breathability of the lung tissue (Figures 7A-C).

[0042] While not wishing to be bound by any theory, the inventors believe that the ability of the nanoparticles of this invention to pass through physiological barriers such as high-density secretions in the airways may be due to the specific surface structure of the nanoparticles that exposes the hydroxyphthalide component to the outside.

[0043] Therefore, in light of the above explanation, the nanoparticles of this invention will be an important and innovative therapeutic tool, particularly in the field of lung diseases.

[0044] In one embodiment of the invention, the diameter of the nanoparticles is 30 to 700 nm, preferably 60 to 200 nm, and more preferably 70 to 150 nm.

[0045] In another embodiment, the zeta potential of the nanoparticles of this invention is -5mV to -60mV.

[0046] In this specification, the term "zeta potential" refers to the electric charge generated at the interface between a solid surface and the liquid in which it is immersed.

[0047] A key advantage of the nanoparticles of this invention is that they do not require additives such as emulsifiers or surfactants to ensure and / or promote the clustering of mycophenolic acid prodrug molecules to form spherical micelle structures and to maintain the stability of the nanoparticles in aqueous suspension.

[0048] Therefore, it is believed that the nanoparticles of this invention contain and deliver a very large amount of mycophenolic acid, namely up to approximately 80% by weight of the total weight of the nanoparticles.

[0049] Furthermore, the absence of the above-mentioned additives means that the nanoparticles are more suitable for all applications requiring that the nanoparticles be non-toxic and stable in physiological media, particularly for therapeutic applications.

[0050] Therefore, in one embodiment, the nanoparticles of this invention consist of a plurality of therapeutic molecules arranged in the form of spherical micelles.

[0051] The nanoparticles of this invention do not require additives, but the scope of this invention also includes embodiments in which the nanoparticles may contain, in addition to a plurality of therapeutic molecules, a surfactant, preferably a surfactant with a low toxicity index or no toxicity. In this embodiment, the surfactant enables improvement of certain chemical-physical properties of the nanoparticles of this invention, for example, reducing and improving their size and stability in bodily fluids, respectively.

[0052] Typical surfactants suitable for use in the particles of this invention include, but are not limited to, phospholipids, PEG complexes having lipophilic molecules such as polyethylene glycol (PEG)-cholesterol complexes, polyoxyethylene / polyoxypropylene copolymers, fatty acids esterified with ethoxylated sorbitan (polysorbate), and combinations thereof.

[0053] In this invention, nanoparticles containing or comprising a plurality of the above-mentioned therapeutic molecules may also contain a lipophilic therapeutic agent inside a hydrophobic core made up of the alkyl chains of the therapeutic molecules.

[0054] Advantageously, in this embodiment, the nanoparticles of the present invention can deliver, in addition to mycophenolic acid, another active ingredient for the treatment of a specific disease.

[0055] Lipophilic therapeutic agents suitable for inclusion in the nanoparticles of this invention include, but are not limited to, immunosuppressants (e.g., tacrolimus, cyclophosphamide, everolimus, sirolimus, methotrexate, azathioprine), anti-inflammatory agents (e.g., dexamethasone, budesonide, beclomethasone), anti-fibrotic agents (e.g., pirfenidone, nintedanib), and antitumor agents (e.g., salakatinib).

[0056] Lipophilic therapeutic agents may be incorporated into the nanoparticles of this invention by non-covalent encapsulation methods, such as nanoprecipitation.

[0057] The selection of the most appropriate sealing method for use in this invention is within the scope of the art of those skilled in the art.

[0058] Due to the beneficial chemical-physical properties described above, and their remarkable ability to pass through dense secretions such as bronchial mucus, the nanoparticles of this invention are particularly suitable for therapeutic use, especially in the treatment of autoimmune diseases, fibrotic diseases, and organ rejection reactions affecting the lungs.

[0059] In a preferred embodiment, the nanoparticles of this invention are suitable for use in the treatment of autoimmune lung diseases and / or fibrous lung diseases.

[0060] In another preferred embodiment, the nanoparticles of this invention are suitable for use in the treatment and / or prevention of organ rejection, preferably lung transplant rejection.

[0061] Autoimmune lung diseases and / or fibrous lung diseases that can be treated with the nanoparticles of this invention include, but are not limited to, systemic sclerosis with interstitial lung disease (SSc-ILD), lymphangioleiomyomatosis (LAM), sarcoidosis, and bronchiolitis obliterans (BOS).

[0062] Pharmaceutical compositions containing the nanoparticles of this invention in combination with at least one pharmaceutically acceptable solvent, excipient, and / or diluent are also within the scope of the invention.

[0063] In this invention, the pharmaceutical composition is suitable for use in the above-mentioned therapeutic applications related to the nanoparticles of this invention.

[0064] The pharmaceutical compositions of this invention can be formulated for administration via appropriate routes, such as enteral (oral or gastrointestinal, rectal, sublingual), parenteral (intravenous, intra-arterial, percutaneous, intramuscular, intradermal, subcutaneous, intraperitoneal), topical (where the drug is in direct contact with the site of action and / or the skin and / or mucous membranes), intraocular, inhalation, and endotracheal routes.

[0065] Preferred oral dosage forms are tablets, capsules, sachets, powders, granules, pellets, gels, syrups, elixirs, oral solutions, suspensions, or emulsions. These dosage forms usually also include additional substances such as lactose, dextrose, mannitol, stearic acid, starch, and / or gelatin.

[0066] Injectable compositions, such as injectable solutions or suspensions in aqueous or oily solutions, can be formulated according to the prior art, and optionally using appropriate dispersants, wetting agents and / or suspending agents.

[0067] Formulations suitable for intranasal administration include, for example, powders with a particle size of 10 to 150 μm containing the nanoparticles of this invention as the active ingredient. These formulations may be administered, for example, by placing a container containing the powder near the nostrils and rapidly inhaling it through the nasal cavity.

[0068] Naturally, the selection of suitable solvents, excipients, and / or diluents is carried out according to the desired dosage form, and this selection is within the scope of the art. The selection of the dose of the active ingredient and the dosage regimen is also within the scope of the art, and this selection depends on several factors, such as the patient's age and weight and the stage of disease progression.

[0069] In one embodiment of the invention, the pharmaceutical composition comprises at least one additional active ingredient. Examples of the at least one additional active ingredient include, but are not limited to, immunosuppressive molecules such as everolimus, tacrolimus, methotrexate, sirolimus, cyclosporine, cyclophosphamide, and azathioprine; anti-inflammatory molecules such as dexamethasone, budesonide, and beclomethasone; antifibrotic agents such as pirfenidone and nintedanib; and antitumor agents such as salakatinib.

[0070] A further object of this invention is a method for producing nanoparticles of this invention, (i) A step of mixing a plurality of the above therapeutic molecules in a polar solvent to obtain a first mixture, wherein the polar solvent is not water; (ii) Adding the first mixture from step (i) to a predetermined amount of water under stirring to obtain a second mixture, wherein the predetermined amount of water is at least 65% by volume of the total volume of the second mixture; and, (iii) The step of removing the polar solvent from the second mixture of step (ii) to obtain nanoparticles containing multiple therapeutic molecules arranged in the form of spherical micelles in an aqueous suspension; This is a method that has [something].

[0071] The most preferred polar solvent is ethanol.

[0072] In the method of this invention, polar solvents can be removed by vacuum evaporation or dialysis.

[0073] The selection of the most appropriate method for evaporating polar solvents within the scope of this invention is within the realm of the art.

[0074] In a preferred embodiment of the invention, the critical micelle concentration (CMC) of the therapeutic molecule in the aqueous suspension of step (iii) is 50 to 150 ng / ml, preferably 50 to 100 ng / ml.

[0075] In this specification, the term "critical micelle concentration" refers to the concentration of a solution of molecules in which a large number of molecules cluster into micelles.

[0076] The method of this invention may also include a step of separating therapeutic molecules that did not participate in the formation of nanoparticles from the aqueous suspension of step (iii).

[0077] Suitable methods for use in the above separation process include, but are not limited to, sedimentation, filtration, and centrifugation.

[0078] Centrifugal separation is particularly preferred.

[0079] The above preferred embodiments can be combined with each other as needed, and the implementation of these combinations is within the scope of the art of those skilled in the art.

[0080] Examples are provided below for illustrative purposes, but these are not intended to limit the scope of the invention as defined in the claims. [Examples]

[0081] Example 1: Chemical and physical properties of nanoparticles of this invention The inventors diligently conducted tests to evaluate the various physicochemical properties of the nanoparticles of this invention, as described below.

[0082] 1. Apparent solubility Apparent solubility refers to the solubility of a solute in a solvent as measured under specific conditions. In this specification, apparent solubility represents the concentration at which the nanoparticles of this invention can form a stable aqueous suspension. A stable suspension refers to an aqueous suspension composed of the nanoparticles of this invention, at a concentration higher than that of a saturated aqueous solution of mycophenolic acid and its derivatives, mofetil salt and sodium salt, where no phase separation and / or precipitation is observed after preparation, and stability is achieved without the use of at least one stabilizer and / or solubilizer.

[0083] 2. Size The analysis was performed using a Zeta-sizer Nano Z instrument (Malvern Instruments, Malvern, UK). Milli-Q water at 25°C was used for the measurements. The technique used to measure the nanoparticle diameter is called dynamic light scattering (DLS). This technique allows for the measurement of the hydrodynamic diameter of nanoparticles dispersed in a liquid. A laser beam is shone onto the sample, and the change in the intensity of the scattered light is measured as a function of time. The intensity change measured by the detector is caused by the Brownian motion of the particles. At the same temperature and viscosity, small particles move rapidly, causing a rapid change in scattering intensity, while large particles move slowly, resulting in a slower change in intensity.

[0084] 3. Zeta potential The analysis was performed using a Zeta-sizer Nano Z instrument (Malvern Instruments, Malvern, UK). All samples were analyzed in Milli-Q water at 25°C. The zeta potential of the nanoparticles of this invention was determined by measuring the electrophoretic mobility of the particles suspended in water. To measure the electrophoretic mobility of the particles, an electric field was applied between the electrodes of a measurement cell in which the sample was placed and a laser beam was irradiated. Charged particles move towards the electrode with the opposite polarity, changing the frequency of the scattered light from the sample, which is proportional to the electrophoretic mobility.

[0085] Example 2: Production of a mycophenolic acid prodrug therapeutic molecule Four therapeutic molecules represented by formula (I) were fabricated, each with a different alkyl chain length R, as follows: (i) (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate pentyl ester, hereafter referred to as PRO-MPA-C5; (ii) (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate octyl ester, hereafter referred to as PRO-MPA-C8; (iii) (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate dodecyl ester, hereafter referred to as PRO-MPA-C12; (iv) (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate hexadecyl ester, hereafter referred to as PRO-MPA-C16.

[0086] Various prodrug molecules were prepared using synthetic methods involving the following two different steps: - Formation of MPA acilcloride; - Formation of the desired ester by reaction with alkyl alcohol.

[0087] In the esterification reaction of the MPA carboxylic acid group with alcohol, alkyl alcohols of various lengths were used: - Pentane alcohol (C5); - Octane alcohol (C8); - Dodecane alcohol (C12); - Hexadecane alcohol (C16).

[0088] PRO-MPA-C5 synthesis The molecule (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate pentyl ester (PRO-MPA-C5) was synthesized by the following method:

[0089] [ka]

[0090] Specifically, mycophenolic acid (700 mg; 2.18 mmol) was dissolved in anhydrous dichloromethane (20 ml). Thionyl chloride (0.48 ml; 3 mol / equivalent) was added, and the solution was stirred at room temperature for 5 hours. The solvent was removed under vacuum. The residue was dissolved in 10 ml of anhydrous dichloromethane and added dropwise to a solution of 1-pentanol (191 mg; 1 mol / equivalent) in anhydrous dichloromethane (5 ml) and pyridine (1.75 ml; 10 mol / equivalent). The resulting mixture was stirred under a nitrogen atmosphere at room temperature for 12 hours, and then examined by HPLC-MS. Water was added to separate the two phases, and the organic phase was further washed with 4% hydrochloric acid, water, and brine. The solvent was removed, and the residue was purified by chromatography (Isolera Biotage; Sfar column; elution: hexane / ethyl acetate 9 / 1). Total yield: 15%.

[0091] PRO-MPA-C8 synthesis The molecule (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate octyl ester (PRO-MPA-C8) was synthesized by the inventors by the following method:

[0092] [ka]

[0093] Mycophenolic acid (700 mg; 2.18 mmol) was dissolved in anhydrous dichloromethane (20 ml). Thionyl chloride (0.48 ml; 3 mol / equivalent) was added, and the solution was stirred at room temperature for 5 hours. The solvent was removed under vacuum. The residue was dissolved in 10 ml of anhydrous dichloromethane and added dropwise to a solution of 1-octanol (283 mg; 1 mol / equivalent) in anhydrous dichloromethane (5 ml) and pyridine (1.75 ml; 10 mol / equivalent). The resulting mixture was stirred under a nitrogen atmosphere at room temperature for 12 hours, and then examined by HPLC-MS. Water was added to separate the two phases, and the organic phase was further washed with 4% hydrochloric acid, water, and brine. The solvent was removed, and the residue was purified by chromatography (Isolera Biotage; Sfar column; elution: hexane / ethyl acetate 9 / 1). Total yield: 19%.

[0094] PRO-MPA-C12 synthesis The molecule (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate dodecyl ester (PRO-MPA-C12) was synthesized by the following method:

[0095] [ka]

[0096] Mycophenolic acid (700 mg; 2.18 mmol) was dissolved in anhydrous dichloromethane (20 ml). Thionyl chloride (0.48 ml; 3 mol / equivalent) was added, and the solution was stirred at room temperature for 5 hours. The solvent was removed under vacuum. The residue was dissolved in 10 ml of anhydrous dichloromethane and added dropwise to a solution of 1-decanol (404 mg; 1 mol / equivalent) in anhydrous dichloromethane (10 ml) and pyridine (1.75 ml; 10 mol / equivalent). The resulting mixture was stirred under a nitrogen atmosphere at room temperature for 12 hours, and then examined by HPLC-MS.

[0097] Water was added to separate the two phases, and the organic phase was further washed with 4% hydrochloric acid, water, and brine. The solvent was removed, and the residue was purified by chromatography (Isolera Biotage; Sfar column; elution: hexane / ethyl acetate 9 / 1). Total yield: 13%.

[0098] PRO-MPA-C16 synthesis The method established by the inventor for the synthesis of the molecule (4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoic acid hexadecyl ester (PRO-MPA-C16) is shown below.

[0099] [ka]

[0100] In short, mycophenolic acid (1 g; 3.12 mmol) was dissolved in anhydrous dichloromethane (40 ml). Thionyl chloride (0.68 ml; 3 mol / equivalent) was added, and the solution was stirred at room temperature for 5 hours. The solvent was removed under vacuum. The residue was dissolved in 10 ml of anhydrous dichloromethane and added dropwise to a solution of 1-hexadecanol (830 mg; 1 mol / equivalent) in anhydrous dichloromethane (10 ml) and pyridine (2.15 ml; 10 mol / equivalent). The resulting mixture was stirred under a nitrogen atmosphere at room temperature for 12 hours, and then examined by HPLC-MS. Water was added to separate the two phases, and the organic phase was further washed with 4% hydrochloric acid, water, and brine. The solvent was removed, and the residue was purified by chromatography (Isolera Biotage; Sfar column; elution: hexane / ethyl acetate 9 / 1). Total yield: 25%.

[0101] The mycophenolic acid prodrug molecules obtained by the above synthesis method were characterized using 1H-NMR and HPLC-MS. For 1H-NMR, a portion of the sample was dissolved in deuterated chloroform and analyzed by nuclear magnetic resonance (300 MHz). HPLC was performed using a Phenomenex Gemini-NX C18 3 μm, 150-2.0 mm chromatography column. The sample was dissolved in acetonitrile / water 1:1 as the mobile phase. HPLC and mass spectrometry (MS ES+) were combined to identify the sample. Sample purity was determined by analyzing the peak area of ​​the chromatography obtained by HPLC.

[0102] Table 1 shows the physicochemical properties of the mycophenolic acid prodrug molecules described above.

[0103] [Table 1]

[0104] Example 3 R derivatized with polyethylene glycol (PEG) 1 Manufacturing of PRO-MPA-C16 containing the component PRO-MPA-C16-OPEG synthesis The method established by the inventor for the synthesis of (4E)-6-(1,3-dihydro-4-carbonyl polyethylene glycol monomethoxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoic acid hexadecyl ester (PRO-MPA-C16-OPEG) is shown in the following figure.

[0105] [ka]

[0106] In short, mycophenolic acid (1 g; 3.12 mmol) was dissolved in anhydrous dichloromethane (40 ml). Thionyl chloride (0.68 ml; 3 mol / equivalent) was added, and the solution was stirred at room temperature for 5 hours. The solvent was removed under vacuum. The residue was dissolved in 10 ml of anhydrous dichloromethane and added dropwise to a solution of 1-hexadecanol (830 mg; 1 mol / equivalent) in anhydrous dichloromethane (10 ml) and pyridine (2.15 ml; 10 mol / equivalent). The resulting mixture was stirred under a nitrogen atmosphere at room temperature for 12 hours, and then examined by HPLC-MS. Water was added to separate the two phases, and the organic phase was further washed with 4% hydrochloric acid, water, and brine. The solvent was removed, and the residue was purified by chromatography (Isolera Biotage; Sfar column; elution: hexane / ethyl acetate 9 / 1). Total yield: 25%.

[0107] Mycophenolate cetyl ester (100 mg; 0,184 mmol) was dissolved in dichloromethane (5 ml) and HOBT (0.2 mol / equivalent; 5 mg), and diisopropylethylamine (2 mol / equivalent; 50 mg), EDC-HCl (1.5 mol / equivalent; 53 mg), and PEG-COOH (MW 970 g / mol; 196 mg; 1.1 mol / equivalent) were added. The mixture was stirred at room temperature (25°C) for 24 hours, washed with water, and the phases were separated. The organic phase was evaporated under vacuum. The oily residue was purified by chromatography using an Isolera Biotage Sfar column (elution: dichloromethane / methanol 95 / 5). Yield: 35%.

[0108] The properties of the mycophenolic acid prodrug molecule obtained by the above synthesis method were evaluated using the method described in Example 2.

[0109] Table 2 shows the physicochemical properties of the PRO-MPA-C16-OPEG molecule.

[0110] [Table 2]

[0111] Example 4: Production of nanoparticles of this invention To produce the nanoparticles of this invention, the inventor employed a solvent injection method (also known as nanoprecipitation). Briefly, the mycophenolic acid prodrug molecule was dissolved in a known amount of water-miscible organic solvent, and the resulting mixture was injected into a known amount of Milli-Q water using an automatic injector. In a typical preparation, the ratio of organic solvent to water was 1:10 (v / v). After the injection was complete, the organic solvent was removed by vacuum evaporation, and the final volume of water was adjusted based on the desired concentration of the molecule.

[0112] More specifically, 2.5 mg of PRO-MPA-C16 was dissolved in 1 ml of ethanol, and the resulting organic mixture was injected into 10 ml of Milli-Q water (40°C) while stirring. Then, to obtain PRO-MPA-C16 nanoparticles with a final concentration of 0.5 mg / ml, the resulting suspension was vacuum evaporated to remove some of the ethanol and water until the final volume was 5 ml.

[0113] Next, the sample was centrifuged at 4000 RPM for 10 minutes to remove non-self-assembled clusters and / or molecules, and the supernatant consisting of the nanoparticles of this invention was subsequently collected and stored at 4°C.

[0114] Using the method described above, the inventors prepared nanoparticles containing four types of mycophenolic acid prodrug molecules, namely PRO-MPA-C5, PRO-MPA-C8, PRO-MPA-C12, and PRO-MPA-C16 nanoparticles. Tables 3 and 4 below show the chemical-physical parameters of the nanoparticles measured immediately after production (T=0) and one week later (i.e., 168 hours later) (T=1 week).

[0115] [Table 3]

[0116] [Table 4]

[0117] As can be seen from the size data shown in Tables 3 and 4, nanoparticles containing PRO-MPA-C16 appear to be more stable. In fact, unlike nanoparticles obtained with other prodrug molecules, no significant increase in size was observed after one week.

[0118] The inventors also used a functionalized polyethylene glycol phospholipid complex (DSPE-PEG2000) as a surfactant to produce the nanoparticles of this invention, particularly PRO-MPA-C16 nanoparticles.

[0119] In this method, 2.5 mg of PRO-MPA-C16 molecules and 0.250 mg of DSPE-PEG2000 were dissolved in 1 ml of ethanol, and the resulting organic mixture was injected into 10 ml of Milli-Q water (40°C) while stirring. Then, to obtain PRO-MPA-C16 nanoparticles with a final concentration of 0.5 mg / ml, the resulting suspension was vacuum evaporated to remove some of the ethanol and water until the final volume was 5 ml.

[0120] Next, the sample (PRO-MPA-C16-P) was centrifuged at 4000 RPM for 10 minutes to remove clusters and / or molecules of the non-self-assembled prodrug, and the supernatant consisting of the nanoparticles of the present invention was collected and stored at 4°C.

[0121] The inventors also manufactured nanoparticles of this invention consisting of PRO-MPA-C16 and PRO-MPA-C16-OPEG.

[0122] In this method, 2.5 mg of PRO-MPA-C16 molecules and 0.250 mg of PRO-MPA-C16-OPEG molecules were dissolved in 1.667 ml of ethanol, and the resulting organic mixture was injected into 5 ml of aqueous buffer [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) while stirring. Then, to obtain nanoparticles with a final concentration of 0.79 mg / ml, the resulting suspension was vacuum evaporated to remove some of the ethanol and water until the final volume was 3.5 ml.

[0123] Next, the sample (PRO-MPA-C16-PRO-MPA-C16-OPEG) was centrifuged at 4000 RPM for 10 minutes to remove clusters and / or molecules of the non-self-assembled prodrug, and the supernatant consisting of the nanoparticles of the present invention was collected and stored at 4°C.

[0124] The properties of PRO-MPA-C16 nanoparticles containing the surfactant DSPE-PEG2000 and PRO-MPA-C16 nanoparticles containing PRO-MPA-C16-OPEG are shown in Table 5 below.

[0125] [Table 5]

[0126] Example 5: Encapsulation of lipophilic active ingredient into nanoparticles of this invention The inventors encapsulated a lipophilic active ingredient in the nanoparticles of this invention using the solvent injection method (also known as nanoprecipitation method) shown in Example 4, through a physical encapsulation process. In the first example, garnicertib molecules were encapsulated within PRO-MPA-C16 nanoparticles.

[0127] In this method, 5 mg of PRO-MPA-C16 molecules and 1 mg of garnicertiveb were dissolved in 1.667 ml of ethanol, and the resulting organic mixture was injected into 5 ml of aqueous buffer [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) while stirring. The resulting suspension was then vacuum evaporated to remove some of the ethanol and water until the final volume was 3.5 ml, in order to obtain PRO-MPA-C16 nanoparticles with a final concentration of 1.43 mg / ml and garnicertiveb with a final concentration of 0.28 mg / ml.

[0128] Next, the sample (PRO-MPA-C16-GAL) was centrifuged at 4000 RPM for 10 minutes to remove unassembled prodrug molecules and / or unencapsulated active ingredients (garnicertib), and the supernatant consisting of the nanoparticles of this invention was then collected.

[0129] In the second example, the active ingredient nintedanib was encapsulated within PRO-MPA-C16 nanoparticles. In this method, 3.5 mg of PRO-MPA-C16 molecules and 0.350 mg of nintedanib were dissolved in 1.667 ml of ethanol, and the resulting organic mixture was stirred and injected into 5 ml of aqueous buffer [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) (pH 9.0). The resulting suspension was then vacuum evaporated to remove some of the ethanol and water until the final volume was 3.5 ml, in order to obtain PRO-MPA-C16 nanoparticles with a final concentration of 1 mg / ml and nintedanib with a final concentration of 0.1 mg / ml.

[0130] Next, the sample (PRO-MPA-C16-NTB) was centrifuged at 4000 RPM for 10 minutes to remove unassembled prodrug molecules and / or unencapsulated active ingredients (nintedanib), and the supernatant consisting of the nanoparticles of this invention was then collected.

[0131] The properties of the nanoparticles obtained in Example 5 are shown in Table 6 below.

[0132] [Table 6]

[0133] Example 6 Evaluation of critical micelle concentration (CMC) The critical micelle concentration (CMC) is the concentration at which a large number of monomers cluster together to form micelles. The CMC of the above-mentioned mycophenolic acid prodrug molecule, more specifically the PRO-MPA-C16 molecule, was determined using multi-angle dynamic light scattering techniques based on the measurement of scattering intensity. Scattering intensity is the intensity of scattered light resulting from the interaction between a light beam and nanoparticles. This is a function of the monomer concentration in the solution. When monomers cluster together to form micelles, the scattering intensity increases significantly.

[0134] Figure 1, a correlation diagram obtained from the analysis of PRO-MPA-C16 nanoparticle samples at various test concentrations in Milli-Q water (25°C), shows a trend where the intercept of the correlation function increases as the test concentration increases. The horizontal line represents the CMC limit of 0.8 g²⁻¹ in the formation of the colloidal structure / CMC.

[0135] Figure 2 shows the scattering intensity (derived count rate) as a function of concentration of the PRO-MPA-C16 nanoparticles tested, along with its trend line.

[0136] Based on the correlation diagram and the combination of scattering intensity test results, the CMC value for PRO-MPA-C16 as a function of the test concentration is 0.075 ± 0.025 μg / ml. This calculated experimental value is considerably lower than conventional micellar systems used in the pharmaceutical industry, such as monomethoxypoly(ethylene glycol)-block-poly(D,L-lactide) used in GENOXOL PM (CMC is 44 μg / ml) and Tween® 80 used in ESTRASORB (CMC is 13-15 μg / ml).

[0137] Advantageously, such a low CMC value ensures high stability of the nanoparticles of this invention after dilution with bodily fluids.

[0138] Example 7: Evaluation of the in vitro enzyme inhibitory effect of the nanoparticles of this invention As is well known, mycophenolic acid is a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH), which is important for the synthesis of purine nucleotides.

[0139] To evaluate the activity of the nanoparticles of this invention, particularly their ability to inhibit the enzyme, the inventors performed in vitro experiments using human bronchial epithelial cells (16HBE14o).

[0140] In the experiment, 16HBE14o cells were exposed to the nanoparticles of this invention at three concentrations (450, 150, and 50 nM) of PRO-MPA-C16 for 6, 4, and 2 hours, respectively. IMPDH activity was measured using a specific test (Assay Genie's IMPDH assay). Samples of 16HBE14o cells treated with mycophenolic acid at the above concentrations and times were used as controls.

[0141] As shown in Figure 3, the nanoparticles of this invention dose-dependently reduced IMPDH, as evidenced by the decrease in IMPDH after treating cells with increasing concentrations and durations of PRO-MPA-C16 nanoparticles or mycophenolic acid. The enzymatic activity of IMPDH was inversely proportional to the concentration of MPA.

[0142] In Figure 3, data are expressed as mean ± SEM, and the amount of IMPDH is normalized to untreated cells. Statistical significance compared to 2h. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, statistical significance of PRO-MPA-C16 versus MPA, #P<0.05, ##P<0.01, and ###P<0.001 were calculated using one-way ANOVA and Bonferroni post-hoc tests.

[0143] Importantly, the inhibition of the IMPDH enzyme was significantly higher when cells were treated with PRO-MPA-C16 nanoparticles compared to when cells were treated with MPA. The maximum enzyme inhibitory effect occurred 6 hours after treatment.

[0144] Example 8 Evaluation of the in vitro enzyme inhibitory effect of nanoparticles of this invention in the presence of mucus To verify whether the nanoparticles of this invention can exert their activity even in physiological secretions, particularly thick and viscous mucus secretions, the inventors performed the above experiment on human bronchial epithelial cells in the presence of a layer of sputum from patients with bronchiectasis / cystic fibrosis.

[0145] Figure 4 shows the results of this experiment, which demonstrate that the inhibitory effect of PRO-MPA-C16 nanoparticles on the IMPDH enzyme is already significantly higher after 2 hours of treatment compared to the mycophenolic acid control.

[0146] This demonstrates that the nanoparticles of this invention can remarkably penetrate / permeate viscous mucus secretions while maintaining enzyme inhibitory activity mediated by their ability to locally release mycophenolic acids.

[0147] Example 9: Evaluation of the in vitro inhibitory effects of the nanoparticles of this invention on immune cells on cell proliferation and enzyme activity. The inventors also conducted in vitro tests using immune cells (PBMCs and T-Jurkat) to evaluate the ability of the nanoparticles of this invention to promote proliferation and inhibit the enzymatic activity of the target enzyme IMPDH.

[0148] Cell proliferation inhibition tests were performed using PBBC ​​cell lines. To evaluate the ability of the PRO-MPA-C16 nanoparticles of this invention to inhibit cell proliferation, PMBC cells were pre-stimulated with 1 μM ionomycin and 2.5 μg / ml phytohemagglutinin (PHA) to induce proliferation.

[0149] Next, the cells were treated for 72 hours with nanoparticles of the present invention consisting of PRO-MPA-C16 at two concentrations (2 μM and 10 μM). 10 μM mycophenolic acid (MPA) was used as a comparison. Cell proliferation was evaluated by quantifying the level of adenosine triphosphate and measuring the number of viable cells (CellTiter-Glo®, Promega).

[0150] As shown in Figure 5, the nanoparticles of this invention inhibited cell proliferation in a dose-dependent manner, as evidenced by the reduction in cell proliferation after 72 hours of treatment with PRO-MPA-C16 nanoparticles or mycophenolic acid.

[0151] The second experiment evaluated the ability of PRO-MPA-C16 nanoparticles to inhibit the enzyme IMPDH, demonstrating that the suppression of cell proliferation described above is indeed mediated by the inhibition of this enzyme. The tests were performed on PMBC (with and without stimulation) and T-Jurkat cell line (without stimulation).

[0152] Cells were treated with two concentrations (2 μM and 10 μM) of PRO-MPA-C16 nanoparticles for 24 hours. 10 μM MPA was used as a comparison. Subsequently, the enzyme IMPDH was extracted and its ability to convert substrates to specific products was evaluated. This test was performed using a special kit (IMPDH assay kit, Biomedical Research Service, University at Buffalo, USA) to evaluate the ability of the enzyme IMPDH to convert the substrate iodine-nitro-tetrazolium (INT) to formazan products. Enzyme activity is proportional to the amount of formazan produced. The latter is evaluated by spectrophotometric measurement at a wavelength of 492 nm.

[0153] As shown in Figure 6, the nanoparticles of this invention dose-dependently reduced IMPDH activity after 24 hours of treatment with PRO-MPA-C16 nanoparticles or mycophenolic acid in PBMCs and T-Jurkat cells. The enzymatic activity of IMPDH was inversely proportional to the concentration of PRO-MPA-C16 nanoparticles.

[0154] In Figures 5 and 6, data are expressed as mean ± SEM. The data shown in Figure 5 are normalized relative to biological controls (cells that were not stimulated). Statistical significance compared to stimulated but untreated cells. * P<0.05, ** P<0.01 was calculated using a one-way ANOVA test.

[0155] Figure 6 shows the statistically significant difference compared to untreated cells. * P<0.05; for 10 μM MPA, p<0.05 was calculated using the Kruskal-Wallis test and Dunnett's post-hoc test.

[0156] Example 10 Evaluation of the in vivo effects of the nanoparticles of this invention in an SSc-ILD model mouse. The in vivo efficacy of the nanoparticles of this invention was evaluated using a mouse model of systemic sclerosis with interstitial lung disease (SSc-ILD). This model, widely described in the literature (Ravanetti et al. “SSC-ILD mouse model induced by osmotic minipump delivered bleomycin: effect of Nintedanib”, Scientific Reports. 2021;11.18513), induces subacute-chronic fibrosis in the lungs and skin by the release of bleomycin (BLM) via an osmotic minipump. Specifically, an osmotic minipump [ALZET 1007D; DURECT, (release rate 0.5 μl / h, 7 days), Cupertino, California, USA] containing 100 μl of saline or BLM (60 U / kg, dissolved in saline) was implanted into a subcutaneous pocket in the intrascapular region of 2-3 month old C57BL / 6 female mice and removed after 8 days. The procedure begins on day 14 and continues until the animals are slaughtered on day 21.

[0157] The animals were treated by intratracheal administration twice daily using a Penn-Century MicroSprayer® Aerosolizer - Model IA-1C and an FMJ-250 High Pressure Syringe, either 50 μl of solvent (TAPS buffer) or 50 μl of nanoparticles consisting of PRO-MPA-C16 of this invention. The latter was administered twice daily at a dose of 2.7 mg / kg body weight (concentration refers to the actual amount of MPA in the nanoparticles).

[0158] Specifically, four groups of animals: 1) Naive (healthy) group treated with solvent (naive + TAPS); 2) Naive (healthy) group treated with PRO-MPA-C16 nanoparticles (naive + PRO-MPA-C16 nanoparticles); 3) BLM (disease) group treated with solvent (BLM + TAPS); 4) BLM (disease) group treated with PRO-MPA-C16 nanoparticles (BLM + PRO-MPA-C16 nanoparticles); I used it.

[0159] Lung ventilation was assessed on days 0, 14, and 21 using microcomputed tomography (micro-CT) techniques with a PerkinElmer Quantum FX μCT instrument (90kV, 88uA, 36FOV for 4 minutes) combined with Analyze 14.0 software. A series of images were acquired, followed by filtering and conversion from gray level to CT count (Hunsfield units - HU). The Hunsfield unit scale is a unit of measurement scale used to quantitatively describe radiation density.

[0160] This transformation is a linear transformation where the density of air is set to -1000 HU and the density of water to 0 HU. Semi-automated segmentation is used to highlight the airways and lungs. For quantitative evaluation of the lung parenchyma, the clinical range of HU was applied to the HU images of the lungs to define whether ventilation was normal [(-900, -500) HU] or insufficient [(-500, -100) HU] (Gattinoni et al. in “What has computed tomography taught us about the acute respiratory distress syndrome?”, Am J Respir Crit Care Med. 2001; 164: 1701-1711, Ravanetti et al. “SSC-ILD mouse model induced by osmotic minipump delivered bleomycin: effect of Nintedanib”, Scientific Reports. 2021; 11.18513).

[0161] The two compartments with different air permeability are expressed as a percentage of the total lung volume. Tissues with insufficient air permeability have a low gas-to-tissue ratio and are used to quantify the progression of pulmonary fibrosis and to evaluate the effectiveness of the PRO-MPA-C16 nanoparticles of this invention.

[0162] Figure 7(A) shows the lung insufficiency levels on day 0 for all animals in the experiment (data shown as mean ± SEM). Specifically, lung insufficiency levels were similar in both healthy animals (naive) and animals subsequently administered bleomycin via osmotic minipump (BLM). Figure 7(B) shows the lung insufficiency levels on day 14 (data shown as mean ± SEM; ** P < 0.01 for naive (unpaired t-test)). Healthy mice (naive) had lung insufficiency levels exactly the same as on day 0, while diseased mice (BLM) showed a decrease in the level of adequately inflated tissue and an increase in the level of poorly inflated tissue, which is understandable as a typical clinical symptom of the disease. This clearly indicates that treatment with BLM induced the disease. Figure 7(C) shows the lung insufficiency levels on day 21 for the four groups of animals. (Data shown as mean ± SEM; * P<0.05 for BLM (ANOVA with Dunnett's post-hoc test); ** P<0.01 for BLM (ANOVA with Dunnett's post-hoc test). It can be seen that the disease group treated with PRO-MPA-C16 nanoparticles (BLM + PRO-MPA-C16 nanoparticles) had completely equivalent airflow levels to the two naive groups (naive + TAPS, naive + PRO-MPA-C16 nanoparticles). In contrast, the lung airflow levels of the untreated disease group (BLM + TAPS) were completely equivalent at day 14.

[0163] The data in Figure 7(C) clearly demonstrates that treatment of diseased mice with PRO-MPA-C16 nanoparticles is particularly effective in restoring lung ventilation to a level completely equivalent to that of non-disease (naive) mice. Specifically, after treatment, an increase in the proportion of adequately ventilated lung tissue was observed, resulting in a decrease in poorly ventilated tissue.

[0164] Importantly, this remarkable remission of the disease is achieved by administering PRO-MPA-C16 nanoparticles via the endotracheal route at a dose approximately 28 times lower than the maximum oral dose reported in the literature (Wollin et al. “The effect of nintedanib versus mycophenolate mofetil in the Fra2 mouse model of systemic sclerosis-associated interstitial lung disease”, Clin Ex Rheumatol. 2021; 39. S134-S141).

[0165] Furthermore, it is interesting that the above treatment was carried out by administering nanoparticles consisting of PRO-MPA-C16 of this invention, which are formulated without the use of excipients and therefore have the characteristic of maximizing the drug load (weight ratio of active ingredient to excipient). This is a major advantage in inhalation administration because it allows for the administration of high doses of the active ingredient. In addition, local administration to the lungs allows for a significant reduction in dosage, leading to a substantial decrease in immunosuppression and associated side effects.

Claims

1. Nanoparticles comprising multiple therapeutic molecules arranged in the form of spherical micelles, wherein the therapeutic molecules are of general formula (I) 【Chemistry 1】 (In the formula, R is a linear or branched saturated or unsaturated alkyl chain having 5 to 22 carbon atoms, R １ (This is an OH molecule bound to a binding molecule selected as needed from the group consisting of polyethylene glycol (PEG), triethylene glycol (TEG), sugars such as hyaluronic acid, mannose, and trehalose, proteins, peptides, antibodies, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), aptamers, and combinations thereof.) Nanoparticles, represented by the term "nanoparticles".

2. The nanoparticle according to claim 1, wherein the diameter of the nanoparticle is 30 to 700 nm, preferably 100 to 200 nm, and / or the zeta potential of the nanoparticle is -5 mV to -60 mV.

3. The nanoparticles according to claim 1 or 2, further comprising a surfactant, preferably the surfactant being selected from the group consisting of phospholipids, PEG complexes having lipophilic molecules such as polyethylene glycol (PEG)-cholesterol, polyoxyethylene / polyoxypropylene copolymers, fatty acids esterified with ethoxylated sorbitan (polysorbate), and combinations thereof.

4. The nanoparticle according to any one of claims 1 to 3, comprising a lipophilic therapeutic agent inside, preferably the lipophilic therapeutic agent being selected from the group consisting of immunosuppressants, anti-inflammatory agents, anti-fibrotic agents, and antitumor agents.

5. A pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 4 and at least one pharmaceutically acceptable solvent, excipient and / or diluent.

6. The pharmaceutical composition according to claim 5, which is a pharmaceutical form suitable for administration via local, oral, rectal, sublingual, intravenous, intra-arterial, transdermal, intramuscular, intradermal, subcutaneous, intraperitoneal, intraocular, inhalation, and endotracheal routes.

7. Nanoparticles according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 or 6 for use in the treatment of autoimmune diseases and / or fibrosis, particularly lung diseases.

8. The nanoparticles or pharmaceutical composition for use according to claim 7, wherein the autoimmune disease and / or fibrosis is a lung disease, and the lung disease is preferably selected from the group consisting of systemic sclerosis with interstitial lung disease (SSc-ILD), lymphangioleiomyomatosis (LAM), sarcoidosis and bronchiolitis obliterans (BOS).

9. Nanoparticles according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 or 6 for use in the treatment and / or prevention of organ rejection, preferably lung transplant rejection.

10. (i) A step of mixing a plurality of therapeutic molecules according to claim 1 in a polar solvent to obtain a first mixture, wherein the polar solvent is not water; (ii) Adding the first mixture from step (i) to a predetermined amount of water under stirring to obtain a second mixture, wherein the predetermined amount of water is at least 65% by volume of the total volume of the second mixture; and, (iii) The step of removing the polar solvent from the second mixture of step (ii) to obtain nanoparticles containing a plurality of therapeutic molecules arranged in the form of spherical micelles in an aqueous suspension; A method for producing nanoparticles according to any one of claims 1 to 6, comprising:

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