Compositions Comprising Small Extracellular Vesicles Derived from Umbilical Cord Blood Mononuclear Cells for Use in the Treatment of Fibrosis

Abstract

The present invention relates to the use of compositions containing specific exosomes secreted by umbilical cord blood mononuclear cells (UCBMNCs) in the prevention and treatment of fibrosis, such as fibrotic skin and lung injury. The exosome compositions of the present invention are active against skin fibrosis and lung disease. Furthermore, these compositions are also effective in ameliorating or preventing disease-related parameters, such as lung disease, and have demonstrated effects against respiratory diseases with inflammatory components, particularly when mediated by macrophages and / or T cells. Therefore, the present invention is in the technical fields of research and development in medicine, medicine, cosmetics, dermocosmetics, cell biology, and their instruments.

Description

[Technical Field]

[0001] The present invention relates to the use of known compositions comprising specific exosomes secreted by umbilical cord blood mononuclear cells (UCBMNC) in the prevention and treatment of fibrotic diseases, such as fibrotic skin and lung injury.

[0002] The exosome compositions described herein as ExoCells of the present invention exhibit activity against skin fibrosis and lung disease.

[0003] Data support the use of these compositions for preventing and treating skin fibrosis, including but not limited to scleroderma, and their known mechanism of action (MoA) and biodistribution patterns suggest potential beneficial effects in liver diseases such as fibrosis. Furthermore, the compositions are effective in ameliorating or preventing disease-related parameters in in vivo models of lung disease, demonstrating potential for respiratory diseases with an inflammatory component, particularly when mediated by macrophages and / or T cells. These may include the development of fibrosis following inflammatory stimuli such as infection, the management of chronic obstructive pulmonary disease (COPD), and the treatment of sarcoidosis.

[0004] Therefore, the present invention is in the technical field of research and development in medicine, medicine, cosmetics, dermocosmetics, cell biology and their instruments. [Background technology]

[0005] Exosomes are liposome-like vesicles (30–200 nm) secreted by most cell types. They form within secretory cells in compartments called multivesicular bodies (MVBs) and are subsequently released by fusion of the endosomal compartment with the plasma membrane, releasing their contents into the extracellular environment. Depending on the type of secretory cell and stimulus, they contain a specific set of proteins, lipids, and RNA rather than a random sample of cytoplasmic contents. They carry genetic material in the form of mRNA and microRNA, a feature that makes them a promising biologically-directed gene delivery system that can be used in therapeutic approaches.

[0006] The use of exosomes secreted by umbilical cord blood mononuclear cells (UCBMNC) is a topic that has been widely discussed in the literature, with several documents already available.

[0007] Fibrosis is characterized by overgrowth and scarring in several tissues and results from the excessive deposition of extracellular matrix (ECM) components.

[0008] Chronic inflammatory response induced by infection, chemical injury or tissue injury can cause fibrosis.Fibrosis can lead to organ failure and death.Examples of fibrotic diseases include pulmonary fibrosis, liver cirrhosis, cardiovascular fibrosis, systemic sclerosis and nephritis.

[0009] Pulmonary fibrosis is described as a chronic lung disease characterized by abnormal accumulation of extracellular matrix (ECM) and remodeling of lung architecture.

[0010] Pulmonary fibrosis caused by scar formation can destroy lung structure in a progressive and irreversible manner, ultimately leading to pulmonary dysfunction, impaired gas exchange, and death due to respiratory failure. Pulmonary fibrosis is difficult to treat and has a high mortality rate. Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease of unknown cause that occurs in middle-aged adults. IPF is associated with a poor prognosis.

[0011] IPF is one of the most common lung diseases seen in pulmonary practice. The incidence of IPF ranges from 0.22 to 7.4 per 100,000 population in Europe and 16.3 to 17.4 per 100,000 population in the United States. The prevalence and incidence of IPF increase with age and appear to be increasing in recent years.

[0012] The pulmonary fibrosis mouse model involves intratracheal or intranasal delivery of a single dose of bleomycin (BLM) to the lungs, which induces lung injury characterized by inflammatory cell infiltration, collagen deposition, and fibrosis.

[0013] The acute lung injury mouse model involves administration of lipopolysaccharide (LPS) to the airways, which causes an acute inflammatory response accompanied by massive cellular infiltration into the lungs.

[0014] US Patent No. 5,999,949 describes compositions containing UCBMNC exosomes for tissue repair, in particular for wound treatment, and respective methods for obtaining these exosomes and compositions thereof.

[0015] US Patent No. 5,929,999 describes concentrated umbilical cord blood mononuclear cell (UCBMNC) small extracellular vesicles (SEVs) and an optimized process for obtaining them with application to autoimmune diseases associated with inflammation. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2017 / 163312 [Patent Document 2] International Publication No. 2020 / 070700 Summary of the Invention [Problem to be solved by the invention]

[0017] However, none of the cited prior art techniques have used UCBMNC exosomes to treat fibrotic diseases, such as fibrotic lung injury. Therefore, there is a need to develop innovative compositions and methods for treating fibrosis. [Means for solving the problem]

[0018] To this end, the present invention provides an exosome composition (ExoCell) disclosed in Patent Documents 1 and 2, which comprises exosomes derived from specific UCBMNCs for the treatment or prevention of fibrosis or disorders, namely skin and lung disorders. [Brief explanation of the drawings]

[0019] [Figure 1] RT-PCR analysis of ACTA2 shows the expression of ACTA2 mRNA in normal human dermal fibroblast (NHDF) cells after treatment with TGF-β1, exosome composition of the present invention (ExoCell), or PBS. Data represent the mean ± SD. Statistical analysis was performed using an unpaired t-test. [Figure 2] Representative microscopic images of normal human dermal fibroblasts (NHDFs) immunostained for α-SMA in untreated (-TGFβ1) and TGF-β1-treated (+TGFp1) normal human dermal fibroblasts (NHDFs) (Figure 2a). The mean fluorescence intensity (MFI) of α-SMA in untreated and treated NHDF cells is shown. Data represent the mean ± SD. Statistical analysis was performed using an unpaired t-test (Figure 2b). [Figure 3]Representative microscopic images of normal human dermal fibroblasts (NHDFs) immunostained for α-SMA (Figure 3a) are shown. Fluorescence intensity (FI) measured along transverse sections of untreated (-TGFβ1), TGF-β1 and vehicle-treated (+TGFβ1 vehicle-PBS), and TGF-β1 and ExoCell-treated cells (depicted in Figure 3a) is shown (Figure 3b). Data represent the average FI from sections of 12 cells (depicted in Figure 3a). Fold change in MFI for vehicle and untreated and treated with ExoCell is shown (Figure 3c). Data represent mean ± SD. Statistical analysis was performed using a one-sample t-test. *p ≤ 0.05, **p ≤ 0.01. [Figure 4] The figure shows the distribution of ExoCells by total radiant efficiency ([p / s] / [μW / cm2]) in the lungs and stomach of C57BL6 male mice 1 hour after intranasal (in), intratracheal (IT), or IT administration of labeled PBS (control) or fluorescently labeled ExoCells of the present invention in combination with a microspray. Organ images were simultaneously captured using an IVIS-Spectrum. Results are expressed as mean ± SD (n=3 for the control group, n=5 for the exosome composition-treated group). *p<0.05. Statistical analysis: Two-way ANOVA, Tukey's multiple comparison post-hoc test. [Figure 5a] This figure shows the effect of the ExoCell composition of the present invention on weight recovery in mice tested in a dose-dependent assay. The weight progression over the course of the experiment is shown for healthy mice (blank), mice treated with ExoCell at doses A or B, PBS used as a negative control, and nintedanib (clinical comparison). Data are presented as the mean ± SD for 9-12 mice in each group. Statistical evaluation of differences between experimental groups was determined using two-way analysis of variance followed by Sidak's multiple comparison post hoc test or Tukey's multiple comparison post hoc test. A p-value of ≤0.05 was considered significant. [Figure 5b] Collagen concentration in lung homogenates on day 14 is shown. Collagen concentration (μg / mL) was increased in mice treated with bleomycin (BLM). [Figure 5c] Ashcroft scores were assessed on day 14. Ashcroft scores decreased with ExoCell treatment in dose group B (1 x 10 particles). Data are presented as the mean ± SD of 8–12 mice in each group. Statistical evaluation of differences between experimental groups was determined using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test. A p-value ≤ 0.05 was considered significant. ***p ≤ 0.001; ****p ≤ 0.0001. [Figure 6a] The effect of ExoCell on weight loss and collagen accumulation is shown. The weight progression over time is shown for healthy mice (blank) or mice treated with the composition of the present invention at dose B, PBS used as a negative control, or nintedanib as a clinical comparison. Data are presented as the mean ± SD of 9-12 mice per group. Statistical evaluation of differences between experimental groups was determined using two-way analysis of variance followed by Sidak's multiple comparison post hoc test and Tukey's multiple comparison post hoc test. A p value of ≤0.05 was considered significant. [Figure 6b] Collagen concentration in lung homogenates on day 14. Collagen concentration (μg / mL) was increased in mice treated with BLM, and this accumulation was reduced or prevented by administration of ExoCell of the present invention (Dose B, 1×10 particles). [Figure 6c] Ashcroft scores were assessed on day 14. Ashcroft scores were reduced upon ExoCell treatment at dose B (1 x 1010 particles). Data are presented as the mean ± SD of 8–12 mice per group. Statistical evaluation of differences between experimental groups was determined using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test. A p value of ≤0.05 was considered significant. [Figure 7]These results demonstrate that IV administration of the composition of the present invention targets T cells. Figure 7a shows the total number of T cells in BALF (Figure 7a). Figure 7b shows the number of Teff (CD8+) cells in BALF (Figure 7b). Figure 7c shows the number of T helper (CD4+) cells in BALF (Figure 7c). Figure 7d shows the percentage of total T cells in BALF (Figure 7d). Figure 7e shows the percentage of Teff (CD8+) cells in BALF (Figure 7e). Figure 7f shows the percentage of T helper (CD4+) cells in BALF (Figure 7f). T cells were identified as CD45+CD3+ monolayer lymphocytes and further separated into CD4+ or CD8+ cells. Data are presented as the mean ± SD for 8–12 mice in each group. Statistical evaluation of differences between experimental groups was determined using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test. A p value of ≤0.05 was considered significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, p ≤ 0.0001. [Figure 8] Absolute cell counts of each leukocyte subset and IL-1β concentration in BALF from a mouse model of acute lung injury are shown. Total cells in BALF (Figure 8a). Total neutrophil cell counts (Figure 8b). Total lymphocyte cell counts (Figure 8c). Total macrophage cell counts in BALF (Figure 8d). IL-1β concentration (pg / mL) in BALF (Figure 8e). Data represent mean ± SD (n=9). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Statistical differences were determined using one-way ANOVA and Bonferroni post-hoc test. [Figure 9] Cytokine levels in BALF from mouse models of acute lung injury are shown. IL-6 concentration (pg / mL) (Fig. 9a). TNF-α concentration (pg / mL) (Fig. 9b). IL17A concentration (pg / mL) (Fig. 9c). IFNγ (pg / mL (Fig. 9d)). Data represent mean ± SD (n=9). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Statistical differences were determined using one-way ANOVA and Tukey's post-hoc test. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to the use of compositions comprising specific exosomes secreted by umbilical cord blood mononuclear cells (UCBMNCs) in the prevention and treatment of fibrotic diseases, such as fibrotic skin and lung injury.

[0021] The specific exosomes secreted by UCBMNC and the composition thereof, hereinafter referred to as ExoCells, are prepared according to the method described in WO 2017 / 163132 or by an optimized method such as that described in WO 2020 / 070700.

[0022] In a preferred embodiment, the exosomes and compositions thereof of the present invention are prepared according to the optimized method as described in WO 2020 / 070700.

[0023] The exosomes of the present invention can be lyophilized as described in WO 2020 / 070700 to produce a storable product and / or to produce a composition.

[0024] The exosomes (ExoCells) of the present invention contain a specific set of proteins, lipids and RNA that carry genetic material in the form of mRNA and microRNA, a feature that makes them a promising biologically-derived gene delivery system that can be used in therapeutic approaches.

[0025] The exosome composition used in the present invention comprises one or more bioactive molecules selected from the following: (a) proteins, (b) miRNAs, and (c) lipids: a) Protein: [Table 1-1] [Table 1-2]

[0026] More preferably, the one or more proteins are selected from the group of ANXA2, ANK1, CD63, CD81, CD9, CD15, and more preferably, the proteins are present in the SEV composition in quantities of CD81≧1%, CD9≧1%, CD63≧40%, CD15≧20% of positive events when SEVs bound to microbeads are measured by flow cytometry, or in quantities of CD63≧5pg / mL or ANXA2≧0.3ng / mL in purified SEVs when measured by ELISA.

[0027] b) miRNA: [Table 2]

[0028] Preferably, the following miRNAs: hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7i-5p, hsa-miR-130a-3p, hsa-miR-144-3p, hsa-miR-144-5p, hsa-miR-181 a-5p, hsa-miR-185-5p, hsa-miR-18a-5p, hsa-miR-205-5p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-m and more preferably, one or more of miR-30b-5p, hsa-miR-3184-3p, hsa-miR-376c-3p, hsa-miR-486-5p, hsa-miR-92a-3p, and hsa-miR-93-5p, and more preferably, miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, and miR-19b, and more preferably, said miRNAs are present in the SEV composition in the following amounts, respectively, as measured by direct quantification: miR-150-5p > 1.3 pg / 10 9 Department, miR-223-3p>1.2pg / 10 9 Part, miR-16-5p>0.5pg / 10 9 Department, miR-142-3p>0.4pg / 10 9 part, miR-19b>0.2pg / 10 9 Department.

[0029] c) Lipids: cholesteryl esters (CE), diacylglycerides (DAG), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SPM), triacylglycerides (TAG), preferably phosphatidylcholine (PC), phosphatidylserine (PS) and sphingomyelin (SPM), more preferably said lipids being present in the SEV composition in the following minimum proportions relative to the total lipid amount: CE≧0.05±0.05, DAG≧1.3±0.4, PA≧3.18±0.6, PC≧30.9±3.3, PE≧13.0±0.5, PG≧0.25±0.1, PI≧3.8±0.3, PS≧29.4±3.7, SPM≧18.0±1.6, TAG≧0.074±0.13.

[0030] In one embodiment, the exosome composition ExoCell comprises: a) Proteins included in the combination of presentation proteins listed above, preferably a combination of proteins selected from the group consisting of ANXA2, ANK1, CD63, CD81, CD9, and CD15, more preferably the following proteins are present in the SEV composition in amounts of CD81≧1%, CD9≧1%, CD63≧40%, and CD15≧20% of positive events when SEVs bound to microbeads are measured by flow cytometry, or in amounts of CD63≧5pg / mL or ANXA2≧0.3ng / mL in purified SEVs when measured by ELISA; b) Combinations of the presented miRNAs listed above, preferably as follows, as measured by direct quantification: miR-150-5p>1.3pg / 10 9 Department, miR-223-3p>1.2pg / 10 9 Part, miR-16-5p>0.5pg / 10 9 Department, miR-142-3p>0.4pg / 10 9 part, miR-19b>0.2pg / 10 9miRNAs in a combination of miRNAs selected from the group consisting of: c) A combination of the lipids listed above, preferably a combination of lipids selected from CE, diacylglyceride (DAG), PA, phosphatidylcholine (PC), PE, PG, PI, phosphatidylserine (PS), sphingomyelin (SPM), triacylglyceride (TAG), more preferably a combination of lipids selected from phosphatidylcholine (PC), phosphatidylserine (PS) and sphingomyelin (SPM), more preferably the following ratios relative to the total lipid amount: CE≧0.05±0.05, DAG≧1.3±0.4, PA≧3.18±0.6, PC≧30.9±3.3, PE≧13.0±0.5, PG≧0.25±0.1, PI≧3.8±0.3, PS≧29.4±3.7, SPM≧18.0±1.6, TAG≧0.074±0.13.

[0031] In another embodiment, the exosome composition ExoCell comprises the following combination of proteins, miRNAs and lipids: -Proteins: ANXA2, CD63, CD81, CD9 and CD15; RNA: preferably miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b, and - Lipids: CE ≥ 0.05 ± 0.05, DAG ≥ 1.3 ± 0.4, PA ≥ 3.18 ± 0.6, PC ≥ 30.9 ± 3.3, PE ≥ 13.0 ± 0.5, PG ≥ 0.25 ± 0.1, PI ≥ 3.8 ± 0.3, PS ≥ 29.4 ± 3.7, SPM ≥ 18.0 ± 1.6, TAG ≥ 0.074 ± 0.13.

[0032] In another embodiment, the exosome composition ExoCell comprises exosomes enriched in miRNA, palmitoylated tripeptides, peptides, DNA, siRNA, growth factors, amino acids, sugars, lipid-soluble molecules, fatty acids and their derivatives, e.g., DHA, EPA, oleic acid, lipid-modified molecules, e.g., GPI-anchored proteins or peptides, and / or other hydrophobic molecules, preferably the SEVs are enriched in miRNA or palmitoylated tripeptides.

[0033] In other embodiments, the exosome composition ExoCell comprises exosomes enriched for one or more miRNAs selected from the group consisting of hsa-miR-150-5p, hsa-miR-16-5p, hsa-miR-142-3p, hsa-miR-223-3p, hsa-let-7g-5p, hsa-miR-21-5p, hsa-let-7f-5p, hsa-miR-19b-3p, hsa-let-7a-5p, hsa-miR-26a-1-5p, hsa-miR-20a-5p, hsa-miR-181a-5p, hsa-miR-451a , hsa-miR-23a-3p, hsa-miR-342-3p, hsa-miR-191-5p, hsa-miR-103a-3p, hsa-miR-15a-5p, hsa-miR-142-5p, hsa-miR-146a-5p, hsa-miR-19a-3p, hsa-miR- 15b-5p, hsa-miR-26b-5p, hsa-miR-30d-5p, hsa-miR-146b-5p, hsa-miR-106b-5p, hsa-miR-29a-3p, hsa-miR-17-5p, hsa-miR-29b-3p, and hsa-miR-101-3p, Preferably, the exosomes are enriched for one or more miRNAs selected from the group consisting of: hsa-miR-181a-5p, hsa-miR-451a, hsa-miR-103a-3p, hsa-miR-15a-5p, hsa-miR-19a-3p, hsa-miR-15b-5p, hsa-miR-26b-5p, hsa-miR-30d-5p, hsa-miR-146b-5p, hsa-miR-106b-5p, hsa-miR-29a-3p, hsa-miR-17-5p, hsa-miR-29b-3p, and hsa-miR-101-3p, Preferably, the exosomes are enriched in the miRNA hsa-miR-150-5p, thereby increasing the concentration of miRNA in the exosome composition.

[0034] An in vitro model developed to treat skin fibrosis (dermal fibroblasts treated with TGF-β1 - transforming growth factor-β1) showed that treatment with the exosome composition of the present invention reduced the expression of α-SMA (alpha smooth muscle actin), a marker of fibrosis. These results indicate the positive effect of the composition in reducing fibrosis.

[0035] In vivo models were developed to evaluate the activity and mechanism of action (MoA) of exosome compositions in the context of pulmonary diseases, namely, both lipopolysaccharide (LPS)-induced acute pulmonary inflammation (ALI) and bleomycin (BLM)-induced pulmonary fibrosis in rodents.

[0036] Exosome compositions administered locally (intratracheally, IT) or systemically (intravenously, IV) to IPF models (dose-dependently) promoted overall recovery or prevented overt disease, as measured by body weight change, and reduced pulmonary fibrosis (assessed biochemically by collagen accumulation in the lung and histologically using the Ashcroft score).

[0037] In vivo, local administration (intranasal, IN) of exosome compositions was shown to specifically target macrophages in a model of LPS-induced acute lung inflammation, resulting in normalization of their numbers in bronchoalveolar lavage fluid (BALF). Furthermore, the exosome compositions reduced the concentrations of pro-inflammatory mediators in BALF, such as IL-1β and IL-17A, in a dose-response manner.

[0038] Available data point to a macrophage- and / or T-cell-dependent MoA, which is determined by the disease context and the route of administration of the compound.

[0039] Finally, given the natural biodistribution of exosome compositions to the liver and the influence of macrophages on the liver fibrosis process, the compositions are potentially effective in preventing, slowing down, or ameliorating liver fibrosis.

[0040] The exosome compositions (ExoCells) of the present invention demonstrated activity in one in vitro model of skin fibrosis and two in vivo models of lung disease. The in vitro data support the use of the compositions for skin fibrosis, including but not limited to scleroderma, and the known MoA and biodistribution pattern indicate potential beneficial effects in liver diseases such as fibrosis. Furthermore, these compositions were effective in ameliorating or preventing disease-related parameters in two in vivo models of lung disease, demonstrating their potential for respiratory diseases with an inflammatory component, particularly when mediated by macrophages and / or T cells. These may include the development of fibrosis following inflammatory stimuli such as infection, the management of COPD, and the treatment of sarcoidosis.

[0041] Therefore, the present invention also relates to pharmaceutical compositions comprising the above-described ExoCell compositions in liquid, suspension, powder, spray, cream, gel or hydrogel form, aerosol form, vapor for inhalation and nebulization.

[0042] In another embodiment, the pharmaceutical composition is in liquid, suspension, powder, spray, cream, gel or hydrogel form, aerosol form, vapor-borne medical device for inhalation and nebulization.

[0043] The exosome compositions and pharmaceutical compositions thereof are useful for the prevention and treatment of fibrotic diseases.

[0044] In one embodiment, the fibrotic disease is a fibrotic lesion associated with inflammation, an autoimmune disease, an infection, trauma, or an idiopathic disorder.

[0045] In this context, inflammation includes inflammatory diseases accompanied by symptoms of skin fibrosis such as keloids and scars.

[0046] Autoimmune diseases include manifestations of skin fibrosis such as scleroderma.

[0047] Pulmonary viral or bacterial infections are associated with pulmonary fibrosis, such as acute respiratory distress syndrome (ARDS).

[0048] Traumatic lesions include manifestations of skin fibrosis such as keloids and scars.

[0049] Fibrotic lesions may include lesions with or without autoimmune and / or inflammatory causes, such as systemic sclerosis, nephrogenic systemic fibrosis and hepatic fibrosis.

[0050] Idiopathic diseases include pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF).

[0051] Similarly, in contrast to what is described in WO 2017 / 163132 and WO 2020 / 070700, which relate to UCBMNC exosomes and their use in therapeutic or prophylactic applications for wound treatment by topical administration and for application to autoimmune diseases associated with inflammation by topical application or subcutaneous, cutaneous, intraperitoneal and intravenous administration, respectively, the exosomes and compositions (ExoCell) of the present invention are applied in different ways to produce advantageous effects in the treatment or prevention of the above-mentioned fibrotic diseases, i.e., fibrotic lesions associated with inflammation, autoimmune diseases, infections, trauma, or idiopathic disorders, by intravenous or topical administration, for example by intratracheal, intranasal or local injection using ExoCell compositions in the form of aerosol, steam inhalation, spray and liquid formulations for this purpose.

[0052] Thus, in one embodiment for treating and preventing idiopathic pulmonary fibrosis (IPF) symptoms, administration is preferably via the intratracheal or intranasal route by introducing the composition directly into the trachea by inserting a needle into the mouth and throat, or intravenously.

[0053] In another embodiment, intranasal administration is suitable for the treatment and prevention of symptoms of acute lung injury. [Example]

[0054] Example 1. Functional Effects of Exosome Compositions In in vitro studies using a skin fibrosis model, primary normal human dermal fibroblasts (NHDFs) were isolated from the dermis of juvenile foreskin or adult skin and treated with 10 ng / ml transforming growth factor beta 1 (TGF-β1) to induce fibrosis (TGF-β1 is a central mediator of fibrogenesis) (Branton and Kopp 1999; Aoudjehane et al. 2016). NHDF cells were cultured in a 24-well platelet-free medium containing TGF-β1 and ExoCell (1 × 10 10Cells were co-incubated with 1000 ng / ml of 10 ... The following conditions were used for qPCR: 95°C for 2 minutes, followed by 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 20 seconds (fluorescence measurement). At least three biological replicates were used. β-actin (ACTB) was used as an endogenous control to normalize each sample. The resulting data were analyzed using Bio-Rad CFX Manager software. The relative expression of the gene of interest was calculated using 2. -ΔΔCt The analysis was performed according to the method described above. Expression of ACTA2 (normalized to β-actin) increased upon TGF-β1 treatment (P<0.05), as expected, and decreased when ExoCells were co-incubated with TGF-β1 (P<0.05), indicating a role for ExoCells in reducing fibrosis by decreasing ACTA2 expression (Figure 1). NHDFs were harvested using trypsin and plated at 20,000 cells / cm in growth medium. 2The cells were seeded in PBS for 24 hours. After 24 hours, the growth medium was replaced with serum-free medium. The next day, the cells were treated with 10 ng / mL TGFβ1 diluted in serum-free medium. 48 hours after TGFβ1 treatment, the cells were crosslinked using 4% paraformaldehyde for 20 minutes and then blocked for 30 minutes in 0.1% BSA in 0.1% PBS-Triton. Next, the cells were immunostained for 3 hours against α-SMA (mouse) antibody diluted in blocking solution. The cells were washed three times with 0.05% PBS-Tween and incubated for 1 hour in blocking solution containing a fluorescently conjugated secondary antibody, anti-mouse. The cells were then washed three times and nuclei were stained with DAPI (1 μg / mL). Images were acquired using an AxioVert microscope (Figure 2a).

[0055] The mean fluorescence intensity was measured using ImageJ. Briefly, cells were selected using a threshold function and the MFI was determined by measuring the fluorescence intensity. The background intensity was then subtracted to obtain the MFI (Figure 2b).

[0056] Harvest NHDFs using trypsin and plate them at 20,000 cells / cm in growth medium. 2Cells were seeded in 0.5% CO2 for 24 hours. After 24 hours, the growth medium was replaced with serum-free medium and pretreated with ExoCell (20,000 particles / cell). The next day, cells were treated with 10 ng / mL TGFβ1 diluted in serum-free medium and a second dose of ExoCell. 48 hours after TGFβ1 treatment, cells were crosslinked with 4% paraformaldehyde for 20 minutes and then blocked for 30 minutes in 0.1% BSA in 0.1% PBS-Triton. Next, cells were immunostained for 3 hours against α-SMA (mouse) antibody diluted in blocking solution. The cells were washed three times with 0.05% PBS-Tween and incubated for 1 hour in blocking solution containing a fluorescently conjugated secondary antibody, anti-mouse. The cells were then washed three times and nuclei were stained with DAPI (1 μg / mL). Images were acquired using an AxioVert microscope (Figure 3a). Mean fluorescence intensity was measured using ImageJ. Briefly, MFI was measured by selecting cells using a threshold function and measuring their fluorescence intensity. Background intensity was then subtracted to obtain MFI. Fiber fluorescence intensity (FI) was measured by drawing a transverse line across all cells, and ImageJ software then measured FI along the cut. This was performed on 12 cells, and the average was then plotted using GraphPad (Figure 3b). The MFI of control, untreated, and ExoCell-treated cells was normalized to the MFI from TGFβ1- and PBS-treated cells.

[0057] Example 2. Functional Effects of ExoCell in Pulmonary Fibrosis As proof of concept for exosome delivery to the lung, exosome compositions were labeled with DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide; D12731, Thermo Fisher Scientific) fluorescent dye the day before administration to mice and stored at 4°C, protected from light, until day 1. After exosome labeling, aliquots were used to measure fluorescence on an IVIS (black-bottom 96-well plate). Animals were treated intranasally, intratracheally, and with a microspray aerosol generator with either vehicle (blank, dye-exposed PBS, 3 animals) or fluorescently labeled exosome compositions (5 animals per condition). 55 μL of ExoCell solution (1 × 10 11 Particles / mL, total 5×l0 9 ) was administered. Blank animals were administered 50 μL of vehicle (labeled PBS) intranasally, intratracheally, and via a microspray aerosol generator. One hour after composition administration, the animals were sacrificed, and individual lungs and stomachs were collected and fluorescence was measured using an IVIS-Spectrum In Vivo Imaging System (Perkin Elmer). Fluorescence was measured separately in the organs. Both IN and IT administration routes resulted in a homogenous distribution of ExoCell in the lungs (Figure 4). Microspray-coupled IT (MS) administration proved inappropriate due to lower particle intensity and high variability.

[0058] 2.1-Pulmonary fibrosis-bleomycin-induced model Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by pulmonary fibrosis. Bleomycin (BLM) is widely used to induce pulmonary fibrosis in rodents, representing an experimental model of human IPF, to study the potential efficacy of novel treatments (Walters and Kleeberger 2008). To prepare the BLM-induced mouse model, 10-week-old C57BL / 6J female mice were subjected to intratracheal administration (insertion of a needle into the mouth and throat, introducing the drug directly into the trachea) of 1.5 mg / kg (2.25–3 U / kg) BLM (bleomycin sulfate, Streptomyces verticillus, Merck Millipore) as described in the literature (Walters and Kleeberger 2008).

[0059] 2.2-Pulmonary Fibrosis-Functional Effects of ExoCell ExoCell was administered intratracheally (IT) (dose A-1 × 10 9 Particles and dose B-1 x 10 10 The mice were treated with ExoCell IT (particles) starting on day 0 (D) and 2 hours before BLM administration until the end of the experiment on day 14 (12 mice). The mice were sacrificed on day 14. The reference test compound (clinical comparator), nintedanib, was administered daily via the oral route from D5 to D13. Healthy mice were used as blanks (8 mice), and mice treated with BLM and PBS were used as negative controls (12 mice). IPF symptoms include severe weight loss, and therefore, mouse weights were assessed and monitored daily. Mice administered high-dose (Dose B) ExoCell IT tended to regain weight faster than control animals (Figure 5a). ExoCell demonstrated a dose-dependent effect in this model.

[0060] Pulmonary fibrosis in the alveolar septa and peribronchial regions was assessed by histological analysis. On day 14, mice were sacrificed by increasing concentrations of CO2 exposure. For lung collagen measurement, after bronchoalveolar lavage (BAL), saline was perfused throughout the lungs through the right ventricle to flush out vascular contents, and the lungs were frozen at -80°C. Lungs were homogenized in PBS using an Ultra Turrax, centrifuged, the supernatant discarded, and the pellet resuspended in 1 mL of PBS containing protease inhibitors (Roche). After centrifugation, collagen content was measured by the Sircol assay (France Biochem Division, France). Collagen content was increased in BLM-treated mice compared with healthy control animals (Figure 5b).

[0061] After BAL and lung perfusion, the left lobe was fixed by direct immersion in 4% buffered formaldehyde (Fisher Scientific, reference number 15225582) for a minimum of 72 hours (h) and embedded in paraffin. Three-micrometer sections were stained with Sirius Red (SR), a marker of collagen fibers. For SR staining, slides were dehydrated in successive alcohol baths of increasing concentration. Slides were immersed in Direct Red solution for 30 minutes, rinsed in a water bath, and then immersed in Weigert's iron hematoxylin solution for 2 minutes. Lung sections were washed with water, hydrated in successive alcohol baths of decreasing concentration, and protected with a coverslip. Collagen deposition, located in the alveolar septum and peribronchial regions, was assessed using a semiquantitative score—the Ashcroft score—graded from 0 to 5 (Table I).

[0062] ExoCell Dose B IT has a lower Ashcroft score at day 14 compared to the negative control and Dose A (Figure 5c). This data demonstrates that the exosome composition of the present invention, when administered intratracheally (IT), dose-dependently promotes recovery in mice with bleomycin (BLM)-induced pulmonary fibrosis. [Table 3]

[0063] The exosome composition was administered intravenously (IV) (dose A - 1 × 10 9 Particles and dose B-1 x 10 10 The mice were treated with ExoCell IV (particles) starting on day 0 (D), 2 hours before BLM administration, and continued until the end of the experiment on day 14 (Figure 6A). Mice were monitored daily for weight and clinical scores. Mice receiving the high dose (Dose B) of ExoCell IV tended to regain weight more quickly than control animals (Figure 6A). Lung collagen levels, measured by the Sircol assay (described above), were reduced in mice treated with Dose B compared to the negative control (PBS-treated BLM mice) (Figure 6B).

[0064] Furthermore, the Ashcroft score tended to decrease in mice treated with the exosome composition (Figure 6c).

[0065] In conclusion, this data demonstrates that intravenous (IV) administration of the exosome composition of the present invention prevents weight loss and collagen accumulation in mice with bleomycin (BLM)-induced pulmonary fibrosis.

[0066] Effect of ExoCell compounds on immune cell composition in bronchoalveolar lavage fluid (BALF) on day 14. Pulmonary fibrosis was induced in C57BL / 6J mice after administration of BLM. 1 x 10 10 The exosome composition was administered by IV route every 2 days from D0 to D12 after BLM administration. Cells (2 × 10) isolated from BALF and digested lungs were then cultured. 6Cells (1000 cells / well) were suspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 3% fetal calf serum (FCS), 2 mM EDTA) and labeled with a live / dead cell dye and specific surface antibodies (CD4 and CD8) and fluorescence minus one (FMO) control. Flow cytometry analysis was performed on a BD Fortessa X-20 flow cytometer, and data were analyzed using FlowJo software (Tree Star). Animals treated with ExoCell IV had significantly lower total numbers (Figure 7a, b, and c) and percentages (Figure 7d, e, and f) of CD4+ and CD8+ T cells in bronchoalveolar lavage fluid (BALF), indicating that the MoA of ExoCell in this model likely targets T cells. CD4+ and CD8+ T cells are associated with pulmonary fibrosis; CD4+ cells produce cytokines that can act to promote fibrosis, and accumulation of CD8+ cells is associated with impaired lung function (Luzina et al. 2008).

[0067] 2.3-Acute pulmonary fibrosis - lipopolysaccharide (LPS)-induced mouse model To simulate human acute respiratory distress syndrome (ARDS), animal models must reproduce acute damage to the epithelial and endothelial barriers in the lungs and acute inflammatory responses in the airways. One of the most widely used models is lipopolysaccharide-induced acute lung injury (LPS-induced ALI). LPS-induced injury is characterized by an acute phase involving leukocyte influx and high levels of proinflammatory cytokines in the BALF. After mice are exposed to an airway challenge with LPS (intranasal), a significant leukocyte infiltration (mainly neutrophils but also lymphocytes and macrophages) in the BALF is induced, peaking 24 hours after challenge. The proinflammatory cytokines TNFα, IL-6, IL-1β, and IL-17A are also increased.

[0068] Example 3. Mouse model preparation Lipopolysaccharide (LPS) was prepared immediately before the procedure for intranasal challenge: 1 mg of LPS was dissolved in 4 mL of PBS to obtain a solution with a concentration of 0.25 mg / mL. Animals were intranasally administered 50 μL of this LPS solution, resulting in a 0.5 mg / kg dose for LPS-challenged mice. Briefly, mice were placed in a supine position and 50 microliters of this LPS solution (0.25 mg / mL) was dripped into the mice via a micropipette, resulting in a 0.5 mg / kg dose for LPS challenge. Animals were treated with 5 mg / kg dexamethasone as a reference compound (positive control) 1 hour before LPS challenge. On day 0, animals were deeply anesthetized with isoflurane and intranasally administered 50 μL of PBS (non-LPS challenged group 1) or 0.5 mg / kg LPS dissolved in PBS. After intranasal instillation, the mice were held upright for 1 minute to ensure proper distribution of LPS to the lungs. After LPS administration, each mouse was observed to monitor normal respiratory behavior. A total of 1 × 10 9Animals were intranasally administered a single dose of ExoCell particles 1 hour before LPS challenge. The administered intranasal volume was 50 μL. Briefly, mice were placed in a supine position, and 50 μL of ExoCell was instilled via a micropipette. BALF samples were obtained by slowly injecting 0.3 mL of PBS (containing 2% fetal bovine serum, FBS) into the lungs three times using a 1 mL syringe (Injekt®-F Luer Solo, Braun) and withdrawing the solution by gentle aspiration 30 seconds later. BALF samples were then centrifuged at 300 g for 8 minutes at 4°C. The supernatant was transferred to a new Eppendorf tube and stored at -80°C for cytokine analysis, and the cell pellet was processed for flow cytometry analysis. Flow cytometry analysis was performed on leukocytes from the BALF samples collected on day 1. Absolute cell counts were measured at the start of treatment, and cells were then resuspended in either 50 or 100 μL of staining buffer, depending on the cell concentration of each sample. Cell suspensions were stained with appropriate antibody combinations to assess the frequency of macrophages (CD45, CD11, F4 / 80) and neutrophils (CD45, CD11b, and Ly6C). Samples were then fixed in PBS containing 1% formaldehyde, kept at 4°C in the dark until the next day, and acquired on a FACS Aria Fusion cytometer (BD Biosciences). Raw data (FCS files) generated by the FACS Aria Fusion flow cytometer (FACS Diva software) were analyzed using FCS Express software v7.0 (DeNovo Software). Macrophages were specifically targeted by ExoCell in this model, and the number of macrophages in BALF was reduced in the ExoCell-treated group compared to the negative control (Figure 8d). Neutrophil and lymphocyte infiltration remained unaffected (Figures 8b and c). Cytokine multiplex analysis of IL-1β, IL-6, IL-10, IL-17A, and TNF-α was performed in BALF and supernatant samples collected 24 h after LPS administration by using a Luminex kit (Panel MCYTOMAG-70k-05 Mouse MAG).Data were acquired and processed using Luminex xPONENT software and analyzed using the MILLIPLEX Analyst 5.1 program. A threshold for adequate acquisition was established (>35 events). Samples meeting this criterion were included in the analysis. If results were below the detection limit (<3.2 pg / ml), the concentration was considered to be 0. The levels of IL-6 (Figure 9a), TNFα (Figure 9b), IL-17A (Figure 9c), and IFNγ (Figure 9d) in BALF were reduced in ExoCell-treated animals compared with the untreated (negative control) group. Furthermore, IL-1β (pro-inflammatory cytokine) secretion was significantly reduced in BALF (Figure 8e).

Claims

1. A pharmaceutical composition (ExoCell) comprising exosomes secreted by umbilical cord blood mononuclear cells (UCBMNCs), which is applied to the prevention and treatment of fibrotic diseases, The exosomes contain the following: (a) proteins, (b) miRNAs, and (c) lipids: a) Protein: Table 1-1 Table 1-2 More preferably, the one or more proteins are selected from the group of ANXA2, ANK1, CD63, CD81, CD9, CD15, and more preferably, said proteins are present in the SEV composition in an amount of CD81≧1%, CD9≧1%, CD63≧40%, CD15≧20% of positive events when measured by flow cytometry of SEV bound to microbeads, or in an amount of CD63≧5pg / mL or ANXA2≧0.3ng / mL in purified SEV when measured by ELISA; b) miRNA: Table 2 Preferably, the following miRNAs: hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7i-5p, hsa-miR-130a-3p, hsa-miR-144-3p, hsa-miR-144-5p, hsa-miR-181a-5p, hsa-miR-185-5p, hsa-miR-18a-5p, hsa-miR-205-5p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-30b-5p, hsa-miR-3184-3p, hsa-miR-376c-3p, hsa-miR-486-5p, hsa-miR-92a-3p and hsa-miR-93-5p, more preferably one or more of miR-150-5p, miR-223-3p, miR-16-5p, miR-142-3p, miR-19b; More preferably, the miRNAs are present in the following amounts, as measured by direct quantification: miR-150-5p>1.3 pg / 10 ９ part, miR-223-3p>1.2pg / 10 ９ part, miR-16-5p>0.5pg / 10 ９ part, miR-142-3p>0.4pg / 10 ９ part, miR-19b>0.2pg / 10 ９ present in the SEV composition at portions; and c) Lipids: cholesteryl esters (CE), diacylglycerides (DAG), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SPM), triacylglycerides (TAG), preferably phosphatidylcholine (PC), phosphatidylserine and sphingomyelin (SPM), more preferably said lipids are present in the SEV composition in the following minimum percentages relative to the total lipid amount: CE≧0.05±0.05, DAG≧1.3±0.4, PA≧3.18±0.6, PC≧30.9±3.3, PE≧13.0±0.5, PG≧0.25±0.1, PI≧3.8±0.3, PS≧29.4±3.7, SPM≧18.0±1.6, TAG≧0.074±0.

13. and one or more biologically active molecules selected from The fibrotic disease is a fibrotic lesion associated with inflammation, an autoimmune disease, an infection, trauma, or an idiopathic disorder. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the inflammation is associated with inflammatory diseases accompanied by symptoms of skin fibrosis such as keloids and scars.

3. 2. The pharmaceutical composition of claim 1, wherein the autoimmune disease includes manifestations of skin fibrosis, such as scleroderma, and pulmonary fibrosis, such as interstitial lung disease.

4. 2. The pharmaceutical composition of claim 1, wherein the pulmonary viral or bacterial infection is associated with pulmonary fibrosis, such as acute respiratory distress syndrome (ARDS).

5. The pharmaceutical composition of claim 1, wherein the traumatic injury includes signs of skin fibrosis such as keloids and scars.

6. The pharmaceutical composition according to claim 1, wherein the fibrotic lesion is systemic sclerosis, nephrogenic systemic fibrosis, or hepatic fibrosis.

7. The pharmaceutical composition according to claim 1, characterized in that the idiopathic disease is pulmonary fibrosis, preferably idiopathic pulmonary fibrosis (IPF).

8. 10. The pharmaceutical composition according to claim 1, characterized in that the composition is in the form of an aerosol, vapor inhalation, mist, nasal spray, oral administration and liquid formulation.

9. 9. The pharmaceutical composition of claim 8, wherein the composition is in liquid or lyophilized form for use in the treatment of idiopathic pulmonary fibrosis (IPF) symptoms via intratracheal, inhalation, oral administration or intravenous injection.

10. A medical device comprising the pharmaceutical composition according to any one of claims 1 to 9, for use in the prevention or treatment of a fibrotic disease, characterized in that the device is a metered dose inhaler, a dry powder inhaler, or a spray container for intranasal administration or nebulization.

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