IT202000017746A1 - PHARMACEUTICAL COMPOSITION COMPRISING MEDIA CONDITIONED BY SECRETOMA OF MESENCHIMAL CELLS OF THE ORAL CAVITY - Google Patents

Description

Description of the patent application for an industrial invention entitled: Pharmaceutical composition comprising medium conditioned by secretome of mesenchymal cells of the oral cavity

Background of the invention

The present invention refers to the pharmaceutical field and more in particular to a composition, preferably for aerosol administration, comprising a conditioned medium deriving from the secretome of mesenchymal cells of the oral cavity, preferably autologous, grown in the absence of serum, which can be used for the treatment of inflammatory pathologies, immune pathologies, neurodegenerative pathologies, for the induction of tissue regeneration.

State of art

Oral stem cells originate from the neural crest and represent a transient population of embryonic pluripotent stem cells that are induced at the lateral edges of the neural plate and migrate extensively to populate a variety of of fabrics.

Human oral tissue stem cells (hOTSCs) express cell surface markers such as cluster of differentiation (CD) 29, CD44, CD73, CD90, CD105 and do not express CD14, CD34, CD45 and human leukocyte antigen (HLA) ? DR (Trubiani, O.; Pizzicannella, J.; Caputi, S.; Marchisio, M.; Mazzon, E.; Paganelli, R.; Paganelli, A.; Diomede, F., Periodontal Ligament Stem Cells: Current Knowledge and Future Perspectives. Stem cells and development 2019, 28, (15), 995-1003).

Human mesenchymal stem cells isolated from oral tissues possess the peculiarities of the mesenchymal cells, as well as the ability? of proliferation and the maintenance of the characteristics of multipotency, which result in the long term useful for the possibility? to be manipulated in vitro and used for clinical purposes (Diomede, F.; Zini, N.; Pizzicannella, J.; Merciaro, I.; Pizzicannella, G.; D'Orazio, M.; Piattelli, A.; Trubiani, O., 5-Aza Exposure Improves Reprogramming Process Through Embryoid Body Formation in Human Gingival Stem Cells. Front Genet 2018, 9, 419; Pizzicannella, J.; Diomede, F.; Merciaro, I.; Caputi, S.; Tartaro, A.; Guarnieri, S.; Trubiani, O., Endothelial committed oral stem cells as modeling in the relationship between periodontal and cardiovascular disease. Journal of cellular physiology 2018, 233, (10), 6734-6747).

Extracellular vesicles are released by almost all cell types, and act as carriers of target molecules through a paracrine mechanism.

The extracellular vesicles avoid the direct use of stem cells avoiding all the limitations connected to the direct use of the same.

The extracellular vesicles can be used as therapeutic agents alone or as controlled delivery systems. Mesenchymal stem cells (MSCs) are one of the most important extracellular vesicles. Furthermore, the peculiar immunomodulatory effect of mesenchymal stem cells gives their extracellular vesicles fundamental characteristics for autologous and allogeneic therapeutic applications (Trubiani, O.; Marconi, G. D.; Pierdomenico, S. D.; Piattelli, A.; Diomede, F.; Pizzicannella, J ., Human Oral Stem Cells, Biomaterials and Extracellular Vesicles: A Promising Tool in Bone Tissue Repair. International journal of molecular sciences 2019, 20, (20)).

Extracellular vesicles are particles bounded by a lipid bilayer capable of crossing biological barriers and being internalized in target cells so as to play an essential role in intercellular communication between cells.

Extracellular vesicles contain different macromolecules of different nature such as lipids, proteins, DNA, mRNA and non-coding RNA (ncRNA) including microRNA (or miRNA).

Transcriptomic analysis of extracellular vesicles of gingival cells showed that they contain transcripts coding for growth factors such as members of the TGF-? family, with anti-inflammatory action, and members of bone morphogenetic proteins (BMPs) involved in osteogenesis. Also present were members of the FGF family and VEGF, which is involved in angiogenesis. Also present were transcripts coding for members of the neurotrophin and GDNF family, involved in neuronal development, mRNA coding for interleukins and transcripts coding for members of the Wnt family, which play a role in tissue regeneration (Silvestro, S.; Chiricosta, L. Gugliandolo, A., Pizzicannella, J., Diomede, F., Bramanti, P., Trubiani, O., Mazzon, E., Extracellular Vesicles Derived from Human Gingival Mesenchymal Stem Cells: A Transcriptomic Analysis. Genes (Basel) 2020 , 11, (2).

Extracellular vesicles can be used as cell-free therapy in regenerative medicine; for example, extracellular vesicles derived from mesenchymal stem cells promote osteoangiogenic processes, reduce inflammation and neuronal damage in in vivo and in vitro models.

AND? It has recently been shown that extracellular vesicles are associated with bone metabolism, bone healing and a potential ability to of mineralization.

The presence of a scaffold (support) made up of biocompatible materials, associated with stem cells, represents a fundamental element in tissue engineering and/or in regenerative medicine applications. The progress of technology and the development of innovative medicine lead to a continuous increase in diversity? of biomaterials, in particular the development of scaffolds (supports) functionalized with cells or with cellular derivatives. The development of biomaterials for 3D printing, including polylactic acid (3D-PLA), enriched with human oral tissue mesenchymal stem cells (hOTSCs) and/or their derivatives, such as extracellular vesicles (EVs), ? was employed in bone regeneration of critical calvaria defects in mouse models, promoting a new regulatory system in the evolution of osteo-angiogenesis. Scaffolds enriched with oral stem cells, with conditioned medium or extracellular vesicles improved bone regeneration by increasing the expression of genes involved in the regulation of ossification processes, genes coding for adhesion molecules and extracellular matrix, such as collagen, genes involved in differentiation of osteoblasts, including genes of the TGF-? and BMPs. The result ? the deposition of newly formed bone and of the extracellular matrix (Diomede, F.; Gugliandolo, A.; Cardelli, P.; Merciaro, I.; Ettorre, V.; Traini, T.; Bedini, R.; Scionti, D.; Bramanti, A.; Nanci, A., et al. Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair. Stem cell research & therapy 2018, 9, 104, doi: 10.1186/s13287-018-0850-0; Diomede, F.; D'Aurora, M.; Gugliandolo, A.; Merciaro, I.; Orsini, T.; Gatta, V.; Piattelli, A.; Trubiani, O .; Mazzon, E. Biofunctionalized Scaffold in Bone Tissue Repair. International journal of molecular sciences 2018, 19, doi:10.3390/ijms19041022; Diomede, F.; D'Aurora, M.; Gugliandolo, A.; Merciaro, I.; Ettorre, V.; Bramanti, A.; Piattelli, A.; Gatta, V.; Mazzon, E.; Fontana, A., et al. A novel role in skeletal segment regeneration of extracellular vesicles released from periodontal-ligament stem cells International journal of nanomedicine 2018, 13, 3805-3825, doi:10.2147/IJN.S162836. Diomedes, F .; Gugliandolo, A.; Scionti, D.; Merciaro, I.; Cavalcanti, M.F.; Mazzon, E.; Trubiani, O. Biotherapeutic Effect of Gingival Stem Cells Conditioned Medium in Bone Tissue Restoration. International journal of molecular sciences 2018, 19, doi:10.3390/ijms19020329.). Another mechanism by which scaffolds enriched with stem cells and their secretome can promote the osteo-angiogenic process? through the modulation of miRNAs. This construct? in fact able to induce an increase in the levels of miR-2861 and -210, involved in the processes of osteogenesis and angiogenesis, as well as of osteogenic markers (Pizzicannella, J.; Diomede, F.; Gugliandolo, A.; Chiricosta, L .; Bramanti, P.; Merciaro, I.; Orsini, T.; Mazzon, E.; Trubiani, O. 3D Printing PLA/Gingival Stem Cells/ EVs Upregulate miR-2861 and -210 during Osteoangiogenesis Commitment.International journal of molecular sciences 2019, 20, doi:10.3390/ijms20133256). Association of enriched scaffolds with stem cells and their secretome also induces the VEGF signaling pathway, increasing levels of both VEGF and its receptor VEGFR2, promoting angiogenesis, which ? fundamental in bone regeneration (Pizzicannella, J.; Gugliandolo, A.; Orsini, T.; Fontana, A.; Ventrella, A.; Mazzon, E.; Bramanti, P.; Diomede, F.; Trubiani, O. Engineered Extracellular Vesicles From Human PeriodontalLigament Stem Cells Increase VEGF/VEGFR2 Expression During Bone Regeneration.Frontiers in physiology 2019, 10, 512, doi:10.3389/fphys.2019.00512).

Membrane vesicles released from eukaryotic cells, such as exosomes, microparticles, microvesicles and apoptotic bodies, can be maintained as a dynamic extracellular vesicular compartment, strategic for their paracrine or autocrine biological effects in tissue metabolism (Rajan, T. S.; Diomede, F. Bramanti, P., Trubiani, O., Mazzon, E., Conditioned medium from human gingival mesenchymal stem cells protects motor-neuron-like NSC-34 cells against scratch-injury-induced cell death.International journal of immunopathology and pharmacology 2017 , 30, (4), 383-394). In particular, extracellular vesicles and their soluble secretion products, also called "secretoma(s)," contain abundant proteins, nucleic acids, lipids, and a pool of soluble cytokines; and they can act as biomarkers in many cellular functions.

Due to their size, diameter between 50 and 1000 nm, extracellular vesicles can circulate in the human body and in particular are present in most biological fluids. Recently, extracellular vesicles produced by gingival mesenchymal stem cells have been characterized through multiparametric analyzes and have been used to functionalize the scaffold for bone tissue regeneration.

The cavity? oral represents an important point of contact between the external and internal environment of the human organism and ? the first entry point to the respiratory and digestive systems. In the cavity? six different types of human oral tissue mesenchymal stem cells (hOTSCs) were isolated: stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSC), stem cells from dental follicle stem cells (DFPCs), gingival mesenchymal stem cells (GMSCs), and dental pulp stem cells (DPSCs) (Yang JW, Shin YY, Seo Y, Kim HS. Therapeutic Functions of Stem Cells from Oral Cavity: An Update. Int J Mol Sci. 2020 Jun 19;21(12):4389. doi: 10.3390/ijms21124389).

AND? the use of the secretome derived from mesenchymal stem cells of oral tissues is known in the treatment of various diseases, such as autoimmune or inflammatory diseases (Chiricosta, L.; Silvestro, S.; Pizzicannella, J.; Diomede, F.; Bramanti, P.; Trubiani, O.; Mazzon, E., Transcriptomic Analysis of Stem Cells Treated with Moringin or Cannabidiol: Analogies and Differences in Inflammation Pathways. Int J Mol Sci 2019, 20, (23); Mammana, S.; Gugliandolo, A.; Cavalli, E.; Diomede, F.; Iori, R.; Zappacosta, R.; Bramanti, P.; Conti, P.; Fontana, A.; Pizzicannella, J.; Mazzon, E., Human gingival mesenchymal stem cells pretreated with vesicular moringin nanostructures as a new therapeutic approach in a mouse model of spinal cord injury. Journal of tissue engineering and regenerative medicine 2019, 13, (7), 1109-1121).

Therapeutic use of secretome released from human oral tissue stem cells (hOTSCs) has low costs, the possibility of to expand ex vivo, the lack of immune response and the absence of carcinogenicity? (Sinjari, B.; Pizzicannella, J.; D'Aurora, M.; Zappacosta, R.; Gatta, V.; Fontana, A.; Trubiani, O.; Diomede, F., Curcumin/Liposome Nanotechnology as Delivery Platform for Anti-inflammatory Activities via NFkB/ERK/pERK Pathway in Human Dental Pulp Treated With 2-HydroxyEthyl MethAcrylate (HEMA) Frontiers in physiology 2019, 10, 633; Ballerini, P.; Diomede, F.; Petragnani, N.; Cicchitti, S.; Merciaro, I.; Cavalcanti, M.; Trubiani, O., Conditioned medium from relapsing-remitting multiple sclerosis patients reduces the expression and release of inflammatory cytokines induced by LPS-gingivalis in THP-1 and MO3.13 cell lines. Cytokine 2017, 96, 261-272).

Human oral tissue stem cells (hOTSCs) have demonstrated the ability to produce a large quantity? of cytokines and extracellular vesicles with a high content of inflammatory mediators and immunomodulators, effective results for the therapeutic strategy in various diseases of autoimmune aetiology. Specifically in their secretome are IL-10, TGF-? and the growth factors NGF and NT-3.

For example ? It has been extensively studied how periodontal ligament stem cells (hPDLSCs) spontaneously produce high levels of IL-7 and stromal cell-derived factor (SDF)-1?, and are also able to support the development and long-term maintenance of bone marrow (BM) progenitor cells similar to human bone marrow stromal cells (Trubiani, O.; Isgro, A.; Zini, N.; Antonucci, I.; Aiuti, F.; Di Primio, R.; Nanci, A.; Caputi, S.; Paganelli, R., Functional interleukin7/interleukin-7Ralpha, and SDF-1alpha/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells. J. Cell. Physiol. 2008, 214, ( 3), 706-13).

Using a metabololipidomic approach ? Periodontal ligament stem cells (hPDLSCs) have been reported to produce and release a broad spectrum of pro-resolving mediators, including resolving D1, D2, D5, and D6; D1 protection; maresins; and lipoxin B4; what? such as prostaglandins D2, E2 and F2?. These mediators regulate tissue trafficking of granulocytes, stimulating efferocytosis and microbial clearance. Periodontal ligament stem cells (hPDLSCs) also produce large amounts of of prostaglandins E2, which ? a key mediator for the business? anti-inflammatory (Cianci, E.; Recchiuti, A.; Trubiani, O.; Diomede, F.; Marchisio, M.; Miscia, S.; Colas, R. A.; Dalli, J.; Serhan, C. N.; Romano, M., Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins.Stem Cells Transl Med 2016, 5, (1), 20-32).

Some in vivo studies demonstrated that the use of periodontal ligament stem cell secretomes (hPDLSCs) in a mouse model of experimental autoimmune encephalomyelitis (EAE), induced an anti-inflammatory and immunosuppressive effect, leading to regression of disease signals and a clinical improvement of guinea pig health.

Multiple sclerosis (MS) ? a chronic crippling inflammatory autoimmune disease characterized by central nervous system (CNS) immune cell infiltration, demyelination, and axonal loss, resulting in the development of neurological symptoms. MS currently affects over 2.5 million people of the general population. Based on the clinical features, the clinical course of multiple sclerosis? been divided into four subtypes: relapsing-remitting, primary progressive, secondary progressive, and progressive relapse, each of these courses could have mild, moderate, or severe progression. Several treatments are available that aim at modulating the immune system for relapsing remitting MS; however, as yet, there is no Food and Drug Administration (FDA) approved treatment (Constantinescu, C. S.; Farooqi, N.; O'Brien, K.; Gran, B., Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS).Brit J Pharmacol 2011, 164, (4), 1079-1106).

Several animal studies demonstrate that the administration of mesenchymal cells (MSCs) can represent a type of alternative strategy for the treatment of multiple sclerosis for their ability? to suppress inflammation and the autoimmune response and at the same time limit the appearance of neurological symptoms (Zappia, E.; Casazza, S.; Pedemonte, E.; Benvenuto, F.; Bonanni, I.; Gerdoni, E.; Giunti, D.; Ceravolo, A.; Cazzanti, F.; Frassoni, F.; Mancardi, G.; Uccelli, A., Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005, 106, (5 ), 1755-1761; Kassis, I.; Petrou, P.; Karussis, D., Mesenchymal stem cells (MSC) derived from mice with experimental autoimmune encephalomyelitis (EAE) suppress EAE and have similar biological properties to MSC from healthy donors. Mult Scler J 2013, 19, (11), 139-139;Trubiani O, Giacoppo S, Ballerini P, Diomede F, Piattelli A, Bramanti P, Mazzon E. Alternative source of stem cells derived from human periodontal ligament: a new treatment for experimental autoimmune encephalomyelitis. Stem Cell Res Ther. 2016 Jan 4;7:1. doi: 10.1186/s13287-015-0253-4).

AND? Mesenchymal stem cells are known to exert their therapeutic effects primarily through paracrine signaling by exosomes/extracellular vesicles. For this reason, the products secreted by mesenchymal stem cells can provide a new regenerative therapeutic approach without the direct use of the cells in various pathologies. This approach avoids some of the limiting factors related to the direct use of stem cell-based therapies, such as immune competence, carcinogenicity, the number of cells to expand ex vivo, and related handling and maintenance costs (Yeo, R. W. Y. ; Lai, R. C.; Zhang, B.; Tan, S. S.; Yin, Y. J.; Teh, B. J.; Lim, S. K., Mesenchymal stem cell: An efficient mass producer of exosomes for drug delivery. Advanced drug delivery reviews 2013, 65, (3 ), 336-341).

In a mouse model of EAE, the secretome of periodontal cells obtained from healthy donors or multiple sclerosis patients showed anti-inflammatory effects (reduction of the proinflammatory cytokines IL-17, IFN-?, IL-1?, IL-6, TNF-?), and immunosuppressive, inducing spinal cord remyelination and reducing disease progression. These effects were mediated by the presence in the extracellular vesicles of the anti-inflammatory cytokines IL-10 and TGF-?, which induce a reduction of the proinflammatory cascade (Rajan, T.S.; Giacoppo, S.; Diomede, F.; Ballerini, P.; Paolantonio, M. .; Marchisio, M.; Piattelli, A.; Bramanti, P.; Mazzon, E.; Trubiani, O. The secretome of periodontal ligament stem cells from MS patients protects against EAE. Scientific reports 2016, 6, 38743, doi: 10.1038/srep38743.).

AND? Is it known that extracellular vesicles collected from the conditioned medium of cells derived from patients with relapsing-remitting multiple sclerosis show positivity? to CD90 leading to the stimulation of IL10 production (Campioni, D.; Rizzo, R.; Stignani, M.; Melchiorri, L.; Ferrari, L.; Moretti, S.; Russo, A.; Bagnara, G. P.; Bonsi, L.; Alviano, F.; Lanzoni, G.; Cuneo, A.; Baricordi, O. R.; Lanza, F., A Decreased Positivity for CD90 on Human Mesenchymal Stromal Cells (MSCs) Is Associated with a Loss of Immunosuppressive Activity by MSCs. Cytom Part B-Clin Cy 2009, 76B, (3), 225-230), which has an important immunoprotective and remyelination role (Klose, J.; Schmidt, N. O.; Melms, A.; Dohi, M. Miyazaki, J., Bischof, F., Greve, B., Suppression of experimental autoimmune encephalomyelitis by interleukin-10 transduced neural stem/progenitor cells. J Neuroinflamm 2013, 10; Yang, J. X.; Jiang, Z. L.; Fitzgerald, D. C.; Ma, C. G.; Yu, S.; Li, H. M.; Zhao, Z.; Li, Y. H.; Ciric, B.; Curtis, M.; Rostami, A.; Zhang, G. X., Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis. Journal of Clinical Investigation 2009, 119, (12), 3678-3691).

AND? the presence of IL-10 and TGF-? in extracellular vesicles derived from human periodontal ligament stem cells from healthy donors and patients with relapsing-remitting multiple sclerosis may have a crucial role in repressing the activation of the inflammatory cascade in the mouse model of multiple sclerosis (Experimental Autoimmune Encephalomyelitis EAE). The mice treated with the extracellular vesicles showed no immune responses, what? indicates that the immunomodulatory role of the extracellular vesicles and the conditioned medium ? of a selective type within the inflammatory microenvironment of the mouse model of multiple sclerosis (Experimental Autoimmune Encephalomyelitis EAE).

AND? It has been demonstrated that human periodontal ligament cells secrete extracellular vesicles containing anti-inflammatory cytokines, such as IL-10 and TGF-?, involved in activity immunosuppressive and are promoters of anti-inflammatory effects in vitro. Conditioned medium and extracellular vesicles of human periodontal ligament cells exert a protective and anti-inflammatory role not only when derived from healthy donor cells, but also when derived from patients with relapsing-remitting multiple sclerosis, thus suggesting a potential autologous use (Ballerini, P.; Diomede, F.; Petragnani, N.; Cicchitti, S.; Merciaro, I.; Cavalcanti, M.; Trubiani, O., Conditioned medium from relapsing-remitting multiple sclerosis patients reduces the expression and release of inflammatory cytokines induced by LPS-gingivalis in THP-1 and MO3.13 cell lines.Cytokine 2017, 96, 261-272).

Furthermore, ? Conditioned medium and extracellular vesicles derived from human periodontal ligament stem cells from relapsing-remitting multiple sclerosis patients and healthy donors have been shown to exert regulatory functions of apoptosis in mice with experimental autoimmune encephalomyelitis (EAE) through activation of pro-inflammatory cytokines. Indeed, proinflammatory cytokines are capable of inducing p53 and are involved in reducing the p53-mediated mechanism of apoptosis. Mice with Experimental Autoimmune Encephalomyelitis (EAE) administered extracellular vesicles and conditioned medium showed a significant reduction of STAT1, p53, C-caspase 3 and Bax expression showing an antiapoptotic effect (Rajan, T. S.; Giacoppo, S .; Diomede, F.; Ballerini, P.; Paolantonio, M.; Marchisio, M.; Piattelli, A.; Bramanti, P.; Mazzon, E.; Trubiani, O., The secretome of periodontal ligament stem cells from MS patients protects against EAE Scientific reports 2016, 6).

Conditioned medium and extracellular vesicles derived from human periodontal ligament stem cells from relapsing-remitting multiple sclerosis patients have a role in modulating NF-?B levels by inhibiting NALP3 activation and exerting a protective action in mice with autoimmune encephalomyelitis experimental (EAE). In multiple sclerosis NALP3 acts as a disease activator. AND? Inhibition of NALP3 inflammasome activation was demonstrated in mice with Experimental Autoimmune Encephalomyelitis (EAE) treated with conditioned medium and extracellular vesicles derived from human periodontal ligament stem cells (Soundara Rajan, T.; Giacoppo, S.; Diomede, F.; Bramanti, P.; Trubiani, O.; Mazzon, E., Human periodontal ligament stem cells secretome from multiple sclerosis patients suppresses NALP3 inflammasome activation in experimental autoimmune encephalomyelitis. Int. J. Immunopathol. Pharmacol. 2017, 30, ( 3), 238-252).

Mesenchymal stem cell therapy can also be used in other neurodegenerative pathologies, such as in spinal trauma which? accompanied by apoptosis, oxidative stress, and inflammation. In an in vitro model of spinal cord injury, conditioned medium derived from gingival mesenchymal stem cells conferred protection from apoptotic process by stimulating the production of the anti-apoptotic protein Bcl2 and reducing caspase 3 and Bax. The conditioned medium also reduced oxidative stress and inflammation by increasing levels of the anti-inflammatory cytokine IL-10. The neuroprotective and anti-inflammatory effects of the conditioned medium may in part depend on the presence of NGF, NT3, IL-10 and TGF-? in the middle of GMSCs (Rajan, T. S.; Diomede, F.; Bramanti, P.; Trubiani, O.; Mazzon, E., Conditioned medium from human gingival mesenchymal stem cells protects motor-neuron-like NSC-34 cells against scratch- injury-induced cell death. International journal of immunopathology and pharmacology 2017, 30, (4), 383-394).

Furthermore, ? Has integrity restoration been evaluated? of tissues through a remyelination thanks to the reduction of pro-inflammatory cytokines such as IL-17, IFN-?, reduction of the JNK pathway and modulation of autophagy by the PI3K/AKT/mTOR pathway, effects that were mediated by the increase of IL-37. At the same time, ? been demonstrated as the release of IL-10, cytokine with activity? anti-inflammatory, NT3, and TGF-b in the secretome of periodontal ligament stem cells maintained under hypoxic conditions could impact experimental autoimmune encephalomyelitis (EAE) through an IL-37-dependent mechanism, improving the prognosis of disease progression ( Giacoppo, S.; Thangavelu, S. R.; Diomede, F.; Bramanti, P.; Conti, P.; Trubiani, O.; Mazzon, E., Anti-inflammatory effects of hypoxia-preconditioned human periodontal ligament cell secretome in an experimental model of multiple sclerosis: a key role of IL-37 FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2017, 31, (12), 5592-5608).

Therefore, the secretome of human oral tissue stem cells due to their properties immunomodulatory and specific anti-inflammatory pu? exert therapeutic effects on patients affected by immunomodulatory and neurodegenerative pathologies (Giacoppo, S.; Thangavelu, S. R.; Diomede, F.; Bramanti, P.; Conti, P.; Trubiani, O.; Mazzon, E., Antiinflammatory effects of hypoxia - preconditioned human periodontal ligament cell secretion in an experimental model of multiple sclerosis: a key role of IL-37. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2017, 31, (12), 5592-5608). This non-invasive technique offers advantages in the treatment of autoimmune and neurodegenerative diseases.

Secretome and extracellular vesicles harvested from human oral tissue stem cells may provide a novel therapeutic approach, thus overcoming the limitations reported by therapies that are based on the direct use of stem cells. Stem cell-directed therapies include a possible abnormal immune response, substantial running costs, more complex handling, and difficult and ethical issues.

Recent, ? been reported that the aerosol technique MicroSprayer? AerosolizerModel IA-1B has been used to deliver airway epithelial cells in acute lung injury. The use of the MicroSprayer device? Aerosolizer IA-1B has demonstrated its efficacy in delivering intratracheal aerosol-based MSCs directly into the airways, inducing acute inflammation suppression in vivo and in vitro (Kardia, E.; Yusoff, N. M.; Zakaria, Z .; Yahaya, B., Aerosol-based delivery of fibroblast cells for treatment of lung diseases. J. Aerosol Med. Pulm. Drug Deliv. 2014, 27, (1), 30-4; Halim, N. S. S.; Ch'ng, E. S.; Kardia, E.; Ali, S. A.; Radzi, R.; Yahaya, B. H., Aerosolised Mesenchymal Stem Cells Expressing Angiopoietin-1 Enhances Airway Repair. Stem Cell Rev Rep 2019, 15, (1), 112-125). This aerosol method based on human stem cell secretome of oral tissues is accessible and low cost, and acting directly on the cavity? oral can be used representing a first protective barrier from external agents.

Technical problem

Systems employing cells, their secretome, conditioned medium or extracellular vesicles derived from them are unable to provide precision therapy that is tailored to the needs of the patient. therapies for individual patients. Furthermore, these systems, which are difficult to find, can trigger immune responses and unwanted allergic reactions that make them usable with limited success.

On the basis of what has been reported, the present inventors have developed a process which allows to obtain a secretome, constituted by a defined group of non-embryonic stem cells, easily available and usable. This system can be advantageously exploited in the prophylaxis, prevention and treatment of inflammatory states with minimal risk and optimal therapeutic effects. In particular, ? capable of significantly reducing the immune response generated by autoimmune diseases and therefore preventing a worsening of clinical conditions.

Furthermore, the present invention also provides two particular methods of administration of increased effectiveness, which allow to always obtain a correct administration regardless of the health and compliance conditions of the treated subject.

Object of the invention

Object of the present invention ? a pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions and suitable pharmacologically acceptable excipients.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions and suitable pharmacologically acceptable excipients in the form of lyophilized dry powder for inhalation administration by aspiration.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions and suitable pharmacologically acceptable excipients in the form of a spray solution for passive inhalation administration.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration such as medicament.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the induction of tissue regeneration.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the treatment of inflammatory pathologies.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the treatment of neurodegenerative diseases.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the treatment of immune pathologies.

Further characteristics will become apparent from the detailed description which follows with reference to the enclosed figures and the reported experimental data.

Brief description of the figures

Figure 1 shows in graph the effect of the secretome on the regeneration of the dental pulp. hDPSCs: mesenchymal stem cells of the dental pulp; S: secretome. P<0.01.

Figure 2 plots the effect of the secretome on osteogenic differentiation in bone defects of the Oral. hPDLSCs: periodontal ligament stem cells; S: secretome. P<0.01.

Detailed description of the invention

Object of the invention ? a liquid pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions, and suitable pharmacologically acceptable excipients.

Preferably oral tissue stem cells are selected from the group consisting of: stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSC), dental follicle stem cells (DFPC) , gingival mesenchymal stem cells (GMSCs) and dental pulp stem cells (DPSCs).

Pi? preferably the stem cells of the oral mucosa are selected from the group consisting of: mesenchymal stem cells of the periodontal ligament (hPDLSCs), mesenchymal stem cells of the pulp (hDPSCs).

Preferably the oral tissue stem cells are autologous.

Preferably when the oral tissue stem cells are autologous alternatively they can be cultured in the presence of autologous serum.

Preferably the liquid pharmaceutical composition comprises the secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions, at least one tonicity corrector and and at least one chelating agent in a concentration of 0 to 0.3% w/v, a buffering agent, and has a pH in the range of 4 to 7.5.

Preferably the? buffering agent ? selected from the group consisting of citrate, phosphate, succinate, acetate, glutamate and mixtures thereof.

Preferably the corrector of tonicity? ? selected from the group consisting of sodium chloride, dextrose, glycerin glutamate and mixtures thereof.

Preferably the chelating agent ? EDTA or a salt thereof.

A further object of the present invention is a liquid pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions or in the presence of autologous human serum, and suitable pharmacologically acceptable excipients, in the form of lyophilized dry powder for administration passive inhalation.

The liquid pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions or in the presence of autologous human serum, and suitable pharmacologically acceptable excipients, in the form of lyophilized dry powder? obtained through a process which includes the following stages:

a) the addition to the secretome of at least one cryoprotectant at a concentration between 0.5 and 5% (w/v), at least one chelating agent at a concentration between 0 and 0.3% w/v, at least one antimicrobial agent /antioxidant in a concentration between 0 and 5% w/v and at least one surfactant, at a pH between 4 and 7.5;

b) filtration

c) freezing

d) freeze-drying

Preferably in stage c) freezing ? at a temperature of -50 ?C.

Preferably in stage d) is freeze-drying? carried out by means of primary drying at 300 microbar and a temperature of -15 ?C followed by secondary drying at 100 microbar and a temperature of 20 ?C, for a total time of about 70 hours and up to humidity values? residual less than 3%.

The freeze-dried composition can be stored at a temperature of 2-8 ?C.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration such as medicament.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the induction of tissue regeneration.

Preferably tissue regeneration ? bone regeneration, in particular of the musculoskeletal system and the odontostomatological system.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the treatment of neurodegenerative diseases.

Preferably the neurodegenerative pathologies are selected from the group consisting of: multiple sclerosis, spinal cord trauma.

A further object of the present invention ? the pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions and suitable pharmacologically acceptable excipients, preferably in the form of lyophilized dry powder for inhalation administration by aspiration or in the form of a spray solution for passive inhalation administration by use for the treatment of immune pathologies.

Examples

Example 1: Cell culture

Periodontal ligament mesenchymal stem cells were isolated from the periodontal ligament and pulp mesenchymal stem cells (hDPSCs) were isolated from the dental pulp of non-carious third molars extracted during orthodontic therapy. I study ? been approved by the ethics committee of our University. All patients enrolled in the study signed informed consent. To be enrolled, the patients present a good general state of health and do not present pathologies of the cavity. Oral.

The periodontal tissue? taken from the horizontal fibers of the periodontal ligament using a Gracey curette. The tissue fragments were maintained in a cell culture plate with MSCBM-CD medium (Lonza Walkersville Inc.) incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 7 days. The terrain ? been changed every two days. The hPDLSCs spontaneously migrated after one week of culture. When the cells have reached 80-90% confluence, as assessed by phase contrast microscopy, they are isolated using 0.1% trypsin solution and then reseeded in polystyrene cell culture plates at a concentration of 5 ? 10<3 >cells/cm<2>. The pulp tissue? was taken using a cylindrical diamond bur (314, ? ISO 014, L.8.0 mm; Intensiv, Grancia, Switzerland) mounted on a high-speed handpiece? (Bora L; Bien - Air, Bienne, Switzerland) with water irrigation during use. The pulp? was removed using a sterile excavator, cut into small pieces and then cultured in MSCBM-CD medium (Lonza Walkersville Inc., Walkersville, MD, USA). After 20 days of culture, numerous colony forming cells (CFU - F) migrated from the explants. Adherent cells, at 80-90% confluence, were then isolated using 0.1% trypsin solution and seeded into polystyrene cell culture plates at 5 ? 10<3 >cm<2> cells. The gum tissue? taken during oral surgery. The fabric? was harvested using a scalpel blade and surgical tweezers, was the gum tissue incised? been placed on a sterile cell culture plate and ? de-epithelialized state. The fabric? it was then rinsed with sterile PBS and then placed in a plate with MSCGM-CD medium (Lonza) maintained in an incubator for 20 days at 37°C in a humidified atmosphere of 5% CO2 in air. After 20 days of culture, numerous colony forming cells (CFU - F) migrated from the explants. Adherent cells, at 80-90% confluence, were then isolated using 0.1% trypsin solution and seeded into polystyrene cell culture plates at 5 ? 10<3 >cm<2> cells.

Primary cultures of hPDLSCs, hDPSCs and hGMSCs consisted of colonies of fibroblastoid-like cells which, after subcultivation, proliferated on a log scale doubling the cell population at 48 hours. The above mentioned cells were grown using MSCGM-CD (chemically defined mesenchymal stem cell growth medium) (Lonza, Basel, Switzerland) and were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO2 in air, the results are shown in figures 1 and 2.

Example 2: EVs purification and flow cytometry

EVs were purified from supernatants of hPDLSCs, hDPSCs, hGMSCs from healthy donors. Briefly, cells were washed with phosphate-buffered saline (PBS) and then cultured in MSCBM for 24 hours. The conditioned medium after 24 hours of culture? was collected and centrifuged at 2000 g for 20 minutes at 4°C to remove cell debris. Then, the conditioned medium (CM) ? was ultracentrifuged at 100,000 g for 70 minutes at 4°C. The pellets were then washed with filtered PBS and ultracentrifuged at 100,000 g for 70 minutes at 4°C and suspended in PBS (double filtered). All samples were ultracentrifuged in polycarbonate tubes (25 mm × 89 mm, Beckman Coulter) which have a volume of 22 mL. The Beckman Coulter ultracentrifuge (Beckman Coulter OptimaL-90K ultracentrifuge; Beckman Coulter, Fullerton, CA, USA) ? been used with a fixed angle rotor type 70ti.

The following monoclonal antibodies were used for flow cytometric immunophenotyping of EVs derived from hPDLSCs, hDPSCs, hGMSCs: CD90 FITC (fluorescein isothiocyanate), CD34 FITC, CD44 PE (phycoerythrin), CD73 PE, CD14 PE, CD63 PE, CD166 PerCP -Cy 5.5 (phycoerythrin-cyanine 5.5), CD34 PerCP-Cy5.5, CD45 PerCP-Cy5.5, HLA-DR PerCP-Cy5.5, CD19 PerCP-Cy5.5, CD81 APCH7 (APC-cyanine tandem dye) , CD45 V500 (BD Horizon V500), CD44 APC (Allophycocyanin) and CD105 APC. All reagents were purchased from BD Biosciences (San Jos?, CA) with the exception of CD44 APC, Cytognos (Salamanca), and CD105 (R&D Systems, Minneapolis, MN, USA) purchased from Cytognos and R&D Systems, respectively.

The FACSCanto II flow cytometer (BD Biosciences San Jose, CA) ? was used for the acquisition of the EVs using the FACSDiva 6.1 software (BD Biosciences). The FACSCanto II flow cytometer ? an 8-channel instrument with 3 lasers, blue (488 nm, air-cooled, 20-mW solid state), red (633 nm, 17-mW HeNe), and violet (405 nm, 30-mW solid state).

For the calibration of the FACSCanto II flow cytometer, did we use the SPHERO calibration particles? Rainbow (Rainbow Calibration, Eight Peaks-Spherotech, Inc. Lake Forest, USA), following the recommendation of the EuroFlow consortium, with adaptation of the channels of the light scattering detectors to correctly identify the EV. For fluorescence compensation, BD? CompBeads (BD Biosciences, San José, CA, USA) were used with generic and fluorochrome-labeled antibodies used in our experiments. Samples were acquired after daily evaluation of instrument performance using Rainbow bead particles. These particles have also been used to identify electronic noise or background, which mainly comes from foreign particles reaching the detector (scattering of light). Samples were acquired after cytometer calibration and compensation.

Before the acquisition of the EVs, the instrument ? was washed with a rinse solution and PBS (through 0.2 µm Millipore membrane filters) after 6 hours of sedimentation, to reduce instrument background noise. The Forward scatter component (FSC) parameter is used to determine the size of the EVs. The PBS double filter ? was acquired with a mix of fluorescent microspheres composed of various diameters (0.5, 0.9 and 3 ?m) Megamix (Biocytex, Marseille, France) and Microbeads (Cytognos, Salamanca, Spain) of 6?6.4 ?m in size. Samples of different sizes were used in all experiments, with this approach ? been validated the tool based on the ability? to discriminate between Megamix microspheres from 0.5 and 0.9 ?m using the FSC parameter, as well as? their background noise. Sample acquisition ? Was it performed only when the number of dual filtered PBS events acquired per second ranged between 25 and 50 at low speed? with threshold settings between 300 and 350. EVs recovered from ultracentrifugation were suspended in PBS and immunofluorescently stained using monoclonal antibodies. For the study of antigen expression, the samples were incubated in the dark with the appropriate combination of monoclonal antibodies for 15 minutes. For the immunophenotypic characterization, the samples were incubated for 15 minutes in the dark with the following panel of monoclonal antibodies: 5 ?l of anti-CD90-FITC /10 ?l of anti-CD73-PE or anti-CD63-PE /10 ? l of anti-CD34-PerCPCy5.5/5 ?l of anti-CD44-APC /5 ?l of anti-CD81-APCH7 /5 ?l of anti-CD45-V500. After incubation, the antibody excess ? was washed with 2000 g PBS for 10 minutes. The final volume? state of 700 ?l.

To analyze whether the above antibodies do not form aggregates, ? was used as negative control the PBS containing each single antibody, as well as? the combination of all the antibodies used. PBS? been colored following the same methodology as the EVs. Data were acquired immediately after staining. In all samples, EVs unlabelled with the different antibodies were used to discriminate the positive and negative population.

A total of 100,000 events (at low speed) were captured. Data were analyzed using the Infinicyt program (Cytognos, Salamanca, Spain).

Example 3: liquid formulation of the filtered and sterilized secretome

The secretome, suitably stabilized by buffering agents based on salts (citrate, phosphate, succinate, acetate, glutamate) in a pH range between 4 and 7.5 of the hOMSCs, corrected for tonicity by using sodium chloride, dextrose or glycerin and adding chelating agents such as EDTA salts in a concentration range of 0-0.3% w/v. The final wording? filtered and prepared for administration using a nebulizer. The sample ? been subjected to a process of reduction of the microbial load through 220 nm Millidisk filters (Millipore), designed for the removal of particles and microorganisms from liquids.

Example 4: dry powder formulation for use

Mannitol (or other cryoprotectant such as sucrose, PEG), ? was added, at a concentration between 0.5 and 5% (w/v) to the purified secretome solution. The resulting solution? been stabilized by the addition of a chelating agent such as EDTA (or its salts) in a concentration range of 0-0.3% w/v, corrected for pH in a range of between 4 and 7.5 by the addition of salts (citrate, phosphate, succinate, acetate, glutamate), addition of antimicrobial/antioxidant agents (such as phenols, benzyl alcohols, methionine, ascorbic acid, BHT, BHA, cysteine, sulfurous acid salts) in the concentration range 0-5% w/ v and surfactants such as Tween or Lecithin. What is the solution? The prepared product is filtered by reducing the microbial load using a 220 nm Millidisk filter (Millipore) and subjected to a freeze-drying process. The solution is frozen at -50 ?C and after suitable standing, freeze-dried in primary drying at 300 microbar and a temperature of -15 ?C followed by secondary drying at 100 microbar and a temperature of 20 ?C, for a total time of about 70 hours and in any case up to humidity values? residual less than 3%. The lyophilized secretome ? been stored at a temperature of 2-8 ?C until reconstitution and use. From the ratio between the total number of cells used for the production and the recovered milligrams of dry powder? it was possible to validate and then define the yield of the freeze-drying process. Were the freeze-dried products analyzed by evaluating their humidity? residual, the composition, the dissolution time upon reconstitution and the pH of the reconstituted solution, the X-ray morphology, the thermal behavior by DSC and the properties? of flow (Ceschan, N.E.; Bucal?, V.; Mateos, M.V.; Smyth, H.D.C.; Ram?rez-Rigo, M.V., Carrier free indomethacin microparticles for dry powder inhalation. Int. J. Pharm. 2018, 549 (1- 2), 169-178).

The moisture content? ? was evaluated using a METTLER TOLEDO Karl Fisher titrator. The densities? upon pouring and after packing, determined by measuring the volume occupied by a quantity? note of the freeze-dried product, were used to calculate the Carr index. The properties? flow rates were determined in accordance with the criteria reported in the USP (USP 42?NF 37).

The freeze-dried product? was formulated through a dry powder aerosol manufacturer. The formulations were made using the dry powder as it is (carrier-free formulation) or after mixing it with a carrier. Among the excipients considered, lactose ? been selected as the carrier of choice because of its non-toxicity? and biocompatibility. The lio-secretoma ? was mixed for 28 minutes, with lactose (in different proportions in the range 1:1 - 1:3) at 42 rpm. The uniformity? of the resulting mixtures ? was evaluated by carrying out random sampling. For these mixtures, the properties? flow rates were determined in accordance with the criteria reported in the USP (USP 42?NF 37).

The stability? of the liquid formulation and the freeze-dried powder ? was studied in two storage conditions T1 = -20+/-3?C and T2 = 5+/-3?C. The samples in an appropriate number and in the final containers (closed glass vial with stopper and ring nut for both formulations) produced on a laboratory scale according to the methods described above, were analyzed as soon as they were produced (t0) and stored for 4 weeks. A sufficient number of samples for characterization analyzes ? been removed from storage conditions and analyzed at different time intervals and the results obtained compared with those of t0.

The stability index? ? was defined by comparison between the conditions at the start of the study (t0) on the basis of 2 subsequent checks:

- t1: after 1 week of storage

- t2: after 4 weeks of storage ? end of study

The results of the analyzes performed on the samples are shown in tables 1-3, so such as the % variations in subsequent samples intended as a difference in absolute value, compared to the results at the beginning of the study (t0).

Table 1 below shows the stability results? 5?C+/-3?C of the lyophilized dry powder, (where * within the variability of the analytical method).

Table 1

Table 2 shows the results of the stability? -20+/-3?C ?C of the liquid solution (where * within the variability of the analytical method).

Table 2

The freeze-dried dry powder? been subjected to stability? accelerated to verify the degradation profile under conditions of thermal stress.

The samples were stored at 25?C+/-3?C for a total of four weeks and analyzed at regular times as shown in table 3. Also in this case the results were compared with the reference sample at t0 just prepared.

Table 3 below shows the results of the stability? 25?C+/-3?C of the lyophilized dry powder (where * within the variability of the analytical method).

Table 3

The storage conditions of the two proposed pharmaceutical forms are:

- room temperature (15-25?C) for the preparation of lyophilized dry powder

- refrigerated temperature (2-8?C) for the liquid solution as a precaution.

Claims (13)

Claims A pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells cultured under serum-free conditions, and suitable pharmacologically acceptable excipients. The pharmaceutical composition according to claim 1 wherein the oral tissue stem cells are autologous. The pharmaceutical composition according to claim 2 wherein autologous oral tissue stem cells are cultured in the presence of autologous serum. The pharmaceutical composition according to claim 1 wherein the oral tissue stem cells are selected from the group consisting of: stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSC) , dental follicle stem cells (DFPC), gingival mesenchymal stem cells (GMSC), and dental pulp stem cells (DPSC). 5. Pharmaceutical composition according to claim 1 comprising: secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions at least a corrector of tonicity? and at least one chelating agent in a concentration of 0 to 0.3% w/v at least one buffering agent and having a pH in the range of 4 to 7.5 6. Pharmaceutical composition according to any one of claims 1 to 5 in the form of lyophilized dry powder for inhalation administration by aspiration. 7. Pharmaceutical composition according to any one of claims 1 to 5 in the form of a spray solution for passive inhalation administration. 8. Pharmaceutical composition according to any one of claims 1 to 7 for use as a medicament. 9. Pharmaceutical composition according to any one of claims 1 to 7 for use in the treatment of inflammatory pathologies. 10. Pharmaceutical composition according to any one of claims 1 to 7 for use in the treatment of immune pathologies. 11. Pharmaceutical composition according to any one of claims 1 to 7 for use in the treatment of neurodegenerative pathologies. The pharmaceutical composition according to any one of claims 1 to 7 for use for the induction of tissue regeneration. 13. A method for preparing a liquid pharmaceutical composition comprising secretome obtained from heterologous or autologous oral tissue stem cells grown under serum-free conditions or in the presence of autologous human serum, and suitable pharmacologically acceptable excipients, in the form of a lyophilized dry powder which involves the following stages: a) addition to the secretome of at least one cryoprotectant at a concentration between 0.5 and 5% (w/v), at least one chelating agent at a concentration between 0 and 0.3% w/v, at least one antimicrobial/antioxidant agent in a concentration between 0 and 5% w/v and at least one surfactant, at a pH between 4 and 7.5; b) filtration c) freezing d) freeze-drying