HK1263117A1 - Human platelet lysate derived extracellular vesicles for use in medicine - Google Patents

Description

Human platelet lysate-derived extracellular vesicles for use in medicine

Background

Human platelet lysate (hPL) is known as an alternative supplement to Fetal Bovine Serum (FBS) in human cell culture, which is commonly used for supplementing basal medium in human mesenchymal Stem cell culture for experimental and Clinical purposes hPL is advantageous compared to the traditionally used FBS because it does not contain xenogens (xenogens) and is therefore suitable for the production of therapeutic products for cell therapy hPL is presented as a turbid, light yellow liquid obtained from lysis of human platelets by freeze/thaw cycles, by virtue of the physiological tissue repair function of blood platelets, which are a rich source of various bioactive molecules such as growth factors, cytokines (including chemokines and interleukins), other metabolites and extracellular vesicles (extracellular vector, EV) which during storage and lysis release a large amount of growth factors and extracellular vesicles (e.g. platelet lysate vesicles) of about 40 to 1000nm in size and which are used in Clinical Medicine for the manufacture of human platelet lysate (e.g. transfer Drug microvesicles) and extracellular vesicles (GMP) for the manufacture of extracellular Drug microvesicles, which are used in Clinical Medicine for the manufacture of human mesenchymal Stem cell cultures, especially as a pharmaceutical Drug for the manufacture of human mesenchymal Stem cells under the Clinical test, which is considered as a human platelet lysate (Clinical Medicine) for the manufacture of human platelet lysate in the Clinical Medicine for the manufacture of human platelet culture.

Although the value of hPL in stem cell culture is well established, the properties of single factors (e.g., growth factors and EV) in hPL have not been analyzed in detail. While EVs from hPL have not been considered clinically significant, EVs derived from different stem cells (e.g., MSCs) are increasingly becoming the focus of clinical and therapeutic interest.

EV is known to transfer information between cells, organs, and even organisms, and has been detected in a variety of bodily fluids (e.g., blood, urine, cerebrospinal fluid, breast milk, and saliva). Exosomes and microvesicles constitute the most prominently described class of EVs. It is surrounded by a phospholipid membrane and comprises a cell type-specific combination of: proteins, including enzymes, growth factors, receptors, and cytokines; and a lipid; coding and non-coding RNA, mRNA, microrna (mirna), or even small amounts of DNA; and metabolites. Exosomes are defined as derivatives of endosomal compartments ranging in size from 70 to 170nm (+/-20nm, which varies according to literature and analytical techniques). Microbubbles represent a class of larger EVs formed by budding outward through the plasma membrane, with an average size of 100 to 1000 nm. In the present application, the term EV encompasses all of the above vesicles.

Currently, only stem cell-derived EV is shown to be widely recognized and discussed as having therapeutic potential according to therapeutic objectives. WO 2012/053976 discloses the use of exosomes secreted by human mesenchymal stem cells to promote hair growth and wound healing. These effects are disclosed in conjunction with an exosome immunomodulatory load. WO 2012/053976 speculates on the immunomodulatory effects of exosome formulations.

WO 2014013029 a1 relates to the use of exosome formulations derived from neonatal or adult human tissue-derived Mesenchymal Stem Cells (MSCs) for the prevention or treatment of inflammatory disorders such as pre-and postnatal acquired brain injury (i.e. neuronal damage) or immune complications following stem cell transplantation ("graft-versus-Host Disease", GvHD).

Recently discussed sources of EV with therapeutic potential were described by Thomas Leneeret et al in 2015 by ISEV (the International Society of Extracellular Vesicles) a recent opinion paper in Journal of Extracellular vehicles (Applying Extracellular Vesicles based on clinical trials in clinical trials.4: 30087). The cell sources under investigation for regenerative medicine are endothelial cells and endothelial colony forming cells, including human endothelial cells and late stage endothelial cells from the umbilical vein. Furthermore, hematopoietic progenitor cells capable of differentiating into myeloid and lymphoid cells may exert angiogenic promoting functions. Neural Stem Cells (NSCs) have been used in preclinical models of a variety of neurological and neuroinflammatory disorders, such as stroke, Multiple Sclerosis (MS), or spinal cord injury. However, it is clear that, like MSCs, NSCs also exert their therapeutic effects in a paracrine and systemic manner rather than by migrating to the site of pathology. In this case, NSC-derived EV is thought to interact with the host's immune system to mediate neuroprotection and immunomodulation. Neuroprotection and nerve regeneration can also be mediated by EV released from resident glial cells of the nervous system. Finally, very recent studies describe EV isolation from induced pluripotent stem cells (ipscs), their ability to transfer RNA and proteins into heart cells, and their ability to heal in vivo in ischemic myocardium. Furthermore, ipscs can be used as a source for expanded culture of somatic stem cells for large-scale EV production, or as a source for obtaining cells that are difficult to obtain from primary donor material as a source of EV (e.g., human NSCs). In this case, EVs from iPSC-derived MSCs have been shown to reduce limb ischemia. It is discussed that combinations of iPSC and EV technologies may provide new treatment options in the future.

To summarize what is given in the above-mentioned recent opinion papers (Journal of excellular veins 2015, 4: 30087), it must be emphasized that the therapeutic potential of EVs derived from hPL is not considered.

EVs derived from human stem cells (e.g., MSC, NSC, or iPSC) are not readily available because stem cell cultures are complex, expensive, and have limited sources for large-scale manufacture of pharmaceuticals.

The possibility that hPL-derived exosomes might be considered as novel effectors of human platelet lysate activity is discussed in Torreggiani et al publication (2014, European Cells and Materials Vol 28, 137-151). Torreggiani et al discusses the use of platelet-derived exosomes for bone regeneration. In their studies, the role of PL derived exosomes was studied in conjunction with MSC cell culture support. However, the formulation described in Torreggiani et al appears to be not compliant with the quality criteria for reliable extraction of EV.

Furthermore, in most clinical studies using MSC-derived EV, MSCs were cultured in hPL-supplemented media, where the potential synergistic effects of hPLEV were not suggested or discussed. In the prior art, the effect observed in the study of MSC-derived EV was attributed to MSC-derived EV, even though hPL was used as a supplement to the basal medium in mesenchymal stem cell culture.

In addition, there are many scientific papers, patent applications and patents on the use of Platelet Rich Plasma (PRP), for example, Eppley BL et al (2006), plant relay Surg 118 (6): 147e-159 e; mishra AK et al (2012), Curr Pharm Biotechnol 13 (7): 1185-1195; and Carlson NE et al (2002), J Am Dent Assoc 133 (10): 1388-1386. PRP consists of concentrated viable platelets and plasma obtained from patient whole blood by centrifugation to remove red blood cells and other unwanted components. It has a higher concentration of growth factors than whole blood and has been used for tissue injection in a number of disciplines including dentistry, orthopedic surgery and sports medicine.

However, the results of basic science and preclinical trials have not been determined in large-scale randomized control trials until now in 2016. In the 2009 review, the scientific literature was systematically screened and only a few randomized control trials were found to adequately evaluate the safety and efficacy of PRP treatment. PRP is considered to be a "promising but unproven therapeutic option for joint, tendon, ligament and muscle injury" (see also Foster et al (2009). Am J Sports Med 37 (11): 2259-72).

Regarding the use of PL, its particular application in the field of wound healing has been described, as in WO20130765507, which describes pharmaceutical compositions comprising platelet lysate and their use for treating wounds, anal fissures, vaginal atrophy or wrinkles. In Fontana et al (2016) ASC Appl Mater Interfaces 8 (1): 988-.

In summary, it is shown that in the PRP related prior art, the use of hPL or hPLEV for clinical applications other than wound healing applications is not described.

Thus, there is an urgent need in the art for means and methods that allow for simple, inexpensive, reliable, and efficient treatment using PRP and PL as sources. The same applies to EV-based therapies that are not based on complex and expensive stem cell culture systems.

The object of the present invention is to meet the above-mentioned needs and to propose hPL-derived EVs as new tools in medicine, in particular in therapeutic, regenerative and prophylactic medicine.

Summary of The Invention

The present invention relates to pharmaceutical preparations comprising human platelet lysate or a fraction enriched in human platelet lysate-derived extracellular vesicles (this fraction is also abbreviated hereinafter as hPLEV) for use in medicine, in particular for the prophylaxis and/or treatment of acute or/and chronic inflammatory and immunological diseases (also including autoimmune diseases and diseases caused by transplant rejection (e.g. GvHD)), neurological and neurodegenerative diseases (stroke, ischemia), skin diseases, cardiovascular diseases, orthopedic diseases (orthopaedic diseases), infectious diseases, cancer diseases, tissue regeneration (solid organs, hollow structures, lesions).

The invention also relates to the cosmetic use of a fraction of human platelet lysate or enriched in extracellular vesicles derived from human platelet lysate. These applications include: cosmetic skin-related anti-inflammatory treatment, skin regeneration after injury or burn, anti-aging treatment of skin, and prevention and treatment of hair loss.

The invention also relates to the diagnostic use of a fraction of human platelet lysate or enriched extracellular vesicles derived from human platelet lysate.

The invention also relates to a method for producing a pharmaceutical or diagnostic or cosmetic preparation, comprising the following steps: adding a human platelet lysate or a fraction enriched in human platelet lysate derived extracellular vesicles to a pharmaceutical or diagnostic or cosmetic preparation.

Although there are many advantages in biological significance, cost and effort by replacing the stem cell source EV with hPLEV, such replacement has never been suggested in the prior art. Since hPL is derived from human blood/plasma, its source is renewable and readily available compared to MSC, ESC, NSC or other stem cell types. Thus, hPLEV is more readily available and less expensive than EV derived from stem cell cultures. Another advantage of the invention with respect to the corresponding stem cell culture source EV is the fact that: platelets do not require clean room conditions and do not require time-consuming and expensive flow cytometry characterization of cellular entities. In addition, the excess human platelet concentrate preparations produced for hospital and clinic applications and which expire after a few days are still available for the production of hPL rather than being discarded.

Furthermore, the invention relates to hPL produced by expired, frozen and stored platelets, in particular not later than seven days after collection, which provides the active components of the platelets immediately rather than in a delayed release of the live platelets. Thus, an important aspect of the invention relates to the use of hPL or hPLEV for medical or cosmetic treatment, which has surprising advantages as an alternative to the use of hPL or hPLEV, which shows surprising advantages over the use of live platelets (e.g. platelet rich plasma therapy).

It is noteworthy that the present invention describes for the first time the direct use of human platelet lysate or fractions enriched in human platelet lysate derived extracellular vesicles in medicine, in particular for the prevention and/or treatment of acute or/and chronic inflammatory and immunological diseases (also including autoimmune diseases and diseases caused by transplant rejection (e.g. GvHD)), neurological and neurodegenerative diseases (stroke, ischemia), skin diseases, cardiovascular diseases, orthopedic diseases, infectious diseases, cancer diseases, tissue regeneration (solid organs, hollow structures, injuries) and diseases in which antimicrobial treatment is advantageous.

The present invention teaches for the first time the use of human platelet lysate or fractions enriched in human platelet lysate derived extracellular vesicles directly as a medicament and for cosmetic applications. These applications include cosmetic skin-related anti-inflammatory treatments, antimicrobial treatments, skin regeneration after injury or burn, anti-aging treatments of the skin, and the prevention and treatment of hair loss.

The present invention proposes for the first time a new use of hPL or hPLEV as a drug, which can be manufactured at low cost, is xeno-and cell-free product of high medical quality, without the need for clinical cell culture intermediates.

Detailed Description

In support of understanding the present invention, several terms are defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the claims, certain exemplary methods and materials are described herein.

Furthermore, unless the context clearly requires that there be one and only one element, the recitation of an element by a noun without the recitation of a number does not exclude the possibility that more than one element is present. Thus, a noun without the numerical modification is generally intended to mean "at least one".

The term "about" means within a statistically significant range of one or more values, such as the recited concentration, length, molecular weight, pH, time frame, temperature, pressure, or volume. Such values or ranges may be on the order of the given values or ranges, typically within 20%, more typically within 10%, and even more typically within 5%. The permissible variations encompassed by "about" depend on the particular system under study.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and includes the limits of the endpoints defining the range, and each separate value is incorporated into the specification as if it were individually recited herein.

In the context of the present invention, hPL means any kind of human platelet lysate/human platelet lysate pool having a beneficial therapeutic or cosmetic effect. Platelets (also known as thrombocytes) are irregular, disc-like elements in blood that contribute to clotting. During normal coagulation, platelets aggregate by aggregation. Although platelets are generally classified as blood cells, they do not have a nucleus. It is derived from large cells called megakaryocytes that are located/localized in the bone marrow. To produce hPL, platelets are lysed and thereby their contents are released into the surrounding plasma. Lysis of platelets can be achieved by freeze and thaw cycles. Suitable solutions can be found in the prior art.

One biological component that is contained in high amounts in human platelet lysates is EV. Since EVs are present in most bodily fluids, plasma-derived EVs can be derived from different cell types, such as leukocytes, erythrocytes, Dendritic Cells (DCs), platelets, mast cells, epithelial cells, endothelial cells, and neurons. In one aspect, hPL may contain plasma-derived EVs that are already present in plasma at the time of donation. On the other hand, hPL may contain a large amount of specific platelet-derived EV, which is secreted by live thrombocytes during the storage time of the platelet concentrate preparation. During the storage period of approximately one week at 20 ℃ to 24 ℃, the cells continue to secrete their specific EV into the surrounding plasma. When the lifetime of the thrombocyte concentrate is reached and the blood product is available for other purposes (e.g. production of platelet lysate preparations), this specific EV enrichment should still be present in the lysate, which can be further processed to obtain an hPLEV enriched fraction. In addition, platelets rupture by lysis and release their soluble internal components into the plasma.

The protein profile of EV varies depending on its cellular origin.

Specific CD markers used to characterize platelets are the surface markers CD9, CD41b, CD42a, CD42b and CD61 that appear on the platelet surface prior to activation.

There are also markers that appear on the surface of platelets during activation, such as PAC-1, CD62P, CD31, CD63 and intrinsic protein (Syntenin).

Exosomes from platelets or endothelial cells can be identified, for example, by expression of typical surface markers (e.g., CD31 ═ platelet endothelial cell adhesion molecule-1 or CD62P ═ P-selectin). These markers correspond to surface markers on the secretory cell entity. Plasma EV may be derived from different cell subsets and may therefore also comprise different subsets of markers.

Another point is that hPL contains EV released from live thrombocytes during the storage period of the thrombocyte concentrate (20 ℃ to 24 ℃) before reaching the expiration date after 4 to 6 days. Thus, hPL contains a large portion of EV derived from human platelets. Platelet lysate produced from expired thrombocyte concentrate is highly enriched in platelet EV compared to normal plasma preparations. At the time point of subsequent lysis with treatment, all soluble paracrine factors of the concentrate are retained while removing cells and other cellular components.

EV is used as a vector for a variety of biomolecules, including lipids, proteins (e.g., transcription factors, cytokines, growth factors), and nucleic acids (e.g., mRNA, micro-rna (mirna), or even small amounts of DNA).

The lipid component of exosomes includes membrane lipids such as sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, ganglioside (GM3), phosphatidylinositol, prostaglandins, and lysodiphosphatidic acid (lysobisphosphaydicacid).

In addition, exosomes may contain nucleic acids, including mRNA, miRNA, and a variety of other small non-coding RNA species, in addition to lipids and proteins, including RNA transcripts that overlap with protein coding regions, repeat sequences, structural RNA, tRNA, rRNA, vault RNA (vault RNA), Y RNA, and short DNA sequences of small interfering RNA (sirna), as well as mitochondrial DNA and retrotransposons (retrotransposons).

The term "nucleic acid" as used herein generally comprises polyribonucleotides (RNA) and polydeoxyribonucleotides (DNA), or mixtures thereof, including hybrid molecules, each in single-and/or double-stranded form, linear or circular. The RNA may include, but is not limited to, messenger RNA (mrna), non-coding RNA (nc-RNA, including antisense RNA), silencing RNA, microrna (mirna), short hairpin RNA (shrna), small interfering RNA (sirna), repeat-associated small interfering RNA (rasirna), ptwi interacting RNA (pirna), Y RNA, long non-coding RNA (long ncRNA, lncRNA), transfer RNA (trna), ribosomal RNA (rrna), small nuclear RNA (snrna), small nucleolar ribonucleic acid (snoRNA), splicing leader RNA (sl RNA). All of the above RNAs are in principle contemplated as EV components and may be utilized in the methods of the present invention.

EV also includes proteins and peptides. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" refers to a protein or peptide that typically contains or consists of at least 20 (and preferably at least 30, e.g., at least 50) amino acids. The term "peptide" refers to an oligomer containing or consisting of at least 2 amino acids to about 19 amino acids.

The EV proteins most commonly recognized are membrane transporters and fusion proteins (e.g., gtpases such as Rab5, annexin and raft protein (flotillin)), heat shock proteins (e.g., HSC70), tetraspanin (e.g., CD9, CD63 and CD81), proteins from MVB-biogenesis (e.g., Alix and TSG101), lipid-associated proteins and phospholipases, cytoskeletal proteins (actin, filaggrin-1 (cofilin-1), ezrin/rootin/moesin (ezrin/radixin/moesin), assembly inhibitory protein-1 (profilein-1) and tubulin), metabolic enzymes and ribosomal proteins. Several proteins are considered as exosome markers, of which tetraspanin (e.g. CD63, CD81) and TSG101 (proteins of the ESCRT complex) are the most commonly used detection markers.

While the latter is often used as a marker for exosomes, it may not be unique to exosomes and may be found on other extracellular vesicles.

In the context of the present invention, hPL comprises any human platelet lysate that may be useful in therapeutic, diagnostic or cosmetic applications. Preferably, it is hPL produced according to GMP conditions, and preferably it is manufactured according to german pharmaceutical Act (AMG). The source of hPL can be derived from platelets from a single donor donation or pooled donor donations. hPL derived from donors of a particular age (e.g., from donors 10 to 60 years old, 18 to 50 years old, 18 to 40 years old, 18 to 30 years old, or 18 to 20 years old) may preferably be used. In the context of precise medicine, it may be advantageous to treat it with human platelet lysate from a patient's own blood donation or a fraction enriched in human platelet lysate-derived extracellular vesicles. According to a preferred embodiment of the invention, the hPL is derived from a healthy individual.

To avoid misunderstandings, hPL is generally enriched in human platelet-derived extracellular vesicles compared to plasma EV prior to any EV enrichment, isolation or purification process. In the context of the present invention, the term "fraction enriched in human platelet lysate derived extracellular vesicles" means that hPL is processed by at least one step in an EV enrichment, isolation or purification process. After this enrichment, isolation or purification step, the "fraction enriched in human platelet lysate-derived extracellular vesicles" contains less non-EV contaminants.

The hPL or hPL enriched fraction may be obtained from platelets that have been incubated prior to lysis with one or more compounds known as thrombocyte activators from the prior art (e.g. related to PRP treatment) that activate the platelets and thus improve the quality of the preparation of the invention.

According to one embodiment of the invention, the hPL may be derived from human umbilical cord blood. Human umbilical cord platelet lysate preparations are known in the art (e.g., US 8501170B 2; Parazzi, v., c. lavazza, et al (2015); or Forte, d., m. cicerello, et al (2015)).

For the purposes of the present invention, the term "isolation" in all its grammatical forms relates to the act of separating or recovering EV from its environment (e.g., a serum or plasma sample). The term "purification" in all grammatical forms relates to the act of (substantially) reducing/depleting (non-EV) contaminants from the desired EV. The term "enriched" in all grammatical forms means to increase the proportion of EV in its respective solvent.

The hPL or hPLEV enriched fractions described herein are useful in medicine. According to one embodiment of the invention the hPL or the hPLEV enriched fraction is used for the prevention and/or treatment of inflammation driven diseases (inflammation driven disease). Inflammatory disorders comprise a large group of disorders underlying a variety of human diseases. The immune system is often involved in inflammatory disorders that exhibit both allergy and some myopathies, many of which cause abnormal inflammation. Non-immune diseases whose etiology originates in inflammatory processes include cancer, arteriosclerosis, and ischemic heart disease. Many proteins are involved in inflammatory processes. Any of which may be modified by genetic mutation, resulting in impaired normal protein function or protein expression or dysregulation. Some examples of inflammation-related disorders include: acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, and interstitial cystitis. Many diseases are thought to be accompanied by inflammation or classified as autoimmune diseases. Some different types of inflammatory diseases include: gout, lupus, asthma, pleuritis, eczema, arthritis, gastritis, splenitis, sinusitis, hepatitis, nephritis, psoriasis, vasculitis, laryngitis, thyroiditis, prostatitis, pharyngitis, sarcoidosis, atherosclerosis, allergy, multiple sclerosis, certain myopathies, rheumatoid arthritis, seborrheic dermatitis, Wegener's granulomatosis, irritable bowel syndrome (IBS; Crohn's disease), ulcerative colitis, diverticulitis.

According to one embodiment of the invention, the hPL or hPLEV enriched fraction is used for the prevention and/or treatment of neurodegenerative diseases such as, but not limited to, Alzheimer's Disease, amyotrophic lateral sclerosis, corticobasal degeneration, frontotemporal dementia, HIV-associated cognitive impairment, Huntington's Disease, Lewy body dementia, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy and vascular dementia.

According to one embodiment of the invention, the hPL or hPLEV enriched fraction is used for the prevention and/or treatment of immune/autoimmune diseases, such as, but not limited to, Addison disease, celiac disease-sprue (spurue) (gluten-sensitive enteropathy), dermatomyositis, graves disease, hashimoto's thyroiditis (hashimoto ' thyroiditis), multiple sclerosis, myasthenia gravis, pernicious anemia, reactive arthritis, rheumatoid arthritis, sjogren's syndrome (r) ((r))syndrome), systemic lupus erythematosus, and type I diabetes.

According to one embodiment of the invention the hPL or hPLEV enriched fraction is used for the prevention and/or treatment of cardiovascular diseases such as, but not limited to, coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism.

According to one embodiment of the invention the hPL or hPLEV enriched fraction is used for the prevention and/or treatment of skin diseases such as, but not limited to, acne, eczema (atopic eczema), nail fungal infections, herpes and psoriasis.

According to one embodiment of the invention, hPL or the hPLEV enriched fraction is used for the prevention and/or treatment of orthopaedic diseases such as, but not limited to, rheumatoid arthritis, bursitis, elbow pain and problems, elbow syndrome, lateral epicondylitis (tennis elbow), medial epicondylitis (golfer's elbow or baseball elbow), fibromyalgia, foot pain and problems, bone fractures, hip fractures, lower back pain, hand pain and problems, carpal tunnel syndrome, knee pain and problems, knee ligament injuries, meniscus tears, kyphosis, neck pain and problems, osteoporosis, paget's disease of the bone, scoliosis, shoulder pain and problems, soft tissue injuries.

According to one embodiment of the invention the hPL or hPLEV enriched fraction is used for prophylaxis and/or therapy in tissue regeneration medicine. Tissue engineering evolved from the field of biomaterial development and refers to the practice of combining scaffolds, cells and bioactive molecules into functional tissues. The goal of tissue engineering is to assemble a functional structure that restores, maintains, or improves damaged tissue or whole organs. Artificial skin and cartilage are examples of engineered tissues that have been approved by the FDA, however, their use in human patients is currently limited. The field of regenerative medicine is extensive, including tissue engineering, but also incorporates studies on self-healing, where the body uses its own system to regenerate cells and reconstruct tissues and organs, sometimes supported by foreign/allogeneic biomaterials. The terms "tissue engineering" and "regenerative medicine" are largely interchangeable, as the art wishes to focus on the cure rather than the treatment of complex and often chronic diseases.

According to another embodiment of the invention, hPL or hPLEV enriched fractions are used for the prevention and/or treatment of tumor diseases such as, but not limited to, bladder cancer, bone cancer, breast cancer, colon/rectal cancer, Hodgkin's disease, leukemia, liver cancer, lung cancer, cutaneous lymphoma, multiple myeloma, nasopharyngeal cancer, Non-Hodgkin's lymphoma (Non-Hodgkin's lymphoma), osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma-adult soft tissue cancer, skin cancer, small intestine cancer and gastric cancer.

According to another embodiment of the invention the hPL or hPLEV enriched fraction is used for the prevention and/or treatment of transplant rejection. According to another embodiment of the invention, the hPL or hPLEV enriched fraction is used for the prevention and/or treatment of stroke, ischemia or graft versus host disease.

According to a preferred embodiment, the disease may be selected from the group of diseases currently treated by cell therapy, such as, but not limited to, allogeneic cell therapy, human embryonic stem cell therapy, neural stem cell therapy, application of mesenchymal stem cell therapy and hematopoietic stem cell transplantation application.

Allogeneic cell therapy attempts to develop products to treat conditions including crohn's disease and various vascular diseases. Human embryonic stem cell research has been used as the basis for many therapeutic applications, including possible treatments for diabetes and Parkinson's disease. Among neural stem cell therapies, Neural Stem Cells (NSCs) are the subject of ongoing research into possible therapeutic applications, such as the treatment of many neurological disorders, such as parkinson's disease and huntington's disease. Mesenchymal stem cell therapy is used in a wide variety of therapies, including immunomodulatory therapies, bone and cartilage regeneration, cardiac muscle regeneration, and the treatment of Hurler syndrome, skeletal and neurological disorders.

According to another embodiment of the invention the hPL or hPLEV enriched fraction is useful for the prevention or treatment of infectious diseases, in particular caused by bacteria, viruses, fungi or parasites. A preferred embodiment is the treatment of infectious diseases in dermatology, such as cellulitis, erysipelas, hidradenitis suppurativa, impetigo and ecthyma, lymphadenitis, lymphangitis, necrotic skin infections or staphylococcal scalded skin syndrome.

Another preferred application of the preparation of the invention is in the context of indications in which platelet therapy or platelet rich plasma therapy has a positive effect as described in the prior art. It is known from the prior art that the important immune and inflammation-related functions of platelets are increasingly appreciated, both in health and disease. Many studies have shown that platelets affect inflammatory processes ranging from atherosclerosis to infectious diseases, making platelets the most abundant circulating cell type with immune function. Platelets interact with leukocytes and vascular endothelial cells directly by contact-dependent mechanisms and indirectly by mechanisms of secreted immune mediators. Thus, platelet-mediated immunity occurs locally at the site of platelet activation and deposition, or systemically at a location remote from the platelet activation itself.

Platelets are well known as a cellular mediator of thrombosis (see Craig N. Morrell et al 2014, "Emergingrolls for platelets as immunes and platelet cells," Blood: 123 (18)). In this article, it is described that the important immunological and inflammatory roles of platelets in both health and disease are now increasingly recognized. Many studies have shown that platelets affect inflammatory processes ranging from atherosclerosis to infectious diseases, making platelets the most abundant circulating cell type with immune function. Platelets interact with leukocytes and vascular endothelial cells directly by contact-dependent mechanisms and indirectly by mechanisms driven by secreted immune mediators. Thus, platelet immunity is noted locally at the site of platelet activation and deposition or systemically at a location remote from the platelet activation itself. Morall et al concluded that platelet interactions with inflammatory cells can mediate pro-inflammatory outcomes, but that these interactions may have evolved to be beneficial in limiting infection. For example, for a breach in the skin, which is exposed to a pathogen, and by combining a thrombotic function with an immune recruitment function, platelets can help with focal hemostasis and an immune response against potential infectious agents to prevent pathogen invasion. However, sustained or long-term interaction of platelets with leukocytes or endothelial cells can lead to adverse effects from excessive immune stimulation and inflammatory injury, see Craig n. It is known from the prior art that platelets have a positive effect in the context of axonal regeneration, wound healing and pain reduction (Kuffler DP et al, Mol Neurobiol.2015 10 months; 52 (2): 990-.

According to the present invention, it has been found that by using hPL or hPLEV instead of live platelets, the important immunological and inflammatory effects of platelets can be partially replaced and the regeneration properties can be improved.

Some preferred indications therefore include wound healing, tissue regeneration, nerve damage, tendonitis, osteoarthritis, myocardial damage, bone repair and regeneration, and orthopedic and oral surgery.

According to another embodiment of the invention, the preparation is cell-free. In the context of the present invention, cell-free means that the preparation is substantially free of living intact cells and cell debris. The hPLEV-fraction of the invention is preferably a cell-free preparation, which is enriched for EV, while the other components are reduced.

According to another embodiment of the invention the hPL or hPLEV enriched fraction is an essential pharmaceutical active ingredient in the formulation.

By essential pharmaceutically active ingredient is meant hPL or a hPLEV enriched fraction which is of therapeutic or other value when administered to a human. Essential pharmaceutically active ingredient means also that the preparation is substantially free of other pharmaceutically active ingredients than human platelet lysate, hPL-fraction or a fraction enriched in human platelet-derived extracellular vesicles.

In addition to the human platelet lysate or the fraction enriched in human platelet lysate derived extracellular vesicles, the preparation according to the invention may also comprise one or more excipients. Such excipients may be natural or synthetic substances formulated with the active ingredient, for example to increase the volume of solid formulations containing potent active ingredients (hence the name "bulking agents", "fillers" or "diluents") for long-term stabilization, or to impart therapeutic enhancements to the active ingredient in the final dosage form, for example to promote drug absorption, reduce viscosity or increase solubility. Excipients may also be used in the manufacturing process to aid in handling the active substance concerned, for example by promoting powder flow ability or non-stick properties; in addition, it may also contribute to in vitro stability, for example to prevent denaturation or aggregation during the expected shelf life. The choice of suitable excipients also depends on the route of administration and the dosage form, as well as the active ingredient and other factors.

According to another embodiment of the invention, the size of the extracellular vesicles in the fraction enriched in human platelet lysate-derived extracellular vesicles (exosomes) is about 70 to 200nm, preferably about 70 to 140nm, or more preferably about 70 to 120 nm. "about" shall mean a deviation of +/-20%. It is also preferred that exosomes are positive for characteristic EV-or exosome-markers, and it is even further preferred that the protein content of the pharmaceutical formulation is higher than 0.5mg/ml, preferably higher than 1 mg/ml.

hPLEV markers include heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD10, CD26, CD53, CD63, CD82), proteins from MVB-biogenesis (e.g., Alix and TSG101), EpCAM or Rab5, but substantially lacking CD81 (which is typically a general EV surface marker), CD2, CD8, CD11a, and CD25(Koliha et al, 2016).

According to another embodiment of the invention, the extracellular vesicles are positive for at least one of the following EV markers or exosome markers: CD9, CD41a, CD41b, CD42b, CD61, CD62P, and CD 63. Preferably, the extracellular vesicles are positive for 2, 3, 4, 5, 6 or 7 of the EV markers or exosome markers described above.

According to the present invention, positive means that the background mediator fluorescence intensity of one of the above-mentioned positive surface markers is higher than that of the comparative example of extracellular vesicles derived from NK cells when the bead-based multiplex platform assay was performed according to the method described by Koliha, Nina et al (2016).

Alternatively, other techniques for specific protein detection of the above markers may be applied, such as Western blot analysis.

According to another embodiment of the invention, the extracellular vesicles are negative for at least one of the following surface markers: CD81, CD3, CD4, CD19, CD20, CD2, CD8, CD11a, and CD 25. Preferably, the extracellular vesicles are negative for 2, 3, 4, 5, 6, 7, 8 or 9 of the above-described extracellular exosome markers.

To demonstrate the quality of the hPLEV-enriched fraction, several general characterization criteria regarding the biological and biophysical properties of the included EVs should be met. The prior art for such characterization is the guidelines and standards recommended by the International Society for Extracellular Vehicles (ISEV). Based on recent scientific knowledge in the field of EVs, these criteria apply to attributing any specific bioburden or function to an EV.

Basic quality criteria/standard:

1. protein content [ mg/ml ] was determined by standard protein quantification methods using BCA assays, similar assays such as Bradford-assays, or instruments based on spectroscopic assays (e.g., "NanoDrop").

2. The mean particle size [ nm ] and size distribution [ curve ] were determined using Nanoparticle Tracking Analysis (NTA) platforms (e.g. Nanosight and Zetaview) or image flow cytometry (e.g. "arnis", which allows Analysis of EV at the single particle level).

3. Semi-quantitative detection of typical EV marker proteins including EV-or exosome proteins (SDS-PAGE, Western blot, detection with specific antibodies, signal detection). Generally, EV content is highly dependent on the cell source, pretreatment and preparation method to generate the cell line. However, EVs (e.g., exosomes) provide a common set of commonly expected markers that can be used for their characterization. The most commonly used markers are tetraspanin (CD9, CD63, CD81), endosome derived or membrane bound proteins (TSG101, annexin, Rab, endostatin, raft) and chaperones (HSC70, HSP 90). In the case of PL-EV, characteristically, it lacks CD 81.

4. Purity was determined by semi-quantitative comparison of cell lysates and PL-EV fractions. The PL-EV fraction should be free of cell residues, such as proteins from the endoplasmic reticulum (e.g. Grp94, calnexin), golgi (e.g. GM130) or mitochondria (e.g. antiproliferative protein, cytochrome C). Such markers may be used as negative markers. In addition, nucleoproteins (histones, argonaute/RISC complexes) may be used as an example of a negative control.

For 3+ 4: analytical methods for detecting typical EV markers may include Western Blotting (WB), high resolution flow cytometry or whole proteomic analysis using mass spectrometry techniques. Additional characterization of PL-EV enriched fractions was based on the following method:

proteomics

Analytical methods for protein mass spectrometry analysis of hPLEV, including Western Blotting (WB), (high resolution) flow cytometry or whole proteomic analysis using mass spectrometry, are also prior art for characterizing other EV markers, such as immune markers, signaling markers, cytokines and other related bioactive protein content.

Microarray analysis of RNA from hPLEV

For profiling of hPLEV RNA, microarray technology can be applied. Microarrays are well-established techniques for analyzing the expression of known nucleic acid fragments using slide-or chip-based media. Microarrays can be used to screen for mRNA, miRNA, and long non-coding rna (lncrna) species.

Lipidomics

Lipids and lipid raft-associated proteins in the vesicle membrane provide extracellular vesicles with stability and structural integrity. The hPL-EV should have a similar lipid composition compared to the cell from which it was derived.

PL-EV may be rich in phosphatidylserine, di-saturated phosphatidylethanolamine, di-saturated phosphatidylcholine, sphingomyelin, gangliosides, and cholesterol. To determine lipid composition and ratio, mass spectrometry, flow cytometry, or other conventional assays can be used.

Cytokine array

Cytokine analysis of EV-enriched fractions based on ELISA assays

The cytokine profile of the hPLEV-containing fraction can be semi-quantitatively analyzed by using, for example, commercially available membrane-based cytokine arrays. Cytokines include chemokines, tumor necrosis factors, interleukins, interferons and colony stimulating factors. This technique provides a very sensitive method to detect many different defined proteins (e.g. 200 cytokines) in parallel. Amounts of protein of only a few picograms can be detected. The assay is based on a membrane to which an immobilized specific primary antibody is bound. Cytokines contained in the surrounding liquid bind to these primary antibodies during incubation. In a subsequent reaction, a so-called "sandwich complex" is formed, in which a biotin-bound secondary antibody is bound to a primary antibody. As a result, the antibody-cytokine-complex is biotin-labeled. The HRP-bound streptavidin or other marker molecule can bind to biotin and thereby make the complex detectable by chemiluminescence, calorimetry (calometrie) or IR light. The detected signal can be compared to signals from known standards, and in the case of chemiluminescence, a density measurement of the signal can be made to compare the proteins being analyzed.

Functional in vitro assay

In vitro assay for analyzing the effects of hPLEV on immune cells

The potential immunomodulatory capacity of the enriched EV fraction should be determined in at least one functional in vitro assay using cells from the human immune system (e.g. PBMCs). The principle of such assays is to analyze the immunomodulatory effects on immune cells in the presence or absence of hPLEV. For this problem, flow cytometry analysis was used. To induce an immune response, cells are stimulated by the addition of e.g. PHA (phytohemagglutinin), PMA (phorbol myristate acetate), ionomycin, monoclonal antibodies, antigens such as candida (Candida) or bacterial proteins or other possible components even from commercially available activation kits. In addition, a method such as mixed-lymphocyte reaction (MLR) may be applicable. Activation can be induced non-specifically (using e.g. PHA) on all PBMCs, or specifically (e.g. selectively) only on T cells or other defined subpopulations of PBMCs. By stimulation, immune cells are activated, which can be detected, inter alia, by changes in the expression profile of cell surface markers. Subsequently, if the stimulation is sufficiently strong, increased proliferative activity (e.g. in the case of T cells) may also be detected. In the presence of the hPLEV-enriched fraction, differences from cell controls without EV (e.g. inhibition of T cell proliferation and expression of activation markers) should be detectable.

Examples of such assays: "PBMC-CFSE-PHA-assay":

the "PBMC-CFSE-PHA-assay" can be used to analyze PHA-induced cell proliferation in the presence of hPLEV-enriched fractions. CFSE (carboxyfluorescein-succinimidyl ester) is a fluorescent dye that can be used for cell tracking using flow cytometry for analysis. The precursor molecule CFSE-SE (carboxyfluorescein-diacetate-succinimidyl ester) passively diffuses into the cell, is cleaved by intracellular enzymes and can be detected by fluorescence. The fluorescence intensity of the proliferating CFSE labeled cells decayed 50% due to each cell division.

Isolated CFSE stained PBMCs were cultured in RPMI media + 10% human AB-serum in 24-well plates for suspension cells for 5 days in the presence or absence of EV. Cell stimulation was induced directly by 200 to 300ng PHA per well (day 0) after the start of culture. The culture volume per well was 400. mu.l of 200000 cells. After 5 days, the cells can be analyzed by flow cytometry. The decrease in fluorescence compared to day 0 indicates a high proliferation rate, and the stable fluorescence indicates inhibition of proliferation due to EV action. In addition, expression of activation markers can also be analyzed at different time points. By staining with specifically conjugated antibodies, subpopulations of immune cells can be differentiated and analyzed individually.

In vitro assay to analyze the effect of hPLEV on angiogenesis:

angiogenesis is a fundamental process in all stages of growth and development, as well as in wound healing and tissue regeneration in ischemic vascular disease. During angiogenesis, new capillary blood vessels are produced by the pre-existing vasculature, and the process is controlled by a sensitive balance of pro-and anti-angiogenic factors. Endothelial cells are activated in response to angiogenic stimuli (e.g., injury, inflammation, and hypoxia).

Tube sample formation assay

One of the most widely used in vitro assays for mimicking the recombination phase of angiogenesis is the tube formation assay. This assay measures the ability of endothelial cells to form capillary-like structures (tubes). Tube formation is usually quantified by measuring the number, length or area of these capillary-like structures in a two-dimensional microscope image of the culture dish.

Wound healing assay

The abrasion assay is used to measure cell migration in vitro. The basic steps include creating "bruises" in a cell monolayer of a homogenous population, capturing images at regular intervals at the beginning and during cell migration to close the bruises, and comparing the images with life imaging microscopy to quantify the mobility (migration rate) of the cells.

These assays can be used to study the effect of hPL-EV on angiogenesis on cell-cell interactions and cell migration that mimics cell migration during wound healing in vivo.

Basic safety guidelines/criteria for the use of hPLEV in the clinic:

in general, the products must be addressed (a) whether autologous, allogeneic or xenogeneic, (b) whether widely or minimally manipulated in vitro, and (c) whether immunologically active or neutral.

In general, the hPL according to the invention is derived from any conceivable human blood sample comprising platelets. According to another embodiment of the invention, the human platelet lysate is derived from a platelet concentrate, such as the so-called Platelet Rich Plasma (PRP). PRP is a platelet concentrate in plasma obtained from patient whole blood by centrifugation to remove red blood cells and other unwanted components. It has a higher concentration of growth factors than whole blood and has been used as a tissue injection in a number of disciplines including dentistry, orthopedic surgery and sports medicine. The platelet concentrate may for example originate from a buffy-coat (buffy-coat) extracted platelet concentrate or from platelet apheresis (platelet apheresis).

According to the invention, the extracellular vesicles may comprise biological agents, such as genetic material, e.g. mRNA, micro rna (mirna), small amounts of DNA; a lipid; and proteins, including transcription factors, cytokines, and growth factors.

The pharmaceutical preparation of the invention is derived from platelets, which typically comprise a plurality of growth factors, in particular one or more, preferably all, of PDGF, VEGF, FGF, EGF, TGF, in particular TGF- β and CTGF. the composition preferably comprises 2, 3, 4, 5 or 6 of these growth factors, platelet-derived growth factors (PDGF) promoting cell growth and production, vascular repair and collagen production Vascular Endothelial Growth Factors (VEGF) promoting growth and production of vascular endothelial cells fibroblast growth factors (fibroblast growth factors, FGF) promoting tissue repair, cell growth, collagen production and hyaluronic acid production epithelial growth factors (epidermal growth factors, EGF) promoting epithelial cell growth, angiogenesis and wound healing factors (transforming growth factors (PDGF) and collagen growth factors (TGF-growth factors) promoting the growth, collagen production and collagen production, and collagen regeneration factors (TGF-fibroblast growth factors) promoting the growth, gf and collagen regeneration factors (TGF-growth factors) and restoring the appearance of the skin, collagen regeneration, collagen.

According to another embodiment, the preparations of the invention (e.g.the hPLEV-enriched fractions of the invention) comprise biological factors, such as proteins, cytokines (e.g.IFN-. gamma., IL-8, IL-10, TGF- β 1 and HLA-G, RANTES, Nap-2) and/or nucleic acids, such as microRNAs.

The preparation according to the invention may contain cytokines with antimicrobial properties. The preparations according to the invention may in particular comprise a high amount of the cytokine RANTES or the cytokine NAP-2 or both cytokines, compared with the levels of other cytokines present.

The antimicrobial properties of the cytokines RANTES and NAP-2 have been described, for example, in article Mariani et al (2015) (BMC Microbiology 15: 149). The antimicrobial properties of the formulations according to the present invention can be measured according to the method shown in fig. 2 to 7 of Mariani et al and this method is used as a reference method.

In the context of the present invention, it is contemplated that the formulation is suitable for, for example, intravenous administration or infusion, or for intraperitoneal injection, subcutaneous injection, intraosseous injection, intracerebroventricular injection, intramuscular injection, intraocular injection, or for topical administration (topicatedadministration).

The invention also relates to a pharmaceutical preparation comprising an enriched fraction of human platelet-derived extracellular vesicles, obtainable by a process comprising the steps of:

a) providing human platelet lysate from platelets from a single donor donation or from pooled donor donations of at least 15 donors (preferably at least 20 or at least 30 or at least 40 donors);

b) enriching extracellular vesicles derived from human platelet lysate;

c) optionally, the in vitro effects of said enriched extracellular vesicles, such as immunomodulating, in particular anti-inflammatory and/or immunosuppressive effects, are determined by e.g. reduced IL-1 β, TNF- α, T-cell proliferation, and

d) optionally, selecting those enriched extracellular vesicle fractions which exhibit said in vitro effect (e.g. an immunomodulatory effect, in particular an anti-inflammatory effect and/or an immunosuppressive effect);

e) optionally, selecting those enriched extracellular vesicles in step b) that exhibit extracellular vesicles negative for EV/exosome marker CD81 and positive for EV/exosome marker CD 9; and

f) optionally, mixing said enriched extracellular vesicles of step b), d) or e) with at least one suitable pharmaceutically acceptable excipient and/or carrier.

According to one embodiment of the invention, the hPL of step a may be derived from platelets that have been incubated prior to lysis with one or more compounds known as thrombocyte activators from the prior art (e.g. related to PRP treatment) that activate the platelets and thus improve the quality of the preparation of the invention.

According to one embodiment of the invention, the hPL of step a may be derived from human umbilical cord blood. Human umbilical cord platelet lysate preparations are known in the art (e.g., US 8501170B 2; Parazzi, v., c. lavazza, et al (2015); or Forte, d., m. cicerello, et al (2015)).

In step a) of the method of the invention as defined above, human platelet lysate derived from platelets from a single donor donation or from pooled donor donations of at least 15 donors (preferably at least 20 or at least 30 or at least 40 donors) should be used.

If precise medication is desired, it may be advantageous to use human platelet lysate derived from the patient's own blood. If a general treatment is desired, it is advantageous to use a pool of at least 15 donors (preferably at least 20 or at least 30 or at least 40 donors) to avoid possible individual deviations of the human platelet lysate compared to human platelet lysate derived from a pool of at least 40 donors.

It may be preferred that the hPL from method step a) as described above is provided by platelet apheresis or by buffy coat platelets, more preferably by platelet apheresis.

The immune modulation mediated by the hPLEV-enriched fraction may be an immunosuppressive effect, which can be detected with the following in vitro assay and corresponding readout. The hPLEV fraction was observed to have the ability to inhibit the proliferation of immune cells and is therefore immunosuppressive. Inhibition of proliferation of stimulated PBMC can be observed in such in vitro assays, as can be observed for subpopulations such as CD3 positive cells (T cells) and CD3 negative cells (including e.g. B cells, NK cells). In addition, there was an inhibitory effect on the expression of the T cell activation marker profile in the presence of the hPLEV fraction (down-regulation of CD69 or CD 25).

The method for producing a pharmaceutical formulation according to the invention comprises a step of specific enrichment of EV. As previously described, EV is found abundant in many body tissues and fluids, and has been successfully purified using differential ultracentrifugation (Raposo, G. et al J.exp.Med.1996; 183 (3): 1161-. Further studies have shown that EV can be isolated using ultracentrifugation in a continuous density gradient of sucrose (Escoria JM et al, J Biol chem.1998, 8.7.v.; 273 (32): 20121-7). Exosomes have also been isolated by immunoaffinity capture methods using lectins or antibodies against common exosome markers (e.g., CD63, CD81, EpCAM or Rab5) (Barr < s C et al, blood.2010, 1/21; 115 (3): 696-.

In general, any suitable purification and/or enrichment method may be used, including, for example, the following: PEG precipitation, monolithic techniques, magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick^TM(Systems Biosciences, CA, USA), chromatography or tangential flow filtration. However, it is important to remember that, depending on the isolation method, different EV subtypes can be enriched and their functional properties can differ even if they originate from the same cell type.

Nevertheless, a method involving precipitation of polyethylene glycol is preferred.

To produce a pharmaceutical formulation according to the invention, a method is preferred wherein the EV enriched fraction is further analyzed in microbiological tests, virulence tests, protein content, pyrogen tests and particle size to determine the most suitable fraction according to the invention.

It was found that the hPLEV enriched fraction is particularly useful in the method according to the invention if it shows a strong immunomodulatory effect in an in vitro activity assay.

It has also been found that the fraction enriched in hPLEV is particularly useful in the method according to the invention if it shows a reduced IL-1 β, TNF- α and/or IFN-gamma cytokine response of the donor effector cells.

Also preferred is a process according to the invention wherein the exosomes have a size of about 70 to 200nm, preferably about 70 to 140nm, or more preferably about 70 to 120 nm. "about" shall mean a deviation of +/-20%. It is also preferred that exosomes are positive for EV/exosome markers, and even further preferred that the protein content of the pharmaceutical formulation is higher than 0.5rng/ml and also preferably higher than 1mg/ml (depending on the final resuspension/elution volume and the initial PL volume processed).

The hPLEV-enriched fraction was found to be particularly useful in the method according to the invention if it shows a strong in vitro immunomodulatory effect in activity assays and a reduced IL-1 β, TNF- α and/or IFN- γ cytokine response of donor effector cells can be found after addition of the EV fraction ELISpot assays show that IL-1 β, TNF- α and/or IFN- γ cytokine responses of effector cells are impaired against allogeneic cells in the presence of an exosome-containing fraction.

The present invention is therefore based on a new concept for improving disease prevention and treatment, in particular in patients suffering from risk of inflammation driven diseases, neurodegenerative diseases, immune/autoimmune diseases, cardiovascular diseases, skin diseases, transplant rejection, GvHD, stroke, and ischemia and related complications, e.g. for avoiding pre-or during surgery inflammatory reactions, and for preventing inflammatory conditions and reactions in patients connected to life support machinery. In one embodiment, the disease may be selected from prenatal or postnatal nervous system injury, such as brain injury associated with hypoxia, inflammation and/or ischemia. In another embodiment, the diseases may be selected from graft versus host disease, or transplant rejection after organ transplantation, respectively.

In a particularly preferred embodiment of the invention, the EV enriched fraction derived from hPL enriched using a polyethylene glycol precipitation protocol is administered prophylactically and/or therapeutically to a patient, in particular a neonate and/or a patient receiving a transplant and/or a patient undergoing surgery.

The pharmaceutical preparation according to the invention is preferably enriched with EVs comprising biological factors such as proteins (e.g. anti-inflammatory cytokines, IL-10, TGF- β 1 and HLA-G), and/or nucleic acids (e.g. mirnas) which achieves the further advantages according to the invention of a) performing multimodal (complex) interventions, b) using biophysical ("self") substances, and c) reducing unwanted side effects of the preparation.

The present invention constitutes multi-modal intervention. Thus, not only are specific factors used (and only interfere with a portion of the cascade (or underlying clinical phenotype)), but also biologically complex and endogenous mediators and regulators are used. These components are present in every human and therefore no significant adverse side effects are expected.

Another aspect of the invention relates to a method for producing a pharmaceutical preparation according to the invention comprising the steps of a) providing hPL, b) enriching said hPL for hPLeV, optionally including polyethylene glycol precipitation, c) determining the in vitro immunomodulatory, in particular anti-inflammatory and/or immunosuppressive effects of said hPLeV enriched fraction by e.g. reduced IL-1 β, TNF- α, T-cell proliferation and/or IFN-gamma cytokine response of donor effector cells, d) selecting those hPLeV enriched fractions exhibiting immunomodulatory, in particular anti-inflammatory and/or immunosuppressive effects, and e) mixing said enriched exosomes of step d) with at least one suitable pharmaceutical excipient and/or carrier.

According to another embodiment of the invention, the administration is suitable for e.g. intravenous administration or infusion, or for intraperitoneal injection, subcutaneous injection, intraosseous injection, intracerebroventricular injection, intramuscular injection, intraocular injection, or for topical administration. Topical application may be applied, for example, by cosmetic skin products or patches (patches), wound pads, and the like, pre-formed or pre-treated or provided with the formulations of the present invention.

Another aspect of the invention is a method for preparing a pharmaceutical or diagnostic or cosmetic formulation comprising the steps of: adding a human platelet lysate or a fraction enriched in human platelet lysate derived extracellular vesicles to a pharmaceutical or diagnostic or cosmetic preparation.

The present invention includes within its scope formulations comprising a therapeutically effective amount of hPL or hPLEV fractions as active ingredients, either alone or in combination with a pharmaceutically acceptable carrier or diluent. The skilled person can select a suitable carrier depending on the desired dosage form. Dosage forms include, for example, tablets, granules, capsules, liquid dosage forms, gels, suppositories, creams, ointments, poultices or patches. A preferred embodiment is the combination of the hPLEV fraction with a suitable polymer matrix, e.g. as described in WO 2013076507. Also preferred is intravenous administration in a 0.9% NaCl solution.

Cosmetic applications of the formulations of the present invention may be formulated with suitable excipients. Generally, the human platelet lysate or the fraction enriched in human platelet lysate derived extracellular vesicles according to the invention may replace PRP when applied to cosmetics. PRP treatment has been applied in many different medical fields, such as cosmetic surgery, dentistry, sports medicine and pain management. PRP has become a highly popular non-surgical procedure, for example, for facial and skin rejuvenation procedures. PRP treatment is a treatment that uses the donor's own platelets to stimulate new cell growth, help improve skin tone, skin texture, and restore lost facial volume.

According to one embodiment of the invention, PRP treatment can be replaced by a cosmetic preparation comprising hPL or hPLEV fractions in place of PRP. Then, autologous hPL or hPLEV fractions from the donor themselves are re-injected into the skin to stimulate collagen and new skin cells. The hPL or hPLEV fraction exploits the beneficial function of the patient's own platelets and thus there is no risk of allergy or rejection of therapy. The hPL or hPLEV fractions can also be successfully used for the treatment of hair thinning and hair loss, especially male pattern baldness. Early initiation of treatment by the patient may be important.

A preferred embodiment of the invention is the use of the hPL or hPLEV fraction in: skin regeneration, for example anti-ageing treatments, sunburn, insect bites, allergies, autoimmune or allergic skin reactions, acne inflammations. The hPL or hPLEV fraction may be part of a cosmetic composition for skin or hair treatment. The present invention provides a regenerative treatment that directly addresses every problem associated with wrinkles and strengthens the skin and underlying scaffold. Treatment with the formulation of the present invention reverses the degenerative cycle of damaged skin to the healthy physiological functions found in normal skin. The formulations of the invention function by rebalancing the cells within the connective tissue, balancing molecular signaling, and restoring the extracellular matrix. The natural healing and tissue regeneration processes result in increased collagen synthesis, regeneration of the collagen extracellular matrix, and proliferation of fibroblasts within the matrix.

Diagnostic use

According to the hPLEV-based invention, in vitro diagnostic tests can be established for diagnostic applications and real-time monitoring of diseases. hPLEV can be used as a diagnostic biomarker for disease by non-invasive blood tests. The specific contents of the individual donor hPLEV preparations can be used as biomarkers for neoplastic diseases or inflammatory disease related diseases.

For a better understanding of the present invention and its advantages, reference is made to the following examples for illustrative purposes only. The examples are not intended to limit the scope of the invention in any way.

Examples

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, DNA recombination, and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, e.g., j.sambrook, e.f. fritsch, and t.maniotis, 1989, Molecular Cloning: ALABORT MANUAL, second edition, volumes 1 to 3, Cold Spring Harbor Laboratory Press; ausubel, F.M. et al (1995 and periodical supplement; Current Protocols in Molecular Biology, chapters 9, 13 and 16, John Wiley & Sons, New York, N.Y.); roe, j.crabtree and a.kahn, 1996, DNAIsolation and Sequencing: essential Techniques, John Wiley & Sons; j.m. polak and James O'd. mcgee, 1990, Oligonucleotide Synthesis: a Practical Approach, IrlPress; D.M.J.Lilley and J.E.Dahlberg, 1992, Methods of Enzymology: DNA structure part a: synthesis and Physical Analysis of DNA Methods in Enzymology, academic Press; use Antibodies: a Laboratory Manual: portable Protocol phase I, Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-; antibodies: a Laboratory Manual, Ed Harbor (eds.), David Lane (eds.) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855; and Lab Ref: a Handbook of drugs, Reagents, and Other references Tools for Use at the bench, Jane Roskams and Linda Rodgers, eds 2002, Cold Spring Harbor Laboratory, JSBN 0-87969-. Each of these general texts is incorporated herein by reference.

Example 1: method for preparing platelet rich plasma

1.1. Platelet concentrates (PRP-PC)

450ml of whole blood was collected in a 450ml triple bag containing CPDA1 anticoagulant (TERUMO PENPO, Ltd. Puliyarakonam, Trivandrum, India). Platelet rich plasma was separated from whole blood by centrifugation through a Heraeus 6000i (germany) refrigerated centrifuge at 1750rpm (1ight spin) for 11 minutes at 21 ℃ with acceleration and deceleration curves of 5 and 4, respectively, and platelets were concentrated by centrifugation through a heavy spin at 3940rpm for 5 minutes at 21 ℃ with acceleration and deceleration curves of 9 and 5, respectively, and subsequent removal of supernatant plasma. The platelet concentrate bag was allowed to stand at room temperature with the label side down for about 1 hour. Platelet poor plasma was rapidly frozen and stored as Fresh Frozen Plasma (FFP) at-30 ℃ or below-30 ℃ for one year.

Method for preparing buffy coat-platelet concentrate (BC-PC)

450ml of whole blood was collected in a 450ml quadruple bag containing 63ml of CPD anticoagulant and additive Solution (SAGM). (TERUMO PENPOL, Ltd., Puliyarakonam, Trivandrum, India). Whole blood was first centrifuged at 3940rpm "hard spin" for 5 minutes at 21 ℃ with acceleration and deceleration curves of 9 and 4, respectively. Whole blood separates into different components according to specific gravity.

Top layer-platelet poor supernatant plasma (150 to 200 ml).

The middle layer, buffy coat, contains about 90% platelets, 70% WBCs and 10% red blood cells.

Bottom layer-packed red blood cells.

The platelet poor supernatant was transferred to one satellite bag (satellite bag) and the buffy coat was transferred to the other satellite bag. Approximately 20 to 30ml of plasma was returned to the buffy coat in order to wash the tubes of residual cells and obtain the appropriate amount of plasma in the BC. SAGM solution was added to red blood cells. The bag containing the red blood cells and plasma is then removed. The red blood cells were placed in a cold chamber at 4 ℃ and the platelet poor plasma was placed as Fresh Frozen Plasma (FFP) in a-40 ℃ deep freezer. The buffy coat was gently mixed with plasma and again "spun" centrifuged at 1,100rpm for 6 minutes at 21 ℃ along an acceleration and deceleration curve of 5 and 4, respectively, along with an empty satellite bag. The supernatant platelet rich plasma was transferred to an empty platelet storage bag and the tube was then sealed. The bag with the residual WBCs and red blood cells was discarded.

1.2. Single Donor Platelet (SDP) -apheresis-PC

The automated cell separator apparatus may be an intermittent flow or continuous flow cell technique using single or double venous access. Automated cell separator using continuous flow, dual venous accessplus，Baxter，Fenwaldivision，Deerfield，1460015，USA。

1. Written consent was obtained from the donors after a detailed explanation of the procedure, the time used and possible risks and benefits.

2. Venous access is an important consideration for apheresis donors, and veins are examined at the time of selection for the following reasons:

long duration of operation

Extended flow rate

Two venipunctures with a continuous flow device are often required.

3. The age of the donor was recorded.

4. Platelet apheresis donors were tested for ABO/Rh typing and infectious disease markers (HIV, HBV, HCV, VDRL and malaria) prior to apheresis procedures.

5. Donors who have taken aspirin or other NSAIDs that may affect platelet function are delayed.

6. Platelet count > 1.5X 10⁵Mu.l of those donors were subjected to thromboapheresis.

1.3. Operation of

This operation is performed in a closed system. The disposable cartridge is mounted to the continuous flow separator and the machine is then started. Donors were prepared by cleaning both venipuncture sites of the antecubital region of both arms with bifida (betadine) and performed a minimally invasive neurophlebotomy (spirit phlebotomy) on the donor. During operation, blood is anticoagulated at the withdrawal point in a controlled manner, and the ratio of whole blood to Anticoagulant (ACD) is maintained at 9: 1 to 11: 1. The anticoagulated blood is pumped into a rotating separation vessel. The red blood cells are concentrated by centrifugal force towards the outer edge of the container and subsequently the red blood cells leave the separation container. The lower density components, such as plasma, platelets, or WBCs, are removed by the plasma pump and passed into a rotating collection container where the platelets are concentrated by centrifugal force toward the outer edge of the container. The separated platelets remain concentrated in the container while the other components of the blood are returned to the donor. At the end of the collection procedure, the platelet collection bag was shaken vigorously to separate the platelets from the bag walls and held at room temperature for 1 hour to make a uniform suspension. This entire operation takes 1.5 to 2.5 hours. The final volume of apheresis-PC is 200 to 300 ml.

Example 2: production of human thrombocyte lysate (hPL)

The production of human thrombocyte lysate (═ human platelet lysate (hPL)) is based on the further processing of expired human thrombocyte concentrate (═ human platelet concentrate (hPC/hTC). to obtain hTC, two different processing methods are generally used, one is defined by the use of pooled buffy coats of whole blood donors from several donors, in this technique, the donor is connected to an extracorporeal cell separator and the thrombocytes are filtered out, while other blood components (e.g., red blood cells, white blood cells, and plasma) are returned to the donor, hi both cases, the resulting blood products are leukocyte depleted TC. these products contain viable thrombocytes for at least 4 to 5 days, and can be used to replace missing thrombocytes, for example in patients suffering from thrombocytopenia.

After the expiration date, too many preparations can be used to produce hPL. By the freeze and thaw cycle (-20 ℃ and RT), the platelets break up/rupture and release their contents into the surrounding liquid (plasma). There are different protocols including a centrifugation step at 3200 to 10000g for about 1 hour. Lysed cells, cell debris and other debris were removed by centrifugation. The result was a clear viscous yellow liquid consisting of platelet lysate and plasma. To sterilize the article after treatment, a 200nm filter may optionally be additionally used. Concurrently, particles larger than 200nm are removed, including extracellular vesicles that do not have exosome size.

Example 3: isolation of peripheral mononuclear cells (PBMC) using Ficoll density gradient centrifugation and in vitro cell culture

Peripheral mononuclear cells are obtained from buffy coat preparations from whole blood donations. PBMC isolation was performed by using Ficoll density gradient centrifugation according to the following description:

1. approximately 100ml of buffy coat preparation was dispensed in equal portions onto three 50ml polypropylene tubes.

The missing volume was replaced with PBS (phosphate buffered saline) if necessary.

2. Three additional 50ml PP tubes were filled with 10ml Ficoll.

3. Two separate layers were formed by carefully pouring 35ml of blood onto 10rnl Ficoll.

4. The density gradient centrifugation was carried out at 900g for 20 minutes at a temperature of 10 ℃ (the interruption was set at 0 or 1).

5. After the gradient was formed, the mesophase containing PBMCs was transferred to a new 50ml PP tube.

6. The collected fractions with PBMCs were filled to a volume of 50ml using PBS.

7. To eliminate platelets, centrifugation was performed at 650g for 5 minutes.

8. The supernatant containing platelets was discarded.

9. For lysis of the erythrocytes, the cells were resuspended in 20 to 25ml of lysis buffer and then incubated for 7 minutes at 4 ℃.

10. The lysis reaction was stopped by PBS by filling PBS to a volume of 50 ml.

11. The lysed debris was depleted by centrifugation at 900g for 5 minutes.

12. The supernatant was discarded and the cells were resuspended in 50ml PBS.

13. The cells are counted manually using a Neubauer chamber or alternatively automatically, for example by using a Sysmex-cytometer.

14. The PBMCs were resuspended in culture medium (RPMI, 10% heat-inactivated hAB-serum, 1% PSG

(penicillin/streptomycin/L-glutamine)), and set the appropriate concentration according to the following in vitro assay.

Example 4: analysis and characterization of extracellular vesicle preparations at the molecular level

1. Determination of the Total protein concentration Using the BCA-assay (or alternatively Standard methods, e.g., Bradford-assay)

The total protein concentration of the EV enriched fraction can be determined by using standard methods. For this purpose, a variety of commercial kits and reagents can be used. For example, BCA protein assay kit from Thermo Scientific was used. Two bicinchoninic acid (BCA) molecules form a chelate complex with one copper ion (1 +). In an alkaline environment, the presence of proteins causes copper reduction. The formation of the chelate complex corresponds to a color change from green to purple in the analyzed liquid. The intensity of the color change can be measured photometrically at an absorption of 562 nm. The protein concentration of the EV fraction can be determined compared to known values for calibrated values for different BSA concentrations.

2. Determination of average particle size [ nm ] and size distribution [ curve ] and particle number (using NTA platforms, e.g. Nanosite or Zetaview)

To characterize the extracellular nanovesicles, Nanoparticle Tracking Analysis (NTA) was used. The physical technique is suitable for tracking particles from a size smaller than the wavelength of light. The method is based on the induction of an electric field in which the particles start to move. Due to this movement, its Brownian motion (Brownian motion) can be tracked during its diffusion through the analytical cell. The size and size distribution of the particles in the fraction can be determined and also give a value for the concentration of the particles. Tracking analysis of brownian motion can be closely noted on a screen connected to a video microscope. Data is digitally converted by software into data. The determination of the size is calculated from the particle diffusion constant and converted into a hydrodynamic particle size. The particle concentration is derived from the analysis of the video portion and is related to the amount of scattered light measured.

Separation method

The separation method can be based on ultracentrifugation (differential centrifugation), size exclusion chromatography (Izon and Exospin columns), polymer-based precipitation (PEG 1000, PEG 6000, PEG 8000EV, Exoquick-Qiagen), membrane affinity (Exoaeasy-Qiagen) and flow filtration. There is no standardized prior art to isolate EVs for therapeutic applications or basic research. The criteria for selecting the purification method are the initial volume of platelet lysate that must be processed and the high purity and recovery of the enriched PL-EV fraction.

Standardization of purification process selection is required for reproducibility, purity, impurities and maintenance of hPLEV functional properties. The method of application should be evaluated in terms of its scalability and reproducibility. The method that yields the highest hPLEV purity is not necessarily optimal for recovering the therapeutically most effective EV fraction due to the fact that: components attached to hPLEV surfaces or co-isolated non-hPLEV-related cofactors may be lost during purification.

EV storage

For storage of EVs, no standardized protocol is currently available. Storage conditions must be verified as they can affect EV stability. Many commonly used solvents and buffers range from sodium chloride to PBS, TRIS-HCI, HEPES and glycerol.

Example 5: principle of centrifugation

Centrifugation is used to separate components, cells, and to separate organelles. Based on the movement of particles in a liquid caused by centrifugal forces. The main component of this technology is the rotor. There are different types, such as fixed angle rotors, vertical rotors or oscillating rotors. Ultracentrifuges belong to the class of high-speed centrifuges. To avoid frictional heat due to aerodynamic drag, a vacuum is provided. According to physical principles, separation of components occurs due to size and density. The particles are accelerated by centrifugal forces in an outward direction. The acceleration depends on the angular velocity of the particles and the distance of the particles from the axis of rotation. Acceleration relates to the force of gravity g.

The sedimentation velocity of spherical particles in a viscous fluid is described by the Svedberg equation. S-value (Svedberg units) from biological material: the sedimentation coefficient s is defined as the sedimentation velocity achieved in a centrifugal field under specific geometric conditions. The unit of the sedimentation coefficient S is defined as the S value. There are a number of centrifugation techniques: differential centrifugation, zonal centrifugation, isopycnic centrifugation, density gradient centrifugation.

Differential centrifugation:

differential centrifugation is based on different particle settling rates. Which serves to enrich the particles and obtain a higher concentration of particles with a reduced volume. A fixed angle rotor is used.

Therefore, it is required that the settling velocities of the centrifuged particles are sufficiently different from each other.

There are the following differences related to the cells and their components, which allow for separation:

first, there was complete cell sedimentation (1000g, 2 min) followed by a high weight cellular component (e.g., nuclei) of larger size (1000g, 5 to 10 min). This was followed by nuclear and plasma membrane sedimentation (1500g, 15 min), followed by golgi (2000g, 20 min), mitochondria, lysosomes and peroxisomes (10000g, 25 min). The microsomes settled at 100000g, 60 minutes or more. These are also EVs, including exosomes. Which is found in the final precipitate.

Purity of the fractions obtained by differential centrifugation:

the fractions could not be isolated and purified to 100%. A sediment consisting of fast settling particles will always contain slow settling particles that are placed near the bottom of the centrifuge tube. Due to this contamination, complete purity cannot be achieved.

Differential centrifugation was used to enrich for EV/exosomes:

in the first centrifugation step, EV-containing liquid (e.g. cell culture supernatant, diluted plasma-containing liquid or diluted hPL) is centrifuged at 2000g for 15 minutes. Cells, dead cells, cell debris (nucleus, nuclear membrane, plasma membrane, golgi apparatus) settle to the bottom and can be removed. In a second centrifugation step at 10000g for 45 minutes at 4 ℃, mitochondrial, lysosomal and peroxisomes in the supernatant of step 1 were depleted. Microsomes (e.g., EVs (including exosomes)) remain in the supernatant and can eventually be precipitated by ultracentrifugation (110000 g, 1 to 2 hours).

PEG-precipitation

For polyethylene glycol precipitation, PEG 6000 may be used. The material is a polymer derived from ethylene glycol and is water soluble, inert and non-toxic. PEG can be used to precipitate high molecular substances such as proteins (also viral particles and EVs). In the presence of PEG, the protein precipitates, while the low molecular species remain soluble. The boundaries defining the high molecular substance and the low molecular substance may vary to some extent depending on the precipitation conditions chosen (molecular weight of PEG, PEG concentration and precipitation temperature). If hydrophilic, uncharged PEG and protein are mixed together in an aqueous solution, water of binding of the protein is present at the same time (hydration water). If a defined PEG concentration is reached, the protein precipitates in a reversible manner. This precipitation represents a very mild precipitation pattern (first described: Polson et al, 1964).

PEG precipitation of EV:

EV-containing liquid diluted, for example, in 0.9% NaCl is incubated overnight (16 hours, 4 ℃) in the presence of 10% v/v PEG 6000 to precipitate the EV. After incubation, the pellet was pelleted at 1500g for 30 minutes (4 ℃). The supernatant was discarded. The pellet is resuspended in, for example, 0.9% NaCl and ultracentrifuged (110000 g, about 2 hours). Alternatively, this step may be repeated as an additional washing step.

In a preferred embodiment, a method for the prevention and/or treatment of a disease selected from the group consisting of: regenerative diseases, inflammation-driven diseases, neurodegenerative diseases, immune/autoimmune diseases, cardiovascular diseases, skin diseases, infectious diseases, transplant rejection, stroke, ischemia or graft-versus-host disease, the method comprising administering to the patient an effective amount of a pharmaceutical formulation according to any one of claims 1 to 13.

Preferably, the method is a method wherein said administration is suitable for intravenous administration or infusion, or for intraperitoneal injection, subcutaneous injection, intraosseous injection, intracerebroventricular injection, intramuscular injection, intraocular injection, or for topical administration.

Preferably, a method of preparing a pharmaceutical or diagnostic or cosmetic formulation is provided, comprising the steps of: adding a human platelet lysate or a fraction enriched in human platelet lysate derived extracellular vesicles to a pharmaceutical or diagnostic or cosmetic preparation.

Reference to the literature

1.Thomas Lener et al.in ISEV position paper in the Journal ofExtracellular Vesicles 2015，4：30087

2.Torreggiani et al.(European Cells and Materials Vol.28，2014 137-151

3.Foster TE，Puskas BL，Mandelbaum BR，Gerhardt MB，Rodeo SA(2009).Am JSports Med 37(11)：2259-72)

4.Koliha，Nina et al.Journal of Extrscellular Vesicles，[S.J.]，Feb.2016.

5.Craig N.Morrell et al.，May 1，2014；Blood：123(18)

6.Kuffler DP et al.in Mol Neurobiol.2015 Oct；52(2)：990-1014.doi：10.1007/s12035-015-9251-x.Epub 2015 Jun 6

7.Raposo，et al.J.Exp，Med.1996；183(3)：1161-1172

8.Escola JM et al.，J Biol Chem.1998 Aug 7；273(32)：20121-7).

9.Barrès C.et al.，Blood.2010 Jan 21；115(3)：696-705

10.Chen，Lab Chip.2010Feb 21；10(4)：505-11).

11.Mariani et al.BMC Microbiology(2015)15：149

12.Eppley，B.L.，W.S.Pietrzak，et al.(2006)Plast Reconstr Surg 118(6)：147e-159e.

13.Mishra，A.，K.Harmon，et al.(2012)Curr Pharrn Biotechnol 13(7)：1185-1195Carlson，N.E.and R.B.Roach，Jr.(2002)J Am Dent Assoc 133(10)：1383-1386.

14.Fontana，F.，M.Mori，et al.(2016)ACS Appl Mater Interfaces 8(1)：988-996.

15.Naaijkens，B.A.，H.W.Niessen，et al.(2012)Cell Tissue Res 348(1)：119-130.

16.Govindasamy，V.，V.S.Ronald，et al.(2011)Cytotherapy 13(10)：1221-1233.

17.Parazzi，V.，C.Lavazza.etal.(2015)，″Extensive Characterization ofPlatelet Gel Releasate From Cord Blood in Regenerative Medicine.″CellTransplant24(12)：2573-2584.

18.Forte，D.，M.Ciciarello，et al.(2015).″Human cord blood-derivedplatelet lysate enhances the therapeutic activity of adipose-derivedmesenchymal stromal cells isolated from Crohn′s disease patients in a mousemodel of colitis.″Stem CellRes Ther 6：170.

Claims (15)

1. A pharmaceutical preparation for use in medicine comprising a fraction enriched in human platelet lysate-derived extracellular vesicles.

2. The formulation according to claim 1 for use in the prevention and/or treatment of inflammation-driven diseases, neurodegenerative diseases, immune/autoimmune diseases, cardiovascular diseases, skin diseases, infectious diseases, transplant rejection, stroke, ischemia or graft-versus-host disease.

3. The preparation of claim 1 or 2, wherein the preparation is acellular.

4. The preparation of any one of claims 1 to 3, wherein an enriched fraction of human platelet lysate-derived extracellular vesicles is an essential pharmaceutically active ingredient in the preparation.

5. The formulation according to any one of claims 1 to 4, wherein the extracellular vesicles in the enriched fraction have a size of 10 to 1000nm, in particular a size of 50 to 200nm, preferably 70 to 140 nm.

6. The formulation of any one of claims 1-5, wherein extracellular vesicles in the enriched fraction are positive for at least one of the following extracellular exosome markers: CD9, CD41a, CD41b, CD42b, CD61, CD62P, CD63, and endogenous proteins.

7. The formulation of any one of claims 1-6, wherein extracellular vesicles in the enriched fraction are negative for at least one extracellular exosome marker of: CD81, CD3, CD4, CD19, CD20, CD2, CD8, CD11a, and CD 25.

8. The formulation of any one of claims 1 to 7 for antimicrobial use, wherein extracellular vesicles in the enriched fraction are positive for the cytokine RANTES or the cytokine NAP-2, or both.

9. The formulation according to any one of claims 1 to 8, wherein the protein content of the pharmaceutical formulation is higher than 0.5 mg/ml.

10. The preparation of any one of claims 1 to 9, wherein the human platelet lysate is derived from platelets from a single donor donation or platelets from pooled donor donations.

11. The preparation of any one of claims 1 to 10, wherein the human platelet lysate is derived from a buffy coat extracted platelet concentrate or from platelet apheresis.

12. The formulation of any one of claims 1-11, wherein the extracellular vesicles comprise biological agents, such as genetic material, e.g., mRNA, microrna (mirna), small amounts of DNA; a lipid; and proteins, including transcription factors, cytokines, growth factors.

13. The formulation according to any one of claims 1 to 12, wherein the formulation comprises a pharmaceutically acceptable carrier, preferably a pharmaceutically acceptable polymer.

14. The formulation of any one of claims 1 to 13, wherein the formulation is suitable for intravenous administration or infusion, for intraperitoneal injection, subcutaneous injection, intraosseous injection, intracerebroventricular injection, intramuscular injection, intraocular injection, or for topical administration.

15. Pharmaceutical preparation comprising an enriched fraction of human platelet-derived extracellular vesicles according to any one of the preceding claims, obtainable by a process comprising the steps of:

a. providing human platelet lysate from platelets from a single donor donation or from pooled donor donations of at least 15 donors, preferably at least 20 or at least 30 or at least 40 donors;

b. enriching extracellular vesicles derived from human platelet lysate;

c. determining in vitro effects, e.g. immunomodulation; and

d. optionally, selecting those enriched extracellular vesicles which exhibit said in vitro effect, such as an immunomodulatory effect, in particular an anti-inflammatory effect and/or an immunosuppressive effect; and

e. optionally, selecting those enriched extracellular vesicles in step b) that exhibit extracellular vesicles negative for the extracellular exosome marker CD81 and positive for the extracellular exosome marker CD 9; and

f. optionally, mixing the human platelet lysate of step a) or the enriched extracellular vesicles of step b), d) or e) with at least one suitable pharmaceutical excipient and/or carrier.