Acellular plasma and uses thereof for treating ocular conditions

Abstract

The invention discloses a pseudoserum derived from acellular plasma at least partially depleted of fibrinogen and fibrin, as well as methods of preparing the pseudoserum of the invention, therapeutic compositions comprising the same, and uses thereof in treating ocular conditions.

Description

[0001] ACELLULAR PLASMA AND USES THEREOF FOR TREATING OCULAR CONDITIONS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to blood-derived compositions, specifically to a plasma derived composition and therapeutic uses thereof for ocular conditions.

[0004] BACKGROUND OF THE INVENTION

[0005] Regenerative medicine is a broad field in which a multitude of technologies are employed to enhance the body's ability to heal itself.

[0006] Blood derived eye drops, whether for example, serum-derived, plasma rich in growth factors (PRGF), umbilical cord blood-derived, or platelet rich plasma (PRP)-based, have all demonstrated clinical efficacy and safety in the treatment of various ophthalmologic al conditions.

[0007] These are mostly manufactured from autologous blood samples or allogenous blood donation(s) (typically, only one donation, in order to reduce the inherent risk of blood-borne viral disease transmission).

[0008] While the precise mechanism of action has not been established, the common belief is that the combined synergistic effects of several growth factors borne by platelet cells and released into the media by activation are a major contributor.

[0009] Serum is generated by passive activation through the coagulation activity initiated in blood maintained ex-vivo without the presence of anticoagulants, through the activity of coagulation enzymes which comprise the "intrinsic" coagulation system.

[0010] Other products such as PRGF are coagulated through activation of the same coagulation pathway by recalcification (typically by adding a calcium salt) of the platelet rich plasma fraction (PRP) from citrated blood. There are numerous highly similar, subtly different, autologous-blood derived products, such as LR-PRP (leukocyte rich PRP) and others that also derive their therapeutic benefit mostly through the enrichment and processing of the platelet cells, these include, but not limited to Advanced Platelet-Rich Fibrin (A-PRF), Autologous Conditioned Plasma (ACP), Autologous Growth Factors (AGF), Autologous Platelet Gel (APG), Clinical Platelet-Rich Plasma (C-PRP), Injectable Platelet-Rich Fibrin (i-PRF), Leukocyte-Poor Platelet-Rich Plasma (LP-PRP), Platelet-derived Factor Concentrate (PFC), Pure Platelet Rich Plasma (P-PRP), Platelet Fibrin Sealant (PFS), Platelet-Leukocyte Gel (PLG), Platelet-Rich Fibrin (PRF), Platelet-Rich Fibrin Matrix (PRFM), and Preparation / plasma Rich in Growth Factors (PRGF).

[0011] The coagulation cascade activation in these products involves platelets and causes degranulation and release of growth-factor containing vesicles from these cells. Other factors also theorized to be involved in the activity of blood products in regenerative medicine, and specifically in ophthalmic applications thereof, may include additional proteins or other molecules such as Vitamin A, and physiological parameters such as optimal osmolarity, pH or nutrient composition.

[0012] Despite the well-established therapeutic benefits of blood and especially platelet containing or derived products or components such as serum, PRP, PRGF, or other similar products, manufacturing of such products from blood is very limited, and several major caveats hamper the large scale GMP-compliant manufacturing, especially of allogenous blood. For example, blood borne viral diseases such as HIV (AIDS), HBV, HCV, and others, may be transmitted from person to person through such allogenous blood derived products, and active viral reduction steps for cellular products are limited and costly as the structural and functional integrity of the cells must be maintained.

[0013] In this regard, cytomegalovirus (CMV) is of special concern in ophthalmic applications as it is capable of causing Uveitis, and other ocular diseases. CMV is especially relevant in the case of patients who are immunocompromised such as essentially all patients suffering from dry eye disease secondary to graft-vs-host-disease (oGVHD).

[0014] In addition, the source material is cell based and therefore very limited in its stability which also dictates virtually insurmountable logistical barriers for large scale manufacturing.

[0015] In contradiction, the acellular component of blood, plasma, is routinely processed in large volumes and under GMP conditions with numerous products produced therefrom and can be subjected to highly efficient viral reduction treatments. Additional advantages are derived from the processing of large numbers of donations such as the reduction of anti-HLA responses for example life-threatening transfusion associated lung injury (TRALI), and the generation of a reproducible product.

[0016] However, there is currently no product, or method to produce a product, which delivers the regenerative benefits of blood, but without the requirement for the cellular components thereof. Platelet lysates, fibrin-depleted platelet lysates, plasma rich in growth factors, and others, are acellular products, but are obtained from (and require the collection of) the plateletcontaining plasma fraction which is activated and then further processed to remove the cellular or cell-derived fraction.

[0017] SUMMARY OF THE INVENTION

[0018] According to some aspects, there are provided a plasma-derived pseudoserum, therapeutic compositions including the same, and kits including the same, as well as methods of preparing the pseudoserum, and uses thereof for treating ophthalmological / ocular conditions.

[0019] According to some embodiments, the pseudoserum is derived from acellular plasma at least partially depleted of fibrinogen and fibrin containing clot, and may be prepared by activating acellular plasma derived from plasma donors or whole blood samples, and removing the formed fibrin containing clot, thereby obtaining the pseudoserum.

[0020] Advantageously, according to some embodiments, the activated and recovered pseudoserum may (i) be tolerant to viral inactivation as the inactivated pseudoserum retains cytokines and growth factors content / compo sition after being inactivated by processes that are applicable in large scale, highly efficient, and in use in commercial products including solvent / detergent (S / D) method and / or heat inactivation method; (ii) be characterized by a hypo-inflammatory composition of cytokines and growth factors following the inactivation; (iii) be further characterized by promoting superior biological activity of cells subjected to the pseudoserum relative to plasma, including enhancement of proliferation, increased resistance to stress, improved cell migration capabilities, and favorable cytokine release profile from the subjected cells; and (iv) exhibit a dose response of biological activity of cells subjected to the pseudoserum. According to some embodiments, it was surprisingly found that while it is commonly accepted in the art that growth factors and other biologically active factors released from platelets are the basis for the biological activity of cells in the presence of platelet-derived products, it is the activation of the plasma component that leads to the generation of factors unknown, that is responsible for the biological activity attributed to such products.

[0021] Furthermore, neither the reduction of platelet-derived growth factor (PDGF), nor the inhibition of platelet proteases processing consensus growth factors such as Epidermal Growth Factor (EGF) or of certain key platelet receptors (Protease Activated Receptor 1; PAR-1) have any consequence on the biological activity of activated plasma, therefore having an effect that is not related to the involvement of platelet-derived extracellular vesicles. Furthermore, as exemplified herein, advantageously, cells subjected to the pseudoserum are as active as cells subjected to platelet lysates, and this activity is superior to that seen in cells exposed to nonprocessed plasma, in several biological assays.

[0022] According to one aspect, there is provided a pseudoserum derived from acellular plasma at least partially depleted of fibrinogen and fibrin; in some embodiments, the pseudoserum comprises less than about 40%, or less than about 30%, of an initial amount / concentration of fibrinogen present in the acellular plasma. Each possibility is a separate embodiment.

[0023] According to some aspects, there is provided a pseudoserum derived from acellular plasma essentially depleted of fibrinogen and fibrin.

[0024] In some embodiments, the acellular plasma is derived from whole blood samples and / or plasma donors. Each possibility is a separate embodiment.

[0025] In some embodiments, the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of activation agent.

[0026] According to some embodiments, the pseudoserum comprises less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma.

[0027] According to some embodiments, the depletion comprises removal of at least about 80% of the amount / volume of a fibrin-containing clot, formed in the presence of an activating agent.

[0028] In some embodiments, the activating agent comprises calcium salt. In some embodiments, the depletion comprises removal of other coagulation factors.

[0029] In some embodiments, the pseudoserum is inactivated for the presence of viruses; and wherein the viral inactivation comprises viral inactivation by a solvent / detergent (S / D) method and / or a heat inactivation method. Each possibility is a separate embodiment.

[0030] According to some embodiments, the pseudoserum may be characterized by a hypo- inflammatory cytokine and / or growth factor content / compo sition of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma; in some embodiments, the hypo-inflammatory cytokine and / or growth factor content / composition comprises reduced amount / concentration of at least one of: IL- 8, Eotaxin, GRO-alpha, IL12p70, MIP-l-beta, VEGF-A, IP-10, TNF-alpha, IL12p40, PDGF AA, MCP-1, PDGF AB / BB RANTES, or any combination thereof; in some embodiments, the reduced amount / concentration of cytokine or growth factor comprise a reduction of more than about 50%. Each possibility is a separate embodiment.

[0031] In some embodiments, the pseudoserum is virally-inactivated.

[0032] In some embodiments, the hypo-inflammatory cytokine and / or growth factor content / composition of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma comprises reduced amount / concentration of cytokine or growth factor comprises reduction of at least VEGF-A; in some embodiments, the reduced amount / concentration of cytokine or growth factor comprise a reduction of more than about 50%.

[0033] In some embodiments, the pseudoserum is virally-inactivated. In some embodiments, the pseudoserum is further characterized by tolerance to viral inactivation.

[0034] In some embodiments, the tolerance to viral inactivation comprises at least partial retention of amount / concentration of cytokine and / or growth factor of the virally-inactivated pseudoserum relative to amount / concentration of cytokine and / or growth factor of noninactivated pseudoserum or non-inactivated plasma; in some embodiments, the retention of the amount / concentration of cytokine and / or growth factor comprises retention of amount / concentration of at least one of: FLT-3L, INF-gamma, IL- IRA, IL-2, IL-5, IL-7, IL- 15, TGF-alpha, IL-9, IL-17A, IL17F, MCP-3, FGF-2, Fractalkine, IL-13, IL-22, IL-l-alpha, IL- 1-beta, EGF, IL-18, TGF-beta-2, IL-17E / IL-25, and TGF-beta-1, or any combination thereof; in some embodiments, the at least partial retention of the amount / concentration of cytokine and / or growth factor comprises a change of less than about 30%. Each possibility is a separate embodiment.

[0035] In some embodiments, the tolerance to viral inactivation comprises at least partial retention of a biological activity of a cell subjected to application of the virally-inactivated pseudoserum relative to respective cell subjected to application of non-inactivated pseudoserum; in some embodiments, the retention of the biological activity of the cell comprises retention of proliferation and / or retention of resistance to hyperosmolar stress; in some embodiments, the retention of proliferation and / or retention of resistance to hyperosmolar stress comprises a change of less than about 50%. Each possibility is a separate embodiment.

[0036] In some embodiments, the pseudoserum diluted to concentration of between 5% and 100%.

[0037] In some embodiments, the cell comprises an epithelial cell, an endothelial cell and / or an immune cell. Each possibility is a separate embodiment. In some embodiments, the cell comprises an epithelial cell and / or an endothelial cell. Each possibility is a separate embodiment.

[0038] According to some embodiments, the pseudoserum characterized by promoting a superior (enhanced) biological activity of a cell subjected to application of the pseudoserum relative to respective cell subjected to application of non-processed plasma; in some embodiments, the superior biological activity comprises increase in cellular proliferation and / or increase in resistance to hyperosmolar stress; in some embodiments, the increase in cellular proliferation and / or increase in resistance to hyperosmolar stress comprises an increase of more than about 30%. Each possibility is a separate embodiment.

[0039] In some embodiments, the pseudoserum is diluted to concentration of between 5% and 100%.

[0040] In some embodiments, the cell comprises an epithelial cell, an endothelial cell and / or an immune cell. Each possibility is a separate embodiment. In some embodiments, the cell comprises an epithelial cell or an endothelial cell. Each possibility is a separate embodiment. According to some embodiments, the pseudoserum characterized by promoting a superior biological activity of a cell subjected to application of the pseudoserum relative to respective cell subjected to application of non-processed plasma; in some embodiments, the superior biological activity comprises increase in concentration / amount of cytokine release of at least one cytokine from the group consisting of IL-6, IL-8, and IL- 10, or any combination thereof; in some embodiments, the increase in concentration / amount of cytokine release comprises more than about 100%. Each possibility is a separate embodiment.

[0041] In some embodiments, the pseudoserum is diluted to a concentration of between 5% and 100%.

[0042] In some embodiments, the cell comprises an epithelial cell, an endothelial cell and / or an immune cell. Each possibility is a separate embodiment.

[0043] In some embodiments, the immune cell comprises an LPS-activated primary blood mononuclear cell (PBMC).

[0044] According to some embodiments, the pseudoserum is characterized by facilitating a dose-dependent biological activity of a cell.

[0045] In some embodiments, the pseudoserum is diluted to a concentration of between 5% and 100%.

[0046] In some embodiments, the cell may include an epithelial cell, an endothelial cell and / or an immune cell. Each possibility is a separate embodiment.

[0047] According to some embodiments, there is provided a composition including the pseudoserum, and at least one pharmaceutical excipient.

[0048] According to some embodiments, the pseudoserum or the composition including the pseudoserum, for use in treating or preventing an ocular disease in a subject.

[0049] According to some embodiments, the pseudoserum or the composition including the pseudoserum may be used in a method for treating or preventing an ocular disease, said method comprises the step of administering to a subject in need thereof a therapeutically effective amount of the pseudoserum or the composition. Each possibility is a separate embodiment. According to some embodiments, the ocular disease may include one or more of: ocular graft vs. host disease (oGVHD), dry eye or other ophthalmic conditions secondary to chronic graft vs. host disease, scleritis, keratitis, corneal ulcer or abrasion, corneal neovascularization, Fuchs dystrophy, keratoconjunctivitis, iritis, uveitis, cataract, chorioretinal inflammation, posterior cyclitis, chorioretinal scars, chorioretinal degeneration, choroidal dystrophy, choroidal haemorrhage or rupture, choroidal detachment, retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration, macular degeneration, retinitis pigmentosa, macular edema, glaucoma, floaters, optic neuropathy, optic disc drusen, amblyopia, scotoma, nyctalopia, red eye, xerophthalmia, or blindness, or any combination thereof. Each possibility is a separate embodiment.

[0050] According to some embodiments, the pseudoserum or the composition comprising the pseudoserum, may include one or more therapeutic ingredient(s). Each possibility is a separate embodiment.

[0051] In some embodiments, the administering may include administering the pseudoserum or the composition comprising the same, concomitantly with the one or more therapeutic ingredient(s) / composition, or separately from the one or more additional therapeutic ingredient(s), in a sequential manner. Each possibility is a separate embodiment.

[0052] In some embodiments, the pseudoserum, or the composition comprising the same, may be administered as a topical agent. Each possibility is a separate embodiment.

[0053] In some embodiments, the pseudoserum, or the composition comprising the same, may be administered to the eye by dropping / dispensing a solution. Each possibility is a separate embodiment.

[0054] According to some embodiments, there is provided a method for treating or preventing an ocular disease, the method comprises administering to a subject in need thereof a therapeutically effective amount of the pseudoserum derived from acellular plasma at least partially depleted of fibrinogen and fibrin, or a therapeutically effective amount of the composition including the same. Each possibility is a separate embodiment.

[0055] According to some aspects, there is provided a method for preparing a pseudoserum, the method comprises the steps of: (i) obtaining acellular plasma from whole blood samples or plasma donations;

[0056] (ii) adding a calcium salt to the acellular plasma, thereby activating the acellular plasma;

[0057] (iii) at least partially depleting fibrinogen and fibrin; and

[0058] (iv) recovering the pseudoserum; thereby obtaining the pseudoserum.

[0059] According to some embodiments, the pseudoserum may include less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma, and wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of calcium salt.

[0060] According to some embodiments, the depletion may include removal of at least about 80% of the amount / volume of a fibrin containing clot, formed in the presence of the calcium salt.

[0061] According to some embodiments, the depletion / removal may include removing other coagulation factors.

[0062] In some embodiments, obtaining acellular plasma comprises pooling acellular plasma from a plurality of whole blood samples and / or plasma donations. Each possibility is a separate embodiment.

[0063] In some embodiments, obtaining acellular plasma comprises apheresis from a plasma donor, or centrifugation of the whole blood samples. Each possibility is a separate embodiment.

[0064] In some embodiments, the calcium salt comprises at least one of calcium chloride, calcium sulphate, calcium gluconate, calcium acetate, calcium carbonate, or any combination thereof. Each possibility is a separate embodiment.

[0065] In some embodiments, the method for preparing a pseudoserum may further include adding at least one activator of intrinsic coagulation cascade, wherein the activator of intrinsic coagulation cascade is added before, during and / or after adding calcium salt. Each possibility is a separate embodiment. In some embodiments, the at least one activator of intrinsic coagulation cascade may include diatomaceous earth, silica, ellagic acid, and / or kaolin, or any combination thereof.

[0066] According to some aspects, there is provided a pseudoserum prepared according to the method for preparing a pseudoserum.

[0067] According to some embodiments, the pseudoserum comprising less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma, and wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of calcium salt.

[0068] According to some aspects, there is provided a kit for treating or preventing an ocular disease, the kit includes a container / holding a composition comprising pseudoserum derived from acellular plasma at least partially depleted of fibrinogen and fibrin; and instructions for administering the composition to the eye of a subject in need thereof.

[0069] According to some embodiments, the kit may further include one or more additional therapeutic ingredient(s), in a separate container, or in the same container as the psuedoserum composition.

[0070] Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the figures, descriptions and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some or none of the enumerated advantages.

[0071] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.

[0072] BRIEF DESCRIPTION OF THE FIGURES

[0073] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures. FIGs. 1A-1E shows pictures presenting pseudoserum (PS) prepared from plasma.

[0074] FIG. 1A presents separation of the clot and the pseudoserum which was generated from plasma by addition of 25 mM CaC12 and incubation of 1-2 hour at room temperature, followed by a centrifugation step performed to sediment the clot thereby enabling retrieval of the liquid fraction, i.e., the pseudoserum.

[0075] FIG. IB presents clotting-in-process achieved after 20 minutes of incubation by accelerating clotting through the addition of both CaCh 25 mM and diatomaceous earth.

[0076] FIG. 1C presents the separation of the pseudoserum from the clot of FIG. IB by centrifugation.

[0077] FIG. ID presents the fibrin clot including the filter-aid remains stuck fast to the test tube after decanting of the pseudoserum.

[0078] FIG. IE presents the fibrin clot including the diatomaceous earth (the filter-aid) that was physically removed from the tube for better visualization, and to demonstrate its cohesiveness.

[0079] FIG. 2 shows a line graph presenting proliferation of A549 epithelial maintained in culture, in growth medium with increasing concentrations (Dose-response) of Fetal Bovine Serum (FBS, continuous line with filled circles), untreated plasma (dashed line with squares), or pseudoserum (dotted line with empty circles). The different treatments were applied to the cells and proliferation was measured using a colorimetric assay (WST1, arbitrary optical density [OD] units). The baseline (thick grey X axis line) was set as the optical density measured under serum-free conditions. Statistical difference - ANOVA followed by post-hoc Tukey analysis. P value for plasma and FBS compared to serum free conditions <0.05 (n=10), P value for Pseudoserum (PS) vs. serum free conditions <0.005 (n=6).

[0080] FIGs. 3A-3C show line graphs presenting proliferation (FIG. 3A) and resistance to hyperosmolar stress measured as proliferation (FIG. 3B) of human primary corneal epithelial cells (hPCECs), and proliferation of human umbilical vein endothelial cells (HUVECs) (FIG. 3C). All cases tested for the ability of increasing concentrations (Dose-response) of FBS, plasma, pseudoserum (PS), or Pseudoserum subjected to standard solvent / detergent protocol (PS + S / D), to induce proliferation or promote rescue from hyperosmolar stress, respectively. In all cases, the X-axis represents serum-free conditions and is marked by a thick grey line.

[0081] Note that non-activated plasma was ineffective in all cases.

[0082] FIGs. 4A-4D show bar charts presenting secretion of interleukins detected by an Enzyme Linked ImmunoSorbent Assay (ELISA), including secretion of: TNF-alpha (TNF-a) (FIG. 4A), Interleukin- 10 (IL-10) (FIG. 4B), IL-8 (FIG. 4C), and IL-6 (FIG. 4D) by primary blood mononuclear cells (PBMCs), after LPS stimulation, in the presence of serum free medium, 30% FBS, 30% plasma, or 30% pseudoserum, and compared with unstimulated control.

[0083] FIGs. 5A-5E show bar charts presenting Multiplex analysis of different cytokines, growth factors, and chemokines (a panel of analyses performed via automated systems) present in plasma, solvent / detergent (S / D)-treated plasma, pseudoserum, or S / D-treated pseudoserum, respectively (bar graphs are grouped in that order). The data were divided into five panels by abundance of the numerous factors assayed, to allow visualization of the large number of tested factors.

[0084] FIGs. 6A-6D present comparisons between 30% concentrations of pseudoserum, plasma and platelet lysates (HPL, or activated HPL) in a proliferation assay (WST1 assay) performed under hyperosmotic stress (FIG. 6A), or dose response (FIG. 6B), and in a cell migration scratch assay (FIGs. 6C-6D). ELAREM and ELAREM-FD are commercial names of human platelet lysate (HPL) and activated HPL, respectively.

[0085] FIG. 6A shows bar charts presenting proliferation of human primary corneal epithelial cells (hPCECs) under hyperosmotic stress in the presence of plasma, pseudoserum, HPL, or activated HPL, that were added at 30% concentrations to supplement free medium, either alone or together with protease inhibitors (PI) at 5-10X of their inhibitory concentration (IC50): GM6001 (0.5nM) or PAR-1 inhibitor (7pM) (Fig. 6A). Protease inhibitors (PI): GM6001 or Protease Activated Receptor 1 (PAR-1) inhibitor have no effect.

[0086] FIG. 6B shows line graphs presenting resistance / rescue of human primary corneal epithelial cells (hPCECs) from hyperosmotic stress by increasing concentrations (Doseresponse 0%, 10%, 20%, and 30%) of plasma, pseudoserum, HPL or activated-HPL.

[0087] FIG. 6C shows line graphs presenting cell migration of A549 cells in a scratch assay The quantification is of the residual scratch area over time. FIG. 6D shows representative images of the cells undergoing the scratch assay. The TO representative image is duplicated 4 times for comparison and is placed above the scratch region in the 21 hours image for ease of visualization.

[0088] FIGs. 7A-7C show bar charts presenting resistance / rescue of human primary corneal epithelial cells (hPCECs) from hyperosmotic stress (FIGs. 7A-7B) and cellular proliferation under normal conditions (FIG. 7C), comparing between the biological activity of cells subjected to pseudoserum treated for viral inactivation by a heat inactivation method, to plasma. Several heat inactivation protocols were performed, including different temperatures for different time periods. As can be seen from the inclusion of several equivalent points in different experiments (similar pseudoserum concentration of 30% and similar heat treatment protocol), pseudoserum protective activity is robust and reproducible.

[0089] DETAILED DESCRIPTION

[0090] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0091] Definitions

[0092] 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 to which the invention pertains. In case of conflict, the patent specification, including definitions, governs.

[0093] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one, or one or more) of the stated object, unless the context clearly dictates otherwise. By way of example, “an element” means one element or more than one element, similarly “a cell” means one cell or more than one cell. As used herein, the term "about" or “approximately” may be used interchangeably when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0094] As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, “inhibit” are used interchangeably.

[0095] As used herein, the terms “enhanced”, “increased”, “elevated”, “improved” may be used interchangeably.

[0096] As used herein, the term “comprising” is synonymous with the terms "including," "containing," or "characterized by," and is inclusive or open-ended i.e. does not exclude additional, unrecited elements. According to some embodiments, the term comprising may be replaced with the term “consisting of’ which excludes any element, step, or ingredient not specified in the claim or with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0097] As used herein, the term “essentially devoid / depleted of’ may refer to a stated material as either entirely absent or present in a residual amount, such as less than about 50%, less than about 40%, less than about 30%, such as less than about 20%, such as less than about 10%, such as less than 5%, or less than 2%, or less than 1%, or less than 0.1% are present, with respect to the initial amount of the stated material. Each possibility is a separate embodiment.

[0098] As used herein, the terms "subject", "patient" or "individual" may be used interchangeably and generally refer to a human, although the methods of the invention are not necessarily limited to humans and should be useful in other mammals.

[0099] As used herein, the term "treatment / treating" refers to an approach for obtaining beneficial or desired results, including clinical results, in a suffering subject. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of disease, stabilization of the state of disease, prevention of deterioration of the disease or condition, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total).

[0100] Additionally, the term “treatment” as used herein refers to both therapeutic treatment and prophylactic or preventative measures in a subject in need hereof. As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, “inhibit”, “alleviate”, may be used interchangeably.

[0101] As used herein, the term a “subject in need” may refer to a subject suffering from / having an ocular disease / condition and / or exhibiting symptoms thereof, and in addition, a subject in need may refer to a subject diagnosed with a risk of developing, or exhibiting symptoms of ocular disease / condition. Hence a subject in need includes a suffering subject as well as a subject at risk of becoming a suffering subject. The term “suffering subject” refers to a subject having an ocular disease / condition and / or exhibiting symptoms thereof. Accordingly, the term subject in need includes the term suffering subject, and may be limited by replacing it with the term suffering subject.

[0102] In some embodiments, the effect of the pseaudoserum, or compositions including the same, disclosed herein may affect or promote regeneration of tissue / cells related to ocular conditions, and may include, for example, proliferation, differentiation, de-differentiation, cytokine secretion and / or migration of cells. Each possibility is a separate embodiment.

[0103] As used herein, the terms “composition for use” or “pharmaceutical composition” or “therapeutic composition” or simply “composition” may be interchangeably used herein, and intended to be used herein in its broader sense to include preparations containing the composition used for therapeutic purposes. Accordingly, the pharmaceutical composition includes a therapeutic amount of the active ingredient, herein namely, the “pseudoserum”. The pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable excipient / carriers, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0104] The term "pharmaceutically acceptable excipient" as used herein is exchangeable with the term "pharmaceutically acceptable carrier" and refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, buffer, a stabilizing agent, and the like that are compatible with pharmaceutical administration. The use of such media and agents in pharmaceutical compositions is well known in the art. The compositions may contain other active compounds / ingredient providing supplemental, additional, or enhanced therapeutic functions.

[0105] As used herein, the term "therapeutically effective amount" is exchangeable with any one of " therapeutically effective dose" or "sufficient / effective amount or dose," and refers to a dose that produces the required therapeutic effects. Specifically, an effective dose generally refers to the amount of the composition disclosed herein sufficient to treat and / or ameliorate / prevent an ocular condition. An effective dose may refer to the amount of the composition sufficient to delay or halt the progression of the disease / condition. An effective dose may also refer to the amount of the composition that provides a therapeutic benefit in the treatment or management of a disease / condition. In addition, an effective dose may be the amount with respect to the composition alone, or in combination with other therapeutic ingredients, that provides a therapeutic benefit in the treatment or management of the condition / disease. The exact effective dose depends inter alia on the purpose of the treatment, the tolerance of the subject and is ascertainable by one skilled in the art using known techniques.

[0106] As used herein, the term “administration / administering” to a subject can be carried out using known procedures, at dosages and for periods of time effective to provide the desired effect. An effective amount of the therapeutic composition necessary to achieve a desired therapeutic effect may vary according to factors such as the age, sex, and weight of the subject, and the ability of the therapeutic composition to treat the condition / disease in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. The administration / administering includes routes of administration which allow the compositions of the invention to perform their intended function. A variety of routes of administration are possible including, but not necessarily limited to, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), injections to a tissue of interest, oral (e.g., dietary), inhalation (e.g., aerosol to lung), topical, nasal, rectal, vaginal, or via slow releasing microcarriers depending on the disease or condition to be treated. With respect to the pseudoserum of the invention, and composition thereof, topical administration and administration by injection, and specifically topical administration to the eyes are preferred modes of administration. The term “topical” may refer to a treatment applied in a localized manner to a particular place on or in the body. A topical treatment may include applying the pseudoserum of the invention and / or compositions comprising the same, to certain locations such as, but not limited to the ocular surface / eyes, using formulations including, for example, but not limited to: drops, creams, foams, gels, lotions, ointments, solutions etc.

[0107] The term “topical agent” refers to the therapeutic composition of the invention applied to the body surface in a localized manner, including for example, but not limited to, drops, creams, ointment, lotions, oils, solutions, etc., applied to for example, but not limited to the eyes.

[0108] In some embodiments, the composition is administered as a topical agent. In some embodiments, the composition is administered to the eye by dropping a solution on the eye which may reach the cornea and / or the conjunctival epithelium. In some embodiments, administering a composition as a topical agent comprises applying the composition to the eyes in a localized manner. Each possibility is a separate embodiment.

[0109] Formulation of a composition to be administered will vary according to the route of administration selected (e.g., solution, emulsion, gels, aerosols, capsule). An appropriate composition to be administered can be prepared in a physiologically acceptable vehicle / excipient or carrier and optional adjuvants and preservatives. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media, sterile water, creams, ointments, lotions, oils, pastes and solid carriers.

[0110] According to some embodiments, the term “carrier” may refer to the part of the composition enabling its delivery. According to some embodiments, the carrier may be water or saline. According to some embodiments, the carrier may be an oil. According to some embodiments, the carrier may be a surfactant. As used herein, the term, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are compatible with the activity of the compound and are physiologically acceptable to the subject. An example of a pharmaceutically acceptable carrier is buffered or unbuffered normal saline (approximately 0.9% which is about 0.15M NaCl). The use of such media and agents for pharmaceutically active substances is well known in the art, except insofar as any conventional media or agent is incompatible with the therapeutic compound, use thereof in the compositions suitable for pharmaceutical administration is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0111] According to one aspect, the present disclosure provides pseudoserum derived from acellular plasma essentially depleted of fibrinogen and fibrin.

[0112] As used herein, the term “acellular plasma” refers to “plasma” as it is known in the art of medicine and biological science. The plasma / acellular plasma may be derived / obtained from whole blood samples (for example, from blood donations), or from plasma donations, or from pools thereof, by means also known in the art, including for example, apheresis in case of plasma donation or centrifugation of the whole blood samples. As such, the plasma / acellular plasma was not activated for coagulation, it contains fibrinogen and other coagulation factors, and therefore is also referred to herein as “non-processed plasma” or “non activated plasma”, and all these terms may be interchangeably used.

[0113] In some embodiments, the acellular plasma is derived from whole blood samples. In some embodiments, the acellular plasma is derived from plasma donors. In some embodiments, the acellular plasma is obtained from plasma donors by apheresis.

[0114] As used herein, the terms “depleted / depletion” or “depleted of fibrinogen and fibrin” refers to at least partial removal of fibrinogen from the acellular plasma, and of the fibrin polymer assembled therefrom.

[0115] The at least partial depletion / removal of fibrinogen (monomeric and oligomeric forms of the protein) from the acellular plasma, and of the fibrin assembled therefrom (polymeric form of the protein), is facilitated once activation of the plasma is performed, and the fibrinogen polymerizes to form a fibrin clot (also referred to herein as fibrin containing clot) through the activity of coagulation factors present in the plasma. Depletion / removal of the clot (solid fraction) may be achieved, in some embodiments by centrifugation, and consequently by separation of the solid fraction at the bottom (fibrin polymer clot) from the upper liquid fraction, thereby recovering the supernatant which is the “pseudoserum”.

[0116] Hence the term “depleted” or “depleted of fibrinogen and fibrin” implies that the plasma was activated (according to the herein provided term “activated” and / or any one of the embodiments related to activation), fibrin polymers were generated from fibrinogen (including monomers thereof and other oligomeric forms of fibrinogen, i.e., dimers, trimers etc.), fibrin containing clot was formed, and that the resulted pseudoserum was separated from the clot and therefore is at least partially devoid of / lacks fibrin and fibrinogen, as well as all other proteins / coagulation factors attached thereto.

[0117] According to some embodiments, depletion of fibrinogen and fibrin comprises activation. According to some embodiments, depletion of fibrinogen and fibrin includes activation using activation agent. According to some embodiments, the activation agent comprises or consists of calcium salt. Each possibility is a separate embodiment.

[0118] As used herein, the terms “other proteins / coagulation factors” or “coagulation factors” may be interchangeably used, and refer to additional proteins that participate in clot formation in response to activation, and may be attached to the clot, along with fibrin polymers. In some embodiments, other coagulation factors include, but are not limited to, for example (examples relate to the proenzyme / zymogen form of the following enzyme proteins, but relate to both the proenzyme / zymogen form and the activated enzyme form): plasminogen, factor XII (Hagemann factor), factor XI (plasma thromboplastin antecedent) , factor II (prothrombin), von Willebrand factor, factor V (proaccelerin), factor X (Stuart-Prower factor), factor IX (antihemophilia factor B), factor VII (proconvertin), prekallikrein, and others.

[0119] According to some embodiments, the depletion comprises removal of at least about 80% of the amount / volume of the fibrin containing clot.

[0120] In some embodiments, the at least partial depletion comprises removal of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%, of the amount / volume of the fibrin containing clot. Each possibility is a separate embodiment.

[0121] In some embodiments, depletion comprises removal of other coagulation factors from acellular plasma. In some embodiments, depletion comprises optionally removing other coagulation factors from acellular plasma. In some embodiments, depletion comprises essentially removing fibrin and fibrinogen from acellular plasma. In some embodiments, depletion comprises essentially removing fibrin and fibrinogen, and optionally other coagulation factors, from acellular plasma. As used herein, the term “pseudoserum” (“PS”) refers to a composition that constitutes the remaining fraction of an acellular plasma that was activated and then depleted, at least by partial removal, of the formed clot (formed in the presence of an activating agent) which contains fibrin polymers and possibly other coagulation factors. Pseudoserum shares some similarities with serum in the manner of its preparation, however it is not true serum as it is derived from acellular plasma. The terms “pseudoserum” and “pseudoserum composition” may be interchangeably used.

[0122] According to some embodiments, the pseudoserum comprises less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma.

[0123] In some embodiments, the at least partial depletion of fibrinogen and fibrin from the pseudoserum results with the pseudoserum comprising less than about 40% of the initial amount / concentration of fibrinogen present in the acellular plasma, wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of activation agent.

[0124] In some embodiments, the pseudoserum comprises less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%, of the initial amount / concentration of fibrinogen present in the acellular plasma (i.e., before activation was performed). Each possibility is a separate embodiment.

[0125] In some embodiments, the pseudoserum comprises between about 0.1% and about 50% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. In some embodiments, the pseudoserum comprises between about 0.1% and about 40% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. In some embodiments, the pseudoserum comprises between about 0.1% and about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation.

[0126] In some embodiments, the pseudoserum comprises between about 0.01% and about

[0127] 50%, between about 0.01% and about 40%, between about 0.01% and about 35%, between about 0.01% and about 30%, between about 0.01% and about 20%, or between about 0.01% and about 10%, between about 0.01% and about 5% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. Each possibility is a separate embodiment.

[0128] In some embodiments, the pseudoserum comprises between about 1% and about 50%, about 1% and about 40%, between about 2% and about 40%, between about 5% and about 40%, between about 10% and about 40%, between about 15% and about 40%, or between about 1.0% and about 30%, between about 5% and about 30%, between about 10% and about 30%, or between about 2% and about 20% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. Each possibility is a separate embodiment.

[0129] As used herein, the term “derived from acellular plasma” refers to the pseudoserum composition being the liquid fraction that remains after an acellular plasma is activated for coagulation and then depleted of the formed clot.

[0130] As used herein, the terms “activation”, “activated”, “biologically activated” may be used interchangeably, and may refer to - according to some embodiments - the activation / stimulation of the plasma for promoting coagulation, or to - in some other embodiments - the resulting pseudoserum after the plasma was stimulated / activated for promoting coagulation. In turn, the coagulation process facilitates formation of clot, including assembly of fibrinogen into fibrin polymer, and transfer thereof from the liquid plasma fraction into the newly formed solid clot fraction.

[0131] Advateagously, the activation is performed by stimulation of plasma components achieved by adding divalent calcium cation (Ca+2), usually in the form of calcium salt such as CaCh, CaSCU, calcium gluconate, calcium acetate, calcium carbonate, or similar) to the acellular plasma. Other activation methods known in the art may be used in addition to activation by the divalent calcium, including for example but not necessarily limited to, by adding to the acellular plasma an activator of the intrinsic coagulation cascade (such as diatomaceous earth, silica, or similar e.g. ellagic acid, or kaolin), and / or coagulation / activation may also be induced by the extrinsic coagulation system e.g. by the addition of activation proteins / enzymes such as tissue Factor, with or without the phospholipid component (also known as Thromboplastin), or factor Vila (active factor VII which is also known as Proconvertin), factor Ila (active factor II, also known as Thrombin), procoagulant snake venom (e.g. Ecarin, Russel's viper venom, Ancrod, Batrotoxin, or others). In some embodiments, the pseudoserum derived from acellular plasma is activated, and essentially depleted of fibrinogen and fibrin.

[0132] In some embodiments, activation is achieved by adding activation agent, i.e., a calcium salt to the acellular plasma, thereby activating the acellular plasma. In some embodiments, activation is achieved by adding a divalent cation to the acellular plasma, thereby activating the acellular plasma.

[0133] In some embodiments, activation / coagulation may be partial (i.e., assembly of more than about 75% of the initial amount / concentration of fibrinogen present in the acellular plasma into fibrin polymer associated with the clot). In some other embodiments, activation / coagulation may be allowed to proceed up to its completion (i.e., up to assembly of more than 99% of the initial amount / concentration of fibrinogen present in the acellular plasma into fibrin polymer associated with the clot).

[0134] According to another aspect, the present disclosure provides a method for preparing the pseudoserum of the invention, the method comprises the steps of (i) obtaining acellular plasma from whole blood samples or plasma donations (e.g. apheresis plasma); (ii) adding calcium salt to the acellular plasma, thereby activating the acellular plasma; (iii) at least partially depleting fibrinogen and fibrin; and (iv) recovering the pseudoserum; thereby obtaining the pseudoserum.

[0135] In some embodiments, the method for preparing the pseudoserum of the invention comprises the steps of adding a divalent cation to the acellular plasma, thereby activating the acellular plasma.

[0136] In some embodiments the pseudoserum comprises less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma.

[0137] In some embodiments, the pseudoserum comprises less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%, of the initial amount / concentration of fibrinogen present in the acellular plasma (i.e., before activation was performed). Each possibility is a separate embodiment. In some embodiments, the pseudoserum comprises less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%, of the initial amount / concentration of fibrinogen present in the acellular plasma (i.e., before activation was performed). Each possibility is a separate embodiment.

[0138] In some embodiments, the pseudoserum comprises between about 0.1% and about 50% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. In some embodiments, the pseudoserum comprises between about 0.1% and about 40% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. In some embodiments, the pseudoserum comprises between about 0.1% and about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation.

[0139] In some embodiments, the at least partial depletion comprises removal of at least about 80% of the amount / volume of the fibrin containing clot.

[0140] In some embodiments, the at least partial depletion comprises removal of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%, of the amount / volume of the fibrin containing clot. Each possibility is a separate embodiment.

[0141] In some embodiments, the at least partial depletion comprises removal of between about 60% and 100%, between about 70% and about 99.9%, between about 80% and about 99.9%, between about 90% and about 99.9% of the amount / volume of the fibrin containing clot. Each possibility is a separate embodiment.

[0142] In some embodiments, the depletion comprises removing other coagulation factors. In some embodiments, the depletion comprises optionally removing other coagulation factors.

[0143] In some embodiments, the obtaining of acellular plasma comprises pooling acellular plasma from a plurality of whole blood samples and / or a plurality of plasma donations. In some embodiments, the obtaining acellular plasma comprises either apheresis from a plasma donor, or centrifugation of the whole blood samples.

[0144] As used herein, the term “plurality” includes two or more, five or more, ten or more, twenty or more, or fifty or more.

[0145] In some embodiments the calcium salt comprises at least one of calcium chloride, calcium sulphate, calcium gluconate, calcium acetate, calcium carbonate, or similar (Ca+2)- based salt, or any combination thereof.

[0146] In some embodiments, the method for preparing the pseudoserum further comprises adding at least one activator of the intrinsic coagulation cascade, wherein the activator of intrinsic coagulation cascade is added before, during and / or after adding the calcium salt. Each possibility is a separate embodiment.

[0147] In some embodiments, the activator of intrinsic coagulation cascade is added before, during and / or after adding the divalent cation.

[0148] In some embodiments, non-limiting examples of the activator of intrinsic coagulation cascade comprises, but are not limited to, at least one of: diatomaceous earth, silica, ellagic acid, and / or kaolin, or any combination thereof. Each possibility is a separate embodiment.

[0149] In some embodiments of the method for preparing the pseudoserum, the method further comprises adding at least one activator of extrinsic coagulation cascade, wherein the activator of extrinsic coagulation cascade is added before, during and / or after adding the calcium salt; and wherein non-limiting examples of the at least one extrinsic activator comprises, but is not limited to, at least one of: tissue factor, tissue factor with the phospholipid component (Thromboplastin), factor Vila (active factor VII which is also known as Proconvertin), factor Ila (active factor II, also known as Thrombin), procoagulant snake venom (e.g. Ecarin, Russel's viper venom, Ancrod, Batrotoxin, or others), or any combination thereof. Each possibility is a separate embodiment.

[0150] According to some embodiments, the pseudoserum is prepared according to the method for preparing a pseudoserum. In some embodiments, the at least partial depletion of fibrinogen and fibrin from the pseudoserum results with the pseudoserum comprising less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma.

[0151] In some embodiments, the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of activation agent.

[0152] In some embodiments, the pseudoserum prepared according to the method for preparing a pseudoserum comprises less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma.

[0153] In some embodiments, the pseudoserum prepared according to the method for preparing a pseudoserum comprises less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%, of the initial amount / concentration of fibrinogen present in the acellular plasma (i.e., before activation was performed). Each possibility is a separate embodiment.

[0154] In some embodiments, the pseudoserum prepared according to the method for preparing a pseudoserum, wherein the depletion comprises removal of at least about 80% of the amount / volume of the fibrin containing clot.

[0155] In some embodiments, the pseudoserum prepared according to the method for preparing a pseudoserum, wherein the at least partial depletion comprises removal of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%, of the amount / volume of the fibrin containing clot. Each possibility is a separate embodiment.

[0156] Reference is now made to FIG. 1 showing the preparation of pseudoserum (PS) from plasma.

[0157] Advantageously, according to some embodiments, the pseudoserum of the invention, including a virally-inactivated pseudoserum, may be characterized by modified / modulated cytokine and / or growth factor content / compo sition and / or by a modified / modulated biological activity of a cell subjected to the pseudoserum, comprising cytokine and / or growth factor secretion, and / or other biological activities such as proliferation of cells and resistance to hyperosmolar stress, or cell migration capabilities. In such embodiment, modification / modulations may include an increase in measured values (as defined in each of the specific embodiments and sometimes is referred to as “superior”), or a decrease in measured values (as defined in each of the specific embodiments), or in some embodiments it can include a change in measured values (i.e., increase or a decrease, as defined in each of the specific embodiments). In some embodiments, the change comprises a retention of the measured values (i.e., a change that is considered relatively small as defined in each of the specific embodiments)

[0158] As used herein, the term “change” includes both increment and decrement. In accordance, the herein % change may be interpreted as (+ / ) % change. For example, a change of less than about 30% includes less than 30% increase or less than 30% decrease in the measured value.

[0159] As used herein, the terms “superior” and “enhanced” may be used interchangeably. The terms refer to the ability of the pseudoserum to promote or to facilitate conditions that allow enhancement, increment, elevation, improvement, favorability, and / or preferability of a biological activity of a cell. In some embodiments, superiority may be determined relative to the biological activity of a respective cell / cell type subjected to application of non-processed plasma. As used herein, the term “biological activity of a cell” may refer to one or more of: proliferation of cells, survival / proliferation / viability of cells under stress condition (i.e., resistance to stress, including but not limited to hyperosmolar stress), cell migration capabilities, and cytokine expression / levels / release / secretion of cells.

[0160] In some embodiments, biological activity of a cell comprises cellular proliferation, resistance to hyperosmolar stress, cell migration capabilities, cytokine express! on / levels / release / secreti on, or any combination thereof. Each possibility is a separate embodiment.

[0161] In some embodiments, biological activity of a cell comprises cellular proliferation, resistance to hyperosmolar stress, cytokine expression / levels / release / secretion, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, biological activity of a cell comprises cellular proliferation, and / or resistance to hyperosmolar stress. Each possibility is a separate embodiment.

[0162] In some embodiments, biological activity of a cell comprises cytokine expres si on / 1 evel s / rel ease / secreti on .

[0163] The terms “cytokine expression”, “cytokine levels”, “cytokine release”, “cytokine secretion” may be used interchangeably and may refer to a biological activity of a cell.

[0164] As used herein the term “cytokine and / or growth factor content / composition” refers to the amount / concentration of cytokine and / or growth factor in the pseudoserum, and therefore reflects the actual content / composition of the cytokine and / or growth factor in the pseudoserum (i.e., without subjecting the pseudoserum to cells and therefore with no relation to a biological activity of a cell that involve cytokine release / secreti on).

[0165] As used herein, the term “cytokine” has the meanings normally ascribed to it in the art, including interleukins (IL), chemokines etc., but herein it may also collectively refer to and include any growth factors. In accordance, in some embodiments used herein the term “cytokine” or “cytokine and / or growth factor” may comprises any one of the following: FLT- 3L, INF-gamma, IL-IRA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-15, M-CSF, TGF- alpha, TNF-beta, Eotaxin, GRO-alpha, IL-12p70, IL-9, IL- 17 A, IL17F, MCP-3, MIP-1 -alpha, MIP-l-beta, VEGF-A, FGF-2, Fractalkine, IL-13, IP- 10, IL-22, TNF-alpha, G-CSF, IFN- alpha-2, IL-l-alpha, IL-12p40, IL-l-beta, EGF, PDGF-AA, MCP-1, IL-18, TGF-beta-2, sCD40L, IL-17E / IL-25, IL-27, MDC, MIG / CXCL9, PDGF-AB / BB, RANTES, TGF-beta-1, or any combination thereof.

[0166] According to some embodiments, the pseudoserum is treated for inactivation of viruses. In some embodiments, the viral inactivation comprises viral inactivation by a solvent / detergent (S / D) method, while in other embodiments the viral inactivation comprises viral inactivation by a heat inactivation method.

[0167] In some of the herein below embodiments, the pseudoserum is a virally inactivated pseudoserum. As used herein, the “solvent / detergent (S / D) method” refers to an industry standard method for inactivation by the addition of solvents and detergents to solubilize enveloped viruses.

[0168] In some embodiments, non-limiting examples of methods of viral inactivation using the solvent / detergent (S / D) method comprises an industry standard solvent / detergent (S / D) treatment protocol, including but not necessarily limited to: adding to the pseudoserum 1% tween-80 and 0.3% of TnBP (w / w), and mixing together for approximately 4 hours, following by removal of the S / D composition by applying the mixture onto a hydrophobic (C-18) chromatographic resin to absorb the S / D. In some embodiments, other non-limiting examples of compositions of solvents and detergents may be used, including Triton X-100, or others.

[0169] In some embodiments, non-limited examples of viral inactivation by a “heat inactivation” method comprises an industry standard heat inactivation treatment protocol, including but not limited to: subjecting the pseudoserum to a temperature of about 50°C to about 60°C for about Ih and up to about lOh. Each possibility is a separate embodiment.

[0170] As used herein, the term “virally-inactivated pseudoserum” refer to a pseudoserum that was treated with the non-limiting solvent / detergent (S / D) treatment protocol and / or with the non-limiting heat inactivation treatment protocol, as opposed to the term “non-virally- inactivated pseudoserum” or “non-inactivated pseudoserum”, which refers to a pseudoserum that was not treated for inactivation of viruses.

[0171] Advantageously, according to some embodiments, the pseudoserum treated for inactivation of viruses is characterized by a hypo-inflammatory cytokine and / or growth factor content / composition, of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma; ; in some embodiments, the hypo-inflammatory cytokine and / or growth factor content / composition comprises reduced amount / concentration of at least one of: IL-8, Eotaxin, GRO-alpha, IL12p70, MIP-l-beta, VEGF-A, IP- 10, TNF- alpha, IL12p40, PDGF AA, MCP-1, PDGF AB / BB RANTES, or any combination thereof; in some embodiments, the reduced amount / concentration of cytokine or growth factor comprise a reduction of more than about 50%. Each possibility is a separate embodiment.

[0172] According to some embodiments, the hypo-inflammatory cytokine and / or growth factor content / composition of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma comprises reduced amount / concentration of at least VEGF-A.

[0173] In some embodiments, reduced amount / concentration of cytokine or growth factor in pseudoserum treated for inactivation of viruses comprise a reduction of more than about 60%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99%, more than about 99.9% or more, of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma. Each possibility is a separate embodiment.

[0174] As used herein, the term "hypo-inflammatory" refers to the cytokine and growth factor content / compo sition of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma, and includes a reduction of at least 50% in the amount / concentration of at least one of the following cytokine and growth factor: IL- 8, Eotaxin, GRO-alpha, IL12p70, MIP-l-beta, VEGF-A, IP-10, TNF-alpha, IL12p40, PDGF AA, MCP-1, PDGF AB / BB, RANTES.

[0175] In some embodiments, the hypo-inflammatory cytokine and growth factor content / compo sition comprises a reduced amount / concentration of at least 2, sometimes at least 3, sometimes at least 4, sometimes at least 5, sometimes at least 6, sometimes at least 7, sometimes at least 8, sometimes at least 9, sometimes at least 10, or more cytokines and / or growth factors.

[0176] Reference is now made to FIG. 5 demonstrating a hypo-inflammatory content / composition of virally inactivated pseudoserum by showing Multiplex analysis of the content / compo sition of cytokines, growth factors, and chemokines present plasma, and pseudoserum before and after viral inactivation treatment.

[0177] Advantageously and surprisingly, according to some embodiments, the pseudoserum is also characterized by being tolerant to viral inactivation, wherein in some embodiments, the viral inactivation comprises viral inactivation by a solvent / detergent (S / D) method, while in other embodiments the viral inactivation comprises viral inactivation by a heat inactivation method. According to some embodiments, the tolerance of the pseudoserum treated for inactivation of viruses comprises retention of amount / concentration of cytokine and / or growth factor of the virally-inactivated pseudoserum relative to amount / concentration of cytokine and / or growth factor of non-inactivated pseudoserum or non-inactivated plasma; in some embodiments, the retention of the amount / concentration of cytokine and / or growth factor comprises retention of amount / concentration of at least one of: FLT-3L, INF-gamma, IL-IRA, IL-2, IL-5, IL-7, IL-15, TGF-alpha, IL-9, IL-17A, IL17F, MCP-3, FGF-2, Fractalkine, IL-13, IL-22, IL-l-alpha, IL-l-beta, EGF, IL-18, TGF-beta-2, IL-17E / IL-25, and TGF-beta-1, or any combination thereof; in some embodiments, the retention of the amount / concentration of cytokine and / or growth factor comprises a change of less than about 30%. Each possibility is a separate embodiment.

[0178] In some embodiments, the retention of the amount / concentration of cytokine and / or growth factor comprises a change of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 7%, less than about 5%, less than about 3%, or less than about 1%. Each possibility is a separate embodiment.

[0179] In some embodiments, the retention of the amount / concentration of cytokine and / or growth factor comprises retention of amount / concentration of at least one, at least 2, sometimes at least 3, sometimes at least 4, sometimes at least 5, sometimes at least 6, sometimes at least 7, sometimes at least 8, sometimes at least 9, sometimes at least 10, or more cytokines and / or growth factors.

[0180] In some embodiments, the pseudoserum content composition is independent of platelets and / or of extracellular vesicles / microvesicles derived therefrom.

[0181] Reference is now made to FIG. 5 demonstrating the tolerance by showing Multiplex analysis of the content / composition of cytokines, growth factors, and chemokines present plasma, and pseudoserum before and after viral inactivation treatment.

[0182] In some embodiments, the tolerance to viral inactivation comprises retention / modification / modulation of the biological activity of the cell subjected to application of the virally-inactivated pseudoserum relative to a respective cell subjected to application of non-inactivated pseudoserum. In some embodiments, the retention of the biological activity of the cell subjected to application of the virally-inactivated pseudoserum relative to a respective cell subjected to application of non-inactivated pseudoserum comprises retention of proliferation and / or retention of resistance to hyperosmolar stress. Each possibility is a separate embodiment.

[0183] In some embodiments, the at least partial retention of a biological activity of a cell subjected to application of the virally-inactivated pseudoserum relative to a respective cell subjected to application of non-inactivated pseudoserum comprises a change of less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 7%, less than about 5%, less than about 3%, or less than about 1%, of the activity before / without the viral inactivation. Each possibility is a separate embodiment.

[0184] According to some embodiments, the pseudoserum is diluted to concentration of between about 5% and about 100%, sometimes between about 5% and about 80%, sometimes between about 5% and about 70%, sometimes between about 5% and about 60%, sometimes between about 5% and about 50%, sometimes between about 10% and about 100%, sometimes between about 10% and about 90%, sometimes between about 10% and about 80%, sometimes between about 10% and about 70%, sometimes between about 10% and about 60%, sometimes between about 10% and about 50%, sometimes between about 10% and about 40%, sometimes between about 10% and about 30%, sometimes between about 10% and about 20%, sometimes between about 20% and about 50%, sometimes between about 25% and about 35%, or sometimes to a concentration of about 30%. Each possibility is a separate embodiment.

[0185] According to some embodiments, the cell comprises an epithelial cell, an endothelial cell, and / or an immune cell. According to some embodiments, the cell comprises an epithelial cell and / or an endothelial cell. Each possibility is a separate embodiment.

[0186] As used herein the term “relative to respective cell(s)” refers to a comparison between cells subjected to the pseudoserum and cells of the same “type” that serve as a control or as a reference point for the comparison. An example of a type of a cell, includes, but is non-limiting limited to: an epithelial type of a cell, an endothelial type of a cell, and an immune type of a cell. Reference is now made to FIG. 3 and FIG. 7 showing the effect of viral inactivation on biological activity of cells, subjected to the pseudoserum, e.g., proliferation or resistance to hyperosmolar stress.

[0187] Advantageously and surprisingly, according to some embodiments, the pseudoserum is further characterized by promoting a superior biological activity of a cell subjected to application of the pseudoserum relative to a respective cell subjected to application of nonprocessed plasma; wherein the superior biological activity comprises increase in cellular proliferation and / or increase in resistance to hyperosmolar stress; in some embodiments, the superior biological activity comprising the increase in proliferation and / or increase in resistance to hyperosmolar stress comprises an increase of more than about 30%, and less than about 1000%.

[0188] In some embodiments, the pseudoserum is further characterized by promoting a superior biological activity of a cell subjected to application of the pseudoserum relative to a respective cell subjected to application of non-processed plasma; wherein the superior biological activity comprises increase in cell migration capabilities.

[0189] In some embodiments, the increase in cell migration comprises an increase of more than about 3% at about 24h,

[0190] In some embodiments, superior biological activity comprising the increase in cellular proliferation and / or increase in resistance to hyperosmolar stress comprises an increase of more than 10%, more than 20%, more than 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 120%, more than about 150%, more than about 175%, more than about 200%, more than about 250%, more than about 300%, more than about 350%, more than about 400%, more than about 500%, more than about 600%, more than about 750%, more than about 900%, or more than about 1100%, In some embodiments, superior activity comprises an increase in activity of more than about 10% and less than about 2000%, of more than about 30% and less than about 2000%, of more than about 30% and less than about 1600%, of more than about 30% and less than about 1300%, of more than about 30% and less than about 1100%, of more than about 30% and less than about 900%, of more than about 30% and less than about 750%, of more than about 30% and less than about 600%, of more than about 30% and less than about 500%, of more than about 30% and less than about 400%. Each possibility is a separate embodiment.

[0191] It is to be understood that, for example, 900% increment in activity is equal to activity that is enhanced by 10-fold. Similarly, 100% increment in activity is equal to activity that is enhanced by 2-fold, and 50% increment in activity is equal to activity that is enhanced by 1.5- fold.

[0192] In some embodiments, resistance to stress comprises survival / proliferation / viability of cells under stress condition, or otherwise rescue from the conditions of stress.

[0193] In accordance with the hereinabove terms the superiority of the pseudoserum is reflected in increased biological activity of cells subjected to the pseudoserum, with respect to the same type of cells (i.e., “relative to respective cells”) subjected to plasma or to human platelet lysate.

[0194] According to some embodiments, the cells comprise epithelial cells, endothelial cells, and / or an immune cell. Each possibility is a separate embodiment.

[0195] According to some embodiments, the cells comprise epithelial cells and / or endothelial cells. Each possibility is a separate embodiment.

[0196] Reference is now made to FIG. 2, FIG. 3 and FIG. 6 showing superior proliferation, resistance to hyperosmolar stress, and cell migration of cells subjected to pseudoserum.

[0197] In some embodiments, the promotion of a superior biological activity of cells subjected to application of the pseudoserum relative to respective cells subjected to application of nonprocessed plasma comprises promoting superior cytokine release of at least one cytokine from the group consisting of IL-6, IL-8, and IL-10, or any combination thereof; in some embodiments, the superior activity comprises an increase in cytokine release activity of more than about 100%. Each possibility is a separate embodiment.

[0198] In some embodiments, the superior activity comprises an increase in secretion activity of more than about 100%, more than about 250%, more than about 500% (6 fold), more than about 750%, more than about 1000%, more than about 2000% (21 fold), more than about 4,000%, more than about 6,000%, more than about 8,000 (81 fold)%, more than about 10000% (110 fold), or more than 20000% (210 fold); but not more than about 100,000%. Each possibility is a separate embodiment.

[0199] In some embodiments, the cells comprise epithelial cells, endothelial cells and / or immune cells. Each possibility is a separate embodiment.

[0200] In some embodiments, the cells comprise immune cells.

[0201] In some embodiments, the immune cell comprises an LPS -activated primary blood mononuclear cell (PBMC)

[0202] Reference is now made to FIG. 4 showing superior cytokine expression of cells subjected to pseudoserum.

[0203] Advantageously, according to some embodiments, the pseudoserum may be further characterized by facilitating a dose-dependent biological activity of cells.

[0204] In some embodiments, the cells may include epithelial cells, endothelial cells and / or immune cells. Each possibility is a separate embodiment.

[0205] According to some embodiments, the present disclosure provides a composition which includes the pseudoserum. According to some embodiments, the present disclosure provides a composition which includes the pseudoserum and at least one pharmaceutical excipient.

[0206] According to some embodiments, the pseudoserum or the composition comprising the same, may be used in a method for treating or preventing an ocular disease in a subject in need thereof, the method includes the step of administering to a subject in need thereof, a therapeutically effective amount of the pseudoserum or the composition including the same.

[0207] According to some embodiments, the pseudoserum or the composition comprising the same, for use in treating or preventing an ocular disease.

[0208] In some embodiments, the treating or preventing of an ocular disease includes the step of administering to a subject in need thereof a therapeutically effective amount of the pseudoserum or the composition comprising the same. Each possibility is a separate embodiment. In some embodiments, the ocular disease / condition may include one or more of: ocular graft vs. host disease (oGVHD), dry eye or other ophthalmic conditions secondary to chronic graft vs. host disease, scleritis, keratitis, corneal ulcer or abrasion, corneal neovascularization, fuchs dystrophy, keratoconjunctivitis, iritis, uveitis, cataract, chorioretinal inflammation, posterior cyclitis, chorioretinal scars, chorioretinal degeneration, choroidal dystrophy, choroidal haemorrhage or rupture, choroidal detachment, retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration, macular degeneration, retinitis pigmentosa, macular edema, glaucoma, floaters, optic neuropathy, optic disc drusen, amblyopia, scotoma, nyctalopia, red eye, xerophthalmia, or blindness, or any combination thereof. Each possibility is a separate embodiment.

[0209] In some embodiments, the effect of the pseaudoserum, or compositions including the same, disclosed herein may affect or promote regeneration of tissue / cells related to ocular conditions. In some embodiments, tissue regeneration may include, for example, proliferation, differentiation, de-differentiation, cytokine secretion and / or migration of cells. Each possibility is a separate embodiment.

[0210] In some embodiments, tissue regeneration comprises proliferation of one or more types of cells composing the tissue; in some embodiments, the one or more type of cells comprises endothelial cells, epithelial cells, and / or immune cells, or any combination thereof. Each possibility is a separate embodiment.

[0211] In some embodiments, tissue regeneration comprises cellular migration of one or more types of cells composing the tissue; in some embodiments, the one or more type of cells comprises endothelial cells and / or epithelial cells. Each possibility is a separate embodiment.

[0212] In some embodiments, when administered, the composition including the pseudoserum, may include at least one additional therapeutic ingredient. Each possibility is a separate embodiment.

[0213] In some embodiments, the pseudoserum or the composition including the same, may be administered concomitantly with one or more therapeutic ingredients / compositions. In some embodiments, the pseudoserum or the composition including the same, may be administered sequentially with one more therapeutic ingredient / composition. Each possibility is a separate embodiment. In some embodiments, the pseudoserum or the composition including the same, may be administered as a topical agent.

[0214] In some embodiments, the pseudoserum or the composition including the same, may be local administered to an eye by dispensing or dropping a solution. In some embodiments, administration to an eye may include any type of suitable administration, including, topical, external (ophthalmic), injection, and the like.

[0215] According to some aspects the present disclosure provides a method for treating or preventing an ocular disease or condition, the method includes administering to a subject in need thereof, a therapeutically effective amount of the pseudoserum or the composition including the same.

[0216] According to another aspect there is provided a kit for treating or preventing an ocular disease, the kit includes a container holding a composition which includes pseudoserum derived from acellular plasma, essentially depleted of fibrinogen and fibrin; and instructions for administering the composition to eye of a subject in need thereof. Each possibility is a separate embodiment.

[0217] In some embodiments, the ocular disease / condition may include one or more of: ocular graft vs. host disease (oGVHD), dry eye or other ophthalmic conditions secondary to chronic graft vs. host disease, scleritis, keratitis, corneal ulcer or abrasion, corneal neovascularization, fuchs dystrophy, keratoconjunctivitis, iritis, uveitis, cataract, chorioretinal inflammation, posterior cyclitis, chorioretinal scars, chorioretinal degeneration, choroidal dystrophy, choroidal haemorrhage or rupture, choroidal detachment, retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration, macular degeneration, retinitis pigmentosa, macular edema, glaucoma, floaters, optic neuropathy, optic disc drusen, amblyopia, scotoma, nyctalopia, red eye, xerophthalmia, or blindness, or any combination thereof. Each possibility is a separate embodiment.

[0218] In some embodiments, the pseudoserum in the kit may include less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma.

[0219] In some embodiments, the pseudoserum comprises less than about 50%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.1%, of the initial amount / concentration of fibrinogen present in the acellular plasma (i.e., before activation was performed). Each possibility is a separate embodiment.

[0220] In some embodiments, the pseudoserum comprises between about 0.01% and about 50%, between about 0.01% and about 40%, between about 0.01% and about 35%, between about 0.01% and about 30%, between about 0.01% and about 20%, or between about 0.01% and about 10%, between about 0.01% and about 5% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. Each possibility is a separate embodiment.

[0221] In some embodiments, the pseudoserum comprises between about 1% and about 50%, about 1% and about 40%, between about 2% and about 40%, between about 5% and about 40%, between about 10% and about 40%, between about 15% and about 40%, or between about 1.0% and about 30%, between about 5% and about 30%, between about 10% and about 30%, or between about 2% and about 20% of the initial amount / concentration of fibrinogen present in the acellular plasma before activation. Each possibility is a separate embodiment.

[0222] In some embodiments, the kit may include one or more therapeutic ingredient / composition(s), either as a separate eomcition, or as part of the pseudoserum composition. Each possibility is a separate embodiment.

[0223] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0224] EXAMPLES

[0225] Example 1 - preparation of pseudoserum composition from plasma

[0226] In a first experiment, approximately 250 liters of plasma from approximately 300 individual plasma donations was pooled and then filtered through a cascade of filters with pore sizes from 70 microns to 10 microns. Samples were obtained and frozen from this plasma pool. A 50 ml tube of pooled and filtered plasma was then thawed, and incubated with 25 mM of CaCh, an activator of coagulation. Coagulation required between 1-2 hours, after which the plasma was centrifuged for 15 minutes at approximately 2600 RCF. This resulted in a clear separation of clot (solid, bottom) from pseudo-serum (liquid supernatant, top), which could be separated by simple decanting.

[0227] In a second experiment, coagulation was accelerated by further adding an activator of the intrinsic coagulation system which is diatomaceous earth / silica particles (Standard Super- Cel, "Filter- Aid") which directly activates the intrinsic coagulation system. The addition of 1% w / w Super-Cel followed by the CaCh addition (25mM) greatly accelerated coagulation so that within approximately 20 minutes a stable clot was formed, that did not change over the next hours. The composition (referred to as Pseudoserum) was separated from the clot and filter-aid by the centrifugation of between approximately 2500 RCF and approximately 4100 RCF (maximal centrifugation speed of the table-top centrifuge used for these experiments). Centrifugation served to extract a protein solution from the clot, while sedimenting the clot on the bottom of the centrifuge tube for easy separation, e.g. by decanting or aspiration of the protein solution (the Pseudoserum) which comprises the top liquid phase. The clot formed was very compact and completely distinct from the pseudoserum, and could therefore easily be separated and discarded (FIG. 1A-1E).

[0228] Example 2 - Enhanced biological activity of pseudoserum over plasma in promoting proliferation of epithelial cells

[0229] The A549 epithelial cell line was used to demonstrate the capacity of Pseudoserum to enhance cellular proliferation. This cell line is typically maintained with fetal bovine serum (FBS) as a source for various nutrients and growth factors. FBS is produced in a similar method to human serum that is used in some clinical applications (e.g. Serum Eye Drops or SEDs for ophthalmological conditions). Different concentrations of FBS, untreated plasma, or Pseudoserum, were applied to the cells and proliferation was measured using a colorimetric assay (the vital dye WST1, arbitrary optical density [OD] units).

[0230] The baseline (thick grey X axis line) was set as the optical density measured under serum-free conditions. FBS, plasma, or Pseudoserum were added at increasing concentrations into the growth medium. As can be seen in FIG. 2, proliferation was most effective when pseudoserum was added (n=6 independent experiments), with FBS second (n=10) and plasma third (n=10). ANOVA followed by post-hoc Tukey analysis demonstrated that at 30% added concentrations only Pseudoserum is significantly different from serum-free control. P value for plasma and FBS compared to serum free conditions <0.05, P value for Pseudoserum (PS) vs. serum free conditions <0.005.

[0231] Advantageously, in some embodiments, pseudoserum enhances cellular proliferation and is superior to plasma.

[0232] Example 3 - Biological activity of Pseudoserum in promoting proliferation and rescue from hyperosmolar stress of epithelial and endothelial cells is superior to plasma, and is maintained after viral inactivation by solvent / detergent treatment

[0233] Human primary corneal epithelial cells (or hPCECs) are derived from human corneas and therefore faithfully represent corneal conditions in a cell-based system. Primary cells are more dependent on the supply of physiological levels of growth factors and other nutrients, as opposed to transformed cell lines such as A549. These hPCECs were tested for their responsiveness to plasma and pseudoserum (and also compared to standard FBS used for culturing cells, and to complete medium (Corneal Epithelial Cell Basal Medium - ATCC PCS- 700-030 plus Corneal Epithelial Cell Growth Kit) containing proprietary factors optimized to these cells). This was done both by assessing their proliferative abilities using the WST1 assay (FIG. 3A) (as was performed for A549 cells in previous Example 2), and under hyperosmolar stress generated by the addition of NaCl to approximately 500 mOsm (FIG. 3B). This stress is considered an analogous model to the "dry eye" condition where the lacrimal glands underproduce the tear fluid, a condition which occurs, for example, in patients suffering from graft vs. host disease after hematopoietic stem cell transplant (HSPC) in which the lacrimal gland (the tear gland) is attacked by the grafted immune system cells. Plasma and pseudoserum were added to assess their ability to protect primary corneal cells from this stress. In addition, pseudoserum was subjected to standard solvent / detergent (S / D) treatment protocol. 1% tween- 80 and 0.3% of TnBP (w / w) were added to the pseudoserum and mixed together for approximately 4 hours. The S / D composition was removed by applying the mixture onto a hydrophobic (C-18) chromatographic resin to absorb the S / D, a standard industry practice. The S / D mixture inactivates dangerous enveloped viruses such as HIV, HBV, and others, by solubilizing their membraneous envelope. This S / D treated pseudoserum was also tested for its ability to still induce proliferation and rescue cells from stress. A second primary cell line, human umbilical vein endothelial cells (HUVECs) was also tested for the ability of pseudoserum and S / D treated-pseudoserum to affect proliferation (FIG. 3C). This was performed to demonstrate the universal effect of the pseudoserum on primary cells of different types and lineages, and its relevance for treatment of various conditions. Endothelial cells are also affected in many ophthalmic indications, e.g., those involving neovascularization of the cornea, or in chronic graft vs. host disease.

[0234] The results shown in FIGs. 3A-3C are typical / representative results (experiments were repeated with similar results).

[0235] Advantageously, these results demonstrate that in both types of primary cells tested, endothelial and epithelial cells, and in both models of proliferation and rescue from hyperosmolar stress, there was a marked effect of the pseudoserum, but not of regular (nonactivated) plasma. FBS was included as a positive reference but not as a quantitative control. This is because historically most cell growth mediums have been developed to work with bovine serum (or sometimes other animal serum such as horse serum) which may lead to a bias in favor of FBS as a cell supportive reagent compared to human blood-derived components. The FBS superiority is also seen when compared to commercially available human platelet lysates growth-promoting effect, even in human cell lines. Still, in some assays the effect of pseudoserum was almost as high as that of the "classic" serum (FBS) derived from whole bovine blood.

[0236] Advantageously, the S / D treatment which is a robust and safe method to dramatically reduce the risk of enveloped viruses such as HIV, HB V, CMV, and others from plasma-derived products, had only a slight effect on the efficacy of the pseudo serum, but the majority of the activity remained, which indicates that S / D treated pseudoserum from pooled plasma would be efficient in clinical applications.

[0237] To conclude, it was herein exemplified that in some embodiments, (i) endothelial and / or epithelial cells, showed superior biological activity, including proliferation and rescue from hyperosmolar stress (i.e., increased resistance), for the pseudoserum relative to regular (nonactivated) plasma; (ii) the biological activity of the endothelial and epithelial cells was retained in S / D treated pseudoserum. Example 4 - altered biological activity of Pseudoserum in promoting cytokine release after activation of PBMCs

[0238] GVHD ocular symptoms are caused by the action of non-self immune cells derived from grafted bone marrow stem cells, on the corneal epithelium. Primary bone marrow cells / primary blood mononuclear cells (PBMCs) were tested for the capacity of pseudoserum to alter their activity after activation. This was measured by activating the PBMCs with lipopolysaccharide (LPS, bacterial endotoxin) which causes the release of a number of cytokines, either with or without the presence of 30% of plasma or pseudoserum (FIG. 4A- 4D).

[0239] The release of cytokines was altered when stimulated PBMCs were incubated with Plasma, FBS, or Pseudoserum. For example, while both plasma and Pseudoserum reduce the level of the pro-inflammatory TNF-a secretion (FIG. 4A), the anti-inflammatory 11-10 was induced by pseudoserum to about 900ng / ml which is a concentration of about more than 100 fold relative to the 11-10 concentration released by LPS-induced PBMCs in the presence of plasma, and about 9-fold relative to the 11-10 concentration released by LPS-induced PBMCs in the presence of serum-free medium (FIG. 4B). The neutrophil- attractant 11-8 is also greatly induced by pseudoserum compared to plasma, reaching about 7-fold difference (FIG. 4C), as is 11-6 which is also a pro-inflammatory cytokine with important biological activities, and which is inducted by about 3-4 fold in pseudoserum compared to plasma (FIG. 4D).

[0240] Hence, plasma activation (of coagulation) into pseudoserum imbues the pseudoserum composition that is derived or results from the activated acellular plasma, pseudoserum, with novel biological properties - whether on epithelial, endothelial, or immune cells.

[0241] Advantageously and surprisingly, these results suggest that the herein disclosed method for preparing a pseudoserum, comprising the step of plasma activation of coagulation into pseudoserum imbues the produced pseudoserum composition with novel biological properties that affect the activity of epithelial, endothelial, or immune cells, compared with non-processed plasma (not activated to generate the pseudoserum).

[0242] Advantageously and surprisingly, these results suggest that the herein disclosed pseudoserum composition derived from activated acellular plasma is characterized by having novel biological properties that affect the activity of epithelial, endothelial, or immune cells, compared with plasma that was not activated to generate pseudoserum. These differences between plasma and pseudoserum are of biological significance. For example, the presence of Ml macrophages can induce scar- free healing in some contexts, including in the cornea, via TGF03. The production of IL-ip and IL-6 from activated macrophages has been shown to upregulate TGFP3 expression in chondrocytes. IL- 10 is considered an anti-inflammatory cytokine, which is induced by LPS stimulation in PBMCs, and further increased by both serum (FBS) and pseudoserum, whereas non-activated plasma actually reduced its levels. The IL- 8 cytokine is present in platelets and is also induced in PBMCs by LPS. It is related to the communication between neutrophils and platelets. Neutrophils play various roles in the eye including undergoing apoptosis to release neutrophil extracellular traps during sleep as to protect the immobile eye from infection. Aggregated NETs have been measured in the eye Rherum (a medial angle accumulated discharge, independent of any disease) and are believed to prevent spreading of inflammation because they degrade inflammatory mediators. Surprisingly, IL-8 levels were slightly reduced by serum and plasma, but upregulated by Pseudoserum. Finally, IL-6 is also induced by LPS and is reduced by plasma and serum, but not by pseudoserum. Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. IL-6 also exerts an effect on chronic inflammation and autoimmunity.

[0243] In conclusion, Pseudoserum modulates cytokine secretion by LPS-activated PBMCs in a differential pattern compared to both serum from whole blood (FBS) and non-activated plasma. These experiments indicate that plasma, serum, and pseudoserum, are three distinct entities in terms of the biological activity of cells subjected to them. In some herein exemplified embodiments, pseudoserum altered cytokine release profile, including enhanced release of at least one of IL-6, IL-8, and / or IL- 10, or any combination thereof.

[0244] Example 5 - independence of the biological effect of pseudoserum of the composition of growth factors or cytokines

[0245] Platelet-based blood components including for example, but not limited to: Platelet Rich Plasma (PRP) and Human Platelet Lysate (HPL), are theorized to exert their biological function through the increased concentration of various growth factors in these preparations, that are stored within and then released from the platelets, demonstrating the drastic effect of plasma conversion into pseudoserum activation on biological activity of cells (see Example 4) the inventors further tested to see whether growth factors were somehow released into the composition by activation. The inventors further tested S / D-treated plasma and S / D-treated pseudoserum to see whether such factors may reside inside microvesicles and would therefore be released into the medium by solvation of these microvesicles, which would be reflected in an increase in their concentration. Indeed, some have theorized that plasma actually includes platelet-derived microvesicles with an activity equivalent to, redundant with, or even different from the original platelets, and having a role in regenerative medicine. Multiplex / multikine analysis (a panel of analyses performed via automated systems) was performed for numerous growth factors, cytokines, and chemokines (FIGs. 5A-5E). EGF and TGF-betal (TGFbl) were analyzed separately using a specific Enzyme Linked Immuno-Sorbent Assay (ELISA) due to their known importance but are presented on the same graph for brevity and simplicity.

[0246] Surprisingly, activation of plasma into pseudoserum did not cause a marked increase in the levels of any specific factor. Although for a few proteins, such as IL- 17, their levels were increased in pseudoserum compared to plasma, this was not deemed to be of major significance. This is because the increase in their levels was relatively minor and / or abrogated after S / D treatment (compare IL- 17 levels in the S / D-treated plasma vs. Pseudoserum, FIG. 5B). Also, IL- 17 levels were not in correlation with the levels of other cytokines that are associated with PRP such as IL-4, IL-8 (FIG. 5A), IL- 13, tumor necrosis factor (TNF)-a and interferon (IFN)-a (FIG. 5C), and Platelet Derived Growth Factor (PDGF) (FIGs. 5D-5E).

[0247] Interestingly, key growth factors assumed to be related to the biologic activity of platelet-derived compositions such as EGF or TGF-beta, were found at either very similar levels in all four test groups, or even somewhat reduced (FIGs. 5D-5E). Some factors were mostly or completely removed by S / D treatment such as Platelet Derived Growth Factor (PDGF) (FIGs. 5D-5E). This factor was also not found at higher levels (prior to S / D treatment) in pseudoserum compared to plasma. This is surprising since, as its name implies, this protein is believed to be one of the major growth factors that are contributed by the platelets and therefore mediate their protective effects in various systems. This is a clear indication that the effects of pseudoserum activation are not dependent on platelets or platelet-derived extracellular vesicles / microvesicles (FIGs. 5D-5E).

[0248] A similar surprising finding was seen for Vascular Endothelial Growth Factor (VEGF), which is also found in greatly reduced levels in S / D-treated plasma or S / D-treated pseudoserum (FIG. 5B). Plasma, prior to S / D treatment, did not stimulate proliferation of vascular endothelial cells (HUVECs, see previous Example 3,), whereas pseudoserum as well as S / D- treated pseudoserum both did and to a similar extent (see FIG. 3C, especially at high concentrations), indicating that this proliferation effect is independent of VEGF levels. In agreement with these findings is an apparent lack of correlation between proliferation of Epithelial cells seen in the Examples 2-3 above (A549 epithelial cell line (FIG. 2) or primary corneal epithelial cells (FIG. 3A), both of which responded to pseudoserum before and after S / D treatment, but significantly less or not at all to plasma), and the Epithelial Growth Factor - EGF concentrations which were measured in these compositions.

[0249] It is still possible that a minor part of the differences in biological activity of subjected cells, between plasma and pseudoserum, or a minor reduction in activity of S / D-treated pseudoserum in some assays, are derived from differences in the combined and potentially synergistic activity of several growth factors and cytokines that their content / composition were assayed hereinabove (or other factors unknown that were not tested).

[0250] Still, the fact is that the differences in the large number of factors tested herein, between plasma, pseudoserum, and S / D-treated pseudoserum, do not correlate with biological activity of the cells. This indicates that there are additional critical factors that are obtained through activation and are required for the biological activity mediated by pseudoserum, thus rendering pseudoserum as a biologically active composition with a distinctive mode of action from other blood products and components.

[0251] Example 6 - distinction of platelet vs. activation - derived biological activity

[0252] In the present disclosure the inventors performed an experiment wherein the pseudoserum was generated as described above herein in Example 1, but in the presence of protease inhibitors: GM6001 which is an inhibitor of the metalloprotease ADAM 10 / 17 that is involved in TNF-a and EGF bioprocessing on the platelet membrane, and an inhibitor of Protease Activated Receptor 1 (PARI), which is a major activator of platelets. These protease inhibitors had no detectable effect on the biological activity of pseudoserum on primary corneal epithelial cells (hPCEC) measured as proliferation using WST1 assay (OD) under hyperosmotic stress of NaCl to approximately 500 mOsm, i.e. measuring resistance and / or protection from hyperosmotic stress (FIG. 6A).

[0253] Furthermore, the effect of a commercially available human platelet lysate (ELAREM-

[0254] HPL, acquired from PL-Biosciences, GmbH) was compared to plasma and pseudoserum with increasing concentrations, at the same conditions of hyperosmotic stress (FIG. 6B). HPL is prepared by freeze / thaw cycles of the platelet blood component. A platelet lysate activated in a similar manner to pseudoserum (ELAREM-FD-Plus) was also included.

[0255] The results of FIG. 6B show that the biological activity of cells exposed to HPL in the hyperosmotic stress model could be detected without activation, but was much lower than that of pseudoserum or activated HPL. The non-existent effect of plasma under these conditions is consistent with previous results of FIG. 3B. The detectable albeit low effect of HPL (not activated), is indicative of that platelet lysates can also exert some effect in this system through a different mechanism. The pseudoserum, and similarly activated HPL have a similar maximal effect in this system.

[0256] An additional comparison was made between HPL and pseudoserum in a migration assay performed in the epithelial cell line A549, in which a scratch is made in a confluent cell layer and a proliferation inhibitor is added. The filling of the gap by migrating cells is measured over time. The results from this assay are presented in FIGs. 6C-6D showing that HPL was superior to both plasma and pseudoserum. Interestingly, a small advantage after 21 hours was seen to pseudoserum over plasma, and to activated HPL over non-activated HPL, respectively. This indicates that in this assay, too, the effects of activation, albeit relatively minor, can be distinguished and differentiated from platelet-derived effects (FIG. 6C and FIG. 6D).

[0257] Combined, these data demonstrate that there are several distinct activities mediated by the pseudoserum vs. platelet-derived compositions such as HPL. Advantageously activation is critical for proliferation and protection from stress, whereas platelet lysates appear to be superior in cell migration, with an additional small but detectable benefit to activation. Either way, the biological activity of cells subjected to pseudoserum is distinct and can be experimentally differentiated from the platelet-derived biological activity.

[0258] Example 7 - Solvent-Detergent (SD) treatment generates a distinctive hypo- inflammatory Pseudoserum composition

[0259] Solvent-detergent (SD) treatment greatly reduced the levels of several cytokines. The basis for this may be a tendency for these proteins to unfold and denature under hydrophobic conditions and associate with the SD fraction, potentially also being removed by the C18 resin treatment. Indeed, MIP-1, MCP-1, Gro, Eotaxin, Rantes, IP- 10 and IL-8, all belong to the same superfamily and share similar structure determinants allowing them to bind to similarly structured receptors, and all are similarly almost completely removed by SD treatment (whether from plasma or from pseudoserum; FIGs. 5A-5E). This finding is construed as a significant advantage of a preparation for the treatment of conditions wherein there is an inflammatory component. This is especially true for ophthalmological preparations and specifically for compositions intended to treat ocular manifestations of graft vs. host disease (oGVHD). The basis for GVHD in this context is an incompatibility between the grafted (donor) immune system, and the host cells. Therefore, the grafted immune cells attack the host cells, and in oGVHD specifically the cornea and associated structures. Inflammation and immune system hyperactivation is also deleterious in several other conditions that are potentially treatable with Pseudoserum (e.g. osteoarthritis).

[0260] MCP-1 and RANTES which are essentially removed by the S / D treatment, mediate acute and chronic inflammation, and are members of the C-C class of the beta chemokine supergene family with inflammatory properties. IL- 12 family cytokines, also reduced after SD treatment, also play major roles in inflammation and autoimmune disorders. The IL- 12 p40 unit, shown to be reduced by SD treatment above, is shared between IL-12 and IL-23, a member of this family.

[0261] Therefore, advantageously, SD treatment serves to produce a distinctive hypo- inflammatory pseudoserum composition, which is an additional beneficial quality as compared to other blood-derived compositions.

[0262] Example 8 - vEGF reduction by Solvent / Detergent treatment provides an additional benefit for treatment of retinal pathologies

[0263] VEGF inhibitors are a first line treatment for retinal pathologies. Age-related macular degeneration (AMD), diabetic retinopathy and retinal vein occlusions (RVOs) are all associated with vascular leakage and proliferation which can lead to retinal edema, hemorrhaging and vision loss. vEGF inhibition has been shown to be able to reduce vascular leakage and proliferation and, in anywhere from 50-95% of eyes, improve vision in these conditions. This is of special importance provided that recently, blood-derived eye drops (e.g. cord blood serum eye drops) have been recently used to treat retinal pathologies. The presence of vEGF in serum eye drops and other products is a potential detrimental aspect in such a treatment. In the case of solvent / detergent treated Pseudoserum, vEGF levels are greatly reduced without the loss of biological activity (FIG. 5B). Therefore, advantageously, SD-treated pseudoserum would be better suited for the treatment of retinal pathologies than "classic" serum or platelet-derived eye drops.

[0264] Example 9 - TGF-b and PDGF levels in plasma are lower than in serum and PRP- derived compositions, leading to a reduced risk of corneal fibrosis and scarring

[0265] An additional advantage of pseudoserum over existing developments is the relatively low levels of TGF-b and PDGF in plasma as compared to serum or PRP-derived compositions such as PRGF. TGF-b is a well-established inducer of epithelial to mesothelial transition (EMT) which is implicated in fibrosis. In the context of corneal healing, TGF-b has been demonstrated to induce myofibroblast differentiation via EMT, a process which is proposed to also cause corneal scarring and vision impairment (corneal "haze"), and also involves PDGF. The levels of TGF-b as well as PDGF in serum and in PRGF (generated from Platelet Rich Plasma - PRP by activation with CaCh) are several fold higher than in plasma. TGF-b levels were reported at levels up to approximately 30 ng / ml for serum, and as high as about 70 ng / ml for PRGF derived from activated PRP, as opposed to the approximately 2-4 ng / ml demonstrated herein (and known in the literature) for plasma and pseudoserum. PDGF is also implicated in this process and its levels were demonstrated to reach levels of up to about 37 ng / ml in PRGF, a platelet-rich-plasma - derived preparation, in the same studies. The levels of PDGF in whole blood derived serum were determined to be approximately 17.5 ng / ml whereas in plasma they were very low (<0.3 ng / ml) to undetectable. The levels detected in the Luminex assay above are higher than this for plasma and pseudoserum, but still much lower than reported for serum or PRGF (between about 2-4 ng / ml), and are greatly reduced (to approximately 0.3- 0.4 ng / ml) after Solvent / Detergent treatment. Platelet lysates also present higher levels than in plasma of PDGF (which is derived from platelets) and TGF-b.

[0266] Thus, in conclusion, pseudoserum, especially after Solvent / Detergent treatment, represents a composition with a lower propensity for fibrosis and corneal scarring compared to existing treatments (serum and platelet-derived compositions). In the context of chronic disease, such as dry eye secondary to graft vs. host disease, in which a patient would receive a long-term (even life-long) treatment of such a composition, this is a key differentiating element of the composition.

[0267] Example 10 - Heat inactivation of Pseudoserum does not impede biological activity Plasma derived medicinal products (PDMPs) are typically required to undergo two orthologous viral inactivation steps. This means that an additional generalist step (i.e. - inactivating viruses based on a physiochemical principle and not targeting specific viruses) may be required to ensure the viral safety of pharmaceutical products that are based on blood, including pseudoserum. Furthermore, S / D-treatment is highly effective against enveloped viruses, but less so against non-enveloped viruses such as the common blood-borne B19 parvovirus, or Hepatitis A. One such mechanism is heat inactivation. Various protocols of heat inactivation have been described for PDMPs, from as low as 50°C for 3-5 hours to completely inactivate viruses in plasma, and up to the commonly used industry standard of 60°C for 10 hours (e.g. Flebogamma® 5% solution).

[0268] Therefore, heat treatment of the pseudoserum was performed at different temperatures and for different time periods within this temperature range (FIGs 7A-7C). To test the bioactivity of the pseudoserum, the heat-inactivated material was tested on primary corneal epithelial cells (hPCEC), testing the ability of the treated material to rescue these cells from hyperosmolar stress and induce proliferation, quantified using the WST1 colorimetric assay as exemplified herein in previous Examples. FBS was included as a positive control, and plasma as a negative control. A single effective concentration of 30% was used.

[0269] Advantageously, as can be seen in FIGs 7A-7C pseudoserum is superior to nonprocessed plasma, and retains biological activity after being subjected to heat-inactivation for about Ih and up to about lOh, at about 50°C to about 60°C, including retaining biological activity related to rescue / enhanced resistance from hyperosmolar stress measured as proliferation, under conditions that include NaCl to approximately 500 mOsm (FIGs 7A-7B), as well as proliferation under normal growth conditions (FIG 7C).

[0270] Therefore, Pseudoserum can be virally inactivated using two orthologous, known in the art methods, as is typically required by regulatory authorities for any plasma-derived medicinal product, and ensuring a higher level of viral safety than any product produced by non-industrial methods.

[0271] A pooled product, produced from the blood plasma of numerous donors, confers additional advantages on such a product such as the reduction (by dilution) of anti-HLA and anti-HNA antibodies and their associated adverse responses, the neutralization of Anti-A or Anti-B isoagglutinin response, and an increased consistency obtained by averaging. The pooling of plasma from numerous donors actually increases the viral safety as many donors carry antibodies against viruses, especially Hepatitis A and B for which an efficient vaccine is available and commonly given. This is an added layer of protection for viruses for which there are no tests available such as emerging or rare viruses. In a large pool there are potential donors who have recovered from such diseases and generated effective antibodies. These are all additional advantages of a pharmaceutical pseudoserum composition that would be derived from numerous blood plasma donations over the typical allogenous serum eye drops that are prepared from single donors or a limited size pool.

[0272] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.

Claims

CLAIMS1. A pseudoserum derived from acellular plasma at least partially depleted of fibrinogen and fibrin, the pseudoserum comprises less than about 30% of an initial amount / concentration of fibrinogen present in the acellular plasma.

2. The pseudoserum of claim 1, wherein the acellular plasma is derived from whole blood samples and / or plasma donors.

3. The pseudoserum of claim 1 or 2, wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of activation agent.

4. The pseudoserum of any one of claims 1-3, wherein the depletion comprises removal of at least about 80% of the amount / volume of a fibrin -containing clot, formed in the presence of an activating agent.

5. The pseudoserum of claim 3 or 4, wherein the activating agent comprises calcium salt.

6. The pseudoserum of any one of claims 1-5, wherein the depletion comprises removal of other coagulation factors.

7. The pseudoserum of any one of claims 1-6, inactivated for a presence of viruses; and wherein the viral inactivation comprises viral inactivation by a solvent / detergent (S / D) method and / or a heat inactivation method.

8. The pseudoserum of claim 7, characterized by a hypo -inflammatory cytokine and / or growth factor content / composition of the virally-inactivated pseudoserum relative to non-inactivated pseudoserum or non-inactivated plasma; and wherein the hypo- inflammatory cytokine and / or growth factor content / composition comprises reduced amount / concentration of at least one of: IL-8, Eotaxin, GRO-alpha, IL12p70, MIP-1- beta, VEGF-A, IP- 10, TNF-alpha, IL12p40, PDGF AA, MCP-1, PDGF AB / BB RANTES, or any combination thereof.

9. The pseudoserum of claim 8, wherein the hypo -inflammatory cytokine and / or growth factor content / composition of the virally-inactivated pseudoserum relative to noninactivated pseudoserum or non-inactivated plasma comprises reducedamount / concentration of cytokine or growth factor comprises reduction of at least VEGF-A.

10. The pseudoserum of any one of claims 7-9, further characterized by tolerance to viral inactivation.

11. The pseudoserum of claim 10, wherein the tolerance to viral inactivation comprises at least partial retention of amount / concentration of cytokine and / or growth factor of the virally-inactivated pseudoserum relative to amount / concentration of cytokine and / or growth factor of non-inactivated pseudoserum or non-inactivated plasma; and wherein the retention of the amount / concentration of cytokine and / or growth factor comprises retention of amount / concentration of at least one of: FLT-3L, INF-gamma, IL-IRA, IL- 2, IL-5, IL-7, IL-15, TGF-alpha, IL-9, IL-17A, IL17F, MCP-3, FGF-2, Fractalkine, IL- 13, IL-22, IL-l-alpha, IL-l-beta, EGF, IL-18, TGF-beta-2, IL-17E / IL-25, and TGF- beta-1, or any combination thereof.

12. The pseudoserum of any one of claims 10-11, wherein the tolerance to viral inactivation comprises at least partial retention of a biological activity of a cell subjected to application of the virally-inactivated pseudoserum relative to respective cell subjected to application of non-inactivated pseudoserum; and wherein the retention of the biological activity of the cell comprises retention of proliferation and / or retention of resistance to hyperosmolar stress.

13. The pseudoserum of any one of claims 1-12, characterized by promoting a superior biological activity of a cell subjected to application of the pseudoserum relative to respective cell subjected to application of non-processed plasma; and wherein the superior biological activity comprises increase in cellular proliferation and / or increase in resistance to hyperosmolar stress.

14. The pseudoserum of any one of claims 11-13, wherein the cell comprises an epithelial cell, an endothelial cell and / or an immune cell.

15. The pseudoserum of any one of claims 11-14, wherein the cell comprises an epithelial cell or an endothelial cell.

16. The pseudoserum of any one of claims 1-15, further characterized by promoting a superior biological activity of a cell subjected to application of the pseudoserum relative to respective cell subjected to application of non-processed plasma; and wherein the superior biological activity comprises increase in concentration / amount of cytokine release of at least one cytokine from the group consisting of IL-6, IL-8, and IL- 10, or any combination thereof.

17. The pseudoserum of claim 16, wherein the cell comprises an epithelial cell, an endothelial cell and / or an immune cell.

18. The pseudoserum of claim 17, wherein the immune cell comprises an LPS-activated primary blood mononuclear cell (PBMC).

19. The pseudoserum of any one of claims 1-18, further characterized by facilitating a dosedependent biological activity of a cell.

20. The pseudoserum of claim 19, wherein the cell comprises an epithelial cell, an endothelial cell and / or an immune cell.

21. A composition comprising the pseudoserum according to any one of claims 1-20, and at least one pharmaceutical excipient.

22. The pseudoserum of any one of claims 1-20, or the composition of claim 21, for use in treating or preventing an ocular disease.

23. The pseudoserum or the composition for use according to claim 22, wherein the ocular disease comprises one or more of: ocular graft vs. host disease (oGVHD), dry eye or other ophthalmic conditions secondary to chronic graft vs. host disease, scleritis, keratitis, corneal ulcer or abrasion, corneal neovascularization, Fuchs dystrophy, keratoconjunctivitis, iritis, uveitis, cataract, chorioretinal inflammation, posterior cyclitis, chorioretinal scars, chorioretinal degeneration, choroidal dystrophy, choroidal haemorrhage or rupture, choroidal detachment, retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration, macular degeneration, retinitis pigmentosa, macular edema, glaucoma, floaters, optic neuropathy, optic disc drusen, amblyopia, scotoma, nyctalopia, red eye, xerophthalmia, or blindness, or any combination thereof.

24. The pseudoserum or the composition for use according to any one of claims 22-23, comprising at least one more therapeutic ingredient and / or composition.

25. The pseudoserum or the composition for use according to claim 24, wherein administration of the pseudoserum or the composition comprises administration with the at least one more therapeutic ingredient / composition concomitantly, or separately in a sequential manner.

26. The pseudoserum or the composition for use according to any one of claims 22-25, administered as a topical agent.

27. The pseudoserum or the composition for use according to any one of claims 22-26, administered to the eye by dropping a solution.

28. A method for treating or preventing an ocular disease, the method comprises administering to a subject in need thereof a therapeutically effective amount of the pseudoserum of any one of claims 1-20, or the composition of claim 21.

29. A method for producing a pseudoserum, the method comprises the steps of:(v) obtaining acellular plasma from whole blood samples or plasma donations;(vi) adding a calcium salt to the acellular plasma, thereby activating the acellular plasma;(vii) at least partially depleting fibrinogen and fibrin; and(viii) recovering the pseudoserum.

30. The method of claim 29, wherein the pseudoserum comprises less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma, and wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of calcium salt.

31. The method of claim 28 or 29, wherein the at least partially depleting comprises removal of at least about 80% of the amount / volume of a fibrin containing clot, formed in the presence of the calcium salt.

32. The method of any one of claims 28-31, wherein the depletion comprises removing other coagulation factors.

33. The method of any one of claims 28-32, wherein obtaining acellular plasma comprises pooling acellular plasma from a plurality of whole blood samples and / or plasma donations.

34. The method of any one of claims 28-33, wherein the obtaining of acellular plasma comprises either apheresis from a plasma donor, or centrifugation of the whole blood samples.

35. The method of any one of claims 28-34, wherein the calcium salt comprises at least one of calcium chloride, calcium sulphate, calcium gluconate, calcium acetate, calcium carbonate, or any combination thereof.

36. The method of any one of claims 28-35, further comprising adding at least one activator of intrinsic coagulation cascade, wherein the activator of intrinsic coagulation cascade is added before, during and / or after adding calcium salt.

37. The method of claim 36, wherein the at least one activator of intrinsic coagulation cascade comprises diatomaceous earth, silica, ellagic acid, and / or kaolin, or any combination thereof.

38. A pseudoserum prepared according to the method of any one of claims 28-37.

39. The pseudoserum of claim 38, comprising less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma, and wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of calcium salt.

40. A kit for treating or preventing an ocular disease, the kit comprising: a container comprising a composition comprising pseudoserum derived from acellular plasma at least partially depleted of fibrinogen and fibrin; and instructions for administering the composition to the eye of a subject in need thereof.

41. The kit of claim 40, wherein the pseudoserum comprises less than about 30% of the initial amount / concentration of fibrinogen present in the acellular plasma, and wherein the initial amount / concentration of fibrinogen in the acellular plasma is an initial amount of fibrinogen in the absence of calcium salt.

42. The kit of any one of claims 40 or 41, further comprising one or more therapeutic ingredients.

43. The kit of claim 42, wherein the one or more therapeutic ingredient is comprised in the container comprising the pseudoserum, or in a separate container.

44. The kit of any one of claims 40-43, wherein the ocular disease comprises one or more of: ocular graft vs. host disease (oGVHD), dry eye or other ophthalmic conditions secondary to chronic graft vs. host disease, scleritis, keratitis, corneal ulcer or abrasion, corneal neovascularization, Fuchs dystrophy, keratoconjunctivitis, iritis, uveitis, cataract, chorioretinal inflammation, posterior cyclitis, chorioretinal scars, chorioretinal degeneration, choroidal dystrophy, choroidal haemorrhage or rupture, choroidal detachment, retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, age-related macular degeneration, macular degeneration, retinitis pigmentosa, macular edema, glaucoma, floaters, optic neuropathy, optic disc drusen, amblyopia, scotoma, nyctalopia, red eye, xerophthalmia, or blindness, or any combination thereof.

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