Compositions of plasma fractions and plasma subfractions, and their use in the treatment of diseases.

Abstract

Methods and compositions for treating and / or preventing diseases are described. The compositions used in these methods include fractions and subfractions of donor-derived plasma that are effective in treating and / or preventing diseases of both the central nervous system and peripheral organs and tissues. Methods for preparing the compositions are also described.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to the filing dates of U.S. Provisional Application No. 63 / 470,325, filed on June 1, 2023, and U.S. Provisional Application No. 63 / 556,021, filed on February 21, 2024, according to 35 U.S.C.§119(e); the disclosures of those applications are incorporated herein by reference.

[0002] Field The present invention relates to the prevention and treatment of diseases. The present invention relates to the use of plasma fractions and their sub - fractions for treating and / or preventing symptoms often associated with aging, such as cognitive impairment, movement disorders, degenerative disorders, and inflammation.

Background Art

[0003] The following is provided only as background information and is not admitted as prior art for the present invention.

[0004] Aging is a significant risk factor for several human diseases, including cognitive impairment, neurodegeneration, cancer, arthritis, vision loss, osteoporosis, diabetes, cardiovascular disease, muscle degeneration, inflammation (including neuroinflammation), and stroke. In addition to normal synaptic loss during natural aging, synaptic loss is a common early pathological event in many neurodegenerative conditions and is most closely correlated with neuronal and cognitive impairments associated with these conditions. Therefore, aging remains the single most dominant risk factor for dementia-related neurodegenerative diseases such as Alzheimer's disease (AD) (Bishop, NA et al., Neural mechanisms of aging and cognitive decline. Nature 464(7288), 529-535 (2010); Heeden, T. et al., Insights into the aging mind: a view from cognitive neuroscience. Nat. Rev. Neurosci. 5(2), 87-96 (2004); Mattson, MP, et al., Ageing and neuronal vulnerability. Nat. Rev. Neurosci. 7(4), 278-294 (2006)). Aging affects all tissues and functions of the body, including the central nervous system (CNS), and neurodegeneration and declines in function, such as cognitive or motor skills, can have a serious impact on quality of life. Treatment of cognitive decline, motor impairment, and neurodegenerative disorders has had limited success in preventing and reversing impairment. Therefore, it is important to identify new treatments to maintain cognitive integrity by protecting against, counteracting, or reversing the effects of aging.

[0005] Furthermore, aging affects not only central nervous system-related diseases but also peripheral system diseases. These include the cardiovascular system (e.g., peripheral artery disease), the musculoskeletal system (e.g., muscle degeneration and osteoporosis), and the immunomodulatory system (e.g., inflammation).

[0006] Plasma fractions have previously been shown to be effective in reversing the effects of certain age-related diseases and associated symptoms in both the central nervous system (CNS) and the periphery (see, for example, U.S. Patent Publication Nos. 20210128693, 20220370568, 20210145875, 20180110839, 20170340671, and 20180311280). However, the supply of such plasma fractions is limited to the supply of offerings from plasma donors, because the therapeutic fraction ultimately derives from this offering pool. The blood fractionation process has become more efficient over the decades since its initial development in the 1940s. However, inefficiencies remain from fractions that produce unused compositions, including blood fraction precipitates. These are sometimes referred to as fractionation waste. One such precipitate is plasma fraction IV-1 paste or precipitate. Therefore, it is necessary to identify the potential therapeutic uses of such underutilized products. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 20210128693 [Patent Document 2] U.S. Patent Application Publication No. 20220370568 [Patent Document 3] U.S. Patent No. 20210145875 [Patent Document 4] U.S. Patent No. 20180110839 [Patent Document 5] U.S. Patent No. 20170340671 [Patent Document 6] U.S. Patent No. 20180311280 [Non-patent literature]

[0008] [Non-Patent Document 1] Bishop,NAet al.,Neural mechanisms of aging and cognitive decline.Nature 464(7288),529-535(2010);Heeden,T.et al.,Insights into the aging mind:a view from cognitive neuroscience.Nat.Rev.Neurosci.5(2),87-96(2004);Mattson,MP,et al.,Aging and neural vulnerability.Nat.Rev.Neurosci.7(4),278-294(2006) [Overview of the Initiative]

[0009] This invention is based on the production and use of blood products for treating and / or preventing diseases, including those related to aging. This invention explores novel efficiencies in the use of plasma fractions. These novel efficiencies include the identification of subfractions of plasma fraction IV-1 paste (or precipitate) that can be used to treat diseases. These subfractions of plasma fraction IV-1 paste also provide compositions for treating diseases.

[0010] One embodiment of the present invention involves treating a subject diagnosed with a disease or disorder by administering to the subject an effective amount of a subfraction of plasma fraction IV-1 paste. Another embodiment of the present invention involves administering an effective amount of a subfraction of plasma fraction IV-1 paste and subsequently monitoring the subject for improvement of symptoms associated with the disease or disorder. Another embodiment of the present invention involves treating a subject diagnosed with a disease or disorder by administering to the subject an effective amount of a subfraction of plasma fraction IV-1 paste, wherein the subfraction of plasma fraction IV-1 paste is administered in a manner that results in improvement of the disease or disorder, including associated symptoms.

[0011] The present invention also recognizes that differences in protein content between different plasma fractions (e.g., fractions, effluents, precipitates / cold precipitates, pastes, plasma protein fractions, human albumin solutions) may cause prevention and / or improvement of certain symptoms or underlying causative factors of diseases, including age-related diseases. For example, such symptoms or underlying causative factors include cognitive or motor impairment and the alleviation of neurodegenerative diseases. Also, as an example rather than an limitation, embodiments of the present invention demonstrate that simply higher concentrations of a single protein factor may not be the driving force behind improvement when treating a disease. For example, a single protein factor (e.g., alpha-1 antitrypsin or antithrombin III) concentrated in and derived from factor IV-1 paste may not have the same therapeutic profile or effect as a subfraction of factor IV-1 paste.

[0012] Blood and plasma from young donors showed improvement and reversal of pre-existing effects of brain aging, including at the molecular, structural, functional, and cognitive levels. (Saul A. Villeda, et al. Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nature Medicine 20 659-663 (2014)). This invention relates to plasma fractions and effluents (some of which have been traditionally used to treat shock in patients), and the discovery that they are effective as methods for treating age-related cognitive impairment, reduced motor function, and neuroinflammatory or neurodegenerative diseases.

[0013] According to aspects of the present invention, a method is provided for treating age-related cognitive impairment, age-related dementia, motor impairment, neuroinflammation, and / or neurodegenerative disease using a plasma blood product fraction. An aspect of the method comprises administering a plasma fraction to an individual who has or is at risk of developing age-related cognitive impairment, motor impairment, neuroinflammation, or neurodegenerative disease. An additional aspect of the method comprises administering a plasma fraction derived from a pool of donors within a specific age range to an individual who has or is at risk of developing age-related cognitive impairment, motor impairment, neuroinflammation, or neurodegenerative disease. A further aspect of the method comprises administering plasma or plasma fractions using a pulsed dosing regimen. Reagents, devices, and kits used to carry out the method are also provided.

[0014] In one embodiment, the plasma fraction may be one of several plasma fractions obtained from a blood fractionation process, such as the Cohn fractionation process described below. In another embodiment, the plasma fraction may be of the type referred to herein as “plasma fraction,” which is a solution containing normal human albumin, alpha and beta globulins, gamma globulin, and other proteins individually or in complex. In yet another embodiment, the plasma fraction may be a type of plasma fraction known to those skilled in the art as “plasma protein fraction” (PPF). In yet another embodiment, the plasma fraction may be a “human albumin solution” (HAS) fraction. In yet another embodiment, the plasma fraction may be one from which substantially all coagulation factors have been removed in order to maintain the effectiveness of the fraction with a reduced risk of thrombosis. Embodiments of the present invention may also include, for example, administering a fraction derived from a young donor or a pool of young donors. Another embodiment of the present invention may include monitoring for cognitive improvement, improved motor function, reduced neuroinflammation, or increased neurogenesis in subjects treated with the plasma fraction.

[0015] Embodiments of the present invention include treating subjects diagnosed with cognitive impairment, neurodegenerative motor disorders, or neuroinflammatory-related diseases by administering an effective amount of plasma or plasma fraction to the subject. Another embodiment of the present invention includes administering an effective amount of plasma or plasma fraction and then monitoring the subject for improvement in cognitive function, improvement in motor function, reduction in neuroinflammation, or increase in neurogenesis. Another embodiment of the present invention includes administering plasma or plasma fraction via a dosing regimen of at least two consecutive days and monitoring the subject for improvement in cognitive function, improvement in motor function, reduction in neuroinflammation, or increase in neurogenesis at least two days after the last dosing day. Further embodiments of the present invention include administering plasma or plasma fraction via a dosing regimen of at least three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen or fourteen days and monitoring the subject for improvement in cognitive function, improvement in motor function, reduction in neuroinflammation, or increase in neurogenesis at least three days after the last dosing day. A further embodiment of the present invention involves administering plasma or plasma fractions via a dosing regimen of at least two consecutive days, and after the last dosing day, and monitoring for cognitive improvement, motor function improvement, neuroinflammation reduction, or neurogenesis increase after the mean half-life of proteins in the plasma or plasma fractions has been reached.

[0016] One embodiment of the present invention involves treating a subject diagnosed with cognitive impairment, motor dysfunction, neuroinflammation, or neurogenesis impairment by administering an effective amount of plasma or plasma fraction to the subject, the subject following an exercise regimen after administration. Another embodiment of the present invention involves the subject following an exercise regimen prescribed to them. Another embodiment of the present invention involves the subject exercising at a higher intensity and / or frequency than they did before administration. Another embodiment of the present invention involves the subject exercising at a similar intensity and / or frequency as they did before administration.

[0017] One embodiment of the present invention includes treating a subject diagnosed with cognitive impairment, motor function impairment, neuroinflammation, or reduced neurogenesis by administering to the subject an effective amount of plasma or a plasma fraction in a subject who has received, is scheduled to receive, or has previously received stem cell therapy. Another embodiment of the present invention includes administering to a subject an effective amount of plasma or a plasma fraction, wherein the subject has received, is scheduled to receive, or has previously received stem cell therapy, and the stem cells used in the treatment can be embryonic stem cells, non-embryonic stem cells, induced pluripotent stem cells (iPSCs), umbilical cord blood stem cells, amniotic fluid stem cells, and the like. Another embodiment of the present invention includes treating a subject diagnosed with traumatic spinal cord injury, stroke, retinal disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, hearing loss, heart disease, rheumatoid arthritis, or severe burns, who has received, is scheduled to receive, or has previously received stem cell therapy, with an effective amount of plasma or a plasma fraction.

[0018] Incorporation by reference All publications and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

[0019] [Figure 1] It is a diagram showing a related manufacturing process for fractionation of plasma. [Figure 2] Reports the dose-response relationship between PPF1 and IV-1 paste. [Figure 3] It is a parallel comparison of the activities of various plasma fractions over in vitro assays. [Figure 4] Shows the separation of IV-1 paste into 13 subfractions by an anion exchange chromatography column. [Figure 5] Coomassie blue staining from 13 subfractions from IV-1 paste. [Figure 6]We report the activity of load (IV-1 paste), flow-through (FT), and 13 subfractions in blood-brain barrier, muscle function, and inflammation assays. [Figure 7] This shows the processing paradigm for isolated primary mouse microglia. [Figure 8] We report the phagocytic activity of treated microglia, quantified by normalized FACS against untreated microglia as shown in Figure 7. [Figure 9] We report the effect of the subfraction of IV-1 paste on a microglial phagocytic assay. [Figure 10] We report the effects of single purified protein products (A1At and ATIII) from IV-1 paste on microglial phagocytic activity. [Figure 11] This is a processing paradigm for analyzing the surface expression of adhesive molecules in HUVEC. [Figure 12] Figure 11 shows the results for individual proteins and plasma fractions in the assay described. [Figure 13] Figure 11 shows the results for individual proteins and plasma fractions in the assay described. [Figure 14] Figure 11 shows the results for individual proteins and plasma fractions in the assay described. [Figure 15] We report the effects of 13 sub-fractions of fraction IV-1 paste on the adhesive molecules shown in Figure 11 (VCAM1). [Figure 16] We report the effects of 13 sub-fractions of fraction IV-1 paste on the adhesive molecules shown in Figure 11 (ICAM1). [Figure 17] We report the effects of 13 sub-fractions of fraction IV-1 paste on the adhesive molecules shown in Figure 11 (CD62E). [Figure 18] We report the effect of single protein products purified from fraction IV-1 paste (A1AT and ATIII) on the surface expression of adhesion molecules. [Figure 19]We report the effect of single protein products purified from fraction IV-1 paste (A1AT and ATIII) on the surface expression of adhesion molecules. [Figure 20] We report the effect of single protein products purified from fraction IV-1 paste (A1AT and ATIII) on the surface expression of adhesion molecules. [Figure 21] We report the effect of single protein products purified from fraction IV-1 paste (A1AT and ATIII) on the surface expression of adhesion molecules. [Figure 22] This describes the processing paradigm for barrier function assays. [Figure 23] We report the effects of fraction IV-1 paste and PPF1 on the normalized relative TEER after pretreatment. [Figure 24] This is a dose-response study of fraction IV-1 paste 72 hours after processing using the TEER assay. [Figure 25] The results of the TEER assay using cells treated with fraction IV-1 paste, flow-through (FT), and sub-fractions 1-13 from fraction IV-1 paste are shown. [Figure 26] The results of TEER assays using cells treated with fraction IV-1 paste, various concentrations of the single protein alpha-1 antitrypsin (A1AT), and various concentrations of the single protein antithrombin III (ATIII) are shown. [Figure 27] This describes a processing paradigm for endothelial cell proliferation assays. [Figure 28] Figure 27 shows the results of the HUVEC endothelial cell proliferation assay. [Figure 29] The results of the time response experiment for HUVEC endothelial cell proliferation assay treated with fraction IV-1 paste are shown. [Figure 30] This describes the processing paradigm for cytokine release assays. [Figure 31] The results of IL-6 release using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) under TNFα stress are shown. [Figure 32] The results of IL-8 release under TNFα stress using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin) are shown. [Figure 33] The results of IL-6 release using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin) under the absence of TNFα stress are shown. [Figure 34] The results of IL-8 release using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin) under the absence of TNFα stress are shown. [Figure 35] The results for 13 Q-Sepharose subfractions from fraction IV-1 paste, tested in a cytokine release assay using a TNFα stressor along with fraction IV-1 paste and flow-through (FT), are shown. [Figure 36] We report the release of IL-8 when 13 (13)Q-Sepharose subfractions were tested in the cytokine release assay described in Figure 30. [Figure 37] We report the release of IL-6 when alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, is tested without the use of a TNFα stressor. [Figure 38] We report the release of IL-8 when alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, is tested without the use of a TNFα stressor. [Figure 39] We report the release of IL-6 when alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, is tested using a TNFα stressor. [Figure 40] We report the release of IL-8 when alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, is tested using a TNFα stressor. [Figure 41]We report the release of IL-6 when antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, is tested without the use of a TNFα stressor. [Figure 42] We report the release of IL-8 when antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, is tested without the use of a TNFα stressor. [Figure 43] We report the release of IL-6 when antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, is tested using a TNFα stressor. [Figure 44] We report the release of IL-8 when antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, is tested using a TNFα stressor. [Figure 45] This is a description of the processing paradigm for the C2C12 myoblast differentiation assay. [Figure 46] Figure 45 shows the results of the effects of 13 subfractions of fraction IV-1 paste on the myotubation assay described above. [Figure 47] We report the effect of alpha-1 antitrypsin (A1AT), a single protein product purified from fraction IV-1 paste, on the myotubation assay described in Figure 45. [Figure 48] We report the effect of antithrombin III (ATIII), a single protein product purified from fraction IV-1 paste, on the myotubation assay described in Figure 45. [Figure 49] This describes the processing paradigm for myotube formation derived from C2C12. [Figure 50] Figure 49 shows the results of the dose-response relationship between PPF1 and fraction IV-1 paste in the glucose utilization assay. [Figure 51] We report the results of the metabolic assay described in Figure 49, using 13 sub-fractions from fraction IV-1 paste. [Figure 52]The results of separating the fraction IV-1 suspension into separate protein pools by Q Sepharose chromatography are shown. [Figure 53] Figure 52 shows the elution pool corresponding to the chromatography. [Figure 54] Figure 52 shows the gel electrophoresis results corresponding to the chromatography. [Figure 55] The following experimental section describes a treatment-paradigm survival assay. [Figure 56] The results of the treatment paradigm survival assay, as illustrated in Figure 55, are provided in the experimental section below. [Figure 57] The following experimental section describes a ROS assay using a processing paradigm. [Figure 58] The results of the processing paradigm ROS assay, as illustrated in Figure 57, are provided in the experimental section below. [Figure 59] The results of survival assays performed on dopaminergic neurons under neurotoxic stress (MPP + 1 mM) using single purified proteins (A1AT and ATIII) are shown. A1AT and ATIII were administered as shown in Figure 55. [Figure 60] The results of a ROS production assay in dopaminergic neurons under peroxide stress (TBHP 50 μM) and single purified proteins (A1AT and ATIII) are shown. A1AT and ATIII were administered as shown in Figure 57. [Figure 61] This shows a heatmap of abundant proteins in fraction IV-1 paste, normalized to the amount of protein in fraction PPF1. [Figure 62] This shows heatmaps of IGF1, IGF2, IGFBP3, and IGFALS proteins in fraction IV-1 paste, normalized to the amount of protein in fraction PPF1. [Modes for carrying out the invention]

[0020] 1. Introduction The present invention relates to the identification and discovery of methods and compositions for treating and / or preventing diseases or physical disorders. Methods and compositions for treating subjects suffering from such diseases and disorders are described herein, and these constitute embodiments of the present invention. Dosage regimens that improve the effectiveness of compositions for treating such diseases / disorders are also described herein. Implementing the present invention involves using one or more fractions or effluents obtained from a blood fractionation process, such as the Cohn fractionation process described below, as a treatment. One embodiment of the present invention involves using a plasma fraction (a solution consisting of normal human albumin, alpha and beta globulins, gamma globulin, and other proteins individually or as a complex (hereinafter referred to as the "plasma fraction")). Another embodiment of the present invention involves using a plasma protein fraction (PPF) as a treatment. Another embodiment of the present invention involves using a human albumin solution (HAS) fraction as a treatment. Yet another embodiment involves using effluents from a blood fractionation process, such as effluent I or effluent II / III described below. Additional embodiments include the use of a fraction IV-1 paste composition, such as a redissolved fraction IV-1 paste, for treating the disease / disorder. Further embodiments include the use of sub-fractions of the redissolved fraction IV-1 paste for treating the disease / disorder. Additional embodiments include the use of one or more of the 13(13) sub-fractions described herein for treating the disease / disorder. Another embodiment of the present invention includes a composition containing sub-fractions of fraction IV-1 paste, including but not limited to the 13 sub-fractions described herein. Further embodiments include a composition obtained from a process of fractionating a fraction IV-1 paste containing the 13 sub-fractions described herein, the process of which is illustrated as described in Example 10.

[0021] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods or compositions described and is therefore naturally subject to change. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0022] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such publications by prior art. Furthermore, the publication dates presented may differ from the actual publication dates and may need to be independently verified.

[0023] Where a range of values ​​is provided, unless explicitly indicated in the context, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit, is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within that stated range is included in the present invention. The upper and lower limits of these smaller ranges may be included in or excluded from the range independently, and each range that includes either limit, does not include either limit, or includes both limits is also included in the present invention, subject to any specifically excluded limits within the stated range. Where a stated range includes one or both limits, the range excluding one or both of the limits that they include is also included in the present invention.

[0024] It should be noted that claims may be drafted to exclude optional elements. Therefore, this statement is intended to serve as a precedent for the use of exclusive terms such as "alone" or "only" in relation to the enumeration of elements in the claims or the use of "negative" limitations.

[0025] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any of the enumerated methods may be performed in the order of the enumerated events, or in any other logically possible order.

[0026] 2.Definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, but some potential and preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference to disclose and describe the relevant methods and / or materials from which the publications are cited. This disclosure is understood to supersede any disclosure in the incorporated publications to the extent that a conflict exists.

[0027] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. For example, a reference to “a cell” includes multiple such cells, and a reference to “the peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0028] In describing the methods of the present invention, the terms “host,” “subject,” “individual,” and “patient” are used interchangeably and refer to any mammal that requires such treatment by the disclosed methods. Such mammals include, for example, humans, sheep, cattle, horses, pigs, dogs, cats, non-human primates, mice, and rats. In certain embodiments, the subject is a non-human mammal. In some embodiments, the subject is livestock. In other embodiments, the subject is a pet. In some embodiments, the subject is a mammal. In certain cases, the subject is a human. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, such as in animal models of diseases), and non-human primates (e.g., chimpanzees and monkeys). Thus, the subjects of the present invention include, but are not limited to, mammals, such as humans and other primates, such as chimpanzees and other apes and monkey species, and in certain embodiments, the subject is a human. The term "subject" also includes any person or organism of any age, weight or other physical characteristics, and the subject may be an adult, child, infant or newborn.

[0029] "Individuals with or at risk of developing age-related cognitive impairment" refers to individuals from approximately 50% to their expected lifespan, for example, from over 60%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or even over 99% to their expected lifespan. The age of the individual depends on the species in question. Therefore, this percentage is based on the expected life expectancy of the species in question. For example, in humans, such individuals are 50 years of age or older, e.g., 60 years of age or older, 70 years of age or older, 80 years of age or older, 90 years of age or older, and usually 100 years of age or younger, e.g., 90 years of age or older, i.e., between approximately 50 and 100 years of age, e.g., 50...55...60...65...70...75...80...85...90...95...100 years of age or older, or any age between 50 and 1000 years of age or older; they still have symptoms of aging-related symptoms, e.g., cognitive impairment. Individuals who are approximately 50 years of age or older, for example 60, 70, 80, or 90 years of age or older, and usually 100 years of age or younger, i.e., between approximately 50 and 100 years of age, for example 50...55...60...65...70...75...80...85...90...95...100 years of age; individuals of any age suffering from cognitive impairment due to age-related disease, as further described below, and individuals of any age diagnosed with age-related disease with cognitive impairment, who have not yet begun to show symptoms of cognitive impairment. The corresponding ages for non-human subjects are publicly known and are intended to be applied herein.

[0030] As used herein, “treatment” means either (i) prevention of disease or disorder, or (ii) reduction or elimination of the symptoms of disease or disorder. Treatment may be performed prophylactically (before the onset of disease) or therapeutically (after the onset of disease). The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. Accordingly, as used herein, the term “treatment” encompasses any treatment of age-related diseases or disorders in mammals, including (a) preventing the onset of disease in subjects who may be predisposed to the disease but have not yet been diagnosed with it; (b) inhibiting the disease, i.e., stopping its onset; or (c) mitigating the disease, i.e., causing disease regression. Treatment may result in a variety of different physical manifestations, such as regulation of gene expression, rejuvenation of tissue or organ, etc. Therapeutic agents may be administered before, during, or after the onset of disease. Treatment of an ongoing disease is of particular interest if the treatment stabilizes or reduces the patient's undesirable clinical symptoms. Such treatment may be performed before complete loss of function in the affected tissue. Targeted therapy may be administered during the symptomatic phase of the disease, and possibly after.

[0031] In some embodiments, the symptom being treated is an impairment of cognitive ability in an individual. Cognitive ability or “cognition” means mental processes including attention and concentration, learning of complex tasks and concepts, memory (acquiring, retaining, and retrieving new information in the short and / or long term), information processing (processing information gathered by the five senses), visuospatial functioning (visual perception, depth perception, use of mental images, copying drawings, constructing objects or shapes), language production and comprehension, language fluency (word discovery), problem solving, decision-making, and executive functioning (planning and prioritizing). “Cognitive decline” means a progressive decline in one or more of these abilities, e.g., a decline in memory, language, thinking, judgment, etc. “Impairment of cognitive ability” and “cognitive impairment” mean a reduction in cognitive ability compared to a healthy individual, e.g., a healthy individual of age-matched age, or to the ability of the individual at an earlier point in time, e.g., two weeks ago, one month ago, two months ago, three months ago, six months ago, one year ago, two years ago, five years ago, or ten years ago or earlier. "Age-related cognitive impairment" typically refers to cognitive impairments associated with aging, including, for example, cognitive impairments associated with the natural aging process, such as mild cognitive impairment (MCI); and cognitive impairments associated with age-related disorders, i.e., impairments that become more frequent with the progression of old age, such as cognitive impairments associated with neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, and vascular dementia.

[0032] In some embodiments, the symptoms being treated are age-related impairments of motor ability in an individual. Motor ability refers to motor processes, including the ability to perform complex muscular and neural actions that produce movements such as fine motor skills (e.g., writing, tying shoelaces) that produce small or precise movements and gross motor skills (e.g., walking, running, kicking) that produce large movements. "Motor decline" means a progressive decrease in one or more of these abilities, e.g., a decline in finding movements or gross motor skills. "Motor impairment" and "motor impairment" mean a reduction in motor ability / skills compared to a healthy individual, e.g., an age-matched healthy individual, or compared to the ability of the individual at an earlier point in time, e.g., two weeks ago, one month ago, two months ago, three months ago, six months ago, one year ago, two years ago, five years ago, or ten years ago or earlier. "Aging-related motor impairment" typically refers to impairment or decline in motor function associated with aging, including, for example, motor impairments associated with the natural aging process and motor impairments or declines associated with aging-related disorders, i.e., disorders that are seen more frequently with the progression of old age, such as neurodegenerative conditions like Parkinson's disease and amyotrophic lateral sclerosis.

[0033] In some embodiments, the symptom being treated is an increase in neuroinflammation in an individual. “Neuroinflammation” refers to the biochemical and cellular response of the nervous system to injury, infection, or neurodegenerative disease. Such a response aims to reduce the triggers by engaging the central nervous system immune system to defend against potential harm. Neurodegeneration occurs in the central nervous system and is characterized by the loss of structure and function of neurons. Neuroinflammatory diseases or neuroinflammation-related symptoms or conditions include, but are not limited to, neurodegenerative diseases such as Alzheimer's disease; Parkinson's disease, multiple sclerosis, and others.

[0034] Blood products containing plasma components. When performing the method, the blood product containing plasma components is administered to an individual in need, for example, an individual who has or is at risk of having cognitive or motor impairment, neuroinflammation, and / or age-related dementia. Accordingly, the method according to embodiments of the present invention involves administering a blood product containing plasma components from an individual ("donor individual" or "donor") to an individual ("recipient individual" or "recipient") who is at risk of having or has at least cognitive or motor impairment, neuroinflammation, neurodegeneration, and / or age-related dementia. "Blood product containing plasma components" means any product derived from blood containing plasma (e.g., whole blood, plasma, or fractions thereof). The term "plasma" is used in its conventional sense and refers to the straw-yellow / pale-yellow liquid component of blood, which consists of about 92% water, 7% proteins (such as albumin, gamma globulin, antihemophilic factors, and other coagulation factors), and 1% mineral salts, sugars, fats, hormones, and vitamins. Non-limiting examples of plasma-containing blood products suitable for use in the methods described include whole blood treated with anticoagulants (e.g., EDTA, citrate, oxalate, heparin), blood products produced by filtering whole blood to remove leukocytes ("leukocyte reduction"), blood products derived from plasma apheresis or plasma derived from apheresis, fresh frozen plasma, blood products essentially consisting of purified plasma, and blood products essentially consisting of plasma fractions. In some cases, the plasma product used is a non-whole blood plasma product, meaning that the product is not whole blood and therefore lacks one or more components found in whole blood, such as red blood cells and leukocytes, to at least the extent that these components are present in whole blood. In some cases, the plasma product is substantially, if not completely, cell-free, in which case the cell content may be less than 5% by volume, e.g., less than 1% (including less than 0.5%), and in some cases, the cell-free plasma fraction is a composition that is completely cell-free, i.e., cell-free.

[0035] Recovery of blood products containing plasma components. Embodiments of the method herein involve the administration of blood products containing plasma components that may originate from donors, including human volunteers. The term “human-derived” may refer to such products. Methods for collecting plasma containing blood products from donors are well known in the art. (See, for example, the AABB Technical Manual, (Mark A. Fung, et al., eds., 18th ed. 2014), incorporated herein by reference).

[0036] In one embodiment, the offering is obtained by venipuncture. In another embodiment, venipuncture is a single vein puncture only. In another embodiment, volume displacement with saline is not used. In a preferred embodiment, plasma apheresis is used to obtain plasma containing the blood product. Plasma apheresis may involve removing a weight-adjusted volume of plasma by returning cellular components to the donor. In a preferred embodiment, sodium citrate is used during plasma apheresis to prevent cellular coagulation. The volume of plasma recovered from the donor is preferably 690–880 mL after citrate administration and preferably in harmony with the donor's body weight.

[0037] 3. Plasma fraction During World War II, there was a need for a stable plasma expander that could be used on the battlefield when soldiers lost large amounts of blood. As a result, a method for preparing lyophilized plasma was developed. However, the use of lyophilized plasma was difficult in combat situations because it required sterile water for reconstitution. As an alternative, Dr. E.J. Cohn suggested the use of albumin and prepared a readily available, stable solution that could be immediately administered for the treatment of shock. (See Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's procedure for purifying plasma fractions utilizes cold ethanol for its denaturing effect and changes in pH and temperature to achieve separation.

[0038] One embodiment of the method described herein involves administering a plasma fraction to a subject. Fractionation is the process by which a specific protein subset is separated from plasma. Fractionation techniques are known in the art and rely on steps developed by Cohn et al. in the 1940s. (Incorporated herein by reference, E. Cohn, Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. 68 J Am Chem Soc 459 (1946)). This process involves several steps, each step involving a specific ethanol concentration, as well as pH, temperature, and gravimetric osmolality shifts that result in selective protein precipitation. The precipitate is also separated by centrifugation or precipitation. The original “Cohn fractionation process” involved the separation of proteins into five fractions, called fraction I, fraction II+III, fraction IV-1, fraction IV-4, and fraction V, via precipitation. Albumin was the first endpoint (fraction V) product identified in this process. According to embodiments of the present invention, each fraction (or effluent from the previous separation step) contains, or potentially contains, a therapeutically useful protein fraction.(See Thierry Burnouf, Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101 (2007); Adil Denizli, Plasma fractionation: conventional and chromatographic methods for albumin purification, 4 J. Biol. & Chem. 315, (2011); and T. Brodniewicz-Proba, Human Plasma Fractionation and the Impact of New Technologies on the Use and Quality of Plasma-derived Products, 5 Blood Reviews 245 (1991), and U.S. Patents 3,869,431, 5110,907, 5219,995, 7531,513 and 8772,461, which are incorporated herein by reference). The above experimental parameters can be adjusted to obtain specific protein fractions.

[0039] More recently, fractionation has reached a greater complexity, and therefore includes additional embodiments of the present invention. This recent increase in complexity arises from: the introduction of chromatography, resulting in the isolation of new proteins from existing fractions, e.g., cold precipitates or pastes, cryo-poor plasma, and Cohn fractions; increased IgG recovery by integrating chromatography and ethanol fractionation processes; and reduction / inactivation / removal of viruses. (Ibid.) Anion exchange chromatography can be utilized to capture proteins at physiological pH and ionic strength (e.g., using Q-Sepharose columns). This maintains the functional activity of the proteins and / or protein fractions. Heparin and monoclonal antibodies are also used in affinity chromatography. Those skilled in the art will recognize that the above parameters can be adjusted in particular to obtain desired plasma protein-containing fractions.

[0040] In one embodiment of the present invention, plasma is fractionated in an industrial environment. Frozen plasma is thawed at 1°C to 4°C. Continuous refrigerated centrifugation is applied to the thawed plasma to isolate the cryo-precipitate. The recovered cryo-precipitate is frozen and stored at -30°C or below. Plasma poor in cryo-precipitate ("cryo-poor") is immediately processed (e.g., via primary chromatography) for the capture of unstable coagulation factors such as factor IX complex and its components, as well as protease inhibitors such as antithrombin and C1 esterase inhibitors. Sequential centrifugation and precipitate isolation can be applied in subsequent steps. Such techniques are known to those skilled in the art and are described, for example, in U.S. Patent Nos. 4,624,780, 5,219,995, 5,288,853, and U.S. Patent Applications Nos. 2014,034,3255 and 2015,034,3025, the disclosures of which are incorporated herein by reference in their entirety.

[0041] In one embodiment of the present invention, the plasma fraction may include a plasma fraction containing a substantial concentration of albumin. In another embodiment of the present invention, the plasma fraction may include a plasma fraction containing a substantial concentration of IgG or intravenous immunoglobulin (IGIV) (e.g., Gamunex-C®). In another embodiment of the present invention, the plasma fraction may include an IGIV plasma fraction, such as Gamunex-C®, in which immunoglobulin (IgG) is substantially depleted by a method well known to those skilled in the art, e.g., protein A-mediated depletion. (See Keshishian, H., et al., Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury, Molecular & Cellular Proteomics, 14 at 2375-93 (2015)). In an additional embodiment, the plasma fraction may be from which substantially all coagulation factors have been removed in order to maintain the effectiveness of the fraction with a reduced risk of thrombosis. For example, the plasma fraction may be a plasma fraction as described in U.S. Patent No. 62 / 376,529, filed on 18 August 2016; its disclosure is incorporated herein by reference in its entirety.

[0042] 4. Albumin products Those skilled in the art will recognize two common categories of albumin plasma preparations ("APPs"): plasma protein fractions ("PPFs") and human albumin solutions ("HASs"). PPFs are derived from processes that yield higher yields than HASs but have lower minimum albumin purity than HASs (over 83% for PPFs and over 95% for HASs). (Production of human albumin solution: a continually developing colloid, P. Matejtschuk et al., British J. of Anaesthesia 85(6):887-95, at 888(2000)). In some cases, PPFs have albumin purity of 83%–95% or alternatively 83%–96%. Albumin purity can be determined by electrophoresis or other quantitative assays such as mass spectrometry. Furthermore, some note that PPFs have drawbacks due to the presence of protein "impurities" such as PKAs. As a result, PPF preparations have lost popularity as albumin plasma preparations and have been deregistered from the pharmacopoeias of certain countries. Contrary to these concerns, the present invention makes beneficial use of these “contaminants.” In addition to α, β, and γ globulins, as well as the aforementioned PKA, the methods of the present invention utilize additional proteins or other factors within the “contaminants” that promote processes such as neurogenesis, neuronal cell survival, improvement of cognitive or motor function, and reduction of neuroinflammation.

[0043] Those skilled in the art will recognize that several commercial sources of PPF exist or have existed ("Commercial PPF Preparations"). These include Plasma-Plex® PPF (Armour Pharmaceutical Co., Tarrytown, NY), Plasmanate® PPF (Grifols, Clayton, NC), Plasmatein® (Alpha Therapeutics, Los Angeles, CA), and Protenate® PPF (Baxter Labs, Inc., Deerfield, IL).

[0044] Those skilled in the art will also recognize that several commercial sources of HAS ("Commercially Available HAS Preparations") exist or have existed. These include Albuminar® (CSL Behring), AlbuRx® (CSL Behring), Albutein® (Grifols, Clayton, NC), Buminate® (Baxatla, Inc., Bannockburn, IL), Flexbumin® (Baxatla, Inc., Bannockburn, IL), and Plasbumin® (Grifols, Clayton, NC).

[0045] a. Plasma protein fraction (human) (PPF) According to the U.S. Food and Drug Administration ("FDA"), "Plasma Protein Fraction (Human)," or PPF, is the formal name of a product defined as "a sterile solution of proteins consisting of albumin and globulin derived from human plasma" (21 CFR 640.90, Code of Federal Regulations "CFR" incorporated herein by reference). The source material for PPF is plasma recovered from whole blood prepared as specified in 21 CFR 640.1–640.5 (incorporated herein by reference), or source plasma prepared as specified in 21 CFR 640.60–640.76 (incorporated herein by reference).

[0046] PPF is tested to determine that it meets the following criteria according to 21 CFR 640.92 (incorporated herein by reference): (a) The final product shall be a 5.0+ / -0.30% protein solution; and (b) The total protein in the final product shall consist of at least 83% albumin and 17% or less globulin. Gamma globulin shall constitute 1% or less of the total protein. The protein composition shall be determined by a method approved by each manufacturer by the Director of the Center for Biologics Evaluation and Research, Food and Drug Administration.

[0047] As used herein, “Plasma Protein Fraction” or “PPF” refers to a sterile solution of proteins composed of albumin and globulin derived from human plasma, which, as determined by electrophoresis, contains at least 83% albumin, 17% or less globulin (including α1, α2, β, and γ globulins) and other plasma proteins, and 1% or less gamma globulin. (Hink, JH, Jr., et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174)(1957)). PPF may also refer to a solid form having a similar composition when suspended in a solvent. The total globulin fraction can be determined by subtracting albumin from the total protein. (Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY, PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, Walker HK, Hall WD, Hurst JD, eds. (1990)).

[0048] b. Albumin (human) (HAS) According to the FDA, “Human Albumin” (also known herein as “HAS”) is the formal name of the product defined as “Sterile Solution of Albumin Derived from Human Plasma” (21 CFR 640.80, Code of Federal Regulations “CFR” incorporated herein by reference). The source material for Human Albumin is plasma recovered from whole blood prepared as specified in 21 CFR 640.1–640.5 (incorporated herein by reference), or source plasma prepared as specified in 21 CFR 640.60–640.76 (incorporated herein by reference). Other requirements for Human Albumin are listed in 21 CFR 640.80–640.84 (incorporated herein by reference).

[0049] Test human albumin to determine if it meets the following criteria according to 21 CFR 640.82.

[0050] (a) Protein concentration. The final product shall conform to one of the following concentrations: 4.0+ / -0.25%; 5.0+ / -0.30%; 20.0+ / -1.2%; and 25.0+ / -1.5% protein solution.

[0051] (b) Protein composition. At least 96% of the total protein in the final product shall be albumin, determined by a method approved by each manufacturer by the Director of the Center for Biologics Evaluation and Research, Food and Drug Administration.

[0052] As used herein, “Albumin (Human)” or “HAS” refers to a sterile solution of proteins composed of albumin and globulin derived from human plasma, containing at least 95% albumin and 5% or less globulin (including α1, α2, β, and γ globulins) and other plasma proteins. HAS may also refer to a solid form having a similar composition when suspended in a solvent. The total globulin fraction can be determined by subtracting albumin from the total protein.

[0053] As can be recognized by those skilled in the art, PPF and HAS fractions can also be lyophilized or in other solid forms. Such preparations can be used, for example, to make tablets, powders, granules, or capsules with appropriate additives. The solid forms can be incorporated into injectable preparations by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher fatty acids, or propylene glycol; and, if necessary, using conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0054] 5. Coagulation factor reduction fraction Another embodiment of the present invention uses a plasma fraction from which substantially all coagulation factors have been removed in order to maintain the effectiveness of the fraction with a reduced risk of thrombosis. Conveniently, the blood product can be derived from a young donor or a pool of young donors and can be made to lack IgM in order to provide a young blood product that is ABO compatible. Currently, the plasma being transfused is ABO compatible because the presence of naturally occurring antibodies against A and B antigens can result in transfusion reactions. IgM appears to be the cause of transfusion reactions when a patient is given plasma that is not ABO matched. Removal of IgM from the blood product or fraction helps to eliminate transfusion reactions in subjects administered with the blood product and plasma fraction of the present invention.

[0055] Accordingly, in one embodiment, the present invention relates to a method for treating or preventing age-related symptoms in a subject, such as cognitive or motor impairment, neuroinflammation, or neurodegeneration. The method comprises administering to a subject a blood product or blood fraction derived from whole blood of an individual or a pool of individuals, wherein the blood product or blood fraction substantially lacks (a) at least one coagulation factor and / or (b) IgM. In some embodiments, the individual(s) from which the blood product or blood fraction is derived is a young individual. In some embodiments, the blood product substantially lacks at least one coagulation factor and IgM. In certain embodiments, the blood product substantially lacks fibrinogen (factor I). In additional embodiments, the blood product substantially lacks red blood cells and / or white blood cells. In further embodiments, the blood product is substantially cell-free. In other embodiments, the blood product is derived from plasma. Such embodiments of the present invention are further supported by U.S. Patent Application No. 62 / 376,529, filed on 18 August 2016, which is incorporated herein by reference in its entirety.

[0056] 6. Processing of protein-enriched plasma protein products Additional embodiments of the present invention use a plasma fraction in which the albumin concentration is reduced compared to PPF, but the amount of globulin and other plasma proteins (some referred to as “impurities”) is increased. As with PPF, HAS, effluent I, and effluent II / III, all embodiments are virtually devoid of coagulation factors. Such plasma fractions are hereafter referred to as “protein-enriched plasma protein products.” For example, one embodiment of the present invention may use a protein-enriched plasma protein product comprising 82% albumin and 18% α, β, and γ globulin and other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein product comprising 81% albumin and 19% α, β, and γ globulin and / or other plasma proteins. Another embodiment of the present invention may use a protein-enriched plasma protein product comprising 80% albumin and 20% α, β, and γ globulin and / or other plasma proteins. Additional embodiments of the present invention may use a protein-enriched plasma protein product comprising 70–79% albumin and corresponding 21–30% α, β, and γ globulin and other plasma proteins. Additional embodiments of the present invention may use a protein-enriched plasma protein product comprising 60-69% albumin and corresponding 31-40% α, β, and γ globulins and other plasma proteins. Additional embodiments of the present invention may use a protein-enriched plasma protein product comprising 50-59% albumin and corresponding 41-50% α, β, and γ globulins and other plasma proteins. Additional embodiments of the present invention may use a protein-enriched plasma protein product comprising 40-49% albumin and corresponding 51-60% α, β, and γ globulins and other plasma proteins. Additional embodiments of the present invention may use a protein-enriched plasma protein product comprising 30-39% albumin and corresponding 61-70% α, β, and γ globulins and other plasma proteins. Additional embodiments of the present invention may use a protein-enriched plasma protein product comprising 20-29% albumin and corresponding 71-80% α, β, and γ globulins and other plasma proteins.An additional embodiment of the present invention may use a protein-enriched plasma protein product comprising 10-19% albumin and corresponding 81-90% α, β, and γ globulins and other plasma proteins. Another additional embodiment of the present invention may use a protein-enriched plasma protein product comprising 1-9% albumin and corresponding 91-99% α, β, and γ globulins and other plasma proteins. A further embodiment of the present invention may use a protein-enriched plasma protein product comprising 0% albumin and 100% α, β, and γ globulins and other plasma proteins.

[0057] The embodiments of the present invention described above may also have a total gamma globulin concentration of 1-5%.

[0058] The specific concentration of protein in a plasma fraction can be determined using techniques well known to those skilled in the art. Examples, but not limited to, include electrophoresis, mass spectrometry, ELISA analysis, and Western blotting analysis.

[0059] 7. Preparation of plasma fractions Methods for preparing PPF and other plasma fractions are well known to those skilled in the art. One embodiment of the present invention allows for the collection of blood used for the preparation of human plasma protein fractions into a flask containing a citrate or anticoagulant dextrose citrate solution to inhibit coagulation, and further separation of fractions I, II+III, IV and PPF according to the method disclosed by Hink et al. (See Hink, JH, Jr., et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174)(1957), incorporated herein by reference.) According to this method, the mixture can be recovered at 2-8°C. The plasma can then be separated by centrifugation at 7°C, removed, and stored at -20°C. The plasma can then be thawed at 37°C and preferably fractionated within 8 hours of removal from storage at -20°C.

[0060] Plasma can be separated from fraction I using 8% ethanol at pH 7.2 and a temperature of -2 to -2.5°C, with a protein concentration of 5.1 to 5.6%. Cold 53.3% ethanol (176 mL / L plasma) containing acetate buffer (200 mL of 4M sodium acetate and 230 mL of glacial acetic acid, diluted to 1 L with appropriate amounts of H2O) can be added using a jet at a rate of, for example, 450 mL / min while lowering the plasma temperature to -2°C. Fraction I can be separated and removed from the effluent (effluent I) by ultracentrifugation. Fibrinogen can be obtained from fraction I according to methods well known to those skilled in the art.

[0061] Fractions II+III can be separated from Eluten I by adjusting the effluent to 21% ethanol at pH 6.8 and temperature -6°C, with a protein concentration of 4.3%. Cold 95% ethanol containing 10M acetic acid (176 mL / L of Eluten I), used for pH adjustment, can be added using a jet at a rate of, for example, 500 mL / min while lowering the temperature of Eluten I to -6°C. The resulting precipitate (Fractions II+III) can be removed by centrifugation at -6°C. Gamma globulin can be obtained from Fractions II+III using methods well known to those skilled in the art.

[0062] Fraction IV-1 can be separated from Effluent II+III ("Effluent II / III") by adjusting the effluent to 19% ethanol at pH 5.2 and a temperature of -6°C, with a protein concentration of 3%. H2O and 10M acetic acid used for pH adjustment can be added using a jet while maintaining Effluent II / III at -6°C for 6 hours. The precipitated fraction VI-1 can be separated from the effluent by allowing it to settle at -6°C for 6 hours, followed by centrifugation at the same temperature. A stable plasma protein fraction can be recovered from Effluent IV-1 by adjusting the ethanol concentration to 30% at pH 4.65 and a temperature of -7°C, with a protein concentration of 2.5%. This can be achieved by adjusting the pH of Effluent IV-1 with cold acid-alcohol (2 parts 2M acetic acid and 1 part 95% ethanol). Add 170 mL of cold ethanol (95%) per liter of adjusted Effluent IV-1 while maintaining a temperature of -7°C. The precipitated protein can be removed by allowing it to settle for 36 hours, followed by centrifugation at -7°C.

[0063] The recovered protein (stable plasma protein fraction) can be dried (e.g., by freeze-drying) to remove alcohol and H2O. The resulting dried powder can be dissolved in sterile distilled water by adjusting the pH of the solution to 7.0 with 1 M NaOH using, for example, 15 liters of water / kg of powder. A final concentration of 5% protein can be achieved by adding sterile distilled water containing sodium acetyltryptophanate, sodium caprylate, and NaCl to adjust the final concentrations to 0.004 M acetyltryptophanate, 0.004 M caprylate, and 0.112 M sodium. Finally, the solution can be filtered at 10°C to obtain a clear solution, which can then be heat-treated at 60°C for at least 10 hours to inactivate the pathogen.

[0064] The fraction IV-1 paste can be redissolved in 0.005 M Tris buffer, followed by heating, and then 0.11 M NaCl can be added. The suspension can then be used for sub-fractionation by anion exchange to maintain protein integrity and functionality for biological testing or processing. A method for redissolving the fraction IV-1 paste is further described in Hoffman DL, AM J Med (1989) 87 (suppl 3B): 23S-26S, which is incorporated herein by reference.

[0065] Fraction IV-1 paste contains significant levels of at least two individual protein products. One is alpha-1 antitrypsin (abbreviated as "A1AT" or "AAT"), also known as alpha-1 antiprotease. The second is antithrombin III ("ATIII").

[0066] Alpha-1 antitrypsin (AAT) is a protein primarily produced in the liver and circulating in the bloodstream. It is part of the serine protease inhibitor family (Serpins) and modulates the activity of certain enzymes in the body. AAT can inhibit the activity of an enzyme called neutrophil elastase, produced by white blood cells, which, if not properly regulated, can damage tissues. AAT deficiency can lead to a condition called alpha-1 antitrypsin deficiency, a genetic disorder that can cause lung and liver diseases. This is due to a lack of AAT, which allows neutrophil elastase to damage lung tissue, causing emphysema, and cirrhosis of liver tissue. Testing AAT levels in the blood can be helpful in diagnosing alpha-1 antitrypsin deficiency.

[0067] Antithrombin III is a proteinase inhibitor and is also associated with serpines. It acts as a regulator of hemostasis and thrombosis. It is useful in treating congenital ATIII deficiency by regulating blood coagulation, primarily through thrombin inhibition.

[0068] Subfractions can be performed by several techniques, including chromatography using anion exchange columns such as Q-Sepharose Fast Flow columns. The fractionation for producing the specific subfractions described herein is further explained in Example 10.

[0069] Those skilled in the art will recognize that each of the different fractions and effluents described above can be used in conjunction with the methods of the present invention to treat diseases. For example, but not limited to, effluent I or effluents II / III may be used to treat diseases such as cognitive, motor, and neurodegenerative disorders, which are embodiments of the present invention.

[0070] The methods described above for preparing plasma fractions and plasma protein fractions (PPFs) are merely illustrative and include only embodiments of the present invention. Those skilled in the art will recognize that these methods can be modified. For example, pH, temperature, and ethanol concentration can be adjusted, among other things, to produce different variations of plasma fractions and plasma protein fractions in different embodiments and methods of the present invention. In another example, an additional embodiment of the present invention involves the use of nanofiltration for the removal / inactivation of pathogens from plasma fractions and plasma protein fractions.

[0071] Additional embodiments of the present invention envision methods and compositions that use and / or include additional plasma fractions. For example, the present invention demonstrates, among other things, that certain concentrations of albumin are not important for improving cognitive or motor activity. Therefore, fractions with reduced albumin concentrations, for example, fractions having less than 83% albumin, are envisioned by the present invention.

[0072] 8. Treatment Embodiments of the methods of the present invention described herein include, for example, treatment of a subject with plasma containing blood products such as plasma fractions, as described above. One embodiment includes treatment of a human subject with plasma containing blood products. Those skilled in the art will recognize that methods of treating a subject with plasma containing blood products are recognized in the art. By example, not by limitation, one embodiment of the methods of the present invention described herein includes administering fresh frozen plasma to a subject for the treatment and / or prevention of cognitive or motor impairment, neuroinflammation, neurodegeneration, or peripheral disease. In one embodiment, the plasma containing blood products is administered immediately to an individual suffering from or at risk of cognitive or motor impairment, neuroinflammation, neurodegeneration, and / or age-related dementia, for example, within about 12 to 48 hours of collection from a donor. In such cases, the product may be stored under refrigeration, for example, at 0 to 10°C. In another embodiment, the fresh frozen plasma is stored frozen at -18°C or lower (cold storage). Before administration, the fresh frozen plasma is thawed and administered to the subject 60 to 75 minutes after thawing and the start of the thawing process. Each subject preferably receives a single unit of fresh frozen plasma (200-250 mL), which preferably originates from a donor within a predetermined age range. In one embodiment of the present invention, the fresh frozen plasma is provided (derived from) a young individual. In another embodiment of the present invention, the fresh frozen plasma is provided (derived from) a donor of the same sex. In yet another embodiment of the present invention, the fresh frozen plasma is provided (derived from) a donor within an age range of 18-22 years.

[0073] In one embodiment of the present invention, plasma containing a blood product is screened by blood type after provision. In another embodiment of the present invention, plasma containing a blood product is screened for infectious disease factors such as HIV I and II, HBV, HCV, HTLV I and II, and anti-HBc, in accordance with the requirements of 21 CFR 640.33 and the recommendations contained in the FDA guidance document.

[0074] In yet another embodiment of the present invention, the subject is treated with a plasma fraction. In one embodiment of the present invention, the plasma fraction is a fraction IV-1 paste (or cold precipitate) redissolved in an aqueous solution. As used below, “fraction IV-1 paste” is synonymous with fraction IV-1 paste redissolved in an aqueous solution. In another embodiment of the present invention, the plasma fraction may be a fractionated subfraction of the fraction IV-1 paste. In another embodiment of the present invention, the plasma fraction may be one of 13 (13) subfractions of the fraction IV-1 paste further described below. In another embodiment of the present invention, the plasma fraction is a PPF or HAS. In a further embodiment of the present invention, the plasma fraction is one of the commercially available PPF preparations of a commercially available HAS preparation. In another embodiment of the present invention, the plasma fraction is a PPF or HAS derived from a pool of individuals of a specific age range, such as young individuals, or a modified PPF or HAS fraction subjected to additional fractionation or processing (e.g., a PPF or HAS from which one or more specific proteins have been partially or substantially removed). In another embodiment of the present invention, the plasma fraction is an IGIV plasma fraction substantially depleted of immunoglobulin (IgG). A blood fraction having a specific protein such as IgG that is "substantially depleted" or "substantially removed" refers to a blood fraction that, when measured using a standard assay well known in the art, contains less than about 50% of the amount that would occur in the reference product or total plasma, e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, less than 0.1%, undetectable levels, or any integer between these values.

[0075] 9. Administration Embodiments of the methods of the present invention described herein include, for example, treatment of a subject with plasma containing a blood product such as plasma or plasma fraction, as described above. One embodiment includes treatment of a human subject with plasma containing a blood product.

[0076] In yet another embodiment of the present invention, a subject is treated with a plasma fraction. Another embodiment of the present invention involves treatment of a human subject with a fraction IV-1 paste. Another embodiment of the present invention involves treatment of a human subject with a subfraction of the fraction IV-1 paste, including, but not limitingly, a third (13) subfraction, which is further described below as an example. In one embodiment of the present invention, the plasma fraction is a PPF or HAS. In a further embodiment of the present invention, the plasma fraction is one of the commercially available PPF preparations of a commercially available HAS preparation. In another embodiment of the present invention, the plasma fraction is a PPF or HAS derived from a pool of individuals in a specific age range, such as young individuals, or a modified PPF or HAS fraction subjected to additional fractionation or processing (e.g., a PPF or HAS from which one or more specific proteins have been partially or substantially removed). In another embodiment of the present invention, the plasma fraction is an IGIV plasma fraction substantially depleted of immunoglobulin (IgG). Blood fractions containing certain proteins such as IgG that are "substantially depleted" or "substantially removed" refer to blood fractions containing less than approximately 50% of the amount present in the reference product or total plasma, e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, less than 0.1%, undetectable levels, or any integer between these values, as measured using standard assays well known in the art.

[0077] Embodiments of the present invention include treating subjects diagnosed with cognitive or motor impairment, neurodegeneration, neuroinflammation, or peripheral disease by administering an effective amount of plasma or plasma fraction to the subject. Another embodiment of the present invention includes administering an effective amount of plasma or plasma fraction and then monitoring the subject for improvement in disease symptoms. Another embodiment of the present invention includes treating subjects diagnosed with disease or impairment by administering an effective amount of plasma or plasma fraction to the subject, wherein the plasma or plasma fraction is administered in such a manner that it results in improvement of disease symptoms or progression after the mean or median half-life of the plasma protein or plasma fraction protein has been reached compared to the most recently administered dose (referred herein to as “pulsed dosing” or “pulse dosed”). Another embodiment of the present invention includes administering plasma or plasma fraction via a dosing regimen of at least two consecutive days and monitoring the subject for improvement in cognitive or motor function, reduction in neuroinflammation, or improvement in neurogenesis at least three days after the last dosing day. Further embodiments of the present invention include administering plasma or plasma fractions via a dosing regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days, and monitoring the subject for improvement of disease symptoms or progression at least 3 days after the last dosing day. Yet another embodiment of the present invention includes administering plasma or plasma fractions via a dosing regimen of at least 2 consecutive days, and after the last dosing day. Yet another embodiment of the present invention includes administering plasma or plasma fractions via a dosing regimen of 2 to 14 non-consecutive days, where each gap between dosings may be 0 to 3 days.

[0078] In some cases, pulsed dosing according to the present invention includes, for example, the administration of a first dose set as described above, followed by a non-dosing period, e.g., a “drug-free period,” followed by the administration of another dose or dose set. The duration of this “drug-free” period can vary, but in some embodiments it is 7 days or more, e.g., 10 days or more, and includes 14 days or more, and in some cases the drug-free period is 15 to 365 days, e.g., 30 to 90 days, and includes 30 to 60 days. Thus, embodiments of the present method include non-chronic (i.e., discontinuous) dosing of plasma preparations, e.g., non-chronic administration. In some embodiments, the pattern of pulsed dosing followed by a drug-free period is repeated multiple times as desired, and in some cases this pattern continues for more than one year, e.g., more than two years, for the lifespan of the subject (and inclusive). Another embodiment of the present invention includes administering plasma or plasma fractions via a dosing regimen of 5 consecutive days, a 2-3 day drug-free period, and then 2-14 consecutive days of dosing.

[0079] Biochemically, the "effective amount" or "effective dose" of an active agent refers to the amount of the active agent that inhibits, antagonizes, reduces, or suppresses approximately 20% or more, for example, 30%, 40%, or 50%, and in some cases, 60%, 70%, 80%, or 90%, and in some cases, approximately 100%, i.e., to a negligible amount, and in some cases reverses the progression of the disease.

[0080] 10. Plasma protein fraction When carrying out the method of the present invention, the plasma fraction is administered to the target. In one embodiment, the plasma fraction is plasma protein fraction (PPF). In an additional embodiment, the PPF is selected from a commercially available PPF preparation.

[0081] In another embodiment, PPF, as determined by electrophoresis, consists of 88% normal human albumin, 12% alpha and beta globulins, and less than 1% gamma globulin. Further embodiments of this embodiment used in carrying out the method of the present invention include, for example, an embodiment as a 5% solution of PPF buffered with sodium carbonate and stabilized with 0.004 M sodium caprylate and 0.004 M acetyltryptophan. Additional formulations, including those that modify the percentage of PPF in the solution (e.g., about 1% to about 10%, about 10% to about 20%, about 20% to 25%, about 25% to 30%) and the concentrations of the solvent and stabilizer, may be used when carrying out the method of the present invention.

[0082] 11. Indications The methods and plasma-containing blood products and plasma fractions are used in treatments for age-related conditions, such as cognitive impairment including (but not limited to) cognitive impairment including age-related dementia, immunological conditions, cancer, and physical and functional decline; and (but not limited to) the prevention of motor disorders such as Parkinson's disease. Individuals who have or are at risk of developing age-related cognitive or motor impairment, neuroinflammation, and / or neurodegeneration, who would benefit from treatment with the plasma-containing blood products disclosed herein, for example, are approximately 50 years of age or older, for example 60 years of age or older, 70 years of age or older, 80 years of age or older, 90 years of age or older, and 100 years of age or older, i.e., between approximately 50 and 100 years of age, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or approximately 100 years of age, and who are at risk of developing natural aging. This includes individuals with cognitive or motor impairments, neuroinflammation, and / or neurodegeneration associated with Seth, such as mild cognitive impairment (MCI); and individuals aged approximately 50 years or older, such as 60, 70, 80, 90, and usually 100 years or younger, i.e., between approximately 50 and 90 years old, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or approximately 100 years old, who have not yet begun to show symptoms of cognitive or motor impairments, neuroinflammation, and / or neurodegeneration. Examples of cognitive and motor impairments, neuroinflammatory disorders, and / or neurodegenerative disorders due to natural aging include:

[0083] a. Mild Cognitive Impairment (MCI). Mild cognitive impairment is a mild cognitive impairment that manifests as problems with memory or other mental functions, such as planning, following instructions, or making decisions, where overall mental function and daily activities are not impaired but worsen over time. Thus, significant neuronal death does not typically occur, but neurons in the aging brain are vulnerable to sublethal age-related changes in synaptic structure, synaptic integrity, and molecular processing, all of which impair cognitive function.

[0084] For example, individuals who have or are at risk of developing age-related cognitive impairment and would benefit from treatment with the plasma-containing blood product or fraction of interest by the methods disclosed herein include individuals of any age who have cognitive impairment due to age-related impairment; and individuals of any age who have been diagnosed with age-related impairment typically accompanied by cognitive impairment, but who have not yet begun to exhibit symptoms of cognitive impairment. Examples of such age-related impairments include:

[0085] b. Alzheimer's disease. Alzheimer's disease is a progressive, irreversible loss of cognitive function associated with an excessive number of senile plaques in the cerebral cortex and subcortical gray matter, including neurofibrillary tangles composed of β-amyloid and tau proteins. The most common form develops in people over 60 years of age, and its incidence increases with age. It accounts for over 65% of dementia in older adults.

[0086] The cause of Alzheimer's disease is unknown. Approximately 15–20% of cases are familial. The remaining so-called sporadic cases have several genetic determinants. The disease exhibits an autosomal dominant inheritance pattern in most early-onset cases and some late-onset cases, although penetrance in later-stage manifestation varies. Environmental factors are a focus of active research.

[0087] During the course of the disease, synapses, and ultimately neurons, are lost in the cerebral cortex, hippocampus, and subcortical structures (including selective cell loss in the nucleus basalis of Meynert), locus coeruleus, and dorsal raphe nuclei. Brain glucose use and perfusion are reduced in several areas of the brain (parietal and temporal cortex in early disease, and prefrontal cortex in later disease). Neurites or senile plaques (composed of neurites, astrocytes, and glial cells surrounding an amyloid core) and neurofibrillary tangles (composed of pairs of spiral filaments) play a role in the pathogenesis of Alzheimer's disease. Senile plaques and neurofibrillary tangles occur with normal aging, but are far more common in people with Alzheimer's disease.

[0088] c. Parkinson's disease. Parkinson's disease (PD) is an idiopathic, slowly progressive, degenerative central nervous system disorder characterized by slow, reduced movement (bradykinesia), muscle rigidity, resting tremor (dystonia), muscle freezing, and postural instability. Originally considered primarily a movement disorder, PD is now recognized as also causing depression and emotional changes. PD can also affect cognition, behavior, sleep, autonomic nervous system function, and sensory function. The most common cognitive impairments include difficulties with attention and concentration, working memory, executive function, language production, and visuospatial function. The characteristic feature of PD is the symptom associated with reduced motor function, which usually precedes the symptom associated with cognitive impairment, and this is helpful in diagnosing the disease.

[0089] In primary Parkinson's disease, pigment neurons in the substantia nigra, locus coeruleus, and other brainstem dopaminergic cell groups degenerate. The cause is unknown. The loss of substantia nigra neurons projecting to the caudate nucleus and putamen leads to depletion of the neurotransmitter dopamine in these areas. Onset generally occurs after age 40, with an increased incidence in older adults.

[0090] Parkinson's disease is newly diagnosed in approximately 60,000 Americans each year, and currently affects about 1 million Americans. While PD itself is not fatal, its complications are the 14th leading cause of death in the United States. Currently, PD is incurable, and treatment is generally prescribed to control symptoms, with surgery prescribed for more severe cases.

[0091] Treatment options for Parkinson's disease (PD) include the administration of medications to help manage movement disorders. These options increase or replace dopamine, a neurotransmitter that PD patients have low brain concentrations of. Such drug therapies include carbidopa / levodopa (which produces more dopamine in the brain); apomorphine, pramipexole, ropinirole, and rotingotine (dopamine agonists); selegiline and rasagiline (MAO-B inhibitors that prevent the breakdown of dopamine); entacapone and tolcapone (catechol-O-methyltransferase [COMT] inhibitors that make more levodopa available in the brain); benztropine and trihexyphenidyl (anticholinergics); and amantadine (which controls tremors and rigidity). Exercise / physical therapy is also commonly prescribed to help maintain physical and mental function.

[0092] Current treatment options address the symptoms of Parkinson's disease (PD) but are not curative and cannot prevent disease progression. Furthermore, current drug therapies tend to lose efficacy in late-stage PD. Levodopa, the most commonly prescribed drug, generally causes adverse effects within 5 to 10 years of starting drug therapy. These adverse effects can be severe and may result in motor variability and unpredictable fluctuations in motor control between doses, as well as seizures / convulsions (dyskinesia), which are difficult to manage and can be as detrimental as the symptoms of PD itself. Therefore, there is still a need for new therapies with novel mechanisms of action that can be administered alongside or in combination with current PD drug therapies.

[0093] d. Parkinsonism. Secondary parkinsonism (also called atypical parkinson's disease or parkinson's disease+) results from the loss or interference of dopamine action in the basal ganglia due to other idiopathic degenerative diseases, drugs, or exogenous toxins. The most common cause of secondary parkinsonism is the ingestion of antipsychotic drugs or reserpine, which cause parkinsonism by blocking dopamine receptors. Less common causes include carbon monoxide or manganese poisoning, hydrocephalus, structural lesions (tumors or infarcts affecting the midbrain or basal ganglia), subdural hematoma, and degenerative disorders, including nigrostriatal degeneration. Certain disorders, such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), and Lewy body dementia (DLB), may present with parkinsonist symptoms before the basic symptoms necessary for a specific diagnosis are acquired, and may therefore be labeled as “parkinsonism.”

[0094] e. Frontotemporal dementia. Frontotemporal dementia (FTD) is a condition resulting from the progressive deterioration of the frontal lobe of the brain. Over time, the degeneration can progress to the temporal lobe. FTD is the second most common disease after Alzheimer's disease (AD) and accounts for 20% of presenteric dementia cases. The symptoms are classified into three groups based on the function of the frontal and temporal lobes affected.

[0095] Symptomatic behavioral variants of FTD (bvFTD) include apathy and loss of spontaneity on the one hand, and disinhibition on the other; progressive non-fluent aphasia (PNFA) where speech fluency is impaired due to dysphonia, phonological and / or syntactic errors, but word comprehension is preserved; and semantic dementia (SD) where the patient remains fluent in normal phonological and syntactic matters, but has increasing difficulties with naming and word comprehension. Other cognitive symptoms common to all FTD patients include impairments in executive function and concentration. Other cognitive abilities, including perception, spatial skills, memory, and conventions, are typically intact. FTD can be diagnosed by the observation of clear frontal and / or anterior-temporal lobe atrophy on structural MRI scans.

[0096] Several forms of FTD exist, all of which can be treated or prevented using targeted methods and compositions. For example, one form of frontotemporal dementia is semantic dementia (SD). SD is characterized by a loss of semantic memory in both the linguistic and non-linguistic domains. Patients with SD often complain of difficulty finding words. Clinical signs include fluent aphasia, anomalism, difficulty understanding the meaning of words, and relevance visual agnosia (inability to match semantically relevant images or objects). As the disease progresses, behavioral and personality changes are often similar to those seen in frontotemporal dementia, although cases have been described as “pure” semantic dementia with little to no late-stage behavioral symptoms. Structural MRI imaging shows a characteristic pattern of temporal lobe atrophy (primarily on the left side), with greater atrophy in the inferior temporal lobe than in the superior temporal lobe, and greater atrophy in the anterior temporal lobe than in the posterior temporal lobe.

[0097] Another example, a different form of frontotemporal dementia, is Pick's disease (PiD, or PcD). A distinctive feature of this disease is the accumulation of tau protein in neurons, which accumulates in silver-stained spherical aggregates known as "Pick bodies." Symptoms include loss of speech (aphasia) and dementia. Patients with orbitofrontal dysfunction may become aggressive and socially inappropriate. They may engage in kleptomania or exhibit compulsive or repetitive stereotyped behaviors. Patients with dorsomedial or dorsolateral frontal dysfunction may exhibit a lack of concern, apathy, or reduced spontaneity. Patients may exhibit a lack of self-monitoring, abnormal self-awareness, and an inability to understand meaning. Patients with gray matter loss in the bilateral posterolateral orbitofrontal cortex and the right anterior insula may exhibit changes in eating behavior, such as a pathological sweet tooth. Patients with more localized gray matter loss in the anterolateral orbitofrontal cortex may develop bulimia. While some symptoms may be initially alleviated, the disease progresses, and patients often die within 2 to 10 years.

[0098] f. Huntington's disease. Huntington's disease (HD) is a hereditary, progressive neurodegenerative disorder characterized by the onset of emotional, behavioral, and psychiatric abnormalities; loss of intellectual or cognitive function; and movement disorders. Classical signs of HD include the onset of chorea (involuntary, rapid, irregular, or spasmodic movements that may affect the face, arms, legs, or trunk), as well as cognitive decline, including a progressive loss of thought processing and acquired intellectual abilities; impairment of memory, abstract thinking, and judgment; disorientation (inappropriate perception of time, place, or identity); increased agitation; and personality changes (disintegration). Symptoms typically become apparent between the ages of 40 and 50, but the age of onset varies, ranging from infancy to late adulthood (e.g., 70s or 80s).

[0099] HD is transmitted within families as an autosomal dominant trait. This disorder results from an abnormally long sequence or "repetition" of the coded instructions in a gene (4p16.3) on chromosome 4. The progressive loss of nervous system function associated with HD is due to the loss of neurons in specific areas of the brain, including the basal ganglia and cerebral cortex.

[0100] g. Amyotrophic lateral sclerosis (ALS). Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, always fatal neurological disease that attacks motor neurons. Signs of muscle weakness and atrophy, as well as anterior horn cell dysfunction, are most frequently seen in the hands initially and less frequently in the feet. The site of onset is random, and progression is asymmetrical. Seizures are common and may precede weakness. Rarely, patients survive for 30 years; 50% die within 3 years of onset, 20% survive for 5 years, and 10% survive for 10 years.

[0101] Diagnostic features include middle- or late-adult onset and progressive generalized motor impairment without sensory disturbances. Nerve conduction velocity remains normal until later stages of the disease. Recent studies have also demonstrated the presentation of cognitive impairment, particularly a decline in immediate verbal memory, visual memory, language, and executive function.

[0102] A decrease in cell body region, synapse number, and total synaptic length has been reported even in normally appearing neurons in ALS patients. It has been suggested that continuous synaptic loss can lead to dysfunction once the plasticity of the active region reaches its limit. Facilitating the formation or new synapse formation, or preventing synaptic loss, may preserve neuronal function in these patients.

[0103] h. Multiple sclerosis. Multiple sclerosis (MS) is characterized by a variety of symptoms and signs of central nervous system (CNS) dysfunction, with remissions and relapsing exacerbations. The most common presenting symptoms are unilateral paresthesia of one or more limbs, trunk, or face; weakness or clumsiness of the legs or hands; or visual impairment, e.g., partial blindness and pain in one eye (retrobulbar optic neuritis), decreased visual acuity, or scotoma. Common cognitive impairments include impairments in memory (acquisition, retention, and retrieval of new information), attention and concentration (especially splitting of attention), information processing, executive function, visuospatial function, and verbal fluency. Common early symptoms include ocular palsy resulting in diplopia, transient weakness of one or more limbs, slight rigidity or abnormal fatigability of the limbs, mild gait disturbance, bladder control difficulties, dizziness, and mild emotional disturbances; all showing scattered CNS involvement and often occurring months or years before the disease is recognized. Excessive fever can worsen symptoms and signs.

[0104] The course of the disease is highly diverse and unpredictable, and in most patients it is relapsing-remitting. Initially, remissions of several months or years may isolate episodes, especially if the disease begins with retrobulbar optic neuritis. However, some patients experience frequent seizures and rapid incapacitation; in some cases, the course can be rapidly progressive.

[0105] i. Glaucoma. Glaucoma is a common neurodegenerative disease affecting retinal ganglion cells (RGCs). Evidence supports the presence of compartmentalized degenerative programs in synapses and dendrites, including RGCs. Recent evidence also shows a correlation between cognitive impairment and glaucoma in older adults (Yochim BP, et al. Prevalence of cognitive impairment, depression, and anxiety symptoms among older adults with glaucoma. J Glaucoma. 2012;21(4):250-254).

[0106] j. Myotonic Dystrophy. Myotonic dystrophy (DM) is an autosomal dominant multiple system disorder characterized by dystrophic muscle weakness and myotonia. The molecular defect is an extended trinucleotide (CTG) repeat in the 3' untranslated region of the myotonin protein kinase gene on chromosome 19q. Symptoms can occur at any age, and the range of clinical severity is wide. Myotonia is prominent in the muscles of the hands, and ptosis is common even in mild cases. In severe cases, marked peripheral muscle weakness occurs, often accompanied by cataracts, premature alopecia, axe-shaped facies, cardiac arrhythmias, testicular atrophy, and endocrine abnormalities (e.g., diabetes mellitus). Intellectual disability is common in severe congenital forms, but age-related declines in frontal and temporal cognitive function, particularly language and executive function, are observed in milder adult forms of the disorder. Severely affected individuals die by their early 50s.

[0107] k. Dementia. Dementia represents a class of disorders characterized by symptoms that affect thinking and social abilities to a degree severe enough to impair daily functioning. In addition to dementia observed in the later stages of age-related disorders as described above, other cases of dementia include vascular dementia and Lewy body dementia, which will be discussed later.

[0108] In vascular dementia, also known as "multiple-infarct dementia," cognitive impairment is caused by problems with blood supply to the brain, typically resulting from a series of minor strokes, or occasionally from a single major stroke preceding or following other smaller strokes. Vascular lesions can be the result of diffuse cerebrovascular disease, such as small vessel disease, or focal lesions, or both. Patients with vascular dementia present with acute or subacute cognitive impairment following an acute cerebrovascular event, followed by progressive cognitive decline. The cognitive impairment is similar to that observed in Alzheimer's disease, including impairments in language, memory, complex visual processing, or executive function, but the associated brain changes are not due to the pathophysiology of AD, but rather to chronic reduced blood flow to the brain, ultimately leading to dementia. Single-photon emission tomography (SPECT) and positron emission tomography (PET) neuroimaging, along with assessments including mental state testing, can be used to confirm the diagnosis of multiple-infarct dementia.

[0109] Lewy body dementia (DLB, also known by various other names including Lewy body dementia, diffuse Lewy body disease, cortical Lewy body disease, and Lewy-type senile dementia) is a type of dementia anatomically characterized by the presence of Lewy bodies (clumps of alpha-synuclein and ubiquitin proteins) in neurons detectable in postmortem brain histology. Its main feature is a decline in cognitive function, particularly executive function. Arousal levels and short-term memory may increase or decrease.

[0110] Persistent or recurrent visual hallucinations with vivid, detailed images are often an early diagnostic symptom. DLB is often confused with Alzheimer's disease and / or vascular dementia in its early stages, but while Alzheimer's disease usually develops very gradually, DLB often has a rapid or acute onset. DLB symptoms also include motor symptoms similar to those of Parkinson's disease. DLB is distinguished from dementia that occurs occasionally in Parkinson's disease by the time frame in which dementia symptoms appear compared to Parkinsonian symptoms. Parkinson's disease with dementia (POD) would be diagnosed when the onset of dementia occurs more than one year after the onset of Parkinson's disease. DLB is diagnosed when dementia symptoms begin simultaneously with or within one year of Parkinsonian symptoms.

[0111] l. Progressive Supranuclear Palsy. Progressive supranuclear palsy (PSP) is a brain disorder that causes serious and progressive problems with gait and balance control, along with problems with complex eye movements and thinking. One of the typical signs of this disorder is the inability to properly direct the eyes, which results from lesions in the brain regions that control eye movements. Some individuals describe this effect as blurred vision. Affected individuals often exhibit mood and behavioral changes, including depression and apathy, as well as progressive mild dementia. The long name of the disorder indicates that the disease starts slowly, continues to worsen (progressive), and causes debilitation (paralysis) by damaging specific parts of the brain (supranuclear) above pea-sized structures called nuclei that control eye movements. PSP was first described as a distinct disorder in 1964 when three scientists published a paper differentiating the symptoms from Parkinson's disease. It is sometimes called Steele-Richardson-Olszewski syndrome, combining the names of the scientists who defined the disorder. While PSP gradually worsens, no one dies from PSP itself.

[0112] m. Ataxia. People with ataxia have problems with coordination because parts of the nervous system that control movement and balance are affected. Ataxia can affect the movement of fingers, hands, arms, legs, body, speech, and eyes. The term ataxia is often used to describe a symptom of coordination disorder that may be associated with infection, injury, other disease, or degenerative changes in the central nervous system. Ataxia is also used to refer to a group of specific degenerative disorders of the nervous system called hereditary and sporadic ataxia, which are the main focus of the National Ataxia Foundation.

[0113] n. Multiple system atrophy. Multiple system atrophy (MSA) is a degenerative neurological disorder. MSA is associated with the degeneration of nerve cells in specific areas of the brain. This cellular degeneration causes problems with movement, balance, and other autonomic nervous system functions of the body, such as bladder control or blood pressure regulation.

[0114] The cause of MSA is unknown, and no specific risk factors have been identified. Approximately 55% of cases occur in men, with a typical age of onset in the late 50s to early 60s. MSA often presents with some of the same symptoms as Parkinson's disease. However, MSA patients generally have a minimal response, if any, to dopamine therapy used for Parkinson's disease.

[0115] o. Frailty. Frailty syndrome ("frailty") is a geriatric syndrome characterized by functional and physical decline, including reduced motor function, muscle weakness, physical retardation, decreased endurance, low physical activity, malnutrition, and involuntary weight loss. Such declines are often concomitant and result of diseases such as cognitive impairment and cancer. However, frailty can occur even without disease. Individuals suffering from frailty have a higher risk of poor prognosis from fractures, accidental falls, handicaps, comorbidities, and premature death. (C. Bugues, et al. Effect of a Prebiotic Formulation on Frailty Syndrome: A Randomized, Double-Blind Clinical Trial, Int. J. Mol. Sci. 2016, 17, 932). Furthermore, individuals suffering from frailty have a higher incidence of medical expenses. (Ibid.)

[0116] Common symptoms of frailty can be determined by certain types of tests. For example, unintentional weight loss is defined as a loss of at least 10 pounds; or more than 5% of the previous year's body weight; muscle weakness can be determined by a minimum 20% reduction in grip strength at baseline (adjusted for sex and BMI); physical stagnation can be based on the time required to walk a distance of 15 feet; decreased endurance can be determined by an individual's self-reported fatigue; and low physical activity can be measured using a standardized questionnaire. (Z. Palace et al., The Frailty Syndrome, Today's Geriatric Medicine 7(1), at 18(2014)).

[0117] In some embodiments, the methods and compositions are used to slow the progression of age-related cognitive, motor, neuroinflammation, or other age-related impairments or symptoms. In other words, cognitive, motor, neuroinflammation, or other abilities or symptoms in an individual will decline more slowly after treatment with the disclosed methods than before or in the absence of treatment with the disclosed methods. In some such cases, the treatment method of the subject includes measuring the progression of decline in cognitive, motor, neuroinflammation, or other age-related abilities or symptoms after treatment and determining that the progression of decline has slowed. In some such cases, the determination is made by comparison with a reference (e.g., the rate of decline in the individual before treatment, which is determined, for example, by measuring cognitive, motor, neuroinflammation, or other age-related abilities or symptoms at two or more time points prior to administration of the blood product of the subject).

[0118] The methods and compositions described herein can also be used to stabilize the cognitive, motor, neuroinflammatory, or other abilities or symptoms of an individual, for example, an individual suffering from or at risk of suffering from age-related cognitive decline. For example, an individual may exhibit some age-related cognitive impairment, and the progression of cognitive impairment observed before treatment by the disclosed method will be halted after treatment by the disclosed method. As another example, an individual may be at risk of developing age-related cognitive decline (for example, the individual may be 50 years of age or older, or may have been diagnosed with age-related impairment), and the individual's cognitive abilities will not be substantially changed after treatment by the disclosed method compared to before treatment by the disclosed method; that is, cognitive decline will not be detectable.

[0119] The methods and compositions are also used to reduce cognitive impairment, motor impairment, neuroinflammatory disorders, or other age-related impairments in individuals suffering from age-related disorders. In other words, the affected abilities are improved in individuals after treatment with the methods. For example, cognitive or motor abilities in an individual increase by, for example, more than 2 times, more than 5 times, more than 10 times, more than 15 times, more than 20 times, more than 30 times, or more than 40 times (including more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, or more than 100 times) after treatment with the methods compared to cognitive or motor abilities observed in the individual before treatment with the methods.

[0120] In some cases, treatment with the methods and compositions restores cognitive, motor, or other abilities in individuals suffering from age-related cognitive or motor decline to the levels they had when they were approximately 40 years of age or younger. In other words, cognitive or motor impairment is suppressed.

[0121] 12. Diagnostic and monitoring methods for improvement In some cases, among various methods for diagnosing and monitoring disease progression and improvement in cognitive disorders, motor disorders, neurodegenerative diseases, and / or neuroinflammatory diseases, the following types of assessments are used, as needed, alone or in combination, in subjects suffering from neurodegenerative diseases. The following types of methods are presented as examples and are not limited to those listed. Any convenient method for monitoring the disease may be used in the implementation of the present invention as needed. These methods are also intended by the methods of the present invention.

[0122] a. General awareness Embodiments of the method of the present invention further include a method for monitoring the effect of a drug therapy or treatment on a subject for treating cognitive impairment and / or age-related dementia, the method including comparing cognitive function before and after treatment. Those skilled in the art will recognize that there are well-known methods for assessing cognitive function. For example, but not limited to, methods may include assessment of cognitive function based on medical history, family history, physical and neurological examinations by a clinician specializing in dementia and cognitive function, laboratory tests, and neuropsychological assessments. Additional embodiments contemplated by the present invention include assessments of mental state, such as the Glasgow Coma Scale (EMV); the Ambidextrous Test Score (AMTS) or Mini-Mental State Examination (MMSE) (Folstein et al., J. Psychiatr. Res 1975;12:1289-198); a global assessment of higher-order functions; and assessments of consciousness, such as those using estimation of intracranial pressure by fundus examination. In one embodiment, monitoring the effect on cognitive impairment and / or age-related dementia includes an improvement of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 points using the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-COG).

[0123] In one embodiment, cognitive function may be assessed using tests of the peripheral nervous system, including any one of the following: sense of smell, visual field and visual acuity, eye movements and pupils (sympathetic and parasympathetic), facial sensory function, strength of facial and shoulder girdle muscles, hearing, taste, pharyngeal movements and reflexes, tongue movements (which can be tested individually; for example, visual acuity can be tested by the Snellen chart; the reflex hammer is used to test reflexes including the masseter, biceps and triceps tendons, patellar tendon, Achilles tendon reflex and plantar flexion (i.e., Babinski sign)); muscle strength, often on an MRC scale of 1-5; signs of muscle tone and stiffness.

[0124] b. Parkinson's disease Embodiments of the present invention further include methods for monitoring the effects of pharmacotherapy or treatment on a subject for treating a motor disorder, the method including comparing motor function before and after treatment. Those skilled in the art will recognize that there are well-known methods for evaluating motor function. For example, but not limited to, methods may include, but are not limited to, assessment of motor function based on medical history, family history, physical and neurological examinations by a clinician specializing in neurodegeneration and motor disorders, laboratory tests, and neurodegenerative assessments. Additional embodiments contemplated by the present invention include the use of evaluation scales discussed below.

[0125] Several assessment scales are used to evaluate the progression of Parkinson's Disease (PD). The most widely used scales include the Unified Parkinson's Disease Rating Scale (UPDRS, introduced in 1987) (J. Rehabil Res. Dev., 2012 49(8):1269-76) and the Hoehn and Yahr scale (Neurology, 1967 17(5):427-42). Further scales include the Movement Disorder Society's (MDS) updated UPDRS scale (MDS-UPDRS) and the Schwab and England Activities of Daily Living (ADL) Scale.

[0126] The UPDRS scale assesses 31 items that contribute to the following three subscales: (1) mental, behavioral, and mood; (2) activities of daily living; and (3) motor skills. The Hoehn and Yahr scale classifies PD into five stages with discrete substages: 0 - no signs of the disease; 1 - symptoms on only one side; 1.5 - symptoms affecting not only one side but also the cervical and spinal areas; 2 - bilateral symptoms without balance impairment; 2.5 - mild bilateral symptoms that resolve on a "pull" test; 3 - balance impairment with mild to moderate disease; 4 - severe handicap but able to walk or stand without assistance; and 5 - wheelchair required or bedridden without assistance. The Schwab and England scale classifies PD into several percentages (from 100% - completely independent to 10% - fully dependent).

[0127] General motor function can be assessed using widely used scales, including the General Motor Function Scale (GMF), which tests three components: dependence, pain, and anxiety. (Aberg AC, et al. (2003) Disabil. Rehabil. 2003 May 6;25(9):462-72.). Motor function can also be assessed using home monitoring or wearable sensors. For example, accelerometers can be used to detect gait (movement speed, variability, leg stiffness); gyroscopes can detect posture (trunk tilt); accelerometers can detect leg movement; accelerometers and gyroscopes can detect hand movement; accelerometers can detect tremor (amplitude, frequency, duration, asymmetry); accelerometers can detect falls; accelerometers can detect gait freeze; accelerometers, gyroscopes, and inertial sensors can detect dyskinesia; accelerometers and gyroscopes can detect bradykinesia (duration and frequency); and microphones can be used to detect aphasia (pitch). (Pastorino M, et al., Journal of Physics: Conference Series 450 (2013) 012055).

[0128] c. Multiple sclerosis In addition to monitoring improvement in cognitive symptoms, the progression or improvement of neurodegeneration associated with multiple sclerosis (MS) can be monitored using techniques well known to those skilled in the art. Examples, but not limited to, that monitoring can be performed through techniques such as cerebrospinal fluid (CSF) monitoring; magnetic resonance imaging (MRI) to detect the development of lesions and demyelinating plaques; evoked potential testing; and gait monitoring.

[0129] CSF analysis can be performed, for example, by lumbar puncture, to obtain pressure, appearance, and CSF content. Normal values ​​are typically within the following ranges: pressure (70-180 mmH2O); appearance is colorless and clear; total protein (15-60 mg / 100 mL); IgG is 3-12% of total protein; glucose is 50-80 mg / 100 mL; cell count is 0-5 white blood cells, with no red blood cells; chloride (110-125 mEq / L). Abnormal results may indicate the presence or progression of MS.

[0130] MRI is another technique that can be used to monitor disease progression and improvement. Typical criteria for monitoring MS using MRI include the appearance of abnormal white matter patches in the cerebral hemispheres and paraventricular regions, and lesions present in the cervical or thoracic regions of the cerebellum and / or brainstem, as well as the spinal cord. Evoked potentials can be used to monitor the progression and improvement of MS in a subject. Evoked potentials measure the delay of electrical impulses such as visual evoked responses (VER), brainstem auditory evoked responses (BAER), and somatosensory evoked responses (SSER). Abnormal responses can help indicate a decrease in the conduction velocity of the central sensory pathway.

[0131] Gait monitoring can also be used to monitor disease progression and improvement in MS patients. MS often involves motor impairment and abnormal gait, partly due to fatigue. Monitoring can be performed, for example, using a mobile monitoring device worn by the patient. (Moon, Y., et al., Monitoring gait in multiple sclerosis with novel wearable motion sensors, PLOS One, 12(2):e0171346(2017)).

[0132] d. Huntington In addition to monitoring improvement in cognitive symptoms, the progression or improvement of neurodegeneration associated with Huntington's disease (HD) can be monitored using techniques well known to those skilled in the art. Monitoring can be performed, but not limited to, through techniques such as motor function; behavior; functional assessment; and imaging.

[0133] Examples of motor function metrics that can be monitored as indicators of disease progression or improvement include chorea and dystonia, rigidity, bradykinesia, oculomotor dysfunction, and changes in gait / balance. Techniques for monitoring these metrics are well known to those skilled in the art. (See Tang C, et al., Monitoring Huntington's disease progression through preclinical and early stages, Neurodegener Dis Manag 2(4):421-35 (2012)).

[0134] The psychiatric effects of HD provide an opportunity to monitor the progression and improvement of the disorder. For example, a psychiatric diagnosis may be made to determine whether the subject is suffering from a psychosis involving depression, irritability, agitation, anxiety, apathy, and paranoia. (Ibid.)

[0135] Functional assessments may be used to monitor disease progression or improvement. A total functional score technique has been reported (ibid.), and in some HD groups, it often decreases by 1 point per year.

[0136] MRI or PET may be used to monitor disease progression or improvement. For example, in HD, there is loss of striatal projection neurons, and changes in the number of these neurons can be monitored in the subject. Techniques for determining neuronal changes in HD subjects include imaging of dopamine D2 receptor binding. (Ibid.)

[0137] e.ALS In addition to monitoring improvement in cognitive symptoms, the progression or improvement of neurodegeneration associated with amyotrophic lateral sclerosis (ALS) can be monitored using techniques well known to those skilled in the art. Examples, but not limited to, monitoring can be carried out through techniques such as functional assessment; determination of muscle strength; measurement of respiratory function; measurement of lower motor neuron (LMN) loss; and measurement of upper motor neuron (UMN) dysfunction.

[0138] Functional assessment can be performed using functional scales well known to those skilled in the art, such as the ALS Functional Rating Scale (ALSFRS-R), which evaluates symptoms related to ocular, limb, and respiratory function. The rate of change is useful in predicting survival and disease progression or improvement. Another scale is the Combined Assessment of Function and Survival (CAFS), which ranks the clinical outcome of a subject by combining survival time and ALSFRS-R changes. (Simon NG, et al., Quantifying Disease Progression in Amyotrophic Lateral Sclerosis, Ann Neurol 76:643-57 (2014)).

[0139] Muscle strength can be tested and quantified using a composite Manual Muscle Testing (MMT) scoring system. This involves averaging measurements obtained from several muscle groups using the Medical Research Council (MRC) muscle strength grading scale. (Ibid.) Among other techniques, handheld dynamometry (HHD) may be used. (Ibid.)

[0140] Respiratory function can be measured using a portable vital capacity measurement unit, which is used to obtain forced vital capacity (FVC) at baseline and predict disease progression or improvement. Furthermore, maximal inspiratory pressure, nasal inspiratory pressure (SNIP), and sipping FVC can be determined and used to monitor disease progression / improvement. (Ibid.)

[0141] Loss of lower motor neurons is another metric that can be used to monitor disease progression or improvement in ALS. Neurophysiological indicators can be determined by measuring compound muscle action potentials (CMAPs) in motor nerve conduction studies, with parameters including CMAP amplitude and F-wave frequency. (Ibid. and de Carvalho M, et al., Nerve conduction studies in amyotrophic lateral sclerosis. Muscle Nerve 23:344-352, (2000)). Lower motor neuron unit count (MUNE) can similarly be estimated. MUNE estimates the number of residual motor axons supplying muscle through estimation of the contribution of individual motor units to the maximal CMAP response and is used to determine disease progression or improvement. (Simon NG et al., previously cited). Further techniques for determining LMN loss include neuronal excitability testing, electrical impedance myography, and the use of muscle ultrasound to detect changes in muscle thickness. (Ibid.)

[0142] Upper motor neuron dysfunction is another metric that can be used to monitor disease progression or improvement in ALS. Techniques for determining dysfunction include performing MRI or PET scans on the brain and spinal cord; transcranial magnetic stimulation; and determining the levels of biomarkers in cerebrospinal fluid (CSF).

[0143] f. Glaucoma In addition to monitoring improvement in cognitive symptoms, the progression or improvement of neurodegeneration associated with glaucoma can be monitored using techniques well known to those skilled in the art. Examples, but not limited to, monitoring can be performed through techniques such as determining intraocular pressure; evaluating the optic disc or optic head for damage; visual field testing for peripheral vision loss; and imaging of the optic disc and retina for topographic analysis.

[0144] g. Progressive supranuclear palsy (PSP) In addition to monitoring improvement in cognitive symptoms, the progression or improvement of neurodegeneration associated with progressive supranuclear palsy (PSP) can be monitored using techniques well known to those skilled in the art. Monitoring can be carried out, but not limited to, through techniques such as functional assessment (activities of daily living, i.e., ADL); motor assessment; determination of psychiatric symptoms; and volumetric measurements and functional magnetic resonance imaging (MRI).

[0145] The functional level of an object in relation to independence, partial or complete dependence on others may be useful in determining disease progression or improvement. (See Duff, K, et al., Functional impairment in progressive supranuclear palsy, Neurology 80:380-84, (2013)). The Progressive Supranuclear Palsy Rating Scale (PSPRS) is an assessment scale containing 28 metrics in six categories: daily activities (as per medical history); behavior; eye, eye movements, limb movements, and gait / midline. The result is a score ranging from 0 to 100. Six items are graded from 0 to 2, and 22 items are graded from 0 to 4, with a possible total of 100. PSPRS scores are a practical measure and a robust predictor of patient survival. They are also sensitive to disease progression and are useful in monitoring disease progression or improvement. (Golbe LI, et al., A clinical rating scale for progressive supranuclear palsy, Brain 130:1552-65, (2007)).

[0146] The ADL section of the UPDRS (Unified Parkinson's Disease Rating Scale) can also be used to quantify the functional activity of individuals with PSP (Duff K et al., previously cited). Similarly, the Schwab & England Activities Daily Living Score (SE-ADL) can be used for independent assessment (ibid.). Furthermore, the motor function section of the UPDRS is useful as a reliable measure for assessing disease progression in PSP patients. The motor section may include, for example, 27 different measures for quantifying motor function in PSP patients. These examples include resting tremor, rigidity, finger tapping, posture, and gait). Disease progression or improvement in an individual can also be assessed by conducting a baseline neuropsychological assessment completed by a trained healthcare professional, which uses the Neuropsychiatric Symptom Assessment (NPI) to determine the frequency and severity of behavioral abnormalities (e.g., delusions, hallucinations, agitation, depression, anxiety, euphoria, apathy, disinhibition, irritability, and abnormal motor behaviors) (ibid.).

[0147] Functional MRI (fMRI) can also be used to monitor disease progression and improvement. fMRI is a technique that uses MRI to measure changes in brain activity in specific areas of the brain, usually based on blood flow to those areas. Blood flow is thought to correlate with brain region activation. Patients with neurodegenerative disorders such as PSP may undergo physical or mental testing before or during an MRI scan. As an example, but not an exhaustive one, a test could be a well-established force control paradigm in which the patient is asked to generate force in the hand most affected by PSP and with maximum voluntary contraction (MVC), and this is measured by fMRI immediately after the test is performed. (Burcius, RG, et al., Distinct patterns of brain activity in progressive supranuclear palsy and Parkinson's disease, Mov. Disord. 30(9):1248-58(2015)).

[0148] Volumetric MRI is a technique in which an MRI scanner determines the volume difference of regional brain volumes. This can be done, for example, by contrasting different disorders or by determining the volume difference of a patient's brain regions over time. Volumetric MRI can be used to determine the progression or improvement of neurodegenerative disorders such as PSP. This technique is well known to those skilled in the art. (Messina D, et al., Patterns of brain atrophy in Parkinson's disease, progressive supranuclear palsy and multiple system atrophy, Parkinsonism and Related Disorders, 17(3):172-76 (2011)). Examples of cerebral regions that can be measured include, but are not limited to, the cranial volume, cerebral cortex, cerebellar cortex, thalamus, caudate nucleus, putamen, globus pallidus, hippocampus, amygdala, lateral ventricles, third ventricle, fourth ventricle, and brainstem.

[0149] h. Neurodevelopment The present invention also aims to treat or improve neurogenesis in subjects with reduced or impaired neurogenesis, which may be evident, for example, by a reduction in cognitive or motor function, or in association with neuroinflammation. One embodiment of the present invention, not limited to but including as an example, involves administering plasma, plasma fractions, or PPF to subjects with reduced or impaired neurogenesis using a pulsed drug administration regimen.

[0150] One embodiment of the present invention also aims to determine the level of neurogenesis before, during, and / or after administration of plasma, plasma fraction, or PPF. Non-invasive techniques for assessing neurogenesis have been reported (Tamura Y. et al., J. Neurosci. (2016) 36(31):8123-31). Positron emission tomography (PET) used with the tracer [18F]FLT, in combination with the BBB transporter inhibitor probenecid, allows for the accumulation of the tracer in neurogenic regions of the brain. Such imaging allows for the assessment of neurogenesis in patients being treated for neurodegenerative diseases.

[0151] i. Neuroinflammation The present invention also aims to treat or improve neuroinflammation in subjects with elevated neuroinflammation, which may manifest, for example, through a reduction in cognitive or motor function, or in association with a reduction in neurogenesis or neurodegeneration. One embodiment of the present invention includes, but is not limited to, the administration of plasma, plasma fractions, or PPF to a subject with neuroinflammation using a pulsed drug administration regimen.

[0152] One embodiment of the present invention also aims to determine the level of neuroinflammation before, during, and / or after administration of plasma, plasma fraction, or PPF. Non-invasive techniques for assessing neuroinflammation, e.g., 11TSPO positron emission tomography (TSPO PET) using C-PK11195 and other such tracers has been reported (see Vivash L, et al., J. Nucl. Med. 2016, 57:165-68; and Janssen B, et al., Biochim. et Biophys. Acta, 2016, 425-41, incorporated herein by reference). Invasive techniques for assessing neuroinflammation include extracting cerebrospinal fluid and detecting the expression levels of neuroinflammation markers or factors such as (but not limited to) prostaglandin E2, cyclooxygenase-2, TNF-alpha, IL-6, IFN-gamma, IL-10, eotaxin, beta-2 microglobulin, VEGF, glial cell line-derived neurotrophic factors, thiotriosidase-1, MMP-9, CXC motif chemokine 13, terminal complement complex, chitinase-3-like protein 1, and osteopontin. (See Vinther-Jensen T, et al., Neruol Neurimmunol Neuroinflamm, 2016, 3(6):e287; and Mishra et al., J. Neuroinflamm., 2017, 14:251, which are incorporated herein by reference.)

[0153] 13. Reagents, devices, and kits Reagents, devices, and kits thereof for performing one or more of the above methods are also provided. The reagents, devices, and kits thereof may vary considerably.

[0154] The reagents and devices of interest include those described above relating to methods for preparing plasma-containing blood products for transfusion to subjects requiring them, such as anticoagulants, cryogenic preservatives, buffers, and isotonic solutions.

[0155] The kit may also include blood collection bags, tubes, needles, centrifuge tubes, etc. In yet another embodiment, the kit described herein includes two or more containers of plasma products, such as plasma protein fractions, e.g., three or more, four or more, five or more (including six or more) containers of plasma products. In some cases, the number of separate containers of plasma products in the kit may be nine or more, twelve or more, fifteen or more, eighteen or more, twenty-one or more, twenty-four or more, thirty or more, and include thirty-six or more, e.g., fourty-eight or more. Each container may be associated with identification information that includes various data about the plasma product it contains, and this identification information may include one or more of the following: the age of the plasma product donor, processing details of the plasma product, e.g., whether the plasma product has been processed to remove proteins exceeding the average molecular weight (such as those described above), blood type details, etc. In some cases, each container in the kit contains identification information about the plasma it contains, and this identification information includes information about the donor age of the plasma product, for example, the identification information provides confirmation of the age-related data of the plasma product donor (such identification information may be the donor's age at the time of collection). In some cases, each container in the kit contains plasma products from donors of substantially the same age, i.e., all containers contain products from donors of substantially the same age, if not identical. Substantially the same age means that the various donors from which the plasma products in the kit are obtained are all different, and in some cases the difference is 5 years or less, e.g., 4 years or less, e.g., 3 years or less, including 2 years or less, e.g., 1 year or less, e.g., 9 months or less, 6 months or less, 3 months or less, including 1 month or less. The identification information can reside on any convenient component of the container, such as a label or RFID chip. The identification information may be human-readable, computer-readable, etc., as needed. The container may have any convenient configuration. The capacity of the container can vary, but in some cases, the capacity is in the range of 10 ml to 5000 ml, for example 25 ml to 2500 ml, or for example 50 ml to 1000 ml (including 100 ml to 500 ml). The container may be rigid or flexible and may be made from any convenient material, such as polymer materials including medical-grade plastic materials.In some cases, the container has a bag or pouch configuration. In addition to the container, such a kit may further include, for example, a dosing device as described above. The components of such a kit may be provided in any suitable packaging, such as a box or similar structure, configured to hold the container and other kit components.

[0156] In addition to the components described above, the kit further includes instructions for carrying out the method. These instructions may be present in the kit in various forms, and one or more of these forms may be present in the kit. One possible form of these instructions is printed information on a suitable medium or substrate, e.g., one or more sheets of paper on which the information is printed, the kit's packaging, or accompanying documents. Yet another means is a computer-readable medium on which the information is recorded, e.g., a diskette, CD, or portable flash drive. Yet another possible means is a website address that can be used via the internet to access the information at the removed site. Any convenient means may be present in the kit. [Examples]

[0157] A. Example 1 - Plasma Fractionation Process The general process for plasma fractionation is well established. Figure 1 details the relevant manufacturing process, starting from pooled plasma. Fraction IV-1 paste is an unused process intermediate in the manufacturing process, yielding alpha-1 antitrypsin (Prolastin® C) and ATIII products. While 40% of IV-1 paste is currently utilized to produce the final product, 60% is considered waste. Fraction IV-1 paste can be utilized by dissolving it in solution by the various methods described above (see Viglio S, et al., Molecules, 25(17):4014(2020); and Chen SX, et al., J Chromatogr A, s02021-9673(97)(1998)). In the following examples, references to IV-1 paste refer to the dissolved IV-1 paste solution.

[0158] B. Example 2 - Plasma fraction activity in various in vitro assays a. Glucose utilization in PPF1 and IV-1 paste and myotubes Figure 2 reports the dose-response relationship between PPF1 and IV-1 paste. PPF1 was previously identified as the active plasma fraction in this assay (U.S. Patent Application Publication No. 20210128693). Myotubes were differentiated into myoblasts as shown in Figure 45. The treatment was added to cells at the following concentrations: 2.5, 1.25, 0.63, and 0.315 mg / mL. The two plasma fractions showed a dose-response relationship to utilized glucose and reflected the degree of myotubular differentiation.

[0159] Unexpectedly, IV-1 paste was 14-fold more active than the positive control PPF1. This suggests that IV-1 paste is an ideal plasma fraction for treating musculoskeletal disorders such as cachexia and frailty that result in muscle weight loss.

[0160] b. Activity of PPF1, HAS1, IV-1 paste, and IV-4 paste between assays Figure 3 shows a parallel comparison of the activity of various plasma fractions across in vitro assays. Heatmaps of various in vitro assays in multiple cell lines are shown. The selected cell lines were endothelial cells (HUVEC), skeletal muscle cells (C2C12), and microglia (primary mouse microglia). The activity of the following plasma fractions was compared with each other: IV-1 paste, IV-4 paste, PPF1, and HAS1 (shown as HSA1 in Figure 3). Recombinant human albumin (rhAlbumin) was included as a protein loading control. Heatmap color coding: Blue indicates potential beneficial activity in the corresponding assay and cell type, white indicates the same activity as the vehicle, and yellow indicates potential harmful activity. (The terms "beneficial activity" and "harmful activity" reflect the more general disease indications that may be treated by the administration of the plasma fractions. For example, in some indications such as infections, increased cytokine release may be found to be beneficial activity in treating those indications.) All plasma fractions were tested at the same protein concentration. The activity was visualized using a prism and normalized against vehicle activity.

[0161] Interestingly, the IV-1 paste was approximately 10 times more potent than PPF1 across multiple cell assays. We found that the fraction IV-1 paste suspension increased barrier function in HUVECs, decreased adhesion molecule surface expression, increased metabolic and regenerative activity in C2C12 cells, and reduced phagocytic activity in activated primary microglia. However, the fraction IV-1 paste suspension also induced the secretion of IL-8 and IL-6. While this suggests a pro-inflammatory response, this data package should be interpreted cautiously depending on the selection of the indication space.

[0162] C. Example 3-IV-1 Subfraction and overall activity of the paste Figure 4 shows the separation of IV-1 paste in 13 compartments using an anion exchange chromatography column. Q-Sepharose was used as the anion exchange matrix. Each sub-fraction was compounded with 0.9% NaCl / 10mM HEPES to prepare test samples. The total protein concentration from each fraction was measured by BCA testing and is listed in Figure 4. Furthermore, the load (material used to load onto the column) and FT (flow-through) were measured for their total protein concentrations.

[0163] Figure 5 shows Coomassie blue staining from 13 subfractions from the IV-1 paste. Each subfraction was loaded onto a gel at the same protein concentration. Proteins were first separated on an SDS-PAGE gel. The gel was then immersed in Coomassie dye to visualize the proteins.

[0164] Figure 6 reports the activity of Load (IV-1 paste), FT, and 13 sub-fractions in brain barrier, muscle function, and inflammation assays. The most active fraction is highlighted with a gray rectangle. For the brain barrier assay, the fraction was concentrated 10-fold, while for the muscle function and inflammation assays, the fraction was tested at 1-fold. Chromatographic sub-fractions 8 and 9 were observed to enhance barrier function; sub-fraction 12 promoted the secretion of IL-6 and IL-8; and sub-fractions 2-5 enhanced muscle activity to a more significant degree compared to the other sub-fractions. Heatmap color coding: blue indicates beneficial activity in the corresponding sub-fraction, white indicates no activity in the sub-fraction, and yellow indicates harmful activity. Each sub-fraction was used at 10% without adjusting protein concentration. Activity was visualized using a prism and normalized to Load activity.

[0165] The abundances of alpha-1-antitrypsin (A1AT) and antithrombin III (ATIII) were compared using RFU values ​​obtained with SomaLogic Aptamer Technology (SomaLogic Operating Co., Inc., Boulder, CO). The heatmap is color-coded as follows: black indicates a high abundance of the indicated protein; gray indicates a low abundance of the protein compared to black; and white indicates the lowest abundance. The abundances were visualized using Prism and normalized against the abundance of the loaded protein. The protein abundances of the final products from the IV-1 paste (i.e., A1AT and ATIII) did not correlate with the activity of the subfractions. This indicates that these final products (i.e., A1AT and ATIII) cannot be the driving factors of activity in these assays, which was surprising since they are the most notable products obtained from the IV-1 paste.

[0166] Surprisingly, the IV-1 paste subfraction resulted in the decoupling of different biological activities of this fraction; demonstrating that multiple therapeutic subfractions with activities unrelated to the known bioactive substances A1AT and ATIII can be developed from the IV-1 paste. It was also observed that increased cytokine release could be isolated from the beneficial effects observed from the IV-1 paste using the subfraction approach. This was an interesting finding, as increased pro-inflammatory cytokines should be avoided in some chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Based on the observed activities of subfractions 8 and 9, they can be used as therapeutic subfractions to treat or even cure vascular dementia caused by symptoms that block blood flow and oxygen supply to the brain and damaged blood vessels in the brain. Subfractions 2-5 can also be used as therapeutic subfractions to treat musculoskeletal disorders such as cachexia and frailty that result in loss of muscle mass. Subfraction 12 can be used as a therapeutic fraction to treat or cure infectious diseases caused by bacteria, viruses, and fungi that can enter the body and cause infection.

[0167] D. Example 4 - Primary mouse microglia phagocytosis Figure 7 shows the processing paradigm of isolated primary mouse microglia. Primary mouse microglia were isolated from co-culture with astrocytes by MACS sorting. Microglia were plated in serum-free medium for 4-5 hours. Microglia are a type of immune cell of the central nervous system (CNS) that plays a role in maintaining brain homeostasis and responding to injury and infection. The degree of phagocytosis is an indicator of neuroinflammation because the main role of microglia is to remove debris for tissue repair and remodeling in the brain. Microglial phagocytosis is one of the processes most affected in neurodegenerative diseases. Hyperactivated microglia phagocytose neurons, neuronal synapses, and myelin in conditions such as aging, multiple sclerosis, and Parkinson's disease. This results in decreased signal conductance and changes in brain physiology. Therefore, reducing phagocytosis from hyperactivated microglia is a strategy that can be used to treat these diseases.

[0168] Microglia were isolated from postnatal mouse pups and purified by MACS isolation. Various treatments were then applied to the microglia overnight. Fluorescently labeled beads were then added to the cells, and after a further 1 hour of culture, the cells were fixed. The number of cells that had incorporated the beads after 1 hour was determined by FACS.

[0169] Figure 8 reports the phagocytic activity of treated microglia, quantified by FACS normalized against untreated microglia as described in Figure 7. Microglia were treated with IV-1 paste in a dose-response pattern ranging from 0.6 mg / mL to 5 mg / mL. Cyto D was used as a positive control. Figure 8 shows that IV-1 paste can reduce phagocytic activity in microglia in a dose-dependent manner. This suggests that in disease situations where microglia are activated, the neuroinflammatory effects of such diseases may be attenuated.

[0170] Figure 9 reports the effects of subfractions of IV-1 paste on a microglial phagocytic assay. IV-1 paste was fractionated into 13 subfractions (Fr 1–Fr 13) using an anionic charge-based Q-Sepharose separation column. The subfractions were tested as shown in Figure 7. Phagocytic activity was determined based on protein isolation. The highest activity was observed in subfractions 7 and 8 (FT = flow-through).

[0171] Figure 10 reports the effects of single purified protein products (A1AT and ATIII) from IV-1 paste on microglial phagocytic activity. A1AT showed significant activity in the phagocytic assay. Furthermore, a dose-response analysis was performed for A1AT. The black bars represent the same amount of protein as the parent IV-1 fraction (i.e., 1X). ATIII did not significantly affect phagocytic activity in microglia, while A1AT showed activity in a dose-response manner. Surprisingly, in terms of the amount present in the IV-1 paste, A1AT had only a slight effect compared to the IV-1 paste itself, suggesting that there is additional biological activity in the IV-1 paste that is not explained by A1AT.

[0172] E. Example 5 - Adhesion Molecule Expression Figure 11 shows a processing paradigm for the analysis of adhesion molecule surface expression in HUVECs. HUVECs (human umbilical vein endothelial cells) are primary cells essential for maintaining vascular integrity. They are a model for studying angiogenesis, vascular permeability, inflammation, and cell adhesion molecule surface expression. The expression levels of specific adhesion molecules on cells are directly related to the ability of T cells and other immune cells to infiltrate tissues, particularly the brain, and induce disease-related inflammation such as peripheral inflammation and neuroinflammation. The recruitment of immune cells and infiltration into the brain are associated with several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, multiple sclerosis, neuromyelitis optica, and acute disseminated encephalomyelitis.

[0173] HUVEC cells were seeded at 10k / well in 96-well plates. Cells were cultured for 48 hours, followed by stress with or without 0.2 ng / mL of TNFα while simultaneously being treated with test proteins or plasma fractions. After 3 hours, the surface expression levels of adhesion molecules were determined by FACS. These included VCAM-1, ICAM-1, and CD62E expression. The amount of adhesion molecules was determined as both the percentage of positive cells and the mean fluorescence intensity (MFI), which quantifies the degree of surface expression of adhesion molecules.

[0174] Figures 12–14 show the results for individual proteins and plasma fractions in the assay described in Figure 11. Both negative control (5 ng / mL) and positive control IKK inhibitors for TNFα were administered to cells in a similar manner to the test protein / plasma fractions described in Figure 11. Several plasma fractions containing PPF1, HAS1, recombinant albumin (rhAlbumin), and IV-1 paste were also tested. All data were normalized to HEPES buffer. IV-1 paste dose-dependently inhibited the expression of VCAM-1 adhesion molecules (0.3 mg / mL to 5 mg / mL). Figure 13 reported similar results for ICAM-1 adhesion molecule expression. Figure 14 reported similar results for CD62E adhesion molecule expression, but the effect was observed only in the highest dose fraction of IV-1 paste.

[0175] Therefore, IV-1 paste showed the highest efficacy in reducing the surface expression of adhesion molecules compared to other tested plasma fractions and proteins. This suggests that in disease situations where the surface expression of adhesion molecules leads to the infiltration of T cells and other immune cells, the effects of such diseases can be reduced more effectively by IV-1 paste than by other plasma fractions.

[0176] Figures 15–17 report the effects of 13 sub-fractions of fraction IV-1 paste on these adhesion molecules, as described in the assay in Figure 11. No sub-fractions showed significantly clear activity in reducing the expression of adhesion molecules VCAM-1, ICAM-1, or CD62E.

[0177] Figures 18–21 report the effects of single protein products purified from fraction IV-1 paste (A1AT and ATIII) on adhesion molecule surface expression. Neither protein appears to contribute to the activity of IV-1 paste that reduces adhesion molecule surface expression (VCAM-1 and ICAM-1). This was observed both under TNFα stress (Figures 18 and 19) and without TNFα stress (Figures 20 and 21).

[0178] F. Example 6 - Barrier Function Assay Figure 22 depicts the processing paradigm for the barrier function assay. HUVEC cells were seeded at 30 k / sq cm in CytoZ 96-well plates and cultured for 3 days. Cells were then treated with plasma fractions or controls, and barrier function was measured for 3 consecutive days using Maestro Pro impedance function at 1 kHz. Endothelial barrier function plays a crucial role in maintaining the integrity of the vascular system and the blood-brain barrier. Disruption of this barrier can lead to the entry of harmful substances into surrounding tissues and contribute to disease progression. Multiple sclerosis, stroke, Alzheimer's disease and Parkinson's disease, as well as HIV-associated neurocognitive disorders (HAND), are some examples of CNS diseases in which endothelial barrier function is disrupted. This disruption promotes the perfusion of inflammatory cells and toxic molecules into the brain parenchyma, leading to neuronal degeneration. Improvement or restoration of barrier function would be beneficial in treating or halting the progression of such diseases. Furthermore, peripheral indications include, for example, loss of barrier function, including acute respiratory distress syndrome (ARDS), sepsis, inflammatory bowel disease (IBD), atherosclerosis, diabetes mellitus, cancer, and allergic reactions.

[0179] Barrier function assays are useful for determining the integrity and function of the endothelial cell barrier formed by HUVEC cells, which is crucial for regulating the passage of molecules and cells between blood flow and other tissues, such as the brain. In the brain, this assay reflects the function and integrity of the blood-brain barrier.

[0180] Transendothelial electrical resistance (TEER) is an assay that can be performed to measure the resistance to the passage of electric current across HUVECs. Resistance to a frequency of 1 kHz is directly proportional to a healthier, more intact, and tighter barrier for endothelial cells.

[0181] Figure 23 reports the effects of fraction IV-1 paste and PPF1 on relative TEER normalized to pretreatment. A83-01, a potent TGF-beta inhibitor, is a positive control that increases barrier function. TNFα is a negative control known to decrease barrier function. Both fraction IV-1 paste and PPF1 increased barrier function 72 hours after treatment, but IV-1 paste was more effective than PPF1. The TEER assay reflects how closely endothelial cells are connected to each other, resulting in less leakage of proteins across the endothelium, the most important component of the blood-brain barrier. Increased blood-brain barrier leakage and loss of integrity are associated with neurodegenerative diseases, particularly those resulting in cognitive impairment.

[0182] Figure 24 shows a dose-response study of fraction IV-1 paste 72 hours after treatment using the TEER assay. A83-01 is a positive control that increases barrier function. TNFα is a negative control known to decrease barrier function. This study demonstrates that fraction IV-1 paste can improve barrier function in a dose-response manner.

[0183] Figure 25 shows the results of a TEER assay using cells treated with fraction IV-1 paste, flow-through (FT), and sub-fractions 1-13 from fraction IV-1 paste. A83-01 is a positive control that increases barrier function. TNFα is a negative control known to decrease barrier function. Thirteen sub-fractions were tested as described in Figure 22. However, these sub-fractions were concentrated 10-fold. Fraction 9 showed the highest activity in the barrier function assay. Fraction 8 also showed significantly increased activity in this assay.

[0184] Figure 26 shows the results of a TEER assay using cells treated with fraction IV-1 paste, different concentrations of the single protein alpha-1 antitrypsin (A1AT), and different concentrations of the single protein antithrombin III (ATIII). A83-01 is a positive control that increases barrier function. TNFα is a negative control known to decrease barrier function. The single proteins A1AT and ATIII showed no significant activity, highlighting that other driving factors should be present in fraction IV-1 paste and 13 specific fraction IV-1 paste subfractions.

[0185] G. Example 7 - Endothelial Cell Proliferation Assay Figure 27 shows the treatment paradigm for the endothelial cell proliferation assay. HUVEC cells were seeded at 4K / well in CytoZ 96-well plates. Cells were cultured for 1 day and then serum-starved for a further 24 hours in low-serum medium (0.1% FBS). Cells were then treated with plasma fractions or sub-fractions for 3 days and continuously measured for Maestro Pro impedance function. A resistance of 41.5 kHz was used to determine the degree of confluence reflecting endothelial cell proliferation. Endothelial cells are the main components of the cardiovascular system that enable the exchange of essential cells and molecules between blood flow and target tissues. Endothelial loss is observed in many cardiovascular metabolic diseases that worsen patient outcomes. Atherosclerosis, type 2 diabetes, and septic shock are some examples in which endothelial cell loss leads to multi-organ (e.g., heart, kidney, eye) failure. Endothelial cell loss is also a common feature of pulmonary hypertension and inflammatory bowel disease (IBD), contributing to lung and intestinal dysfunction. Promoting endothelial cell regeneration may be promising in treating or managing such conditions in which endothelial cell loss constitutes a large part of the disease mechanism.

[0186] Figure 28 shows the results of the HUVEC endothelial cell proliferation assay described in Figure 27. A positive control of the proliferation medium was used. Forskolin and bFGF were also used as positive controls. Fractions IV-1 paste, IV-4 paste, PPF1, and HAS1 were tested side by side. Fraction IV-1 paste showed the highest activity compared to the other fractions after 72 hours.

[0187] Figure 29 shows the results of a time-response experiment of the HUVEC endothelial cell proliferation assay. Cells were administered a growth medium-positive control, fraction IV-1 paste, and vehicle for 3 days, and measurements were taken over time. This indicates that fraction IV-1 paste acts in a time-dependent manner.

[0188] H. Example 8 - Cytokine Release Assay Figure 30 depicts the processing paradigm for the cytokine release assay. HUVEC cells were seeded at 10k / well in 96-well plates. After 24 hours, cells were treated with or without 0.2 ng / mL of TNFα stressor, while simultaneously processing the plasma fraction. After another 24 hours, cytokine release from cells was measured for IL-6 and IL-8 by ELISA. Inducing an inflammatory response can be beneficial in conditions such as injury and infection, where the body needs to activate its immune system. Increased pro-inflammatory cytokine release is a beneficial biological response to infectious agents aimed at mobilizing immune cells and eliminating pathogens. Such infectious agents can be bacterial, viral, or fungal infections. Common infections include sepsis, pneumonia, meningitis, tuberculosis (TB), COVID-19, influenza, HIV, human papillomavirus (HPV), and herpes. Figure 31 shows the results of IL-6 release under TNFα stress using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin). Plasma fractions and rhAlbumin were administered as described in Figure 30. When stressed with TNFα, neither the fractions tested nor rhAlbumin stimulated IL-6 release.

[0189] Figure 32 shows the results of IL-8 release under TNFα stress using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin). Compared with HAS1, PPF1, and rhAlbumin, there was a dose-dependent increase in IL-8 release with IV-1 paste treatment.

[0190] Figure 33 shows the results of IL-6 release using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin) under the absence of TNFα stress. Plasma fractions and rhAlbumin were administered as described in Figure 30. Only IV-1 paste resulted in increased IL-6 release, and this increase was dose-dependent.

[0191] Figure 34 shows the results of IL-8 release using multiple plasma fractions (HAS1, PPF1, and IV-1 paste) or recombinant human albumin (rhAlbumin) under the absence of TNFα stress. Plasma fractions and rhAlbumin were administered as described in Figure 30. Only IV-1 paste resulted in increased IL-8 release, and this increase was dose-dependent.

[0192] Figure 35 shows that 13 (13) Q-Sepharose subfractions, sub-fractionated from fraction IV-1 paste, were tested together with fraction IV-1 paste and FT in a cytokine release assay using a TNFα stressor. Sub-fraction 12 significantly increased IL-6 release, while none of the other fractions increased it. This result suggests that the driving factor for the increase in IL-6 identified in fraction IV-1 paste lies in sub-fraction 12. This indicates that the potentially undesirable effect of fraction IV-1 paste (i.e., inflammation) can be isolated from its more desirable characteristics.

[0193] Figure 36 reports the IL-8 release results from testing 13 (13)Q-Sepharose subfractions in the cytokine release assay described in Figure 30. Subfractions obtained from fraction IV-1 paste were tested in a cytokine release assay using TNFα stressors, together with fraction IV-1 paste and FT. Subfraction 12 significantly increased IL-8 release, while none of the other fractions increased it. From these results, it appears that the driving factor for the increase in IL-6 identified in fraction IV-1 paste lies in subfraction 12. This indicates that the potentially undesirable effect of fraction IV-1 paste (i.e., inflammation) can be isolated from its more desirable characteristics.

[0194] The fact that undesirable pro-inflammatory activity could be fractionated very dramatically and cleanly from other subfractions was unexpected. Eliminating these potentially undesirable features found in subfraction 12 opens up opportunities to use positively active subfractions in the treatment of multiple disease areas.

[0195] Figure 37 reports the results of testing alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, on cytokine release. Without the use of a TNFα stressor, the study demonstrated that A1AT administration did not result in increased IL-6 release in cells.

[0196] Figure 38 reports the results of testing alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, on cytokine release. Without the use of a TNFα stressor, the study demonstrated that there was no increased release of IL-8 in cells induced by A1AT administration.

[0197] Figure 39 reports the results of testing alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, for cytokine release. The TNFα stressor-induced IL-6 release demonstrated that A1AT administration did not result in increased IL-6 release in cells.

[0198] Figure 40 reports the results of testing alpha-1 antitrypsin (A1AT), a single purified protein product derived from IV-1 paste, for cytokine release. The TNFα stressor-induced IL-8 release demonstrated that A1AT administration did not result in increased IL-8 release in cells.

[0199] Figure 41 reports the results of testing antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, for cytokine release. It demonstrated that IL-6 release without the use of a TNFα stressor was not increased by ATIII administration.

[0200] Figure 42 reports the results of testing antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, for cytokine release. It demonstrated that IL-8 release without the use of a TNFα stressor was not increased by ATIII administration.

[0201] Figure 43 reports the results of testing antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, for cytokine release. The TNFα stressor-induced IL-6 release demonstrated that ATIII administration did not result in increased IL-6 release in cells.

[0202] Figure 44 reports the results of testing antithrombin III (ATIII), a single purified protein product derived from IV-1 paste, for cytokine release. The TNFα stressor-induced IL-8 release demonstrated that ATIII administration did not result in increased IL-8 release in cells.

[0203] I. Example 9-C2C12 Myotube Formation Assay Figure 45 depicts the treatment paradigm for the C2C12 myoblast differentiation assay. C2C12 myoblasts were seeded at 8k / well in 96-well plates in DMEM + 4.5 g / L glucose and 10% fetal bovine serum (FBS). After 2 days, differentiation into myotubes was induced by changing the medium to 1 g / L glucose and 0% equine serum. Controls and treatments were added during this differentiation. The medium was changed every other day, and different treatments were added simultaneously as outlined in the timeline. After 6 days, the amount of glucose used by the cells was determined by a quantitative colorimetric assay. The amount of glucose used reflects how well the myoblasts differentiated into myotubes, which are more metabolically active. Highly differentiated and metabolically active myotubes are characteristic of healthy skeletal muscle. Increased myotubogenesis suggests that therapeutic plasma fractions can be used to treat musculoskeletal disorders resulting in muscle mass loss, such as cachexia, frailty, Charcot-Marie-Tooth disease, congenital muscular dystrophy, and congenital fibrous type disparity.

[0204] Figure 46 shows the results of the effects of 13 subfractions of fraction IV-1 paste on the myotubation assay described in Figure 45. Higher degrees of myotubation differentiation correlate with higher metabolic activity and lower values ​​on the y-axis. Subfractions 2-6 showed high activity in promoting myotubation in C2C12 cells. This is unexpected and surprising, as these fractions showed activity even when they differed from their subfractions in HUVEC cells and primary microglia cells, which also showed activity (e.g., cytokine release and microglial phagocytosis assays).

[0205] Figure 47 reports the effect of alpha-1 antitrypsin (A1AT), a single protein product purified from fraction IV-1 paste, on the myotubation assay described in Figure 45. All data were normalized to the A1AT vehicle. A1AT did not show significant activity.

[0206] Figure 48 reports the effect of antithrombin III (ATIII), a single protein product purified from fraction IV-1 paste, on the myotubation assay described in Figure 45. All data were normalized to the ATIII vehicle. ATIII did not show significant activity.

[0207] J. Example 10 - C2C12 Myotube Metabolism Assay Figure 49 depicts the processing paradigm for C2C12-derived myotube formation. C2C12 myoblasts were seeded at 8k / well in 96-well plates in DMEM medium + 4.5 g / L glucose and 10% fetal bovine serum (FBS). After 2 days, differentiation into myotubes was achieved by changing the medium to 1 g / L glucose and 2% equine serum. After 5 days, the cells were fully differentiated into myotubes and treated with various plasma fractions for 24 hours. The amount of glucose utilized by the cells was determined by quantitative colorimetric assay. The amount of glucose utilized reflects the metabolic activity of the cells. Increased cellular metabolism indicates potential treatment for the following diseases: type 2 diabetes, stroke, fatty liver, insulin resistance, certain metabolic myopathy, and cardiovascular disease.

[0208] Figure 50 shows the dose-response relationship between PPF1 and fraction IV-1 paste in the glucose utilization assay described in Figure 49. Glucose utilization was normalized and compared between treatments. Fraction IV-1 paste showed higher efficacy than PPF1, with an effective concentration of 50% (EC2). 50 It is 9.5 times stronger than PPF1.

[0209] Figure 51 reports the results of the metabolic assay described in Figure 49, using 13 sub-fractions from fraction IV-1 paste. Sub-fractions 2-4 and 12 showed significant activity in glucose utilization. In this case, too, it was unexpected that fractions 2-4 were sub-fractions useful for muscle diseases, as it was unclear whether these sub-fractions would isolate bioactive substances with sufficient precision.

[0210] The results observed across this diverse combination of assays were also surprising, as they suggest that each assay can possess its own set of bioactivity-driving factors, and that the resolution in identifying these factors was due to the quality of the subfraction strategy. This makes it possible to develop multiple potential therapeutic fractions from IV-1 paste using this subfraction method. Furthermore, the overall data showed that traditional protein products that may be derived from fraction IV-1 paste (e.g., alpha-1 antitrypsin and antithrombin III) do not significantly contribute to the activity tested in these assays. Instead, the subfractions possess independent factors that can be used to treat disease indications, and in fact, the subfractions themselves can be used in this way.

[0211] K. Example 10 - Subfraction of Fraction IV-1 suspension The fraction IV-1 suspension was separated into separate protein pools by Q Sepharose chromatography. The column was a 5.0 x 19.5 cm (442 ml) Q Sepharose Fast Flow (FF) run, performed at pH 8.0 with 25 mmM Tris-HCl buffer at 75 cm / hour. 450 ml of fraction IV-1 suspension was loaded onto the column, which corresponded to approximately 5 g of protein.

[0212] Figure 52 shows the chromatographic results corresponding to the separation of Q Sepharose from the fraction IV-1 suspension. As indicated by the light blue vertical line at the bottom of the figure, the column FT / wash pool ranged from 400 ml to 2100 ml. Thirteen fractions of 225 ml each were then collected (fractions 1, 6, and 13 are labeled for reference). Each fraction was half the volume of the load. The left vertical axis shows mAU, ranging from 0 to 1600. The left vertical axis corresponds to the signal at a UV wavelength of 280 nm. The right vertical axis shows mS / cm, ranging from 0 to 150. Elution #1 was a 2CV fixed composition elution with 125 mM NaCl, while elution #2 was a 2CV gradient from 125 to 300 mM NaCl, followed by retention in 300 mM NaCl buffer. Elution #3 was a 2M NaCl strip.

[0213] Figure 53 shows the elution pools observed during the experiment. A280 and end values ​​were obtained after HBS pH 7.2 formulation. Maximum values ​​were observed in elution pools 2, 7, 8, 9, and 12, corresponding to the peaks of the mAU signal, as shown in Figure 52.

[0214] Figure 54 shows the results of gel electrophoresis performed on elution pools obtained from the separation of fraction IV-1 suspension by Q Sepharose chromatography. The left column is the MW standard, followed by fraction IV-1 and FT pool samples. Furthermore, elution pools 1-13 were recorded, followed by another fraction IV-1 and MW standard. Significant signals were observed in 116 pools 1 and 2, 66 pools 2-4, 55 pools 6-13, and 22 pools 3-6. Other signals were also found.

[0215] L. Example 11-SH-SY5Y Survival Assay Figure 55 SH-SY5Y cells were seeded at 20k / well in a 96-well plate in serum-free MEM medium. On day 1, differentiation of cells into dopaminergic neurons was initiated by the addition of BDNF (50ng / mL) and retinoic acid (5μM). After 4 days, the complete medium was replaced, and TPA (80nM) was added to serum-free MEM medium. On day 7, after the cells had differentiated into dopaminergic neurons, the cells were stressed with a neurotoxin (MPP + 1mM) for 24 hours, while simultaneously being treated with a plasma fraction. The number of cells surviving the neurotoxic stress was quantified on day 8. Cell viability was quantified using a fluorescent dye (Promega kit). The amount of fluorescence reflects cell viability. Increased dopaminergic neuronal survival indicates a potential treatment for neurodegenerative diseases such as PD.

[0216] Figure 56 shows the results of survival assays performed on dopaminergic cells under neurotoxic stress (MPP + 1 mM) using multiple plasma fractions (HAS1, PPF1, IV-1 paste) or recombinant human albumin (rhAlbumin). Plasma fractions and rhAlbumin were administered as described in Figure 55. IV-1 paste was tested in a dose-response pattern ranging from 0.6 mg / mL to 5 mg / mL. Both negative controls (2 mM MPP+) and positive controls (0.4 mg / mL Apo-transferrin) were administered to cells in a similar manner to the plasma fractions described in Figure 55. Figure 56 shows that dopaminergic neurons stressed by neurotoxicity were dose-dependently rescued by IV-1 paste treatment. This indicates that cell death may be attenuated in disease situations where dopaminergic neurons are dying. This finding is unexpected and surprising, as the beneficial activity of IV-1 paste exceeded the beneficial activity of the positive control and other plasma fractions by several times.

[0217] M. Example 12-SH-SY5Y Reactive Oxygen Species (ROS) Assay Figure 57 SH-SY5Y cells were seeded at 20k / well in a 96-well plate in serum-free MEM medium. On day 1, differentiation of cells into dopaminergic neurons was initiated by the addition of BDNF (50 ng / mL) and retinoic acid (5 μM). After 4 days, the complete medium was replaced, and TPA (80 nM) was added to serum-free MEM medium. On day 7, after the cells had differentiated into dopaminergic neurons, ROS production was increased by stressing the cells with hydroperoxide (TBHP 50 μM) while simultaneously processing the plasma fraction. The increase in ROS production was quantified after 2 hours using a fluorescent dye (Abcam kit). The amount of fluorescence reflects the amount of ROS produced. A decrease in ROS production in dopaminergic neurons indicates a potential treatment for neurodegenerative diseases such as PD.

[0218] Figure 58 shows the results of a ROS production assay in dopaminergic neurons under peroxide stress (TBHP 50 μM) using plasma fraction (IV-1 paste) or recombinant human albumin (rhAlbumin). IV-1 paste and rhAlbumin were administered as described in Figure 57. IV-1 paste was tested in a dose-response mode ranging from 0.6 mg / mL to 5 mg / mL. Both negative control (100 μM TBHP) and positive control (30 μM resveratrol) were administered to cells in the same manner as the plasma fraction described in Figure 57.

[0219] Figure 58 shows that dopaminergic neurons stressed with peroxidase produced less ROS when dose-dependently treated with IV-1 paste. This suggests that the harmful effects of ROS may be attenuated in disease situations where dopaminergic neurons have increased ROS production.

[0220] N. Example 13 - SH-SY5Y survival assay treatment with purified protein products (A1AT and ATIII) Figure 59 shows the results of a survival assay performed on dopaminergic neurons under neurotoxic stress (MPP + 1 mM) using single purified proteins (A1AT and ATIII). A1AT and ATIII were administered as described in Figure 55. The IV-1 paste was tested at 5 mg / mL and showed significant activity. Both negative controls (2 mM MPP+) and positive controls (0.4 mg / mL Apo-transferrin) were administered to cells in a manner similar to the plasma fractions described in Figure 55. A1AT showed slight but insignificant activity in the survival assay. Furthermore, dose-response studies were performed for A1AT and ATIII. Black bars represent the same amount of protein as the parent IV-1 fraction (i.e., 1X). ATIII did not significantly affect the survival of SH-SY5Y cells, but A1AT demonstrated activity in a dose-response manner. Surprisingly, the amount of A1AT present in the IV-1 paste had only a slight effect compared to the IV-1 paste itself, suggesting that there is additional bioactivity in the IV-1 paste that is not explained by A1AT.

[0221] O. Example 14 - SH-SY5Y Reactive Oxygen Species (ROS) Assay Treatment with Purified Protein Products (A1AT and ATIII) Figure 60 shows the results of a ROS production assay in dopaminergic neurons under peroxide stress (TBHP 50 μM) and single purified proteins (A1AT and ATIII). A1AT and ATIII were administered as described in Figure 57. IV-1 paste was tested at 5 mg / mL and showed significant activity. Both negative control (100 μM TBHP) and positive control (30 μM resveratrol) were administered to cells in the same manner as the plasma fractions described in Figure 57. Figure 60 shows that dopaminergic neurons stressed with peroxidase produced less ROS when treated with IV-1 paste. A1AT and ATIII also showed significant activity in the survival assay. Furthermore, dose-response assays were performed for A1AT and ATIII. Black bars represent the same amount of protein as the parent IV-1 fraction (i.e., 1X). A1AT showed activity in a dose-response manner, while ATIII did not. Surprisingly, the amounts of A1AT and ATIII present in the IV-1 paste significantly reduced ROS production in peroxidase-stressed dopaminergic neurons, suggesting that A1AT and ATIII are the bioactive compounds in the IV-1 paste. This further suggests that in disease situations where dopaminergic neurons have increased ROS production, the harmful effects of ROS may be attenuated by treatment with any of the IV-1 paste, A1AT, or ATIII.

[0222] P. SureQuantity of the most abundant protein in IV-1 paste compared to Example 15-PPF1 Figure 61 shows a heatmap of protein-rich proteins in fraction IV-1 paste, normalized to the amount of protein in fraction PPF1. Protein abundance was quantified by a quantitative mass spectrometry-based method known as SureQuant. Compared to PPF1, IV-1 paste is enriched with several beneficial proteins, including A1AT, TF, IGHA1, and APOA2. This suggests that these proteins may contribute to the beneficial effects outlined in Figures 1–60.

[0223] Q. SureQuantity of IGF and related proteins in IV-1 paste compared to Example 16-PPF1 Figure 62 shows heatmaps of IGF1, IGF2, IGFBP3, and IGFALS proteins in fraction IV-1 paste, normalized to the amount of protein in fraction PPF1. Protein abundances were quantified by a quantitative mass spectrometry-based method known as SureQuant. This figure compares the abundances of IGF1, IGF2, and the most important binding proteins in the IGF signaling pathway (IGFBP3 and IGFALS). Compared to PPF1, IV-1 paste is enriched with both IGF1 and IGF2, and enriched with binding proteins (IGFBP3 and IGFALS). This suggests that these proteins may contribute to the beneficial effects outlined in Figures 1–60.

[0224] R. Example 17 - Transcriptome Analysis: IV-1 paste-treated cells showed high differential gene expression across two muscle assays. The IV-1 paste showed the strongest effect compared to other plasma fractions containing PPF1 in differential gene expression across two muscle assays. The plasma fractions were administered to C2C12 cells as described in Figures 45 and 49. Transcriptome analysis showed that fraction IV-1 paste significantly altered gene expression in the following pathways: glucose metabolism, glycolysis, gluconeogenesis, glycogen metabolism, oxidative phosphorylation, extracellular matrix rearrangement, collagen degradation and formation, muscle contraction, cell cycle, proliferation, ion homeostasis, neutrophil degranulation, and inflammation. This suggests that fraction IV-1 paste enriches the protein, which modulates multiple pathways and explains the beneficial effects outlined in Figures 1–60. In conclusion, the transcriptome data support our theory that fraction IV-1 paste is directed toward multimodal activity and polypharmacology.

Claims

1. A method for treating a disease diagnosed as a disease, comprising administering an effective amount of fraction IV-1 subfraction to the subject.

2. The method according to claim 1, wherein the fraction IV-1 sub-fraction is one of the 13 sub-fractions obtained by the process described in Example 10.

3. The method according to claim 1, wherein the fraction IV-1 subfraction is at least one of fraction IV-1 subfractions 2, 3, 4, and 12.

4. The method according to claim 3, wherein the disease is one or more of type II diabetes, stroke, fatty liver disease, insulin resistance, metabolic myopathy, and cardiovascular disease.

5. The method according to claim 1, wherein the fraction IV-1 subfraction is at least one of fraction IV-1 subfractions 7 and 8.

6. The method according to claim 5, wherein the disease is a neurodegenerative disease.

7. The method according to claim 1, wherein the fraction IV-1 subfraction is at least one of fraction IV-1 subfractions 2, 3, 4, 5, 6 and 12.

8. The method according to claim 7, wherein the disease is one or more of cachexia, frailty, Charcot-Marie-Tooth disease, muscular dystrophy, and congenital fibrous disproportion.

9. A composition comprising a fraction IV-1 paste subfraction that can be obtained by the process described in Example 10.

10. The fraction IV-1 paste subfraction according to claim 9, wherein the subfraction is selected from subfraction 1, subfraction 2, subfraction 3, subfraction 4, subfraction 5, subfraction 6, subfraction 7, subfraction 8, subfraction 9, subfraction 10, subfraction 11, subfraction 12, and subfraction 13.

11. The method according to claim 5, wherein the neurodegenerative disease is Parkinson's disease.

12. The method according to claim 11, wherein the Parkinson's disease is associated with the degeneration of dopaminergic neurons having increased production of reactive oxygen species.

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