EA042179B1 - SCHEME OF THE APPLICATION OF BLOOD PLASMA AND BLOOD PLASMA PRODUCTS FOR THE TREATMENT OF COGNITIVE AND MOTOR DISORDERS - Google Patents

Description

This invention relates to the identification and discovery of methods and compositions for the treatment and/or prevention of cognitive and motor disorders, including age-related dementia or decreased motor function, and/or neurodegenerative disease. This document describes

- 7 042179 methods and composition for the treatment of subjects suffering from such disorders, which are aspects of this invention. In addition, this document describes application regimens that stimulate neurogenesis or reduce neuroinflammation, and/or improve cognitive or motor functions in subjects suffering from cognitive or motor impairments. The methods and compositions described herein are useful for: preventing cognitive or motor impairment, age-related dementia, neuroinflammation, and/or neurodegenerative disease; amelioration of symptoms of cognitive or motor impairment, age-related dementia, neuroinflammation and/or neurodegenerative disease; slowing the progression of age-related cognitive or movement disorders, age-related dementia, neuroinflammation and/or neurodegenerative disease; and or reversing the progression of age-related cognitive or motor impairment, age-related dementia, neuroinflammation, and/or neurodegenerative disease. The implementation of the invention includes the use of blood plasma fractions as a treatment, for example, one or more fractions or eluates obtained from blood fractionation processes, such as the Cohn fractionation process described below. An embodiment of the present invention involves the use of a plasma fraction (a solution consisting of normal human albumin, alpha and beta globulins, gamma globulin and other proteins, either alone or as complexes, hereinafter referred to as the plasma fraction). Another embodiment of the present invention involves the use of a plasma protein fraction (PPF) as a treatment. Another embodiment of the present invention involves the use of a human albumin solution (HAS) as a treatment. Another embodiment involves the use of eluates from blood fractionation processes, such as eluate I or eluate II/III, described below. A further embodiment includes a blood plasma fraction from which substantially all clotting factors have been removed so as to retain efficacy while reducing the risk of thrombosis (e.g., see U.S. Patent Applications Nos. 62/236,710 and 63/376,529, which are included as reference in this description in its entirety).

Before describing the present invention in detail, it is to be understood that this invention is not limited to the particular method or composition described, as these may, of course, vary. It should also be understood that the terminology used herein is used only to describe specific embodiments and is not limiting, since the scope of the present invention is limited only by the appended claims.

The publications discussed herein are presented solely in terms of their content prior to the filing date of this application. Nothing herein should be interpreted as an admission that the present invention is not entitled to predate such publication by virtue of the earlier invention. Moreover, the dates of the submitted publications may differ from the actual dates of the publications, which requires independent confirmation.

When a range of values is given, it should be understood that each intermediate value is also described, to the tenth decimal place of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range. Every smaller range between any specified value or intermediate value in the specified range and any other specified or intermediate value in that specified range is covered by this invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and any range in which either, neither, or both limits are included in the smaller ranges is also within the scope of the invention, except for any specifically excluded limit within the specified range. . If the specified range includes one or both of the limits, ranges excluding one or both of these included limits are also included in this invention.

It should be noted that the claims may be amended to remove any optional elements. Therefore, this statement should serve as a preliminary basis for the use of such exhaustive terminology as exclusively, only, etc. in connection with the indication of the elements of the claims, or the use of a negative feature.

As will be appreciated by those skilled in the art upon reading this disclosure, each of the individual embodiments of the invention described and illustrated herein has separate components and features that can be easily separated from or combined with features of any of the other several embodiments of the invention without departure from the scope or essence of the present invention. Any specified method can be carried out using the specified sequence of actions or using any other logically possible sequence.

2. Definitions.

Unless otherwise indicated, all technical and scientific terms used herein have the generally accepted meaning understood by any person skilled in the art to which this invention pertains. Although methods and materials similar or equivalent to opi

- 8 042179 herein may also be used in the practice or testing of the present invention, some possible and preferred methods and materials will now be described. All publications mentioned in this document are incorporated herein by reference for the purpose of disclosing and describing the methods and/or materials in connection with which these publications are cited. It is to be understood that this invention supersedes any disclosure of the publication incorporated herein to the extent that there is a conflict.

It should be noted that the use of the singular herein and in the appended claims includes reference to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to a cell includes a plurality of such cells, and a reference to a peptide includes a reference to one or more peptides and their equivalents, such as polypeptides known to those skilled in the art, and so on.

In describing the methods of the present invention, the terms host, subject, subject, and patient are used interchangeably and refer to any mammal in need of such treatment in accordance with the methods described. Such mammals include, for example, humans, sheep, cows, 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 a farm animal. 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 animals (eg, dogs and cats), livestock (eg, cows, pigs, goats, horses, etc.), rodents (eg, mice, guinea pigs, and rats, eg, as in animal models). diseases) and non-human primates (e.g. chimpanzees and monkeys). Thus, subjects of the invention include, inter alia, mammals such as humans and other primates such as chimpanzees and other apes and apes; and the like, wherein in some embodiments the subjects are humans. The term subject is also intended to include a person or organism of any age, weight, or other physical characteristics, and the subjects may be an adult, child, infant, or neonate.

A young or young individual is understood to mean an individual who is chronologically 40 years old or younger, eg 35 years old or younger, including 30 years old or younger, eg 25 years old or younger or 22 years old or younger. In some instances, the individual who serves as the source of the young plasma blood product is 10 years of age or younger, such as 5 years of age or younger, including 1 year of age or younger. In some cases, the subject is a neonate and the source of the plasma preparation is the umbilical cord, with the plasma preparation collected from the umbilical cord of the newborn. Thus, a young and young individual may refer to a subject whose age ranges from 0 to 40 years old, such as 0, 1, 5, 10, 15, 20, 25, 30, 35, or 40 years old. In other cases, a young and young individual may refer to biological (as opposed to chronological) age, for example, an individual who has not demonstrated plasma levels of inflammatory cytokines that comparatively older individuals exhibit. Conversely, a young and young individual may refer to biological (as opposed to chronological) age, for example, an individual who exhibits higher plasma levels of anti-inflammatory cytokines compared to levels in comparatively older individuals. By way of example, and not limitation, the inflammatory cytokine is eotaxin, and the fold difference between a young subject or a young individual and elderly individuals is at least 1.5-fold. Similarly, the fold difference between older and younger individuals in other inflammatory cytokines can be used to indicate biological age (see US Patent Application No. 13/575437, which is incorporated herein by reference). Typically, the individual is healthy, eg, the individual is free of hematologic malignancies or autoimmune disease at the time of sampling.

By an individual suffering from or at risk of developing age-related cognitive impairment is meant an individual who has lived more than 50% of his life expectancy, such as more than 60%, such as more than 70%, such as more than 75%, 80%, 85%, 90%, 95% or even 99% of his life expectancy. The age of the individual will depend on the species in question. Thus, this percentage is based on the predicted lifespan of the species in question. For example, in the case of a human, such an individual is 50 years of age or older, such as 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 not older than 100 years of age, such as 90 years of age, i.e. age between about 50 and 100, e.g. 50...55...60...65...70...75...80...85...90...95...100 years or older, or any age between 50-1000, and suffering from an age-related pathological condition, as further described below, for example, cognitive impairment associated with the natural aging process; an individual who is about 50 years old or older, such as 60 years old or older, 70 years old or older, 80 years old or older, 90 years old or older, and usually not older than 100 years old, i.e. age around 50-100, for example

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50...55...60...65...70...75...80...85...90...95...100 years old who still hasn't shown symptoms , age-related pathological conditions, such as cognitive impairment; an individual of any age who suffers from a cognitive impairment due to an age-related disease, as further described below, and an individual of any age who has been diagnosed with an age-related disease that is generally accompanied by a cognitive impairment, and the individual has not yet shown symptoms of a cognitive impairment. The appropriate age for non-human subjects is known and applicable herein.

As used herein, the term treatment refers to any of: (i) preventing a disease or disorder, or (ii) reducing or eliminating the symptoms of a disease or disorder. Treatment can be carried out prophylactically (before the onset of the disease) or therapeutically (after the onset of the disease). The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with the disease. Thus, the term treatment as used herein encompasses any treatment of an age-related disease or disorder in a mammal and includes: (a) preventing the onset of a disease in a subject who may be predisposed to the disease but who has not yet been diagnosed with the disease; (b) inhibition of the disease, ie. cessation of its development; or (c) alleviating the disease, i. e. disease regression. Treatment can lead to various physical manifestations, such as modulation of gene expression, rejuvenation of tissues or organs, and so on. The therapeutic agent may be administered before, during or after the onset of the disease. The treatment of the current disease, in which the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment can be performed until complete loss of function in the affected tissues. The therapy according to the invention may be administered during the symptomatic stage of the disease, and, in some cases, after the symptomatic stage of the disease.

In some embodiments, the age-related condition being treated is an age-related impairment in an individual's cognitive ability. Cognitive ability or cognition refers to mental processes that include attention and concentration, learning complex tasks and concepts, memory (acquisition, retention and retrieval of new information in the short and / or long term), information processing (handling information obtained using five senses), visuo-spatial function (visual perception, depth perception, use of mental imagery, pattern copying, creation of objects or shapes), language use and comprehension, verbal fluency (word selection), problem solving, decision making, and executive functions ( planning and prioritization). By a cognitive disorder is meant a gradual decline in one or more of these abilities, such as a disorder in memory, language, thinking, judgment, etc. Cognitive impairment and cognitive impairment refers to a decrease in cognitive ability compared to a healthy person, for example a healthy person in the appropriate age group, or compared to the ability of an individual at an earlier point in time, for example 2 weeks, 1, 2, 3, 6 months , 1, 2 years, 5 or 10 years or more. Age-related cognitive impairment refers to the deterioration in cognitive ability that is generally associated with aging, including, for example, cognitive impairment associated with the natural aging process, such as mild cognitive impairment (MCI); and cognitive impairment associated with age-related disorder, ie. a disorder whose incidence increases with age, such as a neurodegenerative condition such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, vascular dementia, and the like.

In some embodiments, the age-related condition being treated is an age-related movement disorder in an individual. Motor ability refers to motor processes that include the ability to perform complex neuromuscular actions that provide movement, such as fine motor skills for small or precise movements (e.g., writing, tying shoes) and gross motor skills for large movements (e.g., walking, running, kicks). A movement disorder is understood to mean a gradual decline in one or more of these abilities, such as a disorder of movement or gross motor skills, etc. By impaired motor ability and motor impairment is meant a reduction in motor ability/skill compared to a healthy person, e.g. a healthy person in the appropriate age group, or compared to the ability of an individual at an earlier point in time, e.g. 2 weeks, 1, 2, 3 , 6 months, 1, 2 years, 5 or 10 years or more. Age-related movement disorder is understood to mean a deterioration in motor ability that is generally associated with aging, including, for example, movement disorders associated with the natural aging process, and a movement disorder or disorder associated with an age-related disorder, that is, a disorder whose frequency increases with age, e.g. neurodegenerative condition such as Parkinson's disease, amyotrophic lateral

- 10 042179 sclerosis, etc.

In some embodiments, the age-related condition being treated is an age-related increase in neuroinflammation in an individual. Neuroinflammation refers to the biochemical and cellular responses of the nervous system to injury, infection, or neurodegenerative disease. Such responses aim to reduce activating factors by engaging central nervous system immunity to protect against potential harm. Neurodegeneration occurs in the central nervous system and there are signs of loss of structure and function of neurons. Neuroinflammatory diseases or conditions or diseases associated with neuroinflammatory include, by way of example, but not limitation, neurodegenerative diseases such as Alzheimer's disease; Parkinson's disease, multiple sclerosis, etc.

Blood products containing plasma components. In the practice of the methods of this invention, a blood product containing plasma components is administered to an individual in need thereof, for example, an individual suffering from or at risk of developing cognitive or motor impairments, neuroinflammation and/or age-related dementia. In this regard, the methods according to embodiments of the present invention include administering a blood product containing blood plasma components from an individual (donor individual or donor) to an individual who is at least at risk of developing or suffering from a cognitive or motor impairment, neuroinflammation, neurodegeneration and/or age-related dementia (hereinafter referred to as the recipient individual or recipient). By a blood product containing plasma components is meant any blood-derived product that contains plasma (eg, whole blood, blood plasma, or fractions thereof). The term plasma is used in its usual sense to refer to the straw/pale yellow liquid component of blood, consisting of approximately 92% water, 7% proteins such as albumin, gamma globulin, anti-hemophilic factor and other blood clotting factors, and 1% mineral salts, sugars, fats, hormones and vitamins. Non-limiting examples of blood products containing plasma suitable for use in the methods of this invention include whole blood treated with an anticoagulant (e.g., EDTA, citrate, oxalate, heparin, etc.), blood products obtained by filtering whole blood to to remove white blood cells (leukoreduction), blood products consisting of plasma obtained by plasmapheresis or apheresis, fresh frozen plasma, blood products consisting mainly of purified plasma, and blood products consisting mainly of plasma fractions. In some cases, the plasma preparation that is used is not a whole blood plasma preparation, which means that the preparation is not whole blood, therefore it lacks one or more components present in whole blood, such as erythrocytes, leukocytes, etc. ., at least to the extent that these components are present in whole blood. In some cases, the plasma preparation is substantially, if not completely, free of cells, in which case the cell content may be 5% by volume or less, for example, 1% or less, including 0.5% or less, while in some cases, cell-free plasma fractions are those compositions that are completely devoid of cells, i. they do not contain cells.

Collection of blood products containing plasma components. Embodiments of the methods described herein include the administration of blood products containing plasma components that can be obtained from donors, including human volunteers. The term human-derived may refer to such products. Methods for obtaining plasma-containing blood products from donors are well known in the art (see, for example, AABB TECHNICAL MANUAL, (Mark A. Fung, et al., eds., 18th ed. 2014), incorporated herein by reference ).

In one embodiment, blood is obtained by venipuncture. In another embodiment, the venipuncture is a single venipuncture. In another embodiment, blood volume replenishment with saline is not used. In a preferred embodiment, the plasmapheresis method is used to obtain blood products containing plasma. Plasmapheresis may involve the removal of mass-adjusted plasma volume with the return of cellular components to the donor. In a preferred embodiment, sodium citrate is used during plasmapheresis to prevent cell clumping. The volume of plasma collected from a donor is preferably between 690 and 880 ml after citrate administration and is preferably related to the weight of the donor.

3. Plasma fractions.

During World War II, there was a need for a stable plasma expander that could be used on the battlefield when soldiers were losing large amounts of blood. As a result, methods have been developed for obtaining lyophilized plasma. However, the use of lyophilized plasma has been difficult in combat situations because it requires sterile water as an alternative to reconstitute it. Dr. E. Cohn suggested that albumin could be used and prepared a ready-to-use stable solution that could be administered immediately to treat shock (see below). Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., 1st ed. 1991)). In Dr. Kohn's procedure for the purification of plasma fractions

- 11 042179 cold ethanol was used due to its denaturing action and pH and temperature changes were used to achieve separation.

An embodiment of the methods described herein comprises administering plasma fractions to a subject. Fractionation is the process by which certain subgroups of proteins are separated from plasma. Fractionation technology is known in the art and is based on steps developed by Cohn et al. in the 1940s (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), incorporated herein by reference). This process includes several steps, and each step involves the application of certain concentrations of ethanol, as well as shifts in pH, temperature, and osmotic pressure, which result in selective precipitation of the protein. Precipitates are also separated by centrifugation or precipitation. The original Cohn fractionation process involves the separation of proteins through precipitates into five fractions, designated Fraction I, Fraction II+III, Fraction IV-1, Fraction IV-4, and Fraction V. Albumin was originally identified as the end product (fraction V) of this process. . In accordance with embodiments of the invention, each fraction (or eluate from a previous separation step) contains or potentially contains therapeutically useful protein fractions (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 US Pat. In order to obtain specific protein fractions, the above experimental parameters can be adjusted.

More recently, fractionation has reached additional complexity and therefore constitutes further embodiments of the present invention. This recent increase in complexity has come about through the introduction of chromatography, which has led to the isolation of new proteins from existing fractions such as cryoprecipitate, cryo-poor plasma, and Kohn fractions; enhancing IgG recovery by integrating chromatography and ethanol fractionation process; and reduction/inactivation/removal of viruses (Id.) Anion exchange chromatography can be used to capture proteins at physiological pH and ionic strength. This preserves the functional activity of proteins and/or protein fractions. Heparin and monoclonal antibodies are also used in affinity chromatography. One of skill in the art will appreciate that the parameters described above can be modified to obtain fractions containing specific desired plasma proteins.

In one embodiment of the present invention, blood plasma is fractionated under industrial conditions. Frozen plasma is thawed at 1-4°C. Continuous refrigerated centrifugation is applied to the thawed plasma and the cryoprecipitate is isolated. The reconstituted cryoprecipitate is frozen at -30° C. or below and stored. Cryoprecipitate-poor (cryopoor) plasma is immediately processed to capture (by, for example, primary chromatography) labile coagulation factors such as factor IX complex and its components, as well as protease inhibitors such as antithrombin and C1 esterase inhibitor. Sequential centrifugation and separation of the precipitate can be used in subsequent steps. Such methods are known to those skilled in the art and are described, for example, in US Patent Nos. 4,624,780, 5,219,995, 5,288,853 and US Patent Applications Nos. 20140343255 and 20150343025, the contents of which are incorporated herein by reference in their entirety.

In one embodiment of the invention, the plasma fraction may contain a plasma fraction containing a significant concentration of albumin. In another embodiment, the plasma fraction may contain a plasma fraction containing a significant concentration of IgG or intravenous immunoglobulin (IGIV) (eg, Gamunex-C®). In another embodiment, the plasma fraction may comprise an IGIV plasma fraction, such as Gamunex-C®, that has been substantially immunoglobulin (IgG) depleted by methods well known to one of skill in the art, such as, for example, protein-mediated A wasting (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 a further embodiment of the invention, the blood plasma fraction may be one in which substantially all of the clotting factors have been removed in order to retain the effectiveness of the fraction with a reduced risk of thrombosis. For example, the plasma fraction may be the plasma fraction described in US Pat. No. 62/376,529, which was filed Aug. 18, 2016; the contents of which are incorporated herein by reference in their entirety.

4. Albumin preparations.

Those skilled in the art will recognize that there are two main categories of plasma albumin (APP) preparations: plasma protein fraction (PPF) and human albumin solution (HAS). PPF is derived from a higher yield process than HAS but has a lower minimum albumin purity than HAS (>83% for PPF and >95% for HAS) (Production of human albumin solution: a continuously developing colloid, P. Matejtschuk et al., British J. of Anaesthesia 85(6): 887-95, at 888 (2000)). In some cases, PPF has an albumin purity of 83-95% or alternatively 83-96%. The purity of albumin can be determined by electrophoresis or other assays for quantitative determination, such as, for example, mass spectrometry. In addition, some have pointed out that PPF is deficient due to the presence of protein contaminants such as PKA. Id. As a consequence, PPF preparations fell out of favor as plasma albumin preparations, and were even excluded from the Pharmacopeia of some countries. Id. In contrast to these problems, the present invention makes useful use of these impurities. In addition to α-, β- and γ-globulins, as well as the aforementioned PKA, the methods of the invention use additional proteins or other contaminant factors that promote processes such as neurogenesis, nerve cell survival, improved cognitive or motor function, and reduced neuroinflammation.

Those skilled in the art will appreciate that there are, or have been, several commercial sources of PPF (commercial formulations of PPF) 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).

Those skilled in the art will also appreciate that there are, or have been, several commercial sources of HAS (commercial preparations of HAS) 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).

a. Plasma Protein Fraction (Human) (PPF).

According to the U.S. Food and Drug Administration (FDA), Plasma Protein Fraction (Human), or PPF, is the proper name for a drug called a sterile protein solution composed of albumin and globulin derived from human plasma (CFR). CFR 21 CFR 640.90, which is hereby incorporated by reference in its entirety). PPF source material is plasma isolated from whole blood obtained as prescribed in 21 CFR 640.1 - 640.5 (which is incorporated herein by reference) or a plasma source obtained as prescribed in 21 CFR 640.60 - 640.76 (which is included in this document by reference).

PPF is tested to determine compliance with the following standards under 21 CFR 640.92 (which is incorporated herein by reference):

(a) the final product must be a 5.0 +/- 0.30% protein solution and (b) the total protein in the final product must be at least 83% albumin and not more than 17% globulins. No more than 1% of the total amount of protein should be gamma globulin. The composition of the protein is determined according to a method that has been approved for each manufacturer by the Director of the Center for the Evaluation and Study of Biologicals of the Food and Drug Control Commission.

As used herein, a plasma protein fraction or PPF refers to a sterile protein solution composed of albumin and globulin derived from human plasma, with an albumin content of at least 83% with no more than 17% globulins (including globulins α1, α2, β and γ) and other plasma proteins, and not more than 1% gamma globulin as determined by electrophoresis (Hink, J.H., 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 which, when suspended in a solvent, has a similar composition. The fraction of total globulin can be determined by subtracting albumin from total protein (Busher, I, Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY, PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, Walker H.K., Hall W.D., Hurst J.D., eds (1990) ).

b. Albumin (human) (HAS).

According to the FDA, Albumin (Human) (also referred to herein as HAS) is the proper name for a drug called Sterile Human Plasma Albumin Solution (CFR 21 CFR 640.80, which is hereby incorporated by reference in its entirety.) The source material for Albumin (Human) is plasma reconstituted from whole blood as prescribed in 21 CFR 640.1-640.5 (which is incorporated herein by reference) or a plasma source obtained as prescribed in 21 CFR 640.60-640.76 (which incorporated herein by reference). Other requirements for albumin (human) are listed in 21 CFR 640.80 - 640.84 (which is incorporated herein by reference).

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Albumin (Human) is tested to determine compliance with the following 21 CFR standards

640.82:

(a) protein concentration. The final product must correspond to one of the following concentrations: 4.0 +/- 0.25 percent; 5.0 +/- 0.30 percent; 20.0 +/- 1.2% and 25.0 +/- 1.5% protein solution;

(b) protein composition. At least 96% of the total protein in the final product must be albumin as determined by a method that has been approved for each manufacturer by the Director of the FDA's Center for Biological Evaluation and Research.

As used herein, albumin (human) or HAS refers to a sterile protein solution composed of albumin and globulin derived from human plasma, with an albumin content of at least 95%, not more than 5% globulins (including globulins α1, α2, β and γ) and other plasma proteins. HAS may also refer to a solid form which, when suspended in a solvent, has a similar composition. The fraction of total globulin can be determined by subtracting albumin from total protein.

As may be recognized by those of ordinary skill in the art, the PPF and HAS fractions may also be lyophilized or in other solid form. Such preparations, with suitable additives, can be used to prepare, for example, tablets, powders, granules or capsules. The solid form may be formulated into injectables by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent such as vegetable or the like oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

5. Fractions with a reduced number of clotting factors.

In another embodiment of the invention, a blood plasma fraction is used from which substantially all clotting factors have been removed in order to retain the effectiveness of the fraction with a reduced risk of thrombosis. Conveniently, the blood product can be obtained from a young donor or group of young donors and can be freed from IgM in order to provide a young blood product that is AB0 compatible. Currently transfused plasma is AB0 blood type compatible, as the presence of natural antibodies to A and B antigens can lead to transfusion reactions. It appears that IgM is responsible for transfusion reactions when patients receive non-AB0 compatible plasma. Removal of IgM from blood products or fractions helps eliminate transfusion reactions in subjects who are administered the blood products and plasma fractions of the invention.

Accordingly, in one embodiment, the present invention relates to a method for treating or preventing a subject with an age-related pathological condition, such as a cognitive or motor impairment, neuroinflammation, or neurodegeneration. The method includes administering to a subject a blood product or a blood fraction derived from whole blood from an individual or group of individuals, wherein the blood product or blood fraction is substantially devoid of (a) at least one coagulation factor and/or (b) IgM. In some embodiments, the individual(s) from whom the blood product or blood fraction is obtained are young individuals. In some embodiments, the blood product is substantially devoid of at least one coagulation factor and IgM. In certain embodiments, the blood product is substantially devoid of fibrinogen (factor I). In further embodiments, the blood product is substantially devoid of red blood cells and/or white blood cells. In other embodiments, the blood product is substantially devoid of cells. In other embodiments, the blood product is derived from plasma. Such embodiments of the present invention are further supported by US Patent Application No. 62/376,529, filed Aug. 18, 2016, which is incorporated herein by reference in its entirety.

6. Treatment with protein-enriched plasma protein preparations.

In additional embodiments of the invention, a plasma fraction is used with a reduced concentration of albumin compared to PPF, but with an increased amount of globulins and other plasma proteins (those that have been called impurities). Embodiments such as with PPF, HAS, eluate I and eluate II/III are all effectively devoid of clotting factors. Such plasma fractions are hereinafter referred to as protein-enriched plasma protein preparations. For example, in an embodiment of the invention, a protein-enriched plasma protein preparation consisting of 82% albumin and 18% α, β and γ globulins and other plasma proteins can be used. In another embodiment, a protein-enriched plasma protein preparation consisting of 81% albumin and 19% α, β and γ globulins and/or other plasma proteins can be used. In another embodiment of the invention, a protein-enriched plasma protein preparation consisting of 80% albumin and 20% α, β and γ globulins and/or other plasma proteins can be used. In additional options

- 14 042179 implementation of the invention can be used protein-enriched plasma protein preparations, consisting of 70-79% albumin and, respectively, 21-30% globulins α, β and γ and/or other plasma proteins. In additional embodiments of the invention, protein-enriched plasma protein preparations consisting of 60-69% albumin and, respectively, 31-40% α, β and γ globulins and/or other plasma proteins can be used. In further embodiments of the invention, protein-enriched plasma protein preparations consisting of 50-59% albumin and, respectively, 41-50% α, β and γ globulins and/or other plasma proteins can be used. In further embodiments of the invention, protein-enriched plasma protein preparations consisting of 40-49% albumin and, respectively, 51-60% α, β and γ globulins and/or other plasma proteins can be used. In additional embodiments of the invention, protein-enriched plasma protein preparations consisting of 30-39% albumin and, respectively, 61-70% α, β and γ globulins and/or other plasma proteins can be used. In additional embodiments of the invention, protein-enriched plasma protein preparations consisting of 20-29% albumin and, respectively, 71-80% α, β and γ globulins and/or other plasma proteins can be used. In additional embodiments of the invention, protein-enriched plasma protein preparations consisting of 10-19% albumin and, respectively, 81-90% α, β and γ globulins and/or other plasma proteins can be used. In additional embodiments of the invention, protein-enriched plasma protein preparations consisting of 1-9% albumin and, respectively, 91-99% α, β and γ globulins and/or other plasma proteins can be used. In another embodiment of the invention, protein-enriched plasma protein preparations consisting of 0% albumin and 100% α, β and γ globulins and other plasma proteins can be used.

The embodiments of the present invention described above may also have total gamma globulin concentrations of 1-5%.

Specific concentrations of proteins in a plasma fraction can be determined using methods well known to a person of ordinary skill in the art. By way of example, and not limitation, such methods include electrophoresis, mass spectrometry, ELISA, and Western blot analysis.

7. Obtaining plasma fractions.

Methods for obtaining PPF and other plasma fractions are well known to those of ordinary skill in the art. An embodiment of this invention provides for the collection of blood used in the preparation of a human plasma protein fraction in flasks with citrate or anticoagulant nitrate dextrose to inhibit blood coagulation, followed by separation of fractions I, II+III, IV and PPF, according to the method described in Hink et al. (See Hink, J.H., Jr., et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957), which is incorporated herein by reference). According to this method, the mixture can be collected at 2-8°C. Plasma can subsequently be separated by centrifugation at 7°C, removed and stored at -20°C. The plasma can then be thawed at 37°C and fractionated, preferably within 8 hours of removal from storage at -20°C.

Plasma can be separated from Fraction I using 8% ethanol at pH 7.2 and -2-2.5°C with a protein concentration of 5.1-5.6%. Cold 53.3% ethanol (176 ml/L plasma) with acetate buffer (200 ml 4 M sodium acetate solution, 230 ml glacial acetic acid and H ₂ O to 1 L) can be added by pipette at a rate of, for example, 450 ml/min when the plasma temperature drops to -2°C. Fraction I can be separated and removed from the eluate (eluate I) by ultracentrifugation. Fibrinogen can be obtained from fraction I in accordance with methods well known to one of ordinary skill in the art.

Fraction II+III can be separated from eluate I by diluting the eluate to 21% ethanol at pHe 6.8, at -6°C, with a protein concentration of 4.3%. Cold 95% ethanol (176 ml/l eluate I) with 10 M acetic acid used to adjust the pH can be added with a pipette at a rate of, for example, 500 ml/min while lowering the temperature of eluate I to -6°C. The resulting precipitate (fraction II+III) can be removed by centrifugation at -6°C. Gammaglobulin can be obtained from fraction II+III using methods well known to those of ordinary skill in the art.

Fraction IV-1 can be separated from eluate II+III (eluate II/III) by diluting the eluate to 19% ethanol at pH 5.2, at -6°C, with a protein concentration of 3%. H ₂ O and 10 M acetic acid used for pH adjustment can be added continuously with a pipette while maintaining eluate II/III at -6°C for 6 hours. The precipitated fraction VI-1 can be precipitated at -6°C in for 6 h, and then separated from the eluate by centrifugation at the same temperature. A stable plasma protein fraction can be isolated from the IV-1 eluate by adjusting the ethanol concentration to 30% at pH 4.65, -7° C. and a protein concentration of 2.5%. This can be achieved by adjusting the pH of eluate IV-1 with cold alcohol and acid (two parts 2M acetic acid and one part 95% ethanol). While maintaining the temperature -7°C, for each liter of the corrected eluate 1-IV add 170 ml of cold ethanol (95%). Proteins that can be precipitated

- 15 042179 ns, defend for 36 h, and then removed by centrifugation at -7°C.

The isolated proteins (stable fraction of plasma proteins) can be dried (eg by freeze drying) to remove alcohol and H2O. The resulting dried powder can be dissolved in sterile distilled water, for example using 15 l of water/kg of powder, with the solution adjusted to pH 7.0 with 1M NaOH. A final 5% protein concentration can be achieved by adding sterile distilled water containing sodium acetyl tryptophanate, sodium caprylate and NaCl to a final concentration of 0.004 M acetyl tryptophanate, 0.004 M caprylate and 0.112 M sodium. Finally, the solution can be filtered at 10°C to obtain a clear solution and then heat treated to inactivate pathogens at 60°C for at least 10 hours.

One of skill in the art will appreciate that each of the various fractions and eluates described above can be used in the methods of this invention to treat a disease. For example, but not by way of limitation, eluate I or eluates II/III can be used to treat diseases such as cognitive, motor and neurodegenerative disorders and are embodiments of the present invention.

The previous methods for obtaining plasma fractions and plasma protein fractions (PPF) are illustrative only and include only embodiments of the present invention. One skilled in the art will appreciate that these methods may differ. For example, pH, temperature, and ethanol concentration, among other things, can be adjusted to produce different variations in plasma fractions and plasma protein fractions in various embodiments and methods of this invention. In another example, further embodiments of this invention involve the use of nanofiltration to remove/inactivate pathogens from plasma fractions and plasma protein fractions.

An additional embodiment of the present invention provides methods and compositions using and/or containing additional plasma fractions. For example, the invention, among other things, demonstrates that specific concentrations of albumin are not critical for improving cognitive or motor performance. Thus, fractions with a reduced concentration of albumin, such as those fractions having less than 83% albumin, are contemplated in this invention.

8. Treatment.

Aspects of the methods of the invention described herein include treating a subject with a blood product containing plasma, such as a fraction of blood plasma, for example, as described above. An embodiment includes treating a human subject with a blood product containing plasma. One skilled in the art will appreciate that methods for treating patients with a blood product containing plasma are known in the art. By way of example, and not limitation, one embodiment of the methods of the invention described herein includes administering fresh frozen plasma to a subject for the treatment and/or prevention of cognitive or motor impairment, neuroinflammation, neurodegeneration, and/or age-related dementia. In one embodiment, a blood product containing plasma is administered immediately, for example within about 12-48 hours after receipt from a donor, to an individual suffering from or at risk of cognitive or motor impairment, neuroinflammation, neurodegeneration and/or age-related dementia. In such cases, the drug can be stored in a refrigerator, for example, at 0-10°C. In another embodiment, fresh frozen plasma is one that has been stored frozen (cryopreservation) at -18°C or lower. Prior to administration, fresh frozen plasma is thawed, and once thawed, administered to the subject 60-75 minutes after the thawing process has begun. Each subject preferably receives a single portion of fresh frozen plasma (200-250 ml), with fresh frozen plasma preferably obtained from donors of a predetermined age range. In one embodiment of the invention, fresh frozen plasma is provided from (obtained from) young individuals. In another embodiment of the invention, fresh frozen plasma is provided from (obtained from) donors of the same gender. In another embodiment of the invention, fresh frozen plasma is provided from (received from) donors in the age range of 18-22 years.

In one embodiment of the invention, blood products containing plasma are screened for blood type after they have been provided. In another embodiment of the present invention, blood products containing plasma are screened for the presence of infectious disease agents such as HIV-I and II, HBV (hepatitis B virus), HCV (hepatitis C virus), HTLV (human T lymphotropic virus) I and II, antibodies to hepatitis B core antigen (HBc) in accordance with the requirements of 21 CFR 640.33 and recommendations contained in FDA guidelines.

In yet another embodiment of the present invention, a plasma fraction is administered to the subject. In one embodiment of the invention, the plasma fraction is PPF or HAS. In yet another embodiment of the invention, the plasma fraction is one of the commercial PPF preparations or commercial HAS preparations. In another embodiment of this invention

- 16 042179 plasma fraction is PPF or HAS obtained from a group of individuals of a certain age range, such as young people, or is a modified PPF or HAS fraction that has been subjected to additional fractionation or processing (for example, PPF or HAS, in which one or more specific proteins are partially or substantially removed). In another embodiment, the plasma fraction is an IGIV plasma fraction that has been substantially depleted in immunoglobulin (IgG). A blood fraction that is substantially depleted, or in which specific proteins such as IgG are substantially removed, refers to a blood fraction containing less than about 50% of the amount seen in a reference preparation or whole blood plasma, such as less than 45 %, 40%, 35%, 30%, 25%, 20%, 15%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, the undetectable level, or any integer between these values, measured using standard assays well known in the art.

9. Introduction.

Aspects of the methods of the invention described herein include treating a subject with a blood product containing plasma, such as a blood plasma fraction or plasma fraction, for example, as described above. An embodiment includes treating a human subject with a blood product containing plasma. One skilled in the art will appreciate that methods for treating patients with a blood product containing plasma are known in the art. By way of example, and not limitation, one embodiment of the methods of the invention described herein includes administering fresh frozen plasma to a subject for the treatment and/or prevention of cognitive or motor impairment, neuroinflammation, neurodegeneration, and/or age-related dementia. In one embodiment, a blood product containing plasma is administered immediately, for example within about 12-48 hours after receipt from a donor, to an individual suffering from or at risk of cognitive or motor impairment, neuroinflammation, neurodegeneration and/or age-related dementia. In such cases, the drug can be stored in a refrigerator, for example at 010°C. In another embodiment, fresh frozen plasma is one that has been stored frozen (cryopreservation) at -18°C or lower. Prior to administration, fresh frozen plasma is thawed, and once thawed, administered to the subject 60-75 minutes after the thawing process has begun. Each subject preferably receives a single portion of fresh frozen plasma (200-250 ml), with fresh frozen plasma preferably obtained from donors of a predetermined age range. In one embodiment of the invention, fresh frozen plasma is provided from (obtained from) young individuals. In another embodiment of the invention, fresh frozen plasma is provided from (obtained from) donors of the same gender. In another embodiment of the invention, fresh frozen plasma is provided from (received from) donors in the age range of 18-22 years.

In one embodiment of the invention, blood products containing plasma are screened for blood type after they have been provided. In another embodiment of the present invention, blood products containing plasma are screened for the presence of infectious disease agents such as HIV-I and II, HBV (hepatitis B virus), HCV (hepatitis C virus), HTLV (human T lymphotropic virus) I and II, antibodies to hepatitis B core antigen (HBc) in accordance with the requirements of 21 CFR 640.33 and recommendations contained in FDA guidelines.

In yet another embodiment of the present invention, a plasma fraction is administered to the subject. In one embodiment of the invention, the plasma fraction is PPF or HAS. In yet another embodiment of the invention, the plasma fraction is one of the commercial PPF preparations or commercial HAS preparations. In another embodiment of this invention, the plasma fraction is PPF or HAS obtained from a group of individuals of a certain age range, such as young people, or is a modified PPF or HAS fraction that has been subjected to additional fractionation or processing (for example, PPF or HAS, in which one or more specific proteins are partially or substantially removed). In another embodiment, the plasma fraction is an IGIV plasma fraction that has been substantially depleted in immunoglobulin (IgG). A blood fraction that is substantially depleted or in which specific proteins such as IgG have been substantially removed refers to a blood fraction containing less than about 50% of the amount seen in a reference preparation or whole blood plasma, such as less than 45 %, 40%, 35%, 30%, 25%, 20%, 15%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, the undetectable level, or any integer between these values, measured using standard assays well known in the art.

An embodiment of the invention includes treating a subject diagnosed with a cognitive or motor impairment, neurodegeneration, or neuroinflammation by administering to the subject an effective amount of blood plasma or plasma fraction. Another embodiment of the invention includes administering an effective amount of blood plasma or plasma fraction, and then monitoring the subject for improvement in cognitive or motor function, or a decrease in neuro

- 17 042179 inflammation or increased neurogenesis. Another embodiment of the present invention includes treating a subject diagnosed with a cognitive or motor impairment, neurodegeneration, or neuroinflammation by administering to the subject an effective amount of blood plasma or a plasma fraction, wherein the blood plasma or plasma fraction is administered in such a manner that it results in an improvement in cognitive or motor function, a decrease in neuroinflammation or increased neurogenesis after reaching the mean or median half-life of plasma proteins or plasma protein fraction, relative to the most recently administered dose (referred to herein as intermittent use or intermittent dose). Another embodiment of the invention includes administering blood plasma or a plasma fraction according to the schedule for at least two consecutive days and monitoring the subject for improvement in cognitive or motor function, reduction in neuroinflammation, or improvement in neurogenesis for at least 3 days after the date of the last administration. Another embodiment of the invention includes administering blood plasma or a plasma fraction on a schedule for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days and monitoring the subject for improvement cognitive or motor function, decrease in neuroinflammation or increase in neurogenesis for at least 3 days after the date of the last administration. Another embodiment of the invention includes administering blood plasma or plasma fraction according to the regimen for at least 2 consecutive days and after the date of the last administration, monitoring for improvement in cognitive or motor function, reduction in neuroinflammation or increase in neurogenesis, after it has been achieved mean half-life of proteins in plasma or plasma fraction. Another embodiment of the invention involves the administration of blood plasma or plasma fraction according to the scheme of application for 2-14 non-consecutive days, and each gap between doses can be 0-3 days.

In some cases, intermittent use in accordance with the present invention includes the administration of a first set of doses, eg as described above, followed by a period of no use, eg a period of no use, which in turn is followed by the administration of another dose or set of doses. The duration of this period without application may vary, but in some embodiments it is 7 days or longer, for example 10 days or longer, including 14 days or longer, and in some cases, the duration of the period without application ranges from 15 to 365 days, for example from 30 up to 90 days, including from 30 to 60 days. Thus, embodiments of the methods include non-systematic (ie, non-continuous) administration, such as non-systematic administration of a blood plasma preparation. In some embodiments, the intermittent application followed by a period of no application is repeated a number of times, as necessary, and in some cases this regimen is continued for 1 year or more, such as 2 years or more, including the entire life of the subject. Another embodiment of the invention includes the administration of blood plasma or plasma fraction according to the scheme of application for 5 consecutive days, with a period without application equal to 2-3 days, followed by administration for 2-14 consecutive days.

Biochemically, an effective amount or effective dose of an active agent is understood to mean an amount of an active agent that will inhibit, antagonize, decrease, decrease, or suppress by about 20% or more, such as 30% or more, 40% or more, or 50% % or more, in some cases, 60% or more, 70% or more, 80% or more, or 90% or more, in some cases about 100%, i.e. to insignificant amounts, and in some cases, cause reverse progression of cognitive impairment, neuroinflammation, neurodegeneration or age-related dementia.

10. Plasma protein fraction.

In carrying out the methods of this invention, a plasma fraction is administered to the subject. In one embodiment, the plasma fraction is a plasma protein fraction (PPF). In further embodiments, the PPF is selected from commercial PPF preparations.

In another embodiment, the PPF is composed of 88% normal human albumin, 12% alpha and beta globulins, and not more than 1% gamma globulin, as determined by electrophoresis. Other embodiments of this embodiment used in the implementation of the methods of this invention include, for example, an embodiment in the form of 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 with modified percentages of PPF (eg, about 1 to about 10%, about 10 to about 20%, about 20 to 25%, about 25 to 30%) in solution, as well as solvent concentrations and stabilizers can be used in the implementation of the methods according to this invention.

11. Plasma fractions of donors of a specific age.

Additional embodiments of this invention include the administration of a plasma protein fraction derived from the plasma of individuals of certain age ranges. An embodiment involves administering PPF or HAS that have been derived from the plasma of young individuals. In another embodiment of the present invention, the young individuals belong to one particular age or a particular age range. In yet another embodiment of the invention, the average age of the donors is less than that of the subject or less than the average age of the subjects to be treated.

Certain embodiments of the present invention include pooling blood or plasma from individuals of a certain age and fractionating the blood plasma as described above to prepare a plasma protein fraction such as PPF or HAS. In an alternative embodiment of the present invention, the plasma protein fraction, or a specific plasma protein fraction, is obtained from specific individuals within a specified age range.

12. Indications for use.

The methods of the invention and blood products containing plasma and fractions find use in the treatment, including prevention, of age-related pathological conditions, such as cognitive or motor impairments in individuals, for example, cognitive disorders, including (but not limited to) age-related dementia, immunological pathological conditions, cancer, and physical and functional decline; and movement disorders such as (but not limited to) Parkinson's disease. Individuals suffering from or at risk of developing age-related cognitive or motor impairment, neuroinflammation and/or neurodegeneration who benefit from treatment with a blood product containing plasma according to this invention, for example, using the methods described herein, include individuals who are 50 years of age or older, such as 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, that is, between the ages of about 50 and 100, such as 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, or about 100 years old, and suffer from cognitive or motor impairment, neuroinflammation, and/or neurodegeneration associated with the natural aging process, such as mild cognitive impairment (MCI); and individuals who are 50 years of age or older, such as 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 generally not older than 100 years of age, that is, between the ages of about 50 up to 90, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 years old, who have not yet begun to show signs of cognitive or motor impairment, neuroinflammation and/or neurodegeneration. Examples of cognitive and motor, neuroinflammatory and/or neurodegenerative disorders that occur due to natural aging include the following:

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 decision making, that have worsened over time, while general mental function and daily activities have not worsened. Thus, while significant neuronal death does not normally occur, neurons in the aging brain are vulnerable to age-related sublethal changes in structure, synaptic integrity, and molecular processing at the synapse, all of which impair cognitive function.

Individuals suffering from or at risk of developing age-related cognitive impairment who would benefit from treatment with a blood product or blood fraction containing plasma according to this invention, for example, using the methods described herein, also include individuals of any age who suffer from cognitive impairment. function due to age-related disorder, and individuals of any age who have been diagnosed with age-related disorder, which is usually accompanied by cognitive impairment, and the individual has not yet begun to show symptoms of cognitive impairment. Examples of such age-related disorders include the following.

b. Alzheimer's disease.

Alzheimer's disease is a progressive, irreversible loss of cognitive function associated with an excess of senile plaques in the cerebral cortex and subcortical gray matter, which also contains β-amyloid and neurofibrillary tangles composed of tau protein. The common form affects people > 60 years of age and its frequency increases with age. It accounts for more than 65% of dementias in the elderly.

The cause of Alzheimer's disease is not known. The disease occurs in families with a frequency of about 15-20% of cases. The rest, the so-called sporadic cases, have some genetic determinants. The disease has an autosomal dominant genetic profile in cases with the earliest onset and some cases with late onset but with varying penetrance in later life. Environmental factors are the focus of active research.

During the course of the disease, synapses and, ultimately, neurons disappear in the cerebral cortex, hippocampus, and subcortical structures (including selective cell loss in the basal nucleus of Meinert), locus coeruleus, and dorsal raphe nucleus. Cerebral glucose utilization and perfusion are reduced in some areas of the brain (parietal and temporal cortex in early disease, prefrontal cortex in advanced disease). Neuritic or senile plaques (consisting of neurites, astrocytes, and glial cells around the amyloid core) and neurofibrillary tangles (consisting of paired helical filaments) play a role in the pathogenesis of Alzheimer's disease. Senile plaques and neurofibrillary tangles occur with normal aging, but they are much more common in people with Alzheimer's disease.

c. Parkinson's disease.

Parkinson's disease (PD) is an idiopathic, slowly progressive degenerative disease of the CNS characterized by slowing and decreased movement (bradykinesia), muscle rigidity, resting tremor (dystonia), muscle retardation, and postural instability. Originally considered primarily as a movement disorder, PD is now also recognized as a cause of depression and emotional changes. PD can also affect cognition, behavior, sleep, autonomic functions, and sensory functions. The most common cognitive impairments include impaired concentration and attention, working memory, executive function, language use, and visuospatial orientation. A characteristic feature of PD is symptoms associated with decreased motor function, usually preceded by those associated with cognitive impairment, which aids in the diagnosis of the disease.

In primary Parkinson's disease, pigmented neurons in the substantia nigra, locus coeruleus, and other groups of dopaminergic cells in the brainstem degenerate. The reason is not known. The loss of substantia nigra neurons that project to the caudate nucleus and putamen leads to depletion of the neurotransmitter dopamine in these areas. Occurs, as a rule, after 40 years, with an increase in the incidence in older age groups.

Parkinson's disease is first diagnosed in approximately 60,000 Americans each year and currently affects approximately one million Americans. Even if PD is not fatal in itself, its complications are the fourteenth leading cause of death in the United States. Currently, PD cannot be cured and treatment is usually symptomatic, with surgery prescribed for later, severe cases.

Treatment options for PD include the administration of drugs to compensate for motor function deficits. These variants increase or replace the neurotransmitter, dopamine, whose brain concentrations are low in PD patients. Such drugs include carbidopa/levodopa (which create more dopamine in the brain); apomorphine, pramipexolol, ropinirole and rotingotine (dopamine agonists); selegiline and rasagiline (MAO-B inhibitors that prevent the breakdown of dopamine); entacapone and tolcapone (pyrocatechin-O-methyltransferase [COMT] inhibitors that make levodopa more available in the brain); benztropine and trihexyphenidyl (anticholinergics) and amantadine (control of tremor and rigidity). Exercise/physical therapy is also often prescribed to help maintain physical and mental function.

Current therapies that treat the symptoms of PD do not provide a cure, and fail to prevent progression of the disease. In addition, at present, drugs tend to lose their effectiveness in the late stages of PD. The most commonly prescribed drug, levodopa, generally results in adverse effects ranging from 5 to 10 years after the start of the drug. These side effects can be serious and can lead to motor fluctuations and unpredictable fluctuations in motor control between doses, as well as twitching/trembling (dyskinesias), which are difficult to control and are as disabling as the intrinsic symptoms of PD. Thus, there remains a need for new therapies with novel mechanisms of action that can be used together or in combination with existing PD drugs.

d. Parkinsonism.

Secondary parkinsonism (also referred to as atypical Parkinson's disease or Parkinson's plus) is the result of loss or obstruction 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 ingestion of antipsychotic drugs or reserpine, which cause parkinsonism by blocking dopamine receptors. Less common causes include carbon monoxide or manganese poisoning, hydrocephalus, structural damage (tumors, infarcts affecting the midbrain or basal ganglia), subdural hematomas, and degenerative diseases including nigrostriatal degeneration. Some disorders such as progressive supranuclear palsy (PNP), multiple system atrophy (MSA), corticobasal degeneration (CBD), and Lewy body dementia (LBD) may present with symptoms of parkinsonism before the cardinal symptoms needed for a specific diagnosis can occur, and thus may be labeled as parkinsonism.

e. Frontotemporal dementia.

Frontotemporal dementia (FTD) is a condition resulting from progressive degradation of the frontal lobe of the brain. Over time, degeneration can move to the temporal lobe. Second after Alzheimer's disease (AD) in prevalence, FTD accounts for 20% of presenile cases of dementia. Symptoms are divided into three groups depending on the affected

- 20 042179 frontal and temporal lobe functions:

Behavioral variant of FTD (pvFTD), with symptoms, includes lethargy and inspontaneity on the one hand, and disinhibition on the other hand; progressive non-fluent aphasia (PNPA), in which there are interruptions in fluency of speech due to difficulty in articulation, phonological and / or syntactic errors, but understanding of words is preserved; and semantic dementia (SD), in which patients retain fluent and normal phonetics and syntax but have increasing difficulty in naming and understanding words. Other cognitive symptoms common to all patients with FTD include impairments in executive function and the ability to concentrate. Other cognitive abilities, including perception, spatial skills, memory, and practice, tend to remain unaffected. FTD can be diagnosed by observing frontal and/or anterior temporal lobe atrophy on structural MRI scans.

There are a number of forms of FTD, any of which can be treated or prevented using the methods and compositions of this invention. For example, one form of frontotemporal dementia is semantic dementia (SD). SD is characterized by loss of semantic memory in both verbal and non-verbal domains. Patients with DM often complain of difficulty finding words. Clinical features include free aphasia, anomie, impaired understanding of the meaning of words, and associative visual agnosia (inability to match semantically related images or objects). As the disease progresses, there are often behavioral and personality changes similar to those seen in frontotemporal dementia, although cases of pure semantic dementia with few late behavioral symptoms have been described. Structural MRI imaging shows a characteristic atrophy profile in the temporal lobe (predominantly the left), with more inferior than superior involvement and more anterior versus posterior temporal lobe atrophy.

As another example, another form of frontotemporal dementia is Pick's disease (PiD, also PcD). The hallmark of the disease is the concentration of tau proteins in neurons, clustering 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 unacceptable. They may steal or exhibit compulsive or repetitive stereotypical behaviors. Patients with dorsomedial or dorsolateral frontal dysfunction may show anxietylessness, apathy, or reduced spontaneity. Patients may show a lack of self-control, abnormal self-awareness, and an inability to appreciate meaning. Patients with bilateral gray matter loss in the posterolateral orbitofrontal cortex and right anterior insular cortex may show eating behavioral changes such as pathological sugar cravings. Patients with more focal gray matter loss in the anterior orbitofrontal cortex may develop hyperphagia. While some of the symptoms may initially be relieved, the disease progresses and patients often die within two to ten years.

f. Huntington's disease.

Huntington's disease (HD) is a hereditary progressive neurodegenerative disease characterized by the development of emotional, behavioral and mental disorders; loss of intellectual or cognitive functions; and movement disorders (motor disorders). The classic signs of HD include the development of chorea—involuntary, rapid, irregular, jerky movements that can affect the face, arms, legs, or trunk—as well as cognitive decline, including the gradual loss of thought processing and acquired intellectual abilities. There may be deterioration in memory, abstract thinking and judgment; misperception of time, place, or person (disorientation); increased anxiety and personality changes (disintegration of personality). Although symptoms usually become apparent during the fourth or fifth decade of life, age of onset is variable, ranging from early childhood to late adulthood (eg, 70 or 80 years).

HD runs in families as an autosomal dominant trait. The disorder results from abnormally long sequences or repetitions of encoded commands within a gene on chromosome 4 (4p16.3). Progressive loss of nervous system function is associated with HD outcomes from loss of neurons in certain areas of the brain, including the basal ganglia and cerebral cortex.

g. Lateral amyotrophic sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that affects motor neurons. Muscle weakness and atrophy and signs of dysfunction of the cells of the anterior horn of the spinal cord are most often initially noted in the arms, less often in the legs. The site of onset is random and progression is asymmetrical. Seizures are common and may be preceded by weakness. Rarely the patient lives 30 years; 50% die within 3 years of onset, 20% live 5 years, and 10% live 10 years.

Diagnostic features include onset during middle or late adulthood and progressive general motor involvement without sensory impairment. speed nervous

- 21 042179 conduction is normal until the late stage of the disease. Recent studies have demonstrated cognitive impairment, as well as, in particular, a decrease in direct verbal memory, visual memory, language, and executive functions.

A decrease in the cell body area, the number of synapses, and the total length of synapses was observed even in normal neurons in patients with ALS. It has been suggested that when core plasticity reaches its limit, continued loss of synapses can lead to functional impairment. Stimulation of new synapse formation or prevention of synapse loss may maintain neuronal function in these patients.

h. Multiple sclerosis.

Multiple sclerosis (MS) is characterized by various symptoms and signs of CNS dysfunction, with remissions and recurrent exacerbations. The most common symptoms are paresthesias in one or more limbs, in the torso, or on one side of the face; weakness or clumsiness of the leg or arm; or visual disturbances, such as partial blindness and pain in one eye (retrobulbar optic neuritis), blurred vision, or scotomas. Common cognitive impairments include impairments to memory (acquisition, retention, and retrieval of new information), attention and concentration (particularly divided attention), information processing, executive function, visuospatial function, and verbal fluency. Common early symptoms are ocular palsy that results in double vision (diplopia), temporary weakness of one or more limbs, slight stiffness or unusual fatigue of a limb, minor gait disturbances, difficulty with bladder control, dizziness, and mild emotional disturbances; they all indicate diffuse CNS involvement and often occur months or years before the disease is recognized. Excess heat can aggravate symptoms and signs.

The course of the disease is highly variable, unpredictable and, in most patients, relapsing. At first, episodes may be separated by months or years of remission, especially when the disease begins with retrobulbar optic neuritis. However, some patients have frequent seizures and quickly become incapacitated; in some, the course of the disease can be rapidly progressive.

i. Glaucoma.

Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs). The available data indicate the existence of separate patterns of degeneration in synapses and dendrites, including in the GCS. Recent data also indicate a correlation between cognitive impairment and glaucoma in the elderly (Yochim B.P., et al. Prevalence of cognitive impairment, depression, and anxiety symptoms among older adults with glaucoma. J Glaucoma. 2012;21(4):250-254 ).

j. Myotonic dystrophy.

Myotonic dystrophy (MD) is a multisystem autosomal dominant disease characterized by dystrophic muscle weakness and myotonia. The molecular defect is an expanded 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 arms, and ptosis is a common symptom even in mild cases. In severe cases, there is peripheral muscle weakness, often with cataracts, premature baldness, masked face, cardiac arrhythmias, testicular atrophy, and endocrine disorders (eg, diabetes mellitus). Mental retardation is a common symptom in severe congenital forms, while age-related decline in frontal and temporal cognition, particularly language and executive functions, is seen in milder adult forms of the disease. People with a severe lesion die in the region of 50 years.

k. Dementia.

Dementia describes a class of disorders that have symptoms that affect thinking and social abilities severely enough to interfere with daily activities. Other cases of dementia, in addition to the dementia seen in the later stages of the age-related disorders discussed above, include vascular dementia and Lewy body dementia, described below.

In vascular dementia or multi-infarct dementia, cognitive impairment is caused by problems in the blood supply to the brain, usually due to a series of minor strokes, and sometimes due to one large stroke before or after other smaller strokes. Vascular lesions may result from diffuse cerebrovascular disease such as small vessel disease or focal lesions, or both. Patients suffering from vascular dementia have cognitive impairment, acute or subacute, following an acute cerebrovascular event, followed by progressive cognitive decline. Cognitive impairments are similar to those seen in Alzheimer's disease, including impairments in language, memory, complex visual processing, or executive functions, although the corresponding brain changes are not associated with AD pathology, but with a chronic decrease in brain blood flow that, in eventually leads to dementia. Neuroimaging using single photon emission computed tomography (SPECT) and positron emission tomography (PET) can be used to confirm the diagnosis of multi-infarct dementia in combination with assessments that include a mental state examination.

Dementia with Lewy bodies (DLB, also known by various other names, including dementia with Lewy bodies, diffuse Lewy body disease, cortical Lewy body disease, and senile dementia of the Lewy type) is a type of dementia characterized anatomically by the presence of Lewy bodies (clumps of protein alpha-synuclein and ubiquitin) in neurons found on post-mortem histology of the brain. Its main feature is a decrease in cognitive functions, in particular the executive function. Mindfulness and short-term memory will rise and fall.

Persistent or recurring visual hallucinations with vivid and detailed images are often early diagnostic symptoms. LTD is often confused in its early stages with Alzheimer's disease and/or vascular dementia, although Alzheimer's disease usually has a fairly gradual onset, and LTD often has a rapid and acute onset. Symptoms of LTD also include movement symptoms similar to those of Parkinson's disease. DTL differs from dementia, which sometimes occurs in Parkinson's disease, in the time frame in which symptoms of dementia occur compared to symptoms of Parkinson's disease. Parkinson's disease with dementia (PDD) is diagnosed when the onset of dementia is more than one year after the onset of Parkinson's disease. DTL is diagnosed when cognitive symptoms begin at the same time as, or within a year of, symptoms of Parkinson's disease.

1. Progressive supranuclear palsy.

Progressive supranuclear palsy (PNP) is a brain disorder that causes severe and progressive problems with gait control and balance, as well as complex eye movements and problems with thinking. One of the classic signs of the disease is the inability to focus properly, which is due to damage to the area of the brain that coordinates eye movement. Some individuals describe this effect as vagueness. 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 and continues to worsen (progress) and cause weakness (paralysis) by damaging certain parts of the brain above the pea-like structures called nuclei that control eye movements (supranuclear). PNP was first described as a separate disorder in 1964, when three scientists published a paper distinguishing the condition from Parkinson's disease. It is sometimes referred to as the Steele-Richardson-Olszewski syndrome, after the names of the scientists who identified the disorder. Although PNP progresses with deterioration, no one dies from PNP itself.

m. Ataxia.

People with ataxia have problems with coordination because parts of the nervous system that control movement and balance have been affected. Ataxia can affect the fingers, hands, arms, legs, body, speech, and eye movements. The word ataxia is often used to describe the symptoms of incoordination, which can be associated with infections, injuries, other diseases, or degenerative changes in the central nervous system. Ataxia is also used to refer to a group of specific degenerative diseases of the nervous system called hereditary and sporadic ataxias, which are the primary targets of the National Ataxia Foundation.

n. Multiple system atrophy.

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

The cause of MSA is unknown and no specific risk factors have been identified. About 55% of cases occur in men, with a typical age of onset in the late 50s to early 60s. MSA often exhibits some of the same symptoms as Parkinson's disease. However, patients with MSA usually show minimal, if any, response to dopamine drugs used in Parkinson's disease.

o. Decrepitude.

Frailty syndrome (senility) is a geriatric syndrome characterized by functional and physical decline including reduced mobility, muscle weakness, physical sluggishness, poor tolerance, low physical activity, poor nutrition, and involuntary weight loss. This decline is often accompanied by and is a consequence of diseases such as cognitive dysfunction and cancer. However, decrepitude can occur even without illness. Individuals suffering from frailty have an increased risk of poor prognosis from fractures, accidental falls, disability, comorbidities, and premature death (C. Buigues, et al. Effect of a Prebiotic Formulation on Frailty Syndrome: A Randomized, Double-Blind Clinical Trial, Int. J. Mol.

- 23 042179

sci. 2016, 17, 932). In addition, people who suffer from frailty are more likely to have higher health care costs (Id.)

Common symptoms of frailty can be identified through certain types of tests. For example, unintentional weight loss includes losing at least 10 pounds or more than 5% of body weight in the previous year; muscle weakness can be determined by reducing grip strength by 20% from baseline (adjusted for gender and body mass index); physical slowness can be based on the time it takes to walk a distance of 15 feet; weak endurance can be determined by self-reporting exhaustion data; 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)).

In some embodiments, the present methods and compositions find use in slowing the progression of an age-related cognitive, motor, neuroinflammatory, or other age-related disorder or condition. In other words, the individual's cognitive, motor, neuroinflammatory, or other abilities or conditions will decline more slowly after treatment with the described methods than before or without treatment with the described methods. In some such cases, contemplated treatments include measuring the progression of decline in cognitive, motor, neuroinflammatory, or other age-related abilities or symptoms after treatment, and determining that the progression of decline is decreasing. In some such cases, the determination is made by comparison with a reference, for example, the rate of decline in an individual prior to treatment, for example, determined by measuring cognitive, motor, neuroinflammatory or other age-related abilities or conditions for two or more time points before administration of the blood product according to this invention.

The methods and compositions of the invention also find use in stabilizing cognitive, motor, neuroinflammatory, or other abilities or conditions in an individual, such as an individual suffering from age-related cognitive impairment or an individual at risk for age-related cognitive impairment. For example, an individual may exhibit some age-related cognitive impairment, and the progression of cognitive impairment observed before treatment with the methods described will be halted after treatment with the described methods. As another example, an individual may be at risk of developing age-related cognitive impairment (for example, an individual may be aged 50 years or older, or may have been diagnosed with age-related disorder), and the individual's cognitive abilities remain essentially unchanged, that is, a decrease in cognitive ability cannot be detected after treatment with the described methods, compared with the state before treatment with the described methods.

The methods and compositions of this invention also find use in reducing cognitive, motor, neuroinflammatory, or other age-related damage in an individual suffering from an age-related disorder. In other words, after treatment with the methods of the present invention, the impaired ability improves in the individual. For example, cognitive or motor abilities in an individual are increased, for example, 2 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, 30 times or more, or 40 times or more, including 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more or more, after treatment with the methods of this invention compared to the cognitive or motor ability observed in the individual prior to treatment with the methods of this invention.

In some cases, treatment with the methods and compositions of this invention restores cognitive, motor or other abilities in an individual suffering from age-related cognitive or motor impairment, for example, to their level when the person was about 40 years of age or less. In other words, the cognitive or motor impairment is cancelled. Ways to diagnose and monitor for improvement.

13. In some cases, among the various methods for diagnosing and monitoring disease progression and improvement in cognitive diseases, movement disorders, neurodegenerative diseases and/or neuroinflammatory diseases, the following types of assessments are used as necessary, alone or in combination with subjects suffering from neurodegenerative diseases. The following kinds of methods are provided as examples and are not limited to the methods listed. Any convenient means of monitoring the disease, if necessary, can be used in the implementation of the invention. These methods are also provided in the methods of this invention.

a. General knowledge.

Embodiments of the methods of this invention further include methods for monitoring the effect of a drug or treatment on a subject being treated for cognitive impairment and/or age-related dementia, the method comprising comparing cognitive function before and after treatment. Those skilled in the art will recognize that there are known methods for assessing cognitive function. By way of example, and not limitation, the method may include assessing cognitive function based on medical history, family history, physical and neurological examinations by dementia and cognitive physicians, laboratory tests, and neuropsychological assessments. Additional options that are provided in the invention include the assessment of consciousness, for example, using the Glasgow Coma Scale (EMV); a mental health assessment, including an abbreviated intelligence test score (AMTS) or a mini-mental ability test (MMSE) (Folstein et al., J. Psychiatr. Res 1975; 12:1289-198); global assessment of higher functions; assessment of intracranial pressure, for example, by examining the fundus. In one embodiment, monitoring the effect of 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 on a cognitive subscale of the Alzheimer's Disease Severity Rating Scale. (ADAS-COG).

In one embodiment, peripheral nervous system tests can be used to assess cognitive function, including any of the following: smell sensation, visual field and visual acuity, eye and pupil movements (sympathetic and parasympathetic), sensory function of the face, strength of facial muscles and muscles of the shoulder girdle, hearing, taste, pharyngeal movement and reflex, tongue movements, which can be examined separately (for example, visual acuity can be tested using a Snellen chart; a neurological mallet can be used to test reflexes, including chewing reflexes, biceps tendon reflexes, and triceps, knee tendon, Achilles reflex and plantar reflex (i.e. Babinski reflex), muscle strength often on an MRC scale of 1-5, muscle tone and signs of rigidity.

b. Parkinson's disease.

Embodiments of the methods of this invention further include methods for monitoring the effect of a drug or treatment on a subject being treated for movement disorders, the method comprising comparing motor function before and after treatment. Those skilled in the art will recognize that there are known methods for assessing motor function. By way of example, and not limitation, the method may include assessment of motor function based on medical history, family history, physical and neurological examinations by physicians specializing in neurodegeneration and movement disorders, laboratory tests, and neurodegenerative assessments. Additional embodiments contemplated by the invention include the use of the rating scales discussed below.

Several rating scales have been used to assess the progression of PD. The most widely used scales include the Unified Parkinson's Disease Symptom Rating Scale (UPDRS, which was introduced in 1987) (J. Rehabil Res. Dev., 2012 49(8): 1269-76) and the Chen and Yaru scale (Neruology, 1967 17(5): 427-42). Additional scales include the International Movement Disorder Society (MDS) Improved UPDRS Scale (MDS-UPDRS) and the Schwab and England Activity of Daily Activity Scale (ADL).

The UPDRS scores 31 items that make up three subscales: (1) mentality, behavior, and mood; (2) daily activities; and (3) motor activity assessment. The Hen and Yaru scale divides PD into five stages with discrete substages: 0 - no signs of disease; 1 - symptoms on one side only; 1.5 - symptoms on one side, but with involvement of the neck and spine; 2 - symptoms on both sides without any imbalance; 2.5 - mild symptoms on both sides with recovery when performing a resistance test; 3 - imbalance with the degree of disease from mild to moderate; 4 - severe disability, but with the ability to walk or stand without assistance; and 5, needing a wheelchair or bedridden unaided. Schwab and England's daily activity scale classifies PD as a percentage (from 100% complete independence to 10% - complete dependence).

General motor function can be assessed using widely used scales, including the General Motor Function Scale (GMF). It evaluates three components: addiction, pain and insecurity (Aberg A.C., 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, gait (speed of locomotion, variability, stiffness of the legs) can be measured with an accelerometer; posture (torso tilt) - with a gyroscope; leg movement - accelerometer; hand movement - by accelerometer and gyroscope; tremor (amplitude, frequency, duration, asymmetry) - with an accelerometer; fall - accelerometer; slowness of gait - by accelerometer; dyskinesia - accelerometer, gyroscope and inertial sensors; bradykinesia (duration and frequency) with an accelerometer plus a gyroscope, and aphasia (tilt) with a microphone (Pastorino M., et al., Journal of Physics: Conference Series 450 (2013) 012055).

c. Multiple sclerosis.

In addition to monitoring for improvement in cognition-related symptoms, progression or improvement in multiple sclerosis (MS)-associated neurodegeneration can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring can be done using methods such as: cerebrospinal fluid (CSF) monitoring; magnetic resonance imaging (MRI) to detect damage and development of demyelinating plaques; study of evoked potentials; and gait monitoring.

CSF analysis can be performed, for example, by lumbar puncture to obtain CSF pressure, appearance, and content. Normal values are usually in the following range: pressure (70-180 mm H2O); appearance transparent and colorless; total protein (15-60 mg/100 ml); IgG makes up 3-12% of the total protein; glucose 50-80 mg/100 ml; the number of cells 0-5 lymphocytes and the absence of erythrocytes; chloride (110-125 mEq / l). Abnormal results may indicate the presence or progression of multiple sclerosis.

MRI is another method that may be performed to monitor the progression and improvement of the disease. Typical criteria for monitoring PC by MRI include the appearance of patchy areas of abnormal white matter in the cerebral hemisphere and paraventricular regions, the presence of lesions in the cerebellum and/or brainstem, and in the cervical or thoracic spinal cord.

Evoked potentials can be used to monitor the progression and improvement of MS in subjects. Evoked potentials measure the slowing of electrical impulses, for example, in visual evoked response (VER), auditory evoked responses in the brainstem (BAER) and somatosensory evoked responses (SSER). Abnormal responses help to determine the presence of a decrease in conduction velocity in the central sensory pathways.

Gait monitoring can also be used to monitor disease progression and improve disease in subjects with MS. PC is often accompanied by impaired mobility and abnormal gait in part due to fatigue. Monitoring can be performed, for example, using mobile monitoring devices worn by subjects (Moon, Y., et al., Monitoring gait in multiple sclerosis with novel wearable motion sensors, PLOS One, 12(2):e0171346 (2017)).

d. Huntington's disease.

In addition to monitoring for improvement in symptoms associated with cognition, progression or improvement in neurodegeneration associated with Huntington's disease (HD) can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring can be done using methods such as: motor function; behavior; functional evaluation; and visualization.

Examples of motor function that can be monitored as an indicator of disease progression or improvement include chorea and dystonia, rigidity, bradykinesia, oculomotor dysfunction, and gait/balance changes. Methods for monitoring these parameters 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)).

The psychiatric effects of HD provide opportunities for monitoring disease progression and improvement. For example, a psychiatric diagnosis can be made to determine if a subject is suffering from depression, irritability, agitation, anxiety, apathy, and psychosis with paranoia (Id.)

Functional assessment can also be used to monitor disease progression or improvement. Methods for scoring overall functionality (Id.) have been introduced, with scores often declining by one point per year in some HD groups.

MRI or PET may also be used to monitor disease progression or improvement. For example, in HD there is a loss of striatal process neurons, and changes in the number of these neurons can be tracked in subjects. Techniques for determining neuronal change in subjects with HD include imaging of dopamine D2 receptor binding (Id.)

e. ALS.

In addition to monitoring for improvement in cognition-related symptoms, progression or improvement in amyotrophic lateral sclerosis (ALS)-associated neurodegeneration can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring can be carried out using methods such as: functional evaluation; determination of muscle strength; measurement of respiratory function; measurement of lower motor neuron (LMN) loss; and measurement of upper motor neuron (UMN) dysfunction.

Functional assessment can be performed using a functional scale well known to those skilled in the art, such as the ABS Functional Rating Scale (ALSFRS-R), which assesses symptoms associated with bulbar, limbic, and respiratory function. The rate of change is useful in predicting survival as well as disease progression or improvement. Another measure includes the Combined Assessment of Function and Survival (CAFS), ranking the clinical outcomes of subjects by combining survival time with ALSFRS-R change (Simon N.G., et al., Quantifying Disease Progression in Amyotrophic Lateral Sclerosis, Ann Neural 76:643-57 (2014)).

Muscle strength can be examined and quantified through the use of

- 26 042179 evaluation of manual muscle testing (MMT). This involves averaging measurements obtained for several muscle groups using the Medical Research Council (MRC) Muscle Strength Classification Scale (Id.) Hand dynamometry (HHD) can also be used, among other methods (Id.)

Respiratory function can be measured using portable spirometry devices used to obtain forced vital capacity (FVC) at baseline to predict disease progression or improvement. In addition, maximum inspiratory pressure, nasal inspiratory pressure (SNIP), and FVC during meals can be determined and used to monitor disease progression/improvement (Id.)

Loss of lower motor neurons is another indicator that can be used to monitor the progression or improvement of an ABS disease. The neurophysiological index can be determined by measuring electrically evoked muscle response (CMAP) in motor nerve conduction studies whose parameters include CMAP amplitude and F wave frequency (Id. and de Carvalho M., et al., Nerve conduction studies in amyotrophic lateral sclerosis. Muscle Nerve 23:344-352 (2000)). The number of lower motor neurons (MUNE) can also be assessed. MUNE evaluates the number of residual motor axons serving the muscle by evaluating the contribution of individual motor units to the maximum CMAP response and is used to determine disease progression or improvement (Simon N.G., et al., formerly). Additional methods for determining LMN loss include nerve excitability testing, electrical impedance monography, and the application of ultrasound to muscles to detect changes in muscle thickness.

Upper motor neuron dysfunction is another indicator that can be used to monitor the progression or improvement of an ABS disease. Methods for determining dysfunction include performing an MRI or PET scan of the brain and spinal cord, transcranial magnetic stimulation; and determination of biomarker levels in cerebrospinal fluid (CSF).

f. Glaucoma.

In addition to monitoring for improvement in cognition-related symptoms, progression or improvement in glaucoma-related neurodegeneration can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring can be carried out using methods such as the determination of intraocular pressure; assessment of the optic disc or optic nerve head for damage; testing the visual field for peripheral vision loss; and visualization of the optic nerve head and retina for topographic analysis.

g. Progressive supranuclear palsy (PNP).

In addition to monitoring for improvement in symptoms associated with cognition, progression or improvement in neurodegeneration associated with progressive supranuclear palsy (PSP) can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring can be done using methods such as: functional assessment (of daily activities or ADL); assessment of motor functions; identification of psychiatric symptoms; and volumetric and functional magnetic resonance imaging (MRI).

A subject's level of function in terms of independence, partial dependence on others, or complete dependence 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 a rating scale that contains twenty-eight measures in six categories: activities of daily living (by history); behavior; bulbar, ocular motor function, limbic motor function and gait/intermediate function. The result is a score in the range 0-100. Six items were scored 0-2 and twenty-two items were scored 0-4, with a possible total score of 100. PSPRS scores are practical indicators and reliable predictors of patient survival. They are also sensitive to disease progression and useful in monitoring disease progression or improvement (Golbe L.I., et al., A clinical rating scale for progressive supranuclear palsy, Brain 130:1552-65, (2007)).

The ADL section of the UPDRS (Uniform Parkinson's Symptom Rating Scale) can also be used to quantify functional activity in PSP patients (Duff K., et al., formerly). In addition, the Schwab and England Activity of Daily Activity Scale (SE-ADL) can be used to assess independence (Id.). In addition, UPDRS motor function partitions are useful as a reliable measure to assess disease progression in patients with PNP. The motor section may contain, for example, 27 different measures to quantify motor function in patients with PNP. Examples include rest tremor, rigidity, finger tapping, posture and gait). Progression or improvement in a subject's disease may also be assessed by performing a baseline neuropsychological assessment by trained medical personnel, an assessment using the Neuropsychiatric Inventory (NPI) to determine the frequency and severity of behavioral disturbances (eg, delusions, hallucinations, agitation, depression, anxiety, euphoria, apathy, disinhibition, irritability, and aberrant motor behavior).

Functional MRI (fMRI) can be used to monitor disease progression or improvement. fMRI is a technique using MRI to measure changes in brain activity in specific areas of the brain, usually based on blood flow in those regions. It is believed that blood flow correlates with the activation of a brain region. Patients with a neurodegenerative disorder such as PNP may be subjected to physical or mental challenges before or during an MRI scan. By way of example, and not limitation, the tests may be a well-established force management paradigm in which patients are asked to exert force on the arm most affected by the RNP and measure maximal voluntary contraction (MVC) with fMRI immediately after the test is performed. Burciu, R.G., et al., Distinct patterns of brain activity in progressive supranuclear palsy and Parkinson's disease, Mov. Discord. 30(9):1248-58 (2015)).

Volumetric MRI is a technique in which MRI scanners detect differences in the volume of a region of the brain. This can be done, for example, by contrasting different disorders, or by determining differences in the volume of a brain region in a patient over time. Volumetric MRI can be used to determine disease progression or improvement in neurodegenerative disorders such as PNP. The 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 brain regions that can be measured include, but are not limited to, intracranial volume, cerebral cortex, cerebellar cortex, thalamus, caudate nucleus, putamen, pallidum, hippocampus, tonsils, lateral ventricles, third ventricle, fourth ventricle, and brainstem brain.

h. Neurogenesis.

This invention also contemplates the treatment or improvement of neurogenesis in a subject with a decrease or impairment of neurogenesis, which may manifest itself, for example, through a decrease in cognitive or motor function, or through association with neuroinflammation. An embodiment of the present invention includes administering, by way of example, but not limitation, blood plasma, plasma fraction, or PPF to a subject with reduced or impaired neurogenesis using an intermittent application regimen.

An embodiment of the present invention also provides for determining the level of neurogenesis before, during and/or after administration of blood plasma, plasma fraction or PPF. Non-invasive methods for assessing neurogenesis have been reported (Tamura Y. et al., J. Neurosci (2016) 36(31):8123-31). Positron emission tomography (PET) used with an indicator, [18F]FLT, in combination with the BBB transporter inhibitor probenecid, allows the indicator to accumulate in neurogenic areas of the brain. Such imaging allows the assessment of neurogenesis in patients undergoing treatment for neurodegenerative diseases.

i. Neuroinflammation.

This invention also contemplates the treatment or improvement of neuroinflammation in a subject with increased neuroinflammation, which may manifest, for example, through a decrease in cognitive or motor function, or through association with neurogenesis or neurodegeneration. An embodiment of the present invention includes administering, by way of example, but not limitation, blood plasma, plasma fraction, or PPF to a subject with neuroinflammation using an intermittent application schedule.

An embodiment of the present invention also provides for determining the level of neuroinflammation before, during and/or after the administration of blood plasma, plasma fraction or PPF. Non-invasive methods for assessing neuroinflammation have been reported, such as TSPO positron emission tomography (TSPO PET) using ¹¹ C-PK11195 and other similar indicators (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 methods for assessing neuroinflammation include extraction of cerebrospinal fluid and detection, for example, of expression levels of neuroinflammatory 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-derived neurotrophic factor, chiotriosidase-1, MMP-9, chemokine 13 CXC motif, complement terminal fragment 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, incorporated herein by reference).

14. Combination of stem cells and therapy using an intermittent application regimen.

An embodiment of the invention includes treating a subject who has been diagnosed with cognitive impairment, impaired motor function, neuroinflammation, or reduced neurogenesis by administering to the subject an effective amount of blood plasma or a plasma fraction in the case of a subject who is, will be undergoing, or has undergone stem cell therapy. Another variant

- 28 042179 implementation of the invention includes administering to the subject an effective amount of blood plasma or a plasma fraction, while the subject is undergoing, will undergo, or has received stem cell therapy, and the stem cells used in therapy may be embryonic stem cells, not embryonic stem cells, induced pluripotent stem cells (iPSCs), cord blood stem cells, amniotic fluid stem cells, and the like.

Stem cell therapy and methods for performing such therapy are known to those skilled in the art (Andres R.H., et al., Brain 2011, 134; 1777-89; Daadi M.M., et al., Cell Transplant 2013, 22(5):881- 92; Horie N., et al., Stem Cells 2011 29(2): doi: 10.1002/stem.584; Thomsen G. M., et al., Stem Cells 2018, doi: 10.1002/stem.2825; US Patent Application No. No. 09/973198; 12/258210; 12/596884 and 13/290439, all of which are incorporated into this description by reference). Another embodiment includes treating a subject who has been diagnosed with traumatic spinal cord injury, stroke, retinal disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, hearing impairment, heart disease, rheumatoid arthritis, or severe burns, or who is in need of a bone marrow transplant, for the subject is, will be, or has received therapy with stem cells, an effective amount of blood plasma, or a plasma fraction.

15. Methods for Screening Compositions.

Also provided are methods for screening compositions for activity in treating cognitive or movement disorders, reducing neuroinflammation, or enhancing neurogenesis. Such methods are provided in this invention and include those described in the experimental examples below. Compositions that may be screened in embodiments of this invention include: biological compositions (eg, proteins, combinations of proteins, antibodies, small molecule antagonists); plasma fractions or other blood compositions. Results obtained by composition screening methods include, but are not limited to: results of the presence of inflammation/markers of inflammation in the hippocampus (eg, dentate gyrus) or other areas of the CNS; results of cell proliferation in the hippocampus or other areas of the CNS; survival of cells in the hippocampus or other areas of the CNS; developmental direction of cells (eg, astrocytes, new neurons) of proliferating neuronal progenitor cells (NPCs) in the hippocampus or other areas of the CNS; and neurogenesis in the hippocampus or other areas of the CNS.

Additional options for screening compositions for activity in treating cognitive or motor impairment, reducing neuroinflammation or enhancing neurogenesis include determining the acute effects of the compositions on hippocampal inflammation and proliferation, including: administration of 5-7 consecutive daily doses of RDOs with simultaneous administration for 5-7 consecutive days the composition being screened or control (intermittent application) in rodents or other animal models. Up to 10 days (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days) after completion of the intermittent regimen of application of the screening composition, determine the number of cells in the dentate gyrus by staining NOS, and determine the percentage of area with CD-68 staining (an indicator of inflammation).

Another embodiment of methods for screening compositions for activity in treating cognitive or motor impairment, reducing neuroinflammation, or increasing neurogenesis involves administering the NDU for 5 consecutive days (once a day) prior to the start of the intermittent use regimen for 5-7 days of the composition being screened, or control in rodents or other animal models. Four, five, six, seven, eight, nine, ten, eleven, or twelve weeks later, hippocampal cell survival is defined as the number of cells in the dentate gyrus stained with NOS, neurogenesis is defined as the number of cells in the dentate gyrus stained with doublecortin (DCX), and direction development of progenitor cells of neurons that have become astrocytes (associated with aging) or neurons (not associated with aging) are determined by colocalization of ROS with GFAP or Neun markers, respectively.

Another embodiment of methods for screening compositions for activity in treating cognitive or motor disorders, reducing neuroinflammation, or increasing neurogenesis involves administering the NDU and the screening composition or control simultaneously (and daily) for 5-7 days and then determining the degree of neurogenesis by staining DCX in the hippocampus or directing the cellular development of proliferating NPCs as described above.

Another embodiment of methods for screening compositions for activity in treating cognitive or motor impairments, reducing neuroinflammation, or enhancing neurogenesis comprises administering an intermittent application of the screening composition or control and determining improvement in cognitive or motor function in a rodent or other animal model, as described in the examples below.

16. Reagents, devices and kits.

Also provided are reagents, devices, and kits for practicing one or more of the methods described above. The reagents, devices, and kits considered can vary greatly.

Reagents and devices of interest include those mentioned above in relation to methods for obtaining blood products containing plasma for transfusion to a subject in need thereof,

- 29 042179 e.g. anticoagulants, cryopreservatives, buffers, isotonic solutions, etc.

Kits may also include blood collection bags, tubing, needles, centrifuge tubes, and the like. In other embodiments, the kits described herein comprise two or more blood plasma product containers, such as plasma protein fractions, e.g., three or more, four or more, five or more, including six or more blood plasma product containers. In some cases, the number of different blood plasma product containers in the set may be 9 or more, 12 or more, 15 or more, 18 or more, 21 or more, 24 or more, 30 or more, including 36 or more, for example 48 or more. Each container may be associated with identification information that includes various data about the blood plasma product contained in the container, where the identification information may include one or more of the age of the donor of the blood plasma product, details of the processing of the blood plasma product, such as whether the drug was plasma is processed to remove proteins above the average molecular weight (for example, as described above), details of the blood type, etc. In some cases, each container in the kit contains identification information about the blood plasma contained therein, and the identification information includes information about the age of the donor of the blood plasma product, for example, the identification information provides confirmation of the age of the donor of the blood plasma product (for example, the identification information can be age of the donor at the time of blood sampling). In some cases, each container in the kit contains a blood plasma preparation from a donor of substantially the same age, i.e. all containers contain preparation from donors who are approximately the same, if not the same, age. By substantially the same age is meant that the age of the different donors from whom the plasma products for the kits are obtained differs in some cases by 5 years or less, e.g. 4 years or less, e.g. 3 years or less, including 2 years or less , such as 1 year or less, such as 9 months or less, 6 months or less, 3 months or less, including 1 month or less. The identification information may be present on any convenient component of the container, for example in the form of a label, an RFID chip, etc., the identification information may be human readable, machine readable, etc. Containers can have any convenient configuration. While the volume of the containers may vary, in some cases the volumes are in the range of 10 to 5000 ml, such as 25 to 2500 ml, such as 50 to 1000 ml, including 100 to 500 ml. The containers may be rigid or flexible and may be made from any convenient material, such as polymeric materials, including medical grade plastic materials. In some cases, the containers are in a pouch or bag configuration. In addition to containers, such kits may further comprise an insertion device, for example as described above. The components of such kits may be provided in any suitable packaging such as a box or similar structure configured to contain the container and other components of the kit.

In addition to the above components, the kits of this invention may further contain instructions for carrying out the methods of this invention. These instructions may be present in the kits of this invention in various forms, one or more of which may be present in the kit. One form in which these instructions may appear is printed information on an appropriate medium or material, such as the sheet or sheets of paper on which the information is printed, in the package of the kit, in an insert, etc. Another medium would be a computer-readable medium, such as a floppy disk, CD, portable flash drive, etc., on which the information has been recorded. Another means that may be present is a website address that can be used on the Internet to access information at a remote site. Any convenient means can be present in the kits.

17. Exercise.

Exercise can be characterized as aerobic or anaerobic activity and may include high calorie burning activity and moderate calorie burning activity. The exercise may include strength training (such as weight training or isometric exercises). Exercise can also include, for example, running, cycling, walking, dancing, hiking, swimming, yoga, tai chi, balance exercises, leg curls, jumping rope, surfing, rowing, rotating or curling arms or legs, gardening , cleaning, active games such as bowling, aerobics, pilates and martial arts.

An exercise regimen may include performing a single exercise at a specific frequency, or a combination of exercises at a specific frequency. The frequency can be one, two, three, four, five, six or seven times a week. The frequency may vary from week to week. The exercise regimen may remain at the same level of intensity and/or frequency that the subject was practicing prior to administration of the compositions of the invention. The exercise regimen may also be at a higher level of intensity and/or frequency than the levels that the subject was practicing prior to administration of the compositions of the invention. The exercise regimen may be suggested or prescribed by a healthcare professional or fitness trainer, or the exercise regimen may be self-initiated by the subject.

- 30 042179

18. Experimental examples.

A. Example 1

Clarified young human plasma (young plasma) or commercially available PPF (PPF1) was administered to immunocompromised old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). PPF1 is a PPF with approximately 88% normal human albumin (relative to total protein), 12% alpha and beta globulins, and no more than 1% gamma globulin, as determined by electrophoresis. Unless otherwise noted, PPF1 is administered in the examples herein under natural conditions using a 5% solution (w/v, 50 g/l). All mice were divided between treatment groups according to 4 different criteria: home cage nesting score, initial body weight, distance traveled in the open field test, and % time spent in the center of the open field. After group determination, mice were injected intraperitoneally (ip) with BDU (5-bromo-2'-deoxyuridine) in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg for 5 days. Following this, mice were injected intravenously (IV) 3 times a week with 150 μl of PPF1 for 4 weeks. Behavior testing was performed at weeks 5 and 6 when mice received 2 injections per week to avoid injections during concurrent study days. Mice were sacrificed 24 hours after the last iv injection, receiving a total of 16 injections over 6 weeks. Two additional cohorts of mice were injected intravenously (IV) for seven consecutive days with 150 μl of PPF1 or saline (intermittent application). Behavior testing was performed at 5 and 6 weeks at the same time as in the group 3 times a week.

Behavior analyzes were analyzed using CleverSys software (Reston, VA). CleverSys TopScan V3.0 was used to track mouse behavior in Maze Zero, Barnes Maze, Open Field Maze and Y Maze. Barnes' mazes were built by CleverSys. The grip force meter was designed and manufactured by Columbus Instruments of Columbus, Ohio. The Y-maze and open field chambers were built to the specifications of San Diego Instruments (San Diego, CA). Histological analysis of hippocampal sections was performed on a Leica microscope (Buffalo Grove, IL) model DM5500B with a DCF7000T color camera bright field/fluorescent microscope.

Behavior test.

In FIG. 1A shows that the intermittently administered PPF1 groups tended to increase the distance covered in the open field test compared to both the saline control group and the three times weekly PPF1 group. This result indicates a trend towards increased mobility in the intermittent group. In FIG. 1B shows that the intermittently administered PPF1 groups tended to increase the percentage of time spent in the center of the open field compared to both the saline control group and the three times weekly PPF1 group. This result indicates a downward trend in anxiety in the intermittent group.

Body mass.

Fig. 2 is a diagram of the effect of PPF1 on body weight. In both groups receiving PPF1 (via intermittent dosing or three times a week), no adverse effects were found from the injections.

Histology.

In FIG. 3 shows the number of positively labeled DCX cells in the granular layer of the dentate gyrus. There was a significant increase in neurogenesis between the PPF1 intermittent dosing group compared to the thrice weekly treatment group and the saline group. All data are presented as mean ± standard error of the mean. **P < 0.01 one-way ANOVA with Dunnett's multiple comparison post hoc analysis (n: saline = 8, intermittent application PPF1 = 10, PPF1 3x/week = 10). In FIG. 4 shows the number of positively labeled NOS cells in the granular layer of the dentate gyrus of three groups of mice treated separately. There was a significant increase in cell survival between the PPF1 intermittent dosing group compared to the thrice weekly treatment group and the saline group. All data are presented as mean ± standard error of the mean. ****P < 0.0001,* < 0.05 one-way ANOVA with Dunnett's post hoc multiple comparison analysis (n: saline = 8, intermittent dosing PPF1 = 10, PPF1 3x/week = 10).

Hippocampal sections were analyzed using a Leica microscope (Buffalo Grove, IL) model DM5500B with a DCF7000T color camera brightfield/fluorescent microscope. Staining with Ki67 Abcam antibody (ab15580) at a ratio of 1:500 and the secondary antibody was goat anti-rabbit (Alex Fluor 555) (ab150090) at a ratio of 1:300.

b. Example 2

Clarified young person plasma (JP), old person plasma (SP), or

- 31 042179 PPF (PPF1) was administered to immunocompromised old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). All mice were divided between treatment groups according to 4 different criteria: home cage nesting score, initial body weight, distance traveled in the open field test, and % time spent in the center of the open field. After grouping, mice were injected intraperitoneally (ip) with BDU in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg for 5 days. Following this, mice were injected intravenously (IV): 1) three times a week for 6 weeks (3x/week); 2) three times in one week (3x); 3) 7 days within one week, 150 μl of clarified plasma of a young person or PPF1. An additional group of mice were intermittently intravenously injected with saline for 7 days. The last group of mice were injected with old human plasma 3 times in one week or 7 days in one week. All mice were sacrificed 6 weeks after initiation of young or aged plasma, PPF1, or stock solution.

Histology.

In FIG. 5 shows the number of positively labeled DCX cells in the granular layer of the dentate gyrus in nine separately treated groups of mice injected with young human plasma (MP) or aged human plasma (SP) or PPF1 or saline. Mice treated with PPF1 by intermittent administration or treated three times a week showed an increase in neurogenesis compared to other groups. All data are presented as mean ± standard error of the mean; *P < 0.05, Dunnett post hoc ANOVA, PPF1 administration (intermittent or 3x/week) and saline administration (n: saline solution = 4, intermittent administration of PPF1 = 5, PPF1 3x/week = 5, PPF1 3x = 4, MT intermittent = 6, MT 3x/week=6, MT 3x = 4, SP intermittent = 6, SP 3x = 6).

In FIG. 6 shows the number of positively labeled NOS cells in the granular layer of the dentate gyrus in nine separately treated groups of mice injected with young human plasma (MP) or aged human plasma (SP) or PPF1 or saline. Mice treated with PPF1 showed a significant increase in cell survival compared to other groups, with intermittently treated mice with PPF1 showing a greater significant difference than mice treated with PPF1 three times a week. All data are presented as mean ± standard error of the mean; **<0.01, *P<0.05, Dunnett post hoc ANOVA, PPF1 administration (intermittent or 3x/week) and saline administration (n: saline solution = 4, intermittent PPF1 administration = 5, PPF1 3x /week = 5, PPF1 3x = 4, MT intermittent = 6, MT 3x/week = 6, MT 3x = 4, SP intermittent = 6, SP 3x = 6).

c. Example 3

Clarified young human plasma (MP), old human plasma (SP) or commercially available PPF (PPF1) were administered to immunocompromised old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). Mice were administered 7 daily intravenous (IV) doses for 1 week.

All mice were divided between treatment groups according to 4 different criteria: home cage nesting score, initial body weight, distance traveled in the open field test, and % time spent in the center of the open field. After grouping, mice were injected intraperitoneally (ip) with BDU in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg for 5 days. All mice were injected intravenously (IV) for seven consecutive days (called intermittent application) with 150 μl of young or old human plasma, PPF1, or saline. Three weeks after the end of the intermittent application, mice were injected intraperitoneally (ip) with the drug EDU (5-ethyl-2'-deoxyuridine) in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 30 mg/ml. kg for 5 days. The Barnes maze test was run for 8 weeks (6 weeks after the end of intermittent dosing).

Behavior analyzes were analyzed using CleverSys software (Reston, VA). CleverSys TopScan V3.0 was used to track mouse behavior in the Barnes maze. The Barnes Maze was built by CleverSys. Hippocampal slices were analyzed using a Leica microscope (Buffalo Grove, IL) model DM5500B with a DCF7000T color camera brightfield/fluorescent microscope.

Behavior test.

In FIG. 7 shows the delay in finding the target hole per trial per day for each treatment group, as determined using the Barnes maze. Mice receiving intermittent dosing of PPF1 showed a significant reduction in test latency for several individual testing sessions, indicating an improvement in cognitive performance. *P < 0.05 mean ± standard error of the mean; t-test for independent samples (n: saline = 13, PPF1 = 13, SP = 14, MP = 14).

Histology.

In FIG. 8 shows the number of positively labeled DCX cells in the granular layer of the dentate.

- 32 042179 of the gyrus in four separately treated groups of mice injected with young human plasma (MP) or old human plasma (SP), or PPF1, or saline. There was a significant increase in neurogenesis with intermittent PPF1 and intermittent youth plasma compared to saline treatment. All data are presented as mean ± standard error of the mean; ****P < 0.0001, **P<0.01, one-way ANOVA with Dunnett's multiple comparison post hoc analysis (n: saline = 14, PPF1 = 14, SP = 14, MP = 15)

In FIG. 9 shows the number of NDU-labeled cells in the granular layer of the dentate gyrus in mice injected with young human plasma (MP) or aged human plasma (SP) or PPF1 or saline. There was a significant increase in cell survival with intermittent PPF1 and intermittent MP compared to saline treatment. All data are presented as mean ± standard error of the mean; ****P < 0.0001; one-way ANOVA with Dunnett's multiple comparison post hoc analysis (n: saline=14, PPF1=14, SP=14, MP=15).

d. Example 4

Commercially available PPF (PPF1) was administered to immunocompromised old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). Twelve-month-old mice were given 7 daily intravenous (IV) doses by tail vein for 1 week. After treatment, mice were left in their home cage conditions for 4.5 weeks prior to behavior testing. All injections and behavioral testing were performed for 7 weeks for each cohort, for a total duration of 9 weeks. All mice received BDU ip for 5 days prior to the first dosing. Mice were sacrificed one day after the completion of the last behavioral test.

Behavior analyzes were analyzed using CleverSys software (Reston, VA). CleverSys TopScan V3.0 was used to track mouse behavior in the Y Maze.

Behavior test.

In FIG. 10 shows the percentage of the total number of entries made in the familiar or new arm of the total number of entries made in each arm by the treatment group in the Y-maze trial. Mice at twelve months of age were intermittently injected with saline, PPF1, or 5x concentrated PPF1. Mice intermittently injected with PPF1 and PPF1 (5x) showed a significant increase in neo-arm entry compared to the number of neo-arm entries in saline-treated mice, indicative of improved cognitive performance. All data are presented as mean ± standard error of the mean. *P<0.05, paired t-test.

In FIG. 11 shows the ratio of new arm entries compared to the familiar arm of the Y-maze for each treatment group. Mice at twelve months of age were intermittently injected with saline, PPF1, or 5x concentrated PPF1. Mice intermittently injected with PPF1 and PPF1 (5x) showed a trend towards increased entry into the new arm compared to mice injected with saline. All data are presented as mean ± standard error of the mean.

Histology.

In FIG. 12 shows the number of positively labeled NDU cells in all sections of the hippocampus. Mice intermittently injected with PPF1 showed a trend towards increased cell survival compared to mice injected with saline and PPF1 (5x). All data are presented as mean ± standard error of the mean.

In FIG. 13 shows the number of positively labeled DCX cells in sections of the entire hippocampus. Mice intermittently injected with PPF1 and PPF1 (5x) showed a trend towards increased neurogenesis compared to mice injected with saline. All data are presented as mean ± standard error of the mean.

e. Example 5

Commercially available PPF (PPF1) was administered to immunocompromised old (10.5 month old) mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). All mice were assigned to treatment groups according to four different criteria: home cage nesting score, initial body weight, distance traveled in the open field test, and percentage of time spent in the center of the open field. After grouping, mice were injected intraperitoneally (ip) with BDU in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg for 5 days. This was followed by intravenous (IV) administration of PPF1 to mice: 1) 5 consecutive days [PPF1-5d] 2) 7 consecutive days [PPF1-7d] 3) 5 consecutive days with an additional 5 consecutive days of booster (B) dosing for 6 week after completion of initial dosing [PPF1-5d-B] 4) 7 consecutive days with an additional 7 consecutive days of booster (B) dosing at week 6 after completion of initial dosing [PPF1-7d-B]. An additional group was administered saline for 7 consecutive days with an additional 7 consecutive days of dosing 6 weeks after completion of the initial dosing [SAL-7d-B]. Five weeks after intermittent administration, mice were injected ip with EDA (5-ethyl-2'-deoxyuridine) in PBS at a final concentration of 10 mg/ml, dosed at 30 mg/kg for 5 days. All mice were sacrificed 12 weeks after the end of intermittent administration of PPF1 or stock solution.

Hippocampal slices were analyzed using a Leica microscope (Buffalo Grove, IL) model DM5500B with a DCF7000T color camera brightfield/fluorescent microscope. In FIG. 14 shows the number of positively labeled DCX cells in the granular layer of the dentate gyrus in animals treated with PPF1 and saline. These results demonstrate that there is a significant improvement in the group treated for 5 consecutive days followed by a booster dose, which is comparable to the group treated for 7 consecutive days. All data are presented as mean ± standard error of the mean; PPF1-7d, PPF1-5d-B vs. saline *P < 0.05, Dunnett post hoc ANOVA (n: saline = 5, PPF1-5d = 8, PPF1-7d = 7, PPF1-5d-B = 8, PPF1-7d-B = 7).

In FIG. 15 shows the number of positively labeled NDU cells in the granular layer of the dentate gyrus in animals treated with PPF1 and saline. These results demonstrate that, in terms of proliferating cells, stimulation is significantly increased in the group treated for 5 consecutive days followed by a booster dose compared to groups treated for 5 or 7 consecutive days without a subsequent booster dose. In addition, booster treatment significantly increases cell survival in general. All data are presented as mean ± standard error of the mean; PPF1-5d-B, PPF1-7d-B vs. saline ***, P<0.001, *P<0.05, ANOVA with Dunnett's post hoc analysis. PPF1-5d vs. PPF1-5d-B +P<0.05, T-test for independent samples, (n: saline = 7, PPF1-5d = 8, PPF1-7d = 7, PPF15d-B = 8, PPF1 -7d-B = 7).

In FIG. 16 shows the number of positively labeled EDU cells in the granular layer of the dentate gyrus in animals treated with young plasma, PPF1 and saline. These results demonstrate that the effects seen with the booster dose are not related to an increase in the total number of proliferating cells present, but are related to the enhanced survival mechanism induced by the booster dose. All data are presented as mean ± standard error of the mean; (n: saline = 4, PPF1-5d = 7, PPF1-7d = 6, PPF1-5d-B = 7, PPF1-7d-B = 6).

f. Example 6

Commercially available PPF (PPF1) was administered to immunocompromised adult mice (3 and 6 months old) (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). All mice were divided between treatment groups according to four different criteria: home cage nesting score, initial body weight, distance traveled in the open field test, and % time spent in the center of the open field. After grouping, mice were injected intraperitoneally (ip) with BDU in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg for 5 days. Following this, mice were injected with saline or PPF1 intravenously (IV) for 7 consecutive days (intermittent application). A subset of mice injected with both saline and PPF1 received activity wheels in their cages. Mice were sacrificed 3 days, 10 days or 42 days after completion of the intermittent application.

In FIG. 17 shows the number of positively labeled DCX cells in the granular layer of the dentate gyrus in 3-month-old NSG animals treated with PPF1 or saline, with or without activity wheels. All data are presented as mean ± standard error of the mean; activity wheel at 42 days vs. saline at 42 days ***, P<0.0001, *P<0.05, ANOVA with Dunnett's post hoc analysis. Activity wheel against PPF1 at 42 days +++P<0.001, t-test for independent samples (n: saline at 3 days = 8, PPF1 at 3 days = 8, PPF1 at 10 days = 7, stock solution at 42 days = 8 , PPF1 at 42 days = 8, Activity wheel at 42 days = 8, Activity wheel + PPF1 at 42 days = 8). In FIG. 17 also shows the number of positively labeled DCX cells in the granular layer of the dentate gyrus in 6-month-old NSG animals treated with PPF1 or saline, with or without activity wheels. All data are presented as mean ± standard error of the mean; activity wheel at 42 days vs. saline at 42 days ****, P<0.0001, **P<0.01, ANOVA with Dunnett's post hoc analysis. Activity wheel against PPF1 at 42 days +++P<0.001, t-test for independent samples. PPF1 at 42 days vs. saline at 42 days +P<0.05, t-test for independent samples (n: saline at 3 days = 7, PPF1 at 3 days = 8, PPF1 at 10 days = 6, saline after 42 days = 8, PPF1 after 42 days = 6, Activity wheel after 42 days = 8, activity wheel + PPF1 after 42 days = 9).

In FIG. 18 shows the number of positively labeled Ki67 cells in the granular layer of the dentate gyrus in 3-month-old NSG animals injected with PPF1 or saline, with or without activity wheels. All data are presented as mean ± standard error of the mean; wheel

- 34 042179 activities + PPF1 at 42 days vs saline at 42 days ***, P<0.001, ANOVA with Dunnett post hoc analysis (n: saline at 3 days = 6, PPF1 at 3 days = 6, PPF1 at 3 days = 7, saline at 42 days = 8, PPF1 at 42 days = 8, Activity wheel at 42 days = 8, Activity wheel + PPF1 at 42 days = 8).

In FIG. 18 also shows the number of positively labeled Ki67 cells in the granular layer of the dentate gyrus in 6-month-old NSG animals injected with PPF1 or saline, with or without activity wheels. All data are presented as mean ± standard error of the mean; activity wheel at 42 days vs. saline at 42 days ***, P<0.001, *P<0.05, Dunnett post hoc ANOVA (n: saline at 3 days = 7, PPF1 at 3 days = 7, PPF1 after 10 days = 8, saline after 42 days = 8, PPF1 after 42 days = 7, Activity wheel after 42 days = 7, activity wheel + PPF1 after 42 days = 9).

In FIG. 19 shows the number of positively labeled NOS cells in the granular layer of the dentate gyrus in 3-month-old and 6-month-old NSG animals treated with PPF1 or saline, with or without activity wheels. All data are presented as mean ± standard error of the mean; activity wheel + PPF1 at 42 days vs stock solution at 42 days ***, P<0.001, ANOVA with Dunnett's post hoc analysis (**** P < 0.0001; *** P < 0.001; ** P < 0. 01; * P < 0.05, ANOVA with Dunnett's post hoc analysis).

These results demonstrate that there is a significant increase in neurogenesis with PPF1 and activity wheel compared to stock solution at 6 weeks post dosing in 3 month old NSG mice. In addition, there is a significant increase in neurogenesis with PPF1 and the activity wheel compared to the activity wheel alone at 6 weeks post dosing in 3 NSG mice. There is also a significant increase in neurogenesis with PPF1 and activity wheel compared to stock solution at 6 weeks post dosing in 6 month old NSG mice. These results also demonstrate that there is a significant increase in neurogenesis with PPF1 and the activity wheel compared to the activity wheel alone at 6 weeks post dosing in 6 NSG mice. There is also a significant increase in progenitor cell proliferation with PPF1 and activity wheel compared to stock solution at 6 weeks post dosing in 3 and 6 month old NSG mice.

These data in adult NSG mice at 6 months of age indicate possible synergistic effects of exercise and PPF1 administration resulting in a significant increase in neurogenesis compared to exercise or PPF1 treatment alone. This supports the potential utility of PPF1 treatment in combination with an exercise regimen in a clinical setting. Furthermore, these data suggest that there is a significant potential for neurogenesis in the brain that can be achieved through multiple independent or overlapping mechanisms.

g. Example 7

PPF1 or control saline was administered to two treatment groups of 11-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG strain). All mice received intravenous injections of 150 μl of PPF1 or saline per dose for seven consecutive days. The activity wheel (MedAssociates) was placed in cages of mice designated as runners (n=8, n=8 for PPF1 and saline) starting at week 7 of the study. The number of wheel revolutions was recorded for 5 consecutive days, day and night.

In FIG. 20 shows the number of wheel revolutions over given time periods, with a shaded area indicating a dark cycle, an independent sample t-test was used to assess the statistical significance of total mileage for both treated and untreated groups in light and dark cycles. Rhythm profiles were extracted and characterized using time and frequency data analysis for a series of 13 time points, separately for each mouse from treated and untreated groups, with five series of 13 time points per mouse. Period, phase and amplitude were parameters determined for each rhythm and were compared between the two groups using an independent sample t-test. PPF1-treated mice ran significantly longer than untreated animals, which is an indication of increased locomotor activity. The mice were subjected to the hot plate test to monitor for relatively normal pain in their paws. Loss of sensation may have affected previous behavioral readings. Hot plate testing resulted in a slight increase in activity after the activity wheel was returned to the cage, as indicated by the jump in wheel speed indicated in the circled segment of FIG. 20.

h. Example 8

Recombinant human albumin (rhAlbumin, Albumedix, Ltd, Nottingham, United Kingdom), clarified young human plasma (MP), or control saline solution were administered to 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, line NSG). All animals received 50 mg/kg BDU ip for 1 week prior to 7 days of intermittent use. RhAlbumin and MP were diluted to 50 mg/ml in water for injection (WFI, 0.9% saline). All mice received intravenous injections of 150 µl of rhAlbumin, MP, or saline per dose for 7 consecutive days. Mice were sacrificed 6 weeks after the last day of treatment.

In FIG. 21A shows the number of surviving cells in all 3 groups as determined by the number of NOS-labeled cells in the dentate gyrus (DG). Young plasma significantly increased cell survival compared to saline and rhAlbumin, while rhAlbumin had no significant effect on cell survival. All data are presented as mean ± standard error of the mean (***P<0.001, t-test for independent samples).

In FIG. 21B shows the amount of DCX staining in all 3 groups as determined by the amount of DCX-positive labeled cells in the dentate gyrus (DG). Young plasma significantly increased neurogenesis compared to saline and rhAlbumin, while rhAlbumin was associated with reduced neurogenesis compared to control saline. All data are presented as mean ± standard error of the mean (**P< 0.01; ***P<0.001, t-test for independent samples).

i. Example 9

Dissociated mixed neuronal cells derived from E16 mouse cerebral cortex were seeded and grown in a 48-well multi-electrode plate (Axion Biosystems). Each well contains 16 electrodes that are in physical contact with seeded neuronal cells and measure subtle changes in cell membrane properties. This setup allows evaluation of various parameters to obtain information about the neuronal peak activity of heating behavior at the level of a single electrode, as well as information about the degree of neural connectivity by evaluating the synchronism of neural heating properties between multiple electrodes within a well.

Neuronal cultures were maintained in the presence of treatment conditions starting from day 1. Treatment conditions included Neurobasal medium plus B27 supplement containing 10% (v/v): recombinant human albumin ((''rhAlbumin, Albumedix, Ltd, Nottingen, United Kingdom); PPF1 or HAS1. PBS was the control. Neuronal activity was measured on the 7th and 14th day in culture.

In FIG. 22 shows that 7 days of PPF1 treatment resulted in an increase in neural network activity compared to control, rhAlbumin or HAS1 treatment. HAS1 is a commercially available HAS with greater than 95% human albumin (relative to total protein) in 5% solution (w/v, 50 g/l) obtained by cold alcohol fractionation, and derived from pooled human plasma from donors. Both PPF1 and HAS1 were supplied in a 5% solution (w/v, 50 g/l) and diluted 1:10 with Neurobasal medium supplemented with B27. The influence of PPF1 on the activity of the neural network persists for 14 days in culture. This indicates that PPF1 is associated with activation of neural network maturation. Data are presented as mean ± standard error of the mean (*P < 0.05, t-test for independent samples).

j. Example 10

Old human clarified plasma (SP) or sterile saline was administered to 8 week old immunocompromised (young) mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, 'NSG' line). In each experiment, mice were divided by weight into different treatment groups. All mice were injected ip for 5 consecutive days with 150 mg/kg BDU in sterile PBS. After the introduction of the BDU was carried out in / in the introduction of old plasma in various paradigms of treatment at 150 μl per dose. All paradigms are shown in Fig. 23.

Paradigm 1 includes injections twice a week for a total of 10 injections over 5 weeks. Histological analysis was performed 48 hours after the last plasma dose. Paradigm 2 includes injections three times a week for a total of 10 injections over 4 weeks, with histological analysis 48 hours after the last dose. In paradigm 3, mice were administered daily for 7 consecutive days and with histological analysis 48 hours after the last dose. In paradigm 4, mice were administered daily for 7 consecutive days and analyzed 21 days after the last dose. The brains of stale plasma injected mice were analyzed for the endothelial inflammation marker VCAM-1 in the hippocampus and for the number of newly formed neurons as indicated by doublecortin (DCX)-positive cells in the dentate gyrus. VCAM-1 was visualized on Hamamatsu NanoZoomer HT (Hamamatsu) after immunohistochemistry on 30 µm free floating sections and analyzed using Image Pro software (Media Cybernetics). DCX-positive cells in the dentate gyrus were counted alive on a wide-field microscope Leica (Leica).

Analysis of the percentage of VCAM-1 positive area in the hippocampus (Fig. 24) demonstrates that endothelial inflammation increases significantly 48 hours after the last plasma injection with a dosing trend of twice a week (Fig. 24A), and increased significantly after administration of three times a week. week (Fig. 24B) and intermittent application (Fig. 24C). VCAM-1 levels were no longer significantly elevated 21 days after the last plasma dose (FIG. 24D).

An effect on doublecortin was observed only after a 3-4 week time period, so the number of DCX-positive cells was analyzed in the dentate gyrus in paradigms 1, 2, and 4. The analysis demonstrated that there was no effect of aged plasma on neurogenesis at twice-weekly (FIG. 25A) or thrice-weekly (FIG. 25B) dosing paradigms, however, intermittent dosing for 7 consecutive days (FIG. 25C) resulted in a significant reduction in DCX-positive cells. These data suggest that only intermittent use of aged human plasma has a significant effect on neurogenesis.

k. Example 11.

8 week old NSG mice injected with aged human plasma (65-68 years of age) for 7 consecutive days were tested using a modified Barnes maze at 4 weeks after the last injection of aged plasma. In FIG. 26 shows the time course of the Barnes Maze exit delay and indicates the time required to reach and enter the exit port for NSG mice injected with aged plasma and saline. There were no significant differences in exit delay between groups, but on day 4 mice injected with stale plasma performed worse than mice treated with control saline. These data indicate a reduction in learning and memory associated with hippocampal function on a spatial memory task. All data are presented as mean ± standard error of the mean. Two-way ANOVA, Shidak's post hoc test.

In FIG. 27 shows the average exit delay during the last three trials in the Barnes maze on day 4. Mice injected with aged plasma showed a trend towards higher exit latency, indicating impaired memory function. All data are presented as mean ± standard error of the mean, (t-test for independent samples).

In FIG. 28 shows the difference in exit delay between trials 1 and 3 in the Barnes maze and demonstrates that these trials can be used as a measure of one day's learning. Mice injected with aged plasma had a significantly lower difference in exit latency between these studies, indicating reduced learning ability. All data are presented as mean ± standard error of the mean (*P < 0.05, t-test for independent samples).

In FIG. 29 shows the results of qPCR, which was used to quantify mRNA levels of various markers associated with neurogenesis and synaptic function. Relative expression levels of doublecortin (DCX), a marker for newly formed neurons, were reduced according to histological analysis of this marker. In addition, there were downward trends in vglut1 (vesicular glutamate transporter 1), a marker of glutamatergic synapses, synaptic marker syn1 (synapsin 1), tuj1 (beta-tubulin III), and bdnf (brain-derived neurotrophic factor). This decrease indicates a general disruption of the synaptic and neuronal network in the brains of mice injected with old plasma. All data are presented as mean ± standard error of the mean (*P < 0.05, t-test for independent samples).

l. Example 12.

Young (8-week-old) immunocompromised mice (NOD.Cg-Prkdscid Il2grtm 1 Wj1/SzJ, NSG strain) were distributed by weight into different treatment groups. Animals were injected subcutaneously (sc) with 35 mg/kg of kainic acid (Sigma) in sterile saline or control saline. Peripheral administration of kainic acid resulted in acute seizure activity, inflammation in the hippocampus, and, in a subset of mice, also neuronal death in the CA1 region of the hippocampus. 2 hours after the injection of kainic acid, mice were intravenously injected with 150 μl of PPF1 or saline. Administration of PPF1 or saline continued daily for a total of 5 days (FIG. 30). The tissue was collected for analysis on the 6th day. Inflammatory changes in the CA1 region of the hippocampus were analyzed after immunofluorescence staining for microglial (CD68) and astrocyte (GFAP) activation. Sections were visualized on Hamamatsu NanoZoomer HT (Hamamatsu) after immunohistochemistry on 30 μm free floating sections and analyzed using Image Pro software (Media Cybernetics).

Analysis of the percentage of CD68-positive region in the CA1 region of the hippocampus demonstrates that administration of kainic acid results in increased CD68 immunoreactivity, suggesting an increase in microglial activation (FIG. 31A). Five days of PPF1 administration resulted in a significant decrease in the percentage of CD68-positive region and hence a decrease in microglial activation. Similarly, analysis of the percentage of the GFAP positive region (FIG. 31B) shows a significant increase after administration of kainic acid, which decreases significantly after dosing with PPF1. Evidence suggests that PPF1 has an acute anti-inflammatory effect in the brains of mice injected with kainic acid. * P < 0.05 one-way ANOVA with Dunnett's post hoc multiple comparison analysis.

m. Example 13

NSG mice aged 6 months were injected daily for one week (7 days), iv, PPF1 or control saline at a dose of 150 μl (10 mg/ml). All mice were treated with NDU, 50 mg/kg NDU i.p.

- 37 042179 once daily on the same day they received PPF1 or control saline. Mice were divided into two cohorts. The first cohort was sacrificed per day, immediately after 7 days of concurrent treatment with NDU and PPF1. The second cohort was sacrificed 7 days later and the animals received an additional 7 days of daily administration of BDU.

In FIG. 32 shows the number of stained cells in the dentate gyrus of cohorts 1 and 2 (from left to right). Both cohorts showed increased cell proliferation in the dentate gyrus compared to saline control (***p<0.001, t-test for independent samples).

n. Example 14

NSG mice aged 3 or 6 months were administered daily for one week (7 days), iv, PPF1 or basic saline. Mice were then sacrificed 3, 10 or 42 days after 7 daily doses were administered. Brains were stained with Ki67, a nuclear marker that is present only in proliferating cells that labels neural stem and progenitor cells in the blade of the dentate gyrus. In FIG. 33 shows that 6-month-old mice showed an increase in the total number of progenitor cells (Ki67-positive or Ki67+) in the dentate gyrus on day 10 after the end of the 7-day sequential regimen of intermittent PPF1 application. In FIG. 34 shows staining (light areas) for Ki67 in the dentate gyrus on day 10 in NSG mice after the end of a 7-day intermittent PPF1 sequencing regimen. This indicates that one of the possible mechanisms of action of PPF1 in increasing overall cell survival and neurogenesis at day 42 after dosing cessation may be due to an increase in the total number of progenitor cells (neural stem cells).

o. Example 15

Commercially available PPF (PPF1) or saline control was administered to two different populations of 6 and 12 month old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). All animals received 50 mg/kg BDU for 1 week prior to 7 days of intermittent use of the test agent. All mice received intravenous injections of 150 μl of PPF1 or saline per dose for 7 consecutive days. One cohort from each treatment group was used for the proliferation study and sacrificed 6 weeks after the last dose.

In FIG. 35A shows that cohorts of 6-month-old mice injected with PPF1 showed a significant increase in the number of progenitor cells that differentiated into neurons (NeuN+) compared to the saline control, as well as a decrease in the number of progenitor cells that differentiated into astrocytes (GFAP+ ) compared to control. All data are presented as mean ± standard error of the mean (**P < 0.01, t-test for independent samples).

In FIG. 35B shows that cohorts of 12-month-old mice injected with PPF1 showed a significant increase in the number of progenitor cells that differentiated into neurons (NeuN+) compared to the saline control, as well as a statistically non-significant difference in the number of progenitor cells that differentiated into astrocytes compared to control. All data are presented as mean ± standard error of the mean (**P < 0.01, t-test for independent samples).

p. Example 16

Clarified old human blood plasma (old plasma) or sterile saline was administered to 3-month-old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). In each experiment, mice were divided by weight into different treatment groups. All mice were injected ip for 5 consecutive days with 150 mg/kg BDU in sterile saline. Following the NDU injection, stale plasma or sterile saline was administered intravenously at a dose of 150 μl per day for 7 consecutive days and analyzed histologically 4 weeks after the last dose.

In FIG. 36 shows the direction of cellular development of NOS-labeled proliferating neural progenitors 4 weeks after the last dose. In mice injected with stale plasma, the surviving BDU-labeled cells differentiated significantly less into neurons than into astrocytes. This indicates that the plasma of the old person changes the direction of development of neural progenitors in young mice towards the lineage of astrocytes (Fig. 37B) and especially negatively affects the number of newly formed neurons in the dentate gyrus (Fig. 36A) (n = 12 in each group). All data are presented as mean ± standard error of the mean (***P< 0.001; ****P<0.0001, t-test for independent samples).

q. Example 17.

cortical activation.

Old (18-month-old) C57BL/6 mice received daily intravenous injections of 150 μl of PPF1 or 0.9% sterile saline for 7 days. 2.5 hours after the last administration of the test agent, mice were sacrificed by transcardial perfusion with 0.9% saline followed by the addition of 4% formaldehyde under deep anesthesia with ketamine and xylazine. The brain was dissected, fixed, and then processed using the iDisco procedure to visualize cFos-positive

- 38 042179 cells by fluorescence surface illumination microscopy (LSFM) with a voxel resolution of 2x2x3 µm. Visualized brain samples were aligned as 3D volumetric objects, and activated cFos-positive cells were detected by calculation. Statistical comparison between groups was performed using negative binomial regression adjusted for multiple comparisons for false detection rate (* indicates a q value of less than 0.05).

Mouse brain analysis demonstrated an overall increase in the number of cFos-positive cells in the total brain volume as well as in the cerebral cortex and isocortex in 18-month-old PPF1-treated mice (FIG. 37). Using binomial regression adjusted for multiple comparisons, the difference in this increase in total cFos positive cells did not reach significance. However, analysis of more defined cortical regions such as the frontal, orbital, infralimbal, and prelimbal cortices showed a significant increase in cFos-positive cells (Fig. 38), indicating increased neuronal activity. Increased activity in the prefrontal cortex correlates with increased cognitive function, suggesting that PPF1 treatment results in cognitive improvements in aged C57BL/6 mice. A similar significant increase in the number of cFos-positive cells was also found in the adnexal olfactory nucleus and the olfactory tubercle (Fig. 39). These areas are associated with the processing of olfactory information, and an increase in activity indicates increased olfactory function. Voxel imaging based on cFos activation statistics in red demonstrated an increase in cFos signal in the cerebral cortex of mice injected with PPF1 (FIG. 40).

r. Example 18.

Commercially available PPF (PPF1) or control saline was administered to 22-month-old wild type (WT) mice (C57BL/6J, WT, line code 0664, Jackson Labs, Bar Harbor, Maine). All animals received 50 mg/kg BDU at 1 week prior to 7-day intermittent dosing. As a result, all mice received intravenous injections of 150 μl of PPF1 or saline per dose for seven consecutive days. Mice were sacrificed 10 days after the last injection of PPF1 or saline, and brain samples were prepared for histology.

In FIG. 41A shows the percentage of CD68 immunoreactive area in the hippocampus (n = 10, 10). In FIG. 41B shows the percentage of Iba-1 immunoreactive area in the hippocampus (n = 10, 10). In FIG. 41C shows the percentage of GFAP immunoreactive area in the hippocampus (n = 10, 10). All data are presented as mean ± standard error of the mean (*P< 0.05; **P<0.01, t-test for independent samples). These results demonstrate a significant reduction in the microglial markers CD68 and Iba-1 in the hippocampus of old mice injected with PPF1.

s. Example 19.

Commercially available PPF (PPF1) or control saline was administered to 23-month-old wild-type mice (DTC57BL/6J, DT, Line Code 0664, Jackson Labs, Bar Harbor, Maine). All animals received 50 mg/kg BDU at 1 week prior to 7 days of consecutive intermittent dosing. As a result, all mice received intravenous injections of 150 μl of PPF1 or saline per dose for seven consecutive days. One cohort from each treatment group was used to study histological markers of neuroinflammation and sacrificed 6 weeks after the last dose.

In FIG. 42A shows the percentage change in NDU expression compared to saline control at 6, 9 and 12 weeks after dosing, which is an indication of cell survival in the hippocampus. Animals received 7 consecutive days of the intermittent application regimen.

In FIG. 42B shows the percentage change in doublecortin (DCX) expression compared to saline control at 6, 9 and 12 weeks post dosing, which is indicative of cell neurogenesis in the hippocampus. Animals received 7 consecutive days of the intermittent application regimen.

t. Example 20.

Thirty (30) male alpha-synuclein transgenic mice (strain 61, wild background C57BL/6J) aged 4 to 4.5 months were divided into two groups of 15 and treated with PPF1 or stock solution for seven (7) consecutive days. PPF1 was injected intravenously at 5 µl per 1 g of body weight. Alpha synuclein mice served as a transgenic model of Parkinson's disease and overexpressed the alpha synuclein protein. This transgenic model is not immunodeficient, unlike NSG mice.

One day after the last injection of PPF1 or stock solution, all mice were subjected to cognitive and motor testing such as nest building, pasta chewing, wire hanging, spinning rod test, and pole walking. Pasta chewing, wire hanging, and pole walking were performed a second time at the end of the study. Testing was carried out in random order.

Animals were weighed once a week. In FIG. 43A and 43B show that there were no significant differences between alpha-synuclein transgenic mice (Tg) injected with PPF1 and stock solution (veh).

In FIG. 44 shows the results of nest building. Mice were housed individually in cages containing wood chip bedding and one square of pressed cotton (nestlet). No one

- 39 042179 no other bedding material (eg wood fibers) was present. Nestlet was introduced 24 hours prior to nest evaluation approximately 2-3 hours before the dark phase was initiated, and subsequent building behavior was assessed the following day of the experiment, approximately 2-3 hours after the light phase began. The time interval between the introduction of a cotton square and the assessment of the next status was the same for all studies. Nestlet manipulation and nest building were assessed on a five-point scale (Deacon, R.M. 2006, Assessing nest building in mice. Nat Protoc 1:1117-19). As shown in FIG. 44, there was an increase in the tendency in nesting behavior in mice injected with PPF1 compared to mice injected with stock solution.

In FIG. 45A and 45B show that there was a significant increase in pasta nibbling in the PPF1 treated group compared to the stock solution treated group 3 weeks after the last injection, indicating improvement in motor function (FIG. 45B). The test was developed to study motor deficits in small rodents. Animals were transferred to the experimental room at least 2 hours before testing. The cage lid, water bottle and food pellets were removed and a small piece of dry pasta (approx. 5 mm) was placed in the cage. The microphone was placed above a piece of pasta. Recording began as soon as the animal began to eat. The number of bites required for chewing and the frequency of bites were assessed, and the chewing profile was analyzed using Avisoft SASLab Pro software. All data are presented as mean ± standard error of the mean (*P < 0.05, t-test for independent samples).

In FIG. 46 shows the results of a wire hanging test that evaluates neuromuscular motor force abnormalities. There was a significant increase in time to fall in the PPF1 treated group compared to the stock solution treated group 3 weeks after the last injection. In order to perform the test, a metal wire cage cover was used, and adhesive tape was placed around the perimeter to prevent the mouse from walking along the edge. The animal was placed on top of the cage lid. The lid was shaken lightly three times to get the mouse to grab the wire, and then the lid was turned upside down. The lid was fixed at a height of approximately 50-60 cm above the soft padding, high enough to prevent the mouse from jumping down, but not high enough to cause harm if dropped. The delay to fall was measured and a 300 second cutoff was used. Generally, wild-type mice can hang upside down for several minutes.

In FIG. 47A-47C show the results of the pole walking test. In FIG. 47A shows the different shapes and sizes of the pole (square or cylindrical pole) used in five different tests of increasing difficulty. In FIG. 47B shows the results of five trials 72 hours after the last injection. In FIG. 47C shows the results of five trials 3 weeks after the last injection. Mice treated with PPF1 showed significantly higher pole climbing success during Trial 5 of Test 1 (72 hours post-administration) and Trial 4 of Test 2 (3 weeks post-administration). All data are presented as mean ± standard error of the mean (**P < 0.01, binomial test).

In FIG. 48A-48F show histological staining results of the striatum and hippocampus. In FIG. 48A shows CD68 staining in the striatum. In FIG. 48B shows CD68 staining in the hippocampus. In FIG. 48C shows Iba-1 staining in the striatum. In FIG. 48D shows Iba-1 staining in the hippocampus.

In FIG. 48E shows NeuN staining in the striatum. In FIG. 48F shows NeuN staining in the hippocampus. These drawings show that mice injected with PPF1 showed a reduction in microgliosis (neuroinflammation) by Iba-1 and CD68 staining in both the striatum and hippocampus, and an increase in neuronal survival by NeuN staining in the striatum and hippocampus. All data are presented as mean ± standard error of the mean (*P< 0.05, **p< 0.01, ***P<0.001, t test for independent samples).

u. Example 21.

PPF1, HAS1 or control saline were administered to 12-month-old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). HAS1 is a commercially available HAS with greater than 95% human albumin (relative to total protein) in 5% solution (w/v, 50 g/L) obtained by cold alcohol fractionation, and derived from pooled human plasma from donors. Unless otherwise noted, HAS1 is administered in the examples herein in vivo using a 5% solution. Mice were administered intravenously with PPF1, HAS1 or sterile saline at a dose of 150 μl daily for 7 consecutive days and analyzed for behavior 4 weeks after the last dose.

In FIG. 49 shows the Barnes maze exit delay for mouse exit finding for mice treated with PPF1, HAS1, and stock solution. Mice injected with PPF1 found their exit site significantly faster than animals injected with stock solution. These data demonstrate that PPF1 effectively enhances cognitive function in aged animals.

- 40 042179

NSG, while the introduction of HAS1 has no effect on hippocampal-dependent memory.

All data are presented as mean ± standard error of the mean (*P < 0.05, t-test for independent samples).

v. Example 22.

Clinical paradigms using PPF.

(1) Asthma, mild to moderate.

Men and women aged 60 years and older with mild to moderate AD are randomly assigned to receive 100 ml or 250 ml of PPF1 once a day for 5 days (intermittent use) for week 1 and week 13 of the study with a total lasting 6 months. During two 5-day dosing periods, subjects are inpatient and observed to facilitate safety assessment, and all subjects undergo a screening visit, a baseline visit, during treatment visits, follow-up visits, and an end-of-study visit. early termination of participation in the study. Safety and tolerability assessment is carried out at each visit. Neurocognitive and motor functions are assessed during the initial visit and during periodic interim visits after dosing.

The primary endpoints are the safety, tolerability, and feasibility of each application regimen. Safety is determined by the frequency of adverse events that occur during treatment. Tolerability is determined by the number of subjects who completed an 8-week course with at least 5 infusions and subjects who completed a 24-week course with at least 10 infusions. Study feasibility is determined by the number of subjects who completed a course of 5 and 10 infusions. Secondary endpoints define potential effects on cognitive function using various established cognitive measures, including the Alzheimer's Disease Severity Scale Cognitive Subscale. Research endpoints include changes in the composition and distribution of blood biomarkers, and changes in magnetic resonance imaging.

(2) Asthma, mild to moderate.

Two groups of subjects diagnosed with mild to moderate AD are randomized to active treatment in a double-blind manner. All subjects receive one infusion per day of the randomized dose for 5 consecutive days at weeks 1 and 13 for a study duration of 6 months. Subjects are randomized to one of the following two dose levels: 100 ml and 250 ml PPF1. Dosing groups are also separated by gender. The duration of administration is 2-2.5 hours, and the titration rate is chosen according to the dose-specific guidelines so that the entire dose is administered.

Subjects are participating in an additional CSF (cerebrospinal fluid) biomarker study. Such subjects undergo two lumbar punctures to collect CSF, the first before the initial dose and the second after the last dose. Neurocognitive and motor functions are assessed during the initial visit, and assessment at periodic intermediate visits is carried out after dosing.

Determine the safety, tolerability and feasibility of each application regimen. During the study, cognitive assessments are determined and summarized, including the following: Mini-Mental Status Assessment (MMSE); Alzheimer's Disease Severity Assessment Scale 11 Cognitive Subscale (ADAS-Cog/11); test with a board with grooves for inserting pins; categorization speed test (CFT); dementia clinical rating scale - sum of checkboxes (CDR-SOB); Alzheimer's Cooperative Study - Activity of Daily Living (ADCS-ADL); Alzheimer's Cooperative Study - Assessment of Change in Global Clinical Impression Scale (ADCS-CGIC); neuropsychiatric status questionnaire (NPI-Q) and Savonix neurocognitive tests, and repetition of numbers in forward and reverse order.

(3) Parkinson's disease.

Subjects with Parkinson's disease and cognitive impairment are randomized into two groups: 2 periods of active treatment and placebo. Subjects receive one infusion per day of active treatment or placebo for 5 consecutive days (intermittent dosing) during the first week of the study. During week 13, both groups receive active treatment for 5 consecutive days, and the duration of the study is about 7 months. The duration of administration is 2-2.5 hours, and the titration rate is chosen according to the dose-specific guidelines so that the entire dose is administered.

Determine the safety, tolerability and feasibility of each application regimen. During the study determine the cognitive and motor functions, including MoCA; duration and degree of attention, short-term memory and episodic memory in CDR-CCB; MDS-UPDRS3; MDSUPDRS2; SE-ADL and CISI-PD.

The invention is not limited to the specific embodiments described, as these may vary. It should also be understood that the terminology used herein is used only to describe specific aspects and is not limiting, since the scope of this invention is limited only by the appended claims.

When a range of values is given, it is to be understood that this invention covers every intermediate value, to the tenth decimal place of the lower limit, unless the context explicitly states otherwise, between the upper and lower limits of that range, as well as any other specified or intermediate value. value in that specified range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also within the scope of the invention, apart from any specifically excluded limit within the specified range. If the specified range includes one or both of the limits, ranges excluding one or both of these included limits are also included in this invention.

Unless otherwise indicated, all technical and scientific terms used herein have the generally accepted meaning understood by any person skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, typical illustrative methods and materials will now be described.

All publications and patents cited in this specification are incorporated herein by reference as if it were indicated that each individual publication or patent was expressly and individually incorporated by reference into this document for the purpose of disclosing and describing methods and/or materials, in links with which publications are cited. The citation of any publication is provided in relation to its description prior to the filing date and should not be construed as an admission that the present invention is not entitled to predate such publication due to the operation of the earlier invention. Moreover, the dates of the submitted publications may differ from the actual dates of the publications, which requires independent confirmation.

Attention should be drawn to the fact that the use of the singular in this specification and in the appended claims includes references to the plural, unless the context clearly indicates otherwise. It should also be noted that the claims may be amended to remove any optional elements. Therefore, this statement should serve as a preliminary basis for the use of such exhaustive terminology as exclusively, only, etc. in connection with the indication of the elements of the claims, or the use of a negative feature.

As will be appreciated by those skilled in the art upon reading this description, each of the individual aspects of the invention described and illustrated herein has separate components and features that can be easily separated from or combined with features of any of the other several aspects of the invention without departing from scope or essence of the present invention. Any specified method can be carried out using the specified sequence of actions or using any other logically possible sequence.

Although the foregoing invention has been illustrated and described in some detail by means of illustrations and examples for the sake of clarity of understanding, it should be apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims. .

Accordingly, the foregoing merely illustrates the principles of the invention. It should be understood that those skilled in the art will be able to develop various devices which, although not expressly described or shown herein, embody the principles of the present invention and are included within its spirit and scope. In addition, all examples and conventions given herein are primarily intended to help the reader understand the principles of the invention and the concepts introduced by the inventors in the development of the prior art, and should be considered as not limiting such specifically listed examples and conditions. Moreover, all statements made herein that set forth principles, aspects, and aspects of the invention, as well as specific examples thereof, are intended to cover both structural and functional equivalents.

Further, such equivalents are intended to include both currently known equivalents and future developed equivalents, that is, any elements developed that perform the same function, regardless of design. For this reason, the scope of the present invention is not intended to be limited to the exemplary aspects of the invention shown and described herein. Rather, the scope and spirit of this invention is embodied in the appended claims.

Claims (11)

1. A method of treating a subject diagnosed with a cognitive impairment, comprising administering an effective amount of a plasma protein fraction (PPF) using an intermittent application regimen, wherein the PPF contains 83 to 95% albumin and the intermittent application regimen comprises administering the PPF for at least five consecutive days . 2. The method of claim 1, wherein the PPF is a commercially available PPF. 3. The method according to claim 1, wherein the PPF is a protein-rich plasma protein preparation. - 42 042179 4. A method according to any one of the preceding claims, characterized in that the PPF is obtained from plasma from a group of young individuals. 5. A method according to any one of the preceding claims, wherein the PPF is obtained from a mammalian blood product. 6. The method according to claim 5, wherein the mammalian blood product is a human blood product. 7. A method according to any one of the preceding claims, further comprising monitoring the subject for improvement in cognitive function. 8. A method according to any one of the preceding claims, wherein the subject is a mammal. 9. The method of claim 8, wherein the mammal is a human. 10. A method according to any one of the preceding claims, wherein the intermittent application regimen comprises administering PPF for at least seven consecutive days. 11. A method according to any one of the preceding claims, wherein the subject follows the exercise regimen after the complete introduction of the intermittent application regimen, wherein the exercise regimen involves exercising at a higher intensity and/or frequency than if the subject had exercised prior to administration.