EA046284B1 - SCHEME FOR THE APPLICATION OF BLOOD PLASMA AND BLOOD PLASMA PREPARATIONS FOR THE TREATMENT OF COGNITIVE AND MOTOR DISORDERS - Google Patents

Description

Cross references to related applications

Pursuant to 35 USC § 119(e), this application claims filing date priority to: US Provisional Patent Application No. 62/701411, filed July 20, 2018; US Provisional Patent Application No. 62/751434, filed October 26, 2018, and US Provisional Patent Application No. 62/862364, filed June 17, 2019; the contents of which are incorporated herein by reference.

This application is also a continuation in part of U.S. Patent Application No. 15/961618, filed April 24, 2018, which, pursuant to 35 U.S.C. § 119(e), claims filing date priority to: U.S. Provisional Patent Application No. 62 /490519 filed April 26, 2017; US Provisional Patent Application No. 62/584571, filed November 10, 2017; US Provisional Patent Application No. 62/623468, filed January 29, 2018, and US Provisional Patent Application No. 62/641194, filed March 9, 2018; the contents of which are incorporated herein by reference.

Field of technology

This invention relates to the prevention and treatment of age-related diseases. The invention relates to the use of blood products, such as blood plasma fractions, for the treatment and/or prevention of age-related pathological conditions, such as cognitive disorders, movement disorders and neuroinflammatory diseases, using different dosing paradigms.

State of the art

The following is prior art information and is not considered to be prior art for this invention.

Aging is an important risk factor for a variety of human diseases, including cognitive impairment, cancer, arthritis, vision loss, osteoporosis, diabetes, cardiovascular disease and stroke. In addition to the normal loss of synapses during natural aging, synapse loss is an early pathological event common to many neurodegenerative conditions and is the best correlate of neuronal and cognitive impairment associated with these conditions. Thus, aging remains the single dominant risk factor for the development of neurodegenerative diseases associated with dementia, such as Alzheimer's disease (AD) (Bishop, N.A. et al., Neural mechanisms of aging and cognitive decline. Nature 464(7288), 529-535 ( 2010); Heeden, T. et al., Insights into the aging mind: a view from cognitive neuroscience. Nat. Rev. Neurosci. 5(2), 87-96 (2004); Mattson, M. P., et al., Ageing and neuronal vulnerability. Nat. Rev. Neurosci. 7(4), 278-294 (2006). Aging affects all tissues and functions of the body, including the central nervous system, and neurodegeneration and decline in functions such as cognitive or motor skills can have a serious impact on quality of life. Treatments for cognitive decline, movement disorders, and neurodegenerative disorders have limited success in preventing and restoring impaired function. Therefore, it is important to identify new treatments to maintain cognitive integrity by protecting against, counteracting, or reversing the effects of aging. Additionally, when developing new treatment options, it is necessary to explore application paradigms to optimize the effectiveness of such treatment options.

Although parabiosis experiments between old and young mice have demonstrated that cognitive function can be improved in old mice by heterochronic blood exchange with young mice, recent data indicate that there is no enhancement of neurogenesis in old mice solely due to young blood transfusion. (Rebo, J. et al. A single heterochronic blood exchange reveals rapid inhibition of multiple tissues by old blood. Nat. Comm. (2016)). In addition, there are doubts that cognitive functions resulting from young plasma infusions and neurogenesis are related. Thus, until now, no regimen has been described using blood plasma or blood plasma fractions that stimulates neurogenesis and improves cognitive function.

Brief description of the invention

This invention is based on the production and use of blood products for the treatment and/or prevention of age-related disorders such as cognitive impairment, age-related dementia, motor impairment, neuroinflammation and neurodegenerative disease. The present invention recognizes, among other things, the need for new therapeutic options for the treatment and/or prevention of cognitive impairment, age-related dementia, movement disorder, neuroinflammation and neurodegenerative disease. Derived from blood and blood plasma, the compositions of this invention address the inconsistencies and shortcomings of current therapeutic options through the use of blood plasma fractions that demonstrate effectiveness in the treatment and/or prevention of cognitive impairment, age-related dementia, movement disorder, neuroinflammation and neurodegenerative disease.

In this invention, it was determined that blood plasma proteins have an average half-life of 2-3 days. The present invention utilizes a regimen of blood plasma or plasma fraction that optimizes neurogenesis, cell survival, reduces neuroinflammation, and improves cognition or motor function in a subject receiving treatment. It was discovered

- 1 046284 It is clear that the dosage regimen of this invention stimulates all of these processes (neurogenesis, cell survival, improved cognition, reduced neuroinflammation, as well as improved motor function) in subjects, and all of these processes were found to be active even after weeks after the last dose.

An embodiment of the invention includes treating a subject who has been diagnosed with a cognitive impairment by administering to the subject an effective amount of blood plasma or a fraction of plasma. Another embodiment of the invention involves administering an effective amount of blood plasma or a fraction of plasma and then monitoring the subject for improvement in cognitive function. Another embodiment of the invention includes treating a subject who has been diagnosed with a cognitive impairment 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 a manner that results in improved cognitive function or neurogenesis.

An embodiment of the invention includes treating a subject who has been diagnosed with a neurodegenerative movement disorder, such as, by way of example and not limitation, Parkinson's disease, by administering to the subject an effective amount of blood plasma or a fraction of plasma. Another embodiment of the invention involves administering an effective amount of blood plasma or a fraction of plasma and then monitoring the subject for improvement in motor function. Another embodiment of the invention involves treating a subject who has been diagnosed with a neurodegenerative movement disorder 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 a manner that results in improved motor function or neurogenesis.

An embodiment of the invention includes treating a subject who has been diagnosed with neuroinflammation or a disorder associated with neuroinflammation by administering to the subject an effective amount of blood plasma or a fraction of plasma. Another embodiment of the invention involves administering an effective amount of blood plasma or a fraction of plasma and then monitoring the subject for a decrease in neuroinflammation. Another embodiment of the invention includes treating a subject who has been diagnosed with neuroinflammation or a disorder associated with 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 a manner that results in a decrease in neuroinflammation.

Another embodiment of the invention involves administering blood plasma or a fraction of plasma in a regimen of at least two consecutive days. Another embodiment of the invention involves administering blood plasma or a fraction of plasma in a regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days (also referred to herein as document, intermittent use regimen, intermittent use, intermittent use, dose for intermittent use). Another embodiment of the invention involves administering blood plasma or a fraction of plasma for 2 consecutive days and after the date of the last administration. Another embodiment of the invention involves administering blood plasma or a fraction of plasma in a regimen of 2-14 non-consecutive days, with each dose interval being 0-3 days. Another embodiment of the invention includes monitoring the subject for improvement in cognitive or motor function, reduction in neuroinflammation, or improvement in neurogenesis at least 3 days after the date of the last administration. Another embodiment of the invention involves monitoring a subject for improvement in cognitive or motor function, reduction in neuroinflammation, or improvement in neurogenesis after the average half-life of proteins in the blood plasma or plasma fraction has been reached.

In some cases, intermittent use in accordance with this invention involves the administration of a first set of doses, for example, as described above, followed by a period of no use, for example, a period of no use, which in turn is followed by the administration of another dose or set of doses. The duration of this non-use period may vary, but in some embodiments is 7 days or longer, for example, 10 days or longer, including 14 days or longer, with in some cases the duration of the non-use period ranging from 15 to 365 days, for example, from 30 to 90 days, including from 30 to 60 days. Thus, embodiments of the methods include non-systematic (ie, intermittent administration), for example, non-systematic administration of a blood plasma preparation. In some embodiments, the regimen of intermittent use followed by a period of no use is repeated a number of times as needed, and in some cases the regimen continues for 1 year or more, such as 2 years or more, including for the entire life of the subject . Another embodiment of the invention involves administering blood plasma or a fraction of plasma according to a regimen of use for 5 consecutive days, with a period of no use equal to 2-3 days, followed by administration for 2-14 consecutive days.

The present invention also contemplates that differences in protein content between different blood plasma fractions (e.g., fractions, eluate, plasma protein fraction, Albu solution)

- 2,046,284 min human) may be responsible for the prevention and/or improvement of certain cognitive or motor impairments and alleviation of neurodegenerative disease. By way of example and not limitation, embodiments of the present invention demonstrate that the higher concentration of albumin alone in recombinant human albumin or human albumin solution (HAS) preparations is not the driving force behind improved cognition, improved motor function, or reduced neuroinflammation. , cell survival or neurogenesis associated with plasma protein fraction (PPF) preparations with lower albumin concentrations.

Blood and plasma from young donors have demonstrated improvement and reversal of pre-existing effects of brain aging, including at the molecular, structural, functional and cognitive levels. (Saul A. Villeda, et al. Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nature Medicine 20 659-663 (2014)). This invention relates to blood plasma fractions and eluates, some of which are traditionally used to treat a patient in shock, and to the discovery that they are effective as methods for treating age-related cognitive impairment, motor decline and neuroinflammation or diseases associated with neurodegeneration.

In accordance with aspects of the present invention, methods are provided for treating age-related cognitive impairment, age-related dementia, movement disorders, neuroinflammation and/or neurodegenerative diseases using blood plasma fractions of a blood product. Aspects of the methods include administering a blood plasma fraction to a person suffering from or at risk of developing age-related cognitive impairment, movement disorder, neuroinflammation, or neurodegenerative disease. Additional aspects of the methods include administering a fraction of blood plasma obtained from a group of donors of a certain age range to a person suffering from or at risk of developing age-related cognitive impairment, movement impairment, neuroinflammation or neurodegenerative disease. Other aspects of the methods include administering blood plasma or plasma fractions using an intermittent dosing regimen. Also provided are reagents, devices and kits that are used in carrying out the methods of this invention.

In one embodiment, the blood plasma fraction may be, for example, one of several blood plasma fractions obtained in a blood fractionation process, such as the Cohn fractionation process described below. In another embodiment, the blood plasma fraction may be of a type, herein referred to as a plasma fraction, which is a solution consisting of normal human albumin, alpha and beta globulins, gamma globulin and other proteins, either individually or as complexes. In another embodiment, the blood plasma fraction may be a type of blood plasma fraction known to one skilled in the art as a plasma protein fraction (PPF). In another embodiment, the blood plasma fraction may be a human albumin solution (HAS) fraction. In yet another embodiment, the blood plasma fraction may be one in which substantially all of the clotting factors have been removed in order to maintain the effectiveness of the reduced risk of thrombosis fraction. Embodiments of the present invention may also include administering, for example, a fraction obtained from a young donor or group of young donors. Another embodiment of the invention may include monitoring improvement in cognitive performance, improvement in motor function, reduction in neuroinflammation, or increase in neurogenesis in a subject being treated with a blood plasma fraction.

An embodiment of the invention includes treating a subject who has been diagnosed with a cognitive impairment, a neurodegenerative movement disorder, or a disease associated with neuroinflammation by administering to the subject an effective amount of blood plasma or a plasma fraction. Another embodiment of the invention involves administering an effective amount of blood plasma or a fraction of plasma and then monitoring the subject for improved cognitive function, improved motor function, decreased neuroinflammation, or increased neurogenesis. Another embodiment of the invention involves administering blood plasma or a fraction of plasma on a dosing regimen for at least two consecutive days and monitoring the subject for improved cognitive function, improved motor function, decreased neuroinflammation, or increased neurogenesis for at least 2 days after the last date. introduction. Another embodiment of the invention includes administering blood plasma or a fraction of plasma in a regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days and monitoring the subject for improvement in cognitive performance. function, improved motor function, decreased neuroinflammation, or increased neurogenesis for at least 3 days after the date of last administration. Another embodiment of the invention includes administering blood plasma or a fraction of plasma on a dosing schedule for 2 consecutive days and after the date of last administration, monitoring for improvement in cognitive function, improvement in motor function, decrease in neuroinflammation, or increase in neurogenesis after the midpoint has been reached. semi

- 3 046284 life of proteins in blood plasma or plasma fractions.

An embodiment of the invention includes treating a subject who has been diagnosed with cognitive impairment, motor impairment, neuroinflammation, or decreased neurogenesis by administering to the subject an effective amount of blood plasma or a plasma fraction with a post-administration exercise regimen. Another embodiment of the present invention includes a further exercise regimen that is assigned to a subject. Another embodiment of the present invention involves the subject exercising at a higher intensity and/or frequency than if the subject had exercised prior to administration. Another embodiment of the present invention involves the subject exercising at a similar intensity and/or frequency as the subject exercised prior to administration.

An embodiment of the invention includes treating a subject who has been diagnosed with cognitive impairment, motor impairment, neuroinflammation, or decreased neurogenesis by administering to the subject an effective amount of blood plasma, or a plasma fraction in the case of a subject who is undergoing, will be undergoing, or has undergone stem cell therapy. Another embodiment of the invention involves administering to a subject an effective amount of blood plasma or a fraction of plasma, wherein the subject is undergoing, will undergo, or has undergone stem cell therapy, and wherein the stem cells used in the therapy may be embryonic stem cells, other than induced embryonic stem cells. pluripotent stem cells (iPSCs), umbilical cord blood stem cells, amniotic fluid stem cells and the like. Another embodiment of the invention includes treating, with an effective amount of blood plasma or a fraction of plasma, a subject who has been diagnosed with traumatic spinal cord injury, stroke, retinal diseases, Huntington's disease, Parkinson's disease, Alzheimer's disease, hearing loss, heart disease, rheumatoid arthritis, or severe burns. , and the subject is undergoing, will be undergoing, or has undergone stem cell therapy.

Incorporation by reference

All publications and patent applications referenced herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually identified as being incorporated by reference.

Brief description of graphic materials

In fig. 1A depicts the distance traveled in the open field test for mice administered PPF1 using an intermittent and 3 x/week dosing schedule.

In fig. 1B depicts the time spent in the center of the open field for mice administered PPF1 using the intermittent and 3/week dosing schedules.

In fig. 2 depicts the body weight over time of mice administered PPF1 using an intermittent dosing schedule and a 3x/week dosing schedule.

In fig. Figure 3 shows the number of DCX-labeled cells in the granular layer of the dentate gyrus in mice treated with PPF1 using the intermittent and 3/week schedules.

In fig. Figure 4 shows the number of NOS-labeled cells in the granular layer of the dentate gyrus in mice treated with PPF1 using the intermittent and 3/week schedules.

In fig. Figure 5 shows the number of DCX-labeled cells in the granular layer of the dentate gyrus in mice treated with PPF1 using an intermittent dosing regimen and a 3/wk regimen of young adult plasma (YP) or old human plasma (ASP).

In fig. Figure 6 shows the number of NOS-labeled cells in the granular layer of the dentate gyrus in groups of mice that were administered PPF1 using the intermittent and 3x/week MP or SP regimens.

In fig. Figure 7 shows the latency to find the target hole per trial per day for mice treated with PPF1 or MP using an intermittent administration schedule.

In fig. Figure 8 shows the number of DCX-labeled cells in the granular layer of the dentate gyrus in groups of mice treated with young plasma (YP), old plasma (SP), or PPF1 using an intermittent dosing regimen.

In fig. Figure 9 shows the number of NOS-labeled cells in the granular layer of the dentate gyrus in groups of mice treated with young plasma (YP), old plasma (SP), or PPF1 using an intermittent dosing regimen.

In fig. Figure 10 shows the percentage of the total number of entries made into any familiar or novel arm of the total number of entries made into each arm by the treatment group in the Y-maze trial. Mice were intermittently dosed with PPF1 or 5x concentrated PPF1 at twelve months of age.

In fig. Figure 11 shows the ratio of the number of entries into the new arm compared to the familiar arm in the Y-maze test. Mice were intermittently dosed with PPF1 or 5x concentrated PPF1 at twelve months of age.

In fig. Figure 12 shows the number of NOS-labeled cells in a section of the hippocampus of a twelve-month-old

- 4,046,284 mice treated with PPF1 or 5x concentrated PPF1 using an intermittent dosing regimen.

In fig. 13 shows the number of DCX-labeled cells in a hippocampal slice from twelve-month-old mice treated with PPF1 or 5x concentrated PPF1 using an intermittent dosing regimen.

In fig. 14 shows the number of DCX-labeled cells in the granular layer of the dentate gyrus in 10.5-month-old NSG mice treated with PPF1 or saline using an intermittent dosing regimen using one of the following: (1) 5 consecutive days [PPF1-5d ] (2) 7 consecutive days [PPF1-7d] (3) 5 consecutive days with an additional 5 consecutive days of (booster) dosing 6 weeks after completion of the initial dosing [PPF1-5d-B] or (4) 7 consecutive days with additional 7 consecutive days of (booster) dosing at week 6 after completion of initial dosing [PPF1-7d-B].

In fig. Figure 15 shows the number of NOS-labeled cells in the granular layer of the dentate gyrus in 10.5-month-old NSG mice treated with PPF1 or saline using an intermittent dosing regimen using one of the following regimens: (1) 5 consecutive days [PPF1-5d ] (2) 7 consecutive days [PPF1-7d] (3) 5 consecutive days with an additional 5 consecutive days of (booster) dosing 6 weeks after completion of the initial dosing [PPF1-5d-B] or (4) 7 consecutive days with additional 7 consecutive days of (booster) dosing at week 6 after completion of initial dosing [PPF1-7d-B].

In fig. Figure 16 shows the number of EDU-labeled cells in the granular layer of the dentate gyrus in 10.5-month-old NSG mice treated with PPF1 or saline using an intermittent dosing regimen using one of the following regimens: (1) 5 consecutive days [PPF1-5d ] (2) 7 consecutive days [PPF1-7d] (3) 5 consecutive days with an additional 5 consecutive days of (booster) dosing 6 weeks after completion of the initial dosing [PPF1-5d-B] or (4) 7 consecutive days with additional 7 consecutive days of (booster) dosing at week 6 after completion of initial dosing [PPF1-7d-B].

In fig. Figure 17 shows the number of DCX-labeled cells in the granular layer of the dentate gyrus in 3- and 6-month-old NSG animals administered PPF1 or saline, with or without activity wheels.

In fig. Figure 18 shows the number of Ki67-positive cells in the granular layer of the dentate gyrus in 3- and 6-month-old NSG animals administered PPF1 or saline, with or without activity wheels.

In fig. Figure 19 shows the number of positively labeled NOS cells in the granular layer of the dentate gyrus in 3- and 6-month-old NSG animals administered PPF1 or saline, with or without activity wheels.

In fig. 20 shows the number of wheel rotations over predetermined periods of time for 11 month old NSG mice administered PPF1 or saline using an intermittent dosing regimen. Shaded areas indicate the dark cycle, and outlined areas indicate the use of the hot plate test.

In fig. 21A shows the number of NOS-labeled cells in the granular layer of the dentate gyrus in three treatment groups of 10.5-month-old NSG mice treated with young plasma, recombinant human albumin (rhAlbumin), and saline control.

In fig. 21B shows the number of DCX-labeled cells in the granular layer of the dentate gyrus in three treatment groups of 10.5-month-old NSG mice treated with young plasma, recombinant human albumin (rhAlbumin), and saline control.

In fig. 22A-22C show the degree of increase in neural spiking activity (FIGS. 22A and 22B) and neural network activity (FIGS. 22A and 22C) on days 7, 12, 16 and 21 of treatment in MEA wells containing dissociated mixed neuronal cells derived from cerebral cortex of E16 mice treated with control, PPF1 (two different batches), HAS1, or rhAlbumin. In fig. 22A depicts representative spike trains from two MEA wells for each treatment condition, demonstrating a highly synchronous firing pattern in cells treated with PPF1 compared to those treated with control or HAS 1. FIG. 22B depicts the results of quantification of neural spiking activity. In fig. 22C shows bursts in the neural network of cultures of primary cortical neurons of mice maintained in the presence of control, rhAlbumin, PPF1 (two batches) and HAS1 for 21 days. N=25-45 wells from 3 independent experiments ± standard error of the mean, Student's t-test for independent samples *p<0.05, #p<0.01, calculated relative to the base solution treatment at each time point.

In fig. 23 presents four paradigms of aged human clarified plasma (aged plasma) or saline administered to 8-week-old (young) NSG mice.

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In fig. 24A shows VCAM-1-positive labeling in the hippocampus of 8-week-old NSG mice treated with aged plasma twice weekly, 48 hours after the last dose.

In fig. 24B shows VCAM-1-positive labeling in the hippocampus of 8-week-old NSG mice treated with old plasma three times a week, 48 hours after the last dose.

In fig. 24C shows VCAM-1-positive labeling in the hippocampus of 8-week-old NSG mice treated with old plasma using an intermittent dosing regimen, 48 hours after the last dose.

In fig. 24D shows VCAM-1-positive labeling in the hippocampus of 8-week-old NSG mice treated with aged plasma using an intermittent dosing regimen, 21 days after the last dose.

In fig. 25A shows the number of DCX-positive cells in the dentate gyrus of 8-week-old NSG mice treated with aged plasma twice weekly, 48 hours after the last dose.

In fig. 25B shows the number of DCX-positive cells in the dentate gyrus of 8-week-old NSG mice treated with old plasma three times a week, 48 hours after the last dose.

In fig. Figure 25C shows the number of DCX-positive cells in the dentate gyrus of 8-week-old NSG mice treated with old plasma using an intermittent dosing regimen, 21 days after the last dose.

In fig. Figure 26 shows the time course of latency to exit the Barnes maze and indicates the time required to reach and enter the exit hole for 8-week-old (young) NSG mice injected with aged plasma and saline. Mice were treated with old human plasma or saline for 7 consecutive days and tested 4 weeks after the last injection.

In fig. 27 shows the average exit latency over the last three Barnes maze trials on day 4 of testing in 8-week-old (young) NSG mice treated with old human plasma or saline for 7 consecutive days. Testing was carried out 4 weeks after the last injection.

In fig. Figure 28 shows the difference in exit latency between Trials 1 and 3 in the Barnes maze for 8-week-old (young) NSG mice treated with aged plasma or saline for 7 consecutive days. Testing was carried out 4 weeks after the last injection.

In fig. 29 presents the results of quantitative polymerase chain reaction (qPCR), quantitative assessment of the levels of mRNA DCX, vesicular glutamate receptor (vglut1), synapsin 1 (syn1), beta-III tubulin (tuj1), and brain-derived neurotrophic factor (BDNF) in 8-week-old ( young) NSG mice treated with old human plasma or saline for 7 consecutive days.

In fig. 30 depicts a dosing paradigm for 8-week-old (young) NSG mice treated with 35 mg/kg kainic acid or saline, followed by either PPF1 or saline daily for 5 consecutive days.

In fig. 31A shows the percentage of CD68-positive area in the CA1 region of the hippocampus of mice administered in accordance with the paradigm depicted in FIG. 28.

In fig. 31B shows the percentage of GFAP-positive area in the CA1 region of the hippocampus of mice administered in accordance with the paradigm depicted in FIG. 28.

In fig. 32 shows the number of cells stained for NOS in the dentate gyrus of 6-month-old NSG mice treated with PPF1 or saline control using an intermittent dosing regimen for 7 consecutive days with simultaneous administration of NOS. The first two columns represent the cohort analyzed 7 days after the last administration of PPFI/saline control and NOS; the second two columns constitute the cohort analyzed 14 days after the last administration of PPFI/saline control and NOS.

In fig. 33 depicts an increase in the number of proliferating cells (Ki67+) in the dentate gyrus of 6-month-old NSG mice 10 days after completion of PPF1 administration using an intermittent dosing regimen.

In fig. Figure 34 shows sections of the dentate gyrus and subventricular zone in 6-month-old NSG mice 10 days after completion of PPF1 administration using an intermittent dosing regimen.

In fig. 35A illustrates the direction of cell development in the dentate gyrus of 6-month-old NSG mice administered either PPF1 or saline control on a 7-day intermittent regimen in which BDU was administered for 5 consecutive days immediately prior to the start of the intermittent regimen. The degree of colocalization of NeuN+ with NOS staining indicates the extent to which neuronal progenitor cells have become neurons. Degree of colocalization of GFAP+ with

- 6 046284 NOS staining indicates the extent to which neuronal progenitor cells have become astrocytes.

In fig. 35B shows the results of a similar experiment as in FIG. 35A, but in 12 month old NSG mice.

In fig. 36A illustrates the direction of cell development in the dentate gyrus of 3-month-old NSG mice treated with either aged plasma or saline control on a 7-day intermittent regimen in which BDU was administered for 5 consecutive days immediately prior to the start of the intermittent regimen. The degree of colocalization of NeuN+ with NOS staining indicates the extent to which neuronal progenitor cells have become neurons.

In fig. 36B shows the results of the experiment detailed in FIG. 36A, but indicating the degree of colocalization of GFAP+ with NOS staining, indicating the degree to which neuronal progenitor cells have become astrocytes.

In fig. 37A-37C show the number of cFos-positive cells in (A) the whole brain, (B) the cerebral cortex, and (C) the isocortex of 18-month-old mice treated with PPF1 or saline using a 7-day intermittent dosing regimen.

In fig. 38A-38D show the number of cFos-positive cells in (A) frontal cortex, (B) orbital cortex, (C) infralimbal cortex, and (D) prelimbal cortex of 18-month-old mice treated with PPF1 or saline using an intermittent dosing regimen. within 7 days.

In fig. 39A and 39B show the number of cFos-positive cells in (A) the accessory olfactory nucleus and (B) the olfactory tubercle of 18-month-old mice administered PPF1 or saline using a 7-day intermittent dosing regimen.

In fig. 40 depicts voxel-based imaging statistics of cortical local activation in the frontal cortex (FRP), orbital cortex (ORB), infralimbal cortex (ILA), prelimbal cortex (PL), and adnexal olfactory nucleus (AON) in 18-month-old PPF1-injected mice or saline solution using an intermittent dosing regimen for 7 days.

In fig. 41A shows the percentage of CD68 immunoreactive region in the hippocampus of 22-month-old C57BL/6J wild-type mice 10 days after PPF1 or saline administration using a 7-day intermittent dosing regimen.

In fig. 41B shows the percentage of Iba-1 immunoreactive area in the hippocampus of 22-month-old C57BL/6J wild-type mice 10 days after PPF1 or saline administration using a 7-day intermittent dosing regimen.

In fig. 41C shows the percentage of GFAP immunoreactive area in the hippocampus of 22-month-old C57BL/6J wild-type mice 10 days after PPF1 or saline administration using a 7-day intermittent dosing regimen.

In fig. 41D shows the percentage of CD68 immunoreactive region in the hippocampus of 21-month-old C57BL/6J wild-type mice 4 weeks after PPF1 or saline administration using a 7-day intermittent dosing regimen.

In fig. 41E shows the percentage of Iba-1 immunoreactive area in the hippocampus of 21-month-old C57BL/6J wild-type mice 4 weeks after PPF1 or saline administration using a 7-day intermittent dosing regimen.

In fig. 42A shows the percentage change in NOS staining in 23-month-old wild-type C57BL/6J mice treated with PPF1 compared to saline control at weeks 6, 9, and 12 post-administration using an intermittent dosing regimen for seven consecutive days.

In fig. 42B shows the percentage change in DCX staining in 23-month-old wild-type C57BL/6J mice treated with PPF1 compared to saline control at weeks 6, 9, and 12 post-administration using an intermittent dosing regimen for seven consecutive days.

In fig. 43A and 43B show body weight measurements of 4-4.5 month old male alpha-synuclein mice (line 61) (Parkinson's disease model) administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days.

In fig. 44 shows the results of nest building in 4-4.5 month old male alpha-synuclein mice (line 61) (a model of Parkinson's disease) that were administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days.

In fig. 45A and 45B indicate the results of noodle chewing and associated improvement in motor function in 4-4.5 month old male alpha-synuclein mice (line 61) (Parkinson's disease model) administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days.

In fig. 46 shows the results of wire hanging tests in 4-4.5 month old male alpha-synuclein mice (line 61) (Parkinson's disease model) administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days.

In fig. 47A depicts various pole shapes and sizes used in five different pole walking tests of increasing difficulty.

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In fig. 47B shows the results of five different pole walking tests in 4-4.5 month old male alpha-synuclein mice (line 61) (Parkinson's disease model) administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days. Pole walking tests were performed 72 hours after the last administered dose.

In fig. 47C shows the results of five different pole walking tests in 4-4.5 month old male alpha-synuclein mice (line 61) (Parkinson's disease model) administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days. Pole walking tests were performed 3 weeks after the last dose administered.

In fig. 48A-48F indicate histological staining results of the striatum and hippocampus in 44.5-month-old male alpha-synuclein mice (line 61) (Parkinson's disease model) administered PPF1 or control solution using an intermittent dosing regimen for seven consecutive days. Histological markers examined include CD68, Iba-1 and Neun.

In fig. 49 shows exit latency in the Barnes maze in 12-month-old NSG mice treated with PPF1, HAS1, or control using an intermittent dosing regimen for seven consecutive days.

In fig. 50 and 51 show that 24-month-old mice (24M) showed a decrease in the level of the postsynaptic marker PSD-95 compared to young 3-month-old mice (3M) in brain regions containing a significant number of synapses (hippocampus (HP), striatum ( ST) and substantia nigra (SN)), but not in areas of the brain that do not contain synapses (corpus callosum (CC)).

In fig. 52A and 52B indicate that mice receiving intermittent dosing of PPF1 had significantly higher levels of the postsynaptic marker PSD-95 (Fig. 52A) and higher levels of the presynaptic marker SYNAPSYN1 (Fig. 52B). T-tests for independent samples, *p<0.05.

In fig. 53 is a histological image of CA1 hippocampal sections associated with FIG. 55.

In fig. 54A and 54B show a high resolution confocal micrograph (FIG. 54A) and an enlarged inset (FIG. 54B) with synapses indicated by yellow arrows consisting of the presynaptic marker SYNAPSYN1 (red) and the postsynaptic marker PSD-95 (white) aligned with each other friend.

In fig. 55A-55D show that mice treated with intermittent dosing of PPF1 had significantly greater amounts of colocalized pre- and postsynaptic markers that were visible in high-resolution micrographs (FIG. 55A) and quantified (FIG. 55B). The number of SYNAPSIN1 puncta was also greater in mice treated with intermittent dosing of PPF1 compared to controls (Figure 55C). The number of postsynaptic points remained unchanged among treatment groups (Fig. 55D). G-tests for independent samples, *p<0.05.

In fig. 56A depicts primary mouse cortical neurons cultured in the presence of recombinant human albumin (rAlbumin), PPF1, or HAS1 for 14 days, with cellular composition and morphology assessed by immunostaining for Map2 (neurons) and Gfap (astrocytes) and nestin (cells). predecessors).

In fig. 56B depicts primary mouse hippocampal neurons cultured in the presence of recombinant human albumin (rAlbumin), PPF1, or HAS1 for 14 days, with cellular composition and morphology assessed by immunostaining for Map2 (neurons) and Gfap (astrocytes) and Nestin (cells). predecessors).

In fig. 57A shows increased Gfap protein expression relative to betaactin expression in cortical neuron cultures treated with PPF1 or HAS1 for 14 days compared to control or recombinant human albumin treated cultures.

In fig. 57B shows that treatment with PPF1 or HAS1 for 14 days leads to an increase in the population of Sox2-positive cells compared to control treatment. n=3 independent experiments ± standard error of the mean, comparison by paired Student's t-tests, *p<0.05.

In fig. 58A shows exit latency in a 2-choice swim test (T-maze) in 12-month-old NSG mice treated with intermittent administration of PPF1 or saline control 6 weeks prior to testing. Mice treated with PPF1 showed a trend towards shorter exit latency. Saline solution: n=15. PPF1: n=10. All data are presented as mean ± standard error of the mean. Two-way ANOVA.

In fig. 58B depicts correct and incorrect choices in a 2-choice swim test (T-maze) in 12-month-old NSG mice that received intermittent administration of PPF1 or saline control 6 weeks prior to testing. PPF1-treated mice exhibited significantly higher correct choice rates. Saline: n=15 mice. PPF1: n=10. All data are presented as mean ± standard error of the mean. Chi-square test, *P<0.05.

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In fig. Figure 59 shows a representative confocal image of the dentate gyrus in a 12-month-old NSG mouse triple-labeled with BrdU, NeuN, and GFAP. The arrow indicates a cell labeled with BrdU.

In fig. 60A depicts the total number of BrdU/NeuN or BrdU/GFAP double positive cells in the dentate gyrus of mice treated with saline, MP, PPF1 or SP. In mice treated with PPF1, significantly more BrdU/NeuN double-positive cells were observed in the dentate gyrus. Saline: n=13, MP: n=13, PPF1: n=15, SP: n=14. All data are presented as mean ± standard error of the mean. One-way ANOVA, Tukey's post hoc test. **P<0.01.

In fig. 60B depicts the % of BrdU/NeuN or BrdU/GFAP double-positive cells among all BrdU-positive cells in the dentate gyrus of mice treated with saline, MP, PPF1, or SP. There was a trend toward enhancement toward the neuronal lineage after treatment with PPF1. Saline: n=13, MP: n=13, PPF1: n=15, SP: n=14. All data are presented as mean ± standard error of the mean. One-way ANOVA, Tukey's post hoc test.

In fig. 61A shows exit latency in the Barnes maze in 12-month-old NSG mice receiving intermittent administration of saline control, young plasma (MP), or PPF1 6 weeks prior to testing. Mice treated with PPF1 exhibited significantly shortened exit latency compared with animals treated with saline or MP. Saline: n=13, MP=14, PPF1: n=13. All data are presented as mean ± standard error of the mean. Two-way ANOVA. *P<0.05.

In fig. 61B shows the average exit latency in trials 14 and 15 of the Barnes maze test in 12-month-old NSG mice receiving intermittent administration of saline control, young plasma (MP), or PPF1 6 weeks prior to testing. PPF1-treated animals tended to have shorter exit latency compared to saline- or MP-treated mice. Saline: n=13, MP=14, PPF1: n=13. All data are presented as mean ± standard error of the mean. One-way ANOVA, Tukey's post hoc test.

In fig. 62A shows the number of doublecortin (DCS)-positive cells in the dentate gyrus of 4.5-month-olds (saline: n=8, PPF1: n=8), 7.5-month-olds (saline: n=8, PPF5: n=6) and 12 month old (saline: n=15, PPF5: n=14) NSG mice. There was an age-related decrease in the number of DCX-positive cells, which was significantly restored in 7.5- and 12-month-old mice. All data are presented as mean ± standard error of the mean. T-test for independent samples. *P<0.05, ****P<0.0001.

In fig. 62B shows the percentage of doublecortin (DCX)-positive cells compared to the number of DCX-positive cells at the beginning of treatment. Treatment with PPF1 maintains the level of neurogenesis at the same level as during treatment, restoring the age-related decline in DCX numbers. 3 months: n=8, 6 months: n=7, 10.5 months: n=19, 4.5 months (saline: n=8, PPF1: n=8), 7.5- monthly (saline: n=8, PPF5: n=6), 12 months (saline: n=15, PPF5: n=14). All data are presented as mean ± standard error of the mean. T-test for independent samples. *P<0.05, ****P<0.0001.

In fig. 63 represents the % threshold CD68-positive area in the hippocampus of 12-month-old NSG mice treated with saline or PPF1. There was a significant decrease in CD68 immunoreactivity 24 hours after completion of PPF1 treatment. Saline: n=10, PPF1: n=10. T-test for independent samples. *P<0.05.

In fig. 64A shows representative images of CD68 immunoreactivity in saline- or PPF1-treated mice.

In fig. 64B shows representative images of Iba-1 immunoreactivity in saline- or PPF1-treated mice.

In fig. 65A and 65B depict two intermittent booster regimens in NSG mice starting at either 6 months of age (FIG. 65A) or 3 months of age (FIG. 65B). Mice initially treated at 6 months of age received 2 doses of boosters for seven consecutive days, 8 weeks apart. Mice initially treated at 3 months of age received 3 doses of boosters over seven consecutive days at 8, 8, and 7 week intervals. For both groups, behavioral testing was performed 6 weeks after the last intermittent treatment regimen, when all mice were 12 months old.

In fig. 66 depicts the total number of DCX-positive cells in the hippocampus counted in five middle hippocampal slices from 12-month-old NSG mice treated at 3 months of age with: basal solution without exercise; intermittent doses of PPF1 for 7 days; intermittent doses of PPF1 for 7 days, then two subsequent booster regimens with an interval of 8 weeks between regimens; and a baseline exercise solution for the remainder of the study. All data are mean ± SEM, ****P<.0001, ***P<.0001, ANOVA with Dunnett's post hoc test.

In fig. 67 depicts the total number of DCX-positive cells in the hippocampus counted in five middle hippocampal slices from 12-month-old NSG mice treated at 3 months of age: baseline

- 9 046284 solution without exercises; intermittent doses of PPF1 for 7 days; intermittent doses of PPF1 for 7 days, then three subsequent booster regimens with an interval of 8, 8, and 7 weeks between regimens, respectively; and a baseline exercise solution for the remainder of the study.

In fig. 68 depicts Y-maze behavior characteristics of 12-month-old NSG mice compared to mice from the original 6-month cohort (see FIG. 65A and FIG. 66). The percentage of total number of entries into the new arm of the Y-maze in mice receiving PPF1 and PPF1 plus boosters tended to improve spatial learning and memory compared to controls.

In fig. 69A and 69B show that treatment with PPF1 (two different batches) or HAS1 for 96 hours resulted in a significant increase in neurite outgrowth in cortical (FIG. 69A) or hippocampal neurons (FIG. 69B) compared to cells treated with rhAlbumin or control. N=11-16 independent experiments ± standard error of the mean, one-way ANOVA *p<0.05, **p<0.01.

In fig. 70A and 70B depict increased expression of synaptic markers relative to the general neuronal marker Tuj1 at mRNA levels (qPCR) for SYN1 and PSD-95 (FIG. 70A) and protein levels (as determined by Western blotting, FIG. 70B) for Syn1 in primary cortical neurons mice cultured in the presence of PPF1 (two batches) or HAS1 for 14 days, compared with control-treated cells.

In fig. 71 reported that people with mild to moderate Alzheimer's disease who received either 100 ml or 250 ml of PPF1 for 5 consecutive days did not experience significant deterioration in cognitive or functional scores as measured by the ADAS-Cog, ADCS- ADL and CDR-SB over a six-month period.

Detailed Description of the Invention

1. Introduction.

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 decline in motor function, and/or neurodegenerative disease. Described herein are methods and compositions for treating subjects suffering from such disorders, which are aspects of the present invention. In addition, this document describes 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: the prevention of cognitive or motor impairment, age-related dementia, neuroinflammation and/or neurodegenerative disease; reducing symptoms of cognitive or motor impairment, age-related dementia, neuroinflammation and/or neurodegenerative disease; slowing the progression of age-related cognitive or motor impairment, 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 treatments, for example, one or more fractions or eluates obtained from blood fractionation processes, for example, 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 individually or in complexes, hereinafter referred to as the plasma fraction). Another embodiment of the present invention involves the use of plasma protein fraction (PPF) as a treatment. Another embodiment of the present invention involves the use of 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. An additional embodiment includes a fraction of blood plasma from which substantially all clotting factors have been removed so as to maintain efficacy while reducing the risk of thrombosis (for example, see US Patent Applications Nos. 62/236,710 and 63/376,529, which are included by reference to this description in its entirety).

Before describing the present invention in detail, it should be appreciated that this invention is not limited to the specific 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 intended to be limiting, as the scope of the present invention is limited only by the appended claims.

The publications discussed herein are presented solely with respect to their contents prior to the filing date of this application. Nothing herein should be interpreted as an admission that this invention is not entitled to postdate such publication by virtue of its earlier invention. Moreover, publication dates presented may differ from actual publication dates and require independent confirmation.

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When a range of values is given, it should be understood that each intermediate value, to the tenth decimal place of the lower limit, is also described, unless the context clearly requires otherwise, between the upper and lower limits of that range. Every smaller range between any specified value or intermediate value within a specified range and any other specified or intermediate value within that specified range is covered by the present invention. The upper and lower limits of these smaller ranges may be independently 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 in the specified range . If the specified range includes one or both 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 exclude 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 elements of the claims, or the use of a negative feature.

As those skilled in the art will appreciate upon reading this description, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with features of any of the other several embodiments without deviations 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 specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although methods and materials similar or equivalent to those described 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 herein are incorporated herein by reference for the purpose of disclosure and description of the manner and/or materials in connection with which such publications are cited. It should be understood that this invention supersedes any disclosure in the publication included herein to the extent of conflict.

It should be noted that the use of singular forms in this document and in the accompanying claims includes reference to the plural unless the context clearly indicates otherwise. Thus, for example, reference to a cell includes a plurality of such cells, and reference to a peptide includes reference to one or more peptides and their equivalents, e.g., polypeptides known to those skilled in the art, etc.

When describing the methods of this invention, the terms host, subject, individual and patient are used interchangeably and refer to any mammal in need of such treatment in accordance with the described methods. 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 person. Other subjects may include companion animals (eg, dogs and cats), livestock (eg, cows, pigs, goats, horses, etc.), rodents (eg, mice, guinea pigs and rats, such as in animal models diseases) as well as non-human primates (eg chimpanzees and monkeys). Thus, subjects of the invention include, but are not limited to, mammals, such as humans and other primates such as chimpanzees and other species of apes and monkeys; and the like, wherein in some embodiments the subjects are human beings. The term subject is also intended to include a person or organism of any age, weight, or other physical characteristics, and subjects may be an adult, a child, an infant, or a newborn.

A young or young individual is understood to be an individual who is at the chronological age of 40 years or younger, such as 35 years or younger, including 30 years or younger, such as 25 years or younger, or 22 years or younger. In some cases, the individual who serves as the source of the young plasma containing blood product is one whose age is 10 years or younger, such as 5 years or younger, including 1 year or younger. In some cases, the subject is a newborn and the source of the plasma product is the umbilical cord, with the plasma product being collected from the umbilical cord of newborns. Thus, young and young individual can refer to a subject whose age ranges from 0 to 40, for example, is 0, 1, 5,

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10, 15, 20, 25, 30, 35 or 40 years. In other cases, a young and young individual may be of biological (as opposed to chronological) age, such as an individual who has not demonstrated the plasma levels of inflammatory cytokines that comparatively older individuals exhibit. Conversely, a young and young individual may be biologically (as opposed to chronologically) aged, 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 an elderly individual is at least 1.5 times. Likewise, the fold difference between older and younger individuals in other inflammatory cytokines can be used to indicate biological age. (See US Patent Application No. 13/575,437, which is incorporated herein by reference.) The individual is generally healthy, e.g., the individual does not have hematologic malignancies or autoimmune diseases at the time of collection.

An individual suffering from or at risk of developing age-related cognitive impairment is defined as an individual who has lived more than 50% of his life expectancy, e.g. more than 60%, e.g. more than 70%, e.g. 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. Therefore, this percentage is based on the projected lifespan of the species in question. For example, in the case of a person, such individual is aged 50 years or older, such as 60 years or older, 70 years or older, 80 years or older, 90 years or older, and usually not older than 100 years, such as 90 years or older. .e. age between about 50 and 100, for example 50....55...60...65...70...75...80...85...90...95... 100 years of age or older, or any age between 50-1000, and suffers from an age-related condition as further described below, for example, cognitive impairment associated with the natural aging process; an individual who is about 50 years of age or older, such as 60 years or older, 70 years or older, 80 years or older, 90 years or older, and generally not older than 100 years, i.e. age about 50-100, for example, 50....55...60...65...70...75...80...85...90...95...100 years old, who has not yet demonstrated symptoms, age-related pathological conditions, for example, cognitive impairment; an individual of any age who suffers from 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 typically accompanied by cognitive impairment and the individual has not yet demonstrated symptoms of 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 that 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 the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibition of the disease, i.e. cessation of its development; or (c) alleviation of the disease, i.e. regression of the disease. Treatment can lead to various physical effects, such as modulation of gene expression, tissue or organ rejuvenation, etc. The therapeutic agent may be administered before, during, or after the onset of the disease. Treatment of ongoing disease in which the treatment stabilizes or reduces the patient's undesirable clinical symptoms is of particular interest. Such treatment can be performed until complete loss of function in the affected tissues. The therapy of the invention can 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 the individual's cognitive ability. Cognitive ability or cognition refers to mental processes that include attention and concentration, learning complex tasks and concepts, memory (acquiring, retaining and retrieving new information in the short and/or long term), information processing (handling information obtained through the five senses), visuospatial function (visual perception, depth perception, use of mental imagery, copying drawings, creating objects or shapes), language use and understanding, verbal fluency (word selection), problem solving, decision making, and executive functions ( planning and prioritization). Cognitive impairment refers to a gradual decline in one or more of these abilities, such as impairment of memory, language, thinking, judgment, etc. Cognitive impairment and cognitive impairment mean a decrease in cognitive ability compared to a healthy person, for example, a healthy person in the corresponding age group, or

- 12 046284 compared to the individual's ability at an earlier point in time, for example, 2 weeks, 1, 2, 3, 6 months, 1, 2, 5 or 10 years or more. Age-related cognitive impairment refers to the decline in cognitive ability that is typically associated with aging, including, for example, cognitive impairment associated with the natural aging process, such as mild cognitive impairment (MCI); and age-related cognitive impairment, e.g. a disorder whose incidence increases with age, for example, 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 mobility disorder in the individual. Motor ability refers to motor processes that include the ability to perform complex neuromuscular actions that enable 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 defined as a gradual decline in one or more of these abilities, such as a movement or gross motor disorder, etc. Movement impairment and movement impairment refers to a decrease in motor ability/motor skill compared to a healthy individual, e.g., a healthy individual in the appropriate age group, or compared to an individual's ability at an earlier point in time, e.g., 2 weeks, 1, 2 , 3, 6 months, 1, 2, 5 or 10 years or more. Age-related movement disorder refers to the decline in movement ability that is typically associated with aging, including, for example, movement disorders associated with the natural aging process and movement disorder or disorder associated with age-related disorder, i.e. a disorder whose incidence increases with age, for example, a neurodegenerative condition such as Parkinson's disease, amyotrophic lateral sclerosis and the like.

In some embodiments, the age-related condition being treated is an age-related increase in neuroinflammation in the 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 the central nervous system immune system to protect against potential harm. Neurodegeneration occurs in the central nervous system and involves signs of loss of neuronal structure and function. Neuroinflammatory diseases or conditions or diseases associated with neuroinflammation include, by way of example, but not limitation, neurodegenerative diseases such as Alzheimer's disease; Parkinson's disease, multiple sclerosis and the like.

Blood products containing plasma components. In the practice of the methods of the present 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 impairment, neuroinflammation and/or age-related dementia. In this regard, 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 cognitive or motor impairment, neuroinflammation, neurodegeneration and /or age-related dementia (hereinafter referred to as the recipient individual or recipient). A blood product containing plasma components is any product derived from blood that contains plasma (eg, whole blood, blood plasma, or fractions thereof). The term plasma is used in its common sense to refer to the straw-colored/pale yellow liquid component of blood consisting of approximately 92% water, 7% proteins such as albumin, gamma globulin, anti-hemophilic factor and other clotting factors, and 1% mineral salts, sugars, fats, hormones and vitamins. Non-limiting examples of plasma-containing blood products 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 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 product that is used is not a whole blood plasma product, by which it is meant that the product is not whole blood and therefore lacks one or more components present in whole blood, such as red blood cells, white blood cells, 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, such as 1% or less, including 0.5% or less, at In this case, in some cases, cell-free fractions of blood plasma represent those compositions that are

- 13 046284 are completely devoid of cells, i.e. 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 through venipuncture. In another embodiment, the venipuncture is a single venipuncture. In another embodiment, blood volume replacement with saline is not used. In a preferred embodiment, the plasmapheresis method is used to obtain blood products containing plasma. Plasmapheresis may involve removal of a weight-adjusted volume of plasma with 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 the donor is preferably from 690 to 880 ml after administration of citrate 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 for producing lyophilized plasma were developed. However, the use of lyophilized plasma has been difficult in combat situations, as it requires sterile water to reconstitute it. As an alternative, 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 Johan, Current Approaches to the Preparation of Plasma Fractions in (Biotechnology of Blood) 165 (Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's procedure used cold ethanol to purify plasma fractions due to its denaturing effect and used changes in pH and temperature to achieve separation.

An embodiment of the methods described herein involves administering plasma fractions to a subject. Fractionation is a process by which specific subsets of proteins are separated from plasma. The fractionation technology is known in the art and is based on the 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 in as a reference). The process involves several steps, and each step involves the application of specific concentrations of ethanol as well as shifts in pH, temperature, and osmotic pressure that result in selective protein precipitation. 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 initially 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 Patent Nos. 3869431, 5110907, 5219995, 7531513 and 8772461, which incorporated herein by reference). In order to obtain specific protein fractions, the above experimental parameters can be adjusted.

More recently, fractionation has achieved additional complexity, and therefore constitutes additional embodiments of the present invention. This recent increase in complexity has been due to: the introduction of chromatography, which has led to the isolation of new proteins from existing fractions such as cryoprecipitate, cryo-depleted plasma and Cohn fractions; increasing 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 skilled in the art will appreciate that the parameters described above can be modified to obtain fractions containing the specific desired plasma proteins.

Blood plasma fractionation can also be done on the basis of ammonium sulfate. (See, for example, Odunuga OO, Biochem Compounds, 1:3 (2013); Wingfield PT, Curr Protoc Protein Sci, Appx. 3 (2001), incorporated herein by reference.) In addition to obtaining specific blood fractions, ammonium sulfate fractionation is used to reduce excess coli

- 14 046284 quality of proteins from plasma. (Saha S, et al., J. Proteomics Bioinform, 5(8) (2012), incorporated herein by reference).

In one embodiment of the present invention, blood plasma is fractionated in an industrial setting. Frozen plasma is thawed at 1-4°C. Continuous refrigerated centrifugation is applied to the melt plasma and cryoprecipitate is isolated. The reconstituted cryoprecipitate is frozen at -30°C or below and stored. Cryoprecipitate-depleted (cryo-depleted) plasma is immediately processed to capture (eg, primary chromatography) labile coagulation factors such as the factor IX complex and its components, as well as protease inhibitors such as antithrombin and C1 esterase inhibitor. Sequential centrifugation and precipitate separation 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 comprise a plasma fraction containing a significant concentration of albumin. In another embodiment of the invention, the plasma fraction may comprise a plasma fraction containing a significant concentration of IgG or intravenous immunoglobulin (IGIV) (eg, Gamunex-C®). In another embodiment of the invention, the plasma fraction may comprise an IGIV plasma fraction, such as Gamunex-C®, that has been substantially depleted of immunoglobulin (IgG) using methods well known to one skilled in the art, such as, for example, protein-mediated A exhaustion. (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 coagulation factors have been removed in order to maintain the effectiveness of the reduced risk of thrombosis fraction. 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 albumin plasma preparations (APP): plasma protein fraction (PPF) and human albumin solution (HAS). PPF is a derivative process in higher yield 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 using electrophoresis or other quantitative assays such as mass spectrometry. Additionally, some have noted that PPF is deficient due to the presence of protein contaminants such as PKA. Id. As a consequence, PPF preparations lost popularity as plasma albumin preparations, and were even excluded from the Pharmacopoeia of some countries. Id. In contrast to these problems, the present invention makes it useful to use these impurities. In addition to α-, β- and γ-globulins, as well as the aforementioned PKA, the methods of the invention utilize additional proteins or other factors from the impurities 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 PPF formulations). 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 HAS formulations). These include Albuminar™ (CSL Behring), AlbuRx™ (CSL Behring), Albutein™ (Grifols, Clayton, North Carolina ), Buminate™ (Baxatla, Inc., Bannockburn, IL), Flexbumin™ (Baxatla, Inc., Bannockburn, IL), and Plasbumin™ (Grifols, Clayton, NC).

a. Plasma protein fraction (human) (PPF).

According to the US Food and Drug Administration (FDA), plasma protein fraction (human), or PPF, is the proper name for a preparation called a sterile protein solution consisting of albumin and globulin derived from human plasma. (CFR 21 CFR 640.90, which is incorporated herein by reference in its entirety). PPF starting material is plasma isolated from whole blood obtained as prescribed in 21 CFR 640.1-640.5 (which is included in this document).

- 15 046284 document by reference), or a plasma source obtained as prescribed in 21 CFR 640.60-640.76 (which is incorporated herein 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 preparation must consist of at least 83% albumin and no more than 17% globulins. No more than 1% of the total protein should be gamma globulin. Protein composition is determined using a method that has been approved for each manufacturer by the Director of the Center for Biologics Evaluation and Research of the Food and Drug Administration.

As used herein, plasma protein fraction or PPF refers to a sterile protein solution consisting of albumin and globulin derived from human blood plasma, with an albumin content of at least 83%, with no more than 17% globulins (including α1 globulins, 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 total globulin fraction can be determined by subtracting albumin from total protein. (Busher, J., Serum Albumin and Globulin, Clinical Methods: The History, Physical, and Laboratory Examinations, Chapter 10, Walker HK, Hall WD, Hurst JD, eds. (1990)).

b. Albumin (human) (HAS).

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

Albumin (human) is tested to determine compliance with the following standards under 21 CFR 640.82:

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

(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 Food and Drug Administration's Center for Biologics Evaluation and Research.

As used herein, albumin (human) or HAS refers to a sterile protein solution consisting of albumin and globulin derived from human plasma, with an albumin content of at least 95%, no more than 5% globulins (including α1 globulins, 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 total globulin fraction can be determined by subtracting albumin from total protein.

As will 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 appropriate additives, can be used to prepare, for example, tablets, powders, granules or capsules. The solid form can be converted into injectable preparations by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, higher aliphatic acid esters or propylene glycol; and, if desired, with the usual additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

5. Fractions with a reduced amount of coagulation factors.

In another embodiment of the invention, a fraction of blood plasma is used from which substantially all coagulation factors have been removed in order to maintain the effectiveness of the fraction with a reduced risk of thrombosis. Conveniently, the blood product can be obtained from a young donor or group of young donors and can be stripped of IgM to provide a young blood product that is ABO compatible. Currently, plasma that is transfused is ABO blood type compatible, since the presence of natural antibodies to A and B antigens can lead to transfusion reactions. IgM appears to be responsible for transfusion reactions when patients receive non-ABO-compatible plasma. Removal of IgM from drugs or fractions

- 16 046284 tions of blood helps eliminate transfusion reactions in subjects who are administered blood products and blood plasma fractions according to the invention.

Accordingly, in one embodiment, the present invention provides a method for treating or preventing an age-related condition, such as cognitive or motor impairment, neuroinflammation, or neurodegeneration, in a subject. The method includes administering to a subject a blood product or blood fraction obtained 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 which the blood product or blood fraction is obtained are young individuals. In some embodiments, the blood product is substantially devoid of at least one clotting factor and IgM. In certain embodiments, the blood product is substantially devoid of fibrinogen (factor I). In additional 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 August 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 coagulation 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% globulins α, β and γ and other plasma proteins may 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 may be used. In another embodiment, a protein-enriched plasma protein preparation consisting of 80% albumin and 20% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 70-79% albumin and correspondingly 21-30% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 60-69% albumin and correspondingly 31-40% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 50-59% albumin and correspondingly 41-50% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 40-49% albumin and correspondingly 51-60% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 30-39% albumin and correspondingly 61-70% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 20-29% albumin and correspondingly 71-80% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 10-19% albumin and correspondingly 81-90% α, β and γ globulins and/or other plasma proteins may be used. In additional embodiments, protein-enriched plasma protein preparations consisting of 1-9% albumin and correspondingly 91-99% α, β and γ globulins and/or other plasma proteins may be used. In another embodiment of the invention, protein-enriched plasma protein preparations consisting of 0% albumin and 100% globulins α, β and γ and other plasma proteins may 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 one 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 preparing PPF and other plasma fractions are well known to those of ordinary skill in the art. An embodiment of the present invention provides for the collection of blood used in the preparation of the human plasma protein fraction in flasks containing citrate or anticoagulant citrate dextrose solution to inhibit blood clotting, 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 includes

- 17 046284 chen into this document by reference). According to this method, the mixture can be collected at 2-8°C. The 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 eight hours of removal from -20°C storage.

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 4M sodium acetate solution, 230 ml glacial acetic acid and _H2O to 1 L) can be added by pipetting at a rate of e.g. 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 prepared from Fraction I according to methods well known to those of ordinary skill in the art.

Fraction II-III can be separated from eluate I by diluting the eluate to 21% ethanol at pH 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 by pipetting at a rate of, for example, 500 ml/min while reducing the temperature of eluate I to -6°C. The resulting precipitate (fraction II+III) can be removed by centrifugation at -6°C. Gamma globulin 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%. H2O and 10M acetic acid used for pH adjustment can be added continuously by pipetting while maintaining eluate II/III at -6°C for 6 hours. Precipitated fraction VI-1 can be sedimented at -6°C for 6 hours , and then separated from the eluate by centrifugation at the same temperature. A stable plasma protein fraction can be isolated from eluate IV-1 by adjusting the ethanol concentration to 30% at a pH of 4.65, a temperature of -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 at -7°C, add 170 ml of cold ethanol (95%) for each liter of eluate 1-IV adjusted. Proteins that can be precipitated are allowed to settle for 36 hours and then removed by centrifugation at -7°C.

The isolated proteins (stable plasma protein fraction) can be dried (e.g. by freeze drying) to remove alcohol and _H2O . The resulting dried powder can be dissolved in sterile distilled water, e.g. using 15 L water/kg powder, with a 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 skilled 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, movement and neurodegenerative disorders, and are embodiments of the present invention.

The previous methods for preparing 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 of plasma fractions and plasma protein fractions in various embodiments and methods of the present invention. In another example, additional embodiments of the present invention involve the use of nanofiltration to remove/inactivate pathogenic microorganisms 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 demonstrates, among other things, that specific albumin concentrations are not critical for improving cognitive or motor performance. Thus, fractions with reduced albumin concentration, 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 involve treating a subject with a blood product containing plasma, such as a blood 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 of treating patients with Cro

- 18 046284 vi containing plasma are known in the art. By way of example, and not limitation, one embodiment of the methods of the invention described herein involves 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, the blood product containing the plasma is administered immediately, for example, within about 12-48 hours after receipt from the 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 below. Prior to administration, fresh frozen plasma is thawed and, once thawed, is administered to the subject 60-75 minutes after the thawing process has begun. Each subject preferably receives a single unit of fresh frozen plasma (200-250 ml), with the fresh frozen plasma preferably obtained from donors within a predetermined age range. In one embodiment of the invention, the fresh frozen plasma is provided from (derived from) young individuals. In another embodiment of the invention, the 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 tested for the presence of infectious disease agents such as HIV-I and II, HBV (hepatitis B virus), HCV (hepatitis C virus), HTLV (Human 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 a 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 the commercial HAS preparations. In another embodiment of the present invention, the plasma fraction is PPF or HAS obtained from a group of individuals of a certain age range, such as young adults, or is a modified PPF or HAS fraction that has been subjected to additional fractionation or processing (e.g., PPF or HAS, in which one or more specific proteins have been partially or substantially removed). In another embodiment of the invention, the plasma fraction is an IGIV plasma fraction that has been significantly depleted of 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 observed in the reference preparation or whole blood plasma, e.g., less than than 45, 40, 35, 30, 25, 20, 15, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1%, 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 involve 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 of 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 involves 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, the blood product containing the plasma is administered immediately, for example, within about 12-48 hours after receipt from the 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 below. Prior to administration, fresh frozen plasma is thawed and, once thawed, is administered to the subject 60-75 minutes after the thawing process has begun. Each subject preferably receives a single unit of fresh frozen plasma (200-250 ml), with the fresh frozen plasma preferably obtained from donors within a predetermined age range. In one embodiment of the invention, the fresh frozen plasma is provided from (derived from) young individuals. In another embodiment of the invention, the fresh frozen plasma is provided from (obtained from) donors of the same sex. In another embodiment of the invention, the fresh frozen plasma is provided from (received from) a donor

- 19 046284 age range 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 tested for the presence of infectious disease agents such as HIV-I and II, HBV (hepatitis B virus), HCV (hepatitis C virus), HTLV (Human 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 a 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 the commercial HAS preparations. In another embodiment of the present invention, the plasma fraction is PPF or HAS obtained from a group of individuals of a certain age range, such as young adults, or is a modified PPF or HAS fraction that has been subjected to additional fractionation or processing (e.g., PPF or HAS, in which one or more specific proteins have been partially or substantially removed). In another embodiment of the invention, the plasma fraction is an IGIV plasma fraction that has been significantly depleted of 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 observed in the reference preparation or whole blood plasma, e.g., less than than 45, 40, 35, 30, 25, 20, 15, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1%, 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 who has been diagnosed with a cognitive or motor impairment, neurodegeneration, or neuroinflammation by administering to the subject an effective amount of blood plasma or a fraction of plasma. Another embodiment of the invention involves administering an effective amount of blood plasma or a fraction of plasma and then monitoring the subject for improvement in cognitive or motor function or reduction in neuroinflammation or increase in 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 plasma fraction, wherein the blood plasma or plasma fraction is administered in a manner that results in improved cognitive or motor function, decreased neuroinflammation or increased neurogenesis after reaching the mean or median half-life of plasma proteins or plasma protein fractions relative to the most recently administered dose (referred to herein as intermittent dosing or intermittent dosing). Another embodiment of the invention comprises administering blood plasma or a fraction of plasma in a dosage regimen 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 involves administering blood plasma or a fraction of plasma in a regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days and monitoring the subject for improvement. cognitive or motor function, decreased neuroinflammation, or increased neurogenesis for at least 3 days after the date of last administration. Another embodiment of the invention includes administering blood plasma or a fraction of plasma on a regimen of at least 2 consecutive days and after the date of last administration, monitoring for improvement in cognitive or motor function, reduction in neuroinflammation, or increase in neurogenesis, once achieved the average half-life of proteins in blood plasma or plasma fraction. Another embodiment of the invention involves administering blood plasma or a fraction of plasma in a regimen of 2-14 non-consecutive days, with each dose interval being 0-3 days.

In some cases, intermittent use in accordance with this invention involves the administration of a first set of doses, for example, as described above, followed by a period of no use, for example, a period of no use, which in turn is followed by the administration of another dose or set of doses. The length of this no-use period may vary, but in some embodiments it is 7 days or longer, such as 10 days or longer, including 14 days or longer, with some cases ranging from 15 to 365 days, e.g. from 30 to 90 days, including from 30 to 60 days. Thus, embodiments of the methods include non-systematic (ie, non-continuous use), for example, non-systematic administration of a blood plasma preparation. In some embodiments, the regimen of intermittent use followed by a period of no use is repeated a number of times as needed, and in some cases the regimen is continued for 1 year or more, at

- 20 046284 example, 2 years or more, including the entire life of the subject. Another embodiment of the invention involves administering blood plasma or a fraction of plasma according to a regimen of use for 5 consecutive days, with a period of no use equal to 2-3 days, followed by administration for 2-14 consecutive days.

Biochemically, by an effective amount or effective dose of an active agent is meant an amount of an active agent that will inhibit, antagonize, reduce, decrease, or suppress by about 20% or more, such as by 30% or more, by 40% or more, or by 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 trace amounts, and in some cases, cause reversal of cognitive impairment, neuroinflammation, neurodegeneration or age-related dementia.

10. Plasma protein fraction.

When 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 additional embodiments, the PPF is selected from commercial PPF formulations.

In another embodiment, PPF consists of 88% normal human albumin, 12% alpha and beta globulins, and no more than 1% gamma globulin, as determined by electrophoresis. Other embodiments of this embodiment useful in carrying out the methods of this invention include, for example, an embodiment 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 a modified percentage of PPF (e.g., about 1% to about 10%, about 10% to about 20%, about 20% to 25%, about 25% to 30%) in solution, as well as the concentrations of solvent and stabilizers, can be used in the implementation of the methods of this invention.

11. Plasma fractions from donors of a specific age.

Additional embodiments of the present invention include the administration of a plasma protein fraction obtained from the plasma of individuals within certain age ranges. An embodiment involves administering PPF or HAS that has been obtained from the plasma of young individuals. In another embodiment of the present invention, young individuals fall within one specific age or a specific 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 involve pooling blood or plasma from individuals of a certain age and fractionating the blood plasma as described above to produce a plasma protein fraction preparation 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 impairments in the cognitive or motor abilities of 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 plasma-containing blood product according to this invention, for example, using the methods described herein, include individuals who are aged 50 years and above, for example, 60 years or above, 70 years or above, 80 years or above, 90 years or above and 100 years or above, i.e. aged from about 50 to 100, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 years, 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 aged 50 years or older, for example, 60 years or older, 70 years or older, 80 years or older, 90 years or older, and generally not older than 100 years, i.e. between about 50 and 90 years of age, e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 years, 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 impairment of cognition that manifests itself as problems with memory or other mental functions such as planning, following instructions, or making decisions that have worsened over time while overall mental function and daily functioning have not deteriorated. So, although usually

- 21 046284 does not cause significant neuronal death, neurons in the aging brain are vulnerable to sublethal age-related 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 plasma preparation or blood fraction of the present invention, for example, using the methods described herein, also include individuals of any age who suffer from cognitive impairment functions due to an age-related disorder, and individuals of any age who have been diagnosed with an age-related disorder that is typically accompanied by cognitive impairment and the individual has not yet begun to exhibit symptoms of cognitive impairment. Examples of such age-related disorders include the following.

b. Alzheimer's disease.

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

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

Over the course of the disease, synapses and ultimately neurons are lost in the cerebral cortex, hippocampus and subcortical structures (including selective cell loss in the nucleus basalis of Meynert), locus coeruleus, and dorsal raphe nucleus. Cerebral glucose utilization and perfusion are reduced in some brain regions (parietal lobe and temporal cortex in early disease, prefrontal cortex in late disease). Neuritic or senile plaques (composed of neurites, astrocytes and glial cells around the amyloid core) and neurofibrillary tangles (composed of paired helical filaments) play a role in the pathogenesis of Alzheimer's disease. Senile plaques and neurofibrillary tangles occur during 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 central nervous system characterized by slowing and decreased movement (bradykinesia), muscle rigidity, resting tremor (dystonia), muscle inhibition, and postural instability. Originally viewed primarily as a movement disorder, PD is now recognized as a cause of depression and emotional changes as well. PD can also affect cognition, behavior, sleep, autonomic function, and sensory function. The most common cognitive impairments include problems with 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 helps in diagnosing the disease.

In primary Parkinson's disease, pigmented neurons of the substantia nigra, locus coeruleus, and other groups of dopaminergic cells in the brain stem degenerate. The reason is unknown. The loss of substantia nigra neurons that project to the caudate nucleus and putamen leads to depletion of the neurotransmitter dopamine in these areas. It usually occurs after 40 years of age, with an increase in incidence in older age groups.

Parkinson's disease is newly diagnosed in approximately 60,000 Americans each year and currently affects approximately one million Americans. Even though 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 reserved for later, more severe cases.

Treatment options for PD include the administration of medications to compensate for motor deficits. These variants increase or replace the neurotransmitter, dopamine, whose concentrations are low in the brains of PD patients. Such drugs include: carbidopa/levodopa (which creates more dopamine in the brain); apomorphine, pramipexolol, ropinirole and rotingotine (dopamine agonists); selegiline and rasagiline (MAO-B inhibitors that prevent the destruction of dopamine); entacapone and tolcapone (catechol-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.

- 22 046284

Current treatments that treat the symptoms of PD do not provide a cure and are unable to prevent disease progression. In addition, currently, medications tend to lose effectiveness in the later stages of PD. The most prescribed drug, levodopa, usually produces adverse effects within 5 to 10 years of starting the drug. These side effects can be serious and can result in motor fluctuations and unpredictable fluctuations in motor control between doses, as well as twitching/shaking (dyskinesias) that are difficult to control and as disabling as PD's own symptoms. Thus, there remains a need for new treatments with new mechanisms of action that can be used together or in combination with existing drugs against PD.

d. Parkinsonism.

Secondary parkinsonism (also called atypical Parkinson's disease or Parkinson's plus) results from the loss or inhibition of dopamine action in the basal ganglia due to other idiopathic degenerative diseases, drugs, or exogenous toxins. The most common cause of secondary parkinsonism is the ingestion of antipsychotic drugs or reserpine, which cause parkinsonism by blocking dopamine receptors. Less common causes include carbon monoxide or manganese poisoning, hydrocephalus, structural damage (tumors, infarcts affecting the midbrain or basal ganglia), subdural hematomas, and degenerative diseases including nigrostriatal degeneration. Some disorders, such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB), may exhibit parkinsonian symptoms before the cardinal symptoms required for a specific diagnosis can occur, and , thus, may be labeled as parkinsonism.

e. Frontotemporal dementia.

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

Behavioral variant FTD (bvFTD), with symptoms including lethargy and aspontaneity on one side, and disinhibition on the other; progressive non-fluent aphasia (PSA), in which there are interruptions in speech fluency due to difficulty in articulation, phonological and/or syntactic errors, but the understanding of words is preserved; and semantic dementia (SD), in which patients remain fluent with normal phonetics and syntax but have increasing difficulty naming and understanding words. Other cognitive symptoms common to all patients with FTD include impairments in executive function and concentration. Other cognitive abilities, including perception, spatial skills, memory and practice, tend to be unaffected. FTD can be diagnosed by observing atrophy of the frontal lobe and/or anterior temporal lobe 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). DS is characterized by loss of semantic memory in both verbal and nonverbal domains. Patients with diabetes often complain of difficulty finding words. Clinical features include free aphasia, anomia, impaired understanding of the meaning of words, and associative visual agnosia (inability to match semantically related images or objects). As the disease progresses, behavioral and personality changes similar to those seen in frontotemporal dementia are often observed, although cases of pure semantic dementia with few late behavioral symptoms have been described. Structural MRI imaging demonstrates a characteristic pattern of atrophy in the temporal lobe (predominantly in the left), with greater involvement of the inferior than superior and greater atrophy of the anterior versus posterior temporal lobe.

As another example, another form of frontotemporal dementia is Pick's disease (PiD, also PcD). A hallmark of the disease is the concentration of tau proteins in neurons, clustering into 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 behavior. Patients with dorsomedial or dorsolateral frontal dysfunction may demonstrate lack of restlessness, apathy, or decreased spontaneity. Patients may demonstrate 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 demonstrate dietary changes

- 23 046284 behaviors such as pathological cravings for sweets. Patients with more focal gray matter loss in the anterior orbitofrontal cortex may develop hyperphagia. While some of the symptoms may initially be alleviated, the disease progresses and patients often die within two to ten years.

f. Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by the development of emotional, behavioral and mental disorders; loss of intellectual or cognitive functions; and movement disorders (motor disorders). Classic signs of HD include the development of chorea—involuntary, rapid, irregular, jerky movements that can affect the face, arms, legs, or trunk—and cognitive decline, including the gradual loss of thought processing and acquired intellectual abilities. There may be impairment in memory, abstract thinking, and judgment; incorrect perception of time, place, or person (disorientation); increased anxiety and personality changes (personality disintegration). Although symptoms usually become apparent during the fourth or fifth decade of life, the age of onset is variable and ranges 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 repeats of encoded commands within a gene on chromosome 4 (4p16.3). Progressive loss of nervous system function is associated with HD results from neuronal loss in certain brain regions, including the basal ganglia and cerebral cortex.

g. Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and 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. Cramps are common and may be preceded by weakness. Rarely, the patient lives 30 years; 50% die within 3 years of the onset of the disease, 20% live 5 years, and 10% live 10 years.

Diagnostic features include onset during middle or late adulthood and progressive general involvement of motor function without sensory impairment. Nerve conduction velocity is normal until late in the disease. Recent studies have demonstrated cognitive impairment, as well as declines in immediate verbal memory, visual memory, language, and executive functions, among others.

Reductions in cell body area, number of synapses, and total synapse length were observed even in normal-looking neurons in ALS patients. It has been proposed that when active zone plasticity reaches its limit, continued synaptic loss may lead to functional impairment. Stimulating the formation of new synapses or preventing synapse loss may maintain neuronal function in these patients.

h. Multiple sclerosis.

Multiple sclerosis (MS) is characterized by various symptoms and signs of central nervous system dysfunction, with remissions and recurrent exacerbations. The most common symptoms are paresthesias in one or more limbs, the torso, or one side of the face; weakness or clumsiness of a leg or arm; or vision problems, such as partial blindness and pain in one eye (retrobulbar optic neuritis), blurred vision, or scotomas. Common cognitive impairments include impairments in 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 paralysis that results in double vision (diplopia), temporary weakness of one or more limbs, mild stiffness or unusual fatigue of a limb, minor gait disturbances, difficulty controlling the bladder, dizziness, and mild emotional disturbances; all indicate diffuse central nervous system involvement and often occur months or years before the disease is recognized. Excess heat can worsen symptoms and signs.

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

i. Glaucoma.

Laucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs). Available data indicate the existence of distinct patterns of degeneration in synapses and dendrites, including RGCs. Recent data also indicate a correlation between cognitive impairment and glaucoma in older adults (Yochim BP, et al. Prevalence of cognitive impairment, depression, and anxiety symptoms among older adults with glaucoma. J Glaucoma.

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2012;21(4):250-254).

j. Myotonic dystrophy.

Myotonic dystrophy (MD) is a multisystem autosomal dominant disorder 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 arm muscles, and ptosis is a common symptom even in mild cases. Severe cases include peripheral muscle weakness, often with cataracts, premature balding, mask-like facies, cardiac arrhythmia, 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 cognitive functions, particularly language and executive function, is observed in milder adult forms of the disease. People with severe damage die around 50 years of age.

k. Dementia.

Dementia describes a class of disorders with 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 age-related disorders discussed above include vascular dementia and dementia with Lewy bodies, described below.

In vascular dementia or multi-infarct dementia, cognitive impairment is caused by problems in the flow of blood 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 a progressive decline in cognitive ability. 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 blood flow to the brain, which, in ultimately leads to dementia. Neuroimaging with single photon emission computed tomography (SPECT) and positron emission tomography (PET) can be used to confirm the diagnosis of multi-infarct dementia in conjunction with evaluations that include mental status testing.

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 body type) is a type of dementia anatomically characterized by the presence of Lewy bodies (clumps of protein alpha-synuclein and ubiquitin) in neurons found in post-mortem brain histology. Its main feature is a decrease in cognitive functions, in particular executive function. Attention and short-term memory will wax and wane.

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

l. Progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) 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 the eyes properly, which occurs due to damage to the area of the brain that coordinates eye movement. Some individuals describe this effect as vagueness. Affected individuals often exhibit changes in mood and behavior, including depression and apathy, as well as progressive mild dementia. The long name of the disorder indicates that the disease begins slowly and continues to get worse (progressive), and causes weakness (paralysis) by damaging certain parts of the brain above the pisiform structures called nuclei that control eye movements (supranuclear). PSP was first described as a distinct disorder in 1964, when three scientists published a paper distinguishing it from Parkinson's disease. It is sometimes called Steele-Richardson syndrome

- 25 046284

Olypevsky, after the names of those scientists who identified this disorder. Although PSP progresses with worsening, no one dies from PSP itself.

m. Ataxia.

People with ataxia have problems with coordination because the parts of the nervous system that control movement and balance are affected. Ataxia can affect the fingers, hands, arms, legs, body, speech, and eye movements. The word ataxia is often used to describe symptoms of incoherence that may be associated with infections, trauma, 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 the typical age of onset being late 50s to early 60s. MSA often exhibits some of the same symptoms as Parkinson's disease. However, patients with MSA typically show minimal, if any, response to dopamine drugs used in Parkinson's disease.

o. Decrepitude.

Frailty syndrome (frailty) is a geriatric syndrome characterized by functional and physical decline including decreased mobility, muscle weakness, physical slowness, poor tolerance, low physical activity, poor nutrition, and involuntary weight loss. This decline often accompanies and is a consequence of diseases such as cognitive dysfunction and cancer. However, frailty can occur even without illness. Individuals suffering from frailty have an increased risk of poor prognosis from fractures, accidental falls, disability, comorbidities, and premature mortality. (C. Buigues, et al. Effect of a Prebiotic Formulation on Frailty Syndrome: A Randomized, Double-Blind Clinical Trial, Int. J. Mol. Sci. 2016, 17, 932). In addition, people suffering from frailty are more likely to have higher healthcare costs. (Id.).

Common symptoms of frailty can be determined through certain types of tests. For example, unintentional weight loss includes loss of 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% of 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 the individual providing self-reported data on exhaustion; 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, an individual's cognitive, motor, neuroinflammatory or other abilities or conditions will decline more slowly after treatment using the described methods than before or in the absence of treatment using the described methods. In some such cases, the treatments contemplated 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 the decline is improving. In some such cases, the determination is made by comparison with a standard, for example, the rate of decline in an individual before 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 a blood product according to the present invention.

The methods and compositions of the present invention also find use in stabilizing cognitive, motor, neuroinflammatory or other abilities or conditions in an individual, for example, an individual suffering from age-related cognitive disorder 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 described methods will be arrested after treatment with the described methods. As another example, an individual may be at risk for developing age-related cognitive impairment (for example, the individual may be aged 50 years or older, or may have been diagnosed with an age-related disorder), but the individual's cognitive abilities remain essentially unchanged, i.e. . a decrease in cognitive ability cannot be detected after treatment using the described methods, compared to the state before treatment using the described methods.

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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 this invention, the individual experiences an improvement in the impaired ability. For example, the individual's cognitive or motor abilities increase, 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 before 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 individual was about 40 years of age or less. In other words, the cognitive or motor impairment is reversed. Methods of diagnosis and monitoring for improvement.

13. In some cases, among 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 appropriate, alone or in combination with subjects suffering from neurodegenerative diseases. The following types of methods are provided as examples and are not limited to the listed methods. Any convenient methods for monitoring the disease, if necessary, can be used in the practice of the invention. These methods are also provided in the methods of this invention.

a. General cognition.

Embodiments of the methods of the present 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 including 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 by way of limitation, the method may include assessing cognitive function based on medical history, family history, physical and neurological examinations by physicians specializing in dementia and cognitive function, laboratory tests, and neuropsychological assessments. Additional options that are contemplated by the invention include: assessing consciousness, for example using the Glasgow Coma Scale (EMV); mental state examination, including the Abbreviated Mental Test Score (AMTS) or Mini Mental State Examination (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 the cognitive subscale of the Alzheimer's Disease Severity Rating Scale (ADAS-COG).

In one embodiment, peripheral nervous system tests may be used to assess cognitive function, including any of the following: odor sense, visual field and visual acuity, eye and pupil movements (sympathetic and parasympathetic), facial sensory function, facial and brachial muscle strength belt, hearing, taste, pharyngeal movement and reflex, tongue movements, which can be tested individually (eg, visual acuity can be tested with a Snellen chart; a neurological hammer can be used to test reflexes, including chewing reflexes, biceps tendon reflexes and triceps, patellar tendon, Achilles reflex and plantar reflex (i.e. Babinski reflex), muscle strength often MRC 1-5, muscle tone and signs of stiffness.

b. Parkinson's disease.

Embodiments of the methods of the present invention further include methods for monitoring the effect of a drug or treatment on a subject being treated for a movement disorder, the method including 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 assessing 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 operation with 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 Hoehn and Yaru scale (Neruology, 1967 17( 5): 427-42). Additional scales include the UPDRS, improved between

- 27 046284 by the People's Movement Disorder Society (MDS) (MDS-UPDRS), as well as the Schwab and England Activities of Daily Living Scale (ADL).

The UPDRS measures 31 items that comprise three subscales: (1) mentality, behavior, and mood; (2) daily activities and (3) assessment of motor activity. The Hoehn and Yaru scale divides PD into five stages with discrete substages: 0 - no evidence 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 stability test; 3 - imbalance with mild to moderate disease; 4 - severe disability, but with preservation of the ability to walk or stand without assistance; and 5, need for a wheelchair or bedridden without assistance. The Schwab and England Activities of Daily Living Scale classifies PD in % (from 100% - complete independence to 10% - complete dependence).

Gross motor function can be assessed using commonly used scales, including the General Motor Function (GMF) scale. It assesses three components: dependence, pain and uncertainty. (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 (locomotion speed, variability, leg rigidity) can be measured by an accelerometer; posture (body tilt) - with a gyroscope; leg movement - accelerometer; hand movement - accelerometer and gyroscope; tremor (amplitude, frequency, duration, asymmetry) - with an accelerometer; fall - accelerometer; gait retardation - 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 symptoms related to cognition, progression or improvement of neurodegeneration associated with multiple sclerosis (MS) can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring may be accomplished using techniques such as: cerebrospinal fluid (CSF) monitoring; magnetic resonance imaging (MRI) to detect damage and the development of demyelinating plaques; study of evoked potentials; and gait monitoring.

CSF analysis can be performed, for example, by lumbar puncture to obtain the pressure, appearance and content of the CSF. 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; cell count 0-5 lymphocytes and absence of red blood cells; chloride (110-125 mEq/L). Abnormal results may indicate the presence or progression of multiple sclerosis.

MRI is another method that can be performed to monitor the progression and improvement of the disease. Typical criteria for monitoring MS with 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 PC in subjects. Evoked potentials measure the slowing of electrical impulses, such as in the visual evoked response (VER), brainstem auditory evoked responses (BAER), and somatosensory evoked responses (SSER). Abnormal responses help determine the presence of decreased conduction velocity in the central sensory pathways.

Gait monitoring can also be used to monitor disease progression and improve disease in subjects with MS. MS is often accompanied by impaired mobility and abnormal gait, partly 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 related to cognition, progression or improvement of 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 may be accomplished using methods such as: motor function; behavior; functional assessment; and visualization.

Examples of motor function that may be monitored as an indicator of disease progression or improvement include chorea and dystonia, rigidity, bradykinesia, oculomotor dysfunction, and gait/balance changes. Techniques for monitoring these indicators are well known to those skilled in the art. (See Tang C, et al., Monitoring Hunting- 28 046284 ton's disease progression through preclinical and early stages, Neurodegener Dis Manag 2(4):421-35 (2012)).

The psychiatric effects of HD provide opportunities to monitor disease progression and improvement. For example, psychiatric diagnosis can be performed to determine whether the 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 assessing overall functioning (Id.) have been presented, with the score often decreasing by one point per year in some HD groups.

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

e. ALS.

In addition to monitoring for improvement in symptoms related to cognition, progression or improvement of neurodegeneration associated with amyotrophic lateral sclerosis (ALS) can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring may be carried out using methods such as: functional assessment; determination of muscle strength; respiratory function measurement; measuring 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 related to bulbar, limbic and respiratory function. Rate of change is useful for predicting survival and disease progression or improvement. Another measure includes the Composite Assessment of Function and Survival (CAFS), ranking subjects' clinical outcomes by combining survival time with change in ALSFRS-R. (Simon NG, et al., Quantifying Disease Progression in Amyotrophic Lateral Sclerosis, Ann Neurol 76:643-57 (2014)).

Muscle strength can be examined and quantified through the use of manual muscle testing (MMT) assessment. This involves averaging measurements obtained from multiple muscle groups using the Medical Research Council (MRC) muscle strength classification scale. (Id.) Handheld 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 ABS disease. Neurophysiological index can be determined by measuring the electrically evoked muscle response (CMAP) in motor nerve conduction studies, the parameters of which 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 estimates the number of residual motor axons servicing a muscle by assessing the contribution of individual motor units to the maximal CMAP response and is used to determine disease progression or improvement. (Simon NG, et al., earlier). Additional methods to determine LMN loss include neural excitability testing, electrical impedance monographs, and the application of ultrasound to muscles to detect changes in muscle thickness. (Id.).

Upper motor neuron dysfunction is another indicator that can be used to monitor the progression or improvement of ABS disease. Methods for determining dysfunction include performing MRI or PET scans 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 symptoms related to cognition, progression or improvement of neurodegeneration associated with glaucoma can be monitored using methods well known to those skilled in the art. By way of example, and not limitation, monitoring may be accomplished using methods such as: determination of intraocular pressure; assessment of the optic disc or optic nerve head for damage; visual field testing for peripheral vision loss; and visualization of the optic nerve head and

- 29 046284 retina for topographic analysis.

g. Progressive supranuclear palsy (PSP).

In addition to monitoring for improvement in symptoms related to cognition, progression or improvement of 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 may be accomplished using methods such as: functional assessment (activities of daily living 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 can 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 items in six categories: activities of daily living (history); behavior; bulbar, ocular motor function, limbic motor and gait/intermediate function. The result is a score ranging from 0-100. Six items were scored 0–2, and twenty-two items were scored 0–4, for 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 are useful for monitoring disease progression or improvement. (Golbe LI, et al., A clinical rating scale for progressive supranuclear palsy, Brain 130:1552-65, (2007)).

The ADL section of the UPDRS (Unified Parkinson's Disease Symptom Rating Scale) can also be used to quantify functional activity in patients with PSP. (Duff K, et al., earlier). Additionally, the Schwab and England Activities of Daily Living Scale (SE-ADL) can be used to assess independence. (Id.) Additionally, the motor function sections of the UPDRS are useful as a reliable measure for assessing disease progression in patients with PSP. The motor section may contain, for example, 27 different indicators to quantify motor function in patients with PSP. Examples include resting tremor, rigidity, finger tapping, posture and gait). Progression or improvement of a subject's illness may also be assessed by performing a neuropsychological assessment at baseline 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). (Id.).

Functional MRI (fMRI) can be used to monitor disease progression or improvement. fMRI is a technique that uses MRI to measure changes in brain activity in specific regions of the brain, typically based on blood flow in those regions. Blood flow is thought to correlate with the activation of a brain region. Patients with a neurodegenerative disorder such as PSP may be subjected to physical or mental tests before or during an MRI scan. By way of example, and not limitation, tests may represent a well-established force control paradigm where patients are asked to produce force with the hand most affected by the PSP and maximum voluntary contraction (MVC) is measured using fMRI immediately after the test. 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 MPT is a technique in which MRI scanners detect differences in the volume of a brain region. 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 PSP. 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 may 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.

The present invention also contemplates treating or improving neurogenesis in a subject with decreased or impaired neurogenesis, which may manifest itself, for example, through decreased cognitive or motor function, or through association with neuroinflammation. An embodiment of the present invention includes administering, by way of example and not limitation, blood plasma, plasma fraction, or PPF to a subject with reduced or impaired neurogenesis using an intermittent dosing regimen.

An embodiment of the present invention also provides for determining the level of neuroge

- 30 046284 immediately before, during and/or after the 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 the tracer, [18F]FLT, in combination with the BBB transporter inhibitor probenecid, allows accumulation of the tracer in neurogenic areas of the brain. This imaging allows for the assessment of neurogenesis in patients undergoing treatment for neurodegenerative diseases.

i. Neuroinflammation.

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

An embodiment of the present invention also provides for determining the level of neuroinflammation before, during and/or after administration of blood plasma, plasma fraction or PPF. Non-invasive methods for assessing neuroinflammation such as TSPO positron emission tomography (TSPO PET) using ¹¹ C-PK11195 and other similar indicators have been reported. (See Vivash L, et al., J. Nucl. Med. 2016, 57:165-68 and Janssen B, et al., Biochim. et Biophys. Acta, 2016, 425-41, incorporated herein as links). Invasive methods for assessing neuroinflammation include extracting cerebrospinal fluid and detecting, for example, 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 cell line-derived neurotrophic factor, hyotriosidase-1, MMP-9, chemokine CXC motif 13, terminal complement fragment complex, chitinase-3-like protein 1 and osteopontin. (See VintherJensen 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 dosing regimen.

An embodiment of the invention includes treating a subject who has been diagnosed with cognitive impairment, motor impairment, neuroinflammation, or decreased neurogenesis by administering to the subject an effective amount of blood plasma, or a plasma fraction in the case of a subject who is undergoing, will be undergoing, or has undergone stem cell therapy. Another embodiment of the invention involves administering to a subject an effective amount of blood plasma or a fraction of plasma, wherein the subject is undergoing, will undergo, or has undergone stem cell therapy, and wherein the stem cells used in the therapy may be embryonic stem cells, other than induced embryonic stem cells. pluripotent stem cells (iPSCs), umbilical 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 RH, et al., Brain 2011, 134; 1777-89; Daadi MM, et al., Cell Transplant 2013, 22(5):881-92; Horie N, et al., Stem Cells 2011 29(2 ):doi: 10.1002/stem.584; Thomsen GM, et al., Stem Cells 2018, doi: 10.1002/stem.2825; US patent applications No. 09/973198; 12/258210; 12/596884 and 13/290439 , all of which are incorporated herein 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 loss, heart disease, rheumatoid arthritis or severe burns, or who is in need of a bone marrow transplant, when the subject is undergoing, will undergo, or has undergone stem cell therapy, an effective amount of blood plasma, or a fraction of plasma.

15. Methods for screening compositions.

Methods of screening compositions for activity in treating cognitive or motor impairments, reducing neuroinflammation, or increasing neurogenesis are also provided. Such methods are provided in this invention and include the methods described in the experimental examples below. Compositions that may be screened in embodiments of the present 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 inflammation/markers of inflammation in the hippocampus (eg, dentate gyrus) or other areas of the central nervous system; results of cell proliferation in the hippocampus or other areas of the central nervous system; cell survival in the hippocampus or other areas of the central nervous system; the direction of development of cells (eg, astrocytes, new neurons) of proliferating neuronal progenitor cells (NPCs) in the hippocampus or other areas of the central nervous system; and neurogenesis in the hippocampus or other areas of the central nervous system.

Additional options for methods of screening compositions for activity in treating cognitive or motor impairment, reducing neuroinflammation, or enhancing neurogenesis include:

- 31 046284 dividing the acute effects of compositions on hippocampal inflammation and proliferation, including: administering 5-7 consecutive daily doses of NOS with simultaneous administration for 5-7 consecutive days of the composition being screened for, or a control (intermittent use) in rodents or other animal model. 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 composition being screened, the number of cells in the dentate gyrus is determined by staining NOS, and the percentage of area with CD-68 staining (an indicator of inflammation) is determined.

Another embodiment of methods for screening compositions for activity in treating cognitive or motor impairment, reducing neuroinflammation, or increasing neurogenesis involves administering NOS for 5 consecutive days (once daily) before initiating a regimen of intermittent use for 5-7 days of the composition being screened. or control in rodents or other animal model. After four, five, six, seven, eight, nine, ten, eleven, or twelve weeks, hippocampal cell survival is determined as the number of cells in the dentate gyrus stained with BDU, neurogenesis is determined as the number of cells in the dentate gyrus stained with doublecortin (DCX), and direction the development of neuronal progenitor cells that become astrocytes (associated with aging) or neurons (not associated with aging) is determined by colocalizing NOS with the markers GFAP or Neun, respectively.

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

Another embodiment of methods for screening compositions for activity in treating cognitive or motor impairment, reducing neuroinflammation, or increasing neurogenesis involves intermittent administration of the composition being screened or controlled and determined to improve cognitive or motor function in a rodent or other animal model, such as described in the examples below.

16. Reagents, devices and kits.

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

Reagents and devices of interest include those mentioned above with respect to methods for preparing blood products containing plasma for transfusion to a subject in need thereof, for example, anti-coagulants, cryopreservatives, buffers, isotonic solutions, etc.

The kits may also include blood collection bags, tubing, needles, centrifuge tubes, and the like. In other embodiments, the kits described herein contain two or more containers of blood plasma preparations, such as plasma protein fractions, e.g., three or more, four or more, five or more, including six or more containers of blood plasma preparation . In some cases, the number of different blood plasma product containers in a 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, e.g. 48 or more. Each container may be associated with identifying information that includes various data about the blood plasma product contained in the container, where the identifying 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, for example, whether the drug was plasma is processed to remove proteins above average molecular weight (e.g. as described above), detailed blood type data, etc. In some cases, each container in the kit contains identifying information about the blood plasma contained therein, and the identifying information includes information about the age of the donor of the blood plasma product, for example, the identifying information provides evidence of the age of the donor of the blood plasma product (for example, the identifying information may represent is the age of the donor at the time of blood collection). In some cases, each container in the kit contains a blood plasma preparation from a donor of almost the same age, i.e. all containers contain the drug from donors who are almost the same, if not the same age. By substantially the same age it is meant that the ages of the various donors from whom the blood plasma products for the kits are obtained differ in some cases by 5 years or less, for example 4 years or less, for example 3 years or less, including 2 years or less, for example, 1 year or less, for example, 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, RFID chip, etc., the identification information may be human readable, machine readable, etc. Containers can have any convenient configuration. While the volume of containers may vary, in some cases the volumes range from 10 to 5000 ml, for example from 25 to 2500 ml, for example from 50 to 1000 ml, including from 100 to 500 ml. Containers can be tough

- 32 046284 or flexible and can be made from any convenient material, for example, polymeric materials, including medical grade plastic materials. In some cases, containers have a pouch or pouch configuration. In addition to containers, such kits may further contain an administration device, for example, as described above. The components of such kits may be provided in any suitable package, such as a box or similar structure configured to hold 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 the present invention in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is printed information on a suitable medium or material, such as the sheet or sheets of paper on which the information is printed, in the kit package, in an insert, etc. The other medium will 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, which can be used on the Internet to access information on a remote site. Any convenient products can be included in the kits.

17. Exercise.

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

An exercise regimen may involve performing one exercise at a specific frequency, or a combination of exercises at a specific frequency. The frequency may be one, two, three, four, five, six or seven times a week. 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 the subject was practicing prior to administration of the compositions of the invention. The exercise regimen may be suggested or prescribed by a health care professional or fitness trainer, or the exercise regimen may be initiated by the subject himself.

18. Experimental examples.

a. Example 1.

Young human clarified plasma (young plasma) or commercially available PPF (PPF1) was administered to aged immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG strain). 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. Except where otherwise noted, PPF1 is administered in the examples herein under natural conditions using a 5% solution (w/v, 50 g/L). All mice were assigned to 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 (i.p.) with BDU (5-bromo-2'-deoxyuridine) in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg over 5 days. Following this, mice were administered intravenously (i.v.) 3 times per week with 150 μl of PPF1 for 4 weeks. Behavioral 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 h after the last IV injection, receiving a total of 16 injections over 6 weeks. Two additional cohorts of mice were treated intravenously (IV) for seven consecutive days with 150 μl of PPF1 or saline (intermittent application). Behavior testing was carried out on weeks 5 and 6 at the same time as in the group, 3 times a week.

Behavioral analyzes were analyzed using CleverSys software (Reston, VA). CleverSys TopScan V3.0 was used to track mouse behavior in the zero maze, Barnes maze, open field, and Y maze. Barnes mazes were built by CleverSys. The grip force meter was designed and manufactured by Columbus Instruments (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 (Buffalo Grove, IL) model DM5500B microscope with a DCF7000T bright-field/fluorescence microscope color camera.

Behavior test.

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In fig. Figure 1A shows that the intermittently administered PPF1 groups showed a trend towards increased distance traveled in the open field test compared to both the saline control group and the group receiving PPF1 three times per week. This result indicates a trend towards increased mobility in the intermittent group.

In fig. Figure 1B shows that the intermittently administered PPF1 groups showed a trend towards increased % time spent in the center of the open field compared to both the saline control group and the group receiving PPF1 three times per week. This result indicates a trend toward decreased 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 observed from the injections.

G histology.

In fig. Figure 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 intermittent dosing PPF1 group compared to the three times 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 post hoc multiple comparison analysis (n: saline=8, intermittent PPF1=10, PPF1 3x/week=10).

In fig. Figure 4 shows the number of positively labeled NOS cells in the granular layer of the dentate gyrus of three groups of mice that received separate treatment. There was a significant increase in cell survival between the PPF1 intermittent dosing group compared to the three times 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).

Analysis of hippocampal sections was performed on a Leica (Buffalo Grove, IL) model DM5500B microscope with a DCF7000T bright-field/fluorescence microscope color camera. Staining was with Ki67 Abcam antibody (ab15580) at a ratio of 1:500, and the secondary antibody was goat anti-rabbit antibody (Alex Fluor 555) (ab150090) at a ratio of 1:300.

b. Example 2.

Young clarified plasma (YP), old plasma (SP), or commercially available PPF (PPF1) were administered to aged immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG strain). All mice were assigned to 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 determining the group, mice were injected intraperitoneally (i.p.) 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 administered intravenously (i.v.): 1) three times a week for 6 weeks (3x/week); 2) three times within one week (3x); 3) 7 days for one week, 150 μl of clarified plasma of a young person or PPF1. An additional group of mice was intermittently administered saline solution intravenously for 7 days. The last group of mice was injected with old human plasma 3 times over one week or 7 days over one week. All mice were sacrificed 6 weeks after initiation of young or old plasma, PPF1, or basal solution.

Histology.

In fig. Figure 5 shows the number of DCX-positive cells in the granular layer of the dentate gyrus in nine separately treated groups of mice that received young plasma (YP) or old plasma (SP), or PPF1, or saline. Mice treated with PPF1 through intermittent administration or treated three times a week showed increased neurogenesis compared to other groups. All data are presented as mean ± standard error of the mean; *P<0.05, ANOVA with Dunnett's post hoc analysis, PPF1 (intermittent or 3x/week) and saline (n: saline=4, PPF1 intermittent=5, PPF1 3x/week=5, PPF1 3x=4, MP intermittent=6, MP 3x/week=6, MP 3x=4, SP intermittent=6, SP 3x=6).

In fig. Figure 6 shows the number of positive-labeled NOS cells in the granular layer of the dentate gyrus in nine separately treated groups of mice that received young plasma (YP) or old plasma (SP), or PPF1, or saline. Mice injected with PPF1 showed a significant increase in cell survival compared to the other groups, with mice intermittently injected with PPF1 showing a greater significant difference than mice injected with PPF1 three times per week. All data are presented as mean ± standard error of the mean; **P<0.01, *P<0.05, ANOVA with Dunnett's post hoc analysis, introduction

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PPF1 (intermittent administration or 3x/week) and saline administration (n: saline solution = 4, intermittent administration PPF1 = 5, PPF1 3x/week = 5, PPF1 3x = 4, MP intermittent administration = 6, MP 3x/week week=6, MP 3x=4, SP intermittent administration=6, SP 3x=6).

c. Example 3.

Young clarified plasma (YP), old plasma (SP), or commercially available PPF (PPF1) were administered to aged immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG strain). Mice were administered 7 daily intravenous (IV) doses for 1 week.

All mice were assigned to 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 determining the group, mice were injected intraperitoneally (i.p.) 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 treated intravenously (IV) for seven consecutive days (called intermittent administration) with 150 μl of young or old human plasma, PPF1, or saline. Three weeks after completion of intermittent treatment, mice were administered intraperitoneally (ip) with 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 performed for week 8 (6 weeks after the end of intermittent dosing).

Behavioral analyzes were analyzed using CleverSys software (Reston, VA). CleverSys TopScan V3.0 was used to track mouse behavior in the Barnes maze. Barnes' Maze was built by CleverSys. Analysis of hippocampal sections was performed on a Leica (Buffalo Grove, IL) model DM5500B microscope with a DCF7000T bright-field/fluorescence microscope color camera.

Behavior test.

In fig. Figure 7 shows the latency to find the target hole per trial per day for each treatment group, determined using the Barnes maze. Mice treated with intermittent dosing of PPF1 showed significant reductions in test latency across multiple individual testing sessions, indicating improved cognitive performance. *P<0.05 mean ± SEM; t-test for independent samples (n: saline=13, PPF1=13, SP=14, MP=14).

Histology.

In fig. Figure 8 shows the number of DCX-positive cells in the granular layer of the dentate gyrus in four separately treated groups of mice that received young plasma (YP) or old plasma (SP), or PPF1, or saline. There was a significant increase in neurogenesis with intermittent PPF1 and intermittent young 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) If the experiment lasted longer and histology was performed 12 weeks after the initial intermittent application, the separation between PPF1 and YP was more pronounced, with PPF1 more DCX-positively labeled cells were observed.

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

d. Example 4.

A commercially available PPF (PPF1) was administered to aged immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). Twelve-month-old mice were given 7 daily intravenous (IV) doses via tail vein for 1 week. After treatment, mice were kept in their home cage environment for 4.5 weeks before behavioral testing. All injections and behavioral testing were completed over a 7-week period for each cohort, for a total duration of 9 weeks. All mice received BDU i.p. for 5 days before the first dosing. Mice were sacrificed one day after completion of the last behavioral test.

Behavioral 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 into any familiar or new place

- 35,046,284 cho, of the total number of entries made into each arm by the treatment group in the Y-maze trial. Mice were intermittently injected with saline, PPF1, or 5x concentrated PPF1 at twelve months of age. Mice intermittently injected with PPF1 and PPF1(5x) showed a significant increase in neobrachial input compared to the number of neobrachial inputs in saline-injected mice, indicating improved cognitive performance. All data are presented as mean ± standard error of the mean. *P<0.05, paired t test.

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

G histology.

In fig. Figure 12 shows the number of positively labeled NOS cells in all hippocampal slices. Mice intermittently treated with PPF1 showed a trend towards increased cell survival compared to mice treated with saline and PPF1 (5x). All data are presented as mean ± standard error of the mean.

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

e. Example 5.

A commercially available PPF (PPF1) was administered to aged (10.5 months old) immunocompromised 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 determining the group, mice were injected intraperitoneally (i.p.) 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 administered PPF1 intravenously (i.v.): 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 at 6 week after completion of initial dosing [PPF1-5d-B] 4) 7 consecutive days with an additional 7 consecutive days of booster (B) dosing for 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 administered IP EDU (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 basal solution.

Analysis of hippocampal sections was performed on a Leica (Buffalo Grove, IL) model DM5500B microscope with a DCF7000T bright-field/fluorescence microscope color camera.

In fig. Figure 14 shows the number of DCX-positive 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, PPF1-5dB vs. Saline *P<0.05, ANOVA with Dunnett's post hoc test (n: Saline=5, PPF1-5d=8, PPF1-7d=7, PPF1-5d-B=8, PPF1-7d-B=7).

In fig. Figure 15 shows the number of positively labeled BDU 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 boost dose, compared with groups treated for 5 or 7 consecutive days without a subsequent boost dose. In addition, booster treatment significantly increases overall cell survival. 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 test. PPF1-5d vs PPF1-5d-B +P<0.05, independent samples T-test, (n: saline=7, PPF1-5d=8, PPF1-7d=7, PPF1-5d-B=8 , PPF1-7d-B=7).

In fig. Figure 16 shows the number of positively labeled EDU cells in the granular layer of the dentate gyrus in animals receiving young plasma, PPF1 and saline. These results demonstrate that the effects observed with the booster dose are not due to an increase in the total number of

- 36 046284 proliferating cells present, but associated with an increase in the survival mechanism caused by the administration of a 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.

A commercially available PPF (PPF1) was administered to immunocompromised adult mice (3 and 6 months old) (NOD.Cg-Prkdscid Il2rgtm 1 Wj 1/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 % time spent in the center of the open field. After determining the group, mice were injected intraperitoneally (i.p.) with BDU in PBS (phosphate buffered saline) at a final concentration of 10 mg/ml, dosed at 150 mg/kg for 5 days. Subsequently, mice were administered saline or PPF1 intravenously (IV) for 7 consecutive days (intermittent administration). A subset of mice that were injected with both saline and PPF1 had activity wheels installed in their cages. Mice were sacrificed 3 days, 10 days, or 42 days after completion of intermittent administration.

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

In fig. Figure 18 shows the number of Ki67-positive cells in the granular layer of the dentate gyrus in 3-month-old NSG animals administered 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. saline at 42 days ***P<0.001, ANOVA with Dunnett's post hoc test. (n: saline after 3 days=6, PPF1 after 3 days=6, PPF1 after 10 days=7, saline after 42 days=8, PPF1 after 42 days=8, Activity wheel after 42 days=8, activity wheel +PPF1 after 42 days=8).

In fig. Figure 18 also shows the number of Ki67-positive 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 after 42 days vs. saline after 42 days ***P<0.001, *P<0.05, ANOVA with Dunnett's post hoc test (n: saline after 3 days=7, PPF1 after 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. Figure 19 shows the number of NOS-positive cells in the granular layer of the dentate gyrus in 3-month and 6-month NSG animals administered PPF1 or saline, with or without activity wheels. All data are presented as mean ± standard error of the mean; activity wheel +PPF1 after 42 days vs. basal solution after 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 test).

These results demonstrate that there is a significant increase in neurogenesis with PPF1 and activity wheel compared to basal solution at 6 weeks post-dosing in 3-month-old NSG mice. Additionally, 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 basal 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 basal solution at 6 weeks post-dosing in 3-month and 6-month-old NSG mice.

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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 with 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 significant potential for neurogenesis in the brain, which may be achieved through multiple independent or overlapping mechanisms.

g. Example 7.

PPF1 or saline control was administered to two treatment groups of 11-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). All mice received intravenous injections of 150 μl of PPF1 or saline per dose for seven consecutive days. An activity wheel (MedAssociates) was placed in the 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. Figure 20 presents the number of wheel rotations during given periods of time, with a shaded area indicating the dark cycle. An independent samples t test was used to assess the statistical significance of total mileage for both the treated and untreated groups, in light and dark cycles. Rhythmic expression profiles were extracted and characterized using time and frequency data analysis for a series of 13 time points, separately for each mouse in the 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 samples t test. Mice treated with PPF1 ran significantly further than animals that received no treatment, an indicator of increased locomotor activity. The mice were subjected to a hot plate test to monitor for relatively normal pain sensations in their paws. Sensory loss may have affected previous behavioral readings. Hot plate testing resulted in a slight increase in activity after the activity wheel was returned to the cell, as evidenced by the jump in wheel revolutions indicated in the circled segment of Fig. 20.

h. Example 8.

Recombinant human albumin (rhAlbumin, Albumedix, Ltd, Nottingham, UK), young clarified plasma (MP) or saline control was administered to 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, line NSG). All animals received 50 mg/kg BDU i.p. 1 week before 7 days of intermittent administration. RhAlbumin was 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. Figure 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, independent samples t test).

In fig. 21B demonstrates the amount of DCX staining in all 3 groups as determined by the number 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 decreased neurogenesis compared to saline control. All data are presented as mean ± standard error of the mean. (**P<0.01; ***P<0.001, independent samples t test).

i. Example 9.

Dissociated mixed neuronal cells obtained from embryonic mouse cortex were seeded and grown in a 48-well multielectrode plate (Axion Biosystems). Each well contains 16 electrodes that are in physical contact with the seeded neuronal cells and measures subtle changes in the properties of the cell membranes. This setup allows the assessment of various parameters to obtain information about the neural spiking activity of heating behavior at the single electrode level, as well as information about the extent of neural coupling by assessing the synchrony of neural heating properties between multiple electrodes within a well.

Neuronal cultures were maintained in the presence of treatment conditions starting on day 1. Treatment conditions included Neurobasal medium supplemented with B27 and brain-derived neurotrophic factor (BDNF), which contained 10% (v/v): recombinant human albumin ((rhAlbumin, Albumedix, Ltd, Nottingham, UK); PPF1 or HAS1. Basic the solution constituted the control.Neural activity was measured on days 7, 12, 16 and 21.

In fig. 22A-22C depict that although treatment with both PPF1 and HAS1 stimulated

- 38 046284 neural spiking activity (Fig. 22A, two series of peaks with MEA, and Fig. 22B, quantification of neural spiking activity) compared to control or rhAlbumin treatment, only PPF1 treatment increased the number of neural network bursts during culture maturation neurons (Fig. 22C). HAS1 is a commercially available HAS with greater than 95% human albumin (based on total protein) in a 5% solution (w/v, 50 g/L), prepared by cold alcohol fractionation, and obtained from the combined human plasma from donors. Both PPF1 and HAS1 were supplied in 5% solution (w/v, 50 g/L) and diluted 1:10 in Neurobasal medium with B27 supplement. This effect of PPF1 on the activity of the neural network persists for 21 days in culture. This indicates that PPF1 is associated with activation of neural network maturation. Data are presented as the mean of n=25-45 wells from 3 independent experiments ± standard error of the mean, independent samples Student's t test *p<0.05, #p<0.01.

j. Example 10.

Old human clarified plasma (SP) or sterile saline solution was administered to 8-week-old (young) immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj 1/SzJ, NSG line). In each experiment, mice were assigned by weight to different treatment groups. All mice were administered intravenously for 5 consecutive days with 150 mg/kg BDU in sterile PBS. After BDU administration, old plasma was administered intravenously in different treatment paradigms at 150 μl per dose. All paradigms are indicated in Fig. 23.

Paradigm 1 involves injections twice weekly for a total of 10 injections over 5 weeks. Histological analysis was performed 48 hours after the last dose of plasma. Paradigm 2 involves 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 histologically analyzed 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 mice treated with aged plasma were analyzed for the endothelial inflammatory 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 a 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 Leica wide-field microscope (Leica).

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

The effect of 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 old plasma on neurogenesis in the dosing paradigms with administration twice a week (Fig. 25A) or three times a week (Fig. 25B), however, intermittent application for 7 consecutive days (Fig. 25C) resulted in a significant decrease in the number of DCX-positive cells. These data suggest that only intermittent administration of aged plasma has a significant effect on neurogenesis.

k. Example 11.

Eight-week-old NSG mice injected with old human plasma (65–68 year old origin) for 7 consecutive days were tested using a modified Barnes maze at week 4 after the last old plasma injection.

In fig. Figure 26 shows the time course of latency to exit the Barnes maze and indicates the time required to reach and enter the exit hole for NSG mice injected with aged plasma and saline. There were no significant differences in exit latency between groups, but on day 4, mice injected with old plasma performed worse than mice given control saline. These findings indicate decreased learning and memory associated with hippocampal function in a spatial memory task. All data are presented as mean ± standard error of the mean. Two-factor ANOVA, post hoc Šidák test).

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

In fig. Figure 28 presents the difference in exit latency between trials 1 and 3 in the Barnes maze and demonstrates that these trials can be used as a measure of learning over time.

- 39 046284 one day. Mice injected with old plasma had a significantly lower difference in exit latency between these studies, indicating a decrease in learning ability. All data are presented as mean ± standard error of the mean. (*P<0.05, independent samples t test).

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

1. Example 12.

Young (8-week-old) immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj 1/SzJ, NSG line) were divided by weight into different treatment groups. Animals were injected subcutaneously (SC) with 35 mg/kg 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. Two hours after kainic acid injection, mice were intravenously injected with 150 μl of PPF1 or saline. Administration of PPF1 or saline was 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 activation (CD68) and astrocyte activation (GFAP). Sections were imaged on a Hamamatsu NanoZoomer HT (Hamamatsu) after immunohistochemistry on 30-μm free-floating sections and analyzed using Image Pro software (Media Cybernetics).

Analysis of the % CD68 positive area in the CA1 region of the hippocampus demonstrates that kainic acid administration results in increased CD68 immunoreactivity, suggesting increased microglial activation (FIG. 31A). Five days of PPF1 administration led to a significant decrease in the % CD68-positive region and, consequently, a decrease in microglial activation. Likewise, analysis of the % GFAP positive region (Fig. 31B) shows a significant increase following kainic acid administration, which is significantly reduced following PPF1 dosing. Evidence suggests that PPF1 has acute anti-inflammatory effects in the brains of kainic acid-treated mice. *P<0.05 one-way ANOVA with Dunnett's multiple comparison post hoc analysis.

m. Example 13.

NSG mice aged 6 months were administered intravenously PPF1 or saline control at a dose of 150 μl (10 mg/ml) daily for one week (7 days). All mice received BDU, 50 mg/kg BDU i.p. once daily on the same day they received PPF1 or saline control. The mice were divided into two cohorts. The first cohort was sacrificed on the day immediately after 7 days of concomitant treatment with NOS and PPF1. The second cohort was sacrificed 7 days later, and the animals received an additional 7 days of daily BDU administration.

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

n. Example 14.

NSG mice aged 3 or 6 months were treated daily for one week (7 days), i.v., with PPF1 or basal saline. Mice were then sacrificed 3, 10, or 42 days after the 7 daily doses were administered. The brains were stained with Ki67, a nuclear marker present only in proliferating cells that labels neural stem and progenitor cells in the dentate gyrus blade.

In fig. Figure 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 a 7-day sequential regimen of intermittent PPF1 administration.

In fig. Figure 34 shows Ki67 staining (light areas) in the dentate gyrus on day 10 in NSG mice after completing a 7-day sequential PPF1 intermittent regimen. This indicates that one possible mechanism for PPF1 to increase overall cell survival and neurogenesis at 42 days post-dosing may be due to an increase in the total number of progenitor cells (neural stem cells).

o. Example 15.

A commercially available PPF (PPF1) or control saline solution was administered to two different

- 40 046284 populations of mice aged 6 and 12 months with weakened immunity (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 application 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 proliferation studies and sacrificed 6 weeks after the last dose administered.

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

In fig. 35B shows that cohorts of 12-month-old mice treated with PPF1 showed a significant increase in the number of progenitor cells that differentiated into neurons (NeuN+) compared to 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, independent samples t test).

p. Example 16.

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

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

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. Two and a half (2.5) hours after the last test agent administration, mice were killed by transcardial perfusion of 0.9% saline followed by 4% formaldehyde under deep ketamine and xylazine anesthesia. Brains were dissected, fixed, and then processed using the iDisco procedure to visualize cFos-positive cells via planar illumination fluorescence microscopy (LSFM) at 2 x 2 x 3 μm voxel resolution. The imaged brain samples were aligned as 3D volumetric objects, and activated cFos-positive cells were detected computationally. Statistical comparisons between groups were performed using negative binomial regression, corrected for multiple comparisons for false discovery rates. (* indicates a q value less than 0.05).

Analysis of mouse brains 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 of 18-month-old PPF1-treated mice (Figures 37A-37C). Using binomial regression adjusted for multiple comparisons, differences in this increase in total cFos-positive cells did not reach significance. However, analysis of more specific cortical areas, such as the frontal, orbital, infralimbal and prelimbal cortices, demonstrated a significant increase in the number of cFos-positive cells (Fig. 38A-38D), indicating increased neuronal activity. Increased activity in the prefrontal cortex correlates with increased cognitive function, suggesting that PPF1 treatment leads to cognitive improvements in aged C57BL/6 mice. A similar significant increase in the number of cFos-positive cells was also found in the accessory olfactory nucleus and olfactory tubercle (Figs. 39A and 39B). These areas are associated with the processing of olfactory information, and increased activity indicates increased olfactory function. Voxel-based visualizations based on cFos activation statistics in red demonstrated an increase in cFos signal in the cortex of PPF1-injected mice (Figure 40).

r. Example 18.

Commercially available PPF (PPF1) or saline control was administered to 22-month-olds.

- 41 046284 wild type (WT) sham (C57BL/6J, WT, Line Code 0664, Jackson Labs, Bar Harbor, Maine). All animals received 50 mg/kg BDU at 1 week prior to 7 days of 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) 10 days after dosing.

In fig. 41B shows the percentage of Iba-1 immunoreactive area in the hippocampus (n=10, 10) 10 days after dosing. In fig. 41C shows the percentage of GFAP immunoreactive area in the hippocampus (n=10, 10) 10 days after dosing.

In fig. 41D shows the percentage of CD68 immunoreactive area in the hippocampus (n=11, 12) 4 weeks after dosing. In fig. 41E presented an Iba-1 immunoreactive area in the hippocampus (n=11, 12) 4 weeks after dosing. All data are presented as mean ± standard error of the mean. (*P<0.05; **P<0.01; ***P<0.001 by t-test for independent samples). These results demonstrate a significant decrease in microglial markers CD68 and Iba-1 in the hippocampus of aged mice 10 days and 4 weeks after treatment with PPF1.

s. Example 19.

Commercially available PPF (PPF1) or saline control were administered to 23-month-old wild-type ^^'576^6.1 mice. DT, Line Code 0664, Jackson Labs, Bar Harbor, Maine). All animals received 50 mg/kg BDU at 1 week prior to seven days of sequential 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 for examination of histological markers of neuroinflammation and sacrificed 6 weeks after the last dose administered.

In fig. 42A shows the percentage change in NOS expression compared to saline control at 6, 9 and 12 weeks post-dosing, which is an indicator of cell survival in the hippocampus. Animals received a 7-day intermittent dosing 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 an indicator of cell neurogenesis in the hippocampus. Animals received a 7-day intermittent dosing regimen.

t. Example 20.

Thirty (30) male alpha-synuclein transgenic mice (line 61, wild-type C57BL/6J background) aged 4 to 4.5 months were divided into two groups of 15 and treated with PPF1 or basal solution for seven (7) consecutive days. PPF1 was administered intravenously at 5 μl per gram 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 administration of PPF1 or basal solution, all mice were tested for cognitive and motor function, such as nest building, noodle chewing, wire hanging, rotarod test, and pole walking. Noodle 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 (Tg) mice administered PPF1 and veh.

In fig. 44 presents the results of nest building. Mice were housed individually in cages containing wood chip bedding and one square of baled cotton (nestlet). No other litter material (eg, wood fibers) was present. Nestlet was introduced the day before the nest condition assessment, approximately 2–3 h before the dark phase was initiated, and nest-building behavior was assessed on the next day of the experiment for approximately 2–3 h after the light phase began. The time interval between the introduction of the cotton square and the assessment of the next status was the same for all studies. The nestlet manipulation and the structure of the created nest were assessed on a five-point scale (Deacon, RM 2006, Assessing nest building in mice. Nat Protoc 1:1117-19). As shown in FIG. 44, there was an increased trend in nesting behavior in PPF1-injected mice compared with basal solution-injected mice.

In fig. 45A and 45B show that there was a significant increase in noodle chewing in the PPF1-administered group compared to the basal solution group 3 weeks after the last administration, indicating improved 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 noodles (approx. 5 mm) was placed into 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 to gnaw and the frequency of bites were assessed, and the gnaw profile was analyzed using Avisoft SASLab Pro software. All data before

- 42 046284 are given as the mean ± standard error of the mean. (*P<0.05, independent samples t test).

In fig. 46 shows the results of wire hanging tests, which are used to evaluate neuromuscular abnormalities of motor strength. There was a significant increase in time to fall in the PPF1-administered group compared with the basal solution group 3 weeks after the last administration. To perform the test, a cage lid made of metal wire was used, and adhesive tape was placed around the perimeter to prevent the mouse from walking on the edge. The animal was placed on the top of the cage lid. The lid was shaken lightly three times to force the mouse to grasp the wire, and then the lid was turned upside down. The lid was secured at a height of approximately 50-60 cm above the soft pad, high enough to prevent the mouse from jumping down, but not high enough to cause harm if it fell. Latency to fall was measured and a 300-second cutoff was used. Typically, wild-type mice can hang upside down for several minutes.

In fig. 47A, 47B and 47C show the results of a pole walking test. In fig. Figure 47A shows various pole shapes and sizes (square or cylindrical poles) used in five different tests of increasing difficulty. In fig. 47B shows the results of five tests 72 hours after the last administration. In fig. 47C shows the results of five tests 3 weeks after the last administration. PPF1-injected mice showed significantly higher pole-walking success during Test 1 trial 5 (72 h post-administration) and Test 2 trial 4 (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 for 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 figures show that PPF1-treated mice showed decreased microgliosis (neuroinflammation) as measured by Iba-1 and CD68 staining in both the striatum and hippocampus, and increased neuronal survival as measured 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, independent samples t test).

u. Example 21.

PPF1, HAS1, or saline control 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 (based on total protein) in a 5% solution (w/v, 50 g/L), prepared by cold alcohol fractionation, and obtained from the combined human plasma from donors. Except where otherwise noted, HAS1 is administered in the examples herein under natural conditions using a 5% solution. Mice were administered intravenously 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 latency to exit the Barnes maze for the mouse to find the exit hole for mice injected with PPF1, HAS1 and basal solution. Mice injected with PPF1 found the exit hole significantly faster than animals injected with the basal solution. These data demonstrate that PPF1 effectively enhances cognitive function in aged NSG animals, whereas HAS1 administration has no effect on hippocampus-dependent memory. All data are presented as mean ± standard error of the mean. (*P<0.05, independent samples t test).

v. Example 22.

Clinical paradigms using PPF.

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

Primary endpoints are safety, tolerability, and feasibility of each regimen. Safety is determined by the frequency of adverse events occurring during treatment. Tolerability was determined by the number of subjects completing an 8-week course with at least 5 infusions and subjects completing a 24-week course with at least 10 infusions.

- 43 046284 fusions. Study feasibility was determined by the number of subjects completing a course of 5 and 10 infusions. Secondary endpoints measure potential effects on cognitive function using various established cognitive measures, including the cognitive subscale of the Alzheimer's Disease Severity Rating Scale. Exploratory endpoints include assessment of changes in the composition and distribution of blood biomarkers, as well as changes in magnetic resonance imaging.

(2) AD, mild to moderate. Two groups of subjects diagnosed with mild to moderate asthma are randomized to active treatment in a double-blind manner. All subjects received one infusion per day of a 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 divided by gender. The duration of administration is 2-2.5 hours, and the titration rate is selected according to dose-specific guidelines so that the entire dose is administered.

Subjects are participating in an additional study of CSF (cerebrospinal fluid) biomarkers. 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 at the initial visit, and assessments at periodic intermediate visits occur after dosing.

Determine the safety, tolerability, and feasibility of each regimen. During the study, cognitive assessments are determined and summarized, including the following: Mini Mental State Examination (MMSE); Alzheimer's Disease Severity Scale 11-item cognitive subscale (ADAS-Cog/11); test with a board with grooves for inserting pins; Category Speed Test (CFT); Clinical Dementia Rating Scale - Sum of Checkboxes (CDR-SOB); Alzheimer's Disease Cooperative Study - Activities of Daily Living (ADCS-ADL); Alzheimer's Disease Cooperative Study—Change in Clinical Global Impression Scale (ADCS-CGIC); Neuropsychiatric Performance Questionnaire (NPI-Q) and Savonix Neurocognitive Assessments, and forward and backward digit repetition.

(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 approximately 7 months. The duration of administration is 2-2.5 hours, and the titration rate is selected according to dose-specific guidelines so that the entire dose is administered.

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

w. Example 23.

Intermittent application increases the number of synapses and the degree of synaptic connection.

Tissue collection and histology were performed as follows. C57BL/6 mice were deeply anesthetized with Avertin (250 mg/kg i.p.). Mice treated with PPF1 or control were anesthetized 10 days after completion of dosing. Brains were collected after saline perfusion and separated using a midsagittal section and fixed with half a drop of freshly prepared 4% PFA (paraformaldehyde). After 24-48 hours, PFA was replaced with 30% sucrose. After another 24 hours, a second replacement was made with 30% sucrose. The other half was dissected into the hippocampus and cortex and then flash frozen on dry ice. Fixed brain tissue was sectioned and analyzed for the synaptic markers SYNAPSYN1 and PSD-95 and photographed using a Zeiss LSM800 with Airyscan. Z-stacks were analyzed using the synapse counting macro in ImageJ, which quantified the number and size of pre- and postsynaptic puncta. The total number of synapses was assessed by counting the number of colocalized or combined pre- and postsynaptic points. Frozen brain tissue was analyzed for the presence of synaptic markers using qRT-PCR.

Old mice have significant synaptic loss compared to young mice (see Morrison JH et al., The Aging Cortical Synapse: Hallmarks and Implications for Cognitive Decline, Nature Reviews Neuroscience. March 7, 2012; and Shi Q et al., Complement CS-Deficient Mice Failure to Display Age-Related Hippocampal Decline, Journal of Neuroscience, September 23, 2015) FIG. 50 and 51 show that in 24-month-old mice (24M) a decrease in the level of the postsynaptic marker PSD-95 was observed compared with young 3-month-old mice (3M) in brain regions containing a significant number of synapses (in the hippocampus (HP), striatum ( ST), substantia nigra (SN)), but not in areas of the brain that do not contain synapses (corpus callosum (CC)).

In fig. 52A and 52B indicate that mice receiving intermittent dosing of PPF1 had significantly higher levels of the postsynaptic marker PSD-95 (Fig. 52A) and higher levels (statistical trend, p=0.07) of the presynaptic marker SYNAPSYN1 (Fig. 52B) , quantitatively

- 44 046284 determined by measuring the integrated optical density (IOD) in the CA1 hippocampus. T tests for independent samples, *p<0.05.

In fig. 53 is a histological image of CA1 hippocampal sections quantified in FIG. 52.

In fig. 54A and 54B show a high-resolution confocal micrograph (FIG. 54A) and an enlarged inset (FIG. 54B) in which synapses are indicated by yellow arrows, with presynaptic (SYNAPSYN1, red) and postsynaptic (PSD-95, white) markers aligned with a friend. The image was acquired from the same tissue that was quantified in FIG. 52A and 52B.

In fig. 55A-55D indicate that mice receiving intermittent dosing of PPF1 had a significantly higher number of presynaptic puncta than mice receiving control treatment, while the number of postsynaptic puncta (PSD-95) remained unchanged. In fig. 55A shows a set of representative high-resolution micrographs of saline- and PPF1-treated mice with presynaptic (SYNAPSYN1, red) and postsynaptic (PSD-95, white) markers aligned. In fig. 55B shows the results of quantification of combined pre- and postsynaptic markers, and FIG. Figure 55C presents the results of assessing the number of SYNAPSYN1 points. The number of postsynaptic sites remained unchanged among treatment groups. (Fig. 55D). T-tests for independent samples, *p<0.05. From these results, it is clear that PPF1, which was given intermittently, increases the number of synapses and the degree of synaptic connectivity between neurons, which is associated with enhanced cognitive ability.

x. Example 24.

The cortex or hippocampus was dissected from dissected E16 mouse brains and grown separately in PDL-coated 96-well glass-bottom plates at a density of 20 thousand cells per well. Neuronal cultures were maintained in the presence of treatment conditions for 14 days. Treatment conditions included B27 and BDNF supplemented medium that contained 10% (v/v) recombinant human albumin (rhAlbumin, Albumedix, Ltd, Nottingham, UK), PPF1 or HAS1, and medium changes were made twice a week. Cells were fixed after 14 days in culture and immunocytochemistry was performed with the following markers: Hoechst to stain all nuclei, anti-Map2 to detect neurons, anti-Gfap to detect astrocytes, and anti-nestin to detect progenitor cells.Images were obtained using an INCell analyzer 2000 (GE Healthcare).

In fig. 56B and 56B depict primary cortical (FIG. 56A) and hippocampal (FIG. 56B) neurons from mice that were cultured in the presence of recombinant human albumin (rhAlbumin), PPF1, or HAS1 for 14 days, and cellular composition and morphology were assessed by immunostaining for Map2 (neurons), Gfap (astrocytes) and nestin (progenitor cells).

At early stages of development, as well as during neurogenesis, proliferating neuronal precursor cells or radial glial cells take the direction of either the neuronal lineage and the formation of postmitotic neurons, or maturation into astrocytes (Berg AD et al., Radial glial cells in the adult dentate gyrus: what are they and where do they come from?, F1000Research 2018). These ray gliocytes are positive for the marker proteins nestin and Gfap and can further divide and increase in number before they differentiate into more mature astrocytes that express only Gfap and have a more complex morphology. Astrocytes play an important role in maintaining overall brain health and mediate functions such as intracellular and extracellular buffering of ions and metabolites, transport and metabolism of metabolites, binding to the vasculature, regulating the uptake of glutamate, GABA and other neurotransmitters, or mediating antioxidant functions (see Kimelberg HK, et al., Functions of astrocytes and their potential as therapeutic targets, Neurotherapeutics, 7(4):338-53, 2010). However, over the past 20 years, it has been discovered that astrocytes play a more active role than just a supporting cell in the brain. Astrocytes have been described to secrete soluble factors that stimulate the formation, maturation and plasticity of synapses, and are therefore actively involved in the formation of new synapses and maintaining the integrity of the neural network (Baldwin KT et al., Molecular mechanisms of astrocyte-induced synaptogenesis, Curr. Opin. Neurobiol., 45:113-20 2017; and Clarke LE et al., Emerging roles of astrocytes in neural circuit development, Nat. Rev. Neurosci. 14:311-212013.)

In fig. 56A and 56B show that 14 days of treatment with PPF1 results in an increase in the number of nestin- and GFAP-positive cells in cortical culture compared to untreated or rhAlbumin-treated cultures. In the case of HAS1, an increased trend can be observed. Similarly, for the hippocampal cultures in Fig. 57A and 57B show that treatment with PPF1 results in an increase in the number of cells expressing nestin and GFAP in hypocampal cultures compared to untreated or rhAlbumin treated conditions. In addition, nestin and GFAP double positive cells, as well as cells expressing only GFAP, exhibit more complex morphology compared to control-treated cells, suggesting that these gliocytes are more mature under PPF1-treated conditions compared to controls. conditions. In case of processing using

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HAS 1, a similar trend can be observed.

In fig. 57A shows Western blot results showing an increase in Gfap protein expression in cultures of cortical neurons treated for 14 days with PPF1 HAS1. Beta-actin was used as a loading control. Similarly, counting the proportion of Sox2-positive cells relative to the total number of cells in cultured cortical neurons indicates a significant increase in the population of Sox2-positive cells when treated with PPF1 for 14 days (Fig. 57B). When treated with HAS1, a similar trend was observed. Sox2 is a nuclear marker used to identify glial cell precursors as well as mature astrocytes.

The observation that neuronal cultures treated with PPF1 are characterized by an increase in neuronal activity, coupled with the observation that treatment with PPF1 appears to increase the population of glial cell precursors and mature GFAP-positive astrocytes, suggests that treatment with PPF1 tends to increase the formation of a pool of healthy astrocytes, which, in turn, increases the degree of neural communication in vitro.

y. Example 25.

PPF1 or saline control was administered to 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG strain). Mice received IV injections of 150 μl PPF1 (n=10) or saline (n=15) for 7 consecutive days, and cognitive behavior testing in a 2-choice swim test was performed 6 weeks after dosing, when mice were at the age of 12 months. The two-choice swim test measures hippocampus-dependent memory by quantifying the latency to reach the exit platform and cortical-dependent executive memory by recording whether the first direction choice made upon exiting the initial arm of the maze is correct or incorrect. The test consisted of a T-maze that was immersed in opaque water, with an exit platform at the end of one of the T-arms. On the first day, mice were trained with a visible platform, gently placed on the bottom of a T-shape, and allowed to swim to the platform for 4 trials. On the second day, the mice were again placed on the bottom of the T-arm, and the exit platform was immersed just below the surface of the water. The results of the analysis were recorded as correct or incorrect choice when exiting the lower arm and latency to reach the exit platform.

In fig. 58A shows that mice treated with PPF1 have a strong tendency to increase latency to exit on day 2, indicating enhanced hippocampus-dependent cognitive function.

In fig. 58B shows an enhancement of cortical-dependent memory, where animals treated with PPF1 show greater success in correctly choosing the direction to the platform compared to mice treated with saline. These data are significant with a p-value of 0.041 by chi-square test. Overall, the data show that treatment with PPF1 can improve hippocampus-dependent and cortical-dependent memory, suggesting beneficial effects on more than one brain region in aged mice.

z. Example 26.

PPF1, young plasma (YP), saline control, or old plasma (SP) were administered to 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG strain). Mice received 150 mg/kg BDU i.p. daily for 5 days followed by i.v. injections of 150 μl saline (n=13), young plasma (MP, n=13), PPF1 (n=15) or old plasma (SP, 14) for 7 consecutive days and were sacrificed when the mice reached 12 months of age. With NOS injections, all proliferating cells will be labeled, and in the dentate gyrus, neural stem and progenitor cells will be primarily labeled. By assessing the number of NOS-positive cells 6 weeks after the start of treatment and 7 weeks after NOS injection, these indicators can be used to assess cell survival. In addition, stem and neuronal progenitor cells that are labeled at that time will differentiate into either NeuN-positive neurons or GFAP-positive astrocytes. By co-staining for NOS, NeuN and GFAP, it is possible to determine whether there are more new neurons in the brains of treated animals and whether there is a shift in the direction of cell development with treatment. Newly formed neurons in the dentate gyrus can integrate into and strengthen the hippocampal network. In fig. Figure 59 shows a representative image of NOS, NeuN, and GFAP staining in the dentate gyrus of a 12-month-old NSG mouse. In fig. 60A shows the results of determining the total number of cells co-labeled with NOS/NeuN and NOS/GFAP in mice treated with saline control, young plasma (YP), PPF1 or old plasma (SP). Mice treated with PPF1 had significantly more NOS/NeuN double-positive cells than mice treated with saline or SP. There was also a trend toward an increase in the number of NOS/NeuN double-positive cells when comparing MP- and PPF1-treated mice, suggesting that more new neurons appeared after treatment with PPF1 compared to treatment with

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MP, saline or SP. There was no change in the number of NOS/GFAP double positive cells. In fig. 60B shows the same data as FIG. 60A, but quantified as % NOS/NeuN and NOS/GFAP double positive cells from all NOS-labeled cells. This quantification allows us to determine whether treatment alters the direction of neuronal stem or progenitor cell development. In this case, there is a trend towards an increase in the % of double positive NOS/NeuN cells with MP and PPF1, which is slightly more pronounced in animals treated with PPF1. Following treatment with SP, there also tended to be an increase in astrocyte formation at the expense of neuron formation, suggesting that SP has an effect inverse to MP or PPF1. Together, these data demonstrate that PPF1 increases the formation of new neurons more strongly than MP, and that this results in a small shift in the direction of stem and progenitor cell development toward the neuronal lineage. These new neurons become embedded in the hippocampal network and may improve memory function.

aa. Example 27.

PPF1, young plasma (YP), or saline control were administered to 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). Mice received intravenous injections of 150 μl saline (n=13), young plasma (MP, n=14) or PPF1 (n=13) for 7 consecutive days, and cognitive behavior testing in the Barnes maze was performed 6 weeks after dosing when mice were 12 months old. The Barnes maze measures hippocampus-dependent memory by measuring how long it takes mice to reach an exit hole using visual cues placed around the maze. Briefly, in this particular assay, mice are subjected to 5 exit hole search trials per day for 3 consecutive days, and the exit hole is left in the same location for all three days.

In fig. 61A shows that mice treated with PPF1 find the exit hole significantly faster than mice treated with saline or MP, demonstrating improved cognitive function. In fig. Figure 61B shows the mean latency to exit for trials 14 and 15 as a complementary measure to the performance obtained in the Barnes maze, and here PPF1 animals also showed a strong tendency to improve memory compared to mice receiving MP or saline control.

bb. Example 28.

PPF1 or saline control was administered to 3-month-old, 6-month-old, and 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). Mice received IV injections of 150 μl of saline control or PPF1 for 7 consecutive days and were sacrificed after 6 weeks for analysis of neurogenesis by doublecortin immunohistochemistry at 4.5, 7.5, and 12 months of age. Doublecortin marks newly formed neurons and allows us to determine the number of newly formed neurons in the dentate gyrus.

In fig. 62A shows that there is an age-related decrease in the number of doublecortin (DCX)-positive newborn neurons and that treatment with PPF1 significantly increases the number of DCX-positive cells in 7.5- and 12-month-old animals (4.5-month-old: saline n= 8, PPF1 n=8; 7.5 months: saline n=8, PPF1 n=6; 12 months: saline n=15, PPF1 n=14). In addition, there was a trend toward an increase in the number of DCX-positive cells in 4.5-month-old mice. In fig. 62B presents the same data, but in a different format, by comparing the levels of neurogenesis at the time of dosing with the level of neurogenesis at the time of sacrifice. This graph shows that treatment with PPF1 reverses the age-related decline in neurogenesis in 7.5 month and 12 month mice and restores or maintains it at the same level as in mice at the time of dosing at 6 months and 10 months. ,5 months of age, respectively.

cc. Example 29.

PPF1 or saline control was administered to 6-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, NSG line). Mice received intravenous injections of 150 μl of PPF1 (n=10) or saline (n=15) for 7 consecutive days and were sacrificed for analysis 24 hours after the last dose. Inflammatory changes in the hippocampus were quantified using CD68 immunohistochemistry, which labels activated microglia.

In fig. 63 shows the results of quantification of CD68-positive area in the hippocampus of mice treated with saline (n=10) and PPF1 (n=10). There was a significant reduction in CD68 levels, suggesting reduced inflammation in the brains of these mice.

dd. Example 30.

PPF1 or saline control was administered to 24-month-old wild-type (C57BL/6, WT) mice. Mice received intravenous injections of 150 μl of PPF1 (n=10) or saline (n=10) for 7 consecutive days and were sacrificed for analysis 10 days after the last dose. Histological analysis of microglial markers CD68 and Iba-1 was performed using immunofluorescence images obtained with a Hamamatsu slide scanner.

In fig. 64A and 64B are representative images of mice treated with

- 47 046284 left solution or PPF1 in the case of CD68 (Fig. 64A) and Iba-1 (Fig. 64B). Levels of both markers were reduced in the brains of mice treated with PPF1, suggesting decreased age-related microglial activation and therefore reduced inflammation.

ee. Example 31.

Two intermittent booster regimens were administered to NSG mice starting at either 6 months of age (FIG. 65A) or 3 months of age (FIG. 65B). A cohort of mice initially treated intravenously at 6 months of age received 2 doses of boosters for seven consecutive days, 8 weeks apart. A cohort of mice initially treated intravenously at 3 months of age received 3 doses of boosters over seven consecutive days at 8, 8, and 7 week intervals. For both groups, behavioral testing was performed 6 weeks after the last intermittent treatment regimen, when all mice were 12 months old.

In fig. 66 depicts the total number of DCX-positive cells (which are indicative of neurogenesis) in the hippocampus counted in five middle hippocampal slices from 12-month-old NSG mice treated at 3 months of age with: basal solution without exercise; intermittent doses of PPF1 for 7 days; intermittent doses of PPF1 for 7 days, then two subsequent booster regimens with an interval of 8 weeks between regimens; and a baseline exercise solution for the remainder of the study. All data are mean ± SEM, ****P<.0001, ***P<.0001, ANOVA with Dunnett's post hoc test.

This resulted in a significant increase in neurogenesis compared to both single intermittent PPF1 treatment (without booster) and basal solution treatment. The results of this observation indicate that repeated administration of PPF does not deplete neuronal progenitor cell (NPC) pools because the generation of immature neurons from NPCs still occurs regardless of repeated intermittent booster doses every two months.

A similar trend was observed in mice that were first treated at 3 months of age. In fig. 67 depicts the total number of DCX-positive cells (which are indicative of neurogenesis) in the hippocampus counted in five middle hippocampal slices from 12-month-old NSG mice treated at 3 months of age with: basal solution without exercise; intermittent doses of PPF1 for 7 days; intermittent doses of PPF1 for 7 days, then three subsequent booster regimens with an interval of 8, 8, and 7 weeks between regimens, respectively; and a baseline exercise solution for the remainder of the study.

In fig. 68 depicts Y-maze behavior in 12-month-old NSG mice compared to mice from the original 6-month cohort (see FIGS. 65A and 66). The percentage of total number of entries into the new arm of the Y-maze in mice receiving PPF1 and PPF1 plus boosters showed significant increases in spatial learning and memory compared to controls.

ff. Example 32.

Primary mouse cortical neurons (FIG. 69A) and hippocampal neurons (FIG. 69B) were maintained in the presence of control treatment, rhAlbumin, PPF1 (two different batches) and HAS1. Neurite length was assessed after 96 h in culture. Treatment of both types of neurons with PPF1 or HAS1 resulted in a significant stimulation of neurite outgrowth compared with control treatment. Treatment with PPF1 or HAS1 also resulted in increased neurite outgrowth compared to treatment with rhAlbumin. Neurite outgrowth is an important physiological process for the newly formed neuron, which occurs before attachment and integration into the neural network. N=11-16 independent experiments ± standard error of the mean, one-way ANOVA *p<0.05, **p<0.01.

Primary mouse cortical neurons were left untreated or maintained in the presence of control, rhAlbumin, PPF1 (two different batches), and HAS1 treatment for 14 days.

In fig. 70A shows increased mRNA expression of synaptic markers (SYN1 and PSD-95) in cells treated with PPF1 and HAS1 compared to control, with less expression observed with HAS1 treatment for the postsynaptic marker, PSD-95, compared to treatment with PPF1.

In fig. 70B shows increased protein levels of the presynaptic marker SYN1 in neuronal cultures treated with PPF1 and HAS1 compared to control, untreated, and rhAlbumin-treated neurons. Tuj1, a common neuronal marker, was used for normalization.

gg. Example 33.

A phase 2 clinical trial was conducted in patients with Alzheimer's disease using PPF1. The main inclusion criteria were: age 60-90 years; probable AD according to National Institute on Aging-Alzheimer's Association (NIA-AA) criteria (Jack CR, et al., Alzheimers Dement., 14(4):535–62 (2018), incorporated herein by reference); Mini-Mental State Examination (MMSE) score 12-24. The main exclusion criteria were: any neurological disorder other than AD; >2 lacunar strokes on magnetic resonance imaging

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Claims (15)

fii (MRI); change in dose of cholinesterase inhibitor or memantine in the past 3 months. Each subject had a baseline assessment visit, two 5-day dosing periods (IV), each followed by a 3-month treatment-free period, for the entire study duration of 6 months. Subjects were randomized 1:1 to receive 100 ml or 250 ml PPF1, and the assigned dose was unknown to the subjects, caregivers, assessors, and investigators. There was no placebo control group. The primary endpoint was safety and tolerability, while secondary endpoints included the AD Severity Rating Scale (ADAS-Cog) cognitive subscale, Clinical Dementia Rating (CDR) scale, AD Cooperative Study - Activities of Daily Living (ADCS-ADL), MMSE and group Savonix cognitive tests. Exploratory endpoints included blood and cerebrospinal fluid biomarkers and structural and functional MRI. This study was conducted at nine (9) centers in the United States between March 2018 and May 2019. Eighty-nine (89) subjects were screened, fifty-two (52) subjects were randomized, fifty-one (51) subjects received at least 1 dose, forty-three (43) subjects completed the first 5-day dosing period, and forty (40 ) subjects completed both dosing periods. Baseline demographics and levels of cognitive and functional impairment for all subjects who received at least 1 dose (n=51) are summarized in FIG. 71. In fig. 71 shows that there was no significant decline in cognitive or functional scores as measured by the ADAS-Cog, ADCS-ADL, and CDR-SB during the 6-month study period. The expected reduction over a 6-month period in asthma subjects with similar baseline severity treated with placebo in other studies was a 2-3 point worsening on the ADAS-Cog scale and a 3-4 point worsening on the ADCS-ADL scale (Ito et al., Alzheimers Dement., 6:39-53 (2010); Ito K et al., Alzheimers Dement., 7:151-60 (2011); Doody RS et al., N Engl J Med. 70:311 -21 (2014); Relkin NR et al., Neurology, 88:1768-75 (2017), all of which are incorporated herein by reference). Results from this Phase 2 clinical trial in patients with Alzheimer's disease indicate that daily dosing of PPF1 for five (5) consecutive days is safe and well tolerated in this population. They also show that the subjects' disease progressed more slowly than would be expected. CLAIM 1. A method of treating a subject diagnosed with cognitive impairment, comprising administering to the subject an effective amount of a plasma fraction using an intermittent dosing regimen, wherein the plasma fraction is a human albumin solution (HAS) containing more than 95% albumin relative to the total amount of protein. 2. The method of claim 1, wherein the HAS is obtained from plasma from a group of people aged 40 years or younger. 3. A method according to any one of the preceding claims, characterized in that the HAS is obtained from a human blood product. 4. The method of any one of the preceding claims, further comprising monitoring the subject for improvement in cognitive function. 5. A method according to any of the preceding claims, characterized in that the subject is a mammal. 6. The method according to claim 5, characterized in that the mammal is a human. 7. The method according to any of the preceding claims, characterized in that the intermittent application schedule includes the administration of HAS for five to seven consecutive days. 8. Method according to any of the preceding claims, characterized in that the effective amount of HAS is 100 ml. 9. Method according to any one of claims 1 to 7, characterized in that the effective amount of HAS is 250 ml. 10. A method as claimed in any one of the preceding claims, characterized in that the subject follows the exercise regimen after complete administration of the intermittent application regimen. 11. Method according to any one of claims 1 to 10, characterized in that the administration increases the number of synapses in the subject. 12. The method according to any one of claims 1 to 10, characterized in that administration increases synaptic integrity in the subject. 13. The method according to any one of claims 1 to 10, characterized in that the administration increases the integrity of the neural network in the subject. 14. Method according to any one of claims 1 to 10, characterized in that the administration increases the degree of neural connection in the subject. 15. Use of a kit for treating a subject with a cognitive disorder, the kit comprising -