Compositions and Methods for Plasma Feresis

Abstract

Compositions and methods for performing plasma pheresis are described herein. The compositions and methods for performing plasma pheresis are innovative, at least in their use for the treatment and prevention of aging and aging-related conditions. The plasma pheresis compositions and methods described herein are directed to reducing or eliminating aging-related conditions. During the past century, the earth's population has more than doubled. More than 20% of the world's population is estimated to be 65 years of age or older.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 347,124, filed May 31, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Over the past century, the Earth's population has more than doubled. More than 20% of the world's population is estimated to be over 65 years old. The United Nations estimates that this population will increase to over 14 billion by 2050. Older humans almost invariably suffer from one or more disorders associated with chronic aging. These can include Alzheimer's disease, infectious diseases, type II diabetes, atherosclerotic cardiovascular disease, obesity, osteoporosis, and sarcopenia. The cumulative impact is a huge financial burden on any healthcare system.

Summary of the Invention

Means for Solving the Problems

[0003] A method of providing plasmapheresis to an individual to improve the individual's health state, comprising: (a) measuring one or more of the individual's strength, balance, mental state, and walking measurements before performing plasmapheresis, thereby generating an indicator of the individual's pre-treatment health state; (b) performing plasmapheresis on the individual; (c) after step (b), measuring one or more of the individual's strength, balance, mental state, and walking measurements, thereby generating an indicator of the individual's post-treatment health state; and (d) comparing the individual's post-treatment health state with the individual's pre-treatment health state to determine that the individual's health state has been improved as a result of the plasmapheresis. In some embodiments, step (a) is performed within 24 hours of performing plasmapheresis on the individual in step (b). In some embodiments, the individual's strength is measured in step (b) by measuring the individual's grip strength. In some embodiments, the individual's balance is measured in step (a), step (c), or both steps (a) and (c) by having the individual stand on one leg and measuring the time the individual remains standing with one leg raised. In some embodiments, the mental state is measured in step (a), step (c), or both steps (a) and (c) using a survey that includes questions to evaluate emotional stability. In some embodiments, the individual's walking measurements are measured in step (a), step (c), or both steps (a) and (c) by having the individual stand up from a seated position and walk. In some embodiments, the plasmapheresis performed in step (b) exchanges at least one unit of plasma volume. In some embodiments, the plasmapheresis performed in step (b) is performed over a plurality of treatment sessions. In some embodiments, two of the plurality of treatment sessions are performed within 72 hours of each other. In some embodiments, the plasmapheresis performed in step (b) is performed over a single treatment session.In some embodiments, comparing the individual's post-treatment health state with the individual's pre-treatment health state in step (d) results in the determination of a quantitative difference between the individual's post-treatment health state and the individual's pre-treatment health state. In some embodiments, the method further includes repeating steps (b)-(d) until the quantitative difference reaches a specific value. In some embodiments, the method further includes repeating steps (a)-(d) until the quantitative difference reaches a specific value.

[0004] A method for treating a condition associated with aging in an individual, the method comprising: (a) performing plasmapheresis on the individual; and (b) monitoring a change in the condition, is described herein. In some embodiments, the condition associated with aging includes loss of strength. In some embodiments, the strength of the individual is monitored in step (b) by measuring the individual's grip strength. In some embodiments, the condition associated with aging includes loss of balance. In some embodiments, the balance of the individual is monitored in step (b) by measuring the time the individual stands on one leg with the other leg raised. In some embodiments, the condition associated with aging includes a decline in walking ability. In some embodiments, the walking ability of the individual is monitored in step (b) by having the individual stand up from a seated position and walk. In some embodiments, the plasmapheresis performed in step (a) exchanges at least one unit of plasma volume. In some embodiments, the plasmapheresis performed in step (a) is performed over a plurality of treatment sessions. In some embodiments, two of the plurality of treatment sessions are performed within 72 hours of each other. In some embodiments, the plasmapheresis performed in step (a) is performed over a single treatment session. In some embodiments, the method further includes repeating steps (a)-(b) until a change in the condition is achieved.

[0005] A method for treating an individual using plasmapheresis by modulating the expression level of cell surface markers on the cell surface of the individual's white blood cells, the method comprising: (a) measuring the expression level of cell surface markers in the individual's blood prior to performing plasmapheresis; (b) performing plasmapheresis on the individual; and (c) after step (b), measuring the expression level of cell surface markers in the individual's blood and determining that the expression of cell surface markers on the cell surface of the individual's white blood cells is modulated. In some embodiments, the white blood cells include lymphocytes. In some embodiments, the lymphocytes include T cells. In some embodiments, the white blood cells include monocytes. In some embodiments, the white blood cells include basophils. In some embodiments, the white blood cells include neutrophils. In some embodiments, the white blood cells include eosinophils. In some embodiments, modulating the expression level of cell surface markers on the cell surface of the individual's white blood cells is changing the expression level of the cell surface markers to an extent measurable in the individual's blood after performing plasmapheresis in step (b). In some embodiments, the cell surface marker includes CD16. In some embodiments, the cell surface marker includes CD25. In some embodiments, the cell surface marker includes CD27. In some embodiments, the cell surface marker includes CD38. In some embodiments, the cell surface marker includes CD57. In some embodiments, the cell surface marker includes CD80. In some embodiments, the cell surface marker includes HLA-DR. In some embodiments, the cell surface marker includes IgM. In some embodiments, the cell surface marker includes KIR. In some embodiments, the cell surface marker includes KLRG1. In some embodiments, the cell surface marker includes NK1. In some embodiments, the cell surface marker includes NKG2A. In some embodiments, the cell surface marker includes TIGIT. In some embodiments, step (a) is performed within 24 hours of performing plasmapheresis on the individual in step (b).In some embodiments, the plasmapheresis performed in step (b) exchanges at least one unit of plasma volume. In some embodiments, the plasmapheresis performed in step (b) is performed over a plurality of treatment sessions. In some embodiments, two of the plurality of treatment sessions are performed within 72 hours of each other. In some embodiments, the plasmapheresis performed in step (b) is performed over a single treatment session. In some embodiments, the expression of the cell surface marker is measured using flow cytometry. In some embodiments, the expression of the cell surface marker is measured using a fluorescent-conjugated antibody. In some embodiments, the modulation is a measurable decrease between the expression level of the cell surface marker measured in step (a) and the expression level of the cell surface marker measured in step (c). In some embodiments, the method further comprises repeating steps (b) to (c) until a cell surface modulation having a specific value is achieved. In some embodiments, the method further comprises repeating steps (a) to (c) until a cell surface modulation having a specific value is achieved.

[0006] A method for treating aging in an individual by reducing cellular aging in the individual using plasma pheresis, the method comprising: (a) measuring the expression level of a marker associated with cellular aging in the blood of the individual prior to performing plasma pheresis; (b) performing plasma pheresis on the individual; and (c) after step (b), measuring the expression level of the marker associated with cellular aging and determining that cellular aging in the individual has been reduced. In some embodiments, the cells in which cellular aging is reduced include lymphocytes. In some embodiments, the lymphocytes include T cells. In some embodiments, the cells in which cellular aging is reduced include monocytes. In some embodiments, the cells in which cellular aging is reduced include basophils. In some embodiments, the cells in which cellular aging is reduced include neutrophils. In some embodiments, the cells in which cellular aging is reduced include eosinophils. In some embodiments, the marker associated with cellular aging includes senescence-associated beta-galactosidase (“SA-β-gal”). In some embodiments, step (a) is performed within 24 hours of performing plasma pheresis on the individual in step (b). In some embodiments, the plasma pheresis performed in step (b) exchanges at least one unit of plasma volume. In some embodiments, the plasma pheresis performed in step (b) is performed over a plurality of treatment sessions. In some embodiments, two of the plurality of treatment sessions are performed within 72 hours of each other. In some embodiments, the plasma pheresis performed in step (b) is performed over a single treatment session. In some embodiments, the expression of the marker associated with cellular aging is measured using flow cytometry. In some embodiments, the expression of the marker associated with cellular aging is measured using a fluorescently conjugated antibody. In some embodiments, the modulation is a measurable decrease between the expression level of the marker associated with cellular aging measured in step (a) and the expression level of the marker associated with cellular aging measured in step (c).In some embodiments, the method further includes repeating steps (b)-(c) until a specific value for reduction in the expression level of a marker associated with cellular senescence is achieved. In some embodiments, the method further includes repeating steps (a)-(c) until a specific value for reduction in the expression level of a marker associated with cellular senescence is achieved.

[0007] A method of performing plasmapheresis for use in treating or preventing a condition associated with aging in an individual, the method comprising removing at least 70% of the factors associated with aging from the individual's vascular system by performing plasmapheresis on the individual at least twice within a period of 72 hours, thereby treating or preventing the condition associated with aging in the individual, is described herein. In some embodiments of the method, each of the at least two times plasmapheresis is performed includes removing at least one plasma volume from the individual. In some embodiments of the method, the volume of the replacement fluid returned to the individual is equal in volume to the at least one plasma volume withdrawn. In some embodiments of the method, the volume of the replacement fluid returned to the individual is greater in volume than the at least one plasma volume withdrawn. In some embodiments of the method, at least one of the at least two times plasmapheresis is performed includes removing at least one and a half plasma volumes from the individual. In some embodiments of the method, the volume of the replacement fluid returned to the individual is equal to the at least one and a half plasma volumes withdrawn. In some embodiments of the method, the volume of the replacement fluid returned to the individual is greater than the at least one and a half plasma volumes withdrawn. In some embodiments of the method, plasmapheresis includes injecting a replacement fluid into the individual's vascular system, the replacement fluid including at least one of saline, lactated Ringer's, albumin, or a therapeutic agent. In some embodiments of the method, the therapeutic agent includes at least one of an anti-inflammatory agent or an immunomodulatory agent. In some embodiments of the method, the immunomodulatory agent includes intravenous immunoglobulin. In some embodiments of the method, the method includes administering a therapeutic agent to the individual after at least one of the at least two times plasmapheresis is performed. In some embodiments of the method, the therapeutic agent includes at least one of an anti-inflammatory agent or an immunomodulatory agent. In some embodiments of the method, the immunomodulatory agent includes intravenous immunoglobulin.

[0008] A method for performing plasmapheresis for use in treating or preventing conditions associated with aging in an individual, the method comprising: (a) determining the biological age of the individual; (b) performing plasmapheresis on the individual; and (c) repeating steps (a) and (b) until the biological age of the individual is below a threshold, is described herein. In some embodiments of the method, the biological age of the individual is determined using the individual's albumin blood level. In some embodiments of the method, the biological age of the individual is determined using the degree of glycation of albumin in the individual's blood. In some embodiments of the method, the biological age of the individual is determined using the individual's ceruloplasmin blood level. In some embodiments of the method, the biological age of the individual is determined using the level of immunoglobulins in the individual's blood. In some embodiments of the method, the biological age of the individual is determined using the individual's glutathione blood level. In some embodiments of the method, the biological age of the individual is determined using an antibody assay, the antibody assay comprising at least one of an antinuclear antibody screening, a rheumatoid factor assay, a thyroid peroxidase antibody assay, or a quantitative immunoglobulin assay. In some embodiments of the method, the biological age of the individual is determined using a proteomics assay, the proteomics assay comprising at least one of a fibrinogen assay, a creatine kinase assay, or a hemoglobin A1C assay. In some embodiments of the method, the biological age of the individual is determined using a metabolomics assay, the metabolomics assay comprising at least one of a cholesterol assay or a blood glucose assay. In some embodiments of the method, the biological age of the individual is determined using urine analysis. In some embodiments of the method, the biological age of the individual is determined using peripheral blood mononuclear cell analysis. In some embodiments of the method, the biological age of the individual is determined using a cellular senescence assay. In some embodiments of the method, the biological age of the individual is determined using a genomic methylation assay.In some embodiments of the method, the biological age of an individual is determined using inflammatory marker analysis. In some embodiments of the method, the biological age of an individual is determined using at least one of a complete blood count, a total protein assay, a liver function assay, a blood urea nitrogen assay, a creatinine assay, or a C-reactive protein assay.

[0009] A method for performing a plasmapheresis regimen, comprising: (a) collecting whole blood of at least one plasma volume from an individual; (b) separating the whole blood into a cell fraction and a plasma fraction; (c) returning the cell fraction to the individual; (d) injecting a replacement fluid into the individual simultaneously with step (a); and (e) repeating steps (a)-(d) until at least one component found in the individual's plasma is diluted by at least 60% as compared to before initiating the plasmapheresis regimen. The method is described herein.

[0010] A method for treating age-related conditions in an individual, the method comprising performing plasmapheresis on the individual and removing at least one plasma volume from the individual during plasmapheresis, is described herein. In some embodiments, the age-related condition is a decrease in the strength of the individual. In some embodiments, the age-related condition is a decrease in the walking exercise of the individual. In some embodiments, the age-related condition is a decrease in balance in the individual. In some embodiments, the age-related condition is a decrease in the mental state of the individual. In some embodiments, the age-related condition is an increase in inflammation in the individual. In some embodiments, the increase in inflammation in the individual is associated with a change in the expression level of a cell surface protein expressed on the surface of white blood cells. In some embodiments, the white blood cells include lymphocytes. In some embodiments, the lymphocytes include T cells. In some embodiments, the white blood cells include monocytes. In some embodiments, the white blood cells include basophils. In some embodiments, the white blood cells include neutrophils. In some embodiments, the white blood cells include eosinophils. In some embodiments, the cell surface protein includes CD16. In some embodiments, the cell surface protein includes CD25. In some embodiments, the cell surface protein includes CD27. In some embodiments, the cell surface protein includes CD38. In some embodiments, the cell surface protein includes CD57. In some embodiments, the cell surface protein includes CD80. In some embodiments, the cell surface protein includes HLADR. In some embodiments, the cell surface protein includes IgM. In some embodiments, the cell surface protein includes KIR. In some embodiments, the cell surface protein includes KLRG1. In some embodiments, the cell surface protein includes NK1. In some embodiments, the cell surface marker includes NKg2a. In some embodiments, the cell surface protein includes TIGIT.

[0011] A plasmapheresis exchange fluid composition for use in performing plasmapheresis for treating or preventing conditions associated with aging in an individual, wherein the total volume of the plasmapheresis exchange fluid composition is equal to at least one plasma volume of the individual. In some embodiments, the composition comprises 5% albumin. In some embodiments, the composition comprises IVIG. In some embodiments, the condition associated with aging is a decrease in the strength of the individual. In some embodiments, the condition associated with aging is a decrease in the walking exercise of the individual. In some embodiments, the condition associated with aging is a decrease in balance in the individual. In some embodiments, the condition associated with aging is a decrease in the mental state of the individual. In some embodiments, the condition associated with aging is an increase in inflammation in the individual. In some embodiments, the increase in inflammation in the individual is associated with a change in the expression level of cell surface proteins expressed on the surface of white blood cells. In some embodiments, the white blood cells include lymphocytes. In some embodiments, the lymphocytes include T cells. In some embodiments, the white blood cells include monocytes. In some embodiments, the white blood cells include basophils. In some embodiments, the white blood cells include neutrophils. In some embodiments, the white blood cells include eosinophils. In some embodiments, the cell surface proteins include CD16. In some embodiments, the cell surface proteins include CD25. In some embodiments, the cell surface proteins include CD27. In some embodiments, the cell surface proteins include CD38. In some embodiments, the cell surface proteins include CD57. In some embodiments, the cell surface proteins include CD80. In some embodiments, the cell surface proteins include HLA-DR. In some embodiments, the cell surface proteins include IgM. In some embodiments, the cell surface proteins include KIR. In some embodiments, the cell surface proteins include KLRG1. In some embodiments, the cell surface proteins include NK1. In some embodiments, the cell surface proteins include NKG2A.In some embodiments, the cell surface protein includes TIGIT.

[0012] A plasmapheresis exchange fluid composition for use in performing plasmapheresis to reduce cellular aging in an individual, wherein the total volume of the plasmapheresis exchange fluid composition is equal to at least one plasma volume of the individual, is described herein. In some embodiments, the composition includes 5% albumin. In some embodiments, the composition includes IVIG. In some embodiments, the cells in which cellular aging is reduced include lymphocytes. In some embodiments, the lymphocytes include T cells. In some embodiments, the cells in which cellular aging is reduced include monocytes. In some embodiments, the cells in which cellular aging is reduced include basophils. In some embodiments, the cells in which cellular aging is reduced include neutrophils. In some embodiments, the cells in which cellular aging is reduced include eosinophils. In some embodiments, the marker associated with cellular aging includes senescence-associated beta-galactosidase (“SA-β-gal”), and cellular aging is measured by measuring the marker. In some embodiments, the plasmapheresis performed is performed over a plurality of treatment sessions. In some embodiments, two of the plurality of treatment sessions are performed within 72 hours of each other. In some embodiments, the plasmapheresis performed is performed over a single treatment session. In some embodiments, cellular aging is measured by measuring a marker associated with cellular aging, and the expression of the marker is measured using flow cytometry. In some embodiments, the expression of the marker is measured using a fluorescent conjugated antibody.

[0013] The novel features of the disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the disclosure will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the disclosure are utilized and the accompanying drawings (also referred to herein as “Figure” and “FIG”).

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0015] Aging Aging coincides with a progression that affects recognizable and often harmful changes in comfort, fitness, appearance, and cognition. Some of these progressions manifest as easily identifiable changes in appearance (e.g., in humans, skin sagging, increased width of the mouth and nose, and drooping of the eyes), but in particular, the underlying biochemistry, which is thought to be involved in complex and multifaceted changes in molecules and signaling pathways over time, is not yet fully understood. To better understand the science of aging and age-related (e.g., genetic, physiological) changes in different organisms, including humans, at both the micro and macro levels, many studies are currently being conducted. Generally, age-related changes and conditions are considered negative, and the treatments described herein have many benefits in improving an individual's health by addressing age-related changes and conditions.

[0016] Effects of Aging For example, many age-related onset conditions such as hearing and vision loss, arthritis, and loss of cognitive function are not welcome and are considered harmful to the quality of life. This phenomenon is almost ubiquitous across species, and beyond a certain point, aging coincides with a decline in ability and biological function. In addition, for example, aging corresponds to degenerative processes, a decrease in recovery or healing ability, and an increased tendency towards acute stress and immune responses, so certain diseases are highly associated with aging and are likely to be interrelated, all of which create an environment in which a particular disease process can occur. As an example, rheumatoid arthritis is a disease highly associated with aging, and its pathophysiology is associated with tissue degeneration, a decrease in recovery or healing, and an increased tendency towards acute stress and immune responses.

[0017] Physiological effects of aging (i.e., age-related conditions) include decreased strength, decreased mobility (and particularly decreased walking ability), decreased balance, and subjective changes in mental well-being.

[0018] Mechanisms of Aging Mechanisms associated with aging are not fully understood at present, however, several observations provide at least empirical insights into factors associated with aging. Observations have shown, for example, that aging is likely to be affected by several health and lifestyle factors (i.e., ostensibly non-genetic factors) including stress, diet, sleep hygiene, and sun exposure. That age can be affected by these factors would seem to indicate that changes in one or more of these factors can affect the rate and / or severity of aging. For example, influencing diet through moderate calorie restriction is a well-established and reliable means of slowing aging, strongly suggesting that diet is an important component of the aging process.

[0019] Also, similarly, it is strongly thought and supported by certain studies that there are genetic components to aging, that the expression of certain genes drives aging-related processes, and that either overexpression or underexpression of certain genes can result in a delay or acceleration of the aging process. In the context of aging, gene expression can result in the production of peptides that can act on or affect targets that are relatively remote in the body from the cells containing the genes being expressed. Thus, a gene expressed in one cell type or one tissue type can have an impact on cells or tissues that are distant from the location where the gene was first expressed.

[0020] Peptides produced by the expression of genes associated with aging can be modified in the body by other chemical processes and can then affect the function of the peptides. Such chemical processes that modify the peptides include, but are not limited to, methylation, glycosylation, and glycation. The type of chemical modification, as well as the degree of chemical modification of a particular post - translational peptide, can be the cause of age - related changes and can also be markers of aging. For example, the degree of glycation of peptides found in the blood, such as albumin, can be directly correlated with general aging or specific aging processes. The degree of chemical modification of a post - translational peptide can refer to the presence of a particular modification within one or more of the peptides and / or the percentage of peptides in which the degree of modification is found.

[0021] Aging is also associated with an increase in the levels of inflammation - promoting markers in the blood and tissues, which is a strong risk factor for multiple diseases that are a very common and frequent cause of disorders in the elderly. This phenomenon is referred to as "inflammaging". Reducing or even reversing inflammaging in aging patients is a pathway for treating age - associated conditions and even treating or reversing aging itself.

[0022] The effects of aging on lifespan and longevity As used herein, the term "lifespan" is the duration of an individual's life. When measured in a population of individuals, lifespan can be any cumulative measure across the entire population or a subset of the population (i.e., a sub - population), such as, for example, the average lifespan of the population or sub - population, the median lifespan of the population or sub - population, the variation in lifespan of the population or sub - population.

[0023] As used herein, the term "longevity" means that an individual or group of individuals has a lifespan or life expectancy that continues longer than a reference lifespan. For example, medical history data can provide the expected lifespan of a population, which can function as a reference lifespan. As used herein, "life expectancy" may describe any measure of an individual's lifespan or the lifespan within a group, and can be reasonably used as a predictor or marker for the lifespan of another individual or individuals within the group. For example, the life expectancy of an individual with a particular physiology can be obtained by calculating the average lifespan of a group of individuals with the same particular physiology, so the average lifespan can function as a reference lifespan. It should be understood that there are numerous ways to determine a reference lifespan, including using statistical techniques for data of the relevant population such as mean, median, mode, standard deviation, and variance.

[0024] The aging process counteracts or limits longevity in the sense that aging has a shortening effect on lifespan. The aging process is not only associated with harmful physiological changes and an increased likelihood of the onset of life-threatening diseases, but is also, of course, well understood to be a cause of death, either directly or indirectly, that shortens lifespan. It is also well understood that reducing, preventing, halting, and / or reversing the effects of aging promotes an increase in lifespan and thus promotes longevity. Generally speaking, if aging can be significantly counteracted, the ability to avoid death is promoted, and thus one lives longer, thereby increasing lifespan and promoting longevity. This applies to both individuals and groups of individuals. Therefore, in addition to aging itself being a good target for therapy, therapies that address aging are expected to similarly promote longevity.

[0025] Therefore, a therapy that promotes longevity can be defined as a therapy that promotes a relative increase in the lifespan of an individual or a group, and / or a therapy that treats, reduces, and / or prevents the effects of aging. For example, the predicted lifespan of a male human with certain physiological characteristics such as black hair and green eye color can correspond to, for example, 89.4 years, and 89.4 years is the average lifespan of a group of men with black hair and green eyes. If a male human with these same physiological characteristics (i.e., black hair and green eyes) lives, for example, up to 91 years and lives longer than the predicted lifespan, it can be said that he experiences longevity. Similarly, for example, a group of men with these same physiological characteristics from a specific geographical region such as Greece (i.e., a subset of a larger group of men with black hair and green eyes but from a specific geographical region in Greece) can be said to have longevity if all of them individually have a lifespan longer than 89.4 years. Therefore, a therapy that is associated with or results in an individual's lifespan or the lifespan of individuals in a group being longer than the predicted lifespan can be called a therapy that promotes longevity.

[0026] In addition, a therapy that promotes longevity can also be defined as a therapy that causes an increase in an individual's predicted lifespan compared to the existing predicted lifespan of a reference individual or reference group. For example, a person who is a smoker and has an initial predicted lifespan undergoes a treatment to quit smoking, and quitting smoking results in a longer predicted lifespan.

[0027] Biological age As used herein, the term "chronological age" refers to the number of years an individual has existed, which is a duration of time that can be expressed as "age" or "years old". Chronological age is purely a calendar measure of time with a starting point (typically birth) and an ending point at death, and is not determined based on any physical or biological characteristics of the individual. For example, an individual's chronological age does not change based on how old their physical appearance may seem, nor does it change based on family history or genetic characteristics that may suggest a particular lifespan for the individual. On the other hand, as used herein, the term "biological age" is a measure of the aging process and takes into account the physical, biological, genetic, and biochemical characteristics of an individual, including but not limited to biological progression, genetic and epigenetic features, homeostasis measures, disease risk, and various molecular changes associated with the individual.

[0028] Biological age can be expressed as a duration of years, similar to how chronological age is expressed. Biological age can refer to the individual as a whole, or to other aspects such as, for example, an individual's organs, organ systems, or other functional systems. For example, the biological age of an individual as a whole can be 35 years old, and the immune system age can be 27 years old (i.e., the immune system age is a subset or type of biological age).

[0029] It is worth noting that biological age and chronological age can be decoupled and can result in an appearance, energy level, and / or biological profile of an individual that is chronologically older or younger. For example, an individual can be 55 years old (from a chronological age perspective) but have a biological age of 42 years old. Similarly, a 35-year-old individual can have a biological age of 51 years old.

[0030] Biological age can be considered a measurable performance metric in at least some respects, and it is generally preferable for an individual's biological age - overall or with respect to specific characteristics of the individual - to be lower than the individual's chronological age. For example, assuming an individual has their own liver rather than a transplanted liver, an individual with a chronological age of 70 has a liver with a chronological age of 70 as well. The same individual in the example could, for instance, have a biological age of 68 as an individual, based on one or more of the metrics discussed above. And the same individual in the example could, for instance, have a liver with a biological age of 65, based on one or more of the metrics discussed above. That is, in this example, the individual can have an overall biological age of 68 while an organ of the same individual (the liver in this example) has a biological age of 65. Thus, biological age can be considered a measure of an overall scale or a scale of an individual's system, and / or a measure of processes within an individual's body.

[0031] In both examples, biological age can be calculated using one or more markers or factors that correlate with or indicate an individual's biological age. Such markers or factors can be measured and / or detected, for example, through testing of biological samples such as blood, urine, sputum, and sweat.

[0032] In addition to population-level variations, biological aging can proceed at multiple rates within an individual. Due to genetic, lifestyle, health, and environmental factors, separate organs, tissue types, or cells within an individual can exhibit different biological ages. For example, among a number of cumulative and harmful effects, obesity, diabetes, and kidney disease often accelerate the aging of the kidneys, such that the apparent age of the kidneys in such individuals may be much higher than the age of other organs and omics profiles (e.g., plasma proteomics). Even healthy individuals can experience significant biological age variations.

[0033] The biological aging rate appears to respond to a range of genetic and environmental factors, but most organisms appear to follow an innate and encoded aging timeline. Biological aging exhibits some intra-species variability, but the upper and lower limits of the aging rate appear to be primarily determined by the type of species. For example, there are no known cases of humans living beyond 125 years, but bowhead whales routinely reach 200 years, and certain species of clams consistently live beyond 500 years. As another extreme example, African killifish typically live only 4 - 6 months and exhibit signs of accelerated aging as early as 2 months. Several human diseases that underlie the genetic basis of aging alter the biological aging rate, and Hutchinson-Gilford progeria syndrome, Werner syndrome, and Down syndrome increase biological aging by approximately 100 - 800%.

[0034] Biological age markers Aging is consistent with diverse and complex processes at the molecular, cellular, and tissue levels. As disclosed herein, selected aspects of these processes can be monitored to determine the biological age in a subject. Since many markers of aging can also respond to health, lifestyle, and the environment, methods for determining biological age can utilize multiple biological markers and can further use non-age-responsive biomarkers as calibrators.

[0035] Exemplary biomolecules, genetic and epigenetic markers, expression patterns, and related measurement methods that may be useful for diagnosing chronological and biological age are outlined below. The biomarkers outlined in this section are particularly useful but are intended to function as examples of age-diagnostic species and are not intended to be limiting.

[0036] Blood-based biomarkers Many of the molecular and biological changes associated with aging manifest as changes in blood composition. At the population level, aging correlates consistently with changes in blood phenotypes, despite being complex. Some of these changes can be mapped to direct increases or decreases in single biomarkers, such as the progressive increase in levels of age-related inflammatory peptide biomarkers (e.g., interleukin (IL)-6), C-reactive protein, and tumor necrosis factor-α (TNF-α)), but aging can also correlate with changes in biomarker processing (e.g., immunoglobulin glycosylation patterns) and ratios among species groups.

[0037] (i) Albumin For many individuals, albumin, the most abundant serum protein, can function as a robust biomarker for aging. Albumin is a family of globular transport proteins essential for the clearance of lipids, hormones, and metabolites, as well as homeostasis. After a typical peak concentration of 40 - 50 mg / mL in late adolescence, serum albumin concentration often decreases by several hundred μg / mL per year and shows an accelerated rate of decrease in the elderly. Typical serum albumin levels are approximately 45 and 42 mg / mL in 30-year-old men and women, respectively, but by 60 years of age, the average levels decrease to approximately 42 and 40 mg / mL in men and women, respectively.

[0038] Furthermore, albumin often exhibits age - dependent structural changes that can be useful for aging diagnosis. In most humans, the proportion of glycated albumin increases with age and typically leads to a decline in function. Since albumin activity is essential for multiple forms of homeostasis, the combined effects of reduced albumin levels and activity can contribute to the detrimental symptoms of aging (such as reduced energy) and may enhance the rate of biological aging. Albumin glycation can also serve as evidence of other age - related pathologies, including a decrease in the concentration and function of regulatory proteins such as insulin. Thus, serum albumin concentration, isoform ratio, and post - translational modifications (such as the glycation pattern) can not only function as diagnostic markers of age but also serve as evidence of the severity of age - related symptoms.

[0039] (ii) Ceruloplasmin In many individuals, changes in ceruloplasmin levels and forms can be used to quantify biological age. Ceruloplasmin is a class of copper - containing proteins that participate in the oxidation and transport of iron. Thus, ceruloplasmin plays a central role in iron transport and the prevention of reactive oxygen species. Ceruloplasmin exhibits progressive changes in post - translational modifications and the isoform population with aging, which can affect its activity, localization (such as intravascular vs. extravascular distribution), and clearance rate.

[0040] The celluloplasmin consortium typically contains a complex array of isoforms and post-translational modification patterns, but age-related progression often manifests as a detectable change in the celluloplasmin copper center. Such changes can be detected using paramagnetically sensitive spectroscopies such as electron spin resonance and magnetic circular dichroism, and can provide evidence of more extensive changes in structure, isoform ratio, and post-translational modification patterns (see, for example, Musci et al. J Biol Chem, 1993;268(18):13388-95). Celluloplasmin also often exhibits age-dependent carbonylation and net charge, with carbonylation being more than three-fold higher (measured, for example, by mass spectrometry) and the isoelectric point being 0.1 higher (measured, for example, by two-dimensional gel electrophoresis) in subjects aged 65 years compared to those aged 15 years. Thus, celluloplasmin structure, isoform ratio, post-translational modification patterns, and combinations thereof can be used to assess biological age.

[0041] (iii) Immunoglobulin Since immunoglobulins exist in the blood as a complex consortium spanning various structural forms, targets, immunological activities (e.g., effector functions and complement-binding affinities), and glycosylation patterns, fluctuations in the immunoglobulin population can function as a powerful marker of biological aging. Humans express five immunoglobulin isotypes (IgG, IgA, IgM, IgD, and IgE) that span multiple subclasses (e.g., IgG1, IgG2, IgA1, etc.) and differ in structure, concentration, in vivo distribution, and immunomodulatory activity. IgG, IgA, and IgM are, respectively, the second, fifth, and ninth most abundant proteins in serum at resting levels in mg / mL, while IgD and IgE are typically present in serum in amounts of μg / mL and ng / mL, respectively.

[0042] Total immunoglobulin concentrations tend to peak in early adulthood and then decline steadily with age. Nevertheless, only some immunoglobulin isotypes and subclasses exhibit age-dependent changes in serum levels. Recent studies (Ritchie et al. J Clin Lab Anal, 1998, 12:363 - 370) have identified an increase in IgA levels, a decrease in IgM levels, and age-invariance of total IgG concentration with age. However, a follow-up study (Lock and Unsworth. Ann Clin Biochem, 2003;40:143 - 148) determined that in certain subjects, only IgG1 and IgG3 levels are invariant with age, while IgG2 and potentially IgG4 may exhibit age-dependent concentration decreases. In contrast to the trends in IgA, IgG, and IgM concentrations, IgD can peak during the first year of life but then remains relatively stable (Josephs and Buckley, J Pediatr, 1980;96(3):417 - 420). In certain subjects, the ratio between immunoglobulin isotype concentrations and subclass concentrations can provide a powerful diagnostic marker for age. For example, the ratio of serum levels between IgA and IgM, IgA and IgG2, IgA and IgG4, IgM and IgG2, IgM and IgG4, and / or IgG2 and IgG4 can be evidence of age.

[0043] The immunoglobulin consortium can also exhibit age-dependent changes in glycosylation. All five human isotypes exhibit diverse glycan modifications that affect immune regulation and in vivo distribution behavior. Within each isotype, the glycosylation pattern (glycome) exhibits a high degree of heterogeneity, similar to health and population variations. For example, the IgG antibody population typically exhibits over 30 types of glycans at asparagine 297, in addition to variable Fab and hinge region glycosylation, some of which vary with disease state. Nevertheless, age-dependent changes in glycosylation patterns have been observed for all five human isotypes. Within the IgG antibody population, increases in agalactosylation and GlcNAc bisecting, as well as decreases in digalactosylation, sialylation, and afucosylation, are typically observed with aging. Furthermore, there is some evidence that IgG glycosylation not only responds to age but is also a determinant of the rate of biological aging (Gudelj et al. Cellular Immunology, 2018; 333: 65-79).

[0044] (iv) glutathione Glutathione is a highly versatile biomolecule that participates in oxidative homeostasis, nitric oxide signaling, aldehyde catabolism, and multiple forms of assimilation. Glutathione exists in the blood at micromolar (μM) concentrations as a mixture of reduced monomers and oxidized disulfide dimers. The ratio of these two forms not only responds to blood conditions such as reactive oxygen species levels but also changes with age. With the enhancement of this effect, the systemic glutathione level steadily decreases with age. Since glutathione is important for reducing oxidative stress, the decrease in glutathione level may partly contribute to the increase in oxidative stress and the progression of stress-related conditions (e.g., Parkinson's disease) among the elderly. Therefore, the systemic glutathione level and the monomer-dimer ratio can function as powerful diagnostic markers for biological age. In both men and women, serum glutathione concentration steadily decreases from about 1 μM at 20 years of age to about 0.5 μM at 60 years of age (Yang et al. J Chromatogr B Biomed Appl, 1995;674(1):23-30).

[0045] Physical function and appearance Despite the wide variation in physical fitness and appearance among individuals, the decline in physical ability and the change in appearance are universal attributes of aging in humans, including a decrease in strength, ambulation (or walking), and balance.

[0046] Methods for measuring biological age The present disclosure provides a range of methods for determining biological age and the rate of biological aging using biomarker analysis. Based on the molecular complexity of aging, several species change in a predictable manner during aging and thus provide indicators of biological age and chronological age. As an individual ages, certain biomarkers can include an increase or decrease in concentration, a change in state (e.g., glycation of albumin and methylation / demethylation of genomic DNA), a change in morphology (e.g., isoform ratio of a specific protein), a change in activity, or a combination thereof. Thus, as described in further detail herein, the biological age can be determined using an assessment of one or more of these biomarkers (e.g., identifying concentration, state, morphology, and / or activity).

[0047] Some biological age measurements can identify the biological age of an individual, while others can identify the biological age of an individual's cells, tissues, organs, or systems (e.g., the immune or endocrine system). In many individuals, biological aging proceeds in a manner specific to cells, tissues, organs, and / or systems, reflecting distinct environments, stresses, as well as genetic and regulatory architectures. In the absence of trauma or abnormal health, the range of biological ages of an individual's tissues and cells is often small, e.g., less than the experimental error of the age measurement. However, many individuals exhibit multiple different ages. For example, compared to chronological age, an individual may have a young brain age and an advanced immune age. In such individuals, the biological age may reflect a kind of median of the biological ages specific to cells, tissues, organs, and / or systems. Alternatively, the biological age of an individual can be represented as a set of distinct, organ- or system-specific biological ages.

[0048] Biological age determination can utilize one biological age marker or multiple biological age markers. In many cases, as more age markers are utilized in the analysis, the accuracy of the biological age determination method increases. However, for many individuals, the use of a single age marker or a small set of age markers is sufficient to accurately determine biological age and / or the rate of biological aging, for example, with a standard error of less than 7 years, less than 5 years, or less than 3 years. Examples of methods for assessing biological age are provided in Table I.

[0049] Methods for biological age determination can utilize a single assay or multiple assays from Table I, a cytokine inflammation marker panel, a metabolomics assay, peripheral blood mononuclear cell (PBMC) analysis, genomic methylation analysis, inflammation marker analysis, or combinations thereof. The assay or multiple assays can evaluate overall biological age, organ and / or system-specific biological age, or combinations thereof. The determined biological age(s) of a subject can be used to calibrate treatments such as the anti-aging treatments disclosed herein.

[0050] The assay or multiple assays can be performed at regular intervals, for example, once a month, once every three months, once every six months, or once a year. Thus (similar to the aging rate diagnostic method), the determined biological age(s) from the assay or multiple assays can be used for determination of the rate of biological aging in a subject, for example, determination of whether an anti-aging treatment is slowing the rate of aging in the subject, calibration of a treatment method to achieve a target age or rate of aging in the subject, dissociation of disease markers from age-related symptoms (e.g., determination of whether an increase in HbA1c level is due to disease or aging), or combinations thereof. [Table 1]

[0051] Antibody assay Methods for determining biological age can include assessment of antibody concentration, type, and structure. Antibodies exist as a complex consortium spanning multiple isotypes (in humans, IgA, IgD, IgE, IgG, and IgM), paratope structure, and processing (e.g., glycosylation), and variations among these consortia can be useful for diagnosing biological aging. For example, as further detailed herein, the ratio of antibody isotypes typically changes with aging. Additionally, an individual's antibody types, such as antinuclear antibodies and antithyroid peroxidase antibodies, change in concentration with age and can thereby function as markers of aging. Some exemplary antibody assays are outlined below. It is contemplated that additional antibody assays can be used in conjunction with the methods of the present disclosure.

[0052] (i) Antinuclear antibody screening Antinuclear antibody (ANA) screening measures the concentration of cell nucleus-binding antibodies in blood, plasma, or serum. A range of ANA subtypes exist in humans, but most ANA screening measures the total ANA antibody concentration, most commonly using indirect immunofluorescence and enzyme-linked immunosorbent (ELISA) detection after cell binding (e.g., to HEp-2 cells). ANA is associated with a range of disorders, many of which are associated with aging. However, even among healthy individuals, ANA blood concentrations tend to increase with age, and older individuals often exhibit ANA levels more than three-fold higher compared to younger individuals (Xavier et al. Mech Aging Dev, 1995;78(2):145-54). Thus, methods consistent with the present disclosure can utilize measurement of blood ANA concentration to determine biological age or the rate of biological aging.

[0053] (ii) Rheumatoid factor assay An age diagnostic method can evaluate the blood (e.g., whole blood, serum, plasma) levels of autoantibodies against the Fc portion of IgG, which is a rheumatoid factor (RF) factor, involved in several age-related conditions including rheumatoid arthritis and decreased bone density. Rheumatoid factors can exist as combinations of immunoglobulin isotypes (e.g., IgA, IgD, IgE, IgG, and IgM) and Fc epitopes, and a certain range of total rheumatoid factor levels, which are binding affinities, can be identified by several binding assays including indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). In most people, rheumatoid factors typically appear between the ages of 30 and 70 and their concentration gradually increases with age. Thus, a method for measuring biological age consistent with the present disclosure can include measuring the blood RF concentration.

[0054] (iii) Thyroid peroxidase antibody assay The thyroid peroxidase antibody assay measures the concentration of autoantibodies targeting thyroid peroxidase (TPO), an enzyme essential for thyroid hormone production. Since thyroid peroxidase is typically the most common thyroid autoantigen, thyroid peroxidase antibody levels can reflect total anti-thyroid antibody levels. Thyroid antibodies (including thyroid peroxidase antibodies) are involved in several diseases, but thyroid antibody levels also tend to increase spontaneously with age (Chen et al. Endocrinology, 2010; 151(9): 4583 - 4593), thereby enabling them to function as markers of biological aging. Thus, the methods of the present disclosure can utilize blood thyroid antibodies and / or blood thyroid peroxidase antibody levels to determine biological age. Additionally, in some subjects, the target of thyroid peroxidase antibodies changes with age, and anti-domain A antibodies typically increase the morbidity (Czarnocka et al. Clin Endocrinol (Oxf), 1998; 48(6): 803 - 8).

[0055] (iv) Quantitative immunoglobulin assay Quantitative immunoglobulin assays can evaluate the total antibody levels in a subject sample. Typically, quantitative immunoglobulin assays measure the total antibody levels in blood. However, some assays evaluate concentrations by antibody isotype or subtype. For example, a quantitative immunoglobulin assay can measure total IgG and IgA concentrations, or can separately determine the concentrations of an individual's antibody subtypes (e.g., IgG1, IgG2, IgA1, IgA2, etc.). As in many subjects, total antibody, antibody isotype, and antibody subtype concentrations change with age (Crisp and Quinn. Allergy Asthma Proc, 2009;30(6):649-54), and quantitative immunoglobulin assays can be used to determine biological age.

[0056] (v) Glycation assay Biological aging can be demonstrated by the immunoglobulin glycosylation profile. All five human antibody isotypes exhibit glycosylation patterns. The positions of glycosylation depend in part on the isotype, but the types of oligosaccharides or glycans that bind at these positions can affect antibody localization, subcellular partitioning, aggregation, as well as the affinity of Fc and complement receptors. Furthermore, these glycosylation patterns can reflect age, environment, and health status. For example, IgG galactosylation often decreases with age, while fucosylation, sialylation, and bisecting can respond to age in a gender-specific manner (Gudelj et al. Cellular Immunology, 2018;333:65-79). Therefore, methods can utilize the antibody glycosylation profile to evaluate biological age.

[0057] In addition, a glycation assay for use in the evaluation of biological age is currently commercially available as the Glycanage test.

[0058] Proteomics assay Humans are estimated to have between 20,000 and 5 million proteins, depending in part on structural variant classification (e.g., whether a splicing variant constitutes a distinct protein), so the proteomic shifts due to aging and changes in health status are often complex. Nevertheless, since proteins participate in and regulate most biological processes, physiological changes, including those associated with aging, are often reflected in protein expression and activity. To date, age-related changes in the human proteome are thought to include both drivers (e.g., decreased superoxide dismutase and catalase activity) and responses (e.g., increased fibrinogen concentration) of biological aging. Age-related changes in the human proteome include up- and down-regulation of individual proteins, as well as changes across the proteome in abundance, ratio, and activity levels. Despite the complexity of the human proteome, some proteins are known to change with aging in a detectable manner. Thus, methods for determining biological age can involve measuring the abundance, state, distribution, modification, or activity of an individual protein or aggregate of proteins. In many such cases, the method involves measuring the concentration of a few blood proteins.

[0059] However, methods for determining biological age may also take a broad proteomic perspective to assess biological age. Since human blood contains over 5,000 types of proteins, even in the absence of statistically significant individual protein biomarkers, the meta-analysis of dozens, hundreds, or thousands of proteins can often correlate small proteomic shifts with biological age. Advances in high-throughput proteomic analysis, such as liquid chromatography–mass spectrometry, protein sequencing, and multiplex immunoassays, can enable the rapid quantification of hundreds or thousands of proteins from an individual sample.

[0060] (i) Fibrinogen assay Fibrinogen activity and blood levels often change with age. Fibrinogen is a blood-based glycoprotein complex that polymerizes into fibrin to promote blood clotting. Fibrinogen levels can respond to health and inflammation, but baseline fibrinogen levels typically increase with age in many individuals, rising by about 250 μg / mL per decade, or from about 2.2 mg / mL to about 3.2 mg / mL between the ages of 25 and 65 years (Hager et al. Aging (Milano), 1994;6(2):133-8). Since elevated fibrinogen levels have been associated with several age-related conditions, including increased susceptibility to cardiovascular disease and decreased kidney and liver function, fibrinogen levels often correlate with many recognizable qualitative aspects of aging.

[0061] Fibrinogen assays typically evaluate at least one of fibrinogen activity and concentration. Fibrinogen activity is typically measured indirectly by blood clotting tests such as thrombin and prothrombin time tests, thromboelastometry, and qualitative clotting assays. However, these assays may have limited ability to distinguish between low fibrinogen levels and decreased fibrinogen activity. Alternatively, or in addition to activity analysis, some assays directly measure blood fibrinogen concentration using a wide range of commercially available immunoassays for such measurements.

[0062] (ii) Creatinine kinase assay The creatine-based changes that rejuvenate muscle cells with phosphocreatine for rapid energy production can reflect aging, and creatine and associated metabolite levels typically decrease with aging. Nevertheless, creatinine, the primary breakdown product of phosphocreatine catabolism, typically increases in blood concentration with age. This discrepancy is likely due to a decline in the ability to recycle and clear creatinine. Creatinine kinase is an intracellular enzyme that converts creatinine back to phosphocreatine for further use, and its concentration decreases with age, leading to an increase in creatinine levels in blood, muscle, and some extravascular spaces. Thus, blood and intracellular creatine kinase levels may be useful in determining biological age.

[0063] (iii) Hemoglobin A1c assay In circulation, hemoglobin can bind indiscriminately to blood glucose in a process called glycation. The baseline rate of glycation is typically low, but hyperglycemia, insulin resistance, and aging can increase the glycation rate, resulting in higher concentrations of glycated hemoglobin. One of the most common forms of the resulting glycated hemoglobin is HbA1c (also called hemoglobin A1c), which contains glucose bound to one or both of the β-peptide N-terminal valines. High levels of HbA1c are most commonly due to hyperglycemia in diabetes, but age-related changes in glucose tolerance and blood glucose regulation tend to increase the HbA1c baseline regardless of health status, resulting in a small (e.g., 5.4% - 5.6% from 25 years to over 65 years) but nevertheless significant shift related to the age of the HbA1c population. When corrected for health and environmental factors, HbA1c levels can be useful in diagnosing biological age (Masuch et al. BMC Endocrine Disorders, 2019;19,20). HbA1c can be measured by various techniques, including quantitative chromatography (e.g., by high-performance liquid chromatography), immunoassays, and capillary electrophoresis.

[0064] (iv) Cytokine and Inflammatory Marker Panel Methods for assessing biological age may include the analysis of one or more cytokines. In many cases, biological age assessment involves determining the concentration of one or more cytokines in the blood. In many instances, aging, termed inflammaging, often coincides with an increase in blood cytokine levels through increased cytokine production and a decrease in the anti-inflammatory response. Central to this process is that several pro-inflammatory markers, such as C-reactive protein and serum amyloid A, increase several-fold in levels throughout middle age and old age, often leading to an imbalance between pro-inflammatory and anti-inflammatory cytokines. These changes in cytokine levels often promote chronic inflammation, weakening of the immune state, and a decrease in energy metabolism (Salvioli et al. Current Pharmaceutical Design, 2006; 12: 3161 - 71), so cytokine imbalance is likely to be both a cause and a consequence of aging. Thus, blood cytokine concentrations may correlate with the progressive age-related changes in cytokine levels to assess biological age.

[0065] Methods for determining biological age may include measuring the blood concentration of one or more cytokines. The blood concentration of a single cytokine may be sufficient to determine biological age, but in many cases, the method includes measuring the blood concentration of multiple cytokines. As a non-limiting example, methods for determining biological age may assess the levels of one or more of C-reactive protein, soluble tumor necrosis factor receptor 1 (sTNF1), soluble tumor necrosis factor receptor 2 (sTNF2), tumor necrosis factor α (TNF-α), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10), all of which typically increase in concentration with age (Salvioli et al., Glossop et al. Arthritis Research & Therapy, 2005;7:R1227). Among these cytokines, C-reactive protein, TNF-α, IL-1α, IL-1β, IL-6, and IL-10 are involved in the increased pro-inflammatory response with aging and may play a role in atherosclerosis and insulin insensitivity, among other conditions (Salvioli et al.). On the other hand, sTNF1 and sTNF2 are likely to exacerbate arthritis (Glossop et al.). Thus, the blood concentration of one or more cytokines can be evidence of aging and age-related symptoms.

[0066] In addition, a test for inflammatory changes in the context of biological age (i.e., the inflammatory age test) is currently commercially available as the iAge test.

[0067] The expression of certain cell surface proteins can also be similarly associated with the inflammatory process, including CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT. These cell surface proteins can be measured and quantified using flow cytometry.

[0068] Metabolomics assay Since metabolism encompasses the biochemistry of growth, energy production and consumption, and certain forms of signaling, age-related changes are often reflected by the turnover of metabolomics. Similar to proteomic analysis, metabolomics profiling can interrogate individual biomolecules in response to aging and can broadly profile a subset of the target metabolome (e.g., by profiling dozens, hundreds, or thousands of metabolomics biomarkers) or can include combinations thereof. Due to the large number of available metabolites and lower variability compared to cell and tissue profiling, metabolomics analysis often focuses on blood metabolites. In human blood, over 18,000 metabolites have been identified (Adav and Wang. Aging Dis, 2021;12(2):646-661), so biological age measurement can, in theory, accurately determine biological age by a broad untargeted profiling approach, even in the absence of strong diagnostic aging markers. However, several age diagnostic studies have identified robust metabolite biomarkers that can accurately measure biological age either alone or in combination with other measurements.

[0069] (i) Total cholesterol assay Cholesterol is generally complexed into various lipid - protein macromolecular structures, commonly referred to as lipoproteins, for transport through the blood. Lipoproteins differ in terms of several characteristics including size, composition, and receptor affinity. In humans, lipoproteins are mainly divided into five major classes based on density. High - density lipoproteins (HDL) tend to have the lowest volume among the five classes of lipoproteins, as well as the highest protein and phospholipid content. Typically, they have a diameter in the range of 5 - 15 nm, a density greater than 1.063 g / mL, and proteins that account for approximately one - third of their mass. Low - density lipoproteins (LDL) tend to have a slightly lower mass of about 1.019 - 1.063 g / mL, a slightly larger diameter of about 18 - 28 nm, and a higher cholesterol content of nearly 50% (by mass). Intermediate - density lipoproteins (IDL) tend to have a density of 1.006 - 1.019 g / mL, a cholesterol content similar to that of high - density lipoproteins, and a diameter in the range of 25 - 50 nm. Very - low - density lipoproteins (VLDL) are typically characterized by a density of 0.95 - 1.006 g / mL, a relatively low protein content (typically about 10% by mass), and a diameter of about 30 - 80 nm. Finally, chylomicrons, the largest lipoproteins, typically have a density of less than 0.95 g / mL, a protein content of less than 2% (by mass), and a diameter in the range of 75 - 1200 nm.

[0070] When assessing cholesterol levels, cholesterol analysis often differentiates at least some types of lipoproteins. For example, a lipid panel typically differentiates between HDL - bound cholesterol and LDL - bound cholesterol, and sometimes further differentiates VLDL, IDL, and chylomicron cholesterol content. However, in many conditions, the total blood cholesterol content is sufficient for an accurate diagnosis.

[0071] Specifically, in some individuals, total cholesterol content can be an indicator of biological age. In men, blood cholesterol levels tend to steadily increase from about 1.6 mg / mL at age 18 to about 2 mg / mL at age 50, and thereafter, blood cholesterol values tend to decrease by about 0.05 mg / mL every 10 years. In women, a slightly more intermittent trend is observed, and blood cholesterol levels typically increase to about 1.7 - 2.1 mg / mL between ages 18 and 60 and then plateau or decline at a slower rate than men through the progression of aging (Yi et al. Scientific Reports, 2019;9:1596). Thus, to determine biological age, the methods disclosed herein may use blood cholesterol levels.

[0072] To determine cholesterol levels, several enzymatic, chemical, electrochemical, and spectroscopic methods can be used. Generally, cholesterol is pooled or collected from lipoprotein fractions and conjugated to a chromophore or fluorophore through its C3-hydroxyl and quantified spectrophotometrically (Li et al. Journal of Food and Drug Analysis, 2019;27(2):375 - 386).

[0073] (ii) Cholesterol, direct LDL assay In many individuals, low-density lipoprotein (LDL), one of the major carriers of cholesterol in the blood, increases in concentration through middle age and then remains stable or decreases with advancing age (McAuley and Mooney. Med Hypotheses, 2017;104:15-19). In men, LDL tends to peak between 50 and 60 years of age, while in women, this trend persists and typically reaches its maximum concentration between 60 and 70 years of age (Kreisberg and Kasim, 1987;82(1):54-60). In many cases, correction for certain factors such as estrogen levels in women is required, but LDL levels can be a powerful indicator of biological age. Thus, a method for determining biological age can include determining the LDL level of the subject.

[0074] Often, LDL is measured simultaneously with other lipids and lipoproteins as part of a lipid profile or panel. In some panels, LDL is identified indirectly through total cholesterol measurement. LDL measurement may identify LDL, LDL-cholesterol (LDL-C, the amount of cholesterol contained within low-density lipoprotein), or both. Since cholesterol is carried primarily within LDL, high-density lipoprotein (HDL), and very-low-density lipoprotein (VLDL), LDL levels are often determined not by measuring LDL, but rather by subtracting the non-LDL abundance from the measured level of cholesterol. For example, the lipid panel in the United States generally estimates LDL levels by subtracting the measured HDL and triglyceride levels from the measured total blood cholesterol.

[0075] LDL may also be measured directly. In many cases, these methods involve the separation of LDL from other lipoproteins and lipids, including HDL, VLDL, and lipoprotein a (Lp(a)). Common methods to achieve these separations include centrifugation (e.g., ultracentrifugation) that can separate lipoproteins and lipids by density; electrophoresis that can separate lipoproteins and lipids based on size and charge; precipitation that selectively extracts lipoproteins and / or lipids from solution; homogeneous methods that utilize combinations of conditions, binding molecules, and polymers to separate lipoprotein fractions; and combinations thereof. The separated lipoproteins and lipids can then be quantified by a range of enzymatic, electrochemical, chemical, and spectroscopic methods (Nauck et al. Clinical Chemistry, 2002;48(2):236-254).

[0076] (iii) Cholesterol, direct HDL assay Similar to LDL, HDL levels tend to decrease in concentration with advancing age. Due to the complexity of the relationship between HDL and aging, low HDL can be correlated with mortality, potentially obscuring other reliable trends in the abundance of HDL that decline with age (Walter, Arteriosclerosis, Thrombosis and Vascular Biology, 2009;29:1244-1250). Nevertheless, several studies have defined a clear decrease in HDL that correlates with age, particularly among men (Ferrara et al. Circulation, 1997;96:37-43). Thus, HDL levels, either alone or in combination with other cholesterol data (e.g., LDL, total cholesterol, etc.), can be used to assess biological age.

[0077] Similar to LDL-cholesterol, HDL-cholesterol is typically measured through separation followed by cholesterol quantification. However, some methods (especially some homogeneous methods) enable the measurement of HDL-cholesterol simultaneously with other forms of cholesterol.

[0078] (iv) Blood glucose assay In many individuals, aging is associated with an increase in fasting and postprandial glucose levels. Since blood glucose levels respond to multiple age-sensitive regulatory mechanisms including insulin and incretin sensitivity and can contribute to age-related pathologies such as increased HbA1c and albumin glycation, blood glucose levels can capture a wide range of age-related progression and function as a reliable diagnostic marker for aging. In both men and women, fasting and postprandial glucose levels tend to increase at a rate of approximately 7 - 11 μg / mL per decade, while postprandial two-hour levels tend to increase at a more significant rate of 56 - 66 μg / mL per decade (Chia et al. Circulation Research, 2018;123(7):886 - 904). According to these trends, blood glucose levels (either fasting, postprandial, or a combination thereof) can be used to determine biological age.

[0079] Several inexpensive methods are available for measuring blood glucose levels. Most commonly, blood glucose is detected through enzymatic or chemical oxidation (e.g., through hydrogen peroxide generation by glucose oxidase).

[0080] Urine analysis Urine is a complex mixture of chemicals that can accurately reflect age, health, and the environment, although water, urea, and sodium chloride account for over 96% of its mass. More than 450 microbial communities and 3,000 molecules have been identified in urine, 480 of which have not been detected in the blood (Bouatra et al. PLoS One, 2013; 8(9):e73076). Many of the molecules in urine are waste substances, including metabolic by-products and damaged and solubilized biomolecules. Therefore, age-related changes within a subject, particularly the age of the kidneys, liver, and bladder, are often reflected in urine composition (Harpole et al. Expert Rev Proteomics, 2016; 13(6):609-626).

[0081] Peripheral blood mononuclear cell analysis Methods for determining biological age can include peripheral blood mononuclear cell (PBMC) analysis. PBMCs include blood cells with round nuclei such as T lymphocytes, B lymphocytes, natural killer cells, and monocytes (as well as control non-nuclear cells such as red blood cells and cells with monolobed nuclei such as granulocytes). Some PBMC phenotypic distributions have been shown to change with age. In a particular individual, the T cell population shifts from CD28+ to CD95+ with age, suggesting reduced proliferation and increased apoptosis (Li et al. J Int Med Res, 2020; 48(7)). Furthermore, aging often coincides with an increase in monocytes and regulatory T cells, as well as a decrease in the naive T cell population (Huang et al. PNAS, 2021; 118(33):e2013216118). Therefore, the polarization of PBMC populations and subpopulations can be used for biological age analysis. For example, such an analysis to identify T lymphocytes, B lymphocytes, and natural killer cells, as well as CD4, CD8, CD28, naive, effector, and central memory cell subpopulations, can include PBMC immunophenotyping. As an illustrative example, PBMCs can be isolated from whole blood and characterized by flow cytometry, for example, as outlined by Li et al.

[0082] In addition to identifying certain cell types, flow cytometry can also be combined with fluorescently conjugated antibodies so that cell surface proteins can be similarly identified and / or quantified. Examples of cell surface markers (i.e., proteins located on the cell surface) found on cells in the blood that can be identified and quantified using flow cytometry include CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT. Examples of cell types that can have these markers include white blood cells, specifically lymphocytes, neutrophils, monocytes, basophils, and eosinophils. For example, T lymphocytes can include one or more of the cell surface markers that can be identified and / or quantified using flow cytometry.

[0083] Cell senescence assay Biological age measurement can include the analysis of senescent cell populations. Cellular senescence is characterized by the cessation of growth and the end of cell division, but senescence is consistent with changes in the cell phenotype. The senescent phenotype not only is harmful to cell function but also generally induces a decrease in fitness levels (e.g., a decrease in energy metabolism and an increase in oxidative stress), and tends to be consistent with harmful secretory behavior, such as pro-inflammatory exosome production, and senescence-associated secretory phenotype (SASP) secretory behavior. To illustrate this point, recent mouse model experiments have demonstrated that as few as 1 / 10,000 senescent cells in healthy adult mice are sufficient to cause systemic dysfunction, metabolic stress, and accelerated aging (Xu et al. Nat Med, 2018;24(8):1246-1256). Thus, cellular senescence is not only harmful to senescent cells but can also accelerate aging and exacerbate aging symptoms in different cells within an organism.

[0084] The aging phenotype typically affects detectable changes in the expression profile. At the cellular level, non-terminally differentiated senescent cells can often be identified by their inability to replicate or undergo DNA synthesis. At the gene level, senescent cells can also typically be identified based on changes in the regulation of proliferation-related and growth-inhibitory genes (Itahana et al. Methods to Detect Biomarkers of Cellular Senescence. In Methods in Molecular Biology: Biological Aging: Methods and Protocols. Jumana Press Inc.). However, senescent cells are most commonly identified by senescence-associated markers, which can include upregulation of the lysosomal-associated protein β-galactosidase (Dimri et al. Proc. Natl. Acad. Sci, 1995;92:9363-9367); the DNA damage response marker H2AX (Campisi, J. Annu. Rev. Physiol, 2013 75:685-705); the tumor suppressors p16ink4a, p21, and p53 (Rufini et al. Oncogene, 2013;32:5129-5143), as well as changes in the secretion profile (e.g., increased production of pro-inflammatory cytokines, Itahana et al.).

[0085] Markers associated with cellular senescence include senescence-associated β-galactosidase ("SA-β-gal"), which can be measured in the blood and used as a measure of cellular senescence.

[0086] Genome methylation assay DNA methylation is a common epigenetic modification that can strongly influence expression in a subject and the corresponding phenotype. Recent studies have demonstrated that aging coincides with genome-wide DNA methylation and demethylation, and that the prevalence of demethylation is higher than that of methylation during most of human lifespan. Total genomic methylation can be correlated with age, but several recent studies have identified specific sites that can function as aging markers based on their methylation or hydroxymethylation status (Salameh et al. Front. Genet., 2020;10). Therefore, to confirm biological age, the methods of the present disclosure can determine the methylation status at a site or sites of genomic DNA.

[0087] Inflammatory marker analysis In some cases, inflammatory markers can be used to assess the biological age of an individual. As used herein, an inflammatory marker can be a species that causes inflammation or whose concentration increases in response to inflammation. Inflammation tends to increase with age and often reduces immune function, contributes to frailty, and exacerbates conditions such as arthritis, asthma, atherosclerosis, and certain forms of dementia. Nevertheless, only some inflammation-related markers increase in concentration with age. For example, recent studies have identified chemokine (C-X-C motif) ligand 9 (CXCL9), eotaxin, macrophage inflammatory protein (Mip-1α), leptin, IL-1β, interleukin-5 (IL-5), interferon-α (IFN-α), interleukin-4 (IL-4), TNF-related apoptosis-inducing ligand (TRAIL), interferon-γ (IFN-γ), chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-2 (IL-2), transforming growth factor-α (TGF-α), plasminogen activator inhibitor (PAI)-1, and leukemia inhibitory factor (LIF) as potential biomarkers of inflammation, while other inflammatory response markers such as IL-6 and TNF-α have minimal association with aging (Sayed et al. Nature Aging, 2021;1:598-615). Arising from observations of this type, the methods of the present disclosure can utilize one or more inflammatory markers to determine biological age.

[0088] Safety analysis The aging treatments disclosed herein can be combined with safety or health assessments. In addition to, or alternatively to, monitoring the biological age in a subject undergoing aging treatment, one or more health markers can be monitored to ensure the effectiveness of the treatment, to provide a calibration for age measurement, or for a combination thereof. Some aging treatments can cause adverse effects such as a decrease in the number of red blood cells (RBCs) or a decrease in liver function in a particular subject. Monitoring health before or at the same time as the aging treatment can make it possible to adjust the treatment to the subject, determine whether the subject is suitable for the aging treatment, and ensure that the treatment is modified or discontinued after an adverse response.

[0089] Furthermore, in some cases, markers of overall health can be important for determining biological age. Since many age markers vary in response to health status, calibration of the subject's health may be required for accurate biological age assessment. For example, certain conditions can increase autoantibody concentrations to a greater extent than aging, and such autoantibody analysis will only be valid for a particular subject. Non-limiting examples of health assessments are outlined in Table II. Subjects of aging treatment methods can be evaluated by one or more health assessments outlined in Table II or otherwise consistent with the methods disclosed herein.

Table 2

[0090] (i) Blood-based assays Subjects of aging treatment methods can be evaluated by a complete blood count (CBC) assay before, during, and / or after treatment. A complete blood count assay typically quantitatively measures multiple components of blood such as red blood cells, white blood cells, hemoglobin, and platelets, as well as health and aging factors such as the red blood cell to plasma ratio. In addition to an overall health screening, a complete blood count assay can identify adverse responses to some aging treatments such as blood diffusion-based anemia.

[0091] Similarly, the subject of anti-aging treatment can be evaluated by a total protein test to determine the protein level in a biological fluid (e.g., blood). Some total protein tests quantify the concentration and / or ratio of specific proteins such as albumin and globulin (e.g., by immunoassay), while others determine the total protein concentration in a biological fluid (e.g., based on the protein band at 280 nm in a spectrophotometric absorbance assay). Total protein tests can identify some potential side effects of anti-aging treatment, including fatigue, edema, and malnutrition.

[0092] Liver function assays can test for several substances that are indicators of proper liver function and health. Many liver function assays determine the blood levels of liver-based or secreted enzymes, including alanine transaminase, aspartate transaminase, alkaline phosphatase, albumin, and gamma-glutamyl transpeptidase. Liver function assays can also evaluate the levels of metabolites, such as bilirubin, a major heme breakdown product, that are regulated (e.g., cleared) by the liver. Liver function assays can also determine the quality of the blood, such as pH or prothrombin time (the rate of blood clotting). The specific set of evaluations included in a liver function assay can depend on the health of the subject, as well as the type of anti-aging treatment and diagnosis the subject is receiving.

[0093] Blood urea nitrogen (BUN) assays can evaluate kidney function and metabolic function in subjects undergoing anti-aging treatment. BUN assays measure the level of urea in the blood. Blood-based urea, which is mainly derived from proteolysis, is maintained at a low level by kidney filtration. In addition to inappropriate kidney function, high blood urea levels can indicate dehydration (a risk associated with some blood dilution methods), internal bleeding, and shock.

[0094] The creatinine assay provides an additional form of assessment of kidney function. The creatinine assay measures the concentration of creatinine, a catabolic waste product in the blood. The kidneys typically filter creatinine from the blood and thus maintain low levels of creatinine in the blood, but this function is impaired by several conditions that can be identified by the creatinine assay. In certain subjects, the use of the creatinine assay can be important for monitoring kidney health before, during, or after geriatric treatment.

[0095] The C-reactive protein (CRP) assay can function as a measure of inflammation and infection in subjects undergoing geriatric treatment. Since some geriatric treatments increase susceptibility to infection, the C-reactive protein assay can be an important measure of the effectiveness of geriatric treatment and the health of the subject.

[0096] Method for performing plasmapheresis Treatment and prevention of the effects of aging using plasmapheresis As used herein, the term "plasmapheresis" is synonymous with the term "therapeutic plasma exchange," and plasmapheresis is a form of apheresis in which some amount of an individual's plasma is removed from the individual's body. Methods of plasmapheresis for treating and / or preventing symptoms or conditions associated with aging are described herein. In treating and / or preventing symptoms or conditions associated with aging, the plasmapheresis methods described herein may also increase lifespan and promote longevity.

[0097] Plasma pheresis treatment typically involves and begins with collecting whole blood from an individual undergoing plasma pheresis treatment. The collected whole blood is separated into a cellular fraction and a plasma fraction. As used herein, the term "cellular fraction" may refer to or include red blood cells, white blood cells, and platelets. As used herein, the term "plasma fraction" or "plasma" may refer to or include the liquid portion of whole blood that contains, among other things, proteins, electrolytes, vitamins, and hormones. Typically, in plasma pheresis treatment, the separated plasma fraction is removed and the cellular fraction is returned to the individual undergoing plasma pheresis treatment. In the context of performing plasma pheresis (or any other type of apheresis procedure), as used herein, the terms "collect", "collection", "collected", and "collecting" (or any other conjugated form of "collect") mean to (actively or passively) collect blood from the vascular system of an individual undergoing plasma pheresis (or other type of apheresis procedure), which can be accomplished using any suitable vascular access, including but not limited to peripheral venous lines and central lines. In the context of performing plasma pheresis (or any other type of apheresis procedure), as used herein, the terms "return" or "returning" (or any other conjugated form of "return"), or "infuse" or "infusing" (or any other conjugated form of "infuse") mean to (actively or passively) return blood to the vascular system of an individual undergoing plasma pheresis (or any other type of apheresis procedure), which can be accomplished using any suitable vascular access. In the context of performing plasma pheresis (or any other type of apheresis procedure), as used herein, the terms "remove", "removal", "removed", and "removing" (or any other conjugated form of "remove") mean to remove at least a portion of the whole blood collected from an individual undergoing plasma pheresis (or any other apheresis procedure) and not return at least a portion of the whole blood to the individual undergoing plasma pheresis, thereby being removed from the body.As used herein, in the context of plasmapheresis (or any other apheresis procedure), the terms "separate", "separated", or "separating" (or any other conjugation of "separate") mean separating the components of blood from one another. For example, in plasmapheresis, whole blood is drawn and plasma is separated from the cellular fraction of the drawn whole blood. As used herein, the term "plasmapheresis" may be combined with other terms such as "therapy" (i.e., plasmapheresis therapy) or "treatment" (i.e., plasmapheresis treatment) or "procedure" (i.e., plasmapheresis procedure), and unless otherwise indicated, no particular meaning should be considered to result from the use in the context in which one or the other of these terms appears. It should be noted that the term "plasmapheresis" is often used synonymously with therapeutic plasma exchange, and in other cases, is used to denote a form of therapeutic plasma exchange in which less plasma is removed than in therapeutic plasma exchange where the plasma removed is removed. To avoid confusion with terms, the term plasmapheresis is used throughout, and where described and used herein, the term therapeutic plasma exchange is synonymous with the term plasmapheresis. However, the term plasmapheresis should not be considered to limit the scope of the disclosure found herein and may relate to different types of apheresis depending on the context.

[0098] A plasmapheresis therapy session can begin with the first step of collecting whole blood from a patient's blood vessels using an apheresis device. An apheresis device is a well-known machine configured to perform procedures including plasmapheresis. The apheresis device can be configured to collect whole blood from an individual through a venous line, separate the whole blood into its components, and return an infusion to the individual through the venous line. The infusion returned to the individual can include separate components and can include blood from which the plasma component has been removed. Additionally, the infusion given to the individual can similarly include a replacement fluid. The apheresis device can be an ex vivo apheresis system or a machine with one or more centrifugal chambers. The ex vivo apheresis system or machine can also include a return flow controller and one or more sensors for monitoring plasma or blood density. The apheresis device can also be configured to deliver an anticoagulant to the patient during the procedure. In some embodiments, the anticoagulant can be dextrose citrate. However, any method or device for performing plasmapheresis is suitable for use with the methods and formulations described herein, and the provided description should not be construed as limiting the methods or formulations of the present invention described herein to any particular device or method for performing plasmapheresis.

[0099] The replacement fluid is typically administered by plasmapheresis, and the replacement fluid is administered to an individual undergoing plasmapheresis intravascularly (i.e., via intravenous access, e.g., through a peripheral or central line) during plasmapheresis treatment. The replacement fluid can include any fluid suitable for use in intravenous fluid administration. For example, non-limiting examples of fluids suitable for intravascular administration by the practice of plasmapheresis described herein are generally referred to as isotonic fluids and include normal saline (i.e., a 0.9% aqueous saline solution) and lactated Ringer's. Replacement fluid solutions suitable for use in plasmapheresis described herein can further include albumin such as human albumin. For example, the replacement fluid can include a normal saline aqueous solution containing 5% by weight albumin. Typically, the source of albumin is human-derived albumin, also referred to as human serum albumin (HSA). As an example, a replacement fluid suitable for use by plasmapheresis can include a sterile liquid preparation containing an amount of human-derived protein of 50 g per 1000 ml of sterile liquid preparation, wherein at least 96% of the human-derived protein is human serum albumin protein. In addition to the human-derived serum albumin protein, the remaining portion of the HSA preparation can include an aqueous saline solution and a small amount of potassium, N-acetyl-DL-tryptophan, caprylic acid, or combinations thereof. The 5% HSA preparation of the replacement fluid can be an FDA-approved 5% HSA preparation. More specifically, the 5% HSA preparation can be manufactured by FDA-approved procedures such as the Cohn-Oncley cold ethanol fractionation procedure followed by ultrafiltration and pasteurization. Replacing the plasma collected from an individual undergoing plasmapheresis with 5% HSA is useful for regulating and stabilizing the volume of circulating blood within the individual.

[0100] The replacement fluid can be mixed or combined in any suitable manner with the cellular fraction remaining after plasmapheresis of whole blood collected during plasmapheresis and returned to the individual undergoing plasmapheresis during the procedure. The replacement fluid suitable for use in plasmapheresis can also contain one or more therapeutic agents. For example, a therapeutic agent that reduces inflammation can be provided by plasmapheresis, for example, by mixing the therapeutic agent with the replacement fluid. The replacement fluid for plasmapheresis can also contain or can be mixed together with one or more blood products (i.e., not including the cellular fraction to be returned), including but not limited to fresh frozen plasma and platelets. When mixed with the replacement fluid, the therapeutic agent and the blood product are collected at least to some extent from the individual undergoing plasmapheresis, and thus, it should be understood that it may be beneficial to administer or infuse the therapeutic agent and / or the blood product after completion of blood collection from the individual so that the therapeutic agent and / or the blood product are not collected from the individual during the plasmapheresis therapy.

[0101] As described, in a typical plasmapheresis procedure, a volume of whole blood is collected from an individual and a portion is returned to the individual together with the replacement fluid. Typically, the replacement fluid is returned to the individual simultaneously with the collection of whole blood, so the calculations associated with the plasmapheresis process are not entirely simple and straightforward.

[0102] Typically, as described above, the replacement fluid is infused into an individual undergoing plasmapheresis simultaneously with collection of whole blood and removal of plasma, so the replacement fluid essentially reconstitutes a portion of the plasma volume during the procedure, and thus it is not possible to remove the entire plasma volume during a typical plasmapheresis procedure because the plasma is continuously replenished to some extent as it is removed. As used herein, "plasma volume" refers to the total volume of plasma within an individual's whole blood and can be calculated using any of a number of methods well known for calculating plasma volume. In a typical plasmapheresis procedure, a volume of plasma equal to about 1 plasma volume is removed from the individual undergoing plasmapheresis, or in a typical plasmapheresis procedure, up to 1.5 plasma volumes of 1 plasma volume can be removed. Typically, during the plasmapheresis procedure described herein, an amount of replacement fluid essentially equal to the amount of plasma volume collected from the patient is returned to the patient such that the removed plasma volume (not returned to the patient) is "exchanged" for the replacement fluid that replaces the removed plasma volume. In some embodiments, the replacement fluid may be more or less than the plasma volume removed, but again, typically it is essentially equal in volume. For example, if 1 plasma volume is removed from an individual in the method described herein, a volume of replacement fluid equal to (or essentially equal to) 1 plasma volume is returned to the individual.

[0103] As described, since the replacement fluid is infused into an individual simultaneously during the plasmapheresis procedure, removing either 1 to 1.5 plasma volumes during the plasmapheresis procedure typically does not mean that the plasma (and its contents) is completely removed even if a volume equal to 1 to 1.5 plasma volumes is removed. The volume removed contains the replacement fluid infused into the individual, and then the replacement fluid is removed as part of the total volume removed. According to Winters (Hematology Am Soc Hematol Educ Program, 2012; 2012(1):7 - 12), in a typical plasmapheresis treatment where 1 to 1.5 plasma volumes are exchanged, approximately 60 to 70% of the substances present in the plasma at the start of plasmapheresis are removed, which means that approximately 30 to 40% of the substances present in the plasma at the start of plasmapheresis remain in the body of an individual undergoing plasmapheresis after a typical single - session plasmapheresis treatment.

[0104] Depending on the weight of the individual undergoing plasmapheresis, the volume of plasma removed can be approximately 2L to 4L. When 2L to 4L of plasma is removed during plasmapheresis, the volume of whole blood collected from the patient is necessarily more than 2.0L to 4.0L, i.e., the volume of whole blood collected contains the volume of plasma removed, and thus the volume of whole blood collected is necessarily always greater than the volume of plasma removed. The plasma volume in an individual depends on several factors including weight and gender, and thus 2L to 4L is used herein only as a non - limiting example of the range of plasma that can be removed during the plasmapheresis procedure, and in some cases, plasmapheresis can involve the removal of less than 2L or more than 4L of plasma.

[0105] Blood can be drawn from a patient's blood vessels, including peripheral blood vessels, central blood vessels, or combinations thereof. Since the blood flow from the patient to the apheresis device needs to be stable and preferably faster than 50 mL / min, the vascular access site is typically a blood vessel that can withstand high negative pressure without collapsing.

[0106] Furthermore, the vascular access site for receiving the exchange fluid, or a mixture comprising the exchange fluid and one or more other components, is typically another blood vessel (or another peripheral or central access point) capable of withstanding a relatively high positive pressure. In some embodiments of the methods described herein, whole blood can be drawn from a patient's peripheral vein, such as the antecubital fossa, basilica vein, or cephalic vein, using a large-bore needle or cannula. Additionally, if determined by the plasmapheresis provider to be the optimal vascular access point, whole blood can also be drawn by cannulating the radial artery of an individual undergoing plasmapheresis. If determined by the plasmapheresis provider to be the optimal vascular access point, whole blood can be drawn using an intravascular device or an implantable device, such as a central venous catheter (CVC), arteriovenous (AV) shunt, AV fistula, or port CVC. For example, whole blood can be drawn from the internal jugular vein, subclavian vein, or femoral vein or artery of an individual undergoing plasmapheresis. Blood from an individual undergoing plasmapheresis can be drawn at a rate of approximately 90 ml / min. However, in the methods described herein, it is also preferred that blood from an individual undergoing plasmapheresis be drawn at a rate of approximately 90 ml / min to 135 ml / min. It should be understood that under certain conditions, drawing at a rate less than 90 ml / min or greater than 135 ml / min may be optimal for an individual undergoing plasmapheresis. As previously discussed, typically, the vascular access site for receiving the fluid returned to an individual undergoing plasmapheresis, which can include, for example, the exchange fluid, cell fraction, therapeutic agent, or blood product, and mixtures thereof, is different from the vascular access site for the initial blood draw. To deliver the fluid returned to an individual undergoing plasmapheresis, which includes the cell fraction and the exchange fluid, to the blood vessels in the arm, hand, neck, or chest of the individual undergoing plasmapheresis, for example, a cannula or catheter extending from or connected to the apheresis device can be used.

[0107] In some embodiments of the methods described herein, each plasmapheresis treatment session may last approximately 90 minutes to 2 hours. However, the length of the plasmapheresis session can vary based on the purpose of the plasmapheresis treatment, and it should be understood that in the methods and formulations described herein, sessions shorter than 90 minutes or longer than 2 hours may be suitable. Additionally, the duration of the plasmapheresis treatment session can vary depending on certain factors associated with the individual undergoing plasmapheresis, including but not limited to the weight of the individual or the overall health status of the individual.

[0108] In an exemplary method for performing plasmapheresis on an individual, the plasmapheresis method may include one or more plasmapheresis therapies. The plasmapheresis method can begin with the step of identifying an individual in need of plasmapheresis treatment. The treatment method may further include collecting whole blood from the blood vessels of the individual undergoing plasmapheresis using an apheresis device or other technique for collecting blood. The plasmapheresis method may further include separating the collected whole blood into a cell fraction and a plasma fraction using an apheresis device or other technique for separating blood components. The plasmapheresis method may further include injecting back into the individual undergoing plasmapheresis an exchange fluid and the cell fraction while removing the plasma fraction from the individual. The amount of exchange fluid returned to the individual may be approximately equal to the amount of plasma removed. For example, if 1 plasma volume is removed from an individual undergoing plasmapheresis, in certain methods described herein, the amount of exchange fluid returned to the individual will also be approximately equal to 1 plasma volume. Alternatively, the amount of exchange fluid returned may exceed the amount of plasma volume removed. For example, if 1 plasma volume is removed, two or more plasma volumes may be injected back into the individual undergoing plasmapheresis.

[0109] The factors involved in aging are located within the patient's plasma and, thus, the factors involved in aging are removed from the body of an individual undergoing plasmapheresis by removal of plasma from the individual's body using the innovative plasmapheresis method described herein.

[0110] As described, aging is likely a multifaceted process that is not currently fully understood and generally impedes the development of effective therapies for treating and preventing the effects of aging. However, while the overall pathophysiology of aging may not yet be fully clear, there are clear choke points in the pathophysiology of aging where the innovative methods described herein are effective. For example, it is understood that factors affecting aging, including cytokines and peptides associated with aging, can all be found in plasma. As used herein, "plasma contents" describes all of the separate components of plasma, and at least some of the plasma contents are known to have components within them that affect aging (even if it is not known specifically what they are within the plasma contents). These targeted aging-related factor(s) must necessarily pass through the vasculature within the plasma in order to reach the cells and / or tissues upon which they act, and thus, these factor(s) can be effectively collected and removed from the vasculature choke point using the innovative methods described herein. Due to the presence of these aging factors in plasma, plasma becomes a potential choke point in the aging process. Even when it is not necessarily known which factor(s) found in the blood are targeted by age-related therapies, by using the innovative methods and formulations described herein to remove all or as much as possible of the plasma contents, factor(s) affecting aging are necessarily also removed from the body, and thus, the aging activity associated with the factor(s) being removed is reduced or prevented.

[0111] As already explained, a plasmapheresis session typically removes about 60 - 70% of the plasma content, which means that about 30 - 40% of the plasma content, including those factors associated with aging in the body of an individual undergoing a typical single plasmapheresis treatment, remains in the blood of the individual undergoing plasmapheresis after a single plasmapheresis treatment.

[0112] In certain methods described herein, the plasmapheresis protocol removes more plasma than is removed in a typical single plasmapheresis treatment, such that more than 60 - 70% of the plasma content of an individual undergoing plasmapheresis is removed from the individual's body. For example, in certain methods described herein, a first plasmapheresis treatment can remove 60 - 70% of an individual's plasma content, and then the individual undergoes an additional plasmapheresis treatment within a time window for the first plasmapheresis treatment, such that the first plasmapheresis treatment together with the additional plasmapheresis treatment achieves a cumulative removal of more than 60 - 70% of the plasma. In these embodiments, the additional plasmapheresis treatment is performed before the plasma removed from the plasmapheresis - treated individual is fully replenished into the blood of the plasmapheresis - treated individual. In these embodiments, the plasmapheresis therapy method described herein can be performed, and then the subsequent plasmapheresis therapy method described herein can be performed at a time within 72 hours of the first plasmapheresis therapy. Similarly, the plasmapheresis therapy method described herein can be performed, and then the additional plasmapheresis therapy method described herein can be performed at a time within 48 hours of the first plasmapheresis therapy. For example, the plasmapheresis therapy method described herein can be performed, and then the additional plasmapheresis therapy method described herein can be performed at a time within 24 hours of the first plasmapheresis therapy. Similarly, in these methods, an additional plasmapheresis treatment in an individual can also be performed within a time frame that is within 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days of the first plasmapheresis treatment in the individual.

[0113] Another approach to achieve as much removal of plasma content as possible involves collecting more than 1.5 times the plasma volume in a single plasmapheresis session. For example, the methods for performing plasmapheresis described herein include collecting 1.5 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment. For example, the methods for performing plasmapheresis described herein include collecting 1.6 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment. For example, the methods for performing plasmapheresis described herein include collecting 1.7 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment. For example, the methods for performing plasmapheresis described herein include collecting 1.8 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment. For example, the methods for performing plasmapheresis described herein include collecting 1.9 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment. For example, the methods for performing plasmapheresis described herein include collecting 2 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment. Similarly, the methods for performing plasmapheresis described herein may include collecting more than 2 times the plasma volume of an individual undergoing plasmapheresis in a single plasmapheresis treatment.

[0114] In each case where a high percentage of plasma content is removed, to address any increased bleeding risk after plasmapheresis treatment due to the removal of blood coagulation factors and platelets that are removed along with the plasma content, coagulation factors and / or platelets can be infused back into the individual undergoing plasmapheresis to replenish the removed blood coagulation factors and platelets.

[0115] As used herein, "plasmapheresis regimen" includes within its scope any application of plasmapheresis described herein for the treatment or prevention of aging and / or the promotion of longevity. A plasmapheresis regimen may include one or more treatments conducted over a specific period of time and, in certain embodiments, may include therapeutic agents provided in conjunction with plasmapheresis.

[0116] In the innovative methods described herein, plasmapheresis may be used in a plasmapheresis regimen to remove a relatively large amount of plasma contents from an individual undergoing plasmapheresis. In certain plasmapheresis regimens, plasmapheresis may be performed on a schedule of twice a week to achieve a cumulative effect with respect to the plasma content removal described herein, within 24 hours of each other (including within the same day), within 36 hours of each other, within 48 hours of each other, within 60 hours of each other, within 72 hours of each other, within 84 hours of each other, within 96 hours of each other, within 108 hours of each other, within 120 hours of each other, within 134 hours of each other, within 156 hours of each other, or within 168 hours of each other, and delivered to an individual during the period between a first plasmapheresis treatment and a second plasmapheresis treatment. Additional plasmapheresis treatments in an individual may also be performed within a time frame that is within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, or within 14 days of the first plasmapheresis treatment in the individual.

[0117] The described plasmapheresis regimen performed twice a month may perform a total of 8 plasmapheresis treatments per month on a weekly basis, a total of 4 plasmapheresis treatments per month on an every-other-week basis, or a total of 2 treatments per month once a month. The plasmapheresis regimen described herein with twice-weekly treatments may be performed with twice-weekly treatments for a total of 6 months. During that 6-month period, all of the twice-weekly treatments may be performed with the same amount of time between each of the twice-weekly treatments, or the amount of time may vary between the twice-weekly treatments.

[0118] The plasmapheresis regimen described herein may include performing plasmapheresis once a week. For example, the described plasmapheresis regimen performed once a month may perform a total of 4 plasmapheresis treatments per month, once a week for each week of the month, a total of 2 plasmapheresis treatments every other week per month, or a total of 1 treatment per month once a month. Any plasmapheresis regimen described herein with once-a-week treatment may be performed over a total of 6 months.

[0119] In certain plasmapheresis treatments described herein, any subsequent plasmapheresis treatment session can only be performed after 24 days have elapsed since the last plasmapheresis treatment session. In these and other embodiments, the overall treatment method can be terminated after 125 days have elapsed since the first plasmapheresis treatment session. The treatment method can also include continuing the treatment method by repeating the method when at least 24 days have elapsed since the last treatment session. In these embodiments, plasmapheresis treatment sessions should not be performed during this intervening waiting period. For example, various plasmapheresis treatment sessions can be separated by only 24, 25, 26, 27, 28, 29 days, or any combination thereof. Various plasmapheresis treatment sessions can be separated by the same number of days or different numbers of days. As a more specific example, the first plasmapheresis treatment session can be separated from the second plasmapheresis treatment session by only 24 days, and the second plasmapheresis treatment session can be separated from the third plasmapheresis treatment session by 25 or 26 days. In other exemplary methods, the first plasmapheresis treatment session can be separated from the second plasmapheresis treatment session by 26 days, and the second plasmapheresis treatment session can be separated from the third plasmapheresis treatment session by 24 or 25 days. In some embodiments, the treatment method can be completely terminated when 125 days have elapsed since the first treatment session. In other embodiments, the treatment method can be terminated when at least 6 plasmapheresis treatment sessions have been performed, regardless of the number of days elapsed since the first plasmapheresis treatment session. In additional embodiments, the treatment method can be terminated when at least 7 or at least 8 plasmapheresis treatment sessions have been performed, regardless of the number of days elapsed since the first plasmapheresis treatment session. In another treatment method for performing plasmapheresis, the treatment method can include a total of 6 (six) plasmapheresis treatment sessions.The first plasma pheresis treatment session can be performed on the first day of treatment (day 1), the second plasma pheresis treatment session can be performed on the 25th day (day 25) of the treatment method, the third plasma pheresis treatment session can be performed on the 50th day (day 50) of the treatment method, the fourth plasma pheresis treatment session can be performed on the 75th day (day 75) of the treatment method, the fifth plasma pheresis treatment session can be performed on the 100th day (day 100) of the treatment method, and the sixth plasma pheresis treatment session can be performed on the 125th day (day 125) of the treatment method. In this embodiment, no plasma pheresis treatment session is performed during the intervening period between the aforementioned treatment sessions. In other embodiments, the plasma pheresis treatment regimen can be extended as long as necessary and can include as many separate sessions / treatments as necessary to achieve one or more measurable effects.

[0120] Regarding the use of plasmapheresis in the treatment of age - related and aging - associated conditions, anti - inflammatory agents or other immunomodulatory therapeutic agents can be used in conjunction with plasmapheresis treatment in a synergistic manner. More specifically, anti - inflammatory agents or immunomodulatory therapy agents can synergistically modulate, reduce, or eliminate the effects of inflammatory and immune cells and factors (e.g., cytokines) remaining in the blood after plasmapheresis treatment, thereby further reducing the effects of these cells and factors. In certain methods described herein, therapeutic agents are administered to target the age - related pathologies found within the blood of an individual undergoing plasmapheresis treatment. For example, inflammation is a known part of the aging process, sometimes referred to as "inflammaging," and the effects of aging correlate with increased levels of inflammatory substances in the blood. Thus, anti - inflammatory agents delivered in conjunction with plasmapheresis act to reduce inflammatory activity and, therefore, can disrupt the effects of inflammation in age - related conditions. In particular, with plasmapheresis, using the methods described herein, most of the acellular inflammatory factors found within the plasma content are removed from the body of the individual undergoing plasmapheresis treatment, and any remaining inflammatory cells can be further affected by the therapeutic agent, such that a strong synergistic effect exists. Thus, in the example provided, with a single typical plasmapheresis, after a single typical plasmapheresis treatment, approximately 30 - 40% of the inflammatory factors in the plasma content remain. When an anti - inflammatory agent or other immunomodulatory agent is administered either during or immediately after the performance of plasmapheresis, the anti - inflammatory agent or other immunomodulatory agent can act synergistically with the removal of inflammatory factors to further attenuate the effects of the inflammatory cells and factors remaining after plasmapheresis treatment. Applying therapy in this targeted manner produces a cumulative inhibitory effect that increases the benefits to the individual undergoing plasmapheresis.

[0121] The plasmapheresis treatment described herein may further include delivering an intravenous immunoglobulin (IVIG) in a therapeutically effective dosage to the blood vessels of an individual undergoing plasmapheresis treatment. In certain plasmapheresis treatments described herein, after returning the cellular fraction and replacement fluid to the individual undergoing plasmapheresis, the IVIG is delivered to the individual undergoing plasmapheresis. That is, a therapeutically effective dosage of IVIG may be provided to the individual separately from the infusion of the cellular fraction and replacement fluid to the individual undergoing plasmapheresis. For example, since the IVIG can be infused into the individual undergoing plasmapheresis after the plasmapheresis treatment is completed, any infused IVIG will not be removed during the plasmapheresis process. It is also possible to administer the IVIG simultaneously with the performance of plasmapheresis.

[0122] The therapeutically effective dosage of IVIG can be approximately 2.0 g of IVIG per kg of body weight of the individual undergoing plasmapheresis. The IVIG delivered can have a positive effect on the immune system of the individual undergoing plasmapheresis and can contribute to establishing an optimal systemic environment for cell growth, and can contain certain specific antibodies and cytokines. As described above, this is suitable for the administration of therapeutic agents such as IVIG mixed with the replacement fluid and / or cellular fraction during a part of the plasmapheresis procedure when the blood is being drawn, or for administering IVIG to the individual as part of the method for performing plasmapheresis described herein when the blood is no longer being drawn from the individual undergoing plasmapheresis. The therapeutically effective dosage of IVIG can be any FDA-approved IVIG, or an immunoglobulin intravenous (IGIV) infusion or preparation. For example, the IVIG can mainly contain gamma globulin.

[0123] The steps described do not require the specific order shown to achieve the desired result. Further, to achieve the desired result, certain steps or processes can be omitted or performed in parallel.

[0124] Each plasmapheresis treatment session can include steps of collecting whole blood from a patient's blood vessels using an apheresis device, separating the collected whole blood into a cellular fraction and a plasma fraction using the apheresis device, mixing the cellular fraction with an exchange fluid containing albumin derived from human plasma, returning a mixture containing the cellular fraction and the exchange fluid to the patient's blood vessels using the apheresis device, and delivering a therapeutically effective dosage of intravenous immunoglobulin (IVIG) to the blood vessels of an individual undergoing plasmapheresis after returning the mixture containing the cellular fraction and the exchange fluid to the individual's blood vessels.

[0125] Biological age, or any other measurable characteristic or marker of the effectiveness of plasmapheresis for an individual, can be measured using a number of markers and assays described herein. Specifically, biological age, physiological measurements (e.g., strength, gait, or balance), mental evaluations (e.g., mood stability surveys), or markers in an individual's blood (e.g., cell surface markers) measured using any of the analytical techniques, markers, and assays described herein can be used in conjunction with the plasmapheresis regimens described herein, and biological age, physiological measurements, mental evaluations, or markers identified, quantified, or determined to be present are used to influence modification of the plasmapheresis regimen.

[0126] For example, an individual's biological age, physiological measurements, mental evaluations, or blood markers can be measured before and after a plasmapheresis regimen (including one or more plasmapheresis treatments over a period of time) described herein using any of the markers and / or assays described herein, and the plasmapheresis regimen can be extended if a decrease (or, if it is beneficial for the item being measured to neither increase nor decrease, an increase) in the individual's biological age, physiological measurements, mental evaluations, or blood markers (measured prior to administration of the regimen) is not achieved. The plasmapheresis regimen can then be continued until the desired decrease (or, if it is beneficial for the item being measured to neither increase nor decrease, an increase) in biological age, physiological measurements, mental evaluations, or markers is achieved.

[0127] Similarly, an individual's biological age, physiological measurements, mental evaluations, or markers found in the blood can shape the plasmapheresis regimen itself. For example, an individual's biological age, physiological measurements, mental evaluations, or blood markers are measured before and after a single plasmapheresis treatment, and subsequent plasmapheresis treatments are performed until the biological age decreases below the desired level.

[0128] In an exemplary method, the plasmapheresis method can begin with the step of identifying an individual in need of plasmapheresis therapy. A blood sample is taken from the individual, either before the start of plasmapheresis or from the whole blood used to determine the individual's biological age using the markers and / or assays described herein. In some embodiments, physiological and mental evaluations are performed to assess one or more of the individual's strength, walking, balance, or mental state (exemplary techniques for obtaining these measurements are described herein, such as in the following sections describing the pilot study and results). The plasmapheresis method can further include collecting whole blood from the blood vessels of an individual undergoing plasmapheresis using an apheresis device or other technique for collecting blood. The plasmapheresis method can further include separating the collected whole blood into a cell fraction and a plasma fraction using an apheresis device or other technique for separating blood components. The treatment method can further include injecting back into the individual undergoing plasmapheresis the replacement fluid and the cell fraction while removing the plasma fraction from the individual. The amount of replacement fluid returned to the individual may be approximately equal to the amount of plasma removed. For example, if one plasma volume is removed from an individual undergoing plasmapheresis, in certain methods described herein, the amount of replacement fluid returned to the individual will also be approximately equal to one plasma volume. Alternatively, the amount of replacement fluid returned may exceed the amount of plasma volume removed. For example, if one plasma volume is removed, two or more plasma volumes may be injected back into the individual undergoing plasmapheresis. When the plasmapheresis therapy is complete, a second sample is obtained and a second biological age is determined using the markers and / or assays described herein.If one or more of an individual's biological age, physiological measurements, mental evaluations, or markers found in the blood do not decrease or increase sufficiently after plasmapheresis therapy (compared to the biological age, physiological measurements, mental evaluations, or markers determined prior to the start of plasmapheresis therapy), additional plasmapheresis therapy is performed while repeating sample collection and measurement of the biological age, physiological measurements, mental evaluations, or markers until the biological age, physiological measurements, mental evaluations, or markers decrease by the required amount while continuing plasmapheresis.

[0129] Also described herein is a method for treating aging by performing hemodilution. As described herein, plasma contents can be removed and replacement fluids can be added. Removing the plasma contents together while adding replacement fluids (even while partially replacing) typically reduces the concentration of one or more components of the plasma in an individual undergoing plasmapheresis therapy. In essence, the solvent (the liquid portion of the plasma replaced by the replacement fluid) remains the same while the solute (the plasma contents) decreases. Thus, in addition to removing plasma content components, the methods described herein dilute one or more components of the plasma contents remaining in the blood of an individual undergoing plasmapheresis therapy after plasmapheresis.

[0130] Plasma content dilution can achieve a synergistic effect on the removal of plasma contents alone in that at least the dilution causes the plasma composition of an individual undergoing plasmapheresis to resemble that of a biologically younger individual. That is, it has been empirically found that factors found in plasma associated with aging are present at lower concentrations in individuals younger than the elderly. A decrease in the concentration of plasma contents in an individual undergoing plasmapheresis as described herein, as in a younger individual in which aging-related factors found in plasma are less active, further reduces the relative activity of aging-related factors in those individuals undergoing plasmapheresis therapy as described herein.

[0131] (i) Modeling of blood dilution Using several models, the amount of dilution achieved by the plasmapheresis treatment described herein can be calculated. For example, Reverberi and Reverberi (Blood Transfus, 2007; 5(3): 164-174) provided equations for modeling the residual plasma analyte concentrations from which equations (I), (Ia), and (Ib) are derived, [Number] wherein v p is the plasma volume, n is the number of plasma volume units exchanged, and v b is the total blood volume (i.e., whole blood volume).

[0132] As an illustrative example of equation (I), a single plasmapheresis treatment using a plasma volume of 3 L, 1 plasma volume exchanged, and a total volume of 5 L of whole blood is expected to result in a residual plasma content concentration of approximately 54% of the original concentration. With the same plasma volume and total volume of blood, but a plasma exchange of 1.5 plasma volumes, the residual solute concentration is approximately 40%.

[0133] Typically, plasma analytes exhibit an immediate concentration decrease after blood exchange, followed by re-concentration along a logarithmic-like trend back to pre-blood dilution levels. Equation (I) can be modified to equation (Ia) to estimate the number of days n t after plasmapheresis, assuming a half-time d 1 / 2 for the plasma analyte to return to pre-blood dilution levels: [Number] Note that since typical plasma analytes return to pre-blood dilution levels after about 10 days, not all plasma analytes re-establish homeostasis at the same rate. Thus, in many cases, a t of 3-4 days 1 / 2is a preferred estimate. With a half-life of 3 days and measurements 3 days after the first plasma exchange treatment, if 3L, 1 plasma volume, and 5L of whole blood are used again, the residual solute concentration increases from approximately 54% after plasma exchange treatment to approximately 72% after 3 days (i.e., 72 hours).

[0134] Winters (cited above) provides the following simplified equation: Y / Y0 = e -x where Y is the final concentration of the substance, Y0 is the initial concentration, and X is the number of times the patient's plasma volume is exchanged. Continuing with the approximately 72% residual concentration calculated above using Equation (Ia), if plasma exchange is performed again 72 hours after the first plasma exchange, after the second plasma exchange treatment, the residual plasma concentration is Y0 = 72%, x = 1, and Y = 26.5%.

[0135] By further using Equation (Ia) and setting n as above, the degree of plasma analyte clearance after multiple plasma exchanges can be determined. In such applications, Equation (Ia) can be estimated as a sum represented by Equation (Ib), where each plasma exchange event i is attenuated by analyte regeneration over n di days following the plasma exchange event:

Number

[0136] The formulas provided by (I), (Ia), (Ib), and Winters all provide excellent useful approximations of the dilution levels suitable for use by the methods described herein. It should be noted that if higher accuracy should be achieved in the calculation of plasma content dilution, the following factors and issues should also be considered: First, many plasma separation methods only partially separate plasma from cellular components. Multiple separations can increase efficiency, but a single iteration of centrifuge-based plasma separation and membrane-based plasma separation typically achieve plasma separation efficiencies of 80% and 30% respectively (Williams and Balogun. Clin J Am Soc Nephrol, 2014;9(1):181-190). Typically, in a 500 mL blood draw containing approximately 55% (275 mL) of plasma, these efficiencies are interpreted as 220 mL of plasma being removed by centrifugation and 82.5 mL of plasma being removed by filtration. The effect of incomplete plasma separation from cellular components is an accompanying decrease in plasma exchange efficiency. When plasma is centrifugally separated from blood at 80% efficiency, considering the amount of blood separated and exchanged, plasma exchange is typically 80% efficient. Second, many plasma components are actively exchanged into the space outside the vascular system. A typical adult male human has approximately 5 liters of blood, which is the majority of the fluid. Similarly, analytes are contained in the interstitial and intracellular spaces, which constitute approximately 10.5 liters and 28 liters of fluid respectively. As used herein, the intracellular space can include all volumes contained within the cell membranes of an organism, including all fluids within these spaces, and the interstitial space can be shown to be the space surrounding tissues. Since many plasma analytes actively equilibrate between blood and these spaces, a fraction of their total population is contained within the blood at any given time. Removal of such species by plasma exchange can be attenuated by their partitioning outside of the blood. For example, only approximately 60% - 70% of IgG1 immunoglobulin is present in the blood at any given time, so plasma exchange can only target 60% - 70% of the IgG1 population.Furthermore, for example, disruption of homeostasis through hemodilution can affect the osmotic gradient that draws seeds from the extravascular space into the blood, thereby accelerating the return to pre-treatment blood analyte levels. Third, plasma analytes are regenerated at a range of rates. Following a blood composition change event such as hemodilution, blood analyte concentrations tend to return to their original resting levels. Hemodilution can change an individual's resting blood analyte levels (e.g., reducing blood triglyceride levels as outlined in Dehal and Adashek. Case Rep Med, 2018;2018:4017573), while diluted species tend to increase in concentration, and concentrated species (e.g., albumin provided from a high-concentration exchange fluid) tend to decrease in concentration and re-establish pre-dilution levels. Some species (especially many cytokines) exhibit complex re-equilibration patterns, while many follow simple exponential growth or decay curves. However, the rates of these processes can vary significantly between species. For example, IgG immunoglobulins often return to pre-hemodilution levels in about 4 days (Harris et al. Journal of Scleroderma and Related Disorders, 2018;3(2):132-152), while low-density lipoproteins can take over 2 weeks to return to resting levels (McGowan. Journal of Clinical Lipidology, 2013;7(3):S21-S26).

[0137] May et al. (Am J Clin Pathol, 1989;91(6):688-94) presented a model to explain extravascular compartmentalization and species regeneration. This model, fitted as Equation (II) below, captures the rate of plasma analyte loss through plasma exchange and clearance,

Number

[0138] Results of a human pilot study An IRB-approved pilot study was conducted to evaluate the effect of plasmapheresis on aging in human participants. This study included eight human participants over 50 years of age (chronological age) who each received six plasmapheresis treatments over a three-month period. Three of the eight treatment participants were female and five were male. In addition, a group of three participants received three sham plasmapheresis treatments over three months each and served as controls for the eight participants who each received six plasmapheresis treatments. All three participants who received the sham treatment were male.

[0139] The plasmapheresis treatments were performed using a Spectra Optia apheresis separator manufactured by Terumo BCT, Inc., supervised by an approved physician, and FDA-approved for that purpose. The eight participants who received plasmapheresis treatments had at least one plasma volume exchanged with replacement fluid containing 5% albumin over a period of 2 - 3 hours between each of the six plasmapheresis treatments. The plasmapheresis was performed in a clinical setting for the eight participants. The three sham patients used as controls appeared to receive plasmapheresis in a clinical setting but did not actually receive plasmapheresis.

[0140] The collected and analyzed data included macrodata and microdata. The macrodata included measures of strength, balance, walking, and mental and emotional stability. The microdata included blood studies that measured and quantified cell surface markers related to aging.

[0141] Macrodata For the macrodata, data on each participant were collected prior to each of the six plasmapheresis treatments. This included measures of strength, balance, walking, and mental and emotional stability. For strength, grip strength was measured using a grip strength measurement device. For balance, participants were asked to stand on one leg and balance, and the total time they were able to stand on one leg was recorded for up to 120 seconds. For walking, participants stood up from a seated position and walked a certain distance, and the total time it took for the participant to stand up and walk that distance was recorded. For mental and emotional stability, participants answered questions from the SF-12 survey, a recognized large-scale data-developed quality of life questionnaire consisting of questions that measure emotional and mental health.

[0142] Figure 1 shows multiple graphs of macrodata for the first participant in a pilot study (referred to as "PT1"), a female who received six plasma pheresis treatments. Macrodata for PT1 was collected prior to each of the six plasma pheresis treatments. In each plasma pheresis treatment that PT1 received, the replacement fluid for PT1 contained 2 grams of IVIG. The graphs in Figure 1 are titled to indicate which macrodata they present. In each of the five graphs shown in Figure 1, the x-axis has the numbers 1 - 6, indicating the time prior to each of the six plasma pheresis treatments for which the data was collected. In the graph titled "PT1 Grip Strength", the y-axis shows the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of PT1 increased overall over the course of the six treatments. In the graph titled "PT1 Stand and Walk", the y-axis is the measurement in seconds for each of the six times that PT1 stood up from a seated position and walked a certain distance (each participant tested walked the same distance). As can be seen, the time for PT1 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT1 Balance", the y-axis is the time in seconds that PT1 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, PT1 was able to stand for the maximum measured time throughout the study period. In the graph titled "PT1 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT1 showed an overall improvement in score over the course of the six treatments. In the graph titled "PT1 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT1 showed an overall improvement in score over the course of the six treatments.

[0143] Figure 2 shows multiple graphs of the macro data of the second participant (referred to as "PT2") in a pilot study who is male and received six plasmapheresis treatments. Macro data of PT2 was collected before each of the six plasmapheresis treatments. The graphs in Figure 2 are titled to indicate which macro data they present. In each of the five graphs shown in Figure 2, the x-axis has numbers from 1 to 6 indicating the time before each of the six plasmapheresis treatments for which the data was collected. In the graph titled "PT2 Grip Strength", the y-axis shows the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of PT2 increased overall over the course of the six treatments. In the graph titled "PT2 Stand and Walk", the y-axis is the time in seconds it took PT2 to stand from a seated position and walk a certain distance (each participant tested walked the same distance). As can be seen, the time it took PT2 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT2 Balance", the y-axis is the time in seconds PT2 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, PT2 was able to stand for the maximum measured time throughout the study period. In the graph titled "PT2 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT2 showed an overall improvement in score over the course of the six treatments. In the graph titled "PT2 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT2 showed an overall improvement in score over the course of the six treatments.

[0144] Figure 3 shows multiple graphs of the macro data of the third participant in a pilot study (PT3), a female who received six plasma exchange treatments. Macro data for PT3 was collected prior to each of the six plasma exchange treatments. In each plasma exchange treatment that PT3 received, PT3's replacement fluid contained 2 grams of IVIG. The graphs in Figure 3 are titled to indicate which macro data they present. In each of the five graphs shown in Figure 3, the x-axis has the numbers 1 - 6 indicating the time before each of the six plasma exchange treatments for which data was collected. In the graph titled "PT3 Grip Strength", the y-axis shows the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength measurements for PT3 decreased from an initial high measurement, then increased, and plateaued at a value lower than the initial strength measurement over the course of the six treatments. In the graph titled "PT3 Rise and Walk", the y-axis is the time in seconds it took PT3 to rise from a seated position and walk a certain distance (the same distance was walked by each participant tested) for each of the six times the measurement was taken. As can be seen, the time it took PT3 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT3 Balance", the y-axis is the time in seconds that PT3 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, PT3's ability to balance on one leg increased overall over the course of the six treatments. In the graph titled "PT3 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT3 showed an overall improvement in score over the course of the six treatments. In the graph titled "PT3 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT3 showed an overall improvement in score over the course of the six treatments.

[0145] Figure 4 shows multiple graphs of the macro data of the fourth participant (PT4) in a pilot study who is female and received six plasma exchange treatments. Macro data for PT4 was collected prior to each of the six plasma exchange treatments. The graphs in Figure 4 are titled to indicate which macro data they present. In each of the five graphs shown in Figure 4, the x-axis has the numbers 1 to 6 indicating the time before each of the six plasma exchange treatments for which the data was collected. In the graph titled "PT4 Grip Strength", the y-axis shows the measured grip strength in units of Kg as measured by a standard grip strength device. As can be seen, the measured hand strength of PT4 increased overall over the course of the six treatments. In the graph titled "PT4 Stand and Walk", the y-axis is the measurement in seconds for each of the six times that PT4 rose from a seated position and walked a certain distance (each participant tested walked the same distance). As can be seen, the time it took PT4 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT4 Balance", the y-axis is the time in seconds that PT4 was able to stand on one leg, and the maximum time measured was 120 seconds. As can be seen, PT4's ability to balance on one leg increased overall over the course of the six treatments. In the graph titled "PT4 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT4 showed an overall improvement in score over the course of the six treatments. In the graph titled "PT4 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT4 showed an overall improvement in score over the course of the six treatments.

[0146] Figure 5 shows multiple graphs of the macro data of the fifth participant in a pilot study (referred to as "PT5") who is male and received six plasma exchange treatments. Macro data of PT5 was collected before each of the six plasma exchange treatments. The graphs in Figure 5 are titled to indicate which macro data they present. In each of the five graphs shown in Figure 5, the x-axis has the numbers 1 to 6, indicating the time before each of the six plasma exchange treatments for which the data was collected. In the graph titled "PT5 Grip Strength", the y-axis shows the measured grip strength in kilograms, measured by a standard grip strength device. As can be seen, the measured hand strength of PT5 increased overall over the course of the six treatments. In the graph titled "PT5 Stand and Walk", the y-axis is the time in seconds it took PT5 to stand up from a seated position and walk a certain distance (the same distance was walked by each participant tested). As can be seen, the time it took PT to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT5 Balance", the y-axis is the time in seconds that PT5 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, the ability of PT5 to balance on one leg increased overall over the course of the six treatments. In the graph titled "PT5 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT5 showed an overall improvement in score initially and then a decrease over the course of the six treatments. In the graph titled "PT5 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT5 showed an overall improvement in score over the course of the six treatments.

[0147] Figure 6 shows multiple graphs of the macro data of the fourth participant (PT6) in a pilot study who is male and received six plasma exchange treatments. Macro data for PT6 was collected prior to each of the six plasma exchange treatments. In each plasma exchange treatment that PT6 received, the replacement fluid for PT6 contained 2 grams of IVIG. The graphs in Figure 6 are titled to indicate which macro data they present. In each of the five graphs shown in Figure 6, the x-axis has the numbers 1 to 6 indicating the time before each of the six plasma exchange treatments for which the data was collected. In the graph titled "PT6 Grip Strength", the y-axis shows the measured grip strength in units of Kg as measured by a standard grip strength device. As can be seen, the measured hand strength of PT6 first decreased and then increased, so there was no overall measured change over the course of the six treatments. In the graph titled "PT6 Stand and Walk", the y-axis is the time in seconds it took PT6 to stand from a seated position and walk a certain distance (each participant tested walked the same distance). As can be seen, the time it took PT6 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT6 Balance", the y-axis is the time in seconds that PT6 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, PT6's ability to balance on one leg increased overall over the course of the six treatments. In the graph titled "PT6 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT6 showed an overall improvement in score over the course of the six treatments. In the graph titled "PT6 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT6 showed an overall slight decrease in score over the course of the six treatments.

[0148] Figure 7 shows multiple graphs of macrodata for the fourth participant in a pilot study (referred to as "PT7") who is male and received six plasma exchange treatments. Macrodata for PT7 was collected prior to each of the six plasma exchange treatments. The graphs in Figure 7 are titled to indicate which macrodata they present. In each of the five graphs shown in Figure 7, the x-axis has the numbers 1 to 6, indicating the time prior to each of the six plasma exchange treatments for which the data was collected. In the graph titled "PT7 Grip Strength", the y-axis shows the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of PT7 first decreased and then increased, so there was a slight decrease in the overall measured strength over the course of the six treatments. In the graph titled "PT7 Stand and Walk", the y-axis is the time in seconds it took for PT7 to stand from a seated position and walk a certain distance (each participant tested walked the same distance). As can be seen, the time it took PT7 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT7 Balance", the y-axis is the time in seconds that PT7 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, PT7 was able to stand for the maximum measured time throughout the study period. In the graph titled "PT7 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT7 showed an overall improvement in score initially and then a decrease over the course of the six treatments. In the graph titled "PT7 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT7 showed an overall slight decrease in score over the course of the six treatments.

[0149] Figure 8 shows multiple graphs of the macro data of the 8th participant (referred to as "PT8") in a pilot study who is male and received 6 plasma exchange treatments. Before each of the 6 plasma exchange treatments, the macro data of PT8 was collected. In each plasma exchange treatment that PT8 received, the replacement fluid for PT8 contained 2 grams of IVIG. The graphs in Figure 8 are titled to indicate which macro data they present. In each of the 5 graphs shown in Figure 8, the x-axis has the numbers 1 to 6, indicating the time before each of the 6 plasma exchange treatments for which the data was collected. In the graph titled "PT8 Grip Strength", the y-axis shows the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of PT8 first decreased and then increased slightly, but decreased overall over the course of the 6 treatments. In the graph titled "PT8 Stand and Walk", the y-axis is the time in seconds it took for PT8 to stand up from a seated position and walk a certain distance (each participant tested walked the same distance). As can be seen, the time it took PT8 to walk that distance decreased between the first and last measurements, indicating improvement. In the graph titled "PT8 Balance", the y-axis is the time in seconds that PT8 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, PT8's ability to balance on one leg increased overall over the course of the 6 treatments. In the graph titled "PT8 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, PT8 showed an overall improvement in score over the course of the 6 treatments. In the graph titled "PT8 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health. As can be seen, PT8 showed an overall improvement in score over the course of the 6 treatments.

[0150] Figure 9 shows multiple graphs of the macrodata of the first sham participant ("SM1") in a pilot study who was male and received three sham plasmapheresis treatments (i.e., no plasmapheresis treatment was delivered, but the participant was given the illusion that plasmapheresis was being performed). SM1 was the first of three participants who provided the control data presented herein. Macrodata for SM1 were collected prior to each of the three sham plasmapheresis treatments. The graphs in Figure 9 are titled to indicate which macrodata they present. In each of the five graphs shown in Figure 9, the x-axis has the numbers 1 - 3 indicating the time prior to each of the three sham plasmapheresis treatments for which data were collected. In the graph titled "SM1 Grip Strength", the y-axis indicates the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of SM1 decreased overall over the course of the three sham treatments. In the graph titled "SM1 Stand and Walk", the y-axis is the time in seconds it took for SM1 to stand from a seated position and walk a fixed distance that SM1 walked (each of the tested participants, including plasmapheresis treatment patients, walked the same distance). As can be seen, the time it took SM1 to walk that distance remained unchanged over the period during which the sham plasmapheresis treatment was received. In the graph titled "SM1 Balance", the y-axis is the time in seconds that SM1 was able to stand on one leg, and the maximum time measured was 120 seconds. As can be seen, SM1's ability to balance on one leg first decreased, then increased, and ended up not changing overall over the course of the three sham plasmapheresis treatments. In the graph titled "SM1 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, SM1 showed an overall improvement in score over the course of the three sham plasmapheresis treatments. In the graph titled "SM1 SF-12 Mental Health", the y-axis represents the SF-12 survey score for subjective mental health.As can be seen, SM1 showed an overall decrease in score over the course of three pseudo-plasma feresis treatments.

[0151] Figure 10 shows multiple graphs of macrodata for the first sham participant ("SM2") in a pilot study who was male and received three sham plasmapheresis treatments (i.e., no plasmapheresis treatment was delivered, but the participant was given the illusion that plasmapheresis was being performed). SM2 was the second of three participants who provided the control data presented here. Macrodata for SM2 was collected prior to each of the three sham plasmapheresis treatments. The graphs in Figure 10 are titled to indicate which macrodata they present. In each of the five graphs shown in Figure 10, the x-axis has the numbers 1 - 3 indicating the time prior to each of the three sham plasmapheresis treatments for which data was collected. In the graph titled "SM2 Grip Strength", the y-axis shows the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of SM2 decreased overall over the course of the three sham treatments. In the graph titled "SM2 Stand and Walk", the y-axis is the time in seconds it took for SM2 to stand from a seated position and walk a certain distance (each of the tested participants, including plasmapheresis patients, walked the same distance). As can be seen, the time it took SM2 to walk that distance increased over the course of the sham plasmapheresis treatment, indicating an overall deterioration. In the graph titled "SM2 Balance", the y-axis is the time in seconds that SM2 was able to stand on one leg, and the maximum measured time was 120 seconds. As can be seen, SM2 was able to stand for the maximum measured time throughout the course of receiving the sham plasmapheresis treatment. In the graph titled "SM2 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, SM2 showed an overall improvement in score over the course of the three sham plasmapheresis treatments. In the graph titled "SM1 SF-12 Emotional Health", the y-axis represents the SF-12 survey score for subjective emotional health.As can be seen, SM2 showed an overall increase in score over the course of three pseudo-plasma feresis treatments.

[0152] Figure 11 shows multiple graphs of macrodata for the first sham participant ( "SM3") in a pilot study who was male and received three sham plasmapheresis treatments (i.e., no plasmapheresis treatment was delivered, but the participant was given the illusion that plasmapheresis was being performed). SM3 was the third of three participants who provided the control data presented here. Macrodata for SM3 were collected prior to each of the three sham plasmapheresis treatments. The graphs in Figure 11 are titled to indicate which macrodata they present. In each of the five graphs shown in Figure 11, the x-axis has the numbers 1 - 3, indicating the time before each of the three sham plasmapheresis treatments for which the data were collected. In the graph titled "SM3 Grip Strength", the y-axis indicates the measured grip strength in units of Kg, measured by a standard grip strength device. As can be seen, the measured hand strength of SM3 decreased overall over the course of the three sham treatments. In the graph titled "SM3 Stand and Walk", the y-axis is the time in seconds it took for SM3 to stand from a seated position and walk a fixed distance that SM3 walked (each of the tested participants, including plasmapheresis patients, walked the same distance). As can be seen, the time it took SM3 to walk that distance did not change over the course of the sham plasmapheresis treatment. In the graph titled "SM3 Balance", the y-axis is the time in seconds that SM3 was able to stand on one leg, and the maximum time measured was 120 seconds. As can be seen, SM3 had an overall decrease in the time it was able to balance on one leg over the course of the three sham plasmapheresis treatments. In the graph titled "SM3 SF-12 Physical Health", the y-axis represents the SF-12 survey score for subjective physical health. As can be seen, SM3 showed an overall improvement in score over the course of the three sham plasmapheresis treatments. In the graph titled "SM3 SF-12 Mental Health", the y-axis represents the SF-12 survey score for subjective mental health.As can be seen, SM3 showed an overall decrease in score over the course of three pseudo-plasma feresis treatments.

[0153] From the perspective of treatment timing, each of PT1, PT2, PT3, PT4, PT5, PT6, PT7, and PT8 received treatment twice a month for a total of three months, and each of the two monthly treatments for all eight participants was always within a maximum of one week of each other (i.e., between treatment #1 and #2, treatment #3 and #4, and treatment #5 and #6 was a maximum of one week). In addition, in PT3, the time between treatment #3 and treatment #4 was 48 hours. In PT4, the time between treatment #3 and treatment #4 was 48 hours. In PT5, the time between treatment #1 and treatment #2 was 48 hours, the time between treatment #3 and treatment #4 was 48 hours, and the time between treatment #5 and treatment #6 was 48 hours. In PT6, the time between treatment #3 and treatment #4 was 72 hours, and the time between treatment #5 and treatment #6 was 48 hours. In PT7, the time between treatment #1 and treatment #2 was 72 hours, the time between treatment #3 and treatment #4 was 48 hours, and the time between treatment #5 and treatment #6 was 48 hours. In PT5, the time between treatment #1 and treatment #2 was 48 hours, the time between treatment #3 and treatment #4 was 48 hours, and the time between treatment #5 and treatment #6 was 48 hours. Therefore, more than half of the paired monthly treatments delivered in PT1, PT2, PT3, PT4, PT5, PT6, PT7, and PT8 were within 72 hours of each other.

Table 3

[0154] Table III above shows the macro data as presented and described in this specification in FIGS. 1-11, the aggregated macro data for all 11 participants, and their respective attached descriptions. More specifically, the numerical data in Table III is the difference (or delta) between the first data point and the last data point for each macro data parameter measured for all 11 participants. That is, for the 8 participants who received 6 plasma pheresis treatments, each number in Table III is the last measured data value (i.e., before the 6th plasma pheresis treatment) minus the first measured data value (i.e., before the 1st plasma pheresis treatment). Similarly, for each of the 3 sham controls, each number is the last measured data value (i.e., before the 3rd sham plasma pheresis treatment) minus the first measured data value (i.e., before the 1st sham plasma pheresis treatment). In addition, when numbers are in parentheses, these numbers are the first measured values. As can be seen for participants PT1, PT2, PT3, PT4, PT5, PT6, PT7, and PT8, there was a broad overall improvement with respect to multiple different metrics, particularly the standing and walking metrics where all treated patients showed improvement (in standing and walking, a decrease in the value is an improvement). Plasma pheresis strongly showed a positive effect on metrics of strength, walking, balance, and mental well-being compared to these 3 sham controls SM1, SM2, and SM3, where all did not show improvement in strength, walking, or balance and the overall improvement on subjective scales was much lower.

[0155] Microdata For microdata, each study participant had three blood samples collected during the course of the plasmapheresis study, one before the first plasmapheresis treatment, one during the plasmapheresis treatment cycle (i.e., before the 4th plasmapheresis treatment), and one before the last plasmapheresis treatment. Flow cytometry (including the use of fluorescently conjugated antibodies) was performed on the blood samples to evaluate and quantify cell distribution and cell surface protein quantification (i.e., the cell count of cells with cell surface protein phenotypes). Flow cytometry and analysis of the blood samples from participants in the pilot study were performed at the Buck Institute for Aging in Novato, California. For each sample, the expression levels of the following cell surface markers (i.e., cell surface proteins): CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT were measured. In addition, SA-β-gal, an indicator of cellular aging, was also quantified in the same manner.

[0156] Figure 12 shows a graph of a normalized flow cytometry microdata assay of the expression levels of cell surface markers CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT in cells from blood samples collected from participants in the pilot study. In the pilot study, an assay was also performed on the expression of SA-β-gal in cells from blood samples collected from participants. The graph in Figure 12 shows the microdata of the blood samples collected from PT3. Three blood samples were collected from PT3, one before the first plasmapheresis treatment, one before the 4th plasmapheresis treatment, and one before the 6th and last plasmapheresis treatment, which are the x-values 1, 2, and 3 of each graph.

[0157] Figure 13 shows a graph of a normalized flow cytometry microdata assay for the expression levels of cell surface markers CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT in cells from blood samples taken from participants in a pilot study. In the pilot study, an assay was similarly performed for the expression of SA-β-gal in cells from blood samples taken from participants. The graph in Figure 13 shows the microdata of blood samples taken from PT4. Three blood samples were taken from PT4, one before the first plasmapheresis treatment, one before the fourth plasmapheresis treatment, and one before the sixth and final plasmapheresis treatment, which are the x-values 1, 2, and 3 of each graph.

[0158] Figure 14 shows a graph of a normalized flow cytometry microdata assay for the expression levels of cell surface markers CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT in cells from blood samples taken from participants in a pilot study. In the pilot study, an assay was similarly performed for the expression of SA-β-gal in cells from blood samples taken from participants. The graph in Figure 14 shows the microdata of blood samples taken from PT5. Three blood samples were taken from PT5, one before the first plasmapheresis treatment, one before the fourth plasmapheresis treatment, and one before the sixth and final plasmapheresis treatment, which are the x-values 1, 2, and 3 of each graph.

[0159] Figure 15 shows a graph of a normalized flow cytometry microdata assay for the expression levels of cell surface markers CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT in cells from blood samples collected from participants in a pilot study. In the pilot study, an assay was similarly performed for the expression of SA-β-gal in cells from blood samples collected from participants. The graph in Figure 15 shows the microdata of blood samples collected from PT6. Three blood samples were collected from PT6, one before the first plasmapheresis treatment, one before the fourth plasmapheresis treatment, and one before the sixth and final plasmapheresis treatment, which are the x-values 1, 2, and 3 of each graph.

[0160] Figure 16 shows a graph of a normalized flow cytometry microdata assay for the expression levels of cell surface markers CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT in cells from blood samples collected from participants in a pilot study. In the pilot study, an assay was similarly performed for the expression of SA-β-gal in cells from blood samples collected from participants. The graph in Figure 16 shows the microdata of blood samples collected from PT7. Three blood samples were collected from PT7, one before the first plasmapheresis treatment, one before the fourth plasmapheresis treatment, and one before the sixth and final plasmapheresis treatment, which are the x-values 1, 2, and 3 of each graph.

[0161] Figure 17 shows a graph of a normalized flow cytometry microdata assay for the expression levels of cell surface markers CD16, CD25, CD27, CD38, CD57, CD80, HLA-DR, IgM, KIR, KLRG1, NK1, NKG2A, and TIGIT in cells from blood samples taken from participants in a pilot study. In the pilot study, an assay was similarly performed for the expression of SA-β-gal in cells from blood samples taken from participants. The graph in Figure 17 shows the microdata of blood samples taken from PT8. Three blood samples were taken from PT8, one before the first plasma exchange treatment, one before the fourth plasma exchange treatment, and one before the sixth and final plasma exchange treatment, which are the x-values 1, 2, and 3 in each graph. [Table 4] [Table 5] [Table 6]

[0162] Table IV above is divided into three parts for adaptation here, showing the microdata of six participants (PT3, PT4, PT5, PT6, PT7, and PT8), and their microdata is shown in FIGS. 12-17, described herein, with their respective explanations. More specifically, the numerical data in Table IV is the difference (or delta) between the first data point and the last data point for each microdata parameter measured for all of PT3, PT4, PT5, PT6, PT7, and PT8 who received six plasma pheresis treatments. That is, for PT3, PT4, PT5, PT6, PT7, and PT8, each number in Table IV is the last measured data value (i.e., before the 6th plasma pheresis treatment) minus the first measured data value (i.e., before the 1st plasma pheresis treatment). Generally, the consensus for cell surface markers, except for the markers IgM and NKg2a marked with an asterisk to indicate that several studies have shown an increase in the values measured for these two markers, is that a decrease in value indicates improvement (i.e., at least a decrease in inflammation). The values in Table IV indicating improvement over the course of the plasma pheresis treatment delivered to PT3, PT4, PT5, PT6, PT7, and PT8 are shown in bold in Table IV. As shown, all of PT5, PT6, PT7, and PT8 had improved expression of cell surface markers over most of the markers measured between the first and last blood samples. In addition to looking at individual markers, KLRG1 expression decreased in all of the participants shown in Table IV, and CD27, CD57, and SAbGal expression decreased in almost all of the participants in Table IV.

[0163] Overview of the present disclosure Each of the individual variations or embodiments described and illustrated in this specification has distinct components and features and can be readily separated from or combined with the features of any of the other variations or embodiments. Modifications can be made to a particular situation, material, composition of matter, process, process act(s), or step(s) to conform to the purpose(s), spirit, or scope of the invention.

[0164] The methods recited herein can be performed in any order of the recited events that is logically possible, as well as in the recited order of events. For example, the methods described herein do not necessarily require the specific order of steps illustrated to achieve the desired result; rather, one or more of the steps of the methods described herein can be performed in a different order compared to other steps. Further, additional steps or acts can be provided or steps or acts can be eliminated to achieve the desired result.

[0165] Those skilled in the art will understand that all or a portion of the methods disclosed herein can be embodied in a non - transitory machine - readable or accessible medium that includes instructions readable or executable by a processor or processing unit of a computing device or other type of machine.

[0166] Further, when a range of values is provided, all intermediate values between the upper and lower limits of that range, as well as any other recited value or intermediate value within the recited range, are included within the invention. Also, any optional features of the described variations of the invention can be described and claimed independently or in combination with any one or more of the features described herein.

[0167] All existing subject matter (e.g., publications, patents, patent applications, and hardware) referred to in this specification is hereby incorporated by reference in its entirety, except where the subject matter might conflict with the subject matter of the present invention (in which case, what is present in this specification prevails). The items referred to are provided only for their disclosure prior to the filing date of this application.

[0168] References to a single item include the possibility that there may be a plurality of the same items. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an,” “said,” and “the” include plural referents unless the context clearly dictates otherwise. Further note that the claims may be drafted to exclude any optional elements. Accordingly, this description is intended to serve as a precedent for the use of exclusive terminology such as “solely,” “only,” etc., or the use of “negative” limitations with respect to the recitation of elements of the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0169] Although the present disclosure is not intended to be limited to the specific forms described, it is intended to cover alternatives, modifications, and equivalents of the variations or embodiments described herein. Further, the scope of the present disclosure fully encompasses other variations or embodiments that may become apparent to one of ordinary skill in the art in view of the present disclosure.

Claims

[Claim 1] The invention described in the specification.

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