Treatment of senescence by vagus nerve stimulation

Abstract

Methods and apparatuses for treating a senescence related disease, disorder, or pathological condition. These methods may include identifying a cellular senescence status in a senescence related disease, disorder, or pathological condition in a subject; and treating the subject for the senescence-related disease, disorder, or conditional by vagal nerve stimulation.

Description

TREATMENT OF SENESCENCE BY VAGUS NERVE STIMULATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. provisional patent application no. 63 / 700,568, titled “TREATMENT OF SENESCENCE BY VAGUS NERVE STIMULATION,” filed on September 27, 2024, and herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND

[0003] A variety of aging-related diseases, disorders, and conditions are difficult to effectively treat. Reducing senescent cells has emerged as a critical strategy in addressing the root causes of aging and age-related diseases. Senescent cells are damaged or stressed cells that have permanently stopped dividing but remain metabolically active, secreting a mix of inflammatory and tissue-degrading molecules known as the senescence-associated secretory phenotype (SASP). While transient senescence plays a protective role — such as in wound healing or preventing cancer — the chronic accumulation of these cells contributes to tissue dysfunction, impaired regeneration, and systemic inflammation. This buildup is linked to a wide range of conditions including cardiovascular disease, neurodegeneration, arthritis, and metabolic disorders. Studies in animal models have shown that selectively removing senescent cells can lead to significant improvements in health, tissue function, and even reversal of some aging markers.

[0004] Despite the promise, current therapies targeting senescent cells remain largely ineffective or underdeveloped. Most approaches fall into two categories: senolytics, which aim to destroy senescent cells, and senomorphics, which attempt to suppress their harmful secretions without killing them. While compounds like dasatinib and quercetin have shown potential in preclinical models, their efficacy in humans is inconsistent, and they often suffer from off-target effects and limited tissue specificity. Moreover, the biological heterogeneity of senescent cells, which may vary by tissue type, age, and disease context, makes it difficult to develop universal treatments. Exiting or proposed therapies typically lack the precision- 1 -SG Docket No.: 10471-747.600needed to avoid harming healthy cells. As a result, while the concept of senotherapeutics is compelling, the field faces significant scientific and clinical hurdles before these interventions can reliably improve human health and longevity.

[0005] Therefore, it would be desirable to provide additional methods and systems that can be used independently or in conjunction with other therapies to guide treatments.SUMMARY

[0006] Described herein are methods and apparatuses for treating diseases and conditions associated with cellular senescence through nerve stimulation, including in particular, vagus nerve stimulation, VNS. Cellular senescence contributes to aging and a wide range of age- related diseases by secreting inflammatory molecules known as the senescence-associated secretory phenotype (SASP). Current therapeutic approaches, such as senolytics and senomorphics, have shown limited efficacy and specificity. The methods and apparatuses described herein may use nerve stimulation (e.g., VNS) as a targeted, technique, either alone or in combination with senolytics and senomorphics, to reduce senescent cell burden and improve tissue regeneration, particularly in neurodegenerative diseases like multiple sclerosis (MS).

[0007] The methods and apparatuses described herein demonstrate that VNS significantly reduces senescent cells, which may accelerate remyelination and reduces the accumulation of senescent microglia in murine models of spinal cord demyelination. The electrical stimulation described herein was shown to decrease markers of senescence such as yH2AX (pSerl39) and TREM2 expression in both young and aged mice. Thus, VNS directly promotes the clearance of senescent cells, thereby enhancing tissue repair and functional recovery.

[0008] Also described herein are optimal stimulation range for electrical stimulation to reduce senescent cells. In some cases, the stimulation may be applied between 0.1 Hz and 20 Hz for reducing senescent cells. Outside this range, the therapeutic effect on senescent cells diminishes. Devices described in the patent are configured to deliver electrical pulses within this effective frequency range, with specific parameters for current, pulse width, and duration. These devices may be implantable or external and include features such as frequency limiters and programmable dosing schedules to ensure precise and safe application.

[0009] Additionally, the methods may be combined with senolytic or senomorphic drugs to enhance therapeutic outcomes. Co-administration of agents like dasatinib, quercetin, and rapamycin with VNS may provide synergistic effects, allowing for lower drug dosages and improved targeting of senescent cells. The patent outlines various biomarkers for assessing- 2 -SG Docket No.: 10471-747.600cellular senescence status and monitoring treatment efficacy, including blood tests, spinal taps, and imaging techniques.

[0010] For example, described herein are methods of reducing senescent cells in a subject’s body comprising applying a dose of electrical stimulation to a vagus nerve, wherein the dose of electrical stimulation comprises between about 0.5 mA to 2.5 mA at a frequency of less than 20 Hz and a pulse width of between 0.1 ms and 0.5 ms for a duration of between about 0.1 second and 2.5 min. In some examples, these methods may include reducing senescent cells in a subject’s body by applying a dose of electrical stimulation to a vagus nerve, wherein the dose of electrical stimulation comprises between about 0.5 mA to 2.5 mA and a pulse width of between 0.1 ms and 0.5 ms for a duration of between about 0.1 second and 2.5 min and limiting the frequency of the applied pulses to a frequency of less than 20 Hz.

[0011] In some examples a method of reducing senescent cells in a subject’s body may include: applying a dose of electrical stimulation to a vagus nerve, wherein the dose of electrical stimulation comprises between about 0.5 mA to 2.5 mA and a pulse width of between 0.1 ms and 0.5 ms for a duration of between about 0.1 second and 2.5 min and limiting the frequency of the applied pulses to a frequency of less than 20 Hz; and delivering one or more senelytic and / or senomodulator drugs to the subject’s body currently with the dose of electrical stimulation.

[0012] The one or more senelytic drugs may include one or more of: Dasatinib, Quercetin, Fisetin, Navitoclax, 17-DMAG, Catechins, FOXO4-DRI peptides, HSP90 inhibitors, Piped ongumine, Curcumin, ABT-737, UBX0101, Procyanidins, Oleandrin, A1331852, and / or Al 155463. The one or more senomodulator drugs may include one or more of: a tyrosine kinase inhibitors (e.g., Dasatinib), a flavonoid (e.g. Quercetin, Fisetin), a Bcl-2 inhibitor (e.g., Navitoclax, ABT-263), Piperlongumine, an mTor inhibitor (e.g., Rapamycin (Sirolimus), Metformin, Resveratrol, Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN), a SASP Inhibitor, a Janus Kinase (JAK) inhibitor, a IL-1 inhibitor, a NF-xB inhibitor (e.g. BAY 11-7082), a telomerase activators (e.g., TA-65, GRN510, etc.), UBX1325, FOXO4-DRI peptide, or a Heat shock protein 90 (HSP90) inhibitor.

[0013] Any of these methods may include waiting at least one week before administering another dose of electrical stimulation.

[0014] In any of these methods, these method may include identifying a decrease in a level of one or more cellular senescence status biomarkers in the subject’s body following delivery of the dose.- 3 -SG Docket No.: 10471-747.600

[0015] The method may include applying the dose comprises applying the dose non- invasively, or from an implanted microstimulator. In some cases, applying the dose comprises applying the dose from an external microstimulator.

[0016] The one or more cellular senescence biomarkers may comprise one or more cell cycle marker, a cell cycle inhibitor marker, a DNA damage indicator, a chromatin abnormality indicator, a metabolic indicator, or a lysosomal alteration. The one or more cellular senescence status biomarkers may include a biomarker selected from TREM2, cyclin-dependent kinase pl6, cyclin-dependent kinase p21, 53BP1; YH2AX (pSerl39), IL6, IL8, Lamin Bl, senescence-associated beta-galactosidase (SA-P-gal or SABG), KI-67, and phosphorylated Retinoblastoma protein (pRb). In some cases, the one or more cellular senescence status biomarkers comprises TREM2 or YH2AX (pSerl39). In any of these methods, identifying a decrease in a level of one or more cellular senescence status biomarkers may comprise analyzing at least five biomarkers. For example, identifying a decrease in a level of one or more cellular senescence status biomarkers may comprise analyzing senescence biomarkers obtained from a blood test, a spinal tap, and a magnetic resonance image of the subject’s brain or spinal cord.

[0017] The method may further comprise identifying that the patient has a senescence related disease or disorder. The senescence related disease or disorder may comprise one or more of: atherosclerotic diseases, cardiovascular diseases, cerebrovascular diseases, aneurysm, hypertension, cancers, radiotherapy-induced senescence, neurodegenerative diseases, Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington, Frontotemporal dementia, macular degeneration, metabolic disorders, Type 2 diabetes mellitus, obesity, lysosomal storage diseases, inflammatory bowel disease, biliary liver damage, liver fibrosis, fatty liver disease, glaucoma, retinopathy, idiopathic pulmonary fibrosis, intervertebral disc degeneration, osteoarthritis, osteoporosis, pulmonary fibrosis, cystic fibrosis, and / or radiation. Specifically in some examples the senescence related disease or disorder comprises multiple sclerosis. The senescence related disease or disorder may comprise relapsing-remitting multiple sclerosis (RRMS), and in some cases may further comprise administering a therapeutically- effective amount of the vagal nerve treatment for RRMS to the subject when the subject is determined to be undergoing relapse.

[0018] Also described herein are apparatuses. For example, an apparatus for reducing senescent cells in a subject’s body may include: a pair of electrodes configured to apply energy to a vagus nerve; a pulse generator coupled to the electrodes and configured to deliver a dose of electrical stimulation to the vagus nerve from the electrodes, wherein the dose of - 4 -SG Docket No.: 10471-747.600electrical stimulation comprises a plurality of pulses between about 0.5 mA to 2.5 mA having a pulse width of between 0.1 ms and 0.5 ms, for a dose duration of between about 0.1 second and 2.5 min, wherein the pulse generator comprise a frequency limiter configured to limit the frequency of the pulses during the dose to less than 20 Hz.

[0019] The electrodes may be positioned on a housing enclosing the pulse generator. The electrodes may be coupled to the pulse generator by a lead. Any of these apparatuses may include a delay control configured to prevent the apparatus from delivering another dose for at least a week before administering another dose of electrical stimulation. The delay control may be part of the pulse generator. The delay control may be configured to prevent the apparatus for at least two weeks before administering another dose of electrical stimulation.

[0020] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0022] FIGS. 1 A-1B show that vagus nerve stimulation accelerated the rate of remyelination. FIG. 1 A shows the study design. FIG. IB shows that vagus nerve stimulation accelerated the rate of remyelination in a standard murine model of lysolecithin (LPC)- induced focal spinal cord (SC) demyelination (lesions). There was a significant reduction in lesion volume in the vagus nerve stimulation group (n = 4-12 / group) compared with the sham group. ***p<0.001.

[0023] FIGS. 2A-2C show that vagus nerve stimulation limited the accumulation of senescent microglia. The standard murine model of lysolecithin (LPC)-induced focal spinal cord (SC) demyelination (lesions) was subjected to vagus nerve stimulation. The middle panel of FIG. 2A shows that representative 20X confocal images showed significantly fewer microglia (Ibal+ cells, images not shown) expressing yH2AX (pSerl39), a marker for cellular senescence. The bottom panel of FIG. 2 A shows that expression of yH2AX (pSerl39) overlapped with DAPI, a nuclei marker.

[0024] FIGS. 2B1-2B3 are graphs summarizing data showing that vagus nerve stimulation of the lysolecithin (LPC)-induced murine model significantly reduced the average (FIG. 2B1) and maximum (FIG. 2B2) number of senescent microglia per lesion compared to control in young mice (2-4 months). FIG. 2B3 shows that while TREM2 is significantly- 5 -SG Docket No.: 10471-747.600upregulated in lesion versus non-lesion region (data not shown), there is a trend towards lowering TREM2 expression within lesion with vagus nerve stimulation as compared to control. For FIGS. 2B1-2B3, n=3 mice / group. Student’ s t-test, *p<0.05, **p<0.01.

[0025] FIGS. 2C1-2C3 are graphs summarizing data showing that similar trends were also observed in older animals (12 months), showing a significant reduction in the average (FIG. 2C1) and maximum (FIG. 2C2) number of senescent microglia per lesion compared to control in young mice (2-4 months). FIG. 2C3 shows trend towards lowering TREM2 expression within lesion with vagus nerve stimulation as compared to control. For FIGS. 2C1-2C3, n=3 mice / group. Student’s t-test, *p<0.05, **p<0.01.

[0026] FIG. 3 is a graph showing the frequency dependence of stimulation when reducing the number of senescent cells as described herein; between about 0.1 Hz and 20 Hz a consistent reduction in senescent cells was seen, with increasing variability in the mean number of senescent cells as well as an increase in the number of senescent cells after between about 20-100 Hz.

[0027] FIG. 4A schematically illustrates an example of an apparatus for applying electrical stimulation to reduce the number of senescent cells, including an integrated microstimulator and electrodes.

[0028] FIG. 4B schematically illustrates another example of an apparatus for applying electrical energy to reduce the number of senescent cells in which the microstimulator is separated from the electrodes by a lead.DETAILED DESCRIPTION

[0029] Cellular senescence is a process of acquired cellular disabilities and is fundamental to the pathophysiology of many diseases. We have demonstrated for the first time the surprising result that VNS treatment reduces senescence and herein describe methods and apparatuses for specifically eliminating senescent cells while sparing healthy cells. In particular, these methods and apparatuses may apply electrical energy to trigger and / or aid in the destruction of senescent cells.

[0030] For example, multiple sclerosis is a chronic inflammatory disease of the central nervous system (CNS), characterized by multifocal areas of demyelinated lesions that are linked to neurological impairment and progressive disability. Available disease-modifying therapies (DMTs) have mechanisms of action that primarily target peripheral immunocytes to indirectly reduce CNS inflammation and reduce rate of relapses. However, to date, no approved DMTs have yet been demonstrated to promote remyelination of damaged axons or to restore function in MS patients.- 6 -SG Docket No.: 10471-747.600

[0031] Previous study has demonstrated the existence of senescent myeloid cells (microglia and infiltrating macrophages) within chronic active lesions in patients with MS. Furthermore, in a recent preprint, a subset of microglia in LPC-induced lesions in both young and aged mice have been shown to express multiple senescent markers, and the persistence of this population was linked to inefficient remyelination. In addition, while upregulation of microglial TREM2 expression is important in facilitating phagocytosis of myelin debris, senescent microglia appear to express higher TREM2 compared to activated microglia.

[0032] The results described herein surprisingly suggest that vagus nerve stimulation (VNS) promote the elimination of senescent cells, including senescent microglia, thus promoting clearance of myelin debris and subsequently facilitating remyelination. Moreover, the use of VNS may be used to treat other indications not previously associated with VNS (e.g., not associated directly with inflammation or myelination state). Further, the methods described herein may provide treatments in which agents that modulate senescence may be used in combination with VNS to achieve much greater and / or alternative effects, and may be used at different concentrations, routes of administration and / or dosing regimens in combination with VNS.Definitions

[0033] The terms “aging-related disease”, “aging-related disorders”, and “aging-related conditions” refer to diseases, disorders, and conditions arising from senescence. Aging- related diseases and disorders are associated with the presence of senescent cells and may also be referred to as senescence related disease, senescence related disorder, or senescence related condition. Aging-related diseases, disorders, and conditions associated with cellular senescence, include, but are not limited to atherosclerotic diseases (including cardiovascular diseases and cerebrovascular diseases, e.g., aneurysm, hypertension, etc.); cancers and radiotherapy-induced senescence; neurodegenerative diseases (e.g., Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington, Frontotemporal dementia, macular degeneration, etc.); metabolic disorders (e.g., Type 2 diabetes mellitus, obesity, lysosomal storage diseases, etc.); and others (e.g., inflammatory bowel disease, biliary liver damage, liver fibrosis, fatty liver disease, glaucoma, retinopathy, idiopathic pulmonary fibrosis, intervertebral disc degeneration, osteoarthritis, osteoporosis, pulmonary fibrosis, cystic fibrosis, space radiation, etc.)

[0034] Examples “senescent cells (SNC)” in aging-related disease or disorders include astrocytes in Alzheimer’s disease; endothelial cells in aneurysm; airway epithelial cells in cystic fibrosis; pancreatic stellate cells in fibrosis in pancreatitis (in mouse); outflow tract in glaucoma; arterial wall in hypertension; fibroblasts in idiopathic pulmonary fibrosis; crypt - 7 -SG Docket No.: 10471-747.600cells in inflammatory bowel disease; nucleus pulposus in intervertebral disc generation; RPE cells in macular degeneration; articular cartilage in osteoarthritis; white adipose tissue in Type 2 diabetes mellitus (obesity).

[0035] The term “apotosis” refers to a type of cell death in which a series of molecular steps in a cell lead to its death. Apoptosis is one method the body uses to get rid of unneeded or abnormal cells. Apoptosis is sometimes also referred to as programmed cell death.

[0036] The term “biomarker” refers to a biological molecule found in a subject (in a fluid, tissue, sample, etc.) that is a sign of a normal or abnormal process, or of a condition, disorder, or disease. In some examples, a biomarker is YH2AX (pSerl39). In some examples, a biomarker is TREM2 MFI. In some embodiments, a disease, disorder, or (pathological condition) can be diagnosed by one or more (e.g., a panel of two, three, four, or five or more biomarkers). A disease, disorder, or (pathological condition) can be diagnosed by one or more (e.g., two, three, four, or five or more biomarkers) having an increased level or a decreased level relative to a reference value. A reference value can be based on a previous assay performed on a subject or can be based on a previous assay(s) not performed on the subject (e.g., a medical standard, an average, etc.). In some embodiments, an increase or decrease in a level of one or more biomarker in a subject is a measurable increase or decrease that correlates with an increased (or decreased, as the case may be) likelihood of therapeutic benefit for the patient, or for a group of patients, or a patient or group of patients yet to be selected. In some embodiments, a biomarker panel is a characterization of cell types present in the plaque or lesion microenvironment or from a plaque or lesion biopsy. An increase or decrease in a biomarker level can be a statistically significant increase or decrease. The term “statistical significance” is well-known in the art and may be determined using methods known in the art, including such as those described herein. In some embodiments, statistical significance means, e.g., p<0.1, p<0.05, p<0.04, p<0.03, p<0.02, or p<0.01 relative to baseline. In some embodiments, a biomarker is a cell cycle biomarker, such as proteins involved in Gl-S, G2-M cycle transitions. Examples of cell cycle protein markers useful for identifying cell senescence include but are not limited to cyclin-dependent kinases (e.g., cyclin-dependent kinase pl6, cyclin-dependent kinase p21, etc.), DNA damage markers (e.g., 53BP1; YH2AX (pSerl39), etc.; presence of the senescence-associated secretory phenotype (SASP) (e.g., IL6, IL8), loss of Lamin Bl (a structural component of the nuclear lamin), senescence-associated beta-galactosidase (SA-P-gal or SABG), KI-67, markers or measurements for cell size (e.g., increased cell size), and phosphorylated Retinoblastoma protein (pRb) (e.g., decreased).- 8 -SG Docket No.: 10471-747.600

[0037] The term “cellular senescence” refers to a stable state of terminal proliferation arrest. When cells are in a state of cellular senescence, they do not undergo apoptosis. The presence of cellular senescence (e.g., senescent cells) is implicated in a variety of diseases and disorders, which may be referred to as senopathies. Senopathies include age-related conditions. Senescent cells contribute to these diseases through mechanisms such as chronic inflammation, tissue remodeling, and impaired regenerative capacity.

[0038] The term “cellular senescence status” refers to the status of a subject with regards to cell senescence that is thought to contribute to a senescence related disease, senescence related disorder, or a senescence related pathological condition. Cellular senescence status can be determined by analysis of one or more senescent cell biomarkers in one or more samples of cells, tissues, bodily fluid, or organs.

[0039] The term “YH2AX (pSerl39)” refers to histone H2AX with phosphorylation and a serine switch. pTyrl42 (H2AX-pY142) undergoes phosphorylation to pSerl39 (yH2AX) in the DNA damage response (DDR). y-H2A.X is formed by post-translational modifications. YH2AX (pSerl39) is a biomarker useful for assaying cell damage (e.g., DNA double strand breaks, DSBs), especially associated with senescence. YH2AX (pSerl39) can be detected by anti -YH2AX (pSerl39) antibodies. In some examples, YH2AX (pSerl39) can be detected and used to help diagnose multiple sclerosis (MS).

[0040] The term “microglia” refers to specialized cells (mononuclear macrophages), typically found in the central nervous system (CNS). During CNS injury, microglia are responsible for carrying out bodily processes, such phagocytosis and elimination of microbes, dead cells, and protein aggregates, as well as other particulate and soluble antigens that may otherwise endanger the CNS.

[0041] The term “multiple sclerosis” (MS) refers to a chronic, neurological disorder of the central nervous system (brain, spinal cord, optic nerves) in which the myelin (insulating covers) of nerve cells are damaged. MS is thought to reflect possible contributions by autoimmune, genetic, and environmental pathogenetic processes culminating in the formation of demyelinating plaques. Sclerosis, as it relates to MS, refers to medical term for the distinctive areas of scar-like tissue (also called plaques or lesions) that result from the attack on myelin by the immune system. These areas may be sufficiently sized so as to be visible by MRI (magnetic resonance imaging). The patches of scar-like tissue can be as small as the head of a pin or as large as a golfball. Multiple sclerosis is typically indicated by evidence of damage in at least two separate areas of the central nervous system.

[0042] Testing for MS may include blood work, MRIs of brain and spinal cord, and an analysis of spinal fluid.- 9 -SG Docket No.: 10471-747.600

[0043] Relapsing-remitting multiple sclerosis (RRMS) is the most common course of MS. RRMS involves defined relapses of new or increasing neurologic symptoms. These relapses — also called "attacks” or “exacerbations" — are followed by periods of partial or complete recovery (remissions). During remissions, all symptoms may disappear, or some symptoms may continue and become permanent. Approximately 85% of people with MS are initially diagnosed with RRMS.

[0044] The term “nicotinic receptor” refers to a family of ligand-gated ion channels (LGIC) that can be activated by an agonist. An example of an endogenous agonist is the neurotransmitter acetylcholine (Ach). An example of an exogenous agonist is nicotine. Stimulation of the receptor by agonist binding, such as acetylcholine or nicotine, induces a conformational change that results in the opening of the ion channel allowing for the influx of Ca++ and Na+, and consequent membrane depolarization. Nicotinic receptors have 5 subunits and can span the cell membrane. The functional and structural diversity of nicotinic receptors depends on various combinations of these subunits. One example of a nicotinic receptor is the nicotinic acetylcholine receptor.

[0045] The term “senescence” refers to a cellular response or process characterized by stable proliferation arrest without undergoing cell death. Senescence is induced due to cellular damage or stress. Senescence can be induced endogenous or non-experimentally (e.g., as a consequence of disease or aging, such as cellular damage, cellular stress, unrepaired DNA damage, etc.) or exogenously or experimentally (e.g., by additional of an agent such as bleomycin to cells, tissues, or organisms). Over time, large numbers of senescent cells can build up in cells throughout the body. These cells remain active and can release substances. Senescence is typically accompanied by a hypersecretory phenotype referred to as senescence-associated secretory phenotype (SASP) that releases various substances (a secretome). Some released substances can be harmful to nearby healthy cells. Some released substances can cause inflammation and damage to otherwise healthy or nearby cells. Senescent cells and their pro-inflammatory secretome have emerged as contributors to age-related tissue dysfunction and morbidity.

[0046] The term “reference level,” sometimes also referred to herein as reference or control refers to a value or range, which may be employed as a benchmark against which to assess a measured or determined result. A reference level may be predetermined. A reference level can be based on a previous assay performed on the same subject or can be based on a previous assay(s) not performed on the subject (e.g., a medical standard, an average, etc.). In some examples, a reference level may be determined based on a “control group” which refers- 10 -SG Docket No.: 10471-747.600to a group of control subjects. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group.

[0047] The term “sample” or “biological sample”, as used herein, refers to a small part or quantity intended to show what the whole is like. A sample can be obtained or derived from a subject, cell or tissue of interest. Samples include, but are not limited to, tissue, whole blood, serum, or plasma blood serum, plasma, urine, cerebrospinal fluid, saliva, lacrimal fluid, or sweat from a subject. Samples also include, but are not limited to tissue, a cell, or a fluid obtained from a tissue or cell. In some examples, a biological sample can be a biopsy sample. In some examples, a biological sample or biopsy is taken from or adjacent to nervous system tissue of interest.

[0048] A sample can be used directly as obtained from a subject or can be pre-treated, such as by addition of reagents, centrifugation, concentration, distillation, extraction, filtration, inactivation of interfering components, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0049] The term “senescent cells” or SNCs are cells characterized by having senescent characteristic, such as senescence-associated proliferation arrest, senescence-associated secretory phenotype, and morphological changes. The presence of senescent cells is typically associated with a disease, disorder, or condition (e.g., pathological condition).

[0050] The term “senescence-associated secretory phenotype” (abbreviated at SASP) refers to a hypersecretory state associated with senescence. SASP is a major mediator of the paracrine effects of senescent cells in their tissue microenvironment.

[0051] The term “senescence-associated proliferation arrest” (abbreviated as SAP A) refers to cell cycle arrest associated with senescence. A cell typically undergoes cell cycle arrest and further changes during the process of senescence (such as hypersecretion, hypertrophy, and lysosomal changes).

[0052] The term “senopathy” refers to diseases, disorders, and conditions for which senescent cells are implicated. Non-limiting examples of senopathies include multiple sclerosis (in which the myelin (insulating covers of nerve cells) and cells bodies and axons of nerve cells are damaged); cardiovascular disease such as atherosclerosis (in which senescent cells contribute to the formation of atherosclerotic plaques by promoting inflammation and tissue remodeling) and heart disease (in which senescent cells accumulate in the heart, contributing to cardiac dysfunction and heart failure); metabolic diseases (e.g., type 2 diabetes in which senescent cells in adipose tissue and pancreatic P-cells can impair insulin secretion and action, contributing to the development of diabetes and obesity -Related Metabolic Dysfunction in which senescent cells in metabolic tissues exacerbate insulin- 11 -SG Docket No.: 10471-747.600resistance and chronic inflammation); musculoskeletal diseases such as osteoarthritis (in which senescent cells in joint tissues contribute to cartilage degradation and inflammation, leading to osteoarthritis) and muscle wasting (in which senescence in muscle cells is associated with age-related sarcopenia and muscle wasting in chronic conditions like COPD); liver diseases such as liver cirrhosis (in which senescent hepatic cells contribute to fibrosis and cirrhosis, particularly in the context of chronic liver diseases such as hepatitis B); and kidney disease such as chronic kidney disease (in which senescent cells in renal tissues contribute to fibrosis and functional decline in chronic kidney disease) and IgA Nephropathy (in which accelerated senescence of renal tubular cells is linked to disease progression in IgA nephropathy); lung diseases such as Chronic Obstructive Pulmonary Disease (COPD) (in which senescent cells in the lung contribute to chronic inflammation and tissue remodeling seen in COPD); neurodegenerative diseases such as Alzheimer's Disease (in which senescent cells in the brain are implicated in neuroinflammation and neuronal dysfunction, contributing to diseases like Alzheimer's; other conditions such as cancer (in which, while senescence acts as a tumor suppressive mechanism, the accumulation of senescent cells can create a pro- tumorigenic environment through the senescence-associated secretory phenotype (SASP) and glaucoma (in which senescent cells in ocular tissues are associated with the development of glaucoma).

[0053] The term “spinal tap” or “lumbar puncture” refers to a medical procedure in which a small sample of cerebrospinal fluid is removed from the spinal canal for laboratory analysis. This sample can show abnormalities in antibodies that are associated with a senescent disease, disorder, or condition. For example, the presence of oligoclonal bands (a biomarker of proteins called immunoglobulins) can indicate inflammation of the central nervous system.

[0054] The term “sTREM2” refers to “soluble Triggering Receptor Expressed On Myeloid Cells 2”. sTREM2 is typically formed from shedding of the TREM2 ectodomain and can be detected in the cerebrospinal fluid (CSF). sTREM2 levels can be used as a biomarker for microglial activity.

[0055] The term “subject” refers to an organism, such as a mammal, including, but not limited to, humans, non-human primates, and non-primates. A subject may be a laboratory or other experimental animal. In some embodiments, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0056] The term “treatment” is used interchangeably herein with the term “therapeutic method” and refers to both: 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and / or halt progression of a condition, disease, or disorder (any of which- 12 -SG Docket No.: 10471-747.600may be diagnosed pathologic), and 2) prophylactic / preventative measures to prevent or reduce risk of a pathologic condition, disease, or disorder. Those in need of treatment may include a subject (individual) already having a particular medical disease, disorder, or condition as well as those who may ultimately acquire the disease, disorder, or condition (i.e., those at risk or needing preventive measures). In some examples, a treatment for an aging- related disease or senopathy (including but not limited to those described herein) is a vagus nerve stimulation.

[0057] The term “TREM2” or “triggering receptor expressed on myeloid cells 2” refers to a membrane protein receptor biomarker expressed in microglia (in the brain). TREM2 is expressed in microglia and helps maintain microglial metabolic fitness during stress. TREM2 is a senescent cell biomarker. It is highly expressed in multiple sclerosis lesions.

[0058] The term “vagus nerve” refers to one (cranial nerve X) of the paired 12 cranial nerves that connects the brain and body. The vagus nerve extends from the brain stem all the way to the gut and is part of the autonomic parasympathetic nervous system.

[0059] The term “vagus nerve stimulation” (or vagal nerve stimulation) (VNS) refers to a technique that stimulates the vagus nerve. Stimulation may be at least one of electrical, chemical, or manual stimulation. When the vagus nerve is stimulated, electrical impulses are induced. The effect of vagus nerve stimulation is mediated through the cholinergic antiinflammatory pathway of nicotinic receptors. The vagus nerve stimulation can come in various forms, including direct or indirect electromagnetic stimulation, pressure or tactile sensation, or breathing exercises. The vagus nerve can also be stimulated through any of its branches, such as Arnold's nerve. The vagus nerve can be stimulated by applying electrical current from an internal or external current source to a vagus nerve of a patient. The vagus nerve can be stimulated by more electrodes applied on or under the skin, the electrodes being connected to an external current source. Stimulation of the vagus nerve can be performed as a single pulse, or as a train of pulses. Electrical pulses can be applied manually by the patient or healthcare provider, or automatically according to a programmed sequence. Electrical pulses can be applied continuously, periodically, or intermittently to the vagus nerve. Various characteristics of the pulses can be controlled, including pulse amplitude (measured in amperes or joules), pulse duration, pulse train duration, and frequency of pulse or pulse train repetition. The external current source can be a conventional alternating current (AC) power supply.Methods

[0060] Described herein are methods for treating a senescence related disease, disorder, or pathological condition.- 13 -SG Docket No.: 10471-747.600

[0061] Any of the method described herein may include treating a patient to reduce the number of senescent cells by applying electrical stimulation to the patient. In particular the method may include applying electrical stimulation to one or more nerves. In some cases the nerve is a vagus nerve, or a nerve that communicates with the vagus nerve (e.g., the splenic nerve, the celiac ganglion, visceral sensory fibers from gut, liver, and lungs, nucleus tractus solitarius, dorsal motor nucleus of vagus, etc.). The electrical stimulation may include the application of **

[0062] In any of these examples, these methods may include the identifying a cellular senescence status in a senescence related disease, disorder, or pathological condition in a subject and treating the subject for the senescence-related disease, disorder, or conditional by vagal nerve stimulation. The methods may include a step of analyzing a biological sample from the subject for one or more senescence biomarkers, wherein analyzing includes identifying a differentially expressed level of each senescence biomarker relative to a reference level.

[0063] The methods may include wherein the one or more senescence biomarkers includes one or more cell cycle marker, a cell cycle inhibitor marker, a DNA damage indicator, a chromatin abnormality indicator, a metabolic indicator, or a lysosomal alteration.

[0064] The methods may include , wherein the at least one or more senescence biomarkers includes a biomarker selected from TREM2, cyclin-dependent kinase pl 6, cyclin- dependent kinase p21, 53BP1; YH2AX (pSerl39), IL6, IL8, Lamin Bl, senescence- associated beta-galactosidase (SA-P-gal or SABG), KI-67, and phosphorylated Retinoblastoma protein (pRb).

[0065] The methods may include wherein the at least one or more senescence biomarker includes TREM2 or YH2AX (pSerl39).

[0066] The methods may analyzing at least five biomarkers.

[0067] The methods may include wherein analyzing includes analyzing senescence biomarkers obtained from a blood test, a spinal tap, and a magnetic resonance image of the subject’s brain or spinal cord.

[0068] The methods may include wherein identifying a cellular senescence status includes identifying a status in a subject senescence related disease, disorder, or pathological including atherosclerotic diseases, cardiovascular diseases, cerebrovascular diseases, aneurysm, hypertension, cancers, radiotherapy-induced senescence, neurodegenerative diseases, Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington, Frontotemporal dementia, macular degeneration, metabolic disorders, Type 2 diabetes mellitus, obesity, lysosomal storage- 14 -SG Docket No.: 10471-747.600diseases, inflammatory bowel disease, biliary liver damage, liver fibrosis, fatty liver disease, glaucoma, retinopathy, idiopathic pulmonary fibrosis, intervertebral disc degeneration, osteoarthritis, osteoporosis, pulmonary fibrosis, cystic fibrosis, and space radiation.

[0069] The methods may include wherein the disease or disorder includes relapsingremitting multiple sclerosis (RRMS).

[0070] The methods may further include including administering a therapeutically- effective amount of the vagal nerve treatment for RRMS to the subject when the subject is determined to be undergoing relapse.Nerve Stimulation Systems and Devices

[0071] The apparatuses described herein are configured to apply electrical and / or mechanical stimulation to a nerve (e.g., vagus nerve) within the parameter range identified herein specific to reduction in senescent cells. In particular, these apparatuses may be limited to a range of parameters (e.g., charge / unit of time, e.g., charge / min, charge / day) within a frequency range (e.g., between 0.1 Hz and 20 Hz) that result in the reduction of senescent cells. For example, these apparatuses may modify a cholinergic anti-inflammatory pathway (NCAP) electrical stimulation apparatus described, for example, in U.S. 6,838,471, U.S.8,914,114, U.S. 9,211,409, U.S. 6,610,713, U.S. 8,412,338, U.S. 8,996,116, U.S. 8,612,002, U.S. 9,162,064, U.S. 8,855,767, U.S. 8,886,339, U.S. 9,174,041, U.S. 8,788,034 U.S.9,211,410, U.S. 2022 / 0193413 and U.S. 2024 / 0299745, each of which is herein incorporated by reference in its entirety. The methods and apparatuses described herein may modify any of these apparatuses including limiting them to the effective range of stimulation parameters, e.g., frequency, to prevent stimulation outside of the effective range which may otherwise result in less effective treatment and may lead to undesirable side effects.

[0072] In some cases, the methods described herein may be used to trigger electrical stimulation within the effective range of parameters based on a marker for senescence. It has not previously been suggested that cellular senescence status may be used to trigger vagus nerve stimulation. Vagus nerve stimulation, through activation of both efferent and afferent pathways (or primarily through one of the efferent or afferent pathway), may be able to reduce senescence in senescence-related diseases, disorders, and conditions, thereby reducing the severity of the symptoms and / or slowing, stopping, or reversing the progression of the disease, disorder, or condition.

[0073] In some variations the devices described herein are electrical stimulation devices that may be implanted and may be activated to apply current for a proscribed duration, followed by a period without stimulation. As described in the examples that follow, the stimulation protocol may comprise a very limited period of stimulation (e.g., an on-time of- 15 -SG Docket No.: 10471-747.600less than 5 minutes, 2 minutes, 1 minute, etc.) followed by an off-time (during which stimulation is not applied, and may be prevented from being applied) of extensive duration (e.g., greater than 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 12 hours, greater than 20 hours, greater than 24 hours, greater than 36 hours, greater than 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, etc.). The applied energy may be electrical energy that is a fixed voltage and / or current, e.g., within the range of about 0.5 mA to 5 mA (e.g., approximately 2 mA), at a frequency of between about 0.1 Hz and about 100 Hz (e.g., between 1 Hz and 50 Hz, between 1 Hz and 30 Hz, between 1 Hz and 20 Hz, etc.), where the pulses applied have a pulse width of approximately (50-500 usee, e.g., between 0.1 ms and 0.5 ms, between 0.2 ms and 0.5 ms, between 0.1 ms and 0.4 ms, between 0.2 ms and 0.4 ms, etc.). The duration of the stimulation may be between about 0.1 second and 5 minutes, e.g., between 0.1 second and 2.5 min, between 0.1 second and 1 min, between 0.1 second and 30 seconds, between 0.1 second and 20 seconds, between 0.1 seconds and 10 seconds, between 0.1 second and 5 seconds, between 0.1 second and 2.5 seconds, between 0.1 second and 2 seconds, etc.). Thus, the duty-cycle of the applied current may be extremely low, where duty cycle may refer to the ratio of on-time / (on-time plus off-time). The stimulation may be applied at an extremely low duty cycle, where duty cycle may refer to the percent of on-time to the total on-time and off-time for the ongoing treatment. For example, low duty cycle may be less than about 10, 5, 4, 3, 2, 1, or 0.5 percent of on-time to the total on time and off-time. The effect may be seen relatively quickly and may persist over the entire off-time.

[0074] Thus, in any of these methods and apparatuses, the amount of energy applied to the nerve by a system as describe herein in order to enhance the destruction of senescent cells may be between about 0.1 pC and about 1000 pC (e.g., between about 0.1 pC and about 900 pC, between about 0.5 pC and about 750 pC, between about 1 pC and about 500 pC, between about 1 pC and about 300 pC, etc.). This charge may be per dose; in general, the dose may delivered at a single time (e.g., lx per day, or spread out over the day (e.g., 2x per day, 3x per day, etc.). More preferably the dose is delivered at a single period, e.g., of between about 0.1 mA to 1 mA, at between 0.1 Hz to 50 Hz (more preferably 0.1 Hz to 20 Hz), with a pulse width of between about 0.2 ms to 0.5 ms, for between about 1 second to 3 minutes (e.g., 1 second to 2 minutes, 30 seconds to 1.5 minutes, etc.), delivered to the nerve (e.g., to a vagus nerve).

[0075] Any of the methods and apparatuses described herein may be configured to deliver the senolytic electrical energy as part of an intermittent treatment, e.g., over a brief period of time followed by a substantial “off’ time, during which no further senolytic- 16 -SG Docket No.: 10471-747.600electrical treatment is applied. For example, treatment every x days or weeks (e.g., 3 days, every 3.5 days, every 4 days, every week, every two weeks, every month, every 5 weeks, every 6 weeks, every 8 weeks) or longer, between doses. A dose may include a single period of simulation or multiple stimulation periods (e.g., over a treatment period of between 1 second or less and 1 hour, between 1 second and 30 minutes, etc.).

[0076] In particular, described herein are methods of co-administering senolytic drugs with the electrical energy described herein. Co-adminstration may include administering a dose of senolytic drug (as described below) immediately before (e.g., within 1 week, 2 days, 1 day, 12 hours, 3 hours, 2 hours, 1 hour, etc.), at approximately the same time or immediately after (e.g., within 1 week, 2 days, 1 day, 12 hours, 3 hours, 2 hours, 1 hour, etc.).

[0077] In particular, the methods and apparatuses described herein may be applied as needed, e.g., when the subject has a cellular senescence status indicating that treatment is needed.

[0078] Also described herein are methods that include the unexpected combination of VNS and one or more drugs that affect cellular senescence, commonly referred to as senolytics or senomodulators. These drugs may include senolytics such as, but not limited to tyrosine kinase inhibitors (e.g., Dasatinib), flavonoids (e.g. Quercetin, Fisetin), Bcl-2 inhibitors (e.g., Navitoclax, ABT-263, ABT-737), Piperlongumine, etc., and / or senomorphics: mTor inhibitors (Rapamycin (Sirolimus), Metformin, Resveratrol, Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN), SASP Inhibitors, Janus Kinase (JAK) inhibitors, IL-1 inhibitors, NF-KB inhibitors (e.g. BAY 11-7082), telomerase activators (e.g., TA-65, GRN510, etc.), UBX1325, FOXO4-DRI peptide, Heat shock protein 90 (HSP90) inhibitors, 17-DMAG, Catechins, Curcumin, UBX0101, Procyanidins, Oleandrin, A1331852, and / or Al 155463.

[0079] Dasatinib is a tyrosine kinase inhibitor. It has demonstrated senolytic activity, particularly when used in combination with quercetin. Dasatinib targets specific types of senescent cells and has shown benefits in animal models, including improved physical function and reduced markers of aging. Quercetin is a plant-derived flavonoid with antioxidant and anti-inflammatory properties. It has senolytic effects on certain types of senescent cells and is often paired with dasatinib to enhance efficacy. Quercetin works by inhibiting anti-apoptotic pathways that senescent cells rely on for survival. Fisetin is another natural flavonoid found in fruits and vegetables such as strawberries and apples. Navitoclax (ABT-263) is a BCL-2 family inhibitor. It induces apoptosis in senescent cells by blocking survival proteins like BCL-xL. HSP90 inhibitors target heat shock protein 90, which plays a role in maintaining the stability of proteins involved in senescence. Other senolytic agents- 17 -SG Docket No.: 10471-747.600may include UBX0101 and UBX1325. Cardiac glycosides such as ouabain and digoxin, traditionally used for heart conditions, have shown senolytic activity by inducing apoptosis in exhausted cell types. Piperlongumine is a natural compound derived from long pepper. It induces oxidative stress selectively in senescent cells, leading to their death. F0X04-DRI peptides disrupt the interaction between FOXO4 and p53, releasing p53 to trigger apoptosis in senescent cells. These peptides have shown effectiveness in preclinical models and represent a novel approach to senolytic therapy.

[0080] Thus, the methods described herein may include co-administering, including concurrent administering of one or more senolytic drugs with electrical stimulation as described herein. Any of these methods may include co-administering with (including concurrent administration with) one or more of: dasatinib, quercetin, fisetin, navitoclax (ABT-263), HSP90 inhibitors, UBX0101, UBX1325, cardiac glycosides (e.g., ouabain and digoxin), piperlongumine, and / or FOXO4-DRI.

[0081] Also described herein are methods and apparatuses in which one electrical stimulation of the nerve (e.g., vagus nerve) is applied in combination with a senomorphic drug. In general, senomorphic drugs are agents that modify senescent cell behavior, and in particular, drugs that suppress the harmful secretions (SASP) of senescent cells, reducing inflammation and tissue damage. Examples of senomorphic drugs that may be coadministered with any of the treatments (e.g., electrical treatments) described herein may include but are not limited to: Rapamycin and Rapalogs (e.g., mTOR inhibitors), JAK Inhibitors, CAR-T cells and antibody-drug conjugates targeting senescence-specific antigens, metformin, aspirin, and various natural polyphenols, such as quercetin and fisetin.

[0082] These methods may also be used to treat cardiovascular diseases, pulmonary fibrosis, Alzheimer’s disease, chronic kidney disease, and cancer.Example 1

[0083] In the standard inflammatory rodent model of multiple sclerosis, experimental autoimmune encephalomyelitis, we have demonstrated that vagus nerve stimulation (VNS) ameliorated inflammation and disease activity. To understand if this was a direct pro- remyelination effect or merely secondary to reducing inflammation, we investigated the role of VNS in the standard murine model of lysolecithin (LPC)-induced focal spinal cord (SC) demyelination. We observed that VNS significantly accelerated remyelination when applied at the peak of lesion formation in both young and aged (12-19 months) mice (FIGS. 2A-2B). To elucidate the mechanisms underlying pro-myelinating effect of VNS, further experiments were performed. Here, we show that VNS (0.75 mA, 10 Hz, 0.25 ms pulse width, 60 s) curbed the accumulation of senescent microglia and modestly decreases microglial TREM2- 18 -SG Docket No.: 10471-747.600expression in demyelinated lesions (FIG. 1 A). At day 8 post induction (8 DPI) in young mice, VNS significantly decreased the number of microglia that expressed phosphorylated yH2AX at Serl39 (pSerl39), a marker for cellular senescence, within lesions (FIG. IB). Importantly, both the average and maximum senescent cells per area were significantly downregulated with VNS compared to sham treatment. Furthermore, TREM2 mean fluorescent intensity (FMI) within lesion was also reduced with VNS. It should be noted that TREM2 FMI was higher in lesion compared to non-lesion region in both sham and VNS treatment groups. Similar trends were observed at 10 DPI in aged mice (FIG. IB).Example 2

[0084] FIGS. 2 A, 2B1-2B3, and 2C1-2C3 show that vagus nerve stimulation limited the accumulation of senescent microglia. The standard murine model of lysolecithin (LPC)- induced focal spinal cord (SC) demyelination (lesions) was subjected to vagus nerve stimulation or a sham control and the results analyzed. FIG. 2A shows representative 20X confocal images. The top panel of FIG. 2 A shows results from staining for yH2AX (pSerl39), a marker for cellular senescence. The middle panel of FIG. 2A shows results from staining for DAP I, a nuclei marker. The bottom panel of FIG. 2A shows that expression of yH2AX (pSerl39) overlapped with DAP I, a nuclei marker. The results show significantly fewer microglia (Ibal+ cells, images not shown) expressing yH2AX (pSerl39), after VNS compared with sham treated animals. FIGS. 2B1-2B3 shows that vagus nerve stimulation of the lysolecithin (LPC)-induced murine model significantly reduced the average (FIG. 2B1) and maximum (FIG. 2B2) number of senescent microglia per lesion compared to control in young mice (2-4 months). FIG. 2B3 shows that while TREM2 is significantly upregulated in lesion versus non-lesion region (data not shown), there is a trend towards lowering TREM2 expression within lesion with vagus nerve stimulation as compared to control. FIGS. 2C1- 2C3 shows that similar trends as shown in FIGS. 2B1-2B3 were also observed in older animals (12 months). For FIGS. 2A, 2B1-2B3 and 2C1-2C3, n=3 mice / group. Student’s t- test, *p<0.05, **p<0.01.Frequency Dependence

[0085] Surprisingly, the improvement in the reduction of senescent cell described herein may be frequency dependent. In some cases the frequency dependence has not previously been identified in other vagus nerve stimulation techniques. Specifically, the ability of the electrical stimulation (e.g., applying stimulation between about 0.1 pC and about 900 pC charge over a single dose), may be specifically limited to an effective frequency range, which has been found to be between about 0.1 Hz and 30 Hz or less (e.g., 25 Hz or less, 20 Hz or less, less than 20 Hz). This is illustrated in FIG. 3. FIG. 3 is a graph showing the effect of- 19 -SG Docket No.: 10471-747.600various electrical stimulation of the vagus nerve on senescent cells, using the yH2AX (pSerl39) marker for senescent cells for different frequency pulses applied to the nerve. As shown in FIG. 3, this data shows a robust effect of the frequency of pulses used for otherwise-identical doses of electrical stimulation on a vagus nerve. In these experiments, between about 0.1 Hz and just under 20 Hz the number of senescent cells, as detected by the yH2AX markers, in test animals dropped significantly, by less than 70% of the control values, in some cases. At about 20 Hz or greater the number of senescent cells became increasingly variable. Thus, there appears to be a significant frequency dependence for the pulsed energy applied. Thus, effective treatment of senescence may be achieved by maintaining the pulse frequency of the applied energy below about 20 Hz. Outside of this range, the senescence effect may be diminished or lost.Devices

[0086] Also described herein are apparatuses (e.g., devices, systems, etc.) for treatment of senescence, as mentioned above. These apparatuses may be implanted or external. In some cases external application of energy may be applied at the neck, e.g., at the level of the carotid and jugular. In some energy may be applied by implanting a vagus nerve stimulator in communication with the vagus nerve or nerve that communicates with the vagus nerve. External simulation of the vagus nerve may be invasive (e.g., by injection or insertion of an electrode near the vagus nerve) or may be noninvasive (e.g., through one or more electrodes on the outer surface of the skin.

[0087] FIG. 4A shows a first example of an apparatus configured as an implantable senescence microstimulator 401 that may be configured to be inserted into a patient to treat the patient. In this example the apparatus 401 may include hardware, software and / or firmware to control the application of energy to the nerve, as described herein, in order to treat senescence (or one or more disorders associated with senescence.

[0088] In FIG. 4A, the microstimulator 401 may include a pulse generator 407 that is configured to generate and apply pulse energy as described by the parameters taught herein in order to treat senescence. In particular, the pulse generator 407 may include one or more processors 415, and a memory; the memory may store instructions (e.g., control logic) 409 that may control operation of the apparatus to deliver electrical energy effective to treat senescence (e.g., to reduce the number of senescent cells and / or to treat a disorder associated with senescence). Any of these pulse generators may include circuitry for controlling the receipt of energy from a power source or power storage 421 component of the microstimulator and may generate a set of pulses having the dosing properties described herein. For example, the pulse generator may generate a series of pulses of between about 0.5- 20 -SG Docket No.: 10471-747.600mA to 5 mA (e.g., approximately 2 mA), at a frequency of between, e.g., between about 0.1 Hz and about 50 Hz (e.g., between 1 Hz and 50 Hz, between 1 Hz and 30 Hz, between 1 Hz and 20 Hz, less than 20 Hz, etc.), and a pulse width of approximately (50-500 usee, e.g., between 0.1 ms and 0.5 ms, between 0.2 ms and 0.5 ms, between 0.1 ms and 0.4 ms, between 0.2 ms and 0.4 ms, etc.) for a duration of between about 0.1 second and 5 minutes, e.g., between 0.1 second and 2.5 min, between 0.1 second and 1 min, between 0.1 second and 30 seconds, between 0.1 second and 20 seconds, between 0.1 seconds and 10 seconds, between 0.1 second and 5 seconds, between 0.1 second and 2.5 seconds, between 0.1 second and 2 seconds, etc.

[0089] In particular, these apparatuses may include a frequency limiter 411 that limits the applied frequency of the pulses to less than the minimum described above, e.g., less than 20 Hz, 19 Hz or less, 18 Hz or less, 15 Hz or less, etc., including between 0.1 and 20 Hz (or 0.1 and less than 20 Hz), 0.1 and 19 Hz, etc. The limiter may be part of the pulse generator 407 and may be integrated into the control logic 109 or other components of the pulse generator.

[0090] The pulse generator 407 may also include one or more inputs / outputs 417 for communicating with one or more devices or systems outside of the implant. Any of these apparatuses may receive input and / or may provide output to a hand-held device (e.g., tablet, phone, etc.), including a dedicated device (e.g., external programmer / controller) that may be operated by the patient, doctor, technician, etc., and may allow programming, including uploading of programming, triggering the application of a dose (either manually and / or automatically), limiting the dose (e.g., to prevent dosing before a predetermined or adjustable “off time’), and / or adjusting any of these dosing parameters.

[0091] In FIG. 4 A, the microstimulator 401 may include one or more (preferably 2 or more) integrated electrodes 403 that may be placed in communication with the nerve (e.g., vagus nerve) once implanted. The electrodes may be on an outer surface of the implantable microstimulator 401.

[0092] The microstimulator may include a power source 421 that may be a battery, which may be rechargeable, by wireless recharging, e.g., inductive charging or the like, or may provide on-demand power, e.g., configured to deliver power when energy is applied from an outside source (e.g., by an antenna). Any of these power sources 421 may include power control circuitry.

[0093] FIG. 4B shows another example of an implantable senescence microstimulator 401’ that is not integrated (as in FIG. 4A), but that include the electrodes 403’ separate from the microstimulator control circuitry (in the body of the microstimulator 421) and is- 21 -SG Docket No.: 10471-747.600connected by a lead 423. The body of the microstimulator may otherwise be similar or identical tot hat shown in FIG. 4A.

[0094] In use, any of these methods may include inserting / implanting the microstimulator into the body, e.g., implanting the electrodes within the cervical region of the neck in communication with the vagus nerve.Disorders and Diseases

[0095] The methods and apparatuses described herein may be used to treat one or more disorders by electrical stimulation of a nerve, including in particular those identified with or associated with senescence that have not previously been expected to be responsive to vagus nerve stimulation. For example, the methods and apparatuses described herein may be used to treat one or more disorders including, but not limited to: cancer and premalignant lesions, including solid tumors (e.g., breast, prostate, colon, lung, pancreatic, etc.), pre-malignant lesions; cardiovascular / vascular disorders, including hypertension-related vascular remodeling, vascular calcification, abdominal aortic aneurysm; metabolic and endocrine, including Type 2 diabetes mellitus, obesity-associated insulin resistance, NAFLD / NASH, osteoporosis; pulmonary disorders, including idiopathic pulmonary fibrosis (IPF), COPD; neurodegenerative disorders (not limited to Alzheimer’s by your instruction), including Parkinson’s disease, Huntington’s disease, ALS, multiple sclerosis; renal disorders, including chronic kidney disease, diabetic nephropathy, glomerulosclerosis; ophthalmic disorders, including AMD, glaucoma, cataracts; musculoskeletal disorders, including osteoarthritis, intervertebral disc degeneration, sarcopenia, osteoporosis, rheumatoid arthritis; dermatological disorders, including skin aging, chronic wound healing impairment, radiation- induced skin injury; reproductive disorders, including infertility (age-related), premature ovarian failure, placental dysfunction; systemic / immune disorders, including generally aging, frailty, immunosenescence (immune aging), hematopoietic stem cell exhaustion, vaccine unresponsiveness / infection susceptibility due to immune aging, etc. these methods and apparatuses may generally be used to enhance or increase longevity.

[0096] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly - 22 -SG Docket No.: 10471-747.600attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0097] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0098] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0099] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.[000100] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and- 23 -SG Docket No.: 10471-747.600“comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.[000101] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and / or steps may alternatively be exclusive and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps. [000102] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.[000103] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described- 24 -SG Docket No.: 10471-747.600method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.[000104] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.- 25 -SG Docket No.: 10471-747.600

Claims

CLAIMSWhat is claimed is:

1. A method of reducing senescent cells in a subject’s body, the method comprising applying a dose of electrical stimulation to a vagus nerve, wherein the dose of electrical stimulation comprises between about 0.5 mA to 2.5 mA at a frequency of less than 20 Hz and a pulse width of between 0.1 ms and 0.5 ms for a duration of between about 0.1 second and 2.5 min.

2. A method of reducing senescent cells in a subject’s body, the method comprising applying a dose of electrical stimulation to a vagus nerve, wherein the dose of electrical stimulation comprises between about 0.5 mA to 2.5 mA and a pulse width of between 0.1 ms and 0.5 ms for a duration of between about 0.1 second and 2.5 min and limiting the frequency of the applied pulses to a frequency of less than 20 Hz.

3. A method of reducing senescent cells in a subject’s body, the method comprising: applying a dose of electrical stimulation to a vagus nerve, wherein the dose of electrical stimulation comprises between about 0.5 mA to 2.5 mA and a pulse width of between 0.1 ms and 0.5 ms for a duration of between about 0.1 second and 2.5 min and limiting the frequency of the applied pulses to a frequency of less than 20 Hz; and delivering one or more senelytic and / or senomodulator drugs to the subject’s body currently with the dose of electrical stimulation.

4. The method of claim 3, wherein the one or more senelytic drugs includes one or more of: Dasatinib, Quercetin, Fisetin, Navitoclax, 17-DMAG, Catechins, FOXO4-DRI peptides, HSP90 inhibitors, Piperlongumine, Curcumin, ABT-737, UBX0101, Procyanidins, Oleandrin, A1331852, and / or Al 155463.

5. The method of claim 3, wherein the one or more senomodulator drugs includes one or more of: a tyrosine kinase inhibitors (e.g., Dasatinib), a flavonoid (e.g. Quercetin, Fisetin), a Bcl-2 inhibitor (e.g., Navitoclax, ABT-263), Piperlongumine, an mTor inhibitor (e.g., Rapamycin (Sirolimus), Metformin, Resveratrol, Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN), a SASP Inhibitor, a Janus Kinase (JAK) inhibitor, a IL-1 inhibitor, a NF-KB inhibitor (e.g. BAY 11-7082), a telomerase activators (e.g., TA-65, GRN510, etc.), UBX1325, FOXO4-DRI peptide, or a Heat shock protein 90 (HSP90) inhibitor.- 26 -SG Docket No.: 10471-747.6006. The method of any of claims 1-5, further comprising waiting at least one week before administering another dose of electrical stimulation.

7. The method of any of claims 1-6, further comprising: identifying a decrease in a level of one or more cellular senescence status biomarkers in the subject’s body following delivery of the dose.

8. The method of any of claim 1-7, wherein applying the dose comprises applying the dose non-invasively.

9. The method of any of claim 1-7, wherein applying the dose comprises applying the dose from an implanted microstimulator.

10. The method of any of claim 1-7, wherein applying the dose comprises applying the dose from an external microstimulator.

11. The method of claim 7, wherein the one or more cellular senescence biomarkers comprises one or more cell cycle marker, a cell cycle inhibitor marker, a DNA damage indicator, a chromatin abnormality indicator, a metabolic indicator, or a lysosomal alteration.

12. The method of claim 7, wherein the one or more cellular senescence status biomarkers includes a biomarker selected from TREM2, cyclin-dependent kinase pl 6, cyclin- dependent kinase p21, 53BP1; YH2AX (pSerl39), IL6, IL8, Lamin Bl, senescence- associated beta-galactosidase (SA-P-gal or SABG), KI-67, and phosphorylated Retinoblastoma protein (pRb).

13. The method of claim 7, wherein the one or more cellular senescence status biomarkers comprises TREM2 or YH2AX (pSerl39).

14. The method of claim 7, wherein identifying a decrease in a level of one or more cellular senescence status biomarkers comprises analyzing at least five biomarkers.

15. The method of claim 7, wherein identifying a decrease in a level of one or more cellular senescence status biomarkers comprises analyzing senescence biomarkers obtained from a blood test, a spinal tap, and a magnetic resonance image of the subject’s brain or spinal cord.- 27 -SG Docket No.: 10471-747.60016. The method of any of claims 1-15, wherein the method further comprises identifying that the patient has a senescence related disease or disorder.

17. The method of claim 16, wherein the senescence related disease or disorder comprises one or more of: atherosclerotic diseases, cardiovascular diseases, cerebrovascular diseases, aneurysm, hypertension, cancers, radiotherapy-induced senescence, neurodegenerative diseases, Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington, Frontotemporal dementia, macular degeneration, metabolic disorders, Type 2 diabetes mellitus, obesity, lysosomal storage diseases, inflammatory bowel disease, biliary liver damage, liver fibrosis, fatty liver disease, glaucoma, retinopathy, idiopathic pulmonary fibrosis, intervertebral disc degeneration, osteoarthritis, osteoporosis, pulmonary fibrosis, cystic fibrosis, and / or radiation.

18. The method of claim 16, wherein the senescence related disease or disorder comprises multiple sclerosis.

19. The method of claim 16, wherein the senescence related disease or disorder comprises relapsing-remitting multiple sclerosis (RRMS).

20. The method of claim 19, further comprising administering a therapeutically-effective amount of the vagal nerve treatment for RRMS to the subject when the subject is determined to be undergoing relapse.

21. An apparatus for reducing senescent cells in a subject’s body, the apparatus comprising: a pair of electrodes configured to apply energy to a vagus nerve; a pulse generator coupled to the electrodes and configured to deliver a dose of electrical stimulation to the vagus nerve from the electrodes, wherein the dose of electrical stimulation comprises a plurality of pulses between about 0.5 mA to 2.5 mA having a pulse width of between 0.1 ms and 0.5 ms, for a dose duration of between about 0.1 second and 2.5 min, wherein the pulse generator comprise a frequency limiter configured to limit the frequency of the pulses during the dose to less than 20 Hz.- 28 -SG Docket No.: 10471-747.60022. The apparatus of claim 21, wherein the electrodes are positioned on a housing enclosing the pulse generator.

23. The apparatus of claim 21, wherein the electrodes are coupled to the pulse generator by a lead.

24. The apparatus of claim 21, further comprising a delay control configured to prevent the apparatus from delivering another dose for at least a week before administering another dose of electrical stimulation.

25. The apparatus of claim 24, wherein the pulse generator includes the delay control.

26. The apparatus of claim 24, wherein the delay control is configured to prevent the apparatus for at least two weeks before administering another dose of electrical stimulation.- 29 -SG Docket No.: 10471-747.600

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