Exercise and spermidine combination therapy

Abstract

Combination therapies for treating or preventing inflammation in a subject using spermidine, an exercise regime and at least one additional pharmaceutical agent.

Description

241101G-WO (772809: MTST-697PC)EXERCISE AND SPERMIDINE COMBINATION THERAPYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 730,869, filed December 11, 2024, the disclosures of which are hereby incorporated by reference in their entireties.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. R01 CAI 12100 awarded by the National Institutes of Health. The government has certain rights in this invention.BACKGROUND

[0003] Tissue-resident macrophages (RTMs) are seeded in organs during embryogenesis and shaped by local environmental cues into key guardians of tissue health. In youth, RTMs clean and repair tissues, and guide essential physiological processes like neural synapse pruning, muscle growth and regeneration. They signal hematopoiesis to generate new immune cells and then work together with these immune cells to fight cancer and infection. However, these protective RTMs decline with age. In response, the bone marrow ramps up myelopoiesis, releasing progenitors that fail to mature into functional RTMs, instead adopting an inflammatory profile. This imbalance disrupts tissue integrity, drives ‘inflammaging’, spreads senescence systemically, causing many of the problems in aging. Increased myelopoiesis is also linked to aging-related diseases, whereas RTMs enhance tissue protection in infection, myocardial infarction and Alzheimer’s disease.SUMMARY

[0004] One aspect of the disclosure is method of treating or preventing inflammation in a subject, the method comprising: administering a combination therapy comprising: a therapeutically effective amount of spermidine; and a therapeutically effective amount of at least one additional pharmaceutical agent, wherein the additional pharmaceutical agent is81580610v1 1241101G-WO (772809: MTST-697PC) configured to amplify immune, muscle, and / or cognitive function in conjunction with an exercise regime.

[0005] One aspect of the disclosure is the use of spermidine in combination with at least one additional pharmaceutical agent in conjunction with an exercise regime for the manufacture of a medicament for treating or preventing inflammation in a subject.

[0006] One aspect of the disclosure is a pharmaceutical composition comprising: spermidine; at least one additional pharmaceutical agent selected from the group consisting of reverse transcriptase inhibitors, natural fungal antibiotics, and mTOR inhibitors; and a pharmaceutically acceptable carrier, wherein the composition is formulated for administration to a subject undergoing an exercise regime.

[0007] One aspect of the disclosure is a kit for treating or preventing inflammation, comprising:

[0008] a first formulation comprising spermidine; a second formulation comprising at least one additional pharmaceutical agent selected from the group consisting of reverse transcriptase inhibitors, natural fungal antibiotics, and mTOR inhibitors; and instructions for administration in conjunction with an exercise regime.

[0009] In an aspect, the at least one additional pharmaceutical agent is a reverse transcriptase inhibitor, natural fungal antibiotic, or mTOR inhibitor. In an aspect, the at least one additional pharmaceutical agent is lamivudine or rapamycin.

[0010] In an aspect, the combination therapy is administered via a route selected from intratumoral, intraperitoneal, intranodal, peritumoral, subcutaneous, or intravenous delivery. In an aspect, spermidine and the at least one additional pharmaceutical agent are administered simultaneously, concurrently, sequentially, successively, alternatively, or separately.

[0011] In an aspect, the combination therapy is administered for a sustained period. In an aspect, the sustained period is at least about 90 days, alternatively at least about 120 days, alternatively at least about 180 days, or alternatively at least about 210 days.

[0012] In an aspect, at least about 1 mg / day of the spermidine is administered, alternatively at least about 2 mg / day, alternatively at least about 3 mg / day, alternatively at least about 4 mg / day, alternatively at least about 5 mg / day, alternatively at least about 6 mg / day, alternatively at least about 7 mg / day, alternatively at least about 8 mg / day, alternatively at least about 9 mg / day, or alternatively at least about 10 mg / day.

[0013] In an aspect, the lamivudine is administered at least once a day or alternatively at least twice a day. In an aspect, about 300 mg or less of the lamivudine is administered. In an81580610v1 2241101G-WO (772809: MTST-697PC) aspect, the rapamycin is administered at least once a week. In an aspect, between about 1 mg to about 10 mg of the rapamycin is administered.

[0014] In an aspect, the method results in an increase in tissue-resident macrophage (RTM) activity. In an aspect, the method results in an increase and / or recovery of immune, muscle, and / or cognitive functions.

[0015] In an aspect, the subject is a human diagnosed with chronic inflammation, autoimmune disorder, or age-related inflammatory condition.

[0016] In an aspect, the exercise regime comprises aerobic exercise, resistance training, or a combination thereof.

[0017] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be employed to achieve the benefits as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts a study schematic according to an aspect of the disclosure.

[0019] FIG. 2 is a graph showing correlations between serum / plasma CCL20, as an example, and age in several cohorts of healthy and disease patients. Linear regression from NPX values was calculated separately across different Olink kits and lots.DETAILED DESCRIPTIONI. Introduction

[0020] To counteract systemic effects of aging and restore tissue and cellular health, there is a need to revive the cellular and molecular processes critical for maintaining tissue homeostasis. Without being bound to any theory, it is thought that progressive dysregulation and loss of the tissue-resident macrophages (RTMs) responsible for maintaining tissue and cellular homeostasis drives pathogenic aging across multiple systems. TRMs play a crucial role in preserving tissue integrity, repairing damage, and clearing dysfunctional cells, all of which are essential for sustaining tissue homeostasis. However, RTM populations decline significantly with age, leading to pathogenic myelopoiesis, chronic inflammation, and tissue damage.

[0021] Rejuvenating immune, cognitive, and muscular health requires an orthogonal approach for enhancing RTMs and curbing pathogenic myelopoiesis, and restoring the tissue81580610v1 3241101G-WO (772809: MTST-697PC) fitness and metabolic health of organs necessary to maintain this balance. Aging impacts the tissue environments that control RTM survival and their reparative and physiological activities, which necessitates a comprehensive intervention. The inventors leveraged the synergistic effects of exercise, in combination with spermidine for systemic metabolic health and RTM support, and favor rebalancing cellular homeostasis and inhibiting senescence with rapamycin, or inhibiting pathogenic myelopoiesis via lamivudine. These agents are expected to complement each other in restoring RTM function and combating cellular aging across tissues, driving a synergy that promotes systemic rejuvenation and functional recovery in multiple organs and systems.

[0022] This disclosure integrates exercise and metabolites like spermidine to improve cellular fitness and replenish RTM function alongside one of two strategies: reducing cellular aging through rapamycin or curbing excessive myelopoiesis with lamivudine. Together, these interventions aim to restore myeloid homeostasis, enhancing immune, muscle, and cognitive health, and extend healthspan by addressing both the causes and consequences of cellular dysfunction in aging.II. Definitions

[0023] 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 disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0024] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0025] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0026] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be81580610v1 4241101G-WO (772809: MTST-697PC)

[0027] preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0028] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0029] By " consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0030] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three- element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0031] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0032] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," "one embodiment,” "an embodiment,” "certain embodiments," or81580610v1 5241101G-WO (772809: MTST-697PC)"some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0033] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0034] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0035] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0036] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For81580610v1 6241101G-WO (772809: MTST-697PC) example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0037] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, “substantially” may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0038] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0039] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0040] The terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the81580610v1 7241101G-WO (772809: MTST-697PC) progression of the disease or disorder in the subject; and / or decrease the number, frequency, or severity of clinical symptoms and / or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms “treatment of a disease” and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.

[0041] The terms “prophylactic”, “preventive”, “preventing”, and “prevention” refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.

[0042] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0043] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Exercise and Spermidine Combination Therapy

[0044] In certain embodiments, the disclosure provides a method for treating or preventing inflammation in a subject by administering a combination therapy comprising spermidine and at least one additional pharmaceutical agent, in conjunction with an exercise regime. The additional pharmaceutical agent may be selected from reverse transcriptase inhibitors, natural fungal antibiotics, mTOR inhibitors, or combinations thereof. In some embodiments, the additional pharmaceutical agent includes lamivudine, rapamycin, or other compounds that modulate immune, muscle, and cognitive function.

[0045] In some embodiments, the combination therapy comprising spermidine and at least one additional pharmaceutical agent may be administered through a variety of routes to optimize bioavailability and therapeutic effect. Oral administration may be employed for ease81580610v1 8241101G-WO (772809: MTST-697PC) of use and patient compliance, utilizing tablets, capsules, or liquid formulations designed for immediate or controlled release. Intravenous delivery may be used for rapid systemic distribution, particularly in acute inflammatory conditions or when high bioavailability is required. Intraperitoneal administration can provide localized exposure within the abdominal cavity, suitable for systemic inflammatory disorders or experimental models. Intratumoral injection may be utilized for direct targeting of tumor-associated inflammation, while intranodal delivery can enhance immune modulation by delivering agents directly to lymph nodes. Peritumoral administration may be employed to create a localized therapeutic environment surrounding a tumor site, reducing systemic exposure while maximizing local efficacy. Subcutaneous delivery offers a minimally invasive route for sustained release formulations, enabling prolonged therapeutic activity with reduced dosing frequency. In certain embodiments, these routes may be combined or alternated based on patient-specific needs, pharmacokinetic profiles, and desired therapeutic outcomes. In certain embodiments, spermidine and the additional pharmaceutical agent are administered simultaneously, sequentially, or separately, depending on the desired therapeutic outcome. The method may further include implementing an exercise regime comprising aerobic activity, resistance training, or a combination thereof, performed at regular intervals to enhance the therapeutic effect. Exercise may be structured as high-intensity interval training (HIIT), moderate-intensity continuous training, or progressive resistance exercises.

[0046] In some embodiments, the combination therapy is administered for a sustained period. The sustained period may be at least about 90 days, alternatively at least about 120 days, alternatively at least about 180 days, or alternatively at least about 210 days. In certain embodiments, the therapy may be continued beyond 210 days for chronic conditions or longterm healthspan interventions.

[0047] In some embodiments, spermidine is administered orally in an amount sufficient to achieve a therapeutic effect. The amount may be at least about 1 mg / day, alternatively at least about 2 mg / day, alternatively at least about 3 mg / day, alternatively at least about 4 mg / day, alternatively at least about 5 mg / day, alternatively at least about 6 mg / day, alternatively at least about 7 mg / day, alternatively at least about 8 mg / day, alternatively at least about 9 mg / day, or alternatively at least about 10 mg / day. In certain embodiments, spermidine may be administered in a controlled-release formulation to maintain steady plasma levels.

[0048] In certain embodiments, the lamivudine is administered at least once a day or alternatively at least twice a day. In some embodiments, lamivudine is administered in an amount of about 600mg or less per dose, alternatively 300 mg or less per dose, or alternatively81580610v1 9241101G-WO (772809: MTST-697PC)150 mg or less per dose, using its standard FDA-approved formulation. Lamivudine may be administered orally, intravenously, or via other suitable routes, either concurrently with spermidine or in a staggered schedule.

[0049] In certain embodiments, rapamycin is administered at least once per week. The amount of rapamycin may range between about 1 mg to about 10 mg per administration, optionally in a low-dose regimen to avoid immunosuppression while promoting immune rejuvenation. Rapamycin may be delivered orally or via injection, and may be formulated for immediate or sustained release.

[0050] In some embodiments, the method results in an increase in tissue-resident macrophage activity and promotes recovery or enhancement of immune, muscle, and cognitive functions. The therapy may also lead to a reduction in inflammatory biomarkers and improvement in overall physiological resilience.

[0051] Additional embodiments include pharmaceutical compositions comprising spermidine and the additional pharmaceutical agent formulated with pharmaceutically acceptable carriers, as well as kits containing separate formulations of these agents along with instructions for use in conjunction with an exercise regime. Optional components of the kit may include monitoring devices or supplemental nutraceuticals to further support immune and metabolic health.

[0052] Scientific Rationale and Preliminary Data

[0053] Preclinical and clinical studies indicate that spermidine (a metabolic supplement and calorie restriction mimetic) and lamivudine (a reverse transcriptase inhibitor) or rapamycin (a natural fungal antibiotic and mTOR inhibitor) and exercise all independently exhibit broad antiaging effects by enhancing cellular and tissue fitness, leading to improved immune, cognitive, and muscle function. As shown herein exercise, spermidine and lamivudine either boost RTM survival and activity or block pathogenic myelopoiesis, respectively (FIG. 1).

[0054] Combining these agents is hypothesized to maximize RTM activity thereby promoting recovery of immune, muscle, and cognitive functions. Blood biomarkers for pathogenic myelopoiesis or RTM loss may also serve as surrogate measures for aging reversal before functional benefits are observed, in

[0055] Baseline Interventions: Exercise and Spermidine

[0056] Spermidine supplementation and exercise offer complementary benefits in promoting healthy aging, making them a scientifically robust baseline intervention for clinical trials. Exercise is well-documented to reduce chronic disease risk, improve healthspan, and counteract aging-associated declines in aerobic capacity and muscle volume. High-intensity interval training81580610v1 10241101G-WO (772809: MTST-697PC)(HIIT) enhances VO2 max, a critical marker of cardiorespiratory fitness linked to reduced mortality, while resistance training builds muscle strength, mitigates frailty, and improves mobility. Both modalities reduce inflammation, support neurogenesis, and bolster immune function. Together, these exercise types provide a foundation for systemic rejuvenation, making them ideal partners for other anti-aging interventions like spermidine.

[0057] Spermidine, a naturally occurring polyamine, has shown significant potential in supporting healthy aging by enhancing autophagy, mitochondrial health, and immune functions. Aging-associated declines in tissue-resident macrophages (RTMs), key cells involved in tissue repair and maintenance, are likely linked to reduced spermidine synthesis (FIG. 1). Supplementation with spermidine supports RTM renewal, bolsters immune responses, and improves muscle and cognitive performance. It mimics the benefits of calorie restriction, reducing inflammation, preventing age-related diseases, and extending lifespan. With a robust safety profile and FDA approval for trials targeting individuals aged 65-80, spermidine alone is being investigated for effects on vaccination responses, cognitive decline, and inflammation.

[0058] Pairing exercise with spermidine supplementation provides synergistic effects. Exercise promotes muscle growth and systemic benefits that sustain RTM function, while spermidine induces autophagy and enhances mitochondrial fitness to aid recovery and adaptation to physical activity. RTMs are pivotal in muscle regeneration and inflammation modulation, suggesting that this combination may amplify tissue repair, growth, and maintenance. This systemic support enhances exercise benefits, boosting muscle protein synthesis and cardiovascular fitness while promoting tissue homeostasis and long-term resilience. Given its safety and systemic benefits, this dual intervention forms the foundation of our clinical trial, providing participants with a sustainable and scientifically grounded approach to healthy aging.

[0059] Additional Pharmaceutical Agents: Rapamycin and Lamivudine

[0060] Rapamycin and / or lamivudine are included in further embodiments to further amplify immune, muscle, and cognitive function.

[0061] Lamivudine (Epivir) is a drug used typically to treat HIV and Hepatitis B Virus (HBV). Lamivudine targets aging-induced reactivation of transposable elements that drive inflammatory cytokine production, pathogenic myelopoiesis, and stromal damage. Preclinical studies have demonstrated that lamivudine reduces inflammation, reverses myelopoiesis, and restores effective anti-tumor immunity in older mice. It has also shown promising anti-aging effects, including improved cognition and lifespan extension, with ongoing trials exploring its role in reducing cognitive decline in Alzheimer’s patients. Complementing the effects of81580610v1 11241101G-WO (772809: MTST-697PC) spermidine, lamivudine specifically reduces pathogenic myelopoiesis, potentially mitigating the source of inflammatory myeloid cells in aging.

[0062] Rapamycin (Rapamune, Sirolimus) is an FDA-approved drug and indicated for use as an immune modulator. Rapamycin takes a different approach by limiting the ability of myeloid progenitors to undergo senescence and suppressing the senescence-associated secretory phenotype. At low, weekly doses, rapamycin supports immune rejuvenation, in contrast to immunosuppression at high doses. Preclinical and early clinical studies have shown its potential to delay aging markers, enhance immune responses, improve muscle and cognitive function, and promote healthspan. It is being tested alone in trials for its effects on immune, cognitive, muscle, and cardiovascular health.

[0063] Given their distinct modes of action, lamivudine and rapamycin are being trialed separately to determine whether blocking the source of senescent inflammatory myeloid cells (lamivudine) or inhibiting their downstream effects (rapamycin) yields better outcomes. Both drugs have strong safety profiles and FDA approval for other indications, making them suitable for aging-related research. These drugs may be administered individually or in combination with spermidine and exercise to evaluate synergistic effects on inflammation and aging markers.

[0064] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.EXAMPLES

[0065] Trial Design Overview

[0066] The Phase lb two-cohort study investigates the feasibility and safety of combining exercise with pharmacologic interventions (spermidine with lamivudine or rapamycin) to influence systemic inflammation and aging-related biomarkers. The trial will provide preliminary evidence on therapeutics that enhance healthspan by targeting cellular processes linked to inflammation, metabolism, autophagy and senescence. During the Semi-Finals Trial, data collected on feasibility, safety, and intervention compliance will help refine methods and dosage, establishing a foundation for a subsequent Finals Trial. This study builds on prior work81580610v1 12241101G-WO (772809: MTST-697PC) in murine models and humans of specific relevance to XPRIZE Healthspan by addressing core mechanisms involved in aging, such as the inflammatory milieu and cellular metabolism. FIG. 1 depicts an exemplary study schematic.

[0067] Study Design Characteristics

[0068] The study will recruit 20 participants (10 per cohort) aged 65-80 years, current or former smokers with a BMI of 25-32, and elevated baseline systemic inflammation. Enrollment is based on feasibility for a preliminary trial in a hospital setting and the competition guidelines set forth for XPRIZE HEALTHSPAN. This sample size supports estimation of initial safety and compliance, which will inform the design of later trials with larger populations.

[0069] Exclusion criteria include recent cancer treatments, chronic kidney disease, and other major health conditions. Participants will be selected from Mount Sinai's BioMe® a large biobank integrating genomic data with longitudinal electronic health records (EHRs) and housing biospecimens like DNA, plasma, serum, and PBMCs. Reflecting New York City's diverse population, BioMe supports exposomic analyses linking environmental and socioeconomic factors to aging and physiological decline. This integration with EHR data and advanced assays aids in identifying biomarkers for anti-aging interventions and tailoring supportive therapies for specific subpopulations. BioMe facilitates clinical trials and precision medicine by enabling diverse, targeted recruitment.

[0070] Interventions are administered for 180 days. All patients will receive a daily dose of spermidine for 180 days orally (2 mg / day), with one cohort randomized to additionally receive lamivudine and the other rapamycin using their standard FDA-approved formulations.

[0071] Arm A: High-intensity interval training (two 20-minute sessions per week) plus resistance training (three 40-minute sessions weekly), daily spermidine, and lamivudine.

[0072] Arm B: The same exercise and spermidine regimen combined with rapamycin.

[0073] Rapamycin will be given once a week at the low dose of 6mg after an initial dose escalation period from 2mg over 4 weeks to allow for side effects to be monitored. This low dose regimen promotes immune responses rather than the immunosuppressive effects of higher doses of rapamycin, with reduced chance of side effects, and is the regimen being employed in a clinical trial of patients with Long Covid.

[0074] The twice-daily dose of lamivudine is based on the concentration that blocks expression of TEs in the HIV replication cycle during long term treatment of human subjects, without causing significant adverse effects; this dose is currently being utilized in a clinical trial for patients with cancer.

[0075] Outcomes81580610v1 13241101G-WO (772809: MTST-697PC)

[0076] Primary: Feasibility and safety, measured by patient adherence to both pharmacologic and exercise interventions.

[0077] Feasibility will be established by the ability to fully accrue 10 participants into each cohort, and have their documented participation in at least 50% of the exercise intervention, and taking at least 80% of the assigned pharmacologic intervention (both spermidine and the cohort-specific therapy). Patients will be given a diary to record their adherence to the study intervention, and this diary will be the method by which feasibility is assessed.

[0078] Safety will be assessed using standard toxicity assessment metrics; assessed and graded according to the Common Toxicity Criteria for Adverse Events (CTCAE), version 5.0. Adverse events at baseline, and at each subsequent visit during administration of the spermidine and additional therapy, will be documented and attribution to one or more study intervention be ascribed by an investigator.

[0079] Secondary: altered systemic immune profiles, assessed via blood markers like NLR and detailed immune cell analyses.

[0080] Neutrophil-to-lymphocyte ratio (NLR) is assessed using standard clinical CBC laboratory measures and is a crude measure of the balance of myelopoiesis that has been validated as a surrogate of pathologic myelopoiesis across numerous disease states, including cancer and obesity (Romano et al. 2023; Marra et al. 2023). It has a strong association with immune fitness and overall prognostic mortality in cancer and viral infections.

[0081] Peripheral blood cellular composition: Changes to the phenotype of peripheral blood myeloid and lymphoid cells will be assessed using mass cytometry and scRNAseqto characterize changes in functional phenotype at the proteomic and transcriptomic level, respectively (LaMarche et al. 2024; Sahaf et al. 2021). Special attention will be placed on the ratio between na’ive, memory and regulatory T and B cells, the clonality of their repertoire, and the ratio of various myeloid compartments, all known to be affected by age and the interventions involved in the study.

[0082] Peripheral blood serum / plasma constitution: Changes in systemic immune milieu will be assessed using Clink, or the Clink Explore HT proteomic platform that characterizes over 5,400 proteins in 2uL of patient blood serum or plasma. Data from the Clink panel will be used to characterize the SASP index (Schafer et al. 2020; Basisty et al. 2020; St Sauver et al. 2022), vascular and muscle senescence markers, and measure blood biomarkers of BBB breakdown and aging, and circulating markers of muscle function. Preliminary Clink data across various cohorts in the HIMC, including healthy controls with a series of paired specimens collected over a span of 8 years, shows an array of inflammatory soluble analytes that change with age both within81580610v1 14241101G-WO (772809: MTST-697PC) patients and / or across patients (example shown in FIG. 2). Leveraging data generated with Olink Explore HT, the inventors will assess individual marker dynamics for treatment-related changes from baseline using longitudinal linear mixed effect models to adjust for age at enrollment, sex, and other potential confounding variables, and to select candidate proteins with significant longitudinal differences dependent on treatment that would be assessed prospectively as immune fitness-related biomarkers in Phase II. Senescence is associated with aging and tissue-repair, but the detection of senescence is challenging due to the rarity, heterogeneity and tissue-specificity of senescence and poorly defined biologic markers. Senescence detection in blood, before and after interventions such as exercise, has the potential to deepen our understanding of the pathobiologic roles senescence plays in promoting healthy aging and identifying novel age- related biomarkers of response to therapy, as well as serve as new therapeutic targets. We can detect senescence in two compartments, the PBMCs, and the Soluble Senescence-Associated Secretome (Exosomes and Soluble Proteins).

[0083] The primary trial endpoint, assessing feasibility and safety, is 60 days following initiation of intervention, however, patients will receive interventions for 180 days, with subsequent follow-up blood draws 3 and 6 months following cessation of intervention. The inventors will assess changes in blood cell and protein markers of pathogenic myelopoiesis, inflammation and SASP / SSAS to provide a longitudinal profile of each participant’s responses to the intervention that will inform whether the rapamycin or lamivudine (or both) arms of the Semi-Finals Trial are used as the basis for the Finals Trial.

[0084] Laboratory Methods Blood samples will be collected at baseline, 30, 60, 90, 180, 270, and 360 days to assess various biomarkers. Immune cell phenotyping will involve mass cytometry, scRNAseq, and proteomic analysis using the Olink or Olink HT platform, providing insights into cellular and protein changes associated with treatment. The HIMC is a leading center for the systemized collection, processing and storage of blood samples from clinical trials, and has the expertise to perform all the assays, subsequent multi-omics analyses, and procedures for anonymizing data for sharing.

[0085] Statistical Methods

[0086] The primary endpoint of safety will be assessed using Common Toxicity Criteria for Adverse Events (CTCAE), version 5.0. The rate of AE in each treatment cohort over the study period and the proportion of patients experiencing AEs attributed to study intervention in each individual cohort will be reported, along with a corresponding two-sided 95% confidence intervals. With 20 patients in the study the inventors will be able to detect any unforeseen event with incidence of at least 9% with 85% probability. The co-primary endpoint of feasibility will81580610v1 15241101G-WO (772809: MTST-697PC) be analyzed by computing the proportion (and associated 95% confidence intervals) of patients deemed compliant with the exercise regimen and the therapy separately for each treatment cohort. In addition, we will record and report the actual level of adherence with the therapeutic dose and exercise. Non-parametric paired tests will be used to compare baseline with 60 day outcomes.

[0087] Secondary endpoints will be analyzed using descriptive statistics. Changes from baseline and the trajectory of NLR will be reported. Given the small sample size, no formal statistical testing will be performed to compare the two groups. However, group differences and their confidence intervals will be reported and considered preliminary data for subsequent studies.

[0088] Interpretation of Results

[0089]

[0068] The results will offer insights into each intervention’s effects on immune cell markers and inflammatory mediators. Potential findings of reduced inflammatory markers or improved immune cell profiles may inform adjustments to intervention dosing or regimens for the Finals Trial. Agents with the most promising preliminary results (i.e. those achieving clinically relevant changes in systemic immune markers) may be selected for further exploration. Ideally, we expect to see in at least one arm of the trial, an improvement in myelopoiesis indicating that RTMs are likely healthy and pathogenic progenitors are not being produced, accompanied by a reduction in circulating markers of inflammation, the SASP / SSAS, BBB breakdown and declining cognitive, immune and muscle function.

[0090] Moving to the Finals Trial

[0091] In the Semi-Finals Trial, the inventors will establish the safety and feasibility of our interventions, complemented by preliminary immune profiling to identify notable dynamic changes in the two treatment cohorts. This analysis may result in prioritizing one intervention for the Finals Trial. The decision on the optimal intervention for the Finals Trial will be done in collaboration with all the investigators across specialties as well as the advisory board. The Finals trial will be a large crossover design trial powered to determine the benefit of intervention over placebo-control, and the cross-over design where patients in the control arm enter a one-year intervention arm after one year of placebo will enable a confirmatory cohort to further test the biologic efficacy of the chosen intervention.

[0092] Safety Monitoring

[0093] Safety will be monitored using the CTCAE version 5.0 for adverse event grading. Weekly calls will assess adherence and manage any side effects, with the option to adjust dosing or temporarily pause interventions if Grade 3 or higher adverse events occur. All adverse events81580610v1 16241101G-WO (772809: MTST-697PC) will be documented in RedCap, ensuring data is secure and reportable. A Data Safety and Monitoring Board (DSMB) of independent internal and external experts has been invited (details in Team section) to oversee the safety and integrity of participants in our trial and that the results are reliable.

[0094] Scaling And Accessibility

[0095] Economic Scalability of Interventions

[0096] The interventions proposed by the inventors are economically scalable, making them feasible for widespread implementation. Exercise regimens utilizing resistance bands are highly cost-effective, accessible, and require minimal infrastructure. Dietary supplementation with spermidine, a naturally occurring polyamine, is relatively inexpensive and has demonstrated significant promise in promoting cellular health. The pharmaceutical interventions being testing, lamivudine and rapamycin, while not classified as "cheap," are substantially faster to synthesize, and considerably more affordable than many complex biologies or cellular therapies that may be in development for tackling physiological decline and disease in aging, such as recombinant versions of blood-borne rejuvenation factors or CAR-T cells targeting senescent cells.

[0097] Spermidine supplementation via dietary supplementation will be encouraged over over-the-counter supplements for purchase. Both lamivudine and rapamycin are long-term generic agents.

[0098] Additionally, their production processes are well-established and more easily standardized, allowing for greater quality control and reduced risks of contamination compared to more complex interventions. This combination of affordability, scalability, and ease of manufacturing positions these interventions as viable candidates for long-term use in diverse populations. Our focus on blood-based interventions scales most efficiently, and serial blood draws will efficiently capture many key immune-system metrics over time.

[0099] While the present disclosure has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present disclosure or appended claims. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed, but that the present disclosure will include all aspects falling within the scope of the appended claims.

[0100] All patents, patent applications, publications, and descriptions mentioned above are herein incorporated by reference in their entirety.81580610v1 17

Claims

241101G-WO (772809: MTST-697PC)What is claimed is:

1. A method of treating or preventing inflammation in a subject, the method comprising: administering a combination therapy comprising: a therapeutically effective amount of spermidine; and a therapeutically effective amount of at least one additional pharmaceutical agent, wherein the additional pharmaceutical agent is configured to amplify immune, muscle, and / or cognitive function in conjunction with an exercise regime.

2. The method of claim 1, wherein the at least one additional pharmaceutical agent is a reverse transcriptase inhibitor, natural fungal antibiotic, or mTOR inhibitor.

3. The method of claim 1 or claim 2, wherein the at least one additional pharmaceutical agent is lamivudine or rapamycin.

4. The method of any one of claims 1 to 3, wherein the combination therapy is administered via a route selected from intratumoral, intraperitoneal, intranodal, peritumoral, subcutaneous, or intravenous delivery.

5. The method of any one of claims 1 to 4, wherein spermidine and the at least one additional pharmaceutical agent are administered simultaneously, concurrently, sequentially, successively, alternatively, or separately.

6. The method of any one of claims 1 to 5, wherein the combination therapy is administered for a sustained period.81580610v1 18241101G-WO (772809: MTST-697PC)7. The method of claim 6, wherein the sustained period is at least about 90 days, alternatively at least about 120 days, alternatively at least about 180 days, or alternatively at least about 210 days.

8. The method of any one of claims 1 to 7, wherein at least about 1 mg / day of the spermidine is administered, alternatively at least about 2 mg / day, alternatively at least about 3 mg / day, alternatively at least about 4 mg / day, alternatively at least about 5 mg / day, alternatively at least about 6 mg / day, alternatively at least about 7 mg / day, alternatively at least about 8 mg / day, alternatively at least about 9 mg / day, or alternatively at least about 10 mg / day.

9. The method of any one of claims 3 to 8, wherein the lamivudine is administered at least once a day or alternatively at least twice a day.

10. The method of any one of claims 3 to 9, wherein about 300 mg or less of the lamivudine is administered.

11. The method of any one of claims 3 to 10, wherein the rapamycin is administered at least once a week.

12. The method of any one of claims 3 to 11, wherein between about 1 mg to about 10 mg of the rapamycin is administered.

13. The method of any one of claims 1 to 12, wherein the method results in an increase in tissue-resident macrophage (RTM) activity.

14. The method of any one of claims 1 to 13, wherein the method results in an increase and / or recovery of immune, muscle, and / or cognitive functions.81580610v1 19241101G-WO (772809: MTST-697PC)15. The method of any one of claims 1 to 14, wherein the subject is a human diagnosed with chronic inflammation, autoimmune disorder, or age-related inflammatory condition.

16. The method of any one of claims 1 to 15, wherein the exercise regime comprises aerobic exercise, resistance training, or a combination thereof.

17. The use of spermidine in combination with at least one additional pharmaceutical agent in conjunction with an exercise regime for the manufacture of a medicament for treating or preventing inflammation in a subject.

18. A pharmaceutical composition comprising: spermidine; at least one additional pharmaceutical agent selected from the group consisting of reverse transcriptase inhibitors, natural fungal antibiotics, and mTOR inhibitors; and a pharmaceutically acceptable carrier, wherein the composition is formulated for administration to a subject undergoing an exercise regime.

19. The pharmaceutical composition of claim 18, wherein the at least one additional pharmaceutical agent is lamivudine or rapamycin.

20. A kit for treating or preventing inflammation, comprising: a first formulation comprising spermidine; a second formulation comprising at least one additional pharmaceutical agent selected from the group consisting of reverse transcriptase inhibitors, natural fungal antibiotics, and mTOR inhibitors; and instructions for administration in conjunction with an exercise regime.81580610v1 20241101G-WO (772809: MTST-697PC)21. The kit of claim 20, wherein the second formulation comprises lamivudine or rapamycin.81580610v1 21

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