Pilot Study: Pharmacological Feasibility and Preliminary Healthspan Effects of Chronic Oral IL-6 Pathway Antagonist Delivery in Mice

Robin Bartolini, PhD and Victor Joo, PhD

1. Overview

Chronic elevation of interleukin-6 (IL-6) is one of the strongest biological predictors of aging- functional decline in humans¹. Large epidemiological studies consistently show that circulating IL-6 correlates with frailty, multimorbidity, cardiovascular risk, cancer progression, and all-cause mortality^2-5. Clinical evidence also indicates that IL-6 is not merely a passive marker of inflammation but an active driver of systemic inflammatory physiology. In multiple disease settings, targeted IL-6 inhibition reverses severe inflammatory syndromes and improves clinical outcomes, demonstrating that modulation of this pathway can directly influence the patient’s ability to recover from inflammatory stress^6-7. Together, these observations identify IL-6 as a central regulatory node in inflammatory balance. The challenge is that the pharmacological tools currently used to modulate IL-6 signaling are poorly suited for long-term applications, as biologic inhibitors such as tocilizumab are engineered to produce near-complete receptor blockade that lasts for weeks. While this is highly effective in autoimmune disease, it also increases the risk of infection and prevents rapid reversibility⁸. Older individuals are already more vulnerable to infection, making continuous immune suppression an unsafe long-term strategy. A reversible, short-acting antagonist is better aligned with the physiological realities of aging.

An orally active IL-6 antagonist represents a fundamentally different therapeutic approach, as oral delivery allows for scalable administration, adjustable exposure, and rapid recovery after withdrawal. However, most experimental work using oral IL-6 antagonists have relied on repeated gavage in inflammatory models^9-10. Oral gavage introduces stress, injury risk, behavioral confounding, and ethical concerns that make it unsuitable for long-term experiments. Delivering the compound in food (chow) would be both more ethical and scientifically appropriate, but the pharmacology of this approach has not been well characterized. We currently lack data for small molecule inhibitors on their stability in food, palatability, circulating concentrations, signaling inhibition, recovery kinetics, and chronic toxicity.

This proposal supports a six-month pilot study to determine whether chow-based oral delivery of an IL-6 antagonist is pharmacologically viable and biologically safe. In addition to calibrating exposure and safety, we include a preliminary exploratory component to assess whether sustained antagonist exposure produces measurable changes in healthspan-relevant functional endpoints. Because the study duration is limited, lifespan effects cannot be rigorously evaluated; however, healthspan-related measures like functional performance and frailty indices are all measurable within this timeframe. This exploratory arm is not designed to prove efficacy but rather to determine whether a signal exists that justifies a longer and more definitive study.

Facilities: All animal work will be conducted through a contracted CRO operating under established institutional ethics approvals and regulatory frameworks. Ex vivo assays, flow cytometry, and histological analyses will be performed at the applicants’ institution, which maintains established infrastructure for advanced cellular and tissue analysis. Funding will support execution of the pilot experiments, and all raw and processed data, along with analysis scripts, will be shared publicly after publication.

Timeline and Data Sharing: The six-month project period includes five months of pilot experimentation and one final month allocated to data analysis, quality control, and preparation of results for reporting and dissemination.

The primary outcome of this pilot is feasibility rather than efficacy: the study is designed to establish the pharmacological groundwork necessary to support a responsible long-duration aging trial.

2. Introduction

Aging is increasingly understood as a chronic inflammatory state. Persistent low-grade inflammation, often described as inflammaging¹¹, amplifies multiple hallmarks of aging including cellular senescence, metabolic dysfunction, immune exhaustion, and tissue remodeling (Figure 1)¹². Among circulating cytokines, IL-6 is one of the most reproducible biomarkers of biological aging and, as mentioned previously, elevated IL-6 predicts disability, frailty, and mortality across independent populations. Inhibition of the IL-6 pathway has shown therapeutic benefit across a wide range of conditions including rheumatoid arthritis¹³ and atherosclerotic disease¹⁴, demonstrating that this cytokine functions as a shared driver of systemic inflammation rather than a disease-specific signal.

The limitation lies not in the target but in the pharmacology of existing drugs. Biologic inhibitors block the receptor for weeks, and this sustained suppression increases infection risk and prevents rapid adjustment of exposure levels. In elderly populations already vulnerable to infection, such pharmacology is unsafe. A reversible antagonist aligns better with aging physiology by allowing temporary inhibition with rapid recovery.

Despite this theoretical advantage, oral IL-6 antagonists have not been calibrated for chronic experimental use. Gavage-based administration dominates the literature but introduces stress artifacts incompatible with long-term aging studies while chow-based delivery is ethically superior but pharmacologically undefined. Our central research question is therefore:

Can an oral IL-6 antagonist be delivered safely and reproducibly through chow for long-term experimental use, and does preliminary exposure produce measurable signals in healthspan-related endpoints?

Hypotheses

H1: Antagonist-formulated chow produces reproducible systemic inhibition of IL-6 signaling.
H2: The compound remains stable and palatable without suppressing voluntary intake.
H3: IL-6 signaling recovers predictably after withdrawal.
H4: Short-term chronic exposure does not produce overt toxicity.
H5 (exploratory): Sustained exposure produces measurable changes in functional healthspan metrics.

Impact Statement: This study addresses a key methodological gap. If successful, it enables safe and ethical long-term studies of cytokine modulation. If not, it prevents moving forward with an approach that would be unreliable or unsafe. In either case, the results will clearly define the practical limits of oral immune modulation. The exploratory functional arm adds early evidence on whether sustained IL-6 inhibition affects overall physiological performance, helping to shape the design of future aging studies.

3. Methods

A- Participants / Samples

The study will use 15 C57BL/6J mice aged 12–14 months at baseline, randomized into three cohorts (n = 5 per group): (1) control (standard chow), (2) low-dose antagonist in chow, and (3) high-dose antagonist in chow. We deliberately use older mice rather than young adults because the IL-6 inhibitor has already been tested extensively in younger animals whereas the key translational uncertainties for our program relate to aging physiology. This pilot therefore focuses on establishing feasibility, safety signals, and pharmacodynamic engagement in the age group most relevant to future aging and frailty studies while remaining compatible within the six-month timeline.

This sample size is appropriate for a calibration-focused pilot: it is designed to estimate variability in exposure, intake, signaling inhibition, recovery dynamics, and age-specific tolerability rather than to deliver definitive efficacy claims. However, because the same mice will also undergo longitudinal phenotyping (weight trajectory, basic functional readouts, and metabolic markers where feasible), the dataset will additionally provide an early indication of whether sustained exposure in older animals produces measurable physiological shifts that should be prioritized in the subsequent long-duration study.

B- Materials and Procedures

Chow formulation and dose rationale

A commercially available IL-6 antagonist will be incorporated into standardized rodent chow at concentrations extrapolated from published oral gavage literature. Prior studies report effective daily gavage dosing in the range of 0.5–5 mg/kg/day with the upper bound representing robust pathway inhibition. We will use this established pharmacological range as the anchor for chow-based conversion. The high-dose formulation corresponds to approximately 40 µg of antagonist per gram of chow, which is estimated to deliver ~5 mg/kg/day assuming a typical intake of 3–4 g of chow per mouse per day. The low-dose formulation will be set at 15 µg/g chow, representing less than half of the high-dose exposure to capture a submaximal signaling window and allow dose-response interpretation.

The compound will be blended into chow by a commercial formulation service using validated mixing protocols to ensure homogeneity. Stability testing will be performed at baseline and after storage under standard animal facility conditions to detect degradation over time. These measurements ensure that animals receive predictable exposure and that any biological effects can be attributed to pharmacology rather than formulation variability.

Intake monitoring and palatability control: Voluntary chow intake is a critical variable in this study and will be assessed daily at the cage level, with body weight being recorded in parallel. Monitoring intake is essential because caloric restriction has been repeatedly shown to extend lifespan and improve healthspan in rodents¹⁵. If antagonist-formulated chow were less palatable, any downstream physiological changes could be falsely attributed to IL-6 inhibition when they are in fact driven by reduced caloric intake. Signs of aversion, selective pellet avoidance, or altered feeding patterns will trigger immediate review of formulation. If intake suppression is detected, the formulation will be adjusted by including the addition of palatability-enhancing homogenates before proceeding.

Baseline normalization and individual reference states: Each mouse will undergo a full baseline characterization before antagonist exposure. This includes body weight, chow intake, circulating leukocyte counts, and IL-6 signaling responsiveness. Importantly, each animal serves as its own signaling reference, allowing pharmacodynamic changes to be interpreted relative to that animal’s physiological baseline rather than group averages.

Pharmacodynamic assessment of IL-6 signaling: Ex vivo IL-6 stimulation will serve as the primary functional readout of pathway activity and target engagement. Because circulating IL-6 levels do not reliably reflect signaling competence, leukocyte responsiveness will be measured by standardized STAT3 phosphorylation assays. Each mouse will have an individual baseline established prior to antagonist exposure, and all subsequent measurements will be expressed relative to that baseline to reduce inter-individual variability.

At defined timepoints during chow exposure (day 3, week 2, months 1–3), peripheral blood will be collected by tail vein sampling. Whole blood will be divided into matched unstimulated and IL-6–stimulated conditions, incubated for 10 minutes at 37 °C, then fixed and stained for lineage markers (CD45,CD11b, Ly6C, CD3, CD19) and intracellular pSTAT3. Signaling capacity will be quantified as the stimulated-minus-unstimulated response within each leukocyte subset (ΔMFI) and normalized to each animal’s baseline.

After antagonist withdrawal, the assay will be repeated every two days to measure recovery. Recovery is defined as return of the IL-6 induced response to at least 70% of baseline. This threshold is used only to estimate recovery timing and to establish conservative exposure limits for future long-duration studies.

Toxicity screening and systemic tolerability: Short-term exposure will be screened for systemic toxicity using several complementary measures. Body weight and behavior will be tracked over time as early indicators of physiological stress. Blood chemistry panels will evaluate liver and kidney function, glucose regulation and inflammatory markers, allowing detection of organ stress that may not be apparent from behavior alone.

At the study endpoint, animals will undergo necropsy with standardized tissue collection. Liver, kidney, spleen, and adipose tissue will be processed for histological analysis and evaluated for inflammation, fibrosis, cellular injury, and structural disruption. These tissue assessments complement circulating biomarkers and enable detection of organ-specific toxicity. Because the study uses aged mice, toxicity screening is especially important. Older animals may process drugs differently and show increased sensitivity to immune modulation. Establishing tolerability in this population helps define safety boundaries for future long-term studies.

Exploratory healthspan experiments: In addition to the pharmacology goals, we will include an exploratory healthspan assessment in the same aged mice to assess whether long-term oral IL-6 antagonist exposure produces early changes in functional outcomes. This part of the study is not meant to prove efficacy but is instead designed to detect whether measurable shifts appear in areas relevant for aging biology, such as frailty, mobility, strength, endurance, and metabolism. These early signals will help determine whether a longer study is justified and will also provide estimates of variability that improve planning for future experiments.

v Frailty will be measured using a standardized mouse frailty index at baseline and then every 2 weeks. Trained evaluators will score each animal using a consistent checklist of common age-related features such as coat condition, posture, tremor, discharge, and body condition. Frailty scores give an overall picture of multi-system decline and are sensitive to both beneficial and harmful physiological changes.

v Locomotor activity and exploratory behavior will be tested using the open field assay. Each mouse will be placed in a standardized arena with automated tracking for about 10 minutes. Total distance traveled, speed, time spent moving versus resting, and time spent in the center versus edges of the arena will be recorded. Environmental conditions will be kept constant, and the arena will be cleaned between animals. These measures help distinguish general slowing or sickness behavior from improved function.

v Neuromuscular strength will be measured using a forelimb grip strength meter. Each mouse will perform several pulls per session (typically 3–5 trials) using a standardized handling method. The main outcome will be the highest peak force achieved, with the average of the top trials recorded as a secondary measure. Grip strength is a well-established marker of muscle health and age-related decline.

v Endurance will be assessed using a motorized treadmill with a protocol suitable for older animals. We will record time or distance to exhaustion, using predefined criteria to determine when an animal has reached its limit. Reasons for stopping will be documented to separate true fatigue from technical interruptions. This test reflects combined musculoskeletal, metabolic, and motivational capacity.

v Body weight will be tracked throughout the study as both a safety measure and a health indicator. Changes in weight can reveal appetite suppression or systemic stress and help interpret functional results. If early findings suggest metabolic changes or if differences in food intake raise concerns that diet rather than the drug is influencing outcomes, we may add optional metabolic measurements such as glucose or lipid markers to clarify interpretation.

The goal of these exploratory experiments is to identify early functional and tissue-level signals (positive, neutral, or adverse) that can guide the next steps. Even if we do not observe clear improvement, the data remains valuable for estimating variability, confirming feasibility, and choosing doses and endpoints. In short, this pilot is meant to reduce uncertainty and make the subsequent long-duration experiment more precise, efficient, and ethically justified.

C- Planned Data Analyses

Our analysis plan is designed to answer three practical questions:
(1) does the drug administered through chow inhibit IL-6 signaling,
(2) does signaling recover predictably after stopping it, and
(3) is the dose safe and interpretable in aged animals?

IL-6 signaling inhibition: The primary pharmacology endpoint is how much the antagonist administered through chow suppresses IL-6 signaling. Pharmacodynamic target engagement will be quantified as the ex vivo IL-6–induced pSTAT3 response (stimulated minus unstimulated) within each leukocyte subset, normalized to each mouse’s pre-treatment baseline. The primary endpoint will be % suppression versus baseline at predetermined timepoints (day 3, week 2, months 1–3), visualized as per-mouse trajectories with group medians and interquartile ranges. Longitudinal changes will be modeled using mixed-effects analyses with fixed effects for dose/ time/ dose x time and a random intercept for mouse to account for repeated sampling, while assay batch/run day/ operator will be included as covariates. Complementary per-mouse summaries, including maximum suppression and suppression AUC over the exposure period, will also be computed to support dose selection and study planning. Group differences in endpoint and AUC summaries will be quantified as effect sizes with bootstrap confidence intervals and supported by permutation tests. The expectation is straightforward: treated groups should show reduced IL-6-induced pSTAT3 compared to baseline, and the high dose should suppress signaling more than the low dose. We are not trying to prove a clinical effect but confirm that the drug engages its target in aged animals and that the inhibition is dose responsive.

Recovery after withdrawal: After stopping antagonist chow, we will track how quickly signaling returns with daily blood sampling from the tail vain. For each mouse, we define “recovery” as the first timepoint when IL-6 responsiveness returns to at least 70% of its original baseline. We will summarize recovery times across animals and compare low vs high dose. Recovery time will be treated as interval-censored between sampling days, summarized across animals (median, variability/percentile range, and proportion not recovered by end of follow-up), and compared between low- and high-dose groups. These estimates will be used to define a realistic recovery kinetics window and conservative exposure limits to guide dosing strategy in subsequent long-duration studies. The goal is not to prove statistical significance but to estimate a realistic recovery kinetics window and understand how variable it is in aged mice. These recovery curves directly inform dosing strategy for the larger experiment.

Intake and palatability: Because caloric restriction alone can extend lifespan and improve health markers, intake monitoring is treated as a core analysis, not a side metric. Chow intake will be measured daily at the cage level, normalized per animal, and analyzed alongside longitudinal body weight to detect palatability-driven differences between formulations. To mitigate confounding by caloric restriction, intake (and/or weight change) will be incorporated as covariates in mixed-effects models of downstream readouts with random effects for cage and mouse to account for clustering and repeated measures. The expectation is that antagonist chow does not meaningfully reduce intake. If however changes occur, we will explicitly account for them when interpreting functional and metabolic outcomes rather than assuming they reflect IL-6 biology.

Toxicity and tolerability: Clinical chemistry markers, body weight trajectories, and histology will be compared across groups to look for safety signals. Safety readouts will be analyzed using effect-size–focused, descriptive comparisons across groups with repeated measures mixed-effects models to emphasize the detection of consistent patterns of physiological stress or organ injury in aged mice in order to define conservative safety boundaries for subsequent long-duration studies. We are primarily looking for recurring patterns across multiple biomarkers and tissues that indicate stress or organ injury rather than powering the study to detect rare adverse events.

Exploratory functional outcomes: Frailty index, locomotion, grip strength, treadmill endurance, and metabolic markers will be collected longitudinally in the same aged mice during oral antagonist exposure. Because this arm is exploratory, the emphasis is on effect size, direction, and consistency across time rather than binary “significant/not significant” decisions. We are asking whether the data show a consistent trend worth following up in a larger study. Absence of an apparent shift will be treated as inconclusive and used primarily to estimate variability and refine dose/endpoint selection for a subsequent adequately powered long-duration study.

Contingent decisions (IF–THEN framework): This pilot is explicitly designed to guide dose selection and minimize unnecessary animal use in the next phase.

IF ex vivo IL-6 stimulation does not produce a clean baseline signal,
THEN stimulation conditions will be optimized once and locked for the rest of the study.
IF antagonist chow reduces intake or causes sustained weight loss,
THEN formulation will be reconsidered and intake will be treated as a confound in all downstream interpretations.
IF the high dose shows adverse effects (toxicity markers, weight loss, or severe behavioral changes),
THEN the larger experiment will use the lower dose.
IF both doses are safe but the low dose fails to inhibit signaling,
THEN the larger experiment will use the high dose.
IF the high dose only inhibits signaling but suppresses intake,
THEN an intermediate dose will be chosen for the larger experiment to separate pharmacology from dietary restriction effects.
IF the low dose produces measurable inhibition without adverse effects,
THEN it will be prioritized for the long-duration study to maximize safety in aged animals.
IF exploratory functional tests show consistent directional improvement,
THEN those endpoints will be prioritized in the next study.

This decision framework ensures that the pilot study settles on a biologically meaningful and safe dose before committing to a large, long-duration experiment. By resolving dosing and tolerability questions early, we avoid repeating full-scale studies and reduce overall animal use.

4. Ethics and Data Management

All animal procedures will be conducted through a contracted CRO operating under existing institutional animal ethics approvals and regulatory compliance frameworks. The CRO maintains active approval for the planned experimental procedures and follows established welfare, monitoring, and humane endpoint standards. Data generated during the study will be stored on secure servers and, after publication, shared publicly with accompanying metadata, protocols, and analysis scripts.

5. Pilot Data

No prior chow-delivery data exist. This study generates foundational feasibility data.

6. Budget

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6. References

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3) Singh, T. & Newman, A. B. Inflammatory markers in population studies of aging. Ageing Res. Rev. 10, 319–329 (2011).

4) Danesh, J. et al. Long-term interleukin-6 levels and subsequent risk of coronary heart disease: two new prospective studies and a systematic review. PLoS Med. 5, e78 (2008).

5) Heikkilä, K., Ebrahim, S. & Lawlor, D. A. Systematic review of the association between circulating interleukin-6 (IL-6) and cancer. Eur. J. Cancer 44, 937–945 (2008).

6) Petit, P. F. et al. Tocilizumab provides dual benefits in treating immune checkpoint inhibitor-associated arthritis and preventing relapse during ICI rechallenge: the TAPIR study. Ann. Oncol. 36, 43–53 (2025).

7) Daoudlarian, D. et al. Tocilizumab and immune signatures for targeted management of cytokine release syndrome in immune checkpoint therapy. Ann. Oncol. 36, 444–459 (2025).

8) Nishimoto, N. et al. Long-term safety and efficacy of tocilizumab in monotherapy in patients with rheumatoid arthritis: evidence from a 5-year extension study. Ann. Rheum. Dis. 68, 1580–1584 (2009).

9) Liu, Q. Q. et al. A GP130-targeting small molecule, LMT-28, reduces LPS-induced bone resorption around implants in diabetic models by inhibiting IL-6/GP130/JAK2/STAT3 signaling. Mediators Inflamm. 2023, 9330439 (2023).

10) Jeong, Y. J. et al. Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model. PLoS One 19, e0302119 (2024).

11) Franceschi, C. et al. Inflamm-aging: an evolutionary perspective on immunosenescence. Ann. N. Y. Acad. Sci. 908, 244–254 (2000).

12) Franceschi, C. et al. Inflammaging: a new immune–metabolic viewpoint for age-related diseases. Nat. Rev. Endocrinol. 14, 576–590 (2018).

13) Emery, P. et al. IL-6 receptor inhibition with tocilizumab improves treatment outcomes in patients with rheumatoid arthritis refractory to anti-tumour necrosis factor biologicals. Ann. Rheum. Dis. 67, 1516–1523 (2008).

14) Kleveland, O. et al. Effect of a single dose of the interleukin-6 receptor antagonist tocilizumab on inflammation and troponin T release in patients with non-ST-elevation myocardial infarction. Eur. Heart J. 37, 2406–2413 (2016).

15) Di Francesco, A. et al. Dietary restriction impacts health and lifespan of genetically diverse mice. Nature 634, 684–692 (2024).

16) López-Otín, C. et al. Hallmarks of aging: An expanding universe. Cell 186, 243–278 (2023)Proposal Template

Pilot Study: Pharmacological Feasibility and Preliminary Healthspan Effects of Chronic Oral IL-6 Pathway Antagonist Delivery in Mice

Pilot Study: Pharmacological Feasibility and Preliminary Healthspan Effects of Chronic Oral IL-6 Pathway Antagonist Delivery in Mice

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