Senolytic therapy to modulate the progression of Alzheimer’s Disease (SToMP-AD) – Outcomes from the first clinical trial of senolytic therapy for Alzheimer’s disease

Cellular senescence has been identified as a pathological mechanism linked to tau and amyloid beta (Aβ) accumulation in mouse models of Alzheimer’s disease (AD). Clearance of senescent cells using the senolytic compounds dasatinib (D) and quercetin (Q) reduced neuropathological burden and improved clinically relevant outcomes in the mice. Herein, we conducted a vanguard open-label clinical trial of senolytic therapy for AD with the primary aim of evaluating central nervous system (CNS) penetrance, as well as exploratory data collection relevant to safety, feasibility, and efficacy. Participants with early-stage symptomatic AD were enrolled in an open-label, 12-week pilot study of intermittent orally-delivered D+Q. CNS penetrance was assessed by evaluating drug levels in cerebrospinal fluid (CSF) using high performance liquid chromatography with tandem mass spectrometry. Safety was continuously monitored with adverse event reporting, vitals, and laboratory work. Cognition, neuroimaging, and plasma and CSF biomarkers were assessed at baseline and post-treatment. Five participants (mean age: 76±5 years; 40% female) completed the trial. The treatment increased D and Q levels in the blood of all participants ranging from 12.7 to 73.5 ng/ml for D and 3.29–26.30 ng/ml for Q. D levels were detected in the CSF of four participants ranging from 0.281 to 0.536 ng/ml (t(4)=3.123, p=0.035); Q was not detected. Treatment was well-tolerated with no early discontinuation and six mild to moderate adverse events occurring across the study. Cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment. CNS levels of IL-6 and GFAP increased from baseline to post-treatment (t(4)=3.913, p=008 and t(4)=3.354, p=0.028, respectively) concomitant with decreased levels of several cytokines and chemokines associated with senescence, and a trend toward higher levels of Aβ42 (t(4)=−2.338, p=0.079). Collectively the data indicate the CNS penetrance of D and provide preliminary support for the safety, tolerability, and feasibility of the intervention and suggest that astrocytes and Aβ may be particularly responsive to the treatment. While early results are promising, fully powered, placebo-controlled studies are needed to evaluate the potential of AD modification with the novel approach of targeting cellular senescence.

chromatography with tandem mass spectrometry. Safety was continuously monitored with 48 adverse event reporting, vitals, and laboratory work. Cognition, neuroimaging, and plasma and 49 CSF biomarkers were assessed at baseline and post-treatment. Five participants (mean age: 50 76+5 years; 40% female) completed the trial. The treatment increased D and Q levels in the 51 blood of all participants ranging from 12.7 to 73.5 ng/ml for D and 3.29-26.30 ng/ml for Q. D 52 levels were detected in the CSF of four participants ranging from 0.281 to 0.536 ng/ml 53 (t(4)=3.123, p=0.035); Q was not detected. Treatment was well-tolerated with no early 54 discontinuation and six mild to moderate adverse events occurring across the study. Cognitive

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The study is an open-label single-site pilot study of 12-week intermittent senolytic therapy in 116 older adults with early-stage AD with the primary aim of evaluating the central nervous system 117 penetrance of D and Q (NCT04063124) 28 . Secondary trial aims were to 1) evaluate target 118 engagement of D+Q by examining changes markers associated with cellular senescence and 119 the SASP; 2) assess the safety and tolerability of the intervention; 3) examine pre-to post-120 treatment changes in cognition and functional status; and 4) assess changes in neuroimaging 121 and biofluid markers of AD and related dementias (ADRD). The study was conducted in 122 adherence with the Guideline for Good Clinical Practice and the protocol was approved by the 123 local institutional review board. All participants provided written informed consent with 124 appropriate legal representation for individuals lacking capacity to consent.

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Participants: Eligibility for the study included adults aged 65 years and over with a diagnosis of 126 AD based on the criteria for the National Institute on Aging-Alzheimer's Association 29 and a 127 Global Clinical Dementia Rating (CDR) Scale score of 1 30 . Anticholinesterase inhibitors and/or 128 memantine use were allowed following a minimum of a three-month stabilization period. Full 129 eligibility criteria were applied as described in Gonzales et al 28 .

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Study Design: As previously described 28 , the study protocol included completion of 11 study 131 visits over a period of 20 to 24 weeks ( Figure 1). Following obtainment of written informed 132 consent, study candidates completed an in-person screening visit consisting of a blood draw,             Table 1).

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All five participants who enrolled in the intervention completed the trial with a 100% 249 study drug adherence rate.    (Table 1). There was a 269 statistically significant decrease on HVLT-R Immediate Recall. All other cognitive tests, as well as questionnaires assessing neuropsychiatric symptoms and functional status, did not 271 demonstrate any significant changes.

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Neuroimaging: Paired t-tests of pre-versus post-treatment MRIs revealed no significant 273 differences in total brain volume, gray matter or white matter density, or right or left hippocampal 274 volume, indicative of stable brain morphology over the three-month assessment period (Table   275 2).  was not designed or powered to detect efficacy. However, our preliminary data suggests the 294 potential of baseline to post-treatment changes in markers of cellular senescence and ADRD, which will require further exploration and validation in randomized placebo-controlled trials that 296 are presently underway (NCT04685590).

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A primary challenge to conducting trials for AD and other neurological diseases is the 298 determination of the appropriate drug dosing as assessing pharmacokinetics in the CNS is 299 highly invasive. In our study, we selected the combination of D and Q as they are among the

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suggesting that drugs do not continually need to be present to be effective 6,21 . Intermittent 306 dosing further reduces potential toxicity. Our study design of in-clinic administrations on the first 307 day of each drug cycle enabled us to carefully monitor participant safety and was likely 308 supportive of our 100% study drug adherence rate. In plasma, D has been shown to reach peak 309 concentrations within two hours of administration 44 ; however, the absorption in the CNS is less 310 well established. Following oral ingestion of D in mice, a prior study reported D in brain 311 homogenates using HPLC/MS at concentrations that were 12-to 31-fold lower than in plasma 44 .

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In humans, D has demonstrated efficacy for treating ALL and CML with CNS involvement and 313 responses can be maintained for months to years 44 , suggesting a robust CNS treatment effect.

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we observed a similar range of D concentrations in CSF. However, the detectable levels were 320 more readily observed in our population, occurring in 80% (4/5 individuals) of participants. More consistent CSF concentrations may have been observed in our study of individuals with AD due 322 to the disease's impact on blood brain barrier integrity 46 . Future pharmacokinetic studies will be 323 helpful for informing on the optimal dosing for desired CNS effects. However, our study 324 demonstrated that D penetrated the CNS and prior research in oncology has shown that the 325 medication can demonstrate CNS efficacy at low or even subnanomolar concentrations 44,47 .

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In our study, Q was consistently detected in plasma across participants. However, unlike 327 D, Q was not detectable in CSF within our sample. In animal model research, oral 328 administration of Q has been shown to reduce oxidative stress in the brain 48,49 , suggesting a 329 therapeutic effect in the CNS. In a preclinical study of mice that ingested 21.3 grams of Q per 330 day, Q was detectable in brain homogenates assessed using HPLC-tandem mass 331 spectrometry, plateauing after one-week of administration 49 . In culture, Q has been shown to 332 permeate primary brain microvessel endothelial cells and primary astroglia cells 50 , suggesting 333 blood brain barrier penetrance. However, confirmatory studies in humans are lacking. Q is 334 rapidly metabolized in the human intestinal mucosa and liver and it has low bioavailability 51 , 335 which may explain why it was not detectable in CSF within our study. There are ongoing efforts 336 to improve the CNS permeability with the use of nanoparticles and/or chemical modification 49 .

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Further pharmacokinetic studies of Q in humans are warranted.

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As the first-in-human clinical trial of senolytic therapy for AD, our study also provides 339 important preliminary data on safety, tolerability, and feasibility. Throughout the study, a total of 340 six AEs occurred, of which three emerged after treatment initiation. Two of these AEs were mild

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Consistent with AD trials, our study also acquired cognitive and neuroimaging measures.

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Baseline to post-treatment changes were not observed for our pre-specified cognitive endpoints,

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As a secondary outcome, our study also evaluated key ADRD biomarkers in both 389 plasma and CSF at baseline and post-treatment. There were no significant changes in plasma 390 biomarkers, which was anticipated given the small sample size and short follow-up period. In 391 CSF, we observed a significant increase in GFAP levels from baseline to post-treatment. CSF 392 GFAP levels are presumed to reflect reactive astrogliosis 56 and demonstrate elevations early in 393 the neurodegenerative disease process 57 . In our study, it is unclear if increases in GFAP reflect 394 or an acute response to treatment. Coupled with the elevated CSF IL-6 data, it is tempting to 395 speculate that the concomitant increase in GFAP may reflect apoptosis of senescent astrocytes.

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Supporting evidence for this would require additional blood and CSF collections, weeks or months after the end of treatment, to determine if increased GFAP and IL-6 were transient or 398 sustained responses to senolytic treatment. Our preclinical trial of D+Q reported 35% fewer 399 insoluble NFTs in the treatment arm relative to placebo 6 , which may have reflected a reduction 400 in tangle formation and/or an increase in tau clearance. In our study, we did not observe 401 changes in total tau, p-tau-181, or p-tau-231, however, the study was not powered to assess 402 these outcomes. On-going efforts by our team are focused on a more comprehensive analyses 403 of phospho-tau in CSF and post-mortem human brain to identify which tau species best reflect 404 senescence. The results from the Lumipulse assay, but not from the SIMOA assay showed a

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While our study provides the first report of senolytic treatment in humans with AD, there 409 are several important limitations that must be considered. First, our study was designed to 410 evaluate the CNS penetrance of D and Q. Therefore, it was not powered to assess outcomes 411 related to target engagement, cognition, or disease modification. The short trial duration and 412 lack of a placebo group place further restrictions on interpreting these outcomes. Another 413 limitation is the lack of established senescence and SASP markers related to AD. Prior studies 414 have reported that biomarkers of cellular senescence vary significantly across cell types and 415 inducers 58,59 . Therefore, further work is necessary to identify clinically meaningful markers of 416 cellular senescence in AD across specimen types, and is under investigation by our team. Our 417 exploratory findings provide initial data on changes in protein levels following senolytic treatment 418 in older adults with AD, but validation and replication in well-powered randomized controlled 419 studies are critical for advancing therapeutic discovery in the field.

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In summary, we report findings from the first clinical trial of senolytic therapy for AD. In 421 alignment with our primary study aim, we identified support for the CNS penetrance of D, although 422 Q was not detectable in CSF. In our study, the treatment was well-tolerated with excellent adherence to the study drug regimen. Broader assessments of target engagement and treatment-424 related outcomes were assessed to provide early feasibility data. While our study was not 425 designed to evaluate efficacy, the data suggests the potential of treatment-related changes in 426 markers of cellular senescence and AD pathology. Our vanguard study provides initial data on 427 the safety, tolerability, and feasibility of senolytic therapy for AD. While early results are promising,