CMS121,a Novel Drug Candidate for the Treatment of Alzheimer’s Disease and Age-Related Dementia

Abstract

Old age is the major risk factor for sporadic Alzheimer’s disease (AD). However, old age-related changes in brain physiology have generally not been taken into consideration in developing drug candidates for the treatment of AD. This is at least partly because the role of these age-related processes in the development and progression of AD are still not well understood. Nevertheless, we and others have described an association between the oxytosis/ferroptosis non-apoptotic regulated cell death pathway and aging. Based on this association, we incorporated protection against this pathway as part of a cell-based phenotypic screening approach to identify novel drug candidates for the treatment of AD. Using this approach, we identified the fisetin derivative CMS121 as a potent neuroprotective molecule that is able to maintain cognitive function in multiple pre-clinical models of AD. Furthermore, we identified a key target of CMS121 as fatty acid synthase, a protein which had not been previously considered in the context of AD. Herein, we provide a comprehensive description of the development of CMS121, its preclinical activities, and the results of the toxicology testing that led to its IND approval.

Keywords

Acetyl CoA Alzheimer’s disease AMP-activated kinase fatty acid synthase mitochondria oxytosis/ferroptosis

INTRODUCTION

The greatest known risk factor for Alzheimer’s disease (AD) is increasing age [1, 2]. About 1 in 9 people age 65 and older has AD, and the prevalence is 1 in 3 in people over the age of 85 [2]. These data suggest that old age offers excellent targets for treating AD. However, the degenerative processes associated with aging are still poorly understood as are their roles in AD. Aging is a complex process characterized by damage to a number of different molecular systems, including DNA, RNA, epigenome, proteins, lipids, and metabolites. Moreover, it is the accumulation of damage in the form of various molecular species that may define aging through their combinatorial effects [3]. At a functional level, this damage can lead to altered intercellular communication (inflammation), genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, and stem cell exhaustion [4]. The idea is that some of these changes might translate into a failure of specific processes in the brain thus contributing to AD.

Over the last few years, we [5, 6] and others [7, 8] have identified an association between the oxytosis/ferroptosis non-apoptotic regulated cell death pathway and aging. We believe that this pathway offers the potential for developing an entirely new class of therapeutics for AD. Oxytosis is a form of non-apoptotic regulated cell death characterized by glutathione (GSH) depletion and dysregulated production of free radicals from mitochondria that results in lethal lipid peroxidation [6 , 9– 11]. The major steps that define oxytosis were first described in 2001 by Tan et al. [10]. Since then, other laboratories have provided additional key insights into this pathway [12 –15], which was redescribed in fibroblasts in 2012 as an iron-dependent, non-apoptotic form of cell death named ferroptosis [16]. However, since the mechanistic steps are essentially the same, we will be calling it oxytosis/ferroptosis [6, 11].

Oxytosis/ferroptosis (Fig. 1) can be triggered by inhibiting cystine uptake via system x_c^– with either glutamate or erastin, which subsequently depletes intracellular GSH [6 , 9– 11]. GSH depletion leads to inhibition of the GSH-dependent enzyme GSH peroxidase 4 (GPX4), activation of lipoxygenases (LOXs), and dysfunctional mitochondria. GPX4 can also be directly inhibited by the chemical RSL3. Reactive oxygen species and lipid peroxides are generated, which potentiate intracellular calcium (Ca^2 +) influx through store-operated Ca^2 + channels and cell death. Iron plays an important role in oxytosis/ferroptosis as iron chelators prevent oxytosis/ferroptosis-mediated cell death [6, 16]. Iron can generate reactive oxygen species via the Fenton reaction and also promote the activation of the non-heme iron-containing LOX enzymes [13 , 18]. Free polyunsaturated fatty acids (PUFAs) as well as lipids containing esterified PUFAs are particularly susceptible to peroxidation and are required for the execution of oxytosis/ferroptosis [6 , 20]. Oxidized PUFAs are not intrinsically toxic to cells, but the excessive accumulation of oxidized PUFA-containing lipids within cell membranes appears to drive oxytosis/ferroptosis [21]. As a result, the integrity, fluidity, permeability, and function of the biomembranes become compromised [22 –24]. In addition, lipid peroxides can generate a variety of short-chain reactive carbonyl species that can further damage biomolecules such as proteins and DNA [22 –24]. Importantly, all of these processes are also observed in the central nervous system (CNS) with aging and are exacerbated in AD [6 , 25– 28].

Fig. 1

The oxytosis/ferroptosis regulated cell death pathway.

Although one of the hallmarks of AD is the accumulation of amyloid-β (Aβ) in extracellular plaques, Aβ also accumulates inside neurons in AD brain [29 –31]. Recently, we found that intraneuronal accumulation of Aβ induces oxytosis/ferroptosis which is associated with the expression of pro-inflammatory mediators and metabolic alterations characterized by deficits in glucose metabolism and mitochondrial bioenergetics [32].

Despite all this evidence, the therapeutic value of oxytosis/ferroptosis for treating AD remains largely unexplored. Importantly, while cultured cells die rapidly following the induction of oxytosis/ferroptosis, it is likely that with aging and in AD patients, oxytosis/ferroptosis takes place over an extended time period thereby leading to a slow degeneration of basic neuronal functions prior to cell death [6]. This large window of opportunity would have huge implications for the development of therapies.

In order to identify small molecules that can prevent oxytosis/ferroptosis, we used phenotypic screening assays where we exposed cultured nerve cells to inducers of oxytosis/ferroptosis, such as cystine deprivation, glutamate, erastin, and RSL3 [6]. Using this approach, we have identified inhibitors of oxytosis/ferroptosis from a variety of sources, including libraries of natural compounds and natural extracts [33, 34]. Once protective compounds were singled out, they were further improved via medicinal chemistry in order to generate compounds with lower EC₅₀ s against oxytosis/ferroptosis (more potent), that are safe (mutagenicity, chromosomal stability, and cardiac safety), present optimal metabolism and pharmacokinetics (DMPK), and have good brain penetrance. As a guideline, successful CNS drugs should have MW≤400, CLogP≤5, tPSA≤90, HBD≤3 and HBA≤7 for best penetration of the blood-brain barrier [35 –37]. The goal is to improve the drug-like properties of a compound prior to testing it in animal models of AD. Using this combination of phenotypic screening against oxytosis/ferroptosis and medicinal chemistry we have generated several very neuroprotective AD drug candidates including the compound CMS121 that is the topic of this manuscript. Specifically, we will describe the development of CMS121 with a focus on the oxytosis/ferroptosis pathway, the primary phenotypic screening model used to identify CMS121. We will next discuss the results of its testing in animal models of AD and age-related dementia and finally the studies conducted to support Investigational New Drug (IND) approval by the FDA.

RESULTS

Development of CMS121

CMS121 is a derivative of the flavonol fisetin [38]. Originally, we identified the flavonoid fisetin (3,7,3’,4’ tetrahydroxyflavone) as a novel neuroprotective compound using our cell-based phenotypic screening assays for protection against oxytosis/ferroptosis [39]. Importantly, fisetin also prevented learning and memory deficits in APPswe/PS1dE9 (huAPP/PS1) double transgenic AD mice [40] and rapidly aging SAMP8 mice [41]. However, fisetin’s relatively high EC₅₀ in cell-based assays (2– 5μM) as well as its low lipophilicity (cLogP 1.24), high tPSA (107Å) and high number of hydrogen bond donors (HBD = 5) suggested that there was room for medicinal chemical improvement. Using structure-activity relationship-driven iterative chemistry, we synthesized more than 160 derivatives of fisetin based on several different chemical scaffolds. We used a multi-tiered approach to screening that allowed us to identify fisetin derivatives with significantly enhanced neuroprotective activity in two in vitro neuroprotection assays (protection against oxytosis/ferroptosis and protection against in vitro ischemia) while at the same time retaining other key actions of fisetin including maintenance of GSH levels under stress. While all of the fisetin derivatives had improved medicinal chemical properties more consistent with those of known CNS drugs, 20 had greatly enhanced neuroprotective activity. Pharmacokinetic studies on the six most promising derivatives showed that several had peak brain levels following a single oral dose of 20 mg/kg that exceeded their average effective concentration at 50% (EC₅₀) in the in vitro neuroprotection assays as well as good oral bioavailability. In particular, CMS121 was not only highly effective in the two neuroprotection assays but also had a peak brain level of 700 nM and oral bioavailability of 59%, both of which were better than those for the other five fisetin derivatives tested. Importantly, the peak brain level is well above the EC₅₀ s for neuroprotection in the cell culture-based neuroprotection assays. Based on these results, we selected CMS121 for further study in preclinical mouse models of AD.

Testing of CMS121 in mouse models of familial AD and age-related dementia

There are several different types of pre-clinical models used to study the possible beneficial effects of new drug candidates in AD including transgenic mice and rapidly aging mice. Most of the numerous transgenic models of AD are based on mutations associated with the rare genetic form of the disease (familial AD or FAD) (https://www.alzforum.org). These models develop different degrees of cognitive impairment, plaques and tangles, synaptic loss, gliosis, and nerve cell death depending on the type and number of mutations. For our studies, we used the APPswe/PS1ΔE9 (line 85) transgenic mice which express a mouse/human chimeric APPswe and a mutant human presenilin 1 (PS1ΔE9) on a C57BL/6J genetic background. They develop progressive synaptic and neuronal loss, neuroinflammation and cognitive dysfunction (https://www.alzforum.org) beginning at 4– 6 months of age.

For the study with CMS121 in the APPswe/PS1ΔE9 transgenic mouse model of AD, the mice were aged to 9 months, a time when they already show cognitive impairment [40], and then treated for 3 months with CMS121 [42]. CMS121 was incorporated into the mouse food at a dose of 400 ppm which is approximately 34 mg/kg/day based on the average food intake of the mice and their weight. CMS121 treatment reduced lipid peroxidation, a marker of oxytosis/ferroptosis, and neuroinflammation in the brains of the mice while alleviating cognitive decline as assessed in multiple behavioral tests [42]. These tests included the elevated plus maze for disinhibition, the two-day water maze for spatial memory and the fear conditioning test for contextual memory. Both of the latter two cognitive tests are dependent on the hippocampus. Lipid peroxides can be formed non-enzymatically and enzymatically [43]. The most important group of enzymes catalyzing the di-oxygenation of lipids— mainly of PUFAs— to form lipid hydroperoxides, are LOXs. In AD, 15LOX2 has been implicated in inflammation and its levels are increased in human AD patients [44]. CMS121 decreased the levels of PUFAs in the brains of the AD mice (Fig. 2) and was also found to inhibit the activity of multiple LOXs (Table 1). Thus, CMS121 can act at multiple steps to prevent the accumulation of a critical driver of the oxytosis/ferroptosis pathway.

Fig. 2

CMS121 decreases free fatty acid levels in multiple AD-relevant models. Free fatty acid levels (FFA) were determined by metabolomic analysis as described [42, 49].

Table 1

Percent Inhibition of LOX and COX Activity by CMS121 (2μM)

Enzyme	5-LOX	12-LOX	15-LOX	COX1	COX2
CMS121	98	67	81	0	0

Although AD drug discovery has largely focused on FAD models, this form of the disease accounts for only a few percent of the total cases and may be quite distinct from the much more prevalent, old age-associated, sporadic form of AD. Importantly, while many therapies directed against the amyloid pathway are effective in FAD transgenic mice, their success in the clinic, especially in terms of reducing the clinical hallmarks of disease progression such as loss of cognitive function, has been modest. Thus, animal models that incorporate aging into disease development may, in the long-term, provide an important complement to the recently approved anti-amyloid therapies and, possibly in combination with those, might offer a better approach to slowing the clinical hallmarks of disease progression.

One mouse model of aging that also develops characteristics of AD is the senescence-accelerated prone 8 (SAMP8) mouse that was developed in Japan by selective breeding of a rapidly aging phenotype [45 –47]. These mice exhibit a progressive, age-associated decline in brain function similar to human AD patients. As they age, SAMP8 mice develop an early deterioration in learning and memory as well as a number of pathophysiological alterations in the brain including increased oxidative stress, inflammation, vascular impairment, gliosis, Aβ accumulation, and tau hyperphosphorylation. Using an integrative multiomics approach, we previously identified a number of behavioral and physiological changes that are altered with aging in these mice [48].

We tested CMS121 in this model using a similar paradigm to that used with the APPswe/PS1ΔE9 transgenic mice wherein the mice were aged to 9 months, a time when previous studies [48] showed significant cognitive impairment, and then treated with CMS121 in the diet at 400 ppm for 4 months [49]. Three groups of SAMP8 mice were compared [49]. Two groups of mice were aged to 9 months and then fed with control or CMS121 diet for an additional 4 months (to a final age of 13 months) while a third group of 9-month-old mice was used as a control group (9 months). We found that 400 ppm of CMS121 in the diet ( 34 mg/kg/day) of CMS121 reduced metabolic and gene transcription markers of aging in SAMP8 brains while preserving cognition when administered at advanced stages of the aging process, demonstrating that it delays molecular aspects of aging and cognitive loss in this model [49]. We also used two different behavioral tests with these mice to monitor cognitive function: the Barnes maze which measures spatial memory and is dependent on the hippocampus and the elevated plus maze which measures disinhibition which is dependent on the cortex and in AD patients presents as a lack of restraint and. In the 13-month-old mice, CMS121 restored these behaviors to those seen in the 9-month-old control mice [49].

The 13-month-old mice showed a number of proteomic changes in the brain that were prevented by CMS121 and that may play a role in its ability to maintain cognitive function. As shown in Fig. 3, there was a significant decrease in the phosphorylation of acetyl CoA carboxylase 1 (ACC1) in 13-month-old SAMP8 mice compared with 9-month-old SAMP8 mice and this was largely prevented by CMS121. ACC1 is a substrate of AMP-activated protein kinase (AMPK) and is responsible for the synthesis of malonyl CoA, the first step in fatty acid synthesis [50]. Phosphorylation of ACC1 results in inhibition of its activity. In addition, we found that CMS121 prevented the age-dependent decrease in several proteins related to mitochondrial function, inflammation and/or synaptic function including Parkin, SAP102, and pro-BDNF. Parkin regulates mitochondrial dynamics [51] and decreases in Parkin levels or function contribute to neuroinflammation [52, 53] and are associated with AD progression in humans [54]. The post-synaptic scaffold protein SAP102 has also been shown to be decreased in human AD brains with the level of reduction directly correlating with disease severity [55]. Both BDNF and its precursor pro-BDNF play roles in synaptic plasticity [56]. There is growing evidence that one of the mechanisms by which inflammation affects brain function is by reducing BDNF expression [56]. Thus, CMS121 positively modulates several proteins that are negatively impacted by neuroinflammation and thereby may contribute to its beneficial effects on cognitive function in the SAMP8 model of age-related dementia. In addition, in this same model we showed that CMS121 also reduces the age-dependent decreases in the brain in the levels of several proteins related to proteostasis including HSP27, HSP70, HSP90, and p62 [57]. Maintenance of many of these proteins has been associated with better outcomes in mouse models of AD [58, 59].

Fig. 3

Western blotting of hippocampal extracts from untreated and CMS121-treated SAMP8 mice. Equal amounts of hippocampal extracts were analyzed for the indicated proteins by SDS-PAGE and western blotting. Normalization was to the total protein (phospho-ACC1) or actin as appropriate. Results are the average for extracts from 5 mice for each condition (mean±SD). (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Tukey’s post-hoc test).

The effects of CMS121 on ACC1 phosphorylation suggested that it might also cause activation of AMPK and this could contribute to its beneficial effects in the context of both oxytosis/ferroptosis [60] and inflammation. AMPK is a key regulator of energy balance and its activation is associated with healthy aging [5]. Dysregulation of AMPK has been implicated in AD as well as other neurodegenerative diseases [61]. Thus, we looked at the effects of CMS121 on AMPK activation in primary mouse cortical neurons by examining both its phosphorylation and the phosphorylation of two of its substrates, ACC1 and Unc-51-like autophagy activating kinase (ULK1) (Fig. 4). We also looked at the effects of CMS121 on acetyl CoA levels since inhibition of ACC1 will result in increases in acetyl CoA levels because acetyl CoA is a substrate of ACC1. Consistent with its in vivo effects on ACC1 phosphorylation, CMS121 activated AMPK in the primary neurons as indicated by an increase in its phosphorylation, inhibited ACC1 as indicated by an increase in its phosphorylation, activated ULK1 as indicated by an increase in its phosphorylation, and increased acetyl CoA levels. Further studies are underway to determine the mechanism underlying the activation of AMPK by CMS121.

Fig. 4

Analysis of primary neurons treated with CMS121. Primary cortical neurons were treated for 24 h with 1μM CMS121 and then analyzed for acetyl CoA using a commercial kit. Results are the average of 3 independent experiments (mean±SD). For the other experiments, primary cortical neurons were treated for 30 min or 4 h with 1μM CMS121 and then the levels of the indicated proteins were analyzed by SDS-PAGE and western blotting. Samples were run in triplicate. Similar results were obtained in 3 independent experiments. Results are the average of the three experiments (mean±SD). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 one-way ANOVA, Tukey’s post-hoc test).

In summary, CMS121 robustly maintained cognitive function in mouse models of age-related dementia and familial AD even when administered after the animals had developed cognitive impairments. In the brains of the APP/PS1 transgenic mice CMS121 modulated lipid metabolism and reduced both lipid peroxidation and neuroinflammation. In the accelerated aging SAMP8 mice CMS121 improved metabolic and transcriptional markers of aging and prevented the age-dependent changes in expression in proteins associated with mitochondrial and synaptic function as well as neuroinflammation. Many of these effects are likely related to the activation of the AMPK pathway by CMS121.

Characterization of the target of CMS121

To identify the target through which CMS121 mediates its effects, we performed drug affinity responsive target stability (DARTS) analysis on HT22 and HeLa cells [42]. Mass spectrometry identified fatty acid synthase (FASN) as a top hit. In fact, FASN was the only protein present among the top 3 targets in both cell lines. We then measured the enzymatic activity of FASN in HT22 cell lysates and found a CMS121-mediated, dose-dependent partial inhibition of FASN activity [42] similar to what was seen with the known FASN inhibitor triclosan. Importantly, knockdown of FASN in cells phenocopied the protective effect of CMS121 against oxytosis/ferroptosis as well as its anti-inflammatory effects in LPS-stimulated microglia [42] further supporting the idea that FASN is a key target of CMS121. Exactly how inhibition of FASN leads to activation of AMPK is currently under active investigation in the laboratory.

There are a number of age-related physiological changes that have been shown to contribute to the development of AD. Among these are type 2 diabetes mellitus (T2DM) and age-related hearing impairment (ARHI). Thus, we asked if CMS121 might also reduce these risk factors and thereby help to prevent the development and progression of AD.

CMS121 in type 2 diabetes mellitus

T2DM is increasing globally with the World Health Organization predicting 350 million cases by 2030 (http://www.who.int/diabetes/publications/en/screening_mnc03.pdf). There is a strong connection between T2DM and AD with some proposing that T2DM may be an important contributing factor to the development of AD [62, 63]. Furthermore, recent studies have shown numerous associations between AD and metabolic disease. For example, both insulin resistance and impaired glucose metabolism have important roles in the pathophysiology of dementia, cognitive decline and AD [64]. Mitochondrial dysfunction, a problem seen in both AD and T2DM, could lead to energy shortages in the hippocampus, potentially helping to explain the memory impairment commonly found in AD [65]. The anti-inflammatory and other protective effects of CMS121 in animal models of aging [49, 57] and AD [42] led us to test for its beneficial effects in db/db mice, a model of T2DM [66, 67]. The characteristics of this model include, beyond the diabetes phenotype, increased lipid levels and hepatic inflammation [68 –71] and chronic kidney disease (CKD) [72]. We investigated whether a diet containing CMS121 at 400 ppm for 6 months could improve glucose metabolism, lipid status, liver inflammation, and CKD. Importantly, both liver inflammation [73, 74] and CKD [75 –78] increase with age and have been associated with cognitive dysfunction and a risk of developing AD.

Although the db/db mice showed no alterations in overall metabolic activity with the CMS121 diet, their body weight decreased by 5% and glucose and lipid metabolism improved significantly [79]. Furthermore, the CMS121 diet produced a significant improvement in the hepatic inflammatory status, as observed by lower levels of active NF-κB in the nucleus and decreased levels of IL-18 and CRP as well as decreased caspase 3 activity [79].

Diabetic nephropathy is a serious microvascular complication of diabetes that progresses from low-grade renal inflammation to renal fibrosis, sclerosis and end-stage renal disease [80]. Indicators of renal decline include functional measures such as glomerular filtration rate, as well as markers of glomerular integrity (albumin and clusterin) and of tubule damage (KIM1, NGAL) in the urine [81, 82]. The CMS121 diet greatly reduced urine albumin and clusterin levels in the db/db mice suggesting improved glomerular filtration while the decrease in urine NGAL levels indicated strong protection of the distal tubules by CMS121 [79]. The CMS121 diet also partially protected the db/db mice from kidney fibrosis [79]. Further evidence, including the results of urine metabolomic analysis, the normalization of renal mitochondrial protein levels and the reduction of renal NOX4 expression, MDA levels and urinary fumarate, strongly suggested that CMS121 can improve kidney function in the context of T2DM [79]. Since, as noted above, both liver and kidney dysfunction have been associated with increased risks for the development of AD, these results suggest additional, peripheral targets through which CMS121 might have beneficial effects in the context of AD. The idea that the modulation of peripheral targets by AD drug candidates could provide additional benefits in the context of AD is something that deserves more attention as these targets are thought to have a significant impact on AD development and progression.

CMS121 and age-related hearing impairment

Another factor that has been found to contribute to cognitive deficits and the development of AD is ARHI [83, 84]. The SAMP8 mice develop premature ARHI and are a fast and robust model for studying this disorder [85, 86]. In a recent study [87], we tested the impact of CMS121 at 200 ppm in the diet ( 17 mg/kg/day) on premature ARHI in the SAMP8 mice beginning at 4 weeks of age. By 13 weeks of age, auditory brainstem response (ABR) thresholds, particularly in the mid-range region, were significantly higher as compared to 4-week-old mice and these increases were significantly decreased in the mice fed CMS121 diet where the ABR thresholds at all frequencies tested remained relatively constant throughout the course of the study. Consistent with the maintenance of mid-range ABR thresholds in the CMS121-treated mice, the number of paired ribbon receptors were also conserved as compared to the untreated 13-week-old mice.

In summary, not only does CMS121 maintain cognitive function in mouse models of familial AD and age-related dementia but it can also reduce or prevent a number of physiological changes that are associated with an increased risk of developing AD including T2DM, kidney dysfunction, liver inflammation, and ARHI. Thus, these data suggest that CMS121 is able to act both in the CNS as well as in the periphery to reduce the factors that contribute to the development of AD and thus might serve as a new paradigm for AD treatment. The potential role of FASN and AMPK in the development and progression of these AD risk factors warrants further investigation.

Investigational new drug-enabling studies with CMS121

In the US, the testing of a new drug candidate, such as CMS121, in human patients requires prior IND approval from the Food and Drug Administration (FDA). The overall goal of the studies required for IND approval is to establish that the drug candidate will not expose human subjects to unreasonable risks when used in short term, early stage clinical trials. Among the studies required are safety pharmacology (effects on respiratory, cardiac and brain function) and studies on the toxicological effects of the drug candidate in animals and in vitro (e.g., genotoxicity, mutagenic potential). In addition, a key goal of the IND studies is to establish both the maximum tolerated dose which is the highest dose that does not cause unacceptable side effects and the no observed adverse effects limit (NOAEL) dose which is the highest dose that does not cause any side effects for the drug candidate. This information is used to determine the starting dose and the dose escalations for the first-in-human study (Phase 1 clinical trial) and is determined from repeated-dose toxicity studies which need to be done in two species, one rodent (usually rats) and one non-rodent (often dog). These studies must use the same route and schedule of administration as is planned for the clinical use of the drug candidate in humans. The results of these studies with CMS121 are summarized below (Table 2).

Table 2

	Safety pharmacology
Central nervous system	No changes in functional observation battery parameters after a single oral dose of 100, 300, and 1000 mg/kg in rats. No microscopic changes in the brains of the rats or dogs in repeat dose.
Cardiovascular system	No effects on blood pressure, heart rate, or body temperature after a single oral dose of 10, 30, or 100 mg/kg in dogs. No ECG changes (including QT interval) at doses up to 100 mg/kg/day in the 28-day repeat dose in dogs.
Respiratory system	No changes in respiratory parameters (flow function, respiration rate, tidal volume, and minute volume) after a single oral dose of 10, 30, or 100 mg/kg in dogs. No clinical signs of respiratory changes and no microscopic changes in the lungs of rats and dogs in the repeat dose.
	Toxicology
In vitro genotoxicity in a non-mammalian system	No increases in revertant colony formation in a bacterial reverse mutation assay using Salmonella typhimurium and Escherichia coli.
In vitro genotoxicity in a mammalian cell system	No dose-related increases in human peripheral blood lymphocytes with structural chromosomal aberrations. No obvious increases in cells with polyploidy or endoreduplication.
14-day dose-range finding study in rats	Well tolerated at doses up to 1000 mg/kg/day. No unscheduled deaths. Clinical signs limited to reddish feces in females given 1000 mg/kg/day.
	Slight decreases in body weight gain were observed in both male and female rats. Upon necropsy, no gross findings or microscopic changes in the tissues examined. No treatment related changes in clinical pathology.
28-day toxicology study in rats	Well tolerated at doses up to 1000 mg/kg/day. No adverse effects on clinical observations, ophthalmology, clinical pathology (clinical chemistry, hematology, coagulation, urinalysis), macroscopic observations, or microscopic exams. Decrease in weight gain in the male rats, particularly at the higher doses.
14-day dose-range finding study in dogs	Well tolerated at doses up to 300 mg/kg/day. No unscheduled deaths or morbidity. Clinical signs included emesis, thinness, decreased activity, reddish feces, and loose feces at 300 mg/kg/day. Reddish feces noted in the 100 mg/kg/day group. Decreased body weight gain at 300 mg/kg/day. Upon necropsy, gross findings were limited to the 300 mg/kg/day group and included decreases in spleen and thymus size that corresponded to decreased organ weights. Ileal intussusception was noted at 300 mg/kg/day. Microscopic changes included hepatocellular vacuolation, hemorrhage in the ileal mucosa, renal cortical tubular degeneration/regeneration, and lymphoid depletion in the spleen and thymus.
28-day toxicology study in dogs	The high dose of 100 mg/kg/day was reduced to 75 mg/kg/day due to decreased body weights and decreased food consumption. Well tolerated at doses up to 100/75 mg/kg/day No early deaths. No effects on body temperature, blood pressure, ECG, ophthalmology, hematology, coagulation, or urinalysis parameters. Test article-related effects at 100/75 mg/kg/day, including decreases in body weight and food consumption, emesis, watery and/or loose feces along with microscopic alterations in the kidney and increases in plasma levels of creatinine and blood urea nitrogen in one animal. Microscopic kidney findings at 75 mg/kg/day considered to be adverse. At lower doses, the adverse effects were not observed.

Metabolism

Metabolite identification was performed using mouse, rat, dog, monkey, and human hepatocytes. The bulk of the metabolites from all species were glucuronide conjugates. The rat and dog metabolites provided the greatest coverage of the potential human metabolites and so they were chosen as the toxicology species.

Safety pharmacology

CNS. To evaluate potential effects of CMS121 on the CNS, a functional observation battery was conducted in rats (8/sex/group) after a single oral dose of CMS121 at dose levels of 100, 300, and 1000 mg/kg. There were no changes in any of the functional observation battery parameters measured in this study. In addition, in the repeat dose toxicology studies in rats and dogs, there were no microscopic changes in the brains of the rats or dogs.

Cardiovascular system. The cardiovascular effects of CMS121 were examined in telemetered dogs treated with a single oral dose of CMS121 at 10, 30 or 100 mg/kg. Eight dogs implanted with telemetry transmitters were assigned to four groups with 1/sex/group using a Latin-square design. Blood pressure, heart rate, body temperature, and electrocardiography (ECG) were examined over a 24-h period. At doses of CMS121 up to 100 mg/kg there were no effects on blood pressure, heart rate, or body temperature. Furthermore, in the 28-day repeat dose toxicology study in dogs, there were no ECG parameter changes at doses up to 100 mg/kg/day, including no significant increases in the corrected QT interval, the time interval from when the cardiac ventricles begin to contract to when they finish relaxing. Drug-induced changes in the QT interval are a concern for the FDA as they can lead to cardiac problems.

Respiratory system. The effects of CMS121 on respiratory parameters were incorporated into the single dose cardiovascular study in dogs. CMS121 was administered to dogs at a single oral dose of 10, 30, or 100 mg/kg and respiratory parameters were measured 1 h prior to each dosing, as well as 40 min, 70 min, and 24-h post-dose. Respiratory parameters included flow function, respiration rate, tidal volume, and minute volume. There were no changes in respiratory parameters at any CMS121 dose level tested. In addition, in the repeat dose toxicology studies in rats and in dogs, there were no clinical signs of respiratory changes and there were no microscopic changes in the lungs of the CMS121-treated animals.

Toxicology

In vitro genotoxicity in a non-mammalian system. The in vitro mutagenic potential of CMS121 was evaluated in a bacterial reverse mutation assay using Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2 uvrA) with and without mammalian microsomal enzymes (Aroclor 1254-induced rat liver S9) as the metabolic activation system [88]. Based on the results of the initial dose-range-finding assay, the concentrations tested in the definitive mutagenicity assay ranged from 20 to 750μg/plate for the tester strain TA98, 10 to 300μg/plate for the tester strains TA100, TA1535, and TA1537, and 100 to 2000μg/plate for the tester strain WP2 uvrA. The highest concentrations of CMS121 tested in the assays were selected based on bacterial viability or precipitation of the compound. There were no CMS121-induced increases in revertant colony formation at any of the concentrations tested either in the presence or absence of the S9 mix. In contrast, all positive controls (2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, and N-methyl-N-nitro-N-nitrosoguanidine) induced the expected number of revertant colonies. Based on the results of this study CMS121 was considered negative in the bacterial mutation assay.

In vitro genotoxicity in a mammalian cell system. The in vitro clastogenic potential of CMS121 was evaluated in human peripheral blood lymphocytes with and without mammalian microsomal enzymes (Aroclor 1254-induced rat liver S9) as the metabolic activation system [89, 90]. Concentrations for the definitive chromosomal aberration assay were based on an initial range-finding cytotoxicity assay. For the definitive study, duplicate cultures of human peripheral blood lymphocytes were incubated with CMS121 at concentrations of 5, 15, 35, and 60μg/ml for 3 h and 4, 8, and 20μg/ml for 24 h for the non-S9 activated treatment series and at concentrations of 5, 15, and 35μg/ml for the S9-activated treatment series for 3 h. There were no dose-related increases in cells with structural chromosomal aberrations. In contrast, the positive control compounds (i.e., cyclophosphamide monohydrate and mitomycin C) induced significant increases in the percentage of cells with structural aberrations. No obvious increases in cells with polyploidy or endoreduplication were observed in the cultures analyzed when compared with the concurrent solvent/vehicle control. Based on these results, CMS121 was considered to be negative for the induction of structural and numerical chromosomal aberrations in human peripheral blood lymphocytes with or without metabolic activation using rat liver S9.

28-day non-clinical toxicity studies in rats and dogs

The non-human mammalian toxicity of CMS121 was assessed in a series of nonclinical toxicology studies in alignment with ICH M3(R2) guidelines (http://www.fda.gov/regulatory-information/search-fda-guidance-documents/m3r2-nonclinical-safety-studies-conduct-human-clinical-trials-and-marketing-authorization). The Sprague-Dawley rat and Beagle dog were chosen as the toxicology species based on their suitable pharmacokinetic profiles and their in vitro metabolite profiles being similar to humans. The oral route of exposure was selected for these studies as this is the intended route of exposure in humans. The maleate salt form of CMS121 (CMS121-M) was used in the toxicology studies and administered by oral gavage as a suspension using 0.5% methylcellulose as the vehicle.

In the 14-day dose-range finding studies in rats (100, 300, and 1000 mg/kg/day; 3/sex/group), CMS121 was well tolerated at doses up to 1000 mg/kg/day. There were no unscheduled deaths and clinical signs were limited to reddish feces in females given 1000 mg/kg/day. Slight decreases in body weight gain were observed in both male and female rats. Upon necropsy, there were no gross findings or microscopic changes in the limited number of tissues examined and there were no treatment related changes in clinical pathology parameters.

In the 28-day definitive toxicology study in rats (100, 300, and 1000 mg/kg/day; 10/sex/group), CMS121 was well tolerated at doses up to 1000 mg/kg/day and had no adverse effects on clinical observations, ophthalmology, clinical pathology (clinical chemistry, hematology, coagulation, urinalysis), macroscopic observations, or microscopic exams. However, there was a decrease in weight gain in the male rats, particularly at the higher doses. Based on these results, the NOAEL in rats was determined to be 1000 mg/kg/day, the highest dose tested.

In the 14-day dose-range finding study in dogs (30, 100, 300 mg/kg/day; 1/sex/group), CMS121 was well tolerated at doses up to 300 mg/kg/day, the highest dose tested. There were no unscheduled deaths or morbidity. Clinical signs included emesis, thinness, decreased activity, reddish feces, and loose feces in both dogs given 300 mg/kg/day. Reddish feces were also noted in the 100 mg/kg/day group. Decreased body weight gain was observed in the dogs given 300 mg/kg/day. Upon necropsy, gross findings were limited to the 300 mg/kg/day group and included decreases in spleen and thymus size that corresponded to decreased organ weights. Ileal intussusception was noted in the male dog given 300 mg/kg/day. Microscopic changes included hepatocellular vacuolation, hemorrhage in the ileal mucosa, renal cortical tubular degeneration/regeneration, and lymphoid depletion in the spleen and thymus. Based on these findings, 300 mg/kg/day was considered as the maximum tolerated dose in dogs.

In the definitive 28-day study in dogs (10, 30, 100/75 mg/kg/day; 3/sex/group), the high dose of 100 mg/kg/day was reduced to 75 mg/kg/day after the first week of dosing due to decreased body weights and decreased food consumption. After the dose reduction on Day 8, the CMS121-treated dogs gained weight similarly to the control dogs. There were no early deaths in this study and all animals survived to their scheduled sacrifices. Furthermore, no effects of CMS121 on body temperature, blood pressure, ECG, ophthalmology, hematology, coagulation, or urinalysis parameters were observed. However, although CMS121 at daily doses up to 100/75 mg/kg/day for 28 consecutive days was tolerated in Beagle dogs, some CMS121-related effects at the 100/75 mg/kg/day dose were observed which included decreases in body weight and food consumption, emesis, watery and/or loose feces along with microscopic alterations in the kidney and increases in plasma levels of creatinine and blood urea nitrogen in one animal. The microscopic kidney findings at 75 mg/kg/day were considered to be adverse. At lower doses, the adverse effects were not seen. Based on these findings, the NOAEL in the 28-day dog study was established at 30 mg/kg/day.

In summary, the nonclinical safety profile of CMS121 was well-characterized through the conduct of single- and repeat-dose studies of up to 28 days in duration. The major conclusion was that CMS121 was well-tolerated in rats and dogs, with no unscheduled deaths or morbidity in the repeat dose studies. The NOAELs were determined for both rats (1000 mg/kg/day) and dogs (30 mg/kg/day) for the 28-day period of this study and the nonclinical safety profile of CMS121 was adequately characterized to support progression into clinical trials in humans treated for up to 28 days in duration.

SUMMARY

In conclusion, CMS121 is a very potent inhibitor of oxytosis/ferroptosis in nerve cell culture and reduces brain pathology and cognitive dysfunction when administered to transgenic AD and aged SAMP8 mice [42, 49]. It appears to be doing so through several related mechanisms including maintenance of mitochondrial function with aging through the activation of the AMPK/ACC1 pathway [49], reduction of markers of oxytosis/ferroptosis [42, 49] and decreases in indicators of neuroinflammation [42]. It is likely that all of these mechanisms are directly related to the inhibition of oxytosis/ferroptosis by CMS121 although this idea requires further investigation. Its key molecular target is a protein new to the process of cell death in oxytosis/ferroptosis, FASN [42]. CMS121 has recently completed a phase 1 clinical trial for safety and tolerability (NCT05318040) and the final Clinical Study Report is pending. The current plan is to begin a phase 2 trial in 2024 (Fig. 5).

Fig. 5

Summary of CMS121 development.

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This work was funded by grants R42AI104034, R44AI104034, and R01AG074447 to PM and WR, grants RF1AG061296 and R01AG069206 to PM and grant R01AG067331 to AC, all from the National Institutes of Health.

CONFLICT OF INTEREST

The Salk Institute holds the patent for CMS121. PM is on the Scientific Advisory Boards of NOVOS and Dong-A ST. The other authors have no conflicts to report.

References

Scheltens

, De Strooper

, Kivipelto

, Hostege

, Chetelat

, Teunissen

, Cummings

, Van der Flier

(2021) Alzheimer’s disease. Lancet397, 1577–1590.

(2022) Alzheimer’s disease facts and figures. Alzheimers Dement18, 700–789.

Gladyshev

, Kritchevsky

, Clarke

, Cuervo

, Fiehn

, De Magalhaes

, Mau

, Maes

, Moritz

, Niedernhofer

, Van Schaftingen

, Tranah

, Walsh

, Yura

, Zhang

, Cummings

(2021) Molecular damage in aging. Nat Aging1, 1096–1106.

Lopez-Otin

, Blasco

, Partridge

, Serrano

, Kroemer

(2013) The hallmarks of aging. Cell153, 1194–2017.

Schubert

, Currais

, Goldberg

, Finley

, Petrascheck

, Maher

(2018) Geroneuroprotectors: Effective geroprotectors for the brain. Trends Pharmacol Sci39, 1004–1007.

Maher

, Currais

, Schubert

(2020) Using the oxytosis/ferroptosis pathway to understand and treat age-associated neurodegenerative diseases. Cell Chem Biol27, 1456–1471.

Larrick

, Larrick

, Mendelsohn

(2020) Contribution of ferroptosis to aging and fraility. Rejuvenation Res23, 434–438.

Mazhar

, Din

, Ali

, TYang

, Ren

, Wang

, Fan

, Yang

(2021) Implication of ferroptosis in aging. Cell Death Discov7, 149.

Tan

, Sagara

, Liu

, Maher

, Schubert

(1998) The regulation of reactive oxygen species production during programmed cell death. J Cell Biol141, 1423–1432.

10.

Tan

, Schubert

, Maher

(2001) Oxytosis: A novel form of programmed cell death. Curr Top Med Chem1, 497–506.

11.

Lewerenz

, Ates

, Methner

, Conrad

, Maher

(2018) Oxytosis/ferroptosis-(Re-)emerging roles for oxidative stress-dependent non-apoptotic cell death in diseases on the central nervous system. Front Neurosci12, 214.

12.

Albrecht

, Lewerenz

, Dittmer

, Noack

, Maher

, Methner

(2010) Mechanisms of oxidative glutamate toxicity: The glutamate/cystine antiporter system x(c)- as a neuroprotective drug target. CNS Neurol Disord Drug Targets9, 373–382.

13.

Seiler

, Schneider

, Forster

, Roth

, Wirth

, Culmsee

, Plesnila

, Kremmer

, Radmark

, Wurst

, Bornkamm

, Schweizer

, Conrad

(2008) Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab8, 237–248.

14.

Dolga

, POppermann

, Richter

, Honrath

, Neitemeier

, Jelinek

, Ganjam

, Culmsee

(2018) Molecular mechanisms underlying oxytosis. In Apoptosis and Beyond: The Many Ways Cells Die, Radosevich

, ed. Wiley, Hoboken, NJ.

15.

Ratan

(2020) The chemical biology of ferroptosis in the central nervous system. Cell Chem Biol27, 1–20.

16.

Dixon

, Lemberg

, Lamprecht

, Skouta

, Zaitsev

, Gleason

, Patel

, Bauer

, Cantley

, Yang

, Morrison

, Stockwell

(2012) Ferroptosis: An iron-dependent form of non-apoptotic cell death. Cell149, 1060–1072.

17.

Chen

, Yu

, Kang

, Tang

(2020) Iron metabolism in ferroptosis. Front Cell Dev Biol8, 590226.

18.

Bayir

, Anthonymuthu

, Tyurina

, Patel

, Amoscato

, Lamade

, Yang

, Vladimirov

, Philpott

, Kagan

(2020) Achieving life through death: Redox biology of lipid peroxidation in ferroptosis. Cell Chem Biol27, 387–408.

19.

Yang

, Kim

, Gaschler

, Patel

, Shchepinov

, Stockwell

(2016) Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A113, E4966–E4975.

20.

Conrad

, Kagan

, Bayir

, Pagnussat

, Head

, Traber

, Stockwell

(2018) Regulation of lipid peroxidation and ferroptosis in diverse species. Genes Dev32, 602–619.

21.

Stockwell

(2022) Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic implications. Cell185, 2401–2421.

22.

El-Beltagi

, Mohamed

(2013) Reactive oxygen species, lipid peroxidation and antioxidative dense mechanisms. Not Bot Horti Agrobot Cluj Napoca41, 44–57.

23.

Alche

(2019) A concise appraisal of lipid oxidation and lipoxidation in higher plants. Redox Biol23, 101136.

24.

Farmer

, Mueller

(2013) ROS-mediated lipid peroxidation and RES-activated signaling. Annu Rev Plant Biol64, 429–450.

25.

Majernikova

, Den Dunner

WFA

, Dolga

(2021) The potential of ferroptosis-targeting therapies for Alzheimer’s disease: From mechanism to transcriptomic analysis. Front Aging Neurosci13, 745046.

26.

Conrad

, Lorenz

, Proneth

(2021) Targeting ferroptosis: New hope for as-yet-incurable diseases. Trends Mol Med27, 113–122.

27.

Zhang

, Zhang

, Shen

, Wang

, Zhao

, Sun

(2021) The potential role of ferroptosis in Alzheimer’s disease. J Alzheimers Dis80, 907–925.

28.

Ashraf

, Jeandriens

, Parkes

, So

(2020) Iron dyshomeostasis, lipid peroxidation and perturbed expression of cystine/glutamate antiporter in Alzheimer’s disease: Evidence of ferroptosis. Redox Biol32, 101494.

29.

Gouras

, Almeida

, Takahashi

(2005) Intraneuronal Abeta accumulation and origin of plawues in Alzheimer’s disease. Neurobiol Aging26, 1235–1244.

30.

LaFerla

, Green

, Oddo

(2007) Intracellular amyloid-beta in Alzheimer’s disease. Nat Rev Neurosci8, 499–509.

31.

Wirths

, Multhaup

, Bayer

(2004) A modified beta-amyloid hypothesis: Intraneuronal accumulation of the beta-amyloid peptide-the first step of a fatal cascade. J Neurochem91, 513–520.

32.

Huang

, McClatchy

, Maher

, Liang

, Diedrich

, Soriano-Castell

, Goldberg

, Shokhirev

, Yates

, Schubert

, Currais

(2020) Intracellular amyloid toxicity induces oxytosis/ferroptosis regulated cell death. Cell Death Dis11, 828.

33.

Fischer

, Currais

, Liang

, Pinto

, Maher

(2019) Old age-associated phenotypic screening for Alzheimer’s disease drug candidates identifies sterubin as a potent neuroprotective compound from Yerba santa. Redox Biol21, 101089.

34.

Soriano-Castell

, Liang

, Maher

, Currais

(2021) Profiling the chemical nature of anti-oxytotic-ferroptotic compounds with phenotypic screening. Free Radic Biol Med177, 313–325.

35.

Hitchcock

, Pennington

(2006) Structure-brain exposure relationships. J Med Chem49, 7559–7583.

36.

Pajouhesh

, Lenz

(2005) Medicinal chemical properties of successful central nervous system drugs. NeuroRx2, 541–553.

37.

Prior

, Chiruta

, Currais

, Goldberg

, Ramsey

, Dargusch

, Maher

, Schubert

(2014) Back to the future with phenotypic screening. ACS Chem Neurosci5, 503–513.

38.

Chiruta

, Schubert

, Dargusch

, Maher

(2012) Chemical modification of the multi-target neuroprotective compound fisetin. J Med Chem55, 378–389.

39.

Maher

(2021) Preventing and treating neurological disorders with the flavonol fisetin. Brain Plast6, 155–166.

40.

Currais

, Prior

, Dargusch

, Armando

, Ehren

, Schubert

, Quehenberger

, Maher

(2014) Modulation of p25 and inflammatory pathways by fisetin maintains cognitive function in Alzheimer’s disease transgenic mice. Aging Cell13, 379–390.

41.

Currais

, Farrokhi

, Dargusch

, Armando

, Quehenberger

, Schubert

, Maher

(2018) Fisetin reduces the impact of aging on behavior and physiology in the rapidly aging SAMP8 mouse. J Gerontol A Biol Sci Med Sci73, 299–307.

42.

Ates

, Goldberg

, Currais

, Maher

(2020) CMS121, a fatty acid synthase inhibitor, protects against excess lipid peroxidation and inflammation and alleviates cognitive loss in a transgenic model of Alzheimer’s disease. Redox Biol36, 101648.

43.

Ayala

, Munoz

, Arguelles

(2014) Lipid peroxidation: Production, metabolism and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev2014, 360438.

44.

Wang

, Zhu

, Hjorth

, Cortes-Toro

, Eyolfsdottir

, Graff

, Nennesmo

, Palmblad

, Eriksdotter

, Sambamurti

, Fitzgerald

, Serhan

, Granholm

, Schultzberg

(2015) Resolution of inflammation is altered in Alzheimer’s disease. Alzheimers Dement11, 40–50.

45.

Pallas

(2012) Senescence-accelerated mice P8: A tool to study brain aging and Alzheimer’s disease in a mouse model. ISRN Cell Biol2012, 917167.

46.

Cheng

, Zhou

, Zhang

(2014) The behavioral, pathological and therapeutic features of the senescence-accelerated mouse prone 8 strain as an Alzheimer’s disease animal model. Ageing Res Rev13, 13–37.

47.

Pacesova

, Holubova

, Hruba

, Strnadova

, Neprasova

, Pelantova

, Kuzma

, Zelezna

, Kunes

, Maletinska

(2022) Age-related metabolic and neurodegenerative changes in SAMP8 mice. Aging14, 7300–7327.

48.

Currais

, Goldberg

, Farrokhi

, Chang

, Prior

, Dargusch

, Daugherty

, Armando

, Quehenberger

, Maher

, Schubert

(2015) A comprehensive multiomics approach toward understanding the relationship between aging and dementia. Aging7, 937–955.

49.

Currais

, Huang

, Goldberg

, Petrascheck

, Ates

, Pinto-Duarte

, Shokhirev

, Schubert

, Maher

(2019) Elevating acetyl-CoA levels reduces aspects of brain aging. eLife8, e47866.

50.

Herzig

, Shaw

(2018) AMPK: Guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol19, 121–135.

51.

Mouton-Liger

, Jacoupy

, Corvol

J-C

, Corti

(2017) PINK1/Parkin-dependent mitochondrial surveillance: From pleiotropy to Parkinson’s disease. Front Mol Neurosci10, 120.

52.

Frank-Cannon

, Tran

, Ruhn

, Martinez

, Hong

, Marvin

, Hartley

, Trevino

, O’Brien

, Casey

, Goldberg

, Tansey

(2008) Parkin deficiency increases vulnerability to inflammation-related nigral degeneration. J Neurosci28, 10825–10834.

53.

Tran

, Nguyen

, Chang

, Tansey

(2011) Lipopolysaccharide and tumor necrosis factor regulate Parkin expression via nuclear factor-kappa B. PLoS One6, e23660.

54.

, Sun

, Starovoytov

, Cai

(2015) Parkin-mediated mitophagy in mutant hAPP neurons and Alzheimer’s disease patient brains. Hum Mol Genet24, 2938–2951.

55.

Proctor

, Coulson

, Dodd

(2010) Reduction in post-synaptic scaffolding PSD-95 and SAP-102 protein levels in Alzheimer inferior temporal cortex is correlated with disease pathology. J Alzheimers Dis21, 795–811.

56.

Calabrese

, Rossetti

, Racagni

, Gass

, Riva

, Molteni

(2014) Brain-derived neurotrophic factor: A bridge between inflammation and neuroplasticity. Front Cell Neurosci8, 430.

57.

Kepchia

, Currais

, Dargusch

, Finley

, Schubert

, Maher

(2021) Geroprotective effects of Alzheimer’s disease drug candidates. Aging13, 3269–3289.

58.

Toth

, Szegedi

, Varga

, Juhasz

, Horvath

, Borbely

, Csibrany

, Alfoldi

, Klenart

, Penke

, Santha

(2013) Overexpression of Hsp27 ameliorates symptoms of Alzheimer’s disease in APP/PS1 mice. Cell Stress Chaperones18, 759–771.

59.

Bitto

, Lerner

, Nacarelli

, Crowe

, Torres

, Sell

(2014) p62/SQSTM1 at the interface of aging, autophagy, and disease. Age (Dordr)36, 1123–1137.

60.

Currais

, Kepchia

, Liang

, Maher

(2022) The role of AMP-activated protein kinase in oxytosis/ferroptosis: Protector or potentiator?Antioxid Redox Signal, 10.1089/acs.2022.0013.

61.

Peixoto

, de Oliveira

, Araujo

SMR

, Nunes

AKS

(2017) AMPK activation: Role in the signaling pathways of neuroinflammation and neurodegeneration. Exp Neurol298, 31–41.

62.

Jangra

, Tople

(2022) Can Alzheimer’s disease be secondary to type-2 diabetes mellitus?Cureus14, e31273.

63.

Onaolapo

, Ojo

, Adeleye

, Falade

, Onaolapo

(2023) Diabetes mellitus and energy dysmetabolism in Alzheimer’s disease: Understanding the relationships and potential therapeutic targets. Curr Diabetes Rev19, e0201232212333.

64.

Cater

, Holter

(2022) A pathophysiological intersection of diabetes and Alzheimer’s disease. Int J Mol Sci23, 11562.

65.

Carvalho

, Moreira

(2023) Metabolic defects shared by Alzheimer’s disease and diabetes: A focus on mitochondria. Curr Opin Neurobiol79, 102694.

66.

Wang

, Chandrasekara

, Pippin

(2014) Leptin- and leptin receptor-deficient rodent models: Relevance for human type 2 diabetes. Curr Diabetes Rev10, 131–145.

67.

Tesch

, Lim

AKH

(2011) Recent insights into diabetic renal injury from the db/db mouse model of type 2 diabetic nephropathy. Am J Physiol Renal Physiol300, F301–F310.

68.

Chen

, Ding

, Wu

, Tong

, Zhao

, Lv

, Meng

, Liu

, Ren

, Li

, Jian

, Li

(2021) Novel sesquiterpene glycoside from loquat leaf alleviates type 2 diabetes mellitus combined with nonalcoholic fatty liver disease by improving insulin resistance, oxidative stress, inflammation and gut microbiota composition. J Agric Food Chem69, 14176–14191.

69.

Jiang

, Sui

, Li

, Xu

, Yu

, Liu

, Yu

(2021) Ginsenoside Re improves inflammation and fibrosis in hepatic tissue by upregulating PPARgamma expression and inhibiting oxidative stress in db/db mice. Evid Bases Complement Alternat Med2021, 9003603.

70.

Yang

, Zhou

, Wang

(2020) Inhibition of HMGB1 involved in the protective effect of salidroside on liver injury in diabetes mice. Int Immunopharmacol89(Pt A), 106987.

71.

Suriano

, Viera-Silva

, Falony

, Roumain

, Paquot

, Pelicaen

, Regnier

, Delzenne

, Raes

, Muccioli

, Van Hul

, Cani

(2021) Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: Two sides of the same coin. Microbiome9, 147.

72.

Shahzad

, Fatima

, Al-Dabet

, Gadi

, Khawaja

, Ambreen

, Elwakiel

, Kloting

, Buhler

, Nawroth

, Mertens

, Michel

, Jaschinski

, Klar

, Isermann

(2021) CHOP-ASO ameliorates glomerular and tubular damage on top of ACE inhibition in diabetic kidney disease. J Am Soc Nephrol32, 3066–3079.

73.

Estrada

, Ahumada

, Cabrera

, Arab

(2019) Liver dysfunction as a novel player in Alzheimer’s progression: Looking outside the brain. Front Aging Neurosci11, 174.

74.

Cheon

, Song

(2022) Novel insights into non-alcoholic fatty liver disease and dementia: Insulin resistance, hyperammonemia, gut dysbiosis, vascular impairment and inflammation. Cell Biosci12, 99.

75.

Davey

, Elias

, Robbins

, Seliger

, Dore

(2013) Decline in renal functioning is associated with longitudinal decline in global cognitive functioning, abstract reasoning and verbal memory. Nephrol Dial Transplant28, 1810–1819.

76.

Anand

, Johansen

, Tamura

(2014) Aging and chronic kidney disease: The impact on physical function and cognition. J Gerontol A Biol Sci Med Sci69, 315–322.

77.

Kelly

, Rothwell

(2022) Disentangling the relationship between chronic kidney disease and cognitive disorders. Front Neurol13, 830064.

78.

Zhang

C-Y

, He

F-F

, Su

, Zhang

, Meng

X-F

(2020) Association between chronic kidney disease and Alzheimer’s disease: An update. Metab Brain Dis35, 883–894.

79.

Zahid

, Dafre

, Currais

, Yu

J-Y

, Schubert

, Maher

(2023) The geroprotective drug candidate CMS121 alleviates diabetes, liver inflammation and renal damage in tdb/db leptin receptor deficient mice. Int J Mol Sci24, 6828.

80.

Tang

, Li

, Zhang

, Chen

, Wang

, Feng

(2021) Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management. Acta Pharmacol Sin B11, 2749–2767.

81.

Mizdrak

, Kumric

, Kurir

, Bolzic

(2022) Emerging biomarkers for early detection of chronic kidney disease. J Pers Med12, 548.

82.

Ntrinias

, Papasotiriou

, Balta

, Kalavrizioti

, Vamvakas

, Papachristou

, Goumenos

(2019) Biomarkers in progressive chronic kidney disease. Still a long way to go. PRILOZI40, 27–39.

83.

Lin

, Albert

(2014) Hearing loss and dementia-who is listening?Aging Ment Health18, 671–673.

84.

Deal

, Betz

, Yaffe

, Harris

, Purchase-Helzner

, Satterfield

, Pratt

, Govil

, Simonsick

, Lin

(2017) Hearing impairment and incident dementia and cognitive decline in older adults: The health ABC study. J Gerontol A Biol Sci Med Sci72, 703–709.

85.

Menardo

, Tang

, Ladrech

, Lenoir

, Casas

, Michel

, Bourien

, Ruel

, Rebillard

, Maurice

, Puel

, Wang

(2012) Oxidative stress, inflammation and autophagic stress as the key mechanisms of premature age-related hearing loss in SAMP8 mouse cochlea. Antioxid Redox Signal16, 263–274.

86.

Peixoto Pinheiro

, Adel

, Knipper

, Muller

, Lowenheim

(2021) Auditory threshold variability in the SAMP8 mouse model of age-related hearing loss: Functional loss and phenotypic change precede outer hair cell loss. Front Aging Neurosci13, 708190.

87.

Pham

, Boussaty

, Currais

, Maher

, Schubert

, Manor

, Friedman

(2023) Attenuation of age-related hearing impairment in senescence-accelerated mouse prone 8 (SAMP8) mice treated with fatty acid sunthase inhibitor CMS121. J Mol Neurosci73, 307–315.

88.

Maron

, Ames

(1983) Revised method for the Salmonella mutagenicity test. Mutat Res113, 173–215.

89.

Evans

, O’Riordan

(1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutat Res31, 135–148.

90.

Preston

, Au

, Bender

, Brewen

, Carrano

, Heddle

, McFee

, Wolff

, Wassom

(1981) Mammalian in vivo and in vitro cytogenetic assays: A report of the US EPA’s gene-tox program. Mutat Res87, 143–188.