Soticlestat, a novel cholesterol 24-hydroxylase inhibitor shows a therapeutic potential for neural hyperexcitation in mice

Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [3H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24S-hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24S-hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.

the relationship of these polymorphisms with disease. Given that CH24H catalyses the dominant mechanism of the excretion of cholesterol from the brain, its activation has been considered as a therapeutic strategy to stimulate brain cholesterol metabolism. For example, local overexpression of CH24H by a viral vector is beneficial in experimental animal models of AD and Huntington's disease [15][16][17] . More recently, the reverse transcriptase inhibitor efavirenz has been characterized as a CH24H activator for potential treatment for AD [18][19][20] . However, such interventional strategies may be limited by the potential toxicity of 24S-hydroxycholesterol, the enzymatic product of CH24H. Previously known as 'cerebrosterol' , 24S-hydroxycholesterol has been shown to influence various biological functions, including facilitation of N-methyl-d-aspartate (NMDA) signalling, inflammation, oxidative stress and necroptosis [21][22][23][24][25] , implying that lowering brain 24S-hydroxycholesterol levels could also be therapeutic when these factors are driving pathology.
Pharmacological inhibition of CH24H can complement and expand on genetic studies to investigate the therapeutic potential of 24S-hydroxycholesterol lowering. Several molecules are known for CH24H inhibitory activity 26 ; however, there have been few attempts to test the therapeutic potential of such agents, presumably owing to their non-specificity and pleiotropic activity. One of the earliest examples is voriconazole. This antifungal agent was originally developed as an ergosterol synthesis inhibitor 27 . Although voriconazole reportedly reduces brain 24S-hydroxycholesterol, its pharmacological effects are not purely attributable to CH24H inhibition because the drug also interferes with extracerebral cholesterol metabolism. It is, therefore, important to identify a potent, highly-specific and brain-penetrable CH24H inhibitor that merits preclinical and clinical investigations of the effects of perturbating brain cholesterol metabolism. Here, we describe soticlestat ([4-benzyl-4-hydroxypiperidin-1-yl][2,4′-bipyridin-3-yl] methanone, TAK-935, also known as OV935) as a novel CH24H inhibitor. To investigate the pharmacological benefits of soticlestat, we employed a transgenic mouse model carrying human amyloid precursor protein and presenilin 1 (APP/PS1-Tg) 28,29 . Originally developed as an AD model, APP/PS1-Tg is also known for excitatory/inhibitory imbalance and seizure-related sudden death 30,31 . Most importantly, it was shown that cross-breeding of APP/PS1-Tg mice with CH24H-deficient mice extended life-span regardless of zygosity 32 . The observation in conventional knockout mice, however, has left room for the argument that the survival benefit of CH24H insufficiency is attributable to alteration of brain development. Employing a newly identified small-molecule inhibitor, we tested if the high mortality can be reversed by post-development intervention. The present study provides a rationale and supports the hypothesis that pharmacological inhibition of CH24H may have therapeutic relevance to central nervous system (CNS) hyperexcitability.

CH24H inhibitory activity and in vitro/in vivo target engagement of soticlestat. Soticlestat
( Supplementary Fig. S1) was discovered as a result of an iterative medicinal chemistry effort following highthroughput screening of an in vitro enzyme assay (Fig. 1A,B). Soticlestat inhibited the catalytic activity of human CH24H in a concentration-dependent manner with a half-maximal inhibitory concentration (IC 50 ) of 4.5 nmol/L. In our preliminary screening assays for major CNS drug targets and drug-metabolizing enzymes, soticlestat did not show notable activities at a concentration 10,000 times higher than the IC 50 for CH24H inhibition (Supplementary table S1 and S2).
Target engagement specificity was further examined via autoradiography using brain slices collected from wild-type (WT) and CH24H-deficient KO mice (Fig. 1C). The binding of [ 3 H]soticlestat (300 nmol/L) showed a clear contrast between WT and CH24H-KO mice. At this concentration, soticlestat inhibitory activity on CH24H is saturated (Fig. 1B). The data suggest that the molecule is highly specific for CH24H. In WT brain slices, the cerebral cortex, thalamus, midbrain and hypothalamus showed relatively stronger binding than the cerebellum, which appeared to be only weakly labelled, implying a relatively broad expression of the enzyme in the cerebrum. The localisation of [ 3 H]soticlestat signals agrees with immunohistochemistry of CH24H protein in mouse brain, further supporting the binding specificity to the target ( Supplementary Fig. S2A). The lower binding of [ 3 H]soticlestat to cerebellum is consistent with our preliminary assessment of regional levels of 24S-hydroxycholesterol, which found the highest levels in the striatum and the lowest in the cerebellum (Supplementary Fig. S2B). These observations also agree with a recent study indicating a low level of 24S-hydroxycholesterol in the cerebellum 33 , while an earlier immunohistochemistry study detected a relatively high level of CH24H expression in Purkinje cells 34 . To further characterize the CH24H binding specificity of soticlestat, biodistribution experiments were conducted in vivo, comparing CH24H-KO and WT mice following intravenous injection (Fig. 1D). The plasma concentrations of soticlestat 1 h after injection were similar between the two strains at 9.2 ± 4.1 ng/mL and 11.9 ± 2.8 ng/mL (mean ± standard error of measurement [s.e.m.]) for WT and CH24H-KO mice, respectively. In the brain, soticlestat content in WT and CH24H-KO mice was notably different at 26.1 ± 0.8 ng/g and 0.9 ± 0.3 ng/g (mean ± s.e.m.), respectively. These data collectively suggest that soticlestat has an adequately specific affinity to CH24H that deserves further investigation of pharmacodynamics (PD).
Evaluation of pharmacodynamics (PD) of soticlestat in APP/PS1-Tg mice. The first study to demonstrate a therapeutic benefit of CH24H inhibition was reported by Halford et al. 32 . In the study, both heterozygous and homozygous CH24H KO remarkably extended the life-span of APP/PS1-Tg mice, while not affecting the amyloid pathology. Based on this study, we employed a strain of APP/PS1-Tg mice with a different PS-1 mutation 28,29 . When brain levels of 24S-hydroxycholesterol were evaluated, APP/PS1-Tg mice did not show notably higher levels of brain 24S-hydroxycholesterol content than WT animals of the same background strain ( Fig. 2A). To test if soticlestat can reduce 24S-hydroxycholesterol in the brain, APP/PS1-Tg mice were orally treated with the drug for different time periods as indicated in Fig. 2B. A hysteresis was observed in soticlestat effects on 24S-hydroxycholesterol with a temporal pattern that was delayed compared with the pharmacokinetics (PK) data ( Fig. 2B and Supplementary Fig. S3A www.nature.com/scientificreports/ reach maximum levels of brain 24S-hydroxycholesterol lowering (Fig. 2B). Despite a small number of samples, an association was found between PK and PD ( Supplementary Fig. S3B, r = − 0.961). Soticlestat PD effects on brain 24S-hydroxycholesterol reached an apparent steady state after repetitive treatments for 3 days. The dose dependency of 24S-hydroxycholesterol lowering was further assessed in APP/PS1-Tg mice after 3 days of soticlestat treatment (Fig. 2C). Soticlestat showed a dose-dependent reduction of 24S-hydroxycholesterol, reaching a decrease of about 50% at 10 mg/kg. Presumably owing to limited bioavailability in mice, 24S-hydroxycholesterol lowering effects of soticlestat almost reached a plateau at the dose of 10 mg/kg and higher with oral gavage ( Supplementary Fig. S4). This dose was selected as a treatment condition of soticlestat that yields similar levels of enzyme inhibition to that found in heterozygous CH24H KO 5 . The dose dependency of 24S-hydroxycholesterol lowering by soticlestat did not show any noticeable difference in the WT control animals ( Supplementary  Fig. S5). The potential effects of soticlestat on AD pathology were examined in pilot experiments using 3-monthold APP/PS1-Tg mice. No noticeable effects of soticlestat were observed on amyloid pathology ( Supplementary  Fig. S6), while a trend was observed on the improvement in working memory deficits in the Y-maze test (Supplementary Fig. S7).
Originally developed as an AD mouse model, APP/PS1-Tg mice have also been recognized as a model of excitatory/inhibitory imbalance, including seizure-related sudden death 30,31,[35][36][37] . The strain kept in our facility showed approximate 50% mortality over the first 3 months after birth. To test the potential effect on survival, soticlestat intervention (10 mg/kg orally [PO], once daily [QD]) was initiated from 7 weeks of age and maintained for 8 weeks. A significant difference was found in the survival curve between the vehicle arm and the soticlestat treatment arm (Fig. 2D). A total of 14 out of 30 mice died in the control group, while 2 out of 30 mice died in the soticlestat group throughout the intervention period. The hazard ratio of death was 5.849 in the vehicle-treated arm compared with the soticlestat arm. The result indicated that soticlestat has a robust pro-survival benefit in the APP/PS1-Tg model in agreement with the earlier study done in CH24H-KO mice 32 .
Interestingly, enhanced cholesterol 24-hydroxylation reportedly decreases astrocytic glutamate sequestration 38 , suggesting a potential role of CH24H in an impaired glutamate uptake system in AD. The APP/ PS1-Tg model is also recognized for an epileptic phenotype, as well as for impairment of extracellular potassium homeostasis 30,37 . To unmask the hyperexcitability phenotype, KCl was infused through a microdialysis cannula into the hippocampus of freely moving awake animals 39 . With 100 mM KCl perfused, 7 out of 11 APP/ PS1-Tg mice developed lethal seizures, while no obvious behavioural changes were found in WT mice. In pilot experiments, a continuous infusion of KCl led to 100% mortality in APP/PS1-Tg mice unless tetrodotoxin was www.nature.com/scientificreports/ infused to antagonize the KCl-induced depolarization (Fig. 3A). In contrast, no deaths were observed in WT mice. To control the mortality and to quantify the levels of extracellular glutamate elevation, experiments were terminated after 60 min of KCl perfusion. Glutamate levels in the WT and APP/PS1-Tg mice reached 4.0 ± 1.2 and 23.0 ± 4.9 (mean ± s.e.m.) times baseline levels, respectively (Fig. 3B). The data suggested the susceptibility of APP/PS1-Tg mice to catastrophic depolarization events. To assess the potential effects of soticlestat on the hippocampal hyperexcitability of the model, APP/PS1-Tg mice were treated with soticlestat for 2 weeks (10 mg/ kg PO, QD) prior to undergoing microdialysis. It was found that the elevation of extracellular glutamate was greatly suppressed by soticlestat (Fig. 3C,D). Following the initiation of KCl perfusion, seizure-like behaviours became apparent in 50% of the vehicle-treated control mice accompanied by markedly elevated glutamate levels, whereas soticlestat-treated animals showed few behavioural abnormalities. Extracellular glutamate levels peaked at 28.9 ± 8.7 and 1.6 ± 0.4 (mean ± s.e.m.) times baseline levels for control and soticlestat groups, respectively. Meanwhile, soticlestat treatment did not affect baseline glutamate levels before KCl perfusion ( Fig. 3E; 0.33 ± 0.04 μmol/L and 0.33 ± 0.04 μmol/L for the control group and soticlestat group, respectively; mean ± s.e.m).
In a separate experiment, DL-threo-β-benzyloxyaspartate (TBOA), an inhibitor of astrocytic glutamate uptake, was used to determine the role of glutamate transporters in the observed increase in extracellular glutamate. In the presence of TBOA (10 μmol/L), soticlestat had no effect on extracellular glutamate ( Supplementary Fig. S8).
It was also reported that glutamate toxicity can induce CH24H translocation from endoplasmic reticulum to the plasma membrane, thereby causing cholesterol loss 40 , which could apply to the APP/PS1-Tg model. However, loss of cholesterol was not found in a detergent resistant membrane component extracted from APP/PS1-Tg brain compared wild-type animals ( Supplementary Fig. S9). It was also shown that soticlestat treatment had no considerable impact on the global brain cholesterol levels ( Supplementary Fig. S10). Given the intricacy of homeostasis 41 , however, it is possible that impacts of CH24H inhibition of cholesterol levels can vary across different brain regions. Indiscriminate dampening effects on neural excitation may result in adverse pharmacological phenotypes such as sedation. To support clinical development in terms of preclinical safety, naïve ICR mice were   Fig. S11). These data collectively suggest that CH24H inhibition can tip the neural excitatory/inhibitory balance without indiscriminately dampening neural excitation.

Discussion
The brain-specific, cholesterol-catabolic enzyme CH24H has gained increased attention as a potential drug target 18,19 . Clarifying hitherto unproven therapeutic benefits of CH24H inhibitor 26 , the present study provides new insights into the therapeutic relevance of pharmacological modulation of this enzyme [18][19][20]42 . The data shown here suggest that 24S-hydroxycholesterol lowering by soticlestat has therapeutic potential in diseases with underlying excitatory/inhibitory imbalance in the brain. The assessment of soticlestat safety and 'absorption, distribution, metabolism and excretion' profile has already led to clinical translation 43 . www.nature.com/scientificreports/ To identify a disease condition relevant to CH24H inhibition, we employed the APP/PS1-Tg model, following the previous report that suggested a potential survival benefit of conventional CH24H KO 32 . Chemical inhibition of a protein does not necessarily produce a phenotype aligned with conventional KO of the gene. In this sense, it might be emphasized that soticlestat intervention showed a survival benefit in 7-week-old APP/PS1-Tg mice. One obvious difference from conventional KO is that CH24H was postnatally inhibited by soticlestat in our experiments, indicating that the therapeutic window is not necessarily confined to the prenatal or perinatal period of APP/PS1-Tg mice. It is intriguing to contemplate that activity of cholesterol 24-hydroxylation matters to the balance between life and death depending on the state of brain. Among known mortality factors, neural excitatory/inhibitory balance was highlighted as being important in a recent study of RE1-silencing transcription factor 44 . We are tempted to speculate that the survival benefit of soticlestat is also related to restoration of excitatory/inhibitory imbalance. Soticlestat suppressed the potassium-evoked glutamate elevation in APP/PS1-Tg mice (Fig. 3C,D). Interestingly, its effects on glutamate were almost completely abolished by inhibition of glutamate reuptake by TBOA (Supplementary Fig. S8). Interpretation of the pharmacological experiment with TBOA is challenging; however, the data suggest that CH24H inhibition is likely to affect astrocytic function as well as neuronal function. As mentioned earlier, cholesterol 24-hydroxylation in astrocytes can impair the glutamate reuptake function 38 . Furthermore, Na + /K + adenosine triphosphatase activity is impaired in the APP/ PS1-Tg model 30 , which is one of the astrocytic risk factors for sudden unexpected death in epilepsy 45 . Given the importance of potassium homeostasis as a risk factor for seizure-related death 46 , the potential involvement of CH24H enzyme activity in the regulation of release and/or clearance of glutamate and/or potassium is an intriguing hypothesis to pursue.
To focus on the mortality-related phenotypes of APP/PS1-Tg mice, soticlestat was characterized mostly in young animals, instead of in aged animals associated with AD-related pathologies, to circumvent a population bias due to the high mortality. Nevertheless, it may be worthwhile briefly describing our preliminary experiments assessing a potential effect of soticlestat on cognitive functions in APP/PS1-Tg mice. One of the reported phenotypes of CH24H-KO mice is an impairment in learning 47 . Conversely, it has been reported that activation of CH24H has pro-cognitive potential 20,48 . Three-month-old APP/PS1-Tg animals were treated with soticlestat and underwent the Y-maze test. Although a pharmacological experiment in 3-month-old APP/PS1-Tg mice does include a survival bias, our preliminary data, to the contrary, showed an apparent trend on the improvement in working memory (Supplementary Fig. S7). This observation may disagree with the cognitive benefits of efavirenz, a reverse transcriptase inhibitor known for CH24H activation 20,48 . It should be noted that the effects of efavirenz on cognition are apparently associated with improvement in amyloid pathology. In our pilot experiments, however, soticlestat treatment had no notable impact on the amyloid pathology of APP/PS1-Tg mice ( Supplementary Fig. S6). The lack of effects on amyloid pathology is also consistent with the phenotype of CH24H KO 32 . The improving trend of cognition seen in soticlestat could be explained by other mechanisms such as modulation of the excitatory/inhibitory balance.
One of the rationales for pharmacological activation of CH24H is acceleration of brain cholesterol turn-over, which is expected to be beneficial for some neurodegenerative diseases 15,20,49 ; however, this hypothesis may not be readily generalizable to other diseases. Firstly, acceleration of cholesterol 24-hydroxylation can disrupt the integrity of membrane 50 , thereby possibly disturbing physiological functions of neurons 1-3 . Secondly, CH24H activation leads to upregulation of cholesterol biosynthesis, an energy-demanding process that consumes 18 mol of acetyl coenzyme A, 18 mol of adenosine triphosphate and 29 mol of nicotinamide adenine dinucleotide phosphate to produce 1 mol of cholesterol 51 . As the brain is an organ of high-energy demand and 100% selfsufficiency in cholesterol production, activation of this costly mechanism may add an extra burden on the brain 52 . Further studies are needed to understand whether activation or inhibition of CH24H can be therapeutic for each disease of interest. Future studies are planned to clarify whether or not, and how CH24H inhibition can modulate cholesterol homeostasis in the brain. We propose that 24S-hydroxycholesterol lowering can be a therapeutic approach when this oxysterol is driving such pathology as inflammation, oxidative stress and excitatory/inhibitory imbalance 24,25,53 . Soticlestat is being tested in clinical trials for the treatment of epilepsy. In a separate study, soticlestat has been shown to ameliorate seizure progression in correlation with lowering of brain 24S-hydroxycholesterol in a mouse model of epilepsy (r = − 0.682; P < 0.05, Pearson correlation coefficient) 43 . The full set of data will be published elsewhere.
Finally, it is interesting to note that CH24H knockdown can be neurotoxic in other animal models 16,54 . The hypothesis is, however, based on a viral vector-mediated RNA interference introduced locally in the hippocampus. Given the intricate interrelationship of neural circuits, it is possible that a regional somatic cell CH24H knockdown, with uncertain specificity and using viral vectors with a known neuronal tropism, produces a result different from a pharmacological inhibition of the enzyme across all cell types and in the entire brain. Importantly, a nearly full inhibition of CH24H by soticlestat did not cause notable abnormalities in motor coordination and spontaneous locomotor activities (Supplementary Fig. S11). In fact, hyperlocomotion is a typical behavioural phenotype of NMDA receptor (NMDAR) blocker 55 . Reduction of brain 24S-hydroxycholesterol, known as a potentiator NMDAR, may produce a similar phenotype, but this was not the case in our observation. As a matter of fact, the brain level of 24S-hydroxycholesterol is estimated at around 25 μM across mammalian species, 20-fold higher than the reported half-maximal effective concentration on NMDAR 21 . Therefore, it is not surprising that even aggressive CH24H inhibition by soticlestat does not disturb the baseline NMDAR functions.
In summary, the experiments conducted in this study collectively demonstrate the therapeutic potential of soticlestat. Considering the in vitro and in vivo assessment of soticlestat target engagement, it can be concluded that CH24H inhibitor has a therapeutic potential that merits further investigation, not only in the clinical setting, but also as a basic research tool to shed light on the biological implications of 24S-hydroxycholesterol. Catalytic activity of the CH24H enzyme was measured using thin-layer chromatography (TLC). To evaluate the inhibitory activity, 2 μL of serial diluted compounds were incubated with 3 μL of CH24H enzyme in assay buffer ( Measurement of soticlestat exposure levels in plasma and brain. Plasma and brain were collected at the indicated time points to determine the exposure level of soticlestat following single and chronic administration. Whole blood was collected from the inferior vena cava with heparin. To collect brain samples, cerebellum was removed. The collected brain was homogenized with saline for subsequent procedures. The aliquots of plasma and the brain homogenate were mixed with acetonitrile containing deuterated soticlestat analogue as internal standard and then centrifuged. (SCIEX, Redwood City, CA, USA) was used for data acquisition and processing. The lower limit of quantification of soticlestat was 0.1 ng/mL and 0.5 ng/g in plasma and brain, respectively.

Measurement of 24S-hydroxycholesterol levels in brain.
In this study, high-performance liquid chromatography (HPLC)-based assay was employed to improve the throughput of drug discovery campaign instead of LC-MS/MS analysis, which is used for plasma 24S-hydroxycholesterol analysis in clinical trials 57 .
To determine the level of 24S-hydroxycholesterol in the brain, brain homogenate was prepared as described above. 24S-hydroxycholesterol was extracted from brain homogenate by mixing with equal volume (w/v) of 98% acetonitrile containing 2% methanol and 0.2% formic acid, and then centrifuged at 12,000g for 15 min at room www.nature.com/scientificreports/ temperature. The supernatant was filtrated with a 0.45 μm filter and measured by high-performance liquid chromatography (HPLC). A ready-to-use prepacked C18 column (Capcell Pak C18 MGII, 3 µm, 3.0 mm inner diameter [ID] × 75 mm length, Shiseido, Kyoto, Japan) with a precolumn packed with the same material (silica gel) was used for separation. Diluted water with 0.1% trifluoroacetic acid (A) and methanol with 0.1% trifluoroacetic acid (B) were used for the chromatographic run. The composition of the mobile phase was changed according to the following time programme: 0-1 min 20% (A) and 80% (B); 1-21 min 10% (A) and 90% (B); 21-25 min 100% (B); and 25-30 min 100% (B). The flow rate was 0.5 mL/min. Peaks were detected by an ultraviolet (UV) detector at 210 nm. The peak at the retention time of 13 min was extracted by fraction collector and confirmed as 24S-hydroxycholesterol by LC-MS/MS. The peak area was analysed by using the LabSolutions software version 5.57 (Shimadzu, Kyoto, Japan). The HPLC method estimate the baseline 24S-hydroxycholesterol at 30 µg/mg tissue, which roughly agree with that reported in literature. The 24S-hydroxycholesterol peak was not detected from brain samples derived from CH24H-KO mice, suggesting that the HPLC condition sufficiently separate 24S-hydroxycholesterol from other contaminants that would undermine the quantification ( Supplementary  Fig. S13).
Animal models. CH24H-KO mice were purchased (B6. 129S7-Cyp46a1Rus/J, Jackson Laboratory, Sacramento, CA, USA). APP/PS1-Tg mice were obtained by cross-breeding APP-SW mice (Tg2576) 28 with PS-1M146L knock-in mice 29 . The female mice were maintained in house and used for the survival experiment at 7 weeks of age. The animals were housed at a temperature of 22 °C ± 1 °C (mean ± s.d.) with a 12 h light-dark cycle (lights on from 07:00 to 19:00) and allowed free access to food and water. Statistical analysis. The statistical significance between two experimental groups was evaluated by Student's t-test, with probability values (P) less than 5% considered significant (*P < 0.05, **P < 0.01). To evaluate dose dependency, one-tailed Williams' test was used and probability values less than 2.5% were considered significant ( # P < 0.025).

Data availability
The data that support the findings of this study are available on request from the corresponding author (T.N.). The data are not publicly available due to Takeda Pharmaceuticals publication policy. www.nature.com/scientificreports/