24(S)-Hydroxycholesterol targets the N-methyl-D-aspartate receptor (NMDAR) as a positive allosteric modulator. In mouse hippocampal neurons, it potentiates NMDA-induced currents with an EC50 of approximately 1.2 μM. The synthetic analog SGE-201 showed an EC50 of 0.11 μM. [3]
It also targets liver X receptors (LXRs) as an agonist, though it is not a critical activator of LXR target genes in vivo. [2]
It is a substrate for CYP46A1 (the synthesizing enzyme) and is metabolized in the liver. [2]
In retinal protection studies, exogenous 24S-HC at 1 μM prevented pressure-induced axonal injury and apoptotic RGC death. [4]
24(S)-Hydroxycholesterol targets multiple receptors and channels. It is an endogenous agonist for liver X receptors (LXRα and LXRβ) with EC50 values of 4 μM and 3 μM, respectively. It also acts as a positive allosteric modulator (PAM) of NMDA receptors with an EC50 of 1.2 μM. Additionally, it modulates Slo1 BK ion channels in a stereospecific manner. By activating LXRs, it regulates the expression of genes involved in cholesterol homeostasis and efflux. Its modulation of NMDA receptors places it at the intersection of cholesterol homeostasis and synaptic signaling.

24(S)-Hydroxycholesterol (10 μM) significantly potentiated NMDA receptor-mediated currents in cultured mouse hippocampal neurons, while cholesterol and other oxysterols (22(R)-HC, 20(S)-HC) were inactive at ≤10 μM. [3]
In human neuroblastoma SH-SY5Y cells, 50 μM 24S-HC induced cell death, which was suppressed by γ-tocopherol but not by γ-tocotrienol. 24S-HC also induced phosphorylation of CaMKII (Thr286) and RIPK1. α-Tocopherol (50 μM) significantly suppressed 24S-HC-induced CaMKII phosphorylation but not RIPK1 phosphorylation. [1]
In HEK293 cells expressing recombinant NMDARs, 24S-HC (1 μM) potentiated NMDA responses, with synthetic analogues SGE-201 and SGE-301 showing stronger effects. The potentiation was not subunit-selective (GluN2A, 2B, 2C, 2D all showed significant potentiation). [3]
In excised outside-out membrane patches from hippocampal neurons, SGE-201 (0.2 μM) robustly potentiated NMDA channel activity, increasing NPo values (channel number/open probability) without changing single-channel current, indicating direct allosteric modulation. [3]
In ex vivo rat retinas, 75 mmHg hydrostatic pressure increased CYP46A1 expression and 24S-HC levels (measured by LC-MS/MS). Exogenous 1 μM 24S-HC prevented high pressure-induced axonal swelling and RGC apoptosis. Conversely, the CYP46A1 inhibitor voriconazole (10 μM) caused severe excitotoxic retinal damage at normobaric pressure, which was prevented by co-administration of 30 μM 24S-HC. [4]

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In vitro, 24(S)-Hydroxycholesterol is a potent activator of LXRα and LXRβ. It has been shown to activate LXRα with an EC50 of 4 μM and LXRβ with an EC50 of 3 μM. It is also a potent positive allosteric modulator of NMDA receptors with an EC50 of 1.2 μM. As a brain-specific oxysterol, it is used in cell-based assays to study cholesterol homeostasis, LXR signaling, and neuroprotection. Its activity is enantiomer-specific, meaning the 24(S) enantiomer has distinct pharmacological properties compared to the 24(R) enantiomer.

In a transgenic mouse model overexpressing human CYP46A1 (C46-HA mice), brain 24S-HC levels were increased approximately twofold (from ~15 to ~30 ng/mg tissue), plasma levels increased fivefold, and fecal excretion increased four- to sevenfold. Despite elevated 24S-HC, no significant activation of LXR target genes (e.g., Cyp7a1, Abca1) was observed in brain or liver. Brain cholesterol precursor levels (lathosterol, etc.) increased, indicating compensatory upregulation of cholesterol synthesis. [2]
In rats, subchronic PCP treatment (5 mg/kg twice daily for 7 days) caused social interaction deficits and novel object recognition impairment. Acute administration of the synthetic 24S-HC derivative SGE-301 (1, 3, 10 mg/kg, i.p.) significantly reversed these cognitive and social deficits. SGE-301 (3 and 10 mg/kg) increased social interaction time from 32 s (vehicle) to 57 s and 48 s respectively. For novel object recognition, SGE-301 at 1 and 3 mg/kg significantly improved discrimination ratios. [3]
In a mouse Y-maze spontaneous alternation test, MK-801 (0.25 mg/kg, i.p.) impaired performance. SGE-201 (3, 10, 30 mg/kg, i.p.) dose-dependently reversed MK-801-induced deficits. [3]

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In vivo, 24(S)-Hydroxycholesterol is a validated circulating biomarker directly reflecting brain cholesterol turnover. Approximately 6-7 mg of this oxysterol is eliminated from the brain per day, crossing the blood-brain barrier into the periphery. Its levels in the blood are used to assess brain cholesterol metabolism and are altered in various neurodegenerative diseases. It functions as an endogenous signaling molecule that can activate LXRs in peripheral tissues. The compound's in vivo activity is intrinsically linked to its role in cholesterol homeostasis and its function as a signaling molecule.

In vitro receptor binding and functional assays for 24(S)-Hydroxycholesterol measure its activity at LXR and NMDA receptors. For LXR activity, a reporter gene assay is commonly used. Cells are transfected with a luciferase reporter gene under the control of an LXR-responsive element and treated with varying concentrations of 24(S)-Hydroxycholesterol. Luciferase activity is then measured to determine the EC50 for receptor activation. For NMDA receptor modulation, electrophysiological techniques such as patch-clamp are used to measure the potentiation of NMDA-induced currents in neurons or cells expressing NMDA receptors. These assays confirm the compound's activity at its targets.

SH-SY5Y cell viability assay (WST-8) : Cells were treated with 50 μM 24S-HC for 24 h with or without vitamin E analogs. Cell viability was measured using WST-8 reagent. 24S-HC significantly decreased viability, and γ-tocopherol (up to 50 μM) dose-dependently suppressed this effect. [1]
Lactate dehydrogenase (LDH) assay: After 24S-HC treatment, LDH release was measured to assess cell death. CaMKII inhibitors mM3 (20 μg/mL) significantly inhibited 24S-HC-induced cell death, while KN62 (10 μg/mL) did not. [1]
Western blotting: Whole cell lysates were immunoblotted with anti-phospho-CaMKII (Thr286), anti-CaMKII, anti-RIPK1, and anti-β-actin. 24S-HC induced CaMKII phosphorylation (53 kDa band), which was reduced by mM3 and by the ACAT1 inhibitor K-604 (5 μM). RIPK1 knockdown by siRNA reduced total CaMKII levels and its phosphorylation. [1]
Nile red staining for lipid droplet-like structures: SH-SY5Y cells treated with 50 μM 24S-HC for 6 h showed increased lipid droplet-like structures, which were not inhibited by γ-tocopherol (50 μM) or γ-tocotrienol (10 μM). [1]
Whole-cell patch-clamp in hippocampal neurons: Cultured neurons were clamped at -70 mV in Mg²⁺-free saline with 0.5 μM glycine. 24S-HC (0.1-30 μM) was preapplied for 40-90 s before 10 μM NMDA application. Currents were potentiated with an EC50 of 1.2 μM. [3]
Excised outside-out patch recording: Membrane patches were excised from hippocampal neurons. NMDA (300 μM) was applied, and SGE-201 (0.2 μM) added. NPo analysis showed increased channel open probability. [3]
HEK293 recombinant NMDAR assay: HEK293 cells expressing human GluN1 and GluN2 subunits were tested with 30 μM NMDA + 5 μM glycine. 24S-HC (1 μM) significantly potentiated currents. [3]

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In vitro cell-based assays for 24(S)-Hydroxycholesterol are used to study its effects on cholesterol metabolism and neuronal function. In a typical assay, neuronal cells or hepatocytes are treated with the compound, and the expression of LXR target genes, such as ABCA1 and CYP7A1, is measured by quantitative PCR. The compound's ability to promote cholesterol efflux can be assessed by measuring the release of labeled cholesterol from cells. For studies on neuroprotection, neurons are treated with 24(S)-Hydroxycholesterol and then exposed to toxic stimuli, with cell viability being measured as an endpoint.

Transgenic mouse model (C46-HA) : Human CYP46A1 cDNA under chicken β-actin promoter was injected into fertilized mouse eggs. Mice were backcrossed with C57Bl/6Ncrl for 7 generations. Animals were killed by CO₂ inhalation; brain, liver, plasma collected. 24S-HC levels measured by GC-MS. [2]
Rat subchronic PCP model: Male Long-Evans rats received PCP (5 mg/kg, i.p.) twice daily for 7 days, followed by a 7-day washout. On day 14, SGE-301 (3, 10, 30 mg/kg in 30% Captisol + 0.01% Tween 80) was administered i.p. 60 min before social interaction test. For novel object recognition, SGE-301 (1, 3, 10 mg/kg) was given 60 min before training. Positive control: risperidone 0.2 mg/kg i.p. [3]
Mouse Y-maze test: Male Swiss CD-1 mice received SGE-201 (3, 10, 30 mg/kg i.p. in 25% hydroxypropyl-β-cyclodextrin in PBS) 1 hour before testing. MK-801 (0.25 mg/kg i.p.) was given 30 min before testing. Spontaneous alternation was recorded for 10 min. [3]
Rat ex vivo retinal pressure model: Eyecups from 28-32 day old rats were placed in aCSF in a closed pressure chamber at 10, 35, or 75 mmHg for 24 h at 30°C. 24S-HC (1, 10, 30 μM) or voriconazole (1, 10 μM) was added to the aCSF. [4]

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In vivo animal experiments for 24(S)-Hydroxycholesterol are not typically performed as a therapeutic intervention, as it is an endogenous metabolite. Instead, studies focus on measuring its levels in the brain and blood as a biomarker. In animal models of neurodegenerative diseases, such as Alzheimer's disease, the levels of 24(S)-Hydroxycholesterol are measured to assess changes in brain cholesterol turnover. Genetically modified mice lacking CYP46A1, the enzyme that produces it, are used to study the physiological role of this oxysterol. These studies help elucidate the connection between cholesterol metabolism and neurodegeneration.

In C46-HA transgenic mice overexpressing human CYP46A1, brain 24S-HC content was approximately 30 ng/mg tissue (twice control), plasma levels ~150 ng/mL (fivefold control), and fecal excretion of free 24S-HC was 4-7 times higher than wild-type (approximately 1.5-2.5 μg/24h vs. 0.3-0.4 μg/24h). [2]
In rats, following intraperitoneal administration of SGE-301 (10 mg/kg), brain concentrations reached ~200 ng/g and plasma concentrations ~300 ng/mL (determined by LC-MS/MS). [3]
In human brain homogenates, endogenous 24S-HC levels are reported to be in the tens of micromolar concentration range (approximately 25 μM). [4]

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24(S)-Hydroxycholesterol has a molecular weight of 402.70 g/mol and a molecular formula of C27H46O2. It is a solid powder with a purity of >98% (HPLC). It is soluble in DMSO. For storage, it is recommended to keep the powder at -20°C for long-term storage (months to years) or at 0-4°C for short-term storage (days to weeks). As an endogenous oxysterol, its levels are tightly regulated in the body. Its pharmacokinetic properties are not studied as a drug, but as a metabolite, it is continuously produced and cleared.

In SH-SY5Y cells, 50 μM 24S-HC induced cell death via necroptosis-like pathways (RIPK1-dependent, caspase-independent). This was not mediated by ROS generation. [1]
In the rat ex vivo retina, the CYP46A1 inhibitor voriconazole (10 μM) caused severe excitotoxic retinal damage characterized by edematous changes in the inner plexiform layer and bull's eye formation in the inner nuclear layer, which was prevented by co-administration of 30 μM 24S-HC. [4]
No in vivo systemic toxicity data (e.g., LD50) for 24S-HC itself are reported in these studies. The synthetic derivatives SGE-201 and SGE-301 were well-tolerated at the doses used (up to 30 mg/kg i.p. in mice/rats). [3]

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24(S)-Hydroxycholesterol is an endogenous metabolite and is not considered toxic at physiological levels. It is a normal component of cholesterol metabolism in the brain. However, elevated levels have been associated with certain pathological conditions. The compound is for research use only and is not intended for therapeutic or diagnostic use in humans. Standard laboratory safety precautions should be followed when handling the compound. As a research chemical, it should be stored properly and handled with care to avoid contamination.

[1]. Tocopherol suppresses 24(S)-hydroxycholesterol-induced cell death via inhibition of CaMKII phosphorylation. Biochimie. 2018 Oct;153:203-209.

[2]. Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo. J Intern Med. 2011 Oct;270(4):377-87.

[3]. The major brain cholesterol metabolite 24(S)-hydroxycholesterol is a potent allosteric modulator of N-methyl-D-aspartate receptors. J Neurosci. 2013 Oct 30;33(44):17290-300.

[4]. 24(S)-Hydroxycholesterol protects the ex vivo rat retina from injury by elevated hydrostatic pressure. Sci Rep. 2016 Sep 22;6:33886.

(24S)-24-hydroxycholesterol is a 24-hydroxycholesterol with an S configuration at position 24. It is a major metabolic breakdown product of cholesterol in the brain. It is a metabolite in mice, as well as a biomarker and a human serum metabolite. Reports and data exist regarding 24-hydroxycholesterol in humans.

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Biosynthesis: 24S-HC is synthesized from cholesterol by CYP46A1, an enzyme expressed predominantly in neurons of the CNS and retina. [2][4]
Mechanism of NMDAR modulation: 24S-HC acts as a positive allosteric modulator at NMDARs, stabilizing the inactive conformation of the receptor? (It potentiates currents but also shows slow onset and reversibility). It binds to a site distinct from pregnenolone sulfate, as occlusion studies showed 24S-HC did not occlude PREGS potentiation. [3]
Role in retinal protection: Elevated hydrostatic pressure (75 mmHg) increases CYP46A1 expression and 24S-HC synthesis in the retina. Exogenous 24S-HC (1 μM) protects against pressure-induced axonal swelling and RGC apoptosis. Voriconazole, a CYP46A1 inhibitor, is retinotoxic at normobaric pressure, and this toxicity is prevented by 24S-HC. [4]
Neuroprotection vs. neurotoxicity: 24S-HC has dual effects: at high concentrations (≥50 μM) it induces necroptosis-like cell death, while at low micromolar concentrations (1-10 μM) it exerts neuroprotective effects against excitotoxicity and pressure-induced damage. [1][3][4]
Synthetic derivatives: SGE-201 and SGE-301 are synthetic analogs with improved potency (EC50 ~0.11 μM for SGE-201) and in vivo efficacy. SGE-301 has a 3α-methyl group designed to improve bioavailability. [3]

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24(S)-Hydroxycholesterol is a research compound and is not approved for any clinical use. It is an endogenous oxysterol produced in the brain by CYP46A1. It functions as an endogenous agonist for LXRα and LXRβ and as a positive allosteric modulator of NMDA receptors. It is the principal mechanism for eliminating cholesterol from the brain. As a validated circulating biomarker, its levels reflect brain cholesterol turnover. It is used as a research tool to study cholesterol homeostasis, neurodegenerative diseases, and LXR and NMDA receptor signaling. It is not a drug and has no therapeutic applications.

C27H46O2

402.652948856354

402.349

C, 80.54; H, 11.52; O, 7.95

474-73-7

24-Hydroxycholesterol;30271-38-6

121948

White to off-white solid powder

1.0±0.1 g/cm3

513.1±23.0 °C at 760 mmHg

174-176°C (lit.)

213.5±17.2 °C

0.0±3.0 mmHg at 25°C

1.536

7.66

2

2

5

29

624

9

O[C@H]1CC[C@@]2(C)C(C1)=CC[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)CC[C@@H](C(C)C)O)CC[C@H]21

IOWMKBFJCNLRTC-XWXSNNQWSA-N

InChI=1S/C27H46O2/c1-17(2)25(29)11-6-18(3)22-9-10-23-21-8-7-19-16-20(28)12-14-26(19,4)24(21)13-15-27(22,23)5/h7,17-18,20-25,28-29H,6,8-16H2,1-5H3/t18-,20+,21+,22-,23+,24+,25+,26+,27-/m1/s1

(3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5S)-5-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

24(S)-hydroxycholesterol; 474-73-7; 24S-hydroxycholesterol; 24S-OHC; 47IMW63S3F; 24S-OH-cholesterol; 24S-OHC; 24S-HC; Cerebrosterol; 24(S)-Saringosterol; 24S-Saringosterol; 24(S) Saringosterol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~20 mg/mL (~49.67 mM)
DMSO : ~0.1 mg/mL (~0.25 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)