REV-ERBα(EC50 = 0.47 μM)

SR8278 exhibits an EC50 of 0.47μM for REV-ERBα transcriptional inhibition inhibitory activity[1].SR8278 is structurally similar to the agonist but blocks the ability of the GSK4112 to enhance REV-ERBα-dependent repression in a cotransfection assay. Additionally, whereas GSK4112 suppresses the expression of REV-ERBα target genes involved in gluconeogenesis, SR8278 stimulates the expression of these genes. Thus, SR8278 represents a unique chemical tool for probing REV-ERB function and may serve as a point for initiation of further optimization to develop REV-ERB antagonists with the ability to explore circadian and metabolic functions[1].

In 6-OHDA-lesioned mice, SR8278 (slow microinjection; 20 μg/mouse) restores emotion-related behaviors to their circadian rhythms and has antidepressant and anxiolytic effects in a circadian time-dependent manner [1]. ??Slow microinjection (SR8278; 20 μg/mouse) enriches R/N motifs recognized by REV-ERBα and NURR1 while restoring binding activity of both proteins to the tyrosine hydroxylase promoter [1].

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Pharmacological Inhibition of REV-ERBα Activity with SR8278 Restored Circadian Mood-Related Behaviors in PD Mouse Models. SR8278 Microinjection Alters Remaining DAergic Neuron-Specific Transcription Levels of Rev-erbα and Nurr1 in the VTA. SR8278 Microinjection Restores Antagonistic Crosstalk of REV-ERBα and NURR1 Binding Activity to TH Promoter and TH Protein Levels in VTA at Dawn. SR8278 Treatment Induces Enrichments of REV-ERBα and NURR1 Binding Motifs at Dawn.[3]

Stability [2]
Long-term (−80 °C for 10 days) and three freeze-thaw cycle stabilities of SR8278 in plasma were measured. Stabilities were expressed as the concentrations after different operations to the concentration at time zero.

HEK293 cells were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. HepG2 cells were maintained and routinely propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. 24 h prior to transfection, HepG2 cells were plated in 96-well plates at a density of 15 × 103 cells/well. Transfections were performed using LipofectamineTM 2000 (Invitrogen). Sixteen h post-transfection, the cells were treated with vehicle or compound. 24 h post-treatment, the luciferase activity was measured using the Dual-GloTM luciferase assay system. The values indicated represent the means ± S.E. from four independently transfected wells. The experiments were repeated at least three times. The REV-ERBα and reporter constructs have been previously described [1].

Animal/Disease Models: 6-OHDA injured mice [1]
Doses: 20 μg/mouse
Route of Administration: slow microinjection; 20 μg/mouse
Experimental Results: The emotion-related behavioral defects of 6-OHDA injured mice were restored. Altered remaining DAergic neuron-specific REV-ERBα and Nurr1 transcript levels in the VTA. Restoration of REV-ERBα and NURR1 binding activity is associated with antagonistic crosstalk of TH promoter and TH protein levels in the VTA. Dawn induces enrichment of REV-ERBα and NURR1 binding motifs.

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Local Injection of SR8278 [3]
The local injection of SR8278 into the midbrain towards the VTA was performed 3 h before each behavioral test under a dim red light. We followed the time-regiment, for handling animal care and slow microinjection of SR8278 to the VTA 3 h before behavioral tests, as shown previously. SR8278 was dissolved in ethanol to a concentration of 50 µg/µL. SR8278 (20 µg/mouse) or the corresponding vehicle (ethanol) was directly microinfused into the VTA using a 33-gauge injector cannula attached to a 10-µL Hamilton syringe at a rate of 0.1 µL/min. For the microinfusion, mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), mounted on a stereotaxic apparatus, and unilaterally implanted with a 26-guage stainless steel cannula into the midbrain towards VTA (AP −3.2 mm, ML −0.5 mm, DV −3.5 mm). A 32-gauge dummy cannula was inserted into each guide cannula to prevent clogging. Once the jewelry screws were implanted in the skull as anchors, the whole assembly was affixed to the skull with resin cement.[3]

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Pharmacokinetic studies[2]
Before SR8278 administration, a cannula was introduced into the jugular vein for injection of formulations and blood collection followed by previously published procedures. The rats were anesthetized by anesthesia cocktail (Ketamine 50 mg/mL, Xylazine 3.3 mg/mL and Acetopromazine 3.3 mg/mL) prior to the jugular vein surgery and administrated carprofen subcutaneously after the surgery [2].
Rats were dosed with SR8278 at 2 mg/kg through jugular vein cannula. Blood samples (200 μL) were withdrawn at 5, 10, 15, 30, 45 min, and 1, 1.5, 2, 4, and 6 h post dose from jugular vein cannula. The volume of blood removed at each sampling time was replaced with an equal volume of saline. And the blood samples were immediately centrifuged at 4 °C to separate the plasma fraction from the blood cells, and the plasma samples were stored at −80 °C until analysis.[2]

The pharmacokinetic parameters of SR8278 were calculated using a three-compartment model with WinNonlin 3.3 software, and the results are shown in Table 4. The predicted concentrations at each time point are shown in Figure 5. As shown in the figure, the predicted concentrations are close to the measured values, indicating that the three-compartment pharmacokinetic model fits the data well. After intravenous administration, the initial concentrations (C0) of SR8278 in normal rats and diabetic rats were 2410.25 ± 202.36 ng/mL and 3742.11 ± 1300.21 ng/mL, respectively. The lower plasma drug concentrations may be due to the rapid distribution of the drug to the compartments other than the central compartment. The volumes of distribution (V) of SR8278 in normal rats and diabetic rats were 44401.58 ± 2106.63 mL/kg and 43516.94 ± 22982.75 mL/kg, respectively. In addition, due to its fast elimination rate constant (K10), the elimination half-life (t1/2) of SR8278 is also extremely short, only 0.17 ± 0.084 h in normal rats and only 0.11 ± 0.04 h in diabetic rats. The AUC values of SR8278 in normal rats and diabetic rats were 608.33 ± 295.25 ng·h/mL and 598.59 ± 276.92 ng·h/mL, respectively. The limited blood exposure and rapid clearance of SR8278 may be a major problem for its clinical application. [2] The stability of SR8278 in plasma was evaluated at -80 °C for 10 days and after three freeze-thaw cycles (Table 3). The recoveries of all samples were between 90.82% and 95.75% after various stability tests, indicating that SR8278 in plasma samples was stable under storage conditions. In addition, we determined the stability of SR8278 after storage at room temperature in 50% methanol solution for 24 hours. The results showed that SR8278 was stable in 50% methanol solution for more than 24 hours (Table 3), thus providing sufficient time to complete the assay. The good results of the method validation parameters (including recovery and activity) also indicated that SR8278 remained stable during sample preparation and analysis. [2]

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The pharmacokinetic behavior of SR8278 in normal rats and diabetic rats was determined using a validated analytical method. The mean plasma concentration-time curves of SR8278 in the two groups of rats are shown in Figure 5. After intravenous injection of SR8278 (2 mg/kg), the plasma concentration of SR8278 in diabetic rats was slightly higher than that in normal rats at all time points. However, these differences were not statistically significant, indicating that diabetes had no effect on the in vivo behavior of SR8278. [2]

[1]. Identification of SR8278, a synthetic antagonist of the nuclear heme receptor REV-ERB. ACS Chem Biol. 2011 Feb 18;6(2):131-4.

[2]. A validated ultra-performance liquid chromatography-tandem mass spectrometry method to identify the pharmacokinetics of SR8278 in normal and streptozotocin-induced diabetic rats. J Chromatogr B Analyt Technol Biomed Life Sci. 2016 May 1;1020:142-7.

[3]. Pharmacological Rescue with SR8278, a Circadian Nuclear Receptor REV-ERBα Antagonist as a Therapy for Mood Disorders in Parkinson's Disease. Neurotherapeutics. 2022 Mar;19(2):592-607.

link exists between circadian rhythms and metabolic disorders. The active ingredient SR8278 competitively binds to and inhibits the nuclear receptor Rev-erb (a major regulator of the mammalian circadian rhythm system), thereby regulating the organism's metabolism. However, our understanding of the pharmacokinetic (PK) characteristics of SR8278 is limited. This article describes a sensitive and reproducible ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantitative analysis of SR8278 in vivo. The linear range and limit of quantitation (LOQ) of SR8278 are 30–3000 ng/mL and 6 ng/mL, respectively. The inter- and intra-day coefficients of variation are both within 10%. This UPLC-MS/MS method was successfully used to characterize the pharmacokinetic behavior of SR8278 after intravenous injection of a 2 mg/kg dose in normal and diabetic rats. No significant differences in the pharmacokinetic parameters of SR8278 were observed between normal and diabetic rats. Specifically, the area under the plasma concentration-time curve (AUC), initial plasma concentration (C0), elimination half-life (t1/2), and clearance rate (CL) of SR8278 in normal rats and diabetic rats were 608.33 ± 295.25 vs. 598.59 ± 276.92 ng·h/mL, 2410.25 ± 202.36 vs. 3742.11 ± 1300.21 ng/mL, 0.17 ± 0.08 vs. 0.11 ± 0.04 h, and 3330.83 ± 1609.48 vs. 3364.81 ± 1111.38 mL/kg·h, respectively. In summary, we have successfully established and validated a UPLC-MS/MS method for the first time, which has the advantages of wide linear range, low limit of quantitation, small sample volume (10 μL), fast analysis speed (4 min) and high recovery rate (>80%). This method was also used to elucidate the pharmacokinetic characteristics of SR8378 in rats. SR8378 showed the same pharmacokinetic behavior in normal rats and diabetic rats, indicating that diabetes may have little or no effect on the distribution, metabolism and/or elimination of SR8378 in vivo, which is of great significance for future clinical research. [2] Parkinson's disease is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons. Motor disorders in patients with Parkinson's disease have been well established, but non-motor symptoms, including mood disorders associated with circadian rhythm disturbances, are also common features. A common phenomenon is "sunset syndrome", which is characterized by the appearance of neuropsychiatric symptoms at a specific time (dusk), leading to a severe decline in quality of life. This study aims to elucidate the potential mechanism of sunset syndrome in Parkinson's disease and its molecular link with the biological clock. We found that 6-hydroxydopamine (6-OHDA)-damaged mice, serving as a mouse model of Parkinson's disease, exhibited increased depressive and anxiety-like behaviors only at dawn (equivalent to dusk in humans). Administration of the REV-ERBα antagonist SR8278 exerted antidepressant and anti-anxiety effects in a circadian rhythm-dependent manner in 6-OHDA-damaged mice, restoring the circadian rhythms of mood-related behaviors. 6-OHDA damage alters the transcription of dopaminergic-specific Rev-erbα and Nurr1, as well as the atypical binding activity of REV-ERBα and NURR1 (upstream nuclear receptors in the promoter region of tyrosine hydroxylase). SR8278 treatment restored the binding activity of REV-ERBα and NURR1 to the tyrosine hydroxylase promoter and induced the enrichment of R/N motifs recognized by REV-ERBα and NURR1, as confirmed by ATAC sequencing; consequently, tyrosine hydroxylase expression was elevated in the ventral tegmentum of 6-OHDA-injected mice, especially at dawn. These results suggest that REV-ERBα is a potential therapeutic target, and its antagonist SR8278 is a potential drug for treating mood disorders associated with circadian rhythm disturbances, namely sunset syndrome in Parkinson's disease. [3]

C18H19NO3S2

361.47

361.081

C, 59.81; H, 5.30; N, 3.87; O, 13.28; S, 17.74

1254944-66-5

1254944-66-5

53393127

White to light yellow solid powder

3.538

0

5

5

24

473

0

UIEBLUZPSFAFOC-UHFFFAOYSA-N

InChI=1S/C18H19NO3S2/c1-3-22-18(21)14-10-12-6-4-5-7-13(12)11-19(14)17(20)15-8-9-16(23-2)24-15/h4-9,14H,3,10-11H2,1-2H3

ethyl 2-{[5-(methylsulfanyl)thiophen-2-yl]carbonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

SR8278; SR 8278; ethyl 2-{[5-(methylsulfanyl)thiophen-2-yl]carbonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; ethyl 2-(5-methylsulfanylthiophene-2-carbonyl)-3,4-dihydro-1H-isoquinoline-3-carboxylate; CHEMBL4754504; ethyl 2-(5-(methylthio)thiophene-2-carbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; SR-8278

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)

DMSO : ~100 mg/mL (~276.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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