SR8278

REV-ERBα(EC50 = 0.47 μM)

SR8278 对 REV-ERBα 转录抑制活性的 EC50 为 0.47μM[1]。 SR8278在结构上与激动剂相似，但在共转染试验中阻断了GSK4112增强REV-ERBα依赖性抑制的能力。此外，GSK4112抑制参与糖异生的REV-ERBα靶基因的表达，而SR8278刺激这些基因的表达。因此，SR8278代表了一种探测REV-ERB功能的独特化学工具，可以作为进一步优化的起点，以开发具有探索昼夜节律和代谢功能能力的REV-ERB拮抗剂[1]。

在 6-OHDA 损伤的小鼠中，SR8278（慢速微注射；20 μg/小鼠）可恢复其昼夜节律的情绪相关行为，并以昼夜节律时间依赖性方式具有抗抑郁和抗焦虑作用 [1]。慢速显微注射（SR8278；20 μg/小鼠）丰富了 REV-ERBα 和 NURR1 识别的 R/N 基序，同时恢复了两种蛋白与酪氨酸羟化酶启动子的结合活性 [1]。

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SR8278对REV-ERBα活性的药理学抑制恢复了PD小鼠模型中的昼夜节律情绪相关行为。 SR8278微量注射改变VTA中Rev erbα和Nurr1的剩余DA能神经元特异性转录水平。 SR8278微量注射可恢复黎明时VTA中REV-ERBα和NURR1与TH启动子和TH蛋白水平结合活性的拮抗串扰。 SR8278处理在黎明时诱导REV-ERBα和NURR1结合基序的富集。[3]

稳定性[2]
测量了SR8278在血浆中的长期（-80°C下10天）和三次冻融循环稳定性。稳定性表示为不同操作后的浓度与时间零点的浓度。

HEK293细胞在37°C、5%CO2的条件下，在添加了10%胎牛血清的Dulbecco改良Eagles培养基（DMEM）中维持。HepG2细胞在37°C、5%CO2的条件下，在添加了10%胎牛血清的最低必需培养基中维持和常规增殖。转染前24小时，将HepG2细胞以15×103个细胞/孔的密度铺在96孔板上。使用LipofectamineTM 2000（Invitrogen）进行转染。转染后16小时，用载体或化合物处理细胞。治疗后24小时，使用Dual-GloTM萤光素酶测定系统测量萤光素酶活性。所示值代表四个独立转染孔的平均值±S.E。实验重复了至少三次。REV-ERBα和报告子结构已在前面描述[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 PK parameters of SR8278 were obtained by the 3-compartmental model, using WinNonlin 3.3, and the results are shown in Table 4. The predicted concentrations for each time point were plotted in Fig. 5. As it is shown, the predicted concentrations are close to those detected values, which indicated the 3-compartment PK model fits the data very well. After i.v. administration, the initial concentrations (C0) of SR8278 in normal rats and diabetic rats were 2410.25 ± 202.36 vs. 3742.11 ± 1300.21 ng/mL, respectively. The low drug concentration in the plasma could be explained by its rapid distribution into other compartments, except the central compartment. The large distribution volumes (V) of SR8278 in normal rats and diabetic rats were 44401.58 ± 2106.63 and 43516.94 ± 22982.75 mL/kg, respectively. Moreover, it was also noted that the elimination half-lives (t1/2) of SR8278 were really short, which were only 0.17 ± 0.084 (normal rats) and 0.11 ± 0.04 h (diabetic rats), because of the fast elimination rate constant (K10). The AUC values of SR8278 were 608.33 ± 295.25 vs. 598.59 ± 276.92 ng·h/mL, in normal rats and diabetic rats, respectively. The limited blood exposure and fast clearance may represent a major problem in the clinical application of SR8278.[2]

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The stabilities of SR8278 in plasma were 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, which demonstrated that the SR8278 in plasma samples was stable under storage conditions. In addition, we also determined the stability of SR8278 in 50% methanol solution after 24 h storage at room temperature. The results showed that SR8278 was stable in 50% methanol solution for more than 24 h (Table 3), thus allowing adequate time for the completion of the assay. The good results of the method validation parameters, including recovery, viability, also demonstrated the stability of SR8278 during sample preparation and until analysis was completed. [2]

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The validated analytical method was used to identify the PK behaviors of SR8278 in normal rats and diabetic rats. The mean plasma concentration-time curves of SR8278 in the two groups of rats are shown in Fig. 5. Following administration of SR8278 (2 mg/kg, i.v.), the plasma concentrations of SR8278 in the diabetic rats were slightly higher than those in normal rats at each time point. However, these differences were not statistically significant, suggesting that diabetes has 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.

There is a relationship between circadian rhythm and metabolic disorders. The active agent, SR8278, could competitively bind to and inhibit the nuclear receptor, Rev-erb (a major modulator of mammalian circadian clock system), to regulate the metabolism in organisms. However, we had limited knowledge of the pharmacokinetic (PK) characteristics of SR8278. Here, we describe a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify SR8278 in vivo. The linearity range and the limit of quantification (LOQ) for SR8278 were 30-3000 ng/mL and 6 ng/mL, respectively. The inter-day and intra-day variability were within 10%. This UPLC-MS/MS method was successfully used to characterize the PK behaviors of SR8278 in normal and diabetic rats after intravenous (i.v.) injection at a dosage of 2mg/kg. No significant differences were observed in the PK parameters of SR8278 in normal and diabetic rats. Specifically, the values of areas under plasma concentration time curves (AUC), initial plasma concentrations (C0), elimination half-lives (t1/2), and clearances (CL) 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, 3330.83 ± 1609.48 vs. 3364.81 ± 1111.38 mL/kg·h for SR8278 in normal rats vs. diabetic rats, respectively. In conclusion, a UPLC-MS/MS method was successfully developed and validated for the first time, with a wide linearity range, low LOQ, small sample volume (10 μL), rapid analysis (4 min) and excellent recoveries (>80%). It was also used to clarify the PK characteristics of SR8378 in rats. The same PK behaviors of SR8278 in normal and diabetic rats showed that diabetes may have little or no effect on the disposition, metabolism and/or elimination in vivo, which may be of great importance for future clinical studies. [2]

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Parkinson's disease is a neurodegenerative disease characterized by progressive dopaminergic neuronal loss. Motor deficits experienced by patients with Parkinson's disease are well documented, but non-motor symptoms, including mood disorders associated with circadian disturbances, are also frequent features. One common phenomenon is "sundowning syndrome," which is characterized by the occurrence of neuropsychiatric symptoms at a specific time (dusk), causing severe quality of life challenges. This study aimed to elucidate the underlying mechanisms of sundowning syndrome in Parkinson's disease and their molecular links with the circadian clock. We demonstrated that 6-hydroxydopamine (6-OHDA)-lesioned mice, as Parkinson's disease mouse model, exhibit increased depression- and anxiety-like behaviors only at dawn (the equivalent of dusk in human). Administration of REV-ERBα antagonist, SR8278, exerted antidepressant and anxiolytic effects in a circadian time-dependent manner in 6-OHDA-lesioned mice and restored the circadian rhythm of mood-related behaviors. 6-OHDA-lesion altered DAergic-specific Rev-erbα and Nurr1 transcription, and atypical binding activities of REV-ERBα and NURR1, which are upstream nuclear receptors for the discrete tyrosine hydroxylase promoter region. SR8278 treatment restored the binding activities of REV-ERBα and NURR1 to the tyrosine hydroxylase promoter and the induction of enrichment of the R/N motif, recognized by REV-ERBα and NURR1, as revealed by ATAC-sequencing; therefore, tyrosine hydroxylase expression was elevated in the ventral tegmental area of 6-OHDA-injected mice, especially at dawn. These results indicate that REV-ERBα is a potential therapeutic target, and its antagonist, SR8278, is a potential drug for mood disorders related to circadian disturbances, namely sundowning 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

100.15

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.92 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.92 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.92 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。