GABA (γ-aminobutyric acid) receptor

体外活性：Allopregnanolone，也称为 5α-pregnan-3α-ol-20-one 或 3α,5α-四氢孕酮 (3α,5α-THP)，或 SAGE-547、Brexanolone 和 AllotetraHydroprogesterone，是一种黄体酮代谢物，具有作为 GABA（γ-氨基丁酸）受体的变构调节剂。它具有治疗癫痫和抑郁症的潜力。 Allopregnanolone 是一种由黄体酮合成的内源性抑制性孕烷神经甾体。 Allopregnanolone 具有与 GABAA 受体上 GABA 作用的其他正变构调节剂（例如苯二氮卓类药物）相似的作用，包括抗焦虑、镇静和抗惊厥活性。内源性产生的四氢孕酮通过微调 GABAA 受体并调节 GABAA 受体上的几种正变构调节剂和激动剂的作用，发挥关键的神经生理学作用。 Allopregnanolone 以剂量依赖性方式诱导源自大鼠海马的神经祖细胞和源自大脑皮层的人神经干细胞的增殖显着增加。四氢孕酮增加促进有丝分裂的基因的表达并抑制抑制细胞增殖的基因的表达。其生物合成始于孕酮，通过酶 5α-DHP 转化为二氢孕酮，之后，酶 3α-HSOR 催化二氢孕酮还原为别孕酮。细胞测定：四氢孕酮以剂量依赖性方式诱导源自大鼠海马的神经祖细胞和源自大脑皮层的人神经干细胞的增殖显着增加。四氢孕酮增加促进有丝分裂的基因的表达并抑制抑制细胞增殖的基因的表达。其生物合成始于孕酮，通过酶 5α-DHP 转化为二氢孕酮，之后，酶 3α-HSOR 催化二氢孕酮还原为别孕酮。

Allopregnanolone 增加 P 大鼠中 K+ 诱发的 [3H]-谷氨酸和 [3H]-GABA 释放。神经类固醇还可以增加 VO 大鼠中 [3H]-谷氨酸的基础释放，其作用取决于 NMDA 受体的调节，而 Mg2+ 可逆转这一作用。通过皮下或静脉途径给药的治疗剂量下，30 分钟内四异孕酮小鼠血浆水平在 34-51 ng/mL 之间。在阿尔茨海默病小鼠模型中，四氢孕酮诱导的神经发生与学习和记忆功能的恢复相关，并且在老年正常小鼠中也同样有效。

CDC2和PCNA蛋白表达的蛋白质印迹分析。[1]
通过蛋白质印迹分析，进一步验证了APα对基因表达的影响。在1小时的接种期后将APα加入培养物中，并在指定的时间点裂解细胞。用冷PBS洗涤细胞，并在由0.1%SDS、1%Igepal CA-630（非离子、非变性洗涤剂）、0.2 mm苯甲基磺酰氟和0.01‰蛋白酶抑制剂混合物组成的冰冷裂解缓冲液中在4°C下孵育30分钟。

HT-22细胞培养和MuLV-GFP感染。永生化小鼠海马HT-22细胞系（Sagara等人，2002；Mize等人，2003）用作MuLV-GFP标记分裂细胞的阳性对照。细胞在DMEM（高糖，含l-谷氨酰胺，含盐酸吡哆醇；）中培养，补充100 U/ml青霉素、100 mg/ml链霉素和5%FBS（热灭活）。细胞每4天分裂1-10次。分裂后一天，如上所述，在Allopregnanolone（APα）存在或不存在的情况下，用MuLV-GFP病毒颗粒感染细胞。1.

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CDC2和PCNA蛋白表达的蛋白质印迹分析。Western blot分析在蛋白质水平进一步验证了Allopregnanolone（APα）对基因表达的影响。在1小时的接种期后将APα加入培养物中，并在指定的时间点裂解细胞。1.

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基因阵列分析。为了分析细胞周期基因调控，根据制造商的说明，使用了96个细胞周期调控基因和两个看家基因的市售靶向cDNA阵列。简而言之，原代培养物用或不用500nmAllopregnanolone（APα）处理24小时，并使用TRIzol试剂分离总RNA。[1]

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将大鼠海马神经元接种到60mm皮氏培养皿上进行FACS分析，或接种到载玻片室进行荧光显微镜观察，并在接种后1小时，在有或没有500nmAllopregnanolone（APα）、500nm APβ、10μm硝苯地平或硝苯地平加APα的情况下，用MuLV eGFP病毒颗粒（2.5-3.5×106/ml）感染。4小时后，洗涤细胞，并进一步用含有类固醇的新鲜培养基孵育，以使GFP在感染细胞中表达[1]。

The hypothalamic release of glutamate and GABA regulates neurosecretory functions that may control the onset of puberty. This release may be influenced by neurosteroids such as allopregnanolone. Using superfusion experiments we examined the role of allopregnanolone on the K(+)-evoked and basal [(3)H]-glutamate and [(3)H]-GABA release from mediobasal hypothalamus and anterior preoptic area in prepubertal, vaginal opening and pubertal (P) rats and evaluated its modulatory effect on GABAA and NMDA (N-methyl-d-aspartic acid) receptors. Also, we examined the hypothalamic activity and mRNA expression of 3α-hydroxysteroid oxidoreductase (3α-HSOR) - enzyme that synthesizes allopregnanolone - using a spectrophotometric method and RT-PCR, respectively. Allopregnanolone increased both the K(+)-evoked [(3)H]-glutamate and [(3)H]-GABA release in P rats, being the former effect mediated by the modulation of NMDA receptors - as was reverted by Mg(2+) and by the NMDA receptor antagonist AP-7 and the latter by the modulation of NMDA and GABAA receptors - as was reverted by Mg(2+) and the GABAA receptor antagonist bicuculline. The neurosteroid also increased the basal release of [(3)H]-glutamate in VO rats in an effect that was dependent on the modulation of NMDA receptors as was reverted by Mg(2+). On the other hand we show that allopregnanolone reduced the basal release of [(3)H]-GABA in P rats although we cannot elucidate the precise mechanism by which the neurosteroid exerted this latter effect. The enzymatic activity and the mRNA expression of 3α-HSOR were both increased in P rats regarding the other two studied stages of sexual development. These results suggest an important physiological function of allopregnanolone in the hypothalamus of the P rat where it might be involved in the 'fine tuning' of neurosecretory functions related to the biology of reproduction of the female rats.[3]

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Rabbit IV pharmacokinetics study[2]
Allopregnanolone (Allo) was dissolved in 20%w/v HBCD solution at 1.5 mg/ml by brief sonication. The pH was recorded as 7.1. The formulation was filter sterilized using a 0.2 μm filter.[2]

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Rabbit TD pharmacokinetics study[2]
Allo was added to dimethyl sulfoxide, 20 mg/ml, and vigorously mixed on a vortex mixer and sonicated until Allo was visibly dissolved.[2]

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Mouse pharmacokinetics and efficacy studies[2]
Allo was dissolved in 6%w/v HBCD solution at 0.5 mg/ml by brief sonication and was administered intravenously (IV) to mice at 1.5 mg/kg via lateral tail vein. Allo was dissolved in 20%w/v HBCD solution at 2.5 mg/ml by brief sonication and was subcutaneously (SC) injected to mice at 0.5, 1, and 10 mg/kg. Additionally, Allo was dissolved in 6%w/v SBECD solution at 0.5 mg/ml and injected IV to mice at 0.1, 0.5, and 1 mg/kg. HBCD or SBECD alone were included as vehicle controls. Topical transdermal (TD) was applied on the shaved dorsal surface at 50mg/kg using a gel solution of 3.3% Allo (w/w), 45% DMSO, 30% EtOH, 2.5% Klucel MF, 19.2% PEG-300. Intranasal (IN) formulations were prepared in both 100% castor oil and 20% HBCD. Intramuscular (IM) formulation was administered to mice as Allo 1.5 mg/ml in 6% SBECD. As a positive control to our previous studies, SC 10 mg/kg 2.5 mg/ml; PBS/5%EtOH was administered as a suspension formulation. For 24 h cell proliferation studies, the thymidine analogue, 5-Bromo-2’-deoxyuridine (BrdU), incorporated into newly synthesized DNA of replicating cells during the S-Phase of the cell cycle, was dissolved in PBS and intraperitoneally injected at 100 mg/kg 1 h following Allo treatment.[2]

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Rat sedation/formulations study Allo was dissolved in 6%w/v SBECD at 1.5 mg/ml by brief sonication and administered IV to rats at 0.5–2 mg/kg. Additionally, Allo was dissolved in 24%w/v SBECD at 6 mg/ml, 1.5 mg/ml and 24 mg/ml by brief sonication and administered by SC and intramuscular (IM) routes to the rats in doses ranging from 2–8 mg/kg. As a control comparison to our previous Allo efficacy studies, Allo 2.5 mg/ml dissolved in ethanol, was diluted to 5% solution 95% phosphate buffered saline, administered as a suspension SC to rats at 8 mg/kg and a consistent sedation score of 4, no detectable sedation, was obtained (n = 4; data not shown). A serial dilution test in 24% SBECD, found that Allo reaches a saturation point between 8–10 mg/ml coinciding with a molar ratio of 4–5 SBECD molecules per Allo molecule in water at room temperature without pH adjustment.[2]

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Allopregnanolone is dissolved in 20%w/v HBCD solution at 2.5 mg/mL by brief sonication and is subcutaneously (SC) injected to mice at 0.5, 1, and 10 mg/kg.

Mice and rats

Absorption, Distribution and Excretion
It has been determined that brexanolone has a low oral bioavailability of approximately <5% in adults, which suggests infant exposure would also be expected to be low.
Following the administration of radiolabeled brexanolone, it was observed that 47% of the administrated dose was recovered largely as metabolites in the feces and 42% in urine, where less than 1% as recovered as unchanged brexanolone.
The volume of distribution documented for brexanolone is approximately 3 L/kg, a value which suggests relatively extensive distribution into tissues.
The total plasma clearance determined for brexanolone is approximately 1 L/h/kg.

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Metabolism / Metabolites
Brexanolone is extensively metabolized by non-cytochrome (CYP) based pathways by way of three main routes - keto-reduction (via aldo-keto reductases), glucuronidation (via UDP-glucuronosyltransferases), and sulfation (via sulfotransferases). Three predominant circulating metabolites result from such metabolic pathways and they are all pharmacologically inactive and ultimately do not contribute to the overall efficacy of the medication.

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Biological Half-Life
The terminal half-life observed for brexanolone is approximately 9 hours.

Hepatotoxicity
In premarketing studies, liver test abnormalities were uncommon in patients receiving brexanolone (
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because of the low amounts of brexanolone in milk and low oral bioavailability, brexanolone would not be expected to cause any adverse effects in breastfed infants. If brexanolone is required by the mother, it is not a reason to discontinue breastfeeding. Because excessive sedation or sudden loss of consciousness can occur during brexanolone infusion, it is suggested that patients provide a separate caregiver for any child who is present during the infusion.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
In a study of 12 healthy women given a 60-hour infusion of brexanolone, there were no reports of effects on milk production according to the manufacturer.

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Protein Binding
The plasma protein binding recorded for brexanolone is greater than 99% and was determined to be independent of plasma concentrations.

[1]. J Neurosci.2005 May 11;25(19):4706-18;
[2]. PLoS One.2015 Jun 3;10(6):e0128313;
[3]. Neuroscience.2013 Jul 23;243:64-75.

Pharmacodynamics
Brexanolone potentiated GABA-mediated currents from recombinant human GABA(a) receptors in mammalian cells expressing α1β2γ2 receptor subunits, α4β3δ receptor subunits, and α6β3δ receptor subunits. Moreover, it was determined during a Phase 1 randomized, placebo and positive-controlled, double-blind, three-period crossover thorough QT study in 30 healthy adult subjects that brexanolone use did not prolong the QT interval to any clinically relevant extent when administered at 1.9-times the exposure occurring at the highest recommended infusion rate (90 mcg/kg/hour).

C21H34O2

318.49

318.255

C, 79.19; H, 10.76; O, 10.05

516-54-1

516-54-1; 516-55-2 (Sepranolone); 4406-35-3 (racemic mixture)

92786

Typically exists as solid at room temperature

1.1±0.1 g/cm3

431.2±18.0 °C at 760 mmHg

176-178°

183.9±13.8 °C

0.0±2.3 mmHg at 25°C

1.524

4.89

37.3

1

2

1

23

500

8

O=C(C)[C@H]1CC[C@@]2([H])[C@]3([H])CC[C@@]4([H])C[C@H](O)CC[C@]4(C)[C@@]3([H])CC[C@@]21C

AURFZBICLPNKBZ-SYBPFIFISA-N

InChI=1S/C21H34O2/c1-13(22)17-6-7-18-16-5-4-14-12-15(23)8-10-20(14,2)19(16)9-11-21(17,18)3/h14-19,23H,4-12H2,1-3H3/t14-,15+,16-,17+,18-,19-,20-,21+/m0/s1

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

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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));
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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；
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