GABA (γ-aminobutyric acid) receptor

Allopregnanolone increases both the K+-evoked [3H]-glutamate and [3H]-GABA release in P rats. The neurosteroid also increases the basal release of [3H]-glutamate in VO rats in an effect that is dependent on the modulation of NMDA receptors as is reverted by Mg2+. At therapeutic doses by either subcutaneous or intravenous routes, allopregnanolone mouse plasma levels range between 34-51ng/mL by 30min. Allopregnanolone-induced neurogenesis correlates with restoration of learning and memory function in a mouse model of Alzheimers disease and is comparably efficacious in aged normal mice.

Western blot analyses for CDC2 and PCNA protein expression.[1]
The effects of APα on gene expression were further validated at the protein level by Western blot analyses. APα was added to the cultures after a 1 h seeding period, and cells were lysed at the time points as indicated. Cells were washed with cold PBS and incubated in ice-cold lysis buffer consisting of 0.1% SDS, 1% Igepal CA-630 (nonionic, nondenaturing detergent), 0.2 mm phenylmethylsulfonylfluoride, and 0.01‰ protease inhibitor mixture for 30 min at 4°C.

HT-22 cell culture and MuLV-GFP infection. The immortalized mouse hippocampal HT-22 cell line (Sagara et al., 2002; Mize et al., 2003) was used as a positive control for labeling dividing cells by MuLV-GFP. Cells were cultivated in DMEM (high glucose, with l-glutamine, with pyridoxine hydrochloride;) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 5% FBS (heat inactivated). The cells were split 1-10 every 4 d. One day after splitting, the cells were infected with MuLV-GFP viral particles in the presence or absence of Allopregnanolone (APα) as described above.[1]

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Western blot analyses for CDC2 and PCNA protein expression. The effects of Allopregnanolone (APα) on gene expression were further validated at the protein level by Western blot analyses. APα was added to the cultures after a 1 h seeding period, and cells were lysed at the time points as indicated.[1]

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Gene-array assay. To analyze cell-cycle gene regulation, a commercially available targeted cDNA array of 96 cell-cycle regulatory genes and two housekeeping genes were used according to the instructions of the manufacturer. Briefly, primary cultures were treated with or without 500 nm Allopregnanolone (APα) for 24 h, and total RNA was isolated using TRIzol reagent.[1]

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Rat hippocampal neurons were seeded onto 60 mm Petri dishes for FACS analysis or slide chambers for fluorescent microscopy observation and were infected with MuLV-eGFP viral particles (2.5-3.5 × 106/ml) in the presence or absence of 500 nm Allopregnanolone (APα), 500 nm APβ, 10 μm nifedipine, or nifedipine plus APα 1 h after seeding. After 4 h, cells were washed and further incubated with fresh media with steroids to allow for the GFP expression in infected cells[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

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)

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.)