GABAA receptor (IC50 = 2 μM)

BicucuLline (1 and 3 μM) ((+)-BicucuLline; d-BicucuLline) produced the highest responses to GABA. BicucuLline is a competitive antagonist of α1β2η2L GABAA receptors because it parallel shifts the GABA dose-response curve to the right without lowering the GABA maximal response [3].

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The convulsant alkaloid BicucuLline continues to be investigated more than 40 years after the first publication of its action as an antagonist of receptors for the inhibitory neurotransmitter GABA. This historical perspective highlights key aspects of the discovery of bicuculline as a GABA antagonist and the sustained interest in this and other GABA antagonists. The exciting advances in the molecular biology, pharmacology and physiology of GABA receptors provide a continuing stimulus for the discovery of new antagonists with increasing selectivity for the myriad of GABA receptor subclasses. Interesting GABA antagonists not structurally related to bicuculline include gabazine, salicylidene salicylhydrazide, RU5135 and 4-(3-biphenyl-5-(4-piperidyl)-3-isoxazole. Bicuculline became the benchmark antagonist for what became known as GABAA receptors, but not all ionotropic GABA receptors are susceptible to bicuculline. In addition, not all GABAA receptor antagonists are convulsants. Thus there are still surprises in store as the study of GABA receptors evolves.[1]

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Small-conductance calcium-activated potassium channels (SK channels) are gated solely by intracellular calcium ions and their activity is responsible for the slow afterhyperpolarization (AHP) that follows an action potential in many excitable cells. Brain slice studies commonly employ a methyl derivative of BicucuLline (bicuculline-m), a GABAA (gamma-aminobutyric acid) receptor antagonist, to diminish the tonic inhibitory influences of GABAergic synapses, or to investigate the role of these synapses in specialized neural networks. However, recent evidence suggests that bicuculline-m may not be specific for GABAA receptors and may also block the slow AHP. Therefore, the effects of bicuculline-m on cloned apamin-sensitive SK2 and apamin-insensitive SK1 channels were examined following expression in Xenopus oocytes. The results show that at concentrations employed for slice recordings, bicuculline-m potently blocks both apamin-sensitive SK2 currents and apamin-insensitive SK1 currents when applied to outside-out patches. Apamin-insensitive SK1 currents run down in excised patches. The potency of bicuculline-m block also decreases with time after patch excision. Site-directed mutagenesis that changes two residues in the outer vestibule of the SK1 pore that confers apamin sensitivity also reduces run down of the current in patches, and endows stable sensitivity to bicuculline-m indistinguishable from SK2. Therefore, the use of bicuculline-m in slice recordings may mask apamin-sensitive slow AHPs that are important determinants of neuronal excitability. In addition, bicuculline-m-insensitive slow AHPs may indicate that the underlying channels have run down. [2]

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The sesquiterpene trilactone bilobalide is one of the active constituents of the 50:1 Ginkgo biloba leaf extract widely used to enhance memory and learning. Bilobalide was found to antagonise the direct action of gamma-aminobutyric acid (GABA) on recombinant alpha(1)beta(2)gamma(2L) GABA(A) receptors. The effect of bilobalide on the direct action of GABA at alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp method was evaluated and compared with the effects of the classical GABA(A) receptor competitive antagonist BicucuLline and noncompetitive antagonist picrotoxinin. Bilobalide (IC(50)=4.6+/-0.5 microM) was almost as potent as bicuculline and pictrotoxinin (IC(50)=2.0+/-0.1 and 2.4+/-0.5 microM, respectively) at alpha(1)beta(2)gamma(2L) GABA(A) receptors against 40 microM GABA (GABA EC(50)). While bilobalide and picrotoxinin were clearly noncompetitive antagonists, the potency of bilobalide decreased at high GABA concentrations suggesting a component of competitive antagonism [3].

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Bicuculline, picrotoxinin and bilobalide dose-dependently inhibited the Cl− conductance generated by 40 μM GABA (Fig. 2A–C). No effects were observed when these compounds were applied on their own at 100 μM. The inhibition dose–response curves of picrotoxinin and bilobalide on 10 μM GABA (EC10), 40 μM GABA (EC50), 100 μM GABA (EC75), 300 μM GABA (EC90) and 1 mM GABA (EC100) are shown in Fig. 3A–E, respectively. Fig. 3B also includes the inhibition dose–response curve of bicuculline on 40 μM GABA (EC50). The IC50 and nH values for each compound are tabulated in Table 1. [3]

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The GABA concentration–effect curve in the presence of 1 and 3 μM bicuculline (Fig. 5A and B) displayed a parallel right shift and attained the maximal response of GABA. GABA response in the presence of 1 and 3 μM bicuculline was 99.5% (P=0.9415) and 101.0% (P=0.0702) of GABA maximal response, respectively (Table 2). Bicuculline at 1 and 3 μM increased GABA EC50 values: 1.6 times (41.0–67.0 μM) and 3.6 times (36.1–129.0 μM), respectively (Table 2). Bicuculline appears to shift the dose–response curves of GABA in parallel to the right without decreasing GABA maximal response, suggesting that it is a competitive antagonist at α1β2γ2L GABAA receptors [3].

BicucuLline can be used to create convulsion models in animal modeling.[5]

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The effects of BicucuLline, a gamma-aminobutyric acid (GABA) antagonist, were investigated in 258 immature rats between the third and 22nd postnatal days. Behavioral and electrocorticographic events were correlated. BicucuLline induced both behavioral and electrographic seizures as early as the third postnatal day, an age when the CD50 for bicuculline was lowest, and therefore the sensitivity to it was the greatest. Bicuculline may thus be a suitable convulsant for epilepsy studies involving rats during the first postnatal week[4].

Electrophysiogical recording [3]
Receptor activity was measured with two-electrode voltage-clamp techniques 2–8 days after injection. Recording microelectrodes were fabricated with a micropipette puller and filled with 3 M KCl solution. Oocytes were placed in a cell bath and voltage clamped at −60 mV. Cells were continuously superfused with ND96 buffer. The currents elicited in response to the application of drugs were recorded using a Geneclamp 500 amplifier, a Mac Lab 2e recorder, and Chart version 3.5.2 program on a Macintosh Quadra 605 computer. Drugs were tested for direct activation of GABA at GABAA receptors. For measurements of inhibitory action of drugs on receptor activation, drugs were added to the buffer solution containing GABA at the concentration producing 10%, 50%, 75%, 90% and 100% of the effect (GABA EC10, EC50, EC75, EC90 and EC100) at the receptors for constructing GABA inhibition dose–response curves. The same procedure, but with a fixed concentration of antagonists and increasing concentrations of GABA, was applied to construct GABA dose–response curves. A washout period of 3–5 min was allowed between each drug application to prevent receptor desensitisation.

Expression of α1β2γ2L GABAA receptors in Xenopus laevis oocytes [3]
Female X. laevis were anaesthetised with 0.17% ethyl 3-aminobenzoate in saline and a lobe of the ovaries surgically removed. The lobe of ovaries was rinsed with OR-2 buffer that contained 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2·6H2O, 5 mM HEPES, pH 7.4, and suspended in a solution of collagenase A (2 mg/ml in OR-2) for 2 h to separate oocytes from connective tissues and follicular cells. Released oocytes were then thoroughly rinsed in ND96 buffer supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline and 50 μg/ml gentamycin, and stage V to VI oocytes were collected. Human α1, β2 and γ2L cDNAs subcloned in pcDM8 were linearised using the restriction enzyme NOT1. Linearised plasmids containing α1, β2 and γ2L cDNAs were transcribed using T7 RNA Polymerase and capped with 5,7-methylguanosine using the “mMESSAGE mMACHINE” kit. Ten nanograms per 50 nl of a 1:1:1 mixture of α1, β2 and γ2L cRNAs were injected using a 15–20 μm diameter tip micropipette into the cytoplasm of individual defolliculated oocytes by using a Nanoject injector. The oocytes were incubated in ND96 buffer at 16 °C in an orbital shaker with a twice-daily change of buffer.

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Pharmacokinetic study [6]
Male Sprague-Dawley rats (200–220 g) were used. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 h after given BicucuLline (15 mg/kg) by manual gavage. The samples were immediately centrifuged at 3000g for 10 min. The plasma obtained (100 μL) was stored at −20 °C until analysis. Plasma BicucuLline concentration versus time for each rat was analyzed by DAS (Drug and statistics) software.

Bicuculline, a phthalide isoquinoline alkaloid is of current interest as an antagonist of gamma-aminobutyric acid (GABA). A simple and sensitive liquid chromatography mass spectrometry method for determination of bicuculline in rat plasma was developed over the range of 5-500ng/mL. After addition of midazolam as internal standard, protein precipitation with acetonitrile-methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1mm×150mm, 5μm) column with acetonitrile -0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 368 for bicuculline and m/z 326 for the IS. Linear calibration was obtained with correlation coefficients r>0.99. The CV of the precision measurements was less than 13%. The accuracy of the method ranged from 93.6% to 100.5%. Mean recoveries of bicuculline in plasma were in the range of 80.5-91.8%. The method was successfully applied to the pharmacokinetic study after gavage administration of 15mg/kg bicuculline in rats.[6]

Toxicity Summary
The action of bicuculline is primarily on the ionotropic GABAA receptors, which are ligand-gated ion channels concerned chiefly with the passing of chloride ions across the cell membrane, thus promoting an inhibitory influence on the target neuron. These receptors are the major targets for benzodiazepines and related anxiolytic drugs. The half-maximal inhibitory concentration (IC50) of bicuculline on GABAA receptors is 3 μM. In addition to being a potent GABAA receptor antagonist, bicuculline can be used to block Ca2+-activated potassium channels. Sensitivity to bicuculline is defined by IUPHAR as a major criterion in the definition of GABAA receptors.

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mouse LD50 intraperitoneal 8480 ug/kg Current Toxicology., 1(199), 1993

[1]. Johnston GA. Advantages of an antagonist: bicuculline and other GABA antagonists. Br J Pharmacol. 2013;169(2):328-336.

[2]. Bicuculline block of small-conductance calcium-activated potassium channels. Pflugers Arch. 1999;438(3):314-321.

[3]. Bilobalide, a sesquiterpene trilactone from Ginkgo biloba, is an antagonist at recombinant alpha1beta2gamma2L GABA(A) receptors. Eur J Pharmacol. 2003;464(1):1-8.

[4]. Bicuculline induced seizures in infant rats: ontogeny of behavioral and electrocortical phenomena. Brain Res Dev Brain Res. 1990 Dec 15;57(2):291-5.

[5]. Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: differential expression of KROX-24, FOS and JUN proteins. Neuroscience. 1992;48(2):315-24.

[6]. Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Mar 15:953-954:143-6.

Bicuculline is a benzylisoquinoline alkaloid that is 6-methyl-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline which is substituted at the 5-pro-S position by a (6R)-8-oxo-6,8-dihydrofuro[3,4-e][1,3]benzodioxol-6-yl group. A light-sensitive competitive antagonist of GABAA receptors. It was originally identified in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae, and several Corydalis species. It has a role as an agrochemical, a central nervous system stimulant, a GABA-gated chloride channel antagonist, a neurotoxin and a GABAA receptor antagonist. It is an isoquinoline alkaloid, a member of isoquinolines and a benzylisoquinoline alkaloid.
Bicuculline is a light-sensitive competitive antagonist of GABAA receptors. It was originally identified in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae, and several Corydalis species.
Bicuculline has been reported in Corydalis repens, Corydalis decumbens, and other organisms with data available.
Bicuculline is a light-sensitive competitive antagonist of GABAA receptors. It was originally identified in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae, and several Corydalis species. Since it blocks the inhibitory action of GABA receptors, the action of bicuculline mimics epilepsy. This property is utilized in laboratories across the world in the in vitro study of epilepsy, generally in hippocampal or cortical neurons in prepared brain slices from rodents. This compound is also routinely used to isolate glutamatergic (excitatory amino acid) receptor function.
An isoquinoline alkaloid obtained from Dicentra cucullaria and other plants. It is a competitive antagonist for GABA-A receptors.

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Immunocytochemistry with specific antisera was used to assess regional levels of six immediate early gene encoded proteins (KROX-24, c-FOS, FOS B, c-JUN, JUN B and JUN D) in the rat hippocampus after 15 min of bicuculline-induced seizures. Serial sections of the dorsal hippocampus were examined at various postictal recovery periods up to 24 h. The results demonstrate a complex temporal and spatial pattern of immediate early gene synthesis and accumulation. Three major categories of immediate early gene products could best be distinguished in the dentate gyrus: KROX-24 and c-FOS showed a concurrent rapid rise with peak levels at 2 h and a return to baseline levels within 8 h after seizure termination. FOS B, c-JUN and JUN B levels increased more gradually with peak intensities in the dentate gyrus reached at 4 h. These immediate early gene products showed above normal levels in various hippocampal subpopulations up to 24 h. JUN D exhibited the most delayed onset combined with a prolonged increase of seizure-induced immunoreactivity. Irrespective of this differential temporal expression profile of individual transcription factors, the sequence of induction in the hippocampal subpopulations was identical for all immediate early gene-encoded proteins examined: first in the dentate gyrus granule cells followed by CA1 and CA3 neurons, respectively. Our data indicate an asynchronous synthesis of several immediate early gene-encoded proteins in the brain after status epilepticus. FOS and JUN proteins act via homo- or heterodimer complexes at the AP-1 and other DNA binding sites. The different time-courses for individual immediate early gene products strongly suggest, that at different time-points after status epilepticus, different AP-1 complexes are effective. In vitro studies have shown that different AP-1 complexes possess different DNA binding affinities as well as different transcriptional regulatory effects. Our results suggest that these molecular mechanisms are also effective in vivo. [5]

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Bicuculline is a competitive antagonist of GABAA receptors (Akaike et al., 1985). The competitive antagonism of bicuculline and noncompetitive antagonism of picrotoxinin at GABAA receptors are also exemplified at the human α1β2γ2L subunit combination. At α1β2γ2L GABAA receptors, bicuculline displayed the general property of the competitive antagonist, producing a parallel shift of GABA concentration–effect curves and having no effect on the maximal response of GABA. [3]

C20H17NO6

367.35

367.105

C, 65.39; H, 4.66; N, 3.81; O, 26.13

485-49-4

38641-83-7；66016-70-4

10237

Off-white to yellow solid powder

1.5±0.1 g/cm3

542.3±50.0 °C at 760 mmHg

196-198 ºC

281.8±30.1 °C

0.0±1.4 mmHg at 25°C

1.665

2.88

0

7

1

27

615

2

CN1CCC2=CC3=C(C=C2[C@H]1[C@H]4C5=C(C6=C(C=C5)OCO6)C(=O)O4)OCO3

IYGYMKDQCDOMRE-ZWKOTPCHSA-N

InChI=1S/C20H17NO6/c1-21-5-4-10-6-14-15(25-8-24-14)7-12(10)17(21)18-11-2-3-13-19(26-9-23-13)16(11)20(22)27-18/h2-3,6-7,17-18H,4-5,8-9H2,1H3/t17-,18+/m0/s1

(R)-6-((S)-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-[1,3]dioxolo[4,5-e]isobenzofuran-8(6H)-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.81 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 (6.81 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 (6.81 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.)