GABAA receptor (IC50 = 2 μM); (+)-Bicuculline is a competitive antagonist of the GABAA receptor (gamma-aminobutyric acid type A receptor) . It inhibits GABA-induced currents by binding to the GABA recognition site on the receptor in a reversible manner . In electrophysiological studies, it shows an IC50 of 9 μM for inhibiting recombinant α1β1γ2 GABAA channels activated by 3 μM GABA, while its effective concentration range in various assays is typically 1–100 μM . The compound does not target GABAB receptors, which are metabotropic G-protein coupled receptors .

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].

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(+)-Bicuculline is a potent and competitive antagonist of GABAA receptors. In radioligand binding assays, it displaces [³H]muscimol from rat brain membranes, demonstrating its ability to bind to the GABA recognition site . In whole-cell patch-clamp recordings, bicuculline competitively antagonizes GABA-induced inward chloride currents in a concentration-dependent manner, shifting the GABA concentration-response curve to the right without affecting the maximal response . It has an IC50 of approximately 1–3 μM for blocking GABAA-induced currents . The antagonism by (+)-bicuculline is reversible, and its effects can be overcome by increasing GABA concentrations . In bovine chromaffin cells, bicuculline suppresses GABA-evoked transmembrane chloride currents recorded using patch-clamp techniques .

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].

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The in vivo effects of (+)-bicuculline are highly dose-dependent and route-dependent. Systemic administration (e.g., intraperitoneal injection of 3 mg/kg) acts as a potent central nervous system convulsant, inducing tonic/clonic seizures and status epilepticus in animal models by blocking GABAergic inhibition throughout the brain . However, studies have identified that local application of bicuculline methiodide into specific brain regions, such as the striatum, can produce an unexpected anticonvulsant effect, protecting rats against seizures induced by systemic application of bicuculline or other convulsants, indicating a complex, region-specific role of GABAergic signaling in seizure generation and suppression . Additionally, direct injection into the thalamic reticular nucleus has been shown to enhance low-threshold calcium spike bursts in neurons, an effect mediated by the blockade of small conductance (SK) calcium-activated potassium channels rather than GABAA receptors .

Radioligand Binding Assay for GABAA Receptors: To assess the binding affinity of bicuculline, a standard protocol involves the displacement of a selective radioligand, such as [³H]muscimol, from rat brain membrane preparations. Membranes are isolated from rat brains (typically cerebral cortex) by homogenization and centrifugation. The membrane preparation is then incubated with a fixed concentration of [³H]muscimol and varying concentrations of (+)-bicuculline. Non-specific binding is determined in the presence of an excess of unlabeled GABA or a high concentration of bicuculline. After incubation, bound and free radioligand are separated by rapid filtration through glass fiber filters, and the radioactivity retained on the filters is measured by liquid scintillation counting. The concentration of (+)-bicuculline required to displace 50% of the specifically bound [³H]muscimol (IC50) can then be calculated .

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.

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Cell-based assays for (+)-bicuculline typically utilize electrophysiological techniques rather than traditional cell viability assays. Whole-Cell Patch-Clamp Recording: Cells (such as HEK293 cells heterologously expressing GABAA receptors or primary neurons) are voltage-clamped at a holding potential, often near the chloride reversal potential. GABA is rapidly applied to the cell via a fast perfusion system to evoke an inward current. To test antagonism, cells are pre-incubated with (+)-bicuculline for a short period before being co-applied with GABA. The reduction in peak current amplitude in the presence of bicuculline is measured. By applying a range of GABA concentrations with and without a fixed concentration of bicuculline, and plotting the resulting concentration-response curves, one can determine the competitive nature of the antagonism, as bicuculline will cause a parallel rightward shift of the GABA dose-response curve without reducing the maximal response .

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.

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Seizure Induction and Assessment in Rats: To study systemic convulsant activity, adult rats (e.g., Sprague-Dawley or Wistar) are often used. (+)-Bicuculline is prepared as a solution (e.g., dissolved in a small volume of 0.1N HCl, diluted with saline, and pH adjusted to approximately 3.0). Animals receive a single intraperitoneal (i.p.) injection at a dose of 3 mg/kg. Following administration, animals are immediately placed in a clean, transparent cage for observation. The latency to onset, duration, and severity of seizure activity are recorded over a standard observation period (e.g., 30–60 minutes). Seizure severity is typically scored using a modified Racine scale. In some models, a sub-convulsant dose (<3 mg/kg) may be used to lower the seizure threshold for testing potential anticonvulsant drugs. It is important to note that bicuculline is light-sensitive and its solutions should be prepared fresh and protected from light .

Bicuron is a phthaloylisoquinoline alkaloid that has attracted considerable attention as a γ-aminobutyric acid (GABA) antagonist. This study established a simple and sensitive liquid chromatography-mass spectrometry (LC-MS) method for determining the concentration of biscuron in rat plasma, with a detection range of 5–500 ng/mL. Midazolam was used as an internal standard, and protein precipitation with acetonitrile-methanol (9:1, v/v) was used as sample pretreatment. Chromatographic separation was performed on a Zorbax SB-C18 column (2.1 mm × 150 mm, 5 μm) using acetonitrile-0.1% formic acid aqueous solution as the mobile phase with gradient elution. Electrospray ionization (ESI) was operated in positive ion mode; selected ion monitoring (SIM) mode was used for quantitative analysis, with target fragment ions at m/z 368 (biscuron) and m/z 326 (internal standard). The correlation coefficient for linear calibration was > 0.99. The coefficient of variation (CV) for precision measurements was less than 13%. The accuracy of the method ranged from 93.6% to 100.5%. The mean recovery of biscoline in plasma ranged from 80.5% to 91.8%. This method has been successfully applied to pharmacokinetic studies in rats after gavage administration of 15 mg/kg biscoline. [6]

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The pharmacokinetic properties of bicuculline have been characterized primarily in rat models following intravenous and intragastric administration. The compound is rapidly absorbed and also rapidly eliminated from systemic circulation . It has a moderate plasma protein binding ratio and exhibits low oral bioavailability, indicating extensive first-pass metabolism or poor absorption from the gastrointestinal tract . Notably, bicuculline is able to cross the blood-brain barrier, allowing it to exert its central nervous system effects. Following systemic administration, the main tissues of distribution are the kidney, liver, and brain, which are sites of high blood flow where the compound is likely to accumulate and be metabolized .

Toxicity Summary
Bicuronium primarily acts on ionotropic GABAA receptors, ligand-gated ion channels mainly responsible for chloride ion transport across the cell membrane, thereby inhibiting target neurons. These receptors are the main targets of benzodiazepines and related anxiolytics. The half-maximal inhibitory concentration (IC50) of biscuronium for GABAA receptors is 3 μM. In addition to being a potent GABAA receptor antagonist, biscuronium can also block Ca2+-activated potassium channels. The International Union of Pharmacology (IUPHAR) defines biscuronium sensitivity as one of the main criteria for defining GABAA receptors. The intraperitoneal LD50 in mice is 8480 μg/kg. Contemporary Toxicology, 1(199), 1993.

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The most significant toxicity of (+)-bicuculline is its potent central nervous system excitatory activity, which manifests as convulsions and epilepsy at doses only slightly higher than those needed for receptor blockade . This proconvulsant effect is a direct consequence of its on-target pharmacology as a GABAA receptor antagonist and represents its dose-limiting toxicity in research applications. For example, systemic administration of 3 mg/kg i.p. is sufficient to induce tonic/clonic seizures and status epilepticus in rats . Beyond this acute neurotoxicity, specific data on chronic toxicity, genotoxicity, or organ-specific toxicity (e.g., hepatotoxicity) are not detailed in the available research literature, as bicuculline is almost exclusively used as an acute research tool rather than a therapeutic candidate.

[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.

Bikuculin is a benzyl isoquinoline alkaloid, chemically named 6-methyl-5,6,7,8-tetrahydro[1,3]dioxacyclopenteno[4,5-g]isoquinoline, in which the 5-pro-S position is substituted with (6R)-8-oxo-6,8-dihydrofurano[3,4-e][1,3]benzodioxacyclopenten-6-yl. It is a competitive antagonist of the photosensitizing GABA_A receptor. Bikuculin was first discovered in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, plants of the Corydalis family, and various Corydalis species. It is used as an agrochemical, central nervous system stimulant, GABA-gated chloride channel antagonist, neurotoxin, and GABA_A receptor antagonist. It is an isoquinoline alkaloid, belonging to the isoquinoline class of compounds and benzylisoquinoline alkaloids. Bikukulin is a photosensitizing GABAA receptor competitive antagonist. It was first discovered in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, plants of the Fumariaceae family, and several plants of the Corydalis genus. Bikukulin has also been reported in Corydalis repens, Corydalis decumbens, and other organisms with relevant data. Bikukulin is a photosensitizing GABAA receptor competitive antagonist. It was first discovered in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, plants of the Fumariaceae family, and several plants of the Corydalis genus. Because bikucurin blocks the inhibitory effects of GABA receptors, its effects are similar to those of epilepsy. Laboratories around the world utilize this property for in vitro studies of epilepsy, typically in hippocampal or cortical neurons prepared from rodent brain slices. This compound is also commonly used to isolate the function of glutamatergic (excitatory amino acid) receptors. Bikucurin is an isoquinoline alkaloid extracted from Dicentra cucullaria and other plants. It is a competitive antagonist of the GABA-A receptor. Immunocytochemical staining with specific antiserum was used to assess the regional expression 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 15 minutes after bicuculin-induced seizures. Serial sections of the dorsal hippocampus were examined during different post-seizure recovery periods lasting up to 24 hours. The results showed that the synthesis and accumulation of immediate early genes exhibited a complex spatiotemporal pattern. In the dentate gyrus, three major immediate early gene products were most clearly distinguishable: the expression levels of KROX-24 and c-FOS increased rapidly 2 hours after seizure termination and returned to baseline levels within 8 hours. The expression levels of FOS B, c-JUN, and JUN B increased more slowly, reaching a peak in the dentate gyrus 4 hours after seizure termination. These immediate early gene products showed higher-than-normal expression levels in different hippocampal subpopulations over a period of up to 24 hours. JUN D expression appeared latest and was accompanied by a sustained increase in seizure-induced immunoreactivity. Despite differences in the expression time of various transcription factors, the induction order of all the immediate early gene-encoded proteins detected in the hippocampal subgroups was the same: first induced in dentate gyrus granular cells, followed by CA1 and CA3 neurons. Our data suggest that the synthesis of several immediate early gene-encoded proteins in the brain is asynchronous after status epilepticus. FOS and JUN proteins function through homo- or hetero-dimeric complexes at AP-1 and other DNA binding sites. The differences in the expression time progression of different immediate early gene products strongly suggest that different AP-1 complexes function at different time points after status epilepticus. In vitro studies have shown that different AP-1 complexes have different DNA binding affinities and different transcriptional regulatory effects. Our results suggest that these molecular mechanisms also work in vivo. [5]

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Bicoline is a competitive antagonist of the GABAA receptor (Akaike et al., 1985). Bicuronite's competitive antagonistic effect on GABAA receptors and cucurbitacin's non-competitive antagonistic effect on GABAA receptors are also observed in the human α1β2γ2L subunit combination. On the α1β2γ2L GABAA receptor, biscuronite exhibits the general characteristics of a competitive antagonist, causing a parallel shift in the GABA concentration-response curve, and has no effect on the maximum response to GABA. [3]

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(+)-Bicuculline (NSC 3219) has a molecular formula of C20H17NO6, a molecular weight of 367.4, and a purity of >99% (HPLC) for research-grade compounds . The compound is light-sensitive and is typically stored as a powder at -20°C, protected from light, where it is stable for up to 3 years . Its IUPAC name is [R-(R,S)]-6-(5,6,7,8-Tetrahydro-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)furo[3,4-e]-1,3-benzodioxol-8(6H)-one . While it is a classic GABAA antagonist, research has shown that quaternary amine derivatives of bicuculline (e.g., bicuculline methiodide, bicuculline methochloride) can also directly block small-conductance calcium-activated potassium (SK) channels, an off-target effect that should be considered when interpreting experimental results .

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.)