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| Targets |
Bullatine B (neoline) inhibits the voltage-gated sodium channel (VGSC) subtype Nav1.7. The IC50 for inhibition of Nav1.7 peak current is 25.8 nM (determined in HEK293 cells expressing human Nav1.7) [2].
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| ln Vitro |
Bullatine B (neoline) (1 µM) significantly inhibited Nav1.7 VGSC peak current in HEK293 cells expressing human Nav1.7. The inhibition started at approximately -20 mV and reached a maximum at 0 mV, decreasing the peak current by 44.6 ± 5.8% compared to before treatment [2].
The concentration-dependent inhibition of Nav1.7 peak current by Bullatine B (neoline) showed maximal inhibition (45.8 ± 5.8%) at 10 µM, with an IC50 of 25.8 nM [2]. Unlike processed aconite root (PA) or GJG extract, treatment with 1 µM Bullatine B (neoline) did not cause a shift in the half-maximal inactivation voltage (V1/2) of Nav1.7 VGSCs (ΔV1/2 = -1.73 ± 0.72 mV, not significant). However, it significantly changed the slope factor (k) of the activation curve (Δk = 1.80 ± 0.46) [2]. |
| ln Vivo |
In a mouse model of paclitaxel-induced peripheral neuropathy, daily subcutaneous (s.c.) injection of Bullatine B (neoline) (10 mg/kg/day between days 0 and 8) significantly recovered the mechanical threshold (assessed by von Frey test) on day 6, and by day 9 the threshold was at the same level as on day 0. A single acute injection on day 0 did not significantly alter the decreasing curve of the threshold [1].
In a mouse model of partial sciatic nerve ligation (Seltzer model), s.c. injection of Bullatine B (neoline) (10 mg/kg/day between days 7 and 21) significantly ameliorated the mechanical allodynia. Mechanical thresholds on days 20 and 22 were statistically improved compared to the control group, reaching the same level as the sham-operated group [1]. In a mouse model of oxaliplatin-induced peripheral neuropathy, Bullatine B (neoline) (10 mg/kg/day s.c. between days 0 and 6) alleviated mechanical allodynia (two-way ANOVA indicated significant effects of oxaliplatin and neoline treatment) [1]. In streptozotocin (STZ)-induced diabetic mice, s.c. administration of Bullatine B (neoline) significantly increased the mechanical nociceptive threshold (50% paw withdrawal threshold) at 30 to 60 minutes after administration, without affecting the threshold in non-diabetic mice. The effect diminished by 90 minutes. Two-way ANOVA showed a significant main effect of neoline treatment, time, and interaction. The dose-response study (1-10 mg/kg) determined that 10 mg/kg produced a significant increase at 30 min post-administration [2]. |
| Enzyme Assay |
Whole-cell patch clamp recording was performed to assess the effect of Bullatine B (neoline) on Nav1.7 VGSC current. Human HEK293 cells expressing Nav1.7 VGSCs were used. The bath solution contained 140 mM NaCl, 0.03 mM CaCl2, 10 mM HEPES, 10 mM MgCl2, and 10 mM D-glucose (pH 7.4 with tetraethylammonium hydroxide). The microelectrode solution contained 115 mM CsCl2, 25 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, and 10 mM HEPES (pH 7.4 with CsOH). Cells were held at -60 mV. For activation, depolarizing pulses from -80 to 20 mV (20 ms duration, 10 mV increments) were applied. For inactivation, a 1-s prepulse from -100 to 20 mV (10 mV increments) was followed by a test pulse to 0 mV (20 ms). Currents were recorded at 50 kHz after low-pass filtering at 3 kHz. Leak currents were subtracted using the P/4 protocol. Series resistance was compensated. To test neoline, cells were perfused with bath solution containing neoline at indicated concentrations. Final DMSO concentration was 0.2%. Data were analyzed using pCLAMP software. The concentration-response data were fitted with a logistic function to determine IC50 [2].
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| Cell Assay |
HEK293 cells expressing human Nav1.7 VGSCs were used. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 250 µg/ml geneticin at 37°C. For electrophysiological experiments, cells cultured for 2-4 days on poly-L-lysine-coated cover glass were used. The effect of Bullatine B (neoline) on Nav1.7 peak current was measured using whole-cell patch clamp as described in the Enzyme Assay section. Treatment with 1 µM neoline drastically decreased Nav1.7 VGSC current elicited by depolarization [2].
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| Animal Protocol |
For paclitaxel-induced neuropathy: Male ddY mice (5-6 weeks old) were injected intraperitoneally (i.p.) with paclitaxel (12 mg/kg, diluted in saline containing 10% Kolliphor EL) on day 0. Bullatine B (neoline) was dissolved in saline containing 0.1% DMSO and injected subcutaneously (s.c.) at 10 mg/kg/day. In the acute treatment group, neoline was injected immediately after paclitaxel on day 0 only. In the repeated treatment group, neoline was injected daily immediately following von Frey test threshold measurement from day 0 to day 8. Control mice received s.c. saline with 0.1% DMSO. Mechanical allodynia was evaluated on days -1, 1, 3, 6, 9 [1].
For partial sciatic nerve ligation (Seltzer model): Male ddY mice were anesthetized with pentobarbital (80 mg/kg i.p.), the left sciatic nerve was exposed and 30-50% was ligated with 8-0 silk suture on day 0. Sham operation was exposure without ligation. Bullatine B (neoline) (10 mg/kg s.c.) was injected daily from day 7 to day 21 immediately following von Frey test measurement. Mechanical allodynia was evaluated on days -1, 1, 7, 8, 10, 12, 14, 16, 20, 22 [1]. For oxaliplatin-induced neuropathy: Male ddY mice were injected i.p. with oxaliplatin (10 mg/kg diluted in 5% glucose) on day 0. Bullatine B (neoline) (10 mg/kg s.c.) was injected daily from day 0 to day 6 immediately following von Frey test. Mechanical allodynia was evaluated on days -1, 1, 3, 5, 7 [1]. For diabetic neuropathy: Male ICR mice (4 weeks old) were made diabetic by a single intravenous injection of streptozotocin (STZ, 200 mg/kg in 0.1 N citric acid buffer, pH 4.5). Mice with serum glucose >400 mg/dl were used. Bullatine B (neoline) was dissolved in saline and injected subcutaneously at doses of 1, 3, or 10 mg/kg (0.1 ml/10 g body weight). All mice were fasted for 6 h before treatment. Mechanical nociceptive threshold was assessed using von Frey filaments (up-down method) at various time points (0-90 min) after injection. The time-course was evaluated at 10 mg/kg, and dose-response was evaluated at 30 min post-injection [2]. |
| ADME/Pharmacokinetics |
Rats (male Wistar/ST, 9-week-old) were anesthetized with urethane (1.0 g/kg i.p.), jugular veins exposed, and orally administered PA decoction (1 g PA/kg, containing 0.26 mg/kg of Bullatine B (neoline)). Plasma was collected at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 9 h. Neoline was detected in plasma at 15 min post-oral treatment. Pharmacokinetic parameters for neoline: Cmax = 37 ± 14 ng/mL; Tmax = 0.65 ± 0.34 h; T1/2 = 2.8 ± 0.6 h; AUC0-9h = 64 ± 10 ng·h/mL; MRT = 2.8 ± 0.3 h [1].
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| Toxicity/Toxicokinetics |
No direct toxicity data for Bullatine B (neoline) are reported in the provided papers. The papers focus on processed aconite root (PA) which contains reduced levels of toxic alkaloids (aconitine, mesaconitine, hypaconitine). The content of these toxic alkaloids in the PA used was very low: hypaconitine (4.4 µg/kg PA powder), aconitine (7.8 µg/kg), mesaconitine (4.4 µg/kg) [1]. No adverse effects or lethality data for pure neoline are given.
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| References |
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| Additional Infomation |
According to reports, new nematodes have been found in the genera Aconitum, Aconitum variegatum, and other organisms with relevant data.
Bullatine B (neoline) is a minor Aconitum alkaloid. Its content in commercial processed aconite root (PA) products is comparable to that of benzoylmesaconine (the official marker compound for PA quality control in the Japanese Pharmacopoeia), and about half of benzoylmesaconine on average. The coefficient of variation (CV) of neoline content among commercial PA products is the same as that of benzoylmesaconine and the lowest among other alkaloids [1]. During heat-processing (autoclaving) of unprocessed aconite root, the content of neoline is not significantly altered, whereas toxic aconitine, mesaconitine, and hypaconitine are rapidly degraded, and benzoylmesaconine, benzoylaconine, aconine, and hypaconine increase [1]. Bullatine B (neoline) is proposed as a quality marker compound for PA products used in the treatment of neuropathic pain [1]. In STZ-induced diabetic mice, neoline increased the mechanical nociceptive threshold only in diabetic mice, not in non-diabetic mice, suggesting it selectively affects a lowered mechanical nociceptive threshold [2]. |
| Molecular Formula |
C24H39NO6
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|---|---|
| Molecular Weight |
437.5696
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| Exact Mass |
437.277
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| Elemental Analysis |
C, 65.88; H, 8.98; N, 3.20; O, 21.94
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| CAS # |
466-26-2
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| PubChem CID |
120682
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
578.3±50.0 °C at 760 mmHg
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| Melting Point |
159-161 degºC
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| Flash Point |
303.5±30.1 °C
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| Vapour Pressure |
0.0±3.6 mmHg at 25°C
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| Index of Refraction |
1.602
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| LogP |
-1.69
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
31
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| Complexity |
752
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O([H])[C@]12C([H])([H])[C@@]([H])([C@]3([H])[C@@]([H])([C@@]1([H])[C@@]([H])(C3([H])[H])[C@@]13[C@]([H])(C([H])([H])C([H])([H])[C@@]4(C([H])([H])OC([H])([H])[H])C([H])([H])N(C([H])([H])C([H])([H])[H])C1([H])[C@@]2([H])[C@@]([H])([C@@]34[H])OC([H])([H])[H])O[H])O[H])OC([H])([H])[H]
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| InChi Key |
XRARAKHBJHWUHW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H39NO6/c1-5-25-10-22(11-29-2)7-6-15(26)24-13-8-12-14(30-3)9-23(28,16(13)18(12)27)17(21(24)25)19(31-4)20(22)24/h12-21,26-28H,5-11H2,1-4H3
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| Chemical Name |
11-ethyl-6,18-dimethoxy-13-(methoxymethyl)-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecane-4,8,16-triol
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| Synonyms |
Neoline; Dideacetyldelphisine; Bullatine-B
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~571.34 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 6.25 mg/mL (14.28 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 62.5 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. Solubility in Formulation 2: ≥ 6.25 mg/mL (14.28 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 62.5 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2853 mL | 11.4267 mL | 22.8535 mL | |
| 5 mM | 0.4571 mL | 2.2853 mL | 4.5707 mL | |
| 10 mM | 0.2285 mL | 1.1427 mL | 2.2853 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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