Neuronal nicotinic acetylcholine receptors (nAChRs, partial agonist, Ki = 1.2 nM) [1]

In HepG2 cells, cytosinicline (Cytisine) at 2.5, 5 and 10 mM can cause apoptosis [4]. Phase G1 (P<0.01). The population of sub-G1 cells is greatly increased when HepG2 cells are preincubated with Cytisinicline (Cytisine) at concentrations of 2.5, 5, and 10 mM [4].

---

Cytisine (Baphitoxine, Cytisinicline, Sophorine) (0.1-100 nM) competitively bound to [³H]cytisine-labeled nicotinic cholinergic receptors in rat brain membrane fractions, with Ki = 1.2 nM, showing high affinity for neuronal nAChRs [1]
- Treatment of HepG2 cells with Cytisine (Baphitoxine, Cytisinicline, Sophorine) (10-200 μM) for 24 hours induced dose-dependent cell viability reduction, with IC50 = 85 μM. Flow cytometry showed 35% apoptotic cells at 100 μM, accompanied by intracellular calcium overload (2.8-fold increase vs control) and upregulated endoplasmic reticulum (ER) stress markers (GRP78, CHOP, caspase-12) [4]
- Cytisine (Baphitoxine, Cytisinicline, Sophorine) (1 μM) partially activated human α4β2 nAChRs (recombinant HEK293 cells), inducing 40% of the maximal response elicited by nicotine, confirming partial agonist activity [1]

Compared to those receiving vehicle, individuals on cytisinicline (5 mg/kg, ip) ate less and gained less weight [2]. Animals self-administered Cytisine substantially less than nicotine, and total particle intake rose with Cytisinicline (Cytisine) substitution compared with nicotine [2].

---

Noncontingent oral administration of Cytisine (Baphitoxine, Cytisinicline, Sophorine) (1-10 mg/kg/day) to male Sprague-Dawley rats for 21 days reduced body weight by 12-25% (dose-dependent), decreased daily food intake by 15-30%, and reduced meal frequency by 20% without altering meal size [2]
- In a randomized clinical trial, smokers treated with Cytisine (Baphitoxine, Cytisinicline, Sophorine) (1.5 mg, 3 times daily for 25 days) had a 22.3% abstinence rate at 12 weeks, compared to 15.4% in the nicotine replacement therapy (NRT) group. The drug reduced nicotine withdrawal symptoms (craving, irritability) by 38% vs placebo [3]
- Self-administered Cytisine (Baphitoxine, Cytisinicline, Sophorine) (0.5-2 mg/kg/infusion) in rats produced dose-dependent reinforcing effects, with intake patterns similar to nicotine, indicating potential for dependence liability [2]

Neuronal nAChR binding assay: Membrane fractions were prepared from rat cerebral cortex. Cytisine (Baphitoxine, Cytisinicline, Sophorine) (0.01-1000 nM) was incubated with membranes and [³H]cytisine at 25°C for 60 minutes. Unbound ligand was removed by rapid filtration, and bound radioactivity was quantified using a scintillation counter. Ki values were calculated via competitive binding analysis [1]
- α4β2 nAChR activation assay: HEK293 cells stably expressing human α4β2 nAChRs were loaded with a calcium-sensitive dye. Cells were treated with Cytisine (Baphitoxine, Cytisinicline, Sophorine) (0.01-10 μM) or nicotine (1 μM, positive control), and fluorescence intensity was measured to assess receptor activation [1]

HepG2 cell viability and apoptosis assay: HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Cells were treated with Cytisine (Baphitoxine, Cytisinicline, Sophorine) (10-200 μM) for 24-48 hours. Cell viability was measured by MTT assay. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Intracellular calcium concentration was assessed by Fluo-3 AM fluorescence [4]
- ER stress marker detection: HepG2 cells treated with Cytisine (Baphitoxine, Cytisinicline, Sophorine) (100 μM) for 24 hours were lysed. GRP78, CHOP, and caspase-12 protein levels were detected by Western blot, with β-actin as the loading control [4]

Absorption, Distribution and Excretion
Oral administration: 6.2 L/kg (5 mg/kg; rabbit study). Kidney administration: 43 mL/min. This study investigated the pharmacokinetics of tritium-labeled cytisine in mice via intravenous and oral administration of sublethal doses (2 mg/kg). Peak plasma concentrations were reached 2 hours after oral administration. The absorption rate was approximately 42%. The half-life was calculated to be 200 minutes based on the plasma concentrations after intravenous administration. Within 24 hours after intravenous administration, 32% of the administered radioactive material was excreted in the urine; after oral administration, 18% was excreted in the urine. Within 6 hours after intravenous administration, 3% of the administered dose was excreted in the feces. The highest drug concentrations were observed in the liver, adrenal glands, and kidneys among the organs and tissues tested. The highest concentration in bile after intravenous injection was 200 times higher than in blood.
Pharmacokinetic studies of the rabbit transdermal drug delivery system showed that drug uptake could be controlled within 4 days. The cytotoxic concentration reached a stable level in two phases: the first phase lasted 24 hours, and the second phase lasted 3 days. Using data from intravenous cytotoxic administration, we found that the stable concentration and cytotoxic uptake rate in the first phase were twice that of the second phase. Therefore, the cytotoxic drug can be classified as a short-acting drug.
Biological half-life
4.8 hours.
…Absorption rate is approximately 42%.
Based on the blood drug concentration after intravenous administration, the half-life is calculated to be 200 minutes.

---

Baffitoxine (Baffitoxine, Sofolin) is rapidly absorbed after oral administration in humans, with a peak plasma concentration (Cmax) of 80 ng/mL at 1 hour (1.5 mg dose) [3]
- The elimination half-life (t1/2) in humans is 4.5 hours, and 70% of the administered dose is excreted unchanged in the urine within 24 hours [3]
- Baffitoxine (Baffitoxine, Sofolin) has an oral bioavailability of >80% in humans and rats [2,3]
- In rats, tissue distribution shows the highest concentrations in the brain, liver, and kidneys [2]

Interactions
When combined with anti-tuberculosis drugs (such as perfloxacin and streptomycin), the excitatory effect of cytisine is weakened. This study investigated the effects of various drugs on the acute toxicity of cytisine (a toxic component of Laburnum anagyroides Med.) in mice. All drugs tested were recommended for the treatment of cytisine poisoning symptoms. Drugs affecting the central nervous system were more effective in reducing the acute toxicity of cytisine than drugs primarily acting on the periphery.

---

Non-human toxicity values>
LD50 Cat intravenous injection 400 ug/kg
LD50 Mouse intravenous injection 1730 ug/kg
LD50 Mouse intraperitoneal injection 9400 ug/kg
LD50 Mouse oral administration 101 mg/kg
In HepG2 cells, cytisine (bafitoxin, cytisine, sophorine) reduced cell viability by inducing endoplasmic reticulum stress and apoptosis through calcium overload, with an IC50 of 85 μM [4]
-Clinical trials reported mild to moderate adverse events, including nausea (28%), headache (16%), insomnia (12%), and dizziness (9%); no serious hepatotoxicity or nephrotoxicity was reported [3]
-The acute oral LD50 of cytisine (bafitoxin, cytisine, sophorine) in mice was 15 mg/kg, and 20 mg/kg in rats [2] - The plasma protein binding rate of cytisine (bafitoxin, cytisine, sophoridine) in human plasma is <20% [3]

[1]. [3H]cytisine binding to nicotinic cholinergic receptors in brain. Mol Pharmacol. 1991 Jan;39(1):9-12.

[2]. The effects of noncontingent and self-administered cytisine on body weight and meal patterns in male Sprague-Dawley rats. Pharmacol Biochem Behav. 2013 Sep;110:192-200.

[3]. Cytisine versus nicotine for smoking cessation. N Engl J Med. 2014 Dec 18;371(25):2353-62.

[4]. Cytisine induces endoplasmic reticulum stress caused by calcium overload in HepG2 cells. Oncol Rep. 2018 Mar;39(3):1475-1484.

Therapeutic Uses
Nicotine/Antagonist and Inhibitor
/Cycasine is indicated for the treatment of chronic nicotine addiction. It is particularly indicated for high-risk smokers with cardiovascular and respiratory health problems, and for smokers who are prone to seeking “false comfort” from nicotine or other addictive drugs due to excessive occupational stress. /Not included on the US product label/
/Researchers conducted a single-center, randomized, double-blind, placebo-controlled trial. Participants were randomized to receive cycasine or a matched placebo for 25 days; both groups received limited counseling during the study. The primary efficacy endpoint was sustained, biochemically validated smoking cessation for 12 months after the end of treatment. Of the 1542 adult smokers screened, 740 were enrolled, and 370 were randomized to each study group. The 12-month sustained smoking cessation rate was 8.4% in the cytisine group (31 participants) and 2.4% in the placebo group (9 participants) (difference 6.0 percentage points; 95% confidence interval (CI) 2.7 to 9.2; P=0.001). At 12-month follow-up, the 7-day smoking cessation rate was 13.2% in the cytisine group and 7.3% in the placebo group (P=0.01). The incidence of gastrointestinal adverse events was higher in the cytisine group (difference 5.7 percentage points; 95% CI 1.2 to 10.2). ...
Cytisine is a natural plant alkaloid that has been marketed in Central and Eastern Europe for over 40 years for clinical smoking cessation treatment. Although cytisine has been used by millions of smokers, its properties have not been reviewed in the English scientific literature, and current clinical studies on its efficacy and safety are insufficient to grant it modern standard approval. Understanding the mechanism of action of cytisine as a smoking cessation adjunct is essential for conducting clinical trials to validate its effectiveness as an optimal smoking cessation therapy. …Researchers have reviewed current knowledge regarding the pharmacokinetics, pharmacodynamics, toxicity, therapeutic effects, and safety of cytisine and its derivatives under development. Recent pharmacological studies have shown that this drug is a low-efficacy partial agonist of the α4β2 nicotine acetylcholine receptor, which is considered central to nicotine's (NIC) action on the reward pathway. When used alone, this drug reduces the effect of nicotine on dopamine release in the mesolimbic system, while alleviating nicotine withdrawal symptoms associated with smoking cessation attempts. Clinical studies have demonstrated that cytisine is safe and effective as a smoking cessation adjunct. A recent uncontrolled trial showed that, after a standard course of cytisine treatment supplemented with minimal behavioral support, the 12-month carbon monoxide-verified sustained withdrawal rate (13.8%; N = 436) was similar to the withdrawal rate after nicotine replacement therapy. Given the desirable pharmacological properties of cytisine, making it an attractive smoking cessation medication, randomized clinical trials should be pursued. However, more detailed preclinical studies are still needed to assess its pharmacokinetics and safety.
Drug Warnings
Contraindications for cytisine include: advanced atherosclerosis, certain types of schizophrenia, pheochromocytoma, diseases associated with severe cardiovascular damage, and malignant hypertension.
The following adverse reactions are commonly observed during the initial treatment with cytisine: altered taste and appetite, dry mouth, headache, irritability, nausea, constipation, tachycardia, and mild hypertension.
Caution should be exercised when using this medication in patients with acute peptic ulcers. Patients should quit smoking after completing the course of treatment…
The physician should inform the patient that concurrent use of this medication and smoking may exacerbate nicotine side effects (nicotine poisoning). This medication should only be used if the patient is genuinely and firmly determined to quit smoking.
For more complete data on drug warnings for CYTISINE (8 in total), please visit the HSDB record page.

---

Pharmacodynamics
Multiple models have been run to test the affinity of nAChR agonists for receptor subtypes to help identify molecules, groups, and spatial conformations crucial for enhancing affinity. Using the nAChR muscle receptor subtype (α1)2β1δγ model, the following results were obtained: anatoxin > epibastine > acetylcholine > DMPP >> CYTISINE > praziquantel > nicotine > coniine > tubocurarine > lobeline, with anatoxin exhibiting the highest activity and tubocurarine the lowest. On the other hand, despite the higher potency of anatoxin, the acetylcholine-induced receptor open time was much longer. These results suggest that toxin derivatives contribute to understanding the structure-activity relationship (SAR) of the muscle-type nicotine acetylcholine receptor (nAChR) (Cooper et al., 1996).

---

Bifidin (also known as cytisine, cytisine, or sophoridine) is a naturally occurring alkaloid found in legumes and is a partial agonist of the neuronal nicotinic acetylcholine receptor (nAChR) [1,3]. Its clinically approved indication is smoking cessation, where it alleviates nicotine withdrawal symptoms by partially activating nAChR (reducing cravings) and blocking nicotine binding (reducing reward effects) [3]. In addition to smoking cessation, it has shown dose-dependent effects on rat body weight and food intake, likely through central nAChR-mediated appetite regulation [2]. In vitro studies have shown that high concentrations may be hepatotoxic, possibly through mechanisms related to hepatocellular toxicity. Calcium overload induces endoplasmic reticulum stress, but this effect has not been observed at therapeutic doses in humans [3,4].

C11H14N2O

190.24

190.11

485-35-8

10235

White to off-white solid powder

1.2±0.1 g/cm3

413.0±34.0 °C at 760 mmHg

154-156ºC

203.6±25.7 °C

0.0±1.0 mmHg at 25°C

1.623

0.07

1

2

0

14

332

2

C1[C@H]2CNC[C@@H]1C3=CC=CC(=O)N3C2

ANJTVLIZGCUXLD-DTWKUNHWSA-N

InChI=1S/C11H14N2O/c14-11-3-1-2-10-9-4-8(5-12-6-9)7-13(10)11/h1-3,8-9,12H,4-7H2/t8-,9+/m0/s1

(1R,5S)-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocin-8-one

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (10.93 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 20.8 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: ≥ 2.08 mg/mL (10.93 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (10.93 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)