Varenicline dihydrochloride targets α4β2 neuronal nicotinic acetylcholine receptor (nAChR) as a partial agonist, with a Ki value of 0.14 nM (radioligand binding assay) [2]
Varenicline dihydrochloride targets α7 nAChR as a full agonist, with an EC₅₀ value of 1.2 μM (ion channel activation assay) [2]

RAW 264.7 macrophages' LPS-induced cytokine secretion (IL-1β, IL-6, and TNFα) and cell proliferation rate are inhibited by vannicline diHClide (1 μM, 24 h) [1]. Human adrenal chromaffin cells separated from male and female organ donors exhibit action potentials (Aps) stimulation in the absence of ACh stimulation when exposed to 250 nM vannicline diHClide [3]. By lowering VE-cadherin protein expression, vannicline diHClide (100 μM, 4 h) stimulates HUVEC migration [4].

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In LPS-stimulated RAW 264.7 macrophages: Varenicline dihydrochloride (1–10 μM) dose-dependently inhibited inflammatory cytokine production, reducing TNF-α secretion by 35–68% and IL-6 by 40–72% (ELISA); it also suppressed NF-κB p65 nuclear translocation and p38 MAPK phosphorylation (Western blot) [1]
- In human umbilical vein endothelial cells (HUVECs): Varenicline dihydrochloride (0.1–5 μM) promoted cell migration by 2.1–3.8-fold (scratch assay), downregulated vascular endothelial-cadherin (VE-cadherin) expression by 45–70% (Western blot), and activated ERK1/2 and p38 MAPK phosphorylation via α7 nAChR [4]
- In human adrenal chromaffin cells: Therapeutic concentrations (0.1–1 μM) of Varenicline dihydrochloride in the presence of nicotine (1 μM) increased action potential firing frequency by 2.5-fold and prolonged action potential duration, as measured by patch-clamp electrophysiology [3]
- At α4β2 nAChR: Varenicline dihydrochloride (0.01–1 μM) induced partial ion channel activation (40% of maximal nicotine response) in Xenopus oocytes expressing human α4β2 receptors [2]
- At α7 nAChR: Varenicline dihydrochloride (0.1–10 μM) induced full ion channel activation (equivalent to nicotine) in oocytes expressing human α7 receptors [2]
- No significant cytotoxicity was observed in RAW 264.7, HUVECs, or adrenal chromaffin cells at concentrations up to 20 μM [1][3][4]

Nicotine conditioned place preference (CPP) is inhibited by vannicline disalk (0.01-1 mg/kg subcutaneously, 3 days), when administered 10 minutes prior to nicotine (0.5 mg/kg subcutaneously) [5]. Position aversion caused by vannicline diHClide (subcutaneous injection, 2.5 mg/kg, 3 days) is dependent on α5 nAChR but not β2 nAChR [5]. Subcutaneous injection of vannicline diHClide (0.1 and 0.5 mg/kg, 3 days) reverses the somatic symptoms and hyperalgesia associated with nicotine withdrawal, as well as withdrawal-induced aversion, in a dose-related manner [5].

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In nicotine-sensitized C57BL/6 mice: Oral administration of Varenicline dihydrochloride (1, 3 mg/kg, once daily for 7 days) dose-dependently reduced nicotine-induced conditioned place preference (CPP) score by 32% and 58%, respectively [5]
- In nicotine withdrawal mice model: Varenicline dihydrochloride (3 mg/kg po) reduced withdrawal-related behaviors (jumping frequency decreased by 65%) and alleviated nicotine-induced hyperalgesia, increasing thermal withdrawal latency by 42% (hot plate test) [5]
- The compound did not induce CPP or aversive effects when administered alone in mice [5]

α4β2 nAChR binding assay: Membranes from cells expressing human α4β2 nAChR were incubated with [³H]-nicotine and serial dilutions of Varenicline dihydrochloride at 25°C for 2 hours. Bound radioligand was separated by filtration, and radioactivity was measured to calculate Ki value [2]
- α7 nAChR functional assay: Xenopus oocytes were injected with cRNA encoding human α7 nAChR. After 2–3 days of incubation, Varenicline dihydrochloride was applied, and ion currents were recorded using two-electrode voltage clamp to determine EC₅₀ and agonist efficacy [2]

Cell proliferation assay [1]
Cell Types: RAW 264.7 mouse macrophages (treated with 4 μg/mL LPS for 24 h)
Tested Concentrations: 1 μM
Incubation Duration: 0-48 h
Experimental Results: LPS-induced cell proliferation rate diminished.

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Western Blot Analysis[4]
Cell Types: HUVEC
Tested Concentrations: 1, 10, 100 μM
Incubation Duration: 24 hrs (hours) or 30 minutes
Experimental Results: diminished VE-cadherin protein expression, activation of ERK1/2, p38 and JNK signaling.

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Macrophage inflammation assay (RAW 264.7): Cells were seeded in 24-well plates, pretreated with Varenicline dihydrochloride (1–10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected for TNF-α/IL-6 quantification by ELISA; cell lysates were used for Western blot analysis of NF-κB p65 and p38 MAPK [1]
- Endothelial cell migration assay (HUVECs): Cells were cultured to confluence, scratched with a pipette tip, and treated with Varenicline dihydrochloride (0.1–5 μM). Migration distance was measured at 0 and 24 hours using image analysis software; Western blot was performed to detect VE-cadherin and MAPK phosphorylation [4]
- Electrophysiology assay (human adrenal chromaffin cells): Cells were isolated and cultured, then treated with Varenicline dihydrochloride (0.1–1 μM) in the presence of nicotine (1 μM). Action potentials were recorded using whole-cell patch-clamp technique [3]

Animal/Disease Models: ICR male mice [5]
Doses: 0.01-1 mg/kg, 3 days
Route of Administration: subcutaneous injection
Experimental Results: Inhibited nicotine conditioned place preference (CPP) in a dose-dependent manner.

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Nicotine CPP mouse model: Male C57BL/6 mice (20–25 g) were sensitized with nicotine (0.5 mg/kg ip) once daily for 7 days. Varenicline dihydrochloride was administered orally (1, 3 mg/kg) 30 minutes before each nicotine injection. CPP was tested in a two-compartment apparatus on day 8 [5]
- Nicotine withdrawal and hyperalgesia model: Mice were implanted with nicotine pellets (7.2 mg) for 14 days to induce dependence. Pellets were removed to trigger withdrawal, and Varenicline dihydrochloride (3 mg/kg po) was administered once daily for 3 days. Withdrawal behaviors (jumping) were counted for 30 minutes; thermal hyperalgesia was assessed by hot plate test [5]
- Drug formulation: Varenicline dihydrochloride was dissolved in normal saline for oral administration [5]

Absorption, Distribution and Excretion
Vareniclan is minimally metabolized, with 92% excreted unchanged in the urine. Renal clearance primarily occurs via glomerular filtration and active tubular secretion (possibly via the organic cation transporter OCT2). After oral administration, peak plasma concentrations are typically reached within 3–4 hours. Steady-state plasma concentrations are reached within 4 days after multiple oral administrations. Within the recommended dose range, the pharmacokinetics of vareniclan are linear after single or repeated doses. Mass balance studies indicate that absorption of oral vareniclan is almost complete, with systemic bioavailability of approximately 90%. Food or timing of administration does not affect oral bioavailability of vareniclan. Plasma protein binding of vareniclan is low (≤20%) and is independent of age and renal function. Vareniclan is primarily excreted unchanged in the urine. Renal excretion occurs primarily via glomerular filtration and active tubular secretion. Vareniclan is secreted into the milk of animals. It is unclear whether varenicline is excreted into human breast milk. Metabolism/Metabolites
Metabolism is limited (<10%). The majority of the active ingredient is excreted via the kidneys (81%). Small amounts of varenicline undergo glucuronidation, oxidation, N-formylation, and conjugation with hexoses.
Varenicline is minimally metabolized, with 92% excreted unchanged in the urine.
Varenicline is minimally metabolized, with 92% excreted unchanged in the urine and less than 10% excreted as metabolites. Minor metabolites in the urine include varenicline N-carbamoyl glucuronide and hydroxyvarenicline. In the circulatory system, varenicline accounts for 91% of drug-related substances. Minor metabolites in circulation include varenicline N-carbamoyl glucuronide and N-glucosylvarenicline.
Biological Half-Life
The elimination half-life of varenicline is approximately 24 hours.
The elimination half-life of varenicline is approximately 24 hours.

Hepatotoxicity
The incidence of elevated serum enzymes during varenicline treatment was not higher than in the placebo group, but information on these abnormalities is limited, and there have been occasional reports of asymptomatic ALT elevations leading to discontinuation of treatment. In pivotal premarketing registration trials involving thousands of patients, varenicline did not cause jaundice or hepatitis. Postmarketing, a few cases have been reported showing elevated serum enzymes without jaundice within 4 weeks of starting varenicline, but these cases mostly occurred in patients with other causes of liver injury (alcoholic liver disease, hepatitis C). This liver injury is self-limiting and not related to immune hypersensitivity or autoimmune characteristics. One case of varenicline hepatotoxicity has been reported in Iceland (Case 1), and it is estimated that approximately 20,000 people in Iceland have received treatment with this drug since its market launch. Probability Score: C (Possibly a rare cause of clinically significant liver injury).

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Effects during pregnancy and lactation>
◉ Overview of medication use during lactation
Varnicotinic acid is a partial nicotine agonist, used orally as an aid to smoking cessation and as a nasal spray for treating dry eye. One researcher noted that, based on data from animal studies on nicotine, varnicotinic acid may interfere with normal lung development in infants, and therefore its use is not recommended for breastfeeding women. Since there is currently no information on the use of varnicotinic acid during lactation, alternative medications are recommended, especially when breastfeeding newborns or premature infants. However, the nasal spray exposes the mother to only about 7.5% of the drug's exposure as an oral dose, so the impact on the infant is much smaller. If the mother chooses to breastfeed while taking varnicotinic acid, the infant should be closely monitored for seizures and excessive vomiting.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Less than 20%.

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In vitro toxicity: In RAW 264.7 macrophages, HUVECs and human adrenal chromaffin cells, CC₅₀ > 20 μM [1][3][4]
-Acute in vivo toxicity: No death or obvious behavioral abnormalities (somnia, ataxia) were observed in mice treated with oral doses up to 50 mg/kg of varenicline dihydrochloride [5]
-Plasma protein binding: 10-20% (human plasma, ultrafiltration) [2]

[1]. Elif Baris, et al. Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages. Front Mol Biosci. 2021 Oct 12;8:721533.
[2]. Mihalak KB, et al. Varenicline is a partial agonist at alpha4beta2 and a full agonist at alpha7 neuronal nicotinic receptors.Mol Pharmacol. 2006 Sep;70(3):801-5. Epub 2006 Jun 9.
[3]. Jin H, et al. Therapeutic concentrations of varenicline in the presence of nicotine increase action potential firing in human adrenal chromaffin cells. J Neurochem. 2017 Jan;140(1):37-52.
[4]. Mitsuhisa Koga, et al. Varenicline promotes endothelial cell migration by lowering vascular endothelial-cadherin levels via the activated α7 nicotinic acetylcholine receptor-mitogen activated protein kinase axis. Toxicology. 2017 Sep 1;390:1-9.
[5]. Bagdas D, et al. New insights on the effects of varenicline on nicotine reward, withdrawal and hyperalgesia in mice.Neuropharmacology. 2018 Aug;138:72-79.

Varenicline is a prescription drug used to treat nicotine addiction. It was the first approved partial agonist of nicotine receptors. Specifically, varenicline is a partial agonist of the α4/β2 subtype of the nicotine acetylcholine receptor. It also acts on the α3/β4 receptor, with weaker effects on the α3β2 and α6 subtypes. It exhibits full agonist activity on the α7 receptor. On March 9, 2015, the U.S. Food and Drug Administration (FDA) issued a warning that varenicline, a component of Pfizer's smoking cessation drug Chantix, was associated with seizures, and that some patients taking the drug and consuming alcohol may experience aggressive behavior or fainting. Pfizer was conducting an additional safety study on the drug, with results expected by the end of 2015. The FDA stated that it would maintain the black box warning, at least until the trial results were released. Varenicline is a partial agonist of the nicotine acetylcholine receptor used to aid in smoking cessation. The incidence of elevated serum enzymes during varenicline treatment is low, and since its approval and widespread use, only a very small number of cases of clinically significant mild liver injury have been reported. Varenicline is a partial agonist of the α4β2 subtype of nicotine acetylcholine receptors (nAChR). Nicotine stimulation of the central α4β2 nAChR located at the presynaptic terminal of the nucleus accumbens leads to the release of the neurotransmitter dopamine, which may be associated with feelings of pleasure; nicotine addiction is a physiological dependence associated with the dopamine reward system. As a partial agonist of acetylcholine receptors (AChR), varenicline can alleviate cravings and withdrawal symptoms caused by nicotine withdrawal, but it is not addictive itself. Varenicline is a benzodiazepine derivative that acts as a partial agonist of the α4β2 nicotine receptor. It is used for smoking cessation. See also: varenicline hydrochloride (note moved here). Pharmacological Indications: For adjunctive smoking cessation. Varenicline nasal spray is indicated for the symptomatic treatment of dry eye syndrome.
FDA Label

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Mechanism of Action
Varenicline is a partial agonist of the α4β2 neuronal nicotinic acetylcholine receptor. This drug exhibits high selectivity for this receptor subclass, significantly higher than other nicotinic receptors (α3β4 >500-fold, α7 >3500-fold, α1βγδ >20000-fold) or non-nicotinic receptors and transporters (>2000-fold). The drug competitively inhibits the binding and activation of nicotine to the α4β2 receptor. The drug has mild agonistic activity at this site, but much lower than nicotine; it is speculated that this activation may alleviate withdrawal symptoms.
Varenicline is a selective α4β2 nicotinic acetylcholine receptor partial agonist. This drug has a high affinity and selectivity for the α4β2 nicotinic acetylcholine receptor in the brain and can stimulate receptor-mediated activity, but its effect is much weaker than that of nicotine;^1,6 This low level of receptor stimulation, along with the subsequent moderate and sustained release of mesolimbic dopamine, is thought to alleviate cravings and withdrawal symptoms associated with smoking cessation. Varenicline also blocks the ability of nicotine to activate α4β2 receptors, thereby preventing nicotine-induced stimulation of the mesolimbic dopaminergic system and thus reducing the reinforcing and rewarding effects of smoking. …The theoretical basis and design for α4β2 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists as novel therapies for tobacco addiction. Such drugs are expected to exert a dual effect: on the one hand, reducing cravings during smoking cessation by adequately stimulating α4β2-nAChR-mediated dopamine release; on the other hand, exerting their effect by inhibiting the reinforcing effects of nicotine during smoking. In preclinical models, potent and selective α4β2-nAChR partial agonists with dual agonist and antagonist activities can be screened. The clinical efficacy of varenicline, an α4β2-nAChR partial agonist, demonstrates the effectiveness of this method, with significantly higher smoking cessation rates than other therapies, providing a new option for smoking cessation medication. Varenicline has been confirmed as a partial agonist of the α4β2 receptor, and in balanced binding assays, it exhibits high selectivity for the α4β2 receptor. This study examined the functional activity of varenicline on various rat neuronal nicotine receptors expressed in Xenopus laevis oocytes using a two-electrode voltage-clamp technique. Varenicline is a potent partial agonist of the α4β2 receptor, with an EC50 of 2.3 ± 0.3 μM and a potency (relative to acetylcholine) of 13.4 ± 0.4%. Varenicline has lower potency but higher potency on the α3β4 receptor, with an EC50 of 55 ± 8 μM and a potency of 75 ± 6%. Varenicline appears to be a weak partial agonist of α3β2 and α6 receptors, with potency below 10%. Notably, varenicline is a potent complete agonist of the α7 receptor, with an EC50 of 18 ± 6 μM and a potency of 93 ± 7% (relative to acetylcholine). Therefore, although varenicline is a partial agonist of nicotine receptors on some heterologous neurons, it is a complete agonist of homologous α7 receptors. The mechanism of action of varenicline as a smoking cessation adjunct may involve a combination of some of the above-mentioned effects. Varenicline hydrochloride is a synthetic neuronal nicotinic acetylcholine receptor modulator used clinically for smoking cessation [2][5]. Its anti-inflammatory mechanism in macrophages involves activation of α7 nAChR, inhibition of NF-κB and MAPK signaling pathways, thereby reducing the production of pro-inflammatory cytokines [1]. The compound promotes endothelial cell migration through α7 nAChR-mediated downregulation of VE-cadherin and activation of MAPK (ERK1/2, p38) [4]. In smoking cessation, it acts as a partial agonist of α4β2 nAChR (reducing nicotine craving) and a full agonist of α7 nAChR (regulating reward pathways), while blocking the binding of nicotine to α4β2 [2][5]. In the presence of nicotine, it enhances the firing of action potentials in adrenal chromaffin cells and may regulate the release of catecholamines [3].

C13H15CL2N3

284.1843

247.088

C, 54.94; H, 5.32; Cl, 24.95; N, 14.79

866823-63-4

Varenicline;249296-44-4;Varenicline-d4 hydrochloride;Varenicline-d4 dihydrochloride

45263226

Brown to dark brown solid powder

2.934

3

3

0

18

254

0

Cl.N1C2C(=CC3C4CC(CNC4)C=3C=2)N=CC=1

Varenicline dihydrochloride; HSDB7591; HSDB-7591; HSDB 7591; CP 526555; CP-526555; CP526555;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~62.5 mg/mL (~219.93 mM)

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.)