Cytisine: State of the art in pharmacological activities and pharmacokinetics.

Cytisine is a naturally occurring bioactive compound, an alkaloid mainly isolated from legume plants. In recent years, various biological activities of cytisine have been explored, showing certain effects in smoking cessation, reducing drinking behavior, anti-tumor, cardiovascular protection, blood sugar regulation, neuroprotection, osteoporosis prevention and treatment, etc. At the same time, cytisine has the advantages of high efficiency, safety, and low cost, has broad development prospects, and is a drug of great application value. However, a summary of cytisine's biological activities is currently lacking. Therefore, this paper summarizes the pharmacological action, mechanism, and pharmacokinetics of cytisine by referring to numerous databases, and analyzes the new and core targets of cytisine with the help of computer simulation technology, to provide reference for doctors.

Cytisine and cytisine derivatives. More than smoking cessation aids.

Cytisine, a natural bioactive compound that is mainly isolated from plants of the Leguminosae family (especially the seeds of Laburnum anagyroides), has been marketed in central and eastern Europe as an aid in the clinical management of smoking cessation for more than 50 years. Its main targets are neuronal nicotinic acetylcholine receptors (nAChRs), and pre-clinical studies have shown that its interactions with various nAChR subtypes located in different areas of the central and peripheral nervous systems are neuroprotective, have a wide range of biological effects on nicotine and alcohol addiction, regulate mood, food intake and motor activity, and influence the autonomic and cardiovascular systems. Its relatively rigid conformation makes it an attractive template for research of new derivatives. Recent studies of structurally modified cytisine have led to the development of new compounds and for some of them the biological activities are mediated by still unidentified targets other than nAChRs, whose mechanisms of action are still being investigated. The aim of this review is to describe and discuss: 1) the most recent pre-clinical results obtained with cytisine in the fields of neurological and non-neurological diseases; 2) the effects and possible mechanisms of action of the most recent cytisine derivatives; and 3) the main areas warranting further research.

Pharmacokinetics, Tissue Distribution, and Druggability Prediction of the Natural Anticancer Active Compound Cytisine N-Isoflavones Combined with Computer Simulation.

Cytisine N-methylene-(5,7-dihydroxy-4'-methoxy)-isoflavone (CNF2) is a new compound isolated from the Chinese herbal medicine Sophora alopecuroides. Preliminary pharmacodynamic studies demonstrated its activity in inhibiting breast cancer cell metastasis. This study examined the pharmacokinetics, absolute bioavailability, and tissue distribution of CNF2 in rats, and combined computer-aided technology to predict the druggability of CNF2. The binding site of CNF2 and the breast cancer target human epidermal growth factor receptor-2 (HER2) were examined with molecular docking technology. Next, ACD/Percepta software was used to predict the druggability of CNF2 based on the quantitative structure-activity relationship (QSAR). Finally, a simple and effective HPLC method was used to determine plasma pharmacokinetics and tissue distribution of CNF2 in rats. Prediction and experimental results show that compared with the positive control HER2 inhibitor SYR127063, CNF2 has a stronger binding affinity with HER2, suggesting that its efficacy is stronger; and the structure of CNF2 complies with the Lipinski's Rule of Five and has good drug-likeness. The residence time of CNF2 in rats is less than 4 h, and the metabolic rate is relatively fast; But the absolute bioavailability of CNF2 in rats was 6.6%, mainly distributed in the stomach, intestine, and lung tissues, where the CNF2 contents were 401.20, 144.01, and 245.82 µg/g, respectively. This study constructed rapid screening and preliminary evaluation of active compounds, which provided important references for the development and further research of such compounds.

Structure-Based Discovery of SD-36 as a Potent, Selective, and Efficacious PROTAC Degrader of STAT3 Protein.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and an attractive therapeutic target for cancer and other human diseases. Despite 20 years of persistent research efforts, targeting STAT3 has been very challenging. We report herein the structure-based discovery of potent small-molecule STAT3 degraders based upon the proteolysis targeting chimera (PROTAC) concept. We first designed SI-109 as a potent, small-molecule inhibitor of the STAT3 SH2 domain. Employing ligands for cereblon/cullin 4A E3 ligase and SI-109, we obtained a series of potent PROTAC STAT3 degraders, exemplified by SD-36. SD-36 induces rapid STAT3 degradation at low nanomolar concentrations in cells and fails to degrade other STAT proteins. SD-36 achieves nanomolar cell growth inhibitory activity in leukemia and lymphoma cell lines with high levels of phosphorylated STAT3. A single dose of SD-36 results in complete STAT3 protein degradation in xenograft tumor tissue and normal mouse tissues. SD-36 achieves complete and long-lasting tumor regression in the Molm-16 xenograft tumor model at well-tolerated dose-schedules. SD-36 is a potent, selective, and efficacious STAT3 degrader.

Novel cytisine derivatives exert anti-liver fibrosis effect via PI3K/Akt/Smad pathway.

A series of new cytisine derivatives with a unique endocyclic scaffold were synthesized and evaluated for their inhibitory effect on collagen α1 (I) (COL1A1) promotor in human LX2 cells, taking cytisine as the lead. Structure-activity relationship (SAR) revealed that introducing a 12N-benzyl substitution might significantly enhance the activity. Compound 5f exhibited a promising inhibitory potency against COL1A1 with an IC50 value of 12.8 µM in human LX2 cells, and an inspiring inhibition activity against COL1A1 on both mRNA and protein levels. It also effectively inhibited the expression of α smooth muscle actin (α-SMA), connective tissue growth factor (CTGA), matrix metalloprotein 2 (MMP-2), and transforming growth factor ß1 (TGFß1), indicating an extensive inhibitory effect against fibrogenetic proteins. In addition, compound 5f displayed reasonable PK and safety profiles. The primary mechanism study indicated that it might repress the hepatic fibrogenesis via PI3K/Akt/Smad signaling pathway. The results provided powerful information for further structure optimization, and compound 5f was selected as a novel anti-liver fibrosis agent for further investigation.

Ascending single dose pharmacokinetics of cytisine in healthy adult smokers.

1. Cytisine, a partial agonist for the α4ß2-nAChR, is used as a smoking cessation medication. Cytisine's current dosing is complex and involves taking 1.5 mg several times a day. The aim of this study was to explore the effect of dose on the pharmacokinetics and safety of cytisine after a single dose in healthy adult smokers. 2. Participants were assigned to one of three groups (n = 6 in each group) to receive a single oral dose of 1.5, 3 or 4.5 mg of cytisine. Blood samples were collected up to 24 h post dose. Pulse, blood pressure and respiratory rate were measured. Adverse effects were recorded. 3. Cytisine reached peak plasma concentration 1-2 h post dose in all participants irrespective of dose, with no dose-dependent changes in the elimination phase. Mean (SD) cytisine exposure (AUC0-24h) were 81.9 (15.8), 181.9 (40.8) and 254.5 (48.1) ng.h/mL following 1.5, 3 and 4.5 mg, respectively. 4. Cytisine appears to have predictable pharmacokinetics following a single dose of up to 4.5 mg and may be safe given as a single 4.5 mg dose, which is threefold greater than the recommended dose taken at one time. This study is registered in ClinicalTrials.gov (ID:NCT02585024).

Plasma concentrations of cytisine, a commercially available plant-based alkaloid, in healthy adult smokers taking recommended doses for smoking cessation.

1. Cytisine is a plant alkaloid that is a partial agonist for the α4ß2 -nAChRs and is used as an aid to smoking cessation. To date, there are no published data on cytisine concentrations in humans following multiple dosing. The aim of this study was to determine cytisine plasma concentrations after taking recommended doses for smoking cessation and to report on adverse effects. 2. Subjects (n=10) were instructed to follow the 25-day standard dosing regimen of cytisine. Blood was collected at 0, 2, 4, 8 and 10 hours on day 1 then on subsequent visits (days 2, 3, 4, 6, 13, 14, 17, 18, 21, 22, 25 and 26) to measure plasma cytisine concentrations. Plasma concentrations were determined using a validated LC-MS method. 3. Accumulation of cytisine was observed with repeated dosing of cytisine on day 1. Mean ± SEM plasma cytisine concentration measured at 10 hours was 50.8 ± 4.7 ng/mL. Due to dose tapering, there was an overall decrease in plasma cytisine concentration over the whole treatment period. 4. Overall, cytisine was well-tolerated and adverse effects reported were minor, indicating that cytisine is safe at concentrations measured in this study. This study is registered in the Australia and New Zealand Clinical Trials Registry (ACTRN12613000002785).

Biotransformation of RDX and HMX by Anaerobic Granular Sludge with Enriched Sulfate and Nitrate.

RDX and HMX are widely used energetic materials and they are recognized as environmental contaminants at numerous locations. The present study investigated the biotransformation of RDX and HMX by anaerobic granular sludge under sulfate- and nitrate-enriched conditions. The results showed that RDX and HMX could be transformed by anaerobic granular sludge when nitrate was present. However, the biotransformation of RDX and HMX was negatively influenced, especially with high nitrate concentrations. Sulfate-enriched conditions were more favorable for the removal of ammunition compounds by anaerobic granular sludge than nitrate-enriched conditions. The removal of RDX and HMX under both nitrate- and sulfate-enriched conditions was facilitated by the use of glucose as additional substrate. This knowledge may help identify factors required for rapid removal of RDX and HMX in high-rate bioreactors. These results can also be applied to devise an appropriate and practical biological treatment strategy for explosive contaminated wastewater.

The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo.

In the present study, we tested the anti-pancreatic cancer activity by AT406, a small-molecule antagonist of IAP (inhibitor of apoptosis proteins). In established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells, treatment of AT406 significantly inhibited cell survival and proliferation. Yet, same AT406 treatment was non-cytotoxic to pancreatic epithelial HPDE6c7 cells. AT406 increased caspase-3/-9 activity and provoked apoptosis in the pancreatic cancer cells. Reversely, AT406' cytotoxicity in these cells was largely attenuated with pre-treatment of caspase inhibitors. AT406 treatment caused degradation of IAP family proteins (cIAP1 and XIAP) and release of cytochrome C, leaving Bcl-2 unaffected in pancreatic cancer cells. Bcl-2 inhibition (by ABT-737) or shRNA knockdown dramatically sensitized Panc-1 cells to AT406. In vivo, oral administration of AT406 at well-tolerated doses downregulated IAPs (cIAP1/XIAP) and inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) nude mice. Together, our preclinical results suggest that AT406 could be further evaluated as a promising anti-pancreatic cancer agent.

A ten fold reduction of nicotine yield in tobacco smoke does not spare the central cholinergic system in adolescent mice.

The tobacco industry has gradually decreased nicotine content in cigarette smoke but the impact of this reduction on health is still controversial. Since the central cholinergic system is the primary site of action of nicotine, here, we investigated the effects of exposure of adolescent mice to tobacco smoke containing either high or low levels of nicotine on the central cholinergic system and the effects associated with cessation of exposure. From postnatal day (PN) 30 to 45, male and female Swiss mice were exposed to tobacco smoke (whole body exposure, 8h/day, 7 days/week) generated from 2R1F (HighNic group: 1.74mg nicotine/cigarette) or 4A1 (LowNic group: 0.14mg nicotine/cigarette) research cigarettes, whereas control mice were exposed to ambient air. Cholinergic biomarkers were assessed in the cerebral cortex and midbrain by the end of exposure (PN45), at short- (PN50) and long-term (PN75) deprivation. In the cortex, nicotinic cholinergic receptor upregulation was observed with either type of cigarette. In the midbrain, upregulation was detected only in HighNic mice and remained significant in females at short-term deprivation. The high-affinity choline transporter was reduced in the cortex: of HighNic mice by the end of exposure; of both HighNic and LowNic females at short-term deprivation; of LowNic mice at long-term deprivation. These decrements were separable from effects on choline acetyltransferase and acetylcholinesterase activities, suggesting cholinergic synaptic impairment. Here, we demonstrated central cholinergic alterations in an animal model of tobacco smoke exposure during adolescence. This system was sensitive even to tobacco smoke with very low nicotine content.

Predicting chemical bioavailability using microarray gene expression data and regression modeling: A tale of three explosive compounds.

BACKGROUND: Chemical bioavailability is an important dose metric in environmental risk assessment. Although many approaches have been used to evaluate bioavailability, not a single approach is free from limitations. Previously, we developed a new genomics-based approach that integrated microarray technology and regression modeling for predicting bioavailability (tissue residue) of explosives compounds in exposed earthworms. In the present study, we further compared 18 different regression models and performed variable selection simultaneously with parameter estimation. RESULTS: This refined approach was applied to both previously collected and newly acquired earthworm microarray gene expression datasets for three explosive compounds. Our results demonstrate that a prediction accuracy of R(2) = 0.71-0.82 was achievable at a relatively low model complexity with as few as 3-10 predictor genes per model. These results are much more encouraging than our previous ones. CONCLUSION: This study has demonstrated that our approach is promising for bioavailability measurement, which warrants further studies of mixed contamination scenarios in field settings.

Safety, pharmacokinetics, and pharmacodynamic properties of oral DEBIO1143 (AT-406) in patients with advanced cancer: results of a first-in-man study.

PURPOSE: To assess safety/tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of DEBIO1143, an antagonist of inhibitor apoptosis proteins. METHODS: This first-in-man study in patients with advanced cancer used an accelerated dose titration design. DEBIO1143 was given orally once daily on days 1-5 every 2 or 3 weeks until disease progressed or patients dropped out. The starting dose of 5 mg was escalated by 100% in single patients until related grade 2 toxicity occurred. This triggered expansion to cohorts of three and subsequently six patients and reduction in dose increments to 50%. Maximum tolerated dose (MTD) was exceeded when any two patients within the same cohort experienced dose-limiting toxicity (DLT). On days 1 and 5, PK and PD samples were taken. RESULTS: Thirty-one patients received doses from 5 to 900 mg. Only one DLT was reported at 180 mg. No MTD was found. Most common adverse drug reactions were fatigue (26%), nausea (23%), and vomiting (13%). Average t max and T 1/2 was about 1 and 6 h, respectively. Exposure increased proportionally with doses from 80 to 900 mg, without accumulation over 5 days. Plasma CCL2 increased at 3-6 h postdose and epithelial apoptosis marker M30 on day 5; cIAP-1 levels in PBMCs decreased at all doses >80 mg. Five patients (17%) had stable disease as the best treatment response. CONCLUSION: DEBIO1143 was well tolerated at doses up to 900 mg and elicited PD effects at doses greater 80 mg. Limited antitumor activity may suggest development rather as adjunct treatment.

Pharmacokinetics of cytisine, an α4 ß2 nicotinic receptor partial agonist, in healthy smokers following a single dose.

Cytisine, an α4 ß2 nicotinic receptor partial agonist, is a plant alkaloid that is commercially extracted for use as a smoking cessation medication. Despite its long history of use, there is very little understanding of the pharmacokinetics of cytisine. To date, no previous studies have reported cytisine concentrations in humans following its use as a smoking cessation agent. A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed and validated for analysis of Tabex® and nicotine-free oral strips, two commercial products containing cytisine. A sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the quantification of cytisine in human plasma and for the detection of cytisine in urine. Single-dose pharmacokinetics of cytisine was studied in healthy smokers. Subjects received a single 3 mg oral dose administration of cytisine. Cytisine was detected in all plasma samples collected after administration, including 15 min post-dose and at 24 h. Cytisine was renally excreted and detected as an unchanged drug. No metabolites were detected in plasma or urine collected in the study. No adverse reactions were reported.

Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: a model of cholinergic capacity limits.

Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/- mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/- mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/- mice. Additionally, CHT+/- mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4ß2* nAChRs in the cortex of CHT+/- mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/- cholinergic terminals. Further analyses of molecular and functional compensations in the CHT+/- model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders.

A potent bivalent Smac mimetic (SM-1200) achieving rapid, complete, and durable tumor regression in mice.

We have designed, synthesized, and evaluated a series of new compounds based upon our previously reported bivalent Smac mimetics. This led to the identification of compound 12 (SM-1200), which binds to XIAP, cIAP1, and cIAP2 with Ki values of 0.5, 3.7, and 5.4 nM, respectively, inhibits cell growth in the MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines with IC50 values of 11.0 and 28.2 nM, respectively. Compound 12 has a much improved pharmacokinetic profile over our previously reported bivalent Smac mimetics and is highly effective in induction of rapid and durable tumor regression in the MDA-MB-231 xenograft model. These data indicate that compound 12 is a promising Smac mimetic and warrants extensive evaluation as a potential candidate for clinical development.

Quantitative changes of nicotinic receptors in the hippocampus of dystrophin-deficient mice.

Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, ß2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4ß2) and [¹²5I]-α-bungarotoxin ([¹²5I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²5I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²5I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, ß2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients.

Accumulation and effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) exposure in the green anole (Anolis carolinensis).

Environmental contamination by energetic compounds is an increasing international concern, although little is known of their accumulation in and affect on wildlife. Reptiles are often good models for contaminants studies due to natural history traits that increase their potential for exposure. We report a study to assess accumulation and effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX, High Melting Explosive) in green anoles (Anolis carolinensis). Acute oral toxicity (LD(50)) was estimated to exceed 2000 mg/kg body weight in adult male and female anoles using a standard up-and-down method. Accumulation of HMX was assessed in adult females via dietary exposure and into eggs by two routes (directly from the soil and via maternal transfer). HMX readily accumulated into adult females in a dose-dependent manner and into eggs following both exposure pathways. However, total HMX in soil-exposed eggs was up to 40-times greater than those exposed via maternal transfer. Although there was a suggestion of an HMX-induced reduction in body weight in adult females, overall there were no effects observed over the 12 week exposure period. The only significant effect on eggs was a 50% reduction in hatching success for eggs exposed to 2000 mg/kg HMX in the soil during incubation. Growth and survival of hatchlings was not affected by HMX exposure. Our results demonstrate that HMX accumulates through the food chain and into eggs from the soil, but likely poses minimal threat to lizards except to hatching success in eggs incubated in soils with HMX levels near maximum environmental concentrations.

A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment.

We report the discovery and characterization of SM-406 (compound 2), a potent and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins (IAPs). This compound binds to XIAP, cIAP1, and cIAP2 proteins with K(i) of 66.4, 1.9, and 5.1 nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free functional assay, induces rapid degradation of cellular cIAP1 protein, and inhibits cancer cell growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-human primates, and dogs, is highly effective in induction of apoptosis in xenograft tumors, and is capable of complete inhibition of tumor growth. Compound 2 is currently in phase I clinical trials for the treatment of human cancer.

Toxicity and bioconcentration evaluation of RDX and HMX using sheepshead minnows in water exposures.

Lethal effects of the explosives RDX and HMX were assessed using ten-day water exposures to juvenile sheepshead minnows (Cyprinodon variegatus). For RDX, maximum mortality occurred during the first two days of exposure with a 10-d median lethal concentration (LC50) of 9.9 mg L(-1). The RDX 10-d median lethal residue (LR50) was 9.6 mg kg(-1) (34.9 µmol kg(-1)) wet weight (ww), the first RDX critical body residue reported for fish. Previous investigations reported that RDX body residues in marine amphipods up to 96 µmol kg(-1) ww and in marine mussels up to 86 µmol kg(-1) ww failed to result in significant mortality. The highest HMX concentration tested, corresponding to its apparent solubility limit in seawater (2.0 mg L(-1)), and the associated mean body residue (3 mg kg(-1) or 14 µmol kg(-1) ww) resulted in no significant mortality for exposed minnows. The mean 10-d bioconcentration factors for RDX (0.6-0.9 L kg(-1)) and HMX (0.3-1.6 L kg(-1)) were typically lower than 1, reflecting the low bioaccumulative potential for these compounds.

Pre-clinical properties of the alpha4beta2 nicotinic acetylcholine receptor partial agonists varenicline, cytisine and dianicline translate to clinical efficacy for nicotine dependence.

BACKGROUND AND PURPOSE: Smoking cessation trials with three high-affinity partial agonists of alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) have demonstrated differences in their clinical efficacy. This work examines the origin of the differences by taking into account brain exposure and pharmacological effects at human alpha4beta2 nAChRs. EXPERIMENTAL APPROACH: Rat plasma and brain pharmacokinetics were characterized and used to predict human steady-state plasma and brain concentrations following recommended doses of each of the three compounds. The pharmacological characterization included in vitro affinities at different nAChR subtypes, functional efficacies and potencies at the human alpha4beta2 nAChR, as well as in vivo effects on rat mesolimbic dopamine turn-over. KEY RESULTS: A comparison of predicted human brain concentrations following therapeutic doses demonstrated that varenicline and nicotine, but not dianicline and cytisine, can extensively desensitize and, to a lesser extent, activate alpha4beta2 nAChRs. The limited clinical efficacy of dianicline may be accounted for by a combination of weak functional potency at alpha4beta2 nAChRs and moderate brain penetration, while recommended doses of cytisine, despite its high in vitro potency, are predicted to result in brain concentrations that are insufficient to affect alpha4beta2 nAChRs. CONCLUSIONS AND IMPLICATIONS: The data provide a plausible explanation for the higher abstinence rate in smoking cessation trials following treatment with varenicline than with the two other alpha4beta2 nAChR partial agonists. In addition, this retrospective analysis demonstrates the usefulness of combining in vitro and in vivo parameters with estimated therapeutic human brain concentrations for translation to clinical efficacy.