Dta1707

1. Pharmacokinetics of cytisine, an α4β2 nicotinic
receptor partial agonist, in healthy smokers
following a single dose
Soo Hee Jeong,a
* David Newcombe,b
Janie Sheridanc
and Malcolm Tinglea
Cytisine, an α4β2 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 pre-
vious 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 15min post-dose and at 24h. 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. Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: cytisine; α4β2 nicotinic partial agonist; pharmacokinetics; LC-MS; human plasma
Introduction
Smoking is a leading cause of many diseases including cancer, car-
diovascular disease, and respiratory disease.[1,2]
Smoking cessation
improves health risks and survival,[3]
but only 3 to 5% of smokers
who try to quit smoking without the use of medication succeed,
as determined by abstinence at six months.[4]
Cytisine, an alkaloid found in plants such as Golden Rain (Cytisus
laburnum), is an α4β2 nicotinic receptor partial agonist that has been
used for smoking cessation since the 1960s.[5,6]
Cytisine has been
widely used in many countries in Central and Eastern Europe and
Central Asia,[7]
but unlike its pharmacologically related analogue
varenicline, cytisine has not been approved for use as a smoking
cessation medication in many countries including the USA, the
UK, Australia and New Zealand. A systematic review and meta-
analysis of clinical studies with cytisine suggests that cytisine is
effective for smoking cessation.[8]
There is growing interest in
getting cytisine approved as a smoking cessation agent because of
its potential to be a much more affordable medication for smoking
cessation than any other current pharmacotherapy available.[8–11]
In an economic evaluation that compared cytisine to varenicline for
smoking cessation, cytisine was estimated to be more cost-effective
than varenicline.[12]
Despite its long history of use, significant gaps
remain in our knowledge of cytisine. Animal pharmacokinetic data
exist[13,14]
but we have found no publicly available information
about the pharmacokinetics/metabolism of cytisine in humans.
Cytisine is commercially available as an oral tablet marketed by
Sopharma, a Bulgarian pharmaceutical company, under its trade
name Tabex®. The standard dosing schedule of Tabex® is complex
and the rationale is not well understood. One Tabex® tablet con-
tains 1.5mg of cytisine and it is recommended that a person takes
1 tablet every 2 h (maximum of 6 tablets per day) as a starting dose
for the first 3 days of the treatment. The dosing frequency and inter-
val are changed throughout the course of the treatment. The dose
is reduced to 5 tablets per day on days 4–12 (1 tablet every 2.5 h),
4 tablets per day on days 13–16 (1 tablet every 3 h) and 3 tablets
per day on days 17–20 (1 tablet every 5 h). During the last 5 days
of treatment (days 21–25) the recommended dose is 1 or 2 tablets
every 6 h (maximum of 2 tablets per day). Recently, another com-
mercial product of cytisine became available in the form of an oral
strip. This has been marketed in Australia and each strip is reported
to contain 1 mg of cytisine (instead of 1.5mg as in tablets). This
product has a similar dosing schedule to Tabex®.
Several methods for the determination of cytisine have been re-
ported in the literature, including methods that have not been vali-
dated in human tissues[14–16]
and methods that have been shown
to be applicable for herbal intoxication or drug abuse cases[17–19]
;
however, human PK analysis has not been conducted. No methods
have yet studied the commercial forms of cytisine and no method
has quantified cytisine in clinical samples following administration
of these products.
* Correspondence to: Soo Hee Jeong, University of Auckland, Private Bag 92019
Auckland 1142 New Zealand.
E-mail: s.jeong@auckland.ac.nz
a University of Auckland, Pharmacology & Clinical Pharmacology, Auckland,
New Zealand
b University of Auckland, School of Population Health, Auckland, New Zealand
c University of Auckland, School of Pharmacy, Auckland, New Zealand
Drug Test. Analysis (2014) Copyright © 2014 John Wiley & Sons, Ltd.
Research article
Drug Testing
and Analysis
Received: 24 June 2014 Revised: 29 July 2014 Accepted: 30 July 2014 Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1707

2. This paper has two overarching aims:
A. To describe the development and validation of an analytical assay
for cytisine and its use in the analysis of two commercially available
products Tabex® tablets and Quit4Good Nicotine Free Oral Strips.
Plasma and urine samples taken from a healthy subject who had
taken Tabex® were analyzed to determine whether this method
was sensitive enough to detect levels of cytisine following oral
administration. For simplification, a single dose was chosen over
the recommended split dosing. Initially, a high performance liquid
chromatography-ultraviolet (HPLC-UV) assay was developed and
validated for analysis of the two commercial products of cytisine.
However, the sensitivity of this method was insufficient (not fit for
purpose) to detect cytisine in human plasma after the administered
dose. Therefore, it was necessary to develop and validate a more
sensitive method using high performance liquid chromatography
coupled with mass spectrometry (LC-MS) that could be used to
investigate pharmacokinetics of cytisine in subsequent human studies.
B. To describe the pharmacokinetic characteristics of cytisine in
healthy smokers after a single dose, over a 24 h period.
The objectives of the pharmacokinetic study were:
1. To measure the concentrations of cytisine in plasma over a 24 h
period, to determine clearance, volume of distribution and
half-life of cytisine in humans, and to screen for the presence
of metabolite(s) in human plasma and urine in healthy
smokers following a 3 mg single oral dose administration.
2. To measure cytisine’s effect on heart rate, blood pressure and
breathing rate over 24 h following a 3 mg single dose
administration.
Methods – development of analytical assays
Chemicals and reagents
Cytisine (≥99% purity) and sulfanilamide (internal standard, IS)
(p-aminobenzenesulfonamide, ≥99% purity) were purchased from
Sigma Aldrich (Auckland, New Zealand). Methanol (>99%, HPLC
grade, Sigma Aldrich) was used in sample preparation. LC-grade
water (Millipore®, Milli-Q system) and methanol (>99%, HPLC
grade, Sigma Aldrich) were used for the mobile phase in the
HPLC-UV method and LC-grade water and acetonitrile (ACN,
>99%, HPLC-grade, Sigma Aldrich) were used for the mobile phase
in the LC-MS method.
HPLC-UV
Standard solutions
Cytisine stock solutions for calibration standards were prepared in
dimethyl sulfoxide (DMSO). Stock solution was further diluted in
DMSO to give appropriate working solutions. The IS solution was
prepared in DMSO at a concentration 400 μM.
Chromatographic separation was achieved on a Phenomenex
Gemini C18 HPLC column (4.6 mm × 150 mm, 5 μm) with a guard
column (C18, 4.6× 10 mm, 5 μm). Mobile phase consisted of
methanol and 50 mM ammonium acetate buffer adjusted to
pH 6.5 (1 to 5% methanol gradient; flow rate 1.0 mL/min, pressure
120 bar), with UV monitoring of the column effluent. Wavelengths
from 220 to 310nm were monitored and quantification was
performed at absorbance of 310 nm (cytisine) and 280 nm
(sulfanilamide). Signals areas were obtained from chromatograms
using manual integration. Cytisine/IS peak area ratio was calculated
as a quantitative measure to prepare calibration curves.
The assay was validated in accordance to the US FDA guidelines
for bioanalytical methods validation over the range of 130 to
4150 ng on column for selectivity/specificity, precision and accu-
racy and linearity.[20]
Tablet cytisine (external QC)
Ten cytisine tablets (Tabex® 1.5mg film-coated tablets, Sopharma)
were obtained by Sopharma Pharmaceuticals, Sofia, Bulgaria.
Tablets from the same batch (Batch number 10211) were crushed,
weighed and four tubes containing an equivalent weight of one
tablet were prepared. To each tube, 1 mL of DMSO was added.
The tubes were then vortex-mixed for 120 s, left to stand for
20 min at room temperature then centrifuged at 22 000 g for
5 min. The supernatant was transferred to a fresh tube, mixed with
internal standard and vortex-mixed for 120s. An aliquot (10 μL) was
then injected onto the HPLC column to determine the amount of
cytisine on the column and to calculate the amount of cytisine
present in one tablet. The value was then compared to the Quality
Certificate (Sopharma) documentation (analytical certificate No.
324/ 17.03.2011).
Oral strip cytisine (external QC)
Five cytisine oral strips were obtained from an Australian marketer –
Quit4Good (www.quit4good.com.au). Each strip was dissolved in
1 mL DMSO then processed as described above.
LC-MS
Standard solutions
Cytisine stock solutions for calibration standards were prepared in
ACN:formate buffer (20:80, v/v). Stock solution was further diluted
in ACN:formate buffer (20:80, v/v) to give appropriate working solu-
tions. The stock solution of the IS was prepared in methanol and the
working solution was prepared at a concentration 400 μM in meth-
anol. Standard samples for the calibration curve of cytisine were
prepared in human plasma. The final concentrations of cytisine in
standard plasma samples were 1.5, 3, 6, 12, 24, 48, 95, 190, 380,
760, and 1522 ng/mL. Plasma samples used for calibration were
stored in À80°C until analysis.
Chromatographic separation
Chromatography was performed using an Agilent 1100 liquid chro-
matography (LC) system coupled with an Agilent MSD model D
single stage quadrupole mass spectrum (MS) detector. Agilent
ChemStation software (Version B.04.03-SP2) (Agilent Technologies,
Goettingen, Germany) was used to access processed data and
chromatograms. Chromatographic separation was achieved on a
Phenomenex Gemini C18 HPLC column (4.6 mm × 150mm, 5 μm)
with a guard column (C18, 4.6 × 10 mm, 5 μm). A mobile phase of
50mM ammonium formate buffer, pH4.5 (solvent A) and acetoni-
trile (solvent B) with a phase gradient 1% (B) from 0 to 3 min, 10%
from 3 to 9 min and 1% at 10 min was used for separation. MS de-
tection using electrospray ionization (ESI) was performed. Detection
by selective ion monitoring (SIM) (positive ion mode) for each mass
ion was used: m/z 191.2 and 173.2 for cytisine and IS, respectively.
Drying gas flow was 12.0L/min and the nebulizer pressure was
35 psig. The total run time was 10min with a flow rate of 0.5mL/min
and sample injection size was 15 μL. Areas of signals were
obtained from chromatograms using manual integration.
S. H. Jeong et al.
Drug Testing
and Analysis
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3. Validation procedures
The assay was validated in human plasma in accordance with the
US FDA guidelines on bioanalytical method over the range of 2.97
to 3043.84 pg on column for selectivity/specificity, precision
accuracy and linearity.[20]
Selectivity/specificity was examined by using blank plasma
samples collected from seven different individuals to look for any
endogenous peaks that could interfere with peaks for cytisine
and IS. Intra- and inter-day accuracy and precision was evaluated
by analyzing quality control (QC) samples at four concentration
levels, low QC, two mid-QCs, and high QC (1.5, 24, 48,
1522 ng/mL). Five replicates were evaluated per concentration. Ac-
curacy was calculated by comparing the measured concentration
with the true concentrations spiked in plasma. Precision, expressed
as relative standard deviations (RSD, %), was calculated on three
separate days. Calibration standards of 10 concentrations in the
range 1.5 to 1522 ng/mL were prepared in human plasma and an-
alyzed (n=3) in three separate analytical runs. Calibration curves in-
cluded a blank sample (no IS), a zero sample (plasma spiked with IS)
and 10 non-zero samples including the limit of quantification
(LOQ). LOQ was evaluated based on signal to noise ratio of 5:1 with
precision and accuracy within 20% of the nominal value. Linearity
was assessed by preparing calibration curves plotting the peak area
ratios of cytisine to IS against the concentrations of cytisine.
Absolute recovery was assessed by comparing the peak areas of
cytisine obtained from extracted spiked plasma standards with
peak areas from un-extracted standards in ACN:formate buffer
(20:80%, v/v).
Short-term temperature stability of cytisine in plasma was exam-
ined by using QC samples (n=3) and comparing freshly spiked
plasma samples to the same samples left at room temperature for
24 h. Freeze-thaw stability was also studied by comparing freshly
prepared spiked plasma samples to the same samples that were
kept at of À80°C for 24 h and thawed at room temperature and
samples that underwent three freeze-thaw cycles.
For urine, selectivity/specificity was examined by using blank
urine samples collected from six different individuals to look for
any endogenous peaks that could interfere with peaks for cytisine.
LOQ was evaluated with precision and accuracy within 20% of the
nominal value.
Sample handling and preparation
Blood samples were allowed to stand at room temperature for
30 min prior to centrifugation at 3000g for 10 min to separate the
plasma and red blood cell fractions. Plasma samples were stored
frozen at À80°C until analysis. Aliquots (100 μL) of plasma samples
were thawed at room temperature and IS (10 μL of 400 μM) was
added along with ice-cold methanol (2:1v/v). Samples were vortex
mixed for 120 s and left overnight at À20°C to precipitate protein.
Samples were then centrifuged (15min at 22000g) and 200 μL of
the clear supernatant was removed and evaporated to dryness
(SC210A SpeedVac® Plus, medium drying rate). The dry extract
was then reconstituted with 30 μL ACN:formate buffer (20:80, v/v),
centrifuged (5min at 22 000 g) and 15 μL of the final extract was
injected onto column.
Urine samples were stored at À20°C until analysis. Prior to
processing, the samples were thawed at room temperature.
0.5 mL of urine sample was taken, IS was added and diluted
(1:1, v/v) with MilliQ® water. Samples were vortex mixed and
centrifuged for 10 min at 22000 g. Solid-phase extraction (SPE)
column (Alltech Prevail C18) was conditioned with 0.5mL methanol
then equilibrated with 0.5mL MilliQ® water under vacuum. After
loading the sample, the column was washed with 0.5 mL of
methanol:water (5:95, v/v) and dried under full vacuum for 10 mins.
Methanol (0.5mL) was used to elute the compounds of interest and
the methanol eluate was collected and 15 μL was injected.
Method – clinical application
Pilot testing for sensitivity
Initially, one healthy subject took a single 3 mg oral dose (two
Tabex®, film-coated 1.5mg oral tablets) to investigate whether
the assay could be used to quantify drug concentrations in
blood plasma. Whilst this involved a higher dose (double the rec-
ommended dose taken at one time) it was selected to increase
the chance of detecting cytisine in human tissue using an analyt-
ical method described in this paper. Blood samples (6 mL) were
collected in heparinised tubes immediately prior to dosing
(t=0), and then at 2 h post-dose. Urine was also collected up to
390 min after dosing. Samples were processed and analyzed as
described above. The developed LC-MS assay was then used to
study single dose pharmacokinetics of cytisine in seven healthy
participants who were smokers at the time, following a single
3 mg oral dose administration.
Single-dose pharmacokinetics study
Participants
Seven healthy subjects (aged between 20 and 39 years) took part
in the study. Subjects were eligible if they were 18 years or older
and were current cigarette smokers at the time of the study
(confirmed by saliva cotinine with NicAlert® – a commercial
semi-quantitative assay that measures cotinine). On average,
subjects smoked 10.6 cigarettes per day. Two subjects had
previously tried to quit smoking. Subjects were excluded from
the study if they self-reported being pregnant or breastfeeding,
suffering from cardiovascular problems, having been diagnosed
with schizophrenia or were currently using nicotine replacement
products or non-nicotine based medications to aid them quit
smoking. Tests were undertaken to exclude severe renal impair-
ment. There was no restriction on diet or smoking during the
study. Participant demographics were collected and an assess-
ment of nicotine dependence using Fagerström Test for Nicotine
Dependence (FTND)[21]
was undertaken.
Study drug
Subjects received a single 3 mg dose of cytisine given as an oral
tablet form (Tabex®, Sopharma Pharmaceuticals, Sofia, Bulgaria).
Sample collection and analysis
Ten blood samples were collected in heparinised tubes at the fol-
lowing times from each subject: 0 (just before dosing), 0.25, 0.5, 1,
2, 3, 4, 6, 8 and 24 h after cytisine administration. Blood samples
were processed to obtain plasma and stored at À80°C until analysis.
After providing a blood sample at 8 h, subjects went home and
returned the next morning 24 h post-dosing. Subjects also provided
spot urine samples. Plasma and urine samples were processed and
analyzed as described above.
Pharmacokinetics of cytisine in healthy smokers following a single dose
Drug Testing
and Analysis
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4. Estimation of PK parameters
Non-linear mixed effects modelling (NONMEM) was used for
modelling and estimating population pharmacokinetic parameters
(CL, Vd). A single compartment model was fit to the data.
Other measurements
Blood pressure, heart rate and respiratory rate were measured at 0
(just before dosing), 2, 4, 8, and 24 h after drug administration.
Ethical approval and consent
All subjects provided a written consent before taking part in the
study. Ethical approval to carry out this study was granted by the
Northern X Regional Ethics Committee of NZ (NTX/11/05/038).
Results
HPLC-UV method
Linearity was verified for this method by visual inspection and using
values for coefficients of determinations (R2
) obtained from nine lin-
ear standard curves generated on three separate days. Correlation
coefficients (R2
) of calibration curves were all above 0.99. Intra-day
and inter-day accuracy and precision values in the range 130 to
4150 ng on column were less than 15% of the actual values.
Cytisine tablets and oral strips
From the calibration curve generated, cytisine concentration was
calculated to be 1.43 mg/mL, corresponding to 1.43 mg per tablet.
The deviation from the value reported on the Quality Certificate
(1.41mg) was 1.27% which was within acceptable limits.
The amount of cytisine in the each of the oral strips was also
calculated. All strips had on average 1.00 to 1.07 mg of cytisine.
The average amount of cytisine in the five oral strips was calculated
to be 1.03 mg. Although no QC documentation is available, this is
within 3% of the stated 1 mg/strip.
LC-MS
Method validation
Spiked plasma samples showed a symmetrical peak for cytisine and
IS. The retention times for cytisine and IS were 7.6 and 9.0min,
respectively (Figures 1 and 2). Selectivity/specificity was exam-
ined by comparing chromatograms of blank plasma samples
with spiked plasma samples. No peaks were observed at the
retention time of cytisine and sulfanilamide in blank plasma
samples collected from seven different individuals. The RSD
(%) of instrument response of the different sources of blank
plasma was within 15% in the seven independent plasma
samples tested.
Accuracy and precision was assessed using QC samples and
values are reported in Tables 1 and 2. Variation for intra-day
and inter-day accuracy and precision was less than 15% for
QC samples. Linearity was verified for this method by using
values for coefficients of determinations (R2
) obtained from
nine linear standard curves generated on three separate days.
Correlation coefficients (R2
) of calibration curves were all
above 0.99. The LOQ for cytisine for this method is 2.97 pg
on column.
The absolute recovery of cytisine was consistent and, on
average, 75% for the QC samples. Stability of cytisine in
spiked plasma samples after storage in room temperature
for 24 h and after 3 freeze-thaw cycles was examined and is
presented in Table 3.
No endogenous peaks were observed at the retention time of
cytisine in blank urine samples collected from six different individ-
uals. The LOQ in urine was 152 pg on column.
Figure 1. Mean plasma concentrations (ng/mL) of cytisine over 24hours
following a single 3mg oral dose. Values are shown as mean± SEM (n=7).
Figure 2. Log[Cytisine] vs. time showing a single linear elimination phase
after maximum concentration is reached (2hours). Values shown are
mean± SEM (n=7).
Table 1. Intra-day variation between spiked plasma samples (n=5)
Spiked concentration
(ng/mL)
Mean concentration
(ng/mL)
Accuracy
(%)
Precision
(RSD, %)
1.5 1.42 95.34 7.56
24 23.59 99.21 11.58
48 50.35 105.87 1.29
1522 1518.61 99.78 2.22
Table 2. Inter-day variation between spiked plasma samples (n=5) an-
alyzed on 3 separate days
Spiked concentration
(ng/mL)
Mean concentration
(ng/mL)
Accuracy
(%)
Precision
(RSD, %)
1.5 1.97 90.17 9.37
24 23.93 92.35 9.44
48 51.08 104.16 3.40
1522 1529.81 100.51 2.32
S. H. Jeong et al.
Drug Testing
and Analysis
wileyonlinelibrary.com/journal/dta Copyright © 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

5. Clinical application
Pilot testing
Plasma samples collected from a healthy subject immediately prior
to cytisine administration (t=0) did not show any endogenous inter-
ference peaks for cytisine or IS. This assay was able to detect and
quantify cytisine in the plasma sample collected from the subject
at 2 h post-dose following a single 3 mg administration. The plasma
concentration of cytisine was determined to be 23.38 ng/mL. This
assay was also able to detect cytisine in urine samples collected
up to 390 min post dose. Urine samples were collected just before
dosing, 0–90 min, 90–180 min, 180–300min and 300–390 min.
Cytisine was detected in all of the urine samples collected.
Single dose study
Study participants
Participant characteristics are described in Table 4. Subjects were all
males, with a mean age of 26.3 years. On average, subjects had
been smoking for 9.5years and only 2 subjects had previously
made an attempt to quit smoking. Six of the seven subjects in the
study had FTND scores of 4 or below (range 1–7) and the mean
FTND score was 3 (low to moderate dependence).
Pharmacokinetics
The current study is the first to describe the pharmacokinetics of
cytisine in humans. After a single 3 mg oral administration, cytisine
was absorbed into the bloodstream, with cytisine detectable in
plasma as early as 15 min after dosing. Peak plasma concentrations
were typically observed at 2 h after administration. In two subjects,
the peak plasma concentration was observed at 1 h post-dose,
suggesting that the peak plasma concentration may actually have
been achieved between 1 and 2 h for all subjects. The peak concen-
trations (Cmax) of cytisine measured in these subjects were between
23.37 and 32.04 ng/mL. The mean Cmax was 27.76ng/mL. Following
the peak plasma concentration, cytisine concentrations declined in
a monophasic manner after a single oral dose. Cytisine was still de-
tectable in the urine collected at 24h for all subjects (mean 24 h
concentration was 428.15 ng/mL).
Cytisine was detected in urine as an unchanged drug and no
metabolites were detected in plasma or in urine in any of the
subjects in the study.
Data collected in this study were modelled using NONMEM to es-
timate PK parameters of volume of distribution (VD) and clearance
(CL). The values for VD and CL were estimated to be 115 L and
16.7 L/h, respectively with standard error values 0.003. Half-life of
cytisine was calculated to be 4.8h.
Safety
There were no reports of adverse events in the study. Blood pres-
sure, heart rate and respiratory rate did not appear to be adversely
affected after 3 mg single dose administration of cytisine.
Discussion
There is currently limited data on the pharmacokinetics of cytisine
in animal studies and none to date reported for humans. The
animal data that exist describe pharmacokinetic parameters in
rabbits and mice but the doses studied are not clinically relevant
in humans.[13,14]
Although both HPLC-UV and LC-MS methods are commonly
used in the detection and quantification of drugs in biological
fluids, an HPLC-UV method was developed and validated first as it
had the advantages of being a relatively simple and low cost
procedure.
The HPLC-UV assay developed had acceptable intra- and inter-
assay accuracy and precision and was accurate in determining the
amount of cytisine in two commercial forms of cytisine including
Tabex® tablets and oral strips. This method was determined to be
externally valid. The main objective of method validation, however,
is to show that the method can be used for its intended purpose
with acceptable reliability and reproducibility. Unfortunately, the
true usefulness (fit for purpose) of the assay (for pharmacokinetic
analysis in humans) was only able to be determined by obtaining
a ‘real’ sample, that is, a plasma sample collected from an individual
after a therapeutically-relevant dose of cytisine. Only after analyzing
the plasma sample collected following a 3 mg single dose adminis-
tration was it revealed that the HPLC-UV method was not suitable
for the quantification of cytisine with dosages used for smoking
cessation. Therefore, a more sensitive method was required.
The developed LC-MS analytical method for the determination of
cytisine in human plasma is more sensitive compared to both the
HPLC-UV method described in this paper and the previously pub-
lished analytical method.[14]
This new method was found to comply
with limits set by US FDA guidelines including accuracy, precision,
specificity and linearity.[20]
This method has been successfully used
to quantitatively determine the concentration of cytisine in a hu-
man subject following a single 3 mg dose of cytisine at 2 h post-
dose. This method, therefore, has the sensitivity required to study
the pharmacokinetics of cytisine in human smokers at clinically
relevant doses.
Plasma cytisine concentrations declined with an average elimina-
tion half-life of 4.8h. The half-life of cytisine has been previously
reported in two animal species including mice and rabbits and
cytisine has been described as a drug with a short half-life. In rab-
bits, the half-life of cytisine following an oral administration has
been reported to be less than 1 h.[14]
The logarithmic plot of mean cytisine concentration versus time
after Cmax (2h) showed a single elimination phase (i.e., no distinc-
tion could be made between the distribution and elimination phase
Table 3. Recovery of cytisine from spiked plasma samples after storage
in room temperature for 24h and after 3 freeze-thaw cycles
Spiked concentration
(ng/mL)
Recovery after 24h in room
temperature (%)
3 Freeze-thaw
cycles (%)
1.5 91.91 89.56
24 93.00 90.94
48 99.15 92.99
1522 90.29 86.95
Table 4. Participant characteristics
Participant characteristics N = 7
Male, % 100
Age, mean± SD, yr 26.3± 6.58
Smoking history, mean± SD, yr 9.5± 6.98
Previously tried to quit, % 28.6
FTND score, mean± SD 3± 2.08
Pharmacokinetics of cytisine in healthy smokers following a single dose
Drug Testing
and Analysis
Drug Test. Analysis (2014) Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

6. of the drug). This indicates that single dose pharmacokinetics of
cytisine may be described using a one-compartment model. This
may propose two possible explanations for how cytisine is distri-
buted in the body. Cytisine may either stay in the blood compart-
ment or it may distribute to other compartments very rapidly. The
apparent volume of distribution for cytisine estimated for the
population in the study (115 L) is more than three times that of
the blood compartment (35 L), which suggests that the latter expla-
nation is more likely.
These results show that pharmacokinetics of cytisine are differ-
ent from that of its synthetic analogue, varenicline. Cytisine not only
has a much shorter half-life than varenicline (approximately 5 h vs
24 h),[22]
but is also different in the way it is distributed in the body.
Varenicline concentrations in plasma decrease in a biphasic manner
and is best described as a two-compartment model.[23]
The volume
of distribution of varenicline is approximately three-fold greater
than cytisine[23]
which suggests that varenicline is distributed to
tissues more extensively. The effect of these pharmacological
agents is expected to be dependent upon the concentrations
achieved in the brain (centrally acting drugs) and potencies at the
target receptor (α4β2 nAChR) and, therefore, it is desirable to look
at the extent of brain penetration of these drugs. Unlike varenicline,
which is known to readily cross the blood brain barrier,[24]
cytisine
has been shown to have poor entry into the brain in animal
models.[24–27]
In rats, average brain concentration of 145 ng/mL has been
shown 15 min after a subcutaneous (s.c.) injection of 1 mg/kg of
cytisine, which was less than 30% of the plasma concentration.[27]
Interestingly, the acid dissociation constant (pKa) of cytisine is 7.8
(cf. 9.3 for varenicline) which indicates that cytisine exists in its
ionised form in lower levels than varenicline at physiological pH
(7.4). Therefore, ionisation alone does not explain cytisine’s limited
brain entry and suggests that cytisine may be removed or excluded
from the brain via active efflux mechanisms. However, cytisine does
not appear to be a substrate for P-gp and its susceptibility to breast
cancer resistance protein (BCRP) transporters has not been found to
differ significantly from varenicline.[24]
More work is needed to
explore whether cytisine is a substrate for other active efflux
transporters. Cytisine, however, is potent and binds to α4β2 nAChRs
at nanomolar concentrations (Ki values ranging from 0.45 to
2.4 nM)[24,28–30]
which may suggest that even with limited blood
brain barrier penetration, the exposure of cytisine at α4β2
nAChRs in the brain may be sufficient to result in activation of
these receptors.
Animal studies have shown that cytisine is renally eliminated.[13]
Consistent with this, this study demonstrates that cytisine is renally
eliminated in humans and detectable in urine. No metabolites were
detected in any plasma or urine sample obtained in this study. The
metabolism of cytisine has not been studied extensively, but
preclinical studies have found that cytisine undergoes minimal
metabolism with 90–95% of the administered dose excreted
unchanged in the urine[31]
and animal studies in rabbits did not
report the presence of metabolic products.[13]
Consistent with
animal studies, this study found that unchanged cytisine is renally
eliminated in humans. For varenicline, two minor metabolites have
been identified in human urine (hydroxyquinoxaline and
N-carbamoylglucuronide metabolites), but more than 90% of the
administered dose is in the blood and urine as unchanged
varenicline.[32]
As with varenicline, the metabolism appears not to
be a primary route of elimination for cytisine. Therefore, unlike
bupropion or nortriptyline (other drugs used for smoking cessation)
which are extensively metabolised by hepatic enzymes (primarily
by CYP2B6 and CYP2D6, respectively,[33,34]
it is unlikely that cytisine
will have drug-drug interactions (DDIs) due to competition for
hepatic enzymes. Furthermore, even if cytisine is metabolised, its
metabolites will be present in very low concentrations compared
to cytisine and so it is unlikely that the metabolites will be pharma-
cologically active. Hepatic insufficiency is, therefore, unlikely to lead
to changes in the pharmacokinetics of cytisine.
On the other hand, as cytisine is eliminated primarily through re-
nal clearance, renal insufficiency would need to be explored to see
whether renal impairment leads to increased systemic exposure to
cytisine and a prolonged half-life in plasma and whether this leads
to increased adverse effects. For varenicline, severe renal impair-
ment leads to 2.1-fold increase in area under the curve (AUC) and
reduced dosing is recommended for these subjects.[35]
In addition,
if cytisine clearance involves active renal secretion (transporters),
there is potential for DDIs with other renally secreted drugs that
are substrates for the same transporters. Varenicline is excreted
partially via active renal secretion and has been shown to be a
substrate for human organic cation transporter 2 (hOCT2), but
not for other major renal transporters such as the human organic
anion transporters (hOAT1 and hOAT3) and human organic
cation/carnitine transporters (hOCTN1 and hOCTN2).[36]
As cytisine
would partly exist as cations at physiological pH, it is expected that
the drug would also be a substrate for active renal transport involv-
ing organic cation transporters.
Cytisine administration did not appear to adversely affect blood
pressure, heart rate and respiratory rate in this study despite the
dose under study being double the normal amount that is
recommended to be ingested at one time.[31]
No side effects
were reported and 3 mg of cytisine was well-tolerated in all sub-
jects. The most common feature of cytisine toxicity reported in
the literature (both animal studies and clinical studies) includes
distresses in the gastrointestinal (GI) tract such as nausea[37–40]
although a meta-analysis found no significant difference
between cytisine and placebo.[8]
Nausea is a commonly reported
dose-related adverse effect with the use of varenicline.[28,35,41]
A
study found that varenicline is less well-tolerated under fasting
conditions and nausea and vomiting may be reduced when
varenicline is taken with food.[42]
It would be interesting to
explore whether this is the same for cytisine as this study was
done with non-fasting subjects.
A potential safety concern with cytisine, as for any other smoking
cessation drug, is that prolonged use may indirectly affect the phar-
macokinetics of concomitantly administered drugs. This is because
chemical constituents (e.g. polycyclic aromatic hydrocarbons) in
cigarette smoke can interact with drug metabolising enzymes
and the use of cytisine may decrease (or stop) cigarette smoking
which in turn could affect the pharmacokinetics and toxicity of
drugs that are metabolically cleared by such enzymes. The most
well-known example is CYP1A2 induction in smokers.[43,44]
Given
that several antipsychotic drugs are metabolised primarily by
CYP1A2,[45,46]
this would be particularly important for patients with
schizophrenia, a population with a high incidence of smoking.[47]
An interesting feature in this study is the little variability observed
between the drug concentrations measured between subjects.
However, the main limitation of this study is the small number of
subjects and thus the little diversity in the study population. The
subjects in this study were relatively homogenous in terms of sex
and build (all relatively fit and no one was overweight); most of
them were in their 20s and their relatively young age was reflected
in the number of years they had smoked (on average less than
10years). All were screened for adequate renal function.
S. H. Jeong et al.
Drug Testing
and Analysis
wileyonlinelibrary.com/journal/dta Copyright © 2014 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

7. Another limitation of this study was that no data were collected
on the effect of food or drink on the pharmacokinetics of cytisine.
Effects of foods on absorption of cytisine would be an area to
explore, whether food affects the oral bioavailability of cytisine.
Given its water solubility and renal elimination, the use of diuretics
such as caffeine or alcohol may have a greater effect on the phar-
macokinetics of cytisine.
More recently, a buccal strip containing cytisine has been
marketed in Australia. Interestingly, cytisine is not a registered
medication in Australia and no quality assurance documentation
of these products is available. It would be interesting to study
the bioavailability of these two different dosage forms of cytisine
to determine the differences in the absorption profile of the two
formulations and more importantly whether this has an impact
on craving for cigarettes. However, as there is no formal docu-
mentation for these oral strips, it is uncertain whether these
products contain only cytisine.
Future studies will need to look at cytisine pharmacokinetics in
renally impaired patients and other special population groups to
determine whether dose adjustments should be made to promote
safe use of cytisine. The data generated in the present study will aid
the pharmacometric modelling for future multi-dose pharmacoki-
netic studies in humans.
Conclusion
This paper reports two methods for the detection and quantifica-
tion of cytisine, a nicotinic partial agonist that has been used as
an aid to smoking cessation. An HPLC-UV assay was able to quantify
cytisine in the two commercial forms of cytisine with acceptable
accuracy and precision, but was not sensitive enough to quantify
cytisine in human plasma after clinically relevant doses. The
LC-MS bioanalytical assay was therefore developed to support
pharmacological studies of cytisine in humans. This method has
been validated and results are within the acceptable range as set
by the US FDA guidelines. This method was successfully used to
detect and quantify cytisine in human plasma after a single oral
administration of Tabex®.
Cytisine was well tolerated after a 3-mg single dose in smokers
and no safety concerns were identified at this dose. Cytisine
showed a simple pharmacokinetic profile with maximum concen-
trations reached typically at 2 h post-dose. Cytisine was detected
as an unchanged drug in plasma and urine and no metabolites
were detected.
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