Dta1707

Research articleDrug 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

Pharmacokinetics of cytisine, an α4β2 nicotinicreceptor partial agonist, in healthy smokersfollowing a single doseSoo 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 cessationmedication. 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 performanceliquid chromatography-ultraviolet (HPLC-UV) method was developed and validated for analysis of Tabex® and nicotine-free oralstrips, two commercial products containing cytisine. A sensitive liquid chromatography-mass spectrometry (LC-MS) method wasdeveloped and validated for the quantification of cytisine in human plasma and for the detection of cytisine in urine. Single-dosepharmacokinetics of cytisine was studied in healthy smokers. Subjects received a single 3mg oral dose administration of cytisine.Cytisine was detected in all plasma samples collected after administration, including 15min post-dose and at 24h. Cytisine wasrenally 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

* Correspondence to: Soo Hee Jeong, University of Auckland, Private Bag 92019Auckland 1142 New Zealand.E-mail: [email protected]

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

Introduction

Smoking is a leading cause of many diseases including cancer, car-diovascular disease, and respiratory disease.[1,2] Smoking cessationimproves health risks and survival,[3] but only 3 to 5% of smokerswho 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 (Cytisuslaburnum), is an α4β2 nicotinic receptor partial agonist that has beenused for smoking cessation since the 1960s.[5,6] Cytisine has beenwidely used in many countries in Central and Eastern Europe andCentral Asia,[7] but unlike its pharmacologically related analoguevarenicline, cytisine has not been approved for use as a smokingcessation medication in many countries including the USA, theUK, Australia and New Zealand. A systematic review and meta-analysis of clinical studies with cytisine suggests that cytisine iseffective for smoking cessation.[8] There is growing interest ingetting cytisine approved as a smoking cessation agent because ofits potential to be a much more affordable medication for smokingcessation than any other current pharmacotherapy available.[8–11]

In an economic evaluation that compared cytisine to varenicline forsmoking cessation, cytisine was estimated to be more cost-effectivethan varenicline.[12] Despite its long history of use, significant gapsremain in our knowledge of cytisine. Animal pharmacokinetic dataexist[13,14] but we have found no publicly available informationabout the pharmacokinetics/metabolism of cytisine in humans.

Cytisine is commercially available as an oral tablet marketed bySopharma, a Bulgarian pharmaceutical company, under its tradename Tabex®. The standard dosing schedule of Tabex® is complexand the rationale is not well understood. One Tabex® tablet con-tains 1.5mg of cytisine and it is recommended that a person takes1 tablet every 2h (maximum of 6 tablets per day) as a starting dose

Drug Test. Analysis (2014)

for the first 3 days of the treatment. The dosing frequency and inter-val are changed throughout the course of the treatment. The doseis 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 tabletsper day on days 17–20 (1 tablet every 5 h). During the last 5 daysof treatment (days 21–25) the recommended dose is 1 or 2 tabletsevery 6 h (maximum of 2 tablets per day). Recently, another com-mercial product of cytisine became available in the form of an oralstrip. This has been marketed in Australia and each strip is reportedto contain 1mg of cytisine (instead of 1.5mg as in tablets). Thisproduct 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 shownto be applicable for herbal intoxication or drug abuse cases[17–19];however, human PK analysis has not been conducted. No methodshave yet studied the commercial forms of cytisine and no methodhas quantified cytisine in clinical samples following administrationof these products.

Copyright © 2014 John Wiley & Sons, Ltd.

S. H. Jeong et al.

Drug Testing

and Analysis

This paper has two overarching aims:

A. To describe the development and validation of an analytical assayfor cytisine and its use in the analysis of two commercially availableproducts Tabex® tablets and Quit4Good Nicotine Free Oral Strips.

Plasma and urine samples taken from a healthy subject who hadtaken Tabex® were analyzed to determine whether this methodwas sensitive enough to detect levels of cytisine following oraladministration. For simplification, a single dose was chosen overthe recommended split dosing. Initially, a high performance liquidchromatography-ultraviolet (HPLC-UV) assay was developed andvalidated for analysis of the two commercial products of cytisine.However, the sensitivity of this method was insufficient (not fit forpurpose) to detect cytisine in human plasma after the administereddose. Therefore, it was necessary to develop and validate a moresensitive method using high performance liquid chromatographycoupled with mass spectrometry (LC-MS) that could be used toinvestigate pharmacokinetics of cytisine in subsequent human studies.

B. To describe the pharmacokinetic characteristics of cytisine inhealthy 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 hperiod, to determine clearance, volume of distribution andhalf-life of cytisine in humans, and to screen for the presenceof metabolite(s) in human plasma and urine in healthysmokers following a 3mg single oral dose administration.

2. To measure cytisine’s effect on heart rate, blood pressure andbreathing rate over 24h following a 3mg single doseadministration.

Methods – development of analytical assays

Chemicals and reagents

Cytisine (≥99% purity) and sulfanilamide (internal standard, IS)(p-aminobenzenesulfonamide, ≥99% purity) were purchased fromSigma Aldrich (Auckland, New Zealand). Methanol (>99%, HPLCgrade, Sigma Aldrich) was used in sample preparation. LC-gradewater (Millipore®, Milli-Q system) and methanol (>99%, HPLCgrade, Sigma Aldrich) were used for the mobile phase in theHPLC-UV method and LC-grade water and acetonitrile (ACN,>99%, HPLC-grade, Sigma Aldrich) were used for the mobile phasein the LC-MS method.

HPLC-UV

Standard solutions

Cytisine stock solutions for calibration standards were prepared indimethyl sulfoxide (DMSO). Stock solution was further diluted inDMSO to give appropriate working solutions. The IS solution wasprepared in DMSO at a concentration 400μM.Chromatographic separation was achieved on a Phenomenex

Gemini C18 HPLC column (4.6mm×150mm, 5μm) with a guardcolumn (C18, 4.6× 10mm, 5μm). Mobile phase consisted ofmethanol and 50mM ammonium acetate buffer adjusted topH6.5 (1 to 5% methanol gradient; flow rate 1.0mL/min, pressure120bar), with UV monitoring of the column effluent. Wavelengthsfrom 220 to 310nm were monitored and quantification wasperformed at absorbance of 310 nm (cytisine) and 280 nm(sulfanilamide). Signals areas were obtained from chromatograms

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usingmanual integration. Cytisine/IS peak area ratio was calculatedas a quantitative measure to prepare calibration curves.

The assay was validated in accordance to the US FDA guidelinesfor bioanalytical methods validation over the range of 130 to4150ng 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 onetablet were prepared. To each tube, 1mL of DMSO was added.The tubes were then vortex-mixed for 120 s, left to stand for20 min at room temperature then centrifuged at 22 000g for5min. The supernatant was transferred to a fresh tube, mixed withinternal standard and vortex-mixed for 120 s. An aliquot (10μL) wasthen injected onto the HPLC column to determine the amount ofcytisine on the column and to calculate the amount of cytisinepresent in one tablet. The value was then compared to the QualityCertificate (Sopharma) documentation (analytical certificate No.324/ 17.03.2011).

Oral strip cytisine (external QC)

Five cytisine oral strips were obtained from an Australianmarketer –Quit4Good (www.quit4good.com.au). Each strip was dissolved in1mL DMSO then processed as described above.

LC-MS

Standard solutions

Cytisine stock solutions for calibration standards were prepared inACN:formate buffer (20:80, v/v). Stock solution was further dilutedin ACN:formate buffer (20:80, v/v) to give appropriate working solu-tions. The stock solution of the IS was prepared inmethanol and theworking solution was prepared at a concentration 400μM in meth-anol. Standard samples for the calibration curve of cytisine wereprepared in human plasma. The final concentrations of cytisine instandard plasma samples were 1.5, 3, 6, 12, 24, 48, 95, 190, 380,760, and 1522ng/mL. Plasma samples used for calibration werestored in �80°C until analysis.

Chromatographic separation

Chromatography was performed using an Agilent 1100 liquid chro-matography (LC) system coupled with an Agilent MSD model Dsingle stage quadrupole mass spectrum (MS) detector. AgilentChemStation software (Version B.04.03-SP2) (Agilent Technologies,Goettingen, Germany) was used to access processed data andchromatograms. Chromatographic separation was achieved on aPhenomenex Gemini C18 HPLC column (4.6mm×150mm, 5μm)with a guard column (C18, 4.6 × 10mm, 5μm). A mobile phase of50mM ammonium formate buffer, pH4.5 (solvent A) and acetoni-trile (solvent B) with a phase gradient 1% (B) from 0 to 3min, 10%from 3 to 9min and 1% at 10min was used for separation. MS de-tection using electrospray ionization (ESI) was performed. Detectionby selective ion monitoring (SIM) (positive ion mode) for each massion was used: m/z 191.2 and 173.2 for cytisine and IS, respectively.Drying gas flow was 12.0 L/min and the nebulizer pressure was35 psig. The total run time was 10min with a flow rate of 0.5mL/minand sample injection size was 15 μL. Areas of signals wereobtained from chromatograms using manual integration.

14 John Wiley & Sons, Ltd. Drug Test. Analysis (2014)

http://www.quit4good.com.au

Pharmacokinetics of cytisine in healthy smokers following a single dose

Drug Testing

and Analysis

Validation procedures

The assay was validated in human plasma in accordance with theUS FDA guidelines on bioanalytical method over the range of 2.97to 3043.84 pg on column for selectivity/specificity, precisionaccuracy and linearity.[20]

Selectivity/specificity was examined by using blank plasmasamples collected from seven different individuals to look for anyendogenous peaks that could interfere with peaks for cytisineand IS. Intra- and inter-day accuracy and precision was evaluatedby analyzing quality control (QC) samples at four concentrationlevels, 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 concentrationwith the true concentrations spiked in plasma. Precision, expressedas relative standard deviations (RSD, %), was calculated on threeseparate days. Calibration standards of 10 concentrations in therange 1.5 to 1522ng/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 spikedwith IS)and 10 non-zero samples including the limit of quantification(LOQ). LOQ was evaluated based on signal to noise ratio of 5:1 withprecision and accuracy within 20% of the nominal value. Linearitywas assessed by preparing calibration curves plotting the peak arearatios of cytisine to IS against the concentrations of cytisine.

Absolute recovery was assessed by comparing the peak areas ofcytisine obtained from extracted spiked plasma standards withpeak 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 spikedplasma samples to the same samples left at room temperature for24 h. Freeze-thaw stability was also studied by comparing freshlyprepared spiked plasma samples to the same samples that werekept at of �80°C for 24 h and thawed at room temperature andsamples that underwent three freeze-thaw cycles.

For urine, selectivity/specificity was examined by using blankurine samples collected from six different individuals to look forany endogenous peaks that could interfere with peaks for cytisine.LOQ was evaluated with precision and accuracy within 20% of thenominal value.

Sample handling and preparation

Blood samples were allowed to stand at room temperature for30min prior to centrifugation at 3000g for 10min to separate theplasma and red blood cell fractions. Plasma samples were storedfrozen at �80°C until analysis. Aliquots (100μL) of plasma sampleswere thawed at room temperature and IS (10μL of 400μM) wasadded along with ice-cold methanol (2:1 v/v). Samples were vortexmixed for 120 s and left overnight at �20°C to precipitate protein.Samples were then centrifuged (15min at 22000g) and 200μL ofthe clear supernatant was removed and evaporated to dryness(SC210A SpeedVac® Plus, medium drying rate). The dry extractwas then reconstituted with 30μL ACN:formate buffer (20:80, v/v),centrifuged (5min at 22 000g) and 15μL of the final extract wasinjected onto column.

Urine samples were stored at �20°C until analysis. Prior toprocessing, 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 andcentrifuged for 10min at 22000g. Solid-phase extraction (SPE)column (Alltech Prevail C18) was conditionedwith 0.5mLmethanol

Drug Test. Analysis (2014) Copyright © 2014 John Wiley

then equilibrated with 0.5mL MilliQ® water under vacuum. Afterloading the sample, the column was washed with 0.5mL ofmethanol:water (5:95, v/v) and dried under full vacuum for 10mins.Methanol (0.5mL) was used to elute the compounds of interest andthe methanol eluate was collected and 15μL was injected.

Method – clinical application

Pilot testing for sensitivity

Initially, one healthy subject took a single 3mg oral dose (twoTabex®, film-coated 1.5mg oral tablets) to investigate whetherthe assay could be used to quantify drug concentrations inblood plasma. Whilst this involved a higher dose (double the rec-ommended dose taken at one time) it was selected to increasethe chance of detecting cytisine in human tissue using an analyt-ical method described in this paper. Blood samples (6mL) werecollected in heparinised tubes immediately prior to dosing(t=0), and then at 2 h post-dose. Urine was also collected up to390min after dosing. Samples were processed and analyzed asdescribed above. The developed LC-MS assay was then used tostudy single dose pharmacokinetics of cytisine in seven healthyparticipants who were smokers at the time, following a single3mg oral dose administration.

Single-dose pharmacokinetics study

Participants

Seven healthy subjects (aged between 20 and 39 years) took partin the study. Subjects were eligible if they were 18 years or olderand were current cigarette smokers at the time of the study(confirmed by saliva cotinine with NicAlert® – a commercialsemi-quantitative assay that measures cotinine). On average,subjects smoked 10.6 cigarettes per day. Two subjects hadpreviously tried to quit smoking. Subjects were excluded fromthe study if they self-reported being pregnant or breastfeeding,suffering from cardiovascular problems, having been diagnosedwith schizophrenia or were currently using nicotine replacementproducts or non-nicotine based medications to aid them quitsmoking. Tests were undertaken to exclude severe renal impair-ment. There was no restriction on diet or smoking during thestudy. Participant demographics were collected and an assess-ment of nicotine dependence using Fagerström Test for NicotineDependence (FTND)[21] was undertaken.

Study drug

Subjects received a single 3mg dose of cytisine given as an oraltablet 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 24h after cytisine administration. Blood sampleswere processed to obtain plasma and stored at�80°C until analysis.After providing a blood sample at 8 h, subjects went home andreturned the nextmorning 24h post-dosing. Subjects also providedspot urine samples. Plasma and urine samples were processed andanalyzed as described above.

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Figure 2. Log[Cytisine] vs. time showing a single linear elimination phaseafter maximum concentration is reached (2hours). Values shown aremean±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, %)

S. H. Jeong et al.

Drug Testing

and Analysis

Estimation of PK parameters

Non-linear mixed effects modelling (NONMEM) was used formodelling 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 24h after drug administration.

Ethical approval and consent

All subjects provided a written consent before taking part in thestudy. Ethical approval to carry out this study was granted by theNorthern X Regional Ethics Committee of NZ (NTX/11/05/038).

Results

HPLC-UV method

Linearity was verified for this method by visual inspection and usingvalues for coefficients of determinations (R2) obtained fromnine lin-ear standard curves generated on three separate days. Correlationcoefficients (R2) of calibration curves were all above 0.99. Intra-dayand inter-day accuracy and precision values in the range 130 to4150ng on column were less than 15% of the actual values.

Cytisine tablets and oral strips

From the calibration curve generated, cytisine concentration wascalculated to be 1.43mg/mL, corresponding to 1.43mg 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 1.07mg of cytisine.The average amount of cytisine in the five oral strips was calculatedto be 1.03mg. Although no QC documentation is available, this iswithin 3% of the stated 1mg/strip.

LC-MS

Method validation

Spiked plasma samples showed a symmetrical peak for cytisine andIS. 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 sampleswith spiked plasma samples. No peaks were observed at the

Figure 1. Mean plasma concentrations (ng/mL) of cytisine over 24hoursfollowing a single 3mg oral dose. Values are shown as mean±SEM (n=7).


retention time of cytisine and sulfanilamide in blank plasmasamples collected from seven different individuals. The RSD(%) of instrument response of the different sources of blankplasma was within 15% in the seven independent plasmasamples tested.

Accuracy and precision was assessed using QC samples andvalues are reported in Tables 1 and 2. Variation for intra-dayand inter-day accuracy and precision was less than 15% forQC samples. Linearity was verified for this method by usingvalues for coefficients of determinations (R2) obtained fromnine linear standard curves generated on three separate days.Correlation coefficients (R2) of calibration curves were allabove 0.99. The LOQ for cytisine for this method is 2.97 pgon column.

The absolute recovery of cytisine was consistent and, onaverage, 75% for the QC samples. Stability of cytisine inspiked plasma samples after storage in room temperaturefor 24 h and after 3 freeze-thaw cycles was examined and ispresented in Table 3.

No endogenous peaks were observed at the retention time ofcytisine in blank urine samples collected from six different individ-uals. The LOQ in urine was 152pg on column.

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


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


Table 3. Recovery of cytisine from spiked plasma samples after storagein room temperature for 24h and after 3 freeze-thaw cycles


Recovery after 24h in roomtemperature (%)

3 Freeze-thawcycles (%)

1.5 91.91 89.56

24 93.00 90.94

48 99.15 92.99

1522 90.29 86.95


Drug Testing

and Analysis

Clinical application

Pilot testing

Plasma samples collected from a healthy subject immediately priorto cytisine administration (t=0) did not show any endogenous inter-ference peaks for cytisine or IS. This assay was able to detect andquantify cytisine in the plasma sample collected from the subjectat 2 h post-dose following a single 3mg administration. The plasmaconcentration of cytisine was determined to be 23.38 ng/mL. Thisassay was also able to detect cytisine in urine samples collectedup to 390min post dose. Urine samples were collected just beforedosing, 0–90min, 90–180min, 180–300min and 300–390min.Cytisine was detected in all of the urine samples collected.

Single dose study

Study participants

Participant characteristics are described in Table 4. Subjects were allmales, with a mean age of 26.3 years. On average, subjects hadbeen smoking for 9.5 years and only 2 subjects had previouslymade an attempt to quit smoking. Six of the seven subjects in thestudy had FTND scores of 4 or below (range 1–7) and the meanFTND score was 3 (low to moderate dependence).

Pharmacokinetics

The current study is the first to describe the pharmacokinetics ofcytisine in humans. After a single 3mg oral administration, cytisinewas absorbed into the bloodstream, with cytisine detectable inplasma as early as 15min after dosing. Peak plasma concentrationswere typically observed at 2h after administration. In two subjects,the peak plasma concentration was observed at 1 h post-dose,suggesting that the peak plasma concentration may actually havebeen achieved between 1 and 2h for all subjects. The peak concen-trations (Cmax) of cytisinemeasured in these subjects were between23.37 and 32.04 ng/mL. Themean Cmax was 27.76ng/mL. Followingthe peak plasma concentration, cytisine concentrations declined ina monophasic manner after a single oral dose. Cytisine was still de-tectable in the urine collected at 24h for all subjects (mean 24hconcentration was 428.15 ng/mL).

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


Cytisine was detected in urine as an unchanged drug and nometabolites were detected in plasma or in urine in any of thesubjects in the study.

Data collected in this study weremodelled 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 and16.7 L/h, respectively with standard error values 0.003. Half-life ofcytisine was calculated to be 4.8 h.

Safety

There were no reports of adverse events in the study. Blood pres-sure, heart rate and respiratory rate did not appear to be adverselyaffected after 3mg single dose administration of cytisine.

Discussion

There is currently limited data on the pharmacokinetics of cytisinein animal studies and none to date reported for humans. Theanimal data that exist describe pharmacokinetic parameters inrabbits and mice but the doses studied are not clinically relevantin humans.[13,14]

Although both HPLC-UV and LC-MS methods are commonlyused in the detection and quantification of drugs in biologicalfluids, an HPLC-UV method was developed and validated first as ithad the advantages of being a relatively simple and low costprocedure.

The HPLC-UV assay developed had acceptable intra- and inter-assay accuracy and precision and was accurate in determining theamount of cytisine in two commercial forms of cytisine includingTabex® tablets and oral strips. This method was determined to beexternally valid. The main objective of method validation, however,is to show that the method can be used for its intended purposewith acceptable reliability and reproducibility. Unfortunately, thetrue usefulness (fit for purpose) of the assay (for pharmacokineticanalysis in humans) was only able to be determined by obtaininga ‘real’ sample, that is, a plasma sample collected from an individualafter a therapeutically-relevant dose of cytisine. Only after analyzingthe plasma sample collected following a 3mg single dose adminis-tration was it revealed that the HPLC-UV method was not suitablefor the quantification of cytisine with dosages used for smokingcessation. Therefore, a more sensitive method was required.

The developed LC-MS analytical method for the determination ofcytisine in human plasma is more sensitive compared to both theHPLC-UV method described in this paper and the previously pub-lished analytical method.[14] This newmethodwas found to complywith limits set by US FDA guidelines including accuracy, precision,specificity and linearity.[20] This method has been successfully usedto quantitatively determine the concentration of cytisine in a hu-man subject following a single 3mg dose of cytisine at 2 h post-dose. This method, therefore, has the sensitivity required to studythe pharmacokinetics of cytisine in human smokers at clinicallyrelevant doses.

Plasma cytisine concentrations declinedwith an average elimina-tion half-life of 4.8h. The half-life of cytisine has been previouslyreported in two animal species including mice and rabbits andcytisine has been described as a drug with a short half-life. In rab-bits, the half-life of cytisine following an oral administration hasbeen reported to be less than 1h.[14]

The logarithmic plot of mean cytisine concentration versus timeafter Cmax (2 h) showed a single elimination phase (i.e., no distinc-tion could bemade between the distribution and elimination phase


S. H. Jeong et al.

Drug Testing

and Analysis

of the drug). This indicates that single dose pharmacokinetics ofcytisine may be described using a one-compartment model. Thismay 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. Theapparent volume of distribution for cytisine estimated for thepopulation in the study (115 L) is more than three times that ofthe 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 onlyhas a much shorter half-life than varenicline (approximately 5h vs24 h),[22] but is also different in the way it is distributed in the body.Varenicline concentrations in plasma decrease in a biphasicmannerand is best described as a two-compartment model.[23] The volumeof distribution of varenicline is approximately three-fold greaterthan cytisine[23] which suggests that varenicline is distributed totissues more extensively. The effect of these pharmacologicalagents is expected to be dependent upon the concentrationsachieved in the brain (centrally acting drugs) and potencies at thetarget receptor (α4β2 nAChR) and, therefore, it is desirable to lookat the extent of brain penetration of these drugs. Unlike varenicline,which is known to readily cross the blood brain barrier,[24] cytisinehas been shown to have poor entry into the brain in animalmodels.[24–27]

In rats, average brain concentration of 145 ng/mL has beenshown 15min after a subcutaneous (s.c.) injection of 1mg/kg ofcytisine, 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 itsionised form in lower levels than varenicline at physiological pH(7.4). Therefore, ionisation alone does not explain cytisine’s limitedbrain entry and suggests that cytisinemay be removed or excludedfrom the brain via active effluxmechanisms. However, cytisine doesnot appear to be a substrate for P-gp and its susceptibility to breastcancer resistance protein (BCRP) transporters has not been found todiffer significantly from varenicline.[24] More work is needed toexplore whether cytisine is a substrate for other active effluxtransporters. Cytisine, however, is potent and binds to α4β2 nAChRsat nanomolar concentrations (Ki values ranging from 0.45 to2.4 nM)[24,28–30] which may suggest that even with limited bloodbrain barrier penetration, the exposure of cytisine at α4β2nAChRs in the brain may be sufficient to result in activation ofthese receptors.Animal studies have shown that cytisine is renally eliminated.[13]

Consistent with this, this study demonstrates that cytisine is renallyeliminated in humans and detectable in urine. Nometabolites weredetected in any plasma or urine sample obtained in this study. Themetabolism of cytisine has not been studied extensively, butpreclinical studies have found that cytisine undergoes minimalmetabolism with 90–95% of the administered dose excretedunchanged in the urine[31] and animal studies in rabbits did notreport the presence of metabolic products.[13] Consistent withanimal studies, this study found that unchanged cytisine is renallyeliminated in humans. For varenicline, two minor metabolites havebeen identified in human urine (hydroxyquinoxaline andN-carbamoylglucuronide metabolites), but more than 90% of theadministered dose is in the blood and urine as unchangedvarenicline.[32] As with varenicline, the metabolism appears not tobe a primary route of elimination for cytisine. Therefore, unlikebupropion 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 cytisinewill have drug-drug interactions (DDIs) due to competition forhepatic enzymes. Furthermore, even if cytisine is metabolised, itsmetabolites will be present in very low concentrations comparedto cytisine and so it is unlikely that the metabolites will be pharma-cologically active. Hepatic insufficiency is, therefore, unlikely to leadto 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 seewhether renal impairment leads to increased systemic exposure tocytisine and a prolonged half-life in plasma and whether this leadsto increased adverse effects. For varenicline, severe renal impair-ment leads to 2.1-fold increase in area under the curve (AUC) andreduced 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 thatare substrates for the same transporters. Varenicline is excretedpartially via active renal secretion and has been shown to be asubstrate for human organic cation transporter 2 (hOCT2), butnot for other major renal transporters such as the human organicanion transporters (hOAT1 and hOAT3) and human organiccation/carnitine transporters (hOCTN1 and hOCTN2).[36] As cytisinewould partly exist as cations at physiological pH, it is expected thatthe drug would also be a substrate for active renal transport involv-ing organic cation transporters.

Cytisine administration did not appear to adversely affect bloodpressure, heart rate and respiratory rate in this study despite thedose under study being double the normal amount that isrecommended to be ingested at one time.[31] No side effectswere reported and 3mg of cytisine was well-tolerated in all sub-jects. The most common feature of cytisine toxicity reported inthe literature (both animal studies and clinical studies) includesdistresses in the gastrointestinal (GI) tract such as nausea[37–40]

although a meta-analysis found no significant differencebetween cytisine and placebo.[8] Nausea is a commonly reporteddose-related adverse effect with the use of varenicline.[28,35,41] Astudy found that varenicline is less well-tolerated under fastingconditions and nausea and vomiting may be reduced whenvarenicline is taken with food.[42] It would be interesting toexplore whether this is the same for cytisine as this study wasdone with non-fasting subjects.

A potential safety concern with cytisine, as for any other smokingcessation drug, is that prolonged usemay indirectly affect the phar-macokinetics of concomitantly administered drugs. This is becausechemical constituents (e.g. polycyclic aromatic hydrocarbons) incigarette smoke can interact with drug metabolising enzymesand the use of cytisine may decrease (or stop) cigarette smokingwhich in turn could affect the pharmacokinetics and toxicity ofdrugs that are metabolically cleared by such enzymes. The mostwell-known example is CYP1A2 induction in smokers.[43,44] Giventhat several antipsychotic drugs are metabolised primarily byCYP1A2,[45,46] this would be particularly important for patients withschizophrenia, a population with a high incidence of smoking.[47]

An interesting feature in this study is the little variability observedbetween the drug concentrations measured between subjects.However, the main limitation of this study is the small number ofsubjects and thus the little diversity in the study population. Thesubjects in this study were relatively homogenous in terms of sexand build (all relatively fit and no one was overweight); most ofthem were in their 20s and their relatively young age was reflectedin the number of years they had smoked (on average less than10years). All were screened for adequate renal function.



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Another limitation of this study was that no data were collectedon the effect of food or drink on the pharmacokinetics of cytisine.Effects of foods on absorption of cytisine would be an area toexplore, whether food affects the oral bioavailability of cytisine.Given its water solubility and renal elimination, the use of diureticssuch as caffeine or alcohol may have a greater effect on the phar-macokinetics of cytisine.

More recently, a buccal strip containing cytisine has beenmarketed in Australia. Interestingly, cytisine is not a registeredmedication in Australia and no quality assurance documentationof these products is available. It would be interesting to studythe bioavailability of these two different dosage forms of cytisineto determine the differences in the absorption profile of the twoformulations and more importantly whether this has an impacton craving for cigarettes. However, as there is no formal docu-mentation for these oral strips, it is uncertain whether theseproducts contain only cytisine.

Future studies will need to look at cytisine pharmacokinetics inrenally impaired patients and other special population groups todetermine whether dose adjustments should be made to promotesafe use of cytisine. The data generated in the present study will aidthe 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 asan aid to smoking cessation. AnHPLC-UV assay was able to quantifycytisine in the two commercial forms of cytisine with acceptableaccuracy and precision, but was not sensitive enough to quantifycytisine in human plasma after clinically relevant doses. TheLC-MS bioanalytical assay was therefore developed to supportpharmacological studies of cytisine in humans. This method hasbeen validated and results are within the acceptable range as setby the US FDA guidelines. This method was successfully used todetect and quantify cytisine in human plasma after a single oraladministration of Tabex®.

Cytisine was well tolerated after a 3-mg single dose in smokersand no safety concerns were identified at this dose. Cytisineshowed a simple pharmacokinetic profile with maximum concen-trations reached typically at 2 h post-dose. Cytisine was detectedas an unchanged drug in plasma and urine and no metaboliteswere detected.

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Dta1707

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