Clinical trial NCT03268343
Conditions: Smoking Cessation
Sponsor: Achieve Life Sciences
Start date: 2017-08-08; Completion date: 2017-09-01
Enrollment: 24 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
This will be an open-label, randomised, 2-period, single-dose crossover study to determine the comparative bioavailability of cytisine following single-dose administration in healthy male and female subjects under fed and fasted conditions.

The study will be comprised of a pre-study screen, followed by 2 treatment periods (1 and 2) and a post-study follow-up.

Detailed description:

Arms: arm 1: Schedule A (12 subjects):

* Period 1: Cytisine (2 x 1.5 mg tablets) will be administered 30 minutes after the start of a high fat breakfast (fed state).
* Period 2: Cytisine (2 x 1.5 mg tablets) will be administered after an overnight fast of at least 10 hours (fasting state). arm 2: Schedule B (12 subjects):

* Period 1: Cytisine (2 x 1.5 mg tablets) will be administered after an overnight fast of at least 10 hours (fasting state).
* Period 2: Cytisine (2 x 1.5 mg tablets) will be administered 30 minutes after the start of a high fat breakfast (fed state).
Interventions: intervention 1: Cytisine
Intervention descriptions: intervention 1: Cytisine 1.5 mg Film-Coated Tablets

Protocol documents:
Prot_000.pdf: 
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Protocol ACH-CYT-01, 19 May 2017, Version 1.0 
 
Cytisine 
 CONFIDENTIAL 
 
 
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SYNOPSIS   
NAME OF COMPANY: Achieve Life Sciences, Inc 
NAME OF INVESTIGATIONAL MEDICINAL PRODUCT (IMP):  
Cytisine 1.5 mg film-coated tablet 
TITLE OF STUDY: A phase 1 open label, randomized, two-way crossover study in healthy 
volunteers to investigate the effect of food on the bioavailability of cytisine 
CHIEF INVESTIGATOR: 
 
STUDY CENTRE: Simbec Research Ltd (Simbec) 
     Merthyr Tydfil, CF48 4DR, UK 
CLINICAL PHASE: I 
OBJECTIVES: 
Primary:  
• To compare the bioavailability (Cmax and AUC0-∞) of cytisine in healthy subjects 
under fed and fasted conditions, following administration of 3 mg cytisine (2 x 1.5 
mg cytisine tablets). 
Secondary:  
• To compare the AUC0-t, T½ and Tmax of cytisine in subjects under fed and fasted 
conditions, following administration of 3 mg cytisine (2 x 1.5 mg cytisine tablets). 
• To assess the safety and tolerability of cytisine at the 3 mg dose level under fed and 
fasted conditions. 
• To assess the renal elimination of cytisine via measurement of urinary concentrations 
of cytisine. 
• To explore possible effects of other study parameters on the bioavailability of cytisine 
(e.g. gender, BMI). 
• To explore for cytisine effects on QT/QTc interval prolongation and cardiac safety. 
 
METHODOLOGY:  
This will be an open-label, randomised, 2-period, single-dose crossover study to determine the 
comparative bioavailability of cytisine following single-dose administration in healthy male and 
female subjects under fed and fasted conditions.   
The study will be comprised of a pre-study screen, followed by 2 treatment periods (1 and 2) and 
a post-study follow-up.   
Screening (Day -28 to Day -2): Screening assessments will be carried out within 28 days before 
first administration of IMP. Eligible subjects will be asked to return for the treatment periods.  
Continued eligibility will be confirmed pre-dose during each treatment period. 
Treatment Periods (Day -1 to Day 2):  Eligible subjects will receive a single-dose of IMP 
under fed and fasted conditions over 2 treatment periods.  
Each treatment period will be approximately 2 days’ duration and will require 2 overnight stays 
in the Clinical Unit.  Each period will begin the afternoon before dosing on Day -1 until 24 hours 


Protocol ACH-CYT-01, 19 May 2017, Version 1.0 
 
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(h) postdose (Day 2).   
During each treatment period, subjects will arrive at the Clinical Unit on Day -1 and will 
undergo an overnight fast of at least 10 h prior to dosing on Day 1. Holter monitoring will also 
be initiated. Randomisation will occur on the morning of Day 1, Period 1, at least 1 hour prior to 
dosing, and IMP will be administered under fasted (after an overnight fast of at least 10 h) or fed 
(after a high fat breakfast) conditions.  Subjects will be discharged 24 h postdose (Day 2).   
Pharmacokinetic (PK) samples (plasma and urine) will be collected predose and up to 24 h 
postdose (Day 2) during each period (17 plasma and 6 urine samples per period) for the 
measurement of plasma and urine cytisine concentrations. Safety will also be evaluated at 
specified times.   
Continuous ECG measurements will be obtained via Holter monitor that will be initiated on Day-
1 prior to dosing and continue until approximately 24 hours post dosing.   
Final blood draw for repeat haematology and chemistries will be obtained just prior to discharge 
on Day 2. 
There will be at least 72 hours between dose administrations. 
Post Study: Post study telephone follow-up will be conducted 6 to 8 days after last dose of IMP. 
NUMBER OF SUBJECTS: 24 subjects (approximately 12 males and 12 females) will be 
randomised/enrolled to complete the study. 
MAIN INCLUSION CRITERIA: 
Healthy males or females (non-pregnant/non-lactating) aged 18 - 55 years, with no allergy or 
sensitivity to cytisine or any of its excipients, who unless surgically sterile, abstaining from 
sexual intercourse or of non-child bearing potential, are willing to use an effective method of 
contraception from the first dose and for 3 months after the last dose of IMP. 
IMP ADMINISTRATION: 
Each subject will receive the IMP over 2 treatment periods under fed and fasted conditions in 
accordance with the randomisation schedule:   
Schedule A (12 subjects):  
• Period 1: Cytisine (2 x 1.5 mg tablets) will be administered 30 minutes after the start of a 
high fat breakfast (fed state). 
• Period 2: Cytisine (2 x 1.5 mg tablets) will be administered after an overnight fast of at 
least 10 hours (fasting state). 
Schedule B (12 subjects):  
• Period 1: Cytisine (2 x 1.5 mg tablets) will be administered after an overnight fast of at 
least 10 hours (fasting state). 
• Period 2: Cytisine (2 x 1.5 mg tablets) will be administered 30 minutes after the start of a 
high fat breakfast (fed state). 
240 mL water will be given with the dose. 
CRITERIA FOR EVALUATION: 
PK:  
PK Endpoints:   
Plasma: The following PK endpoints will be derived from plasma cytisine concentration versus 
time data following administration of 3 mg cytisine (2 x 1.5 mg) under fed and fasted conditions: 
Maximum concentration (Cmax), time to Cmax (Tmax), elimination rate constant (kel), terminal 


Protocol ACH-CYT-01, 19 May 2017, Version 1.0 
 
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elimination halflife (t1/2), area under the concentration versus time curve (AUC) from time of 
dosing to last measurable concentration (AUC0t), AUC extrapolated to infinity (AUC0inf) and 
AUC% extrapolated (residual area). 
Urine: Amount excreted in urine over time (Ae) and percent of drug excreted in urine (Ae%) 
will be calculated. 
Safety:  
Safety Endpoints: AEs, vital signs (supine systolic/diastolic pressure, pulse and oral 
temperature), 12 lead ECG (heart rate, PR interval, QRS width, QT interval and QT interval 
corrected using Fridericia’s (QTcF interval) formula), laboratory safety (haematology, 
biochemistry and urinalysis). 
STATISTICAL METHODS:   
All statistical analysis will be performed using SAS® version 9.1.3 or higher. 
Demographic and Background Data: All demographic and background data will be listed. 
Demographic data will be summarised descriptively (age, height, weight and BMI) by gender 
and overall subject disposition and analysis sets (safety, PK) will also be listed and summarised 
by frequency. 
PK:  All plasma and urine cytisine concentrations and derived PK data following administration 
of cytisine (2 x 1.5 mg) under fed and fasted conditions will be listed and the PK data 
summarised descriptively.  Individual and mean concentration time data will also be plotted.   
Following logarithmic transformation Cmax, AUC0-t and AUC0-inf values will be subjected to an 
analysis of variance (ANOVA) including fixed effects for sequence, period, treatment and 
subject nested within sequence.  Point estimates and 90% confidence intervals (CI) will be 
constructed for the contrasts between treatments using the residual mean square error obtained 
from the ANOVA. The point and interval estimates will be back-transformed to give estimates of 
the ratios of the geometric least squares means (LSmeans) and corresponding 90% CI. Estimated 
geometric means will also be presented for each treatment.  An assessment of Tmax will be 
performed using the Wilcoxon Signed-Rank test. In addition, the Hodges-Lehman estimate of the 
median difference in Tmax and corresponding 95% CI will be presented.  
Safety: All safety data will be listed.   
AEs will be coded using Medical Dictionary for Regulatory Activities (MedDRA) version 20.0.  
The incidence of treatment emergent AEs (TEAE) will be summarised by organ system, 
preferred term, severity and relationship to study drug.  Abnormal laboratory safety results will 
be listed.   
QT/QTc:  Triplicate ECGs, extracted at pre-dose and 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12h and 24h 
post dose for both Period 1 and Period 2 obtained from the Holter recordings will be listed and 
analysed.  Recordings collected from Day -1 to pre-dose may be analysed if large heart rate 
effect is observed during the trial. 
 
DURATION OF STUDY: 
Approximately 6 weeks for each individual (from screening to 
post-study follow-up). 


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TABLE OF CONTENTS 
SYNOPSIS  . ....................................................................................................................................3 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ................................................11 
1. 
ETHICS ......................................................................................................................12 
1.1. 
Research Ethics Committee ........................................................................................12 
1.2. 
Subject Information and Consent ...............................................................................12 
1.3. 
Indemnity Arrangements ............................................................................................13 
2. 
INTRODUCTION AND BACKGROUND ...............................................................13 
2.1. 
Smoking and Health ...................................................................................................13 
2.2. 
Cytisine .......................................................................................................................13 
3. 
RATIONALE FOR THE STUDY ..............................................................................15 
4. 
STUDY OBJECTIVES ..............................................................................................15 
4.1. 
Primary Objective .......................................................................................................15 
4.2. 
Secondary Objectives .................................................................................................15 
5. 
INVESTIGATIONAL PLAN .....................................................................................16 
5.1. 
Study Design ...............................................................................................................16 
5.2. 
Discussion of Study Design ........................................................................................17 
6. 
SELECTION OF STUDY POPULATION ................................................................17 
6.1. 
Inclusion Criteria ........................................................................................................17 
6.1.1. 
To be Confirmed at Screening ....................................................................................17 
6.1.2. 
To be Re-Confirmed Prior to Dosing .........................................................................18 
6.2. 
Exclusion Criteria .......................................................................................................19 
6.2.1. 
To be Confirmed at Screening ....................................................................................19 
6.2.2. 
To be Re-Confirmed Prior to Dosing: ........................................................................19 
6.3. 
Removal of Subjects from Therapy or Assessment ....................................................20 
7. 
RETRICTIONS PRIOR AND DURING THE STUDY ............................................20 
7.1. 
Confinement ...............................................................................................................20 
7.2. 
Diet and Fluid Restrictions .........................................................................................21 
7.2.1. 
Meal Times .................................................................................................................21 
7.2.2. 
Fluid Intake .................................................................................................................21 
7.2.3. 
Alcohol Intake ............................................................................................................21 


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7.2.4. 
Caffeine .......................................................................................................................22 
7.2.5. 
Poppy Seeds ................................................................................................................22 
7.2.6. 
Grapefruit Juice and Other Restrictions .....................................................................22 
7.3. 
Other Life-Style Restrictions ......................................................................................22 
7.3.1. 
Strenuous Exercise ......................................................................................................22 
7.3.2. 
Blood Donation ...........................................................................................................22 
7.3.3. 
Contraception ..............................................................................................................22 
7.3.4. 
Sperm Donation ..........................................................................................................23 
8. 
INVESTIGATIONAL MEDICAL PRODUCT (IMP) ..............................................23 
8.1. 
Cytisine 1.5 mg Film-Coated Tablets .........................................................................23 
8.2. 
Receipt and Storage ....................................................................................................23 
8.3. 
Assembly and Certification ........................................................................................24 
8.4. 
Administration ............................................................................................................24 
8.5. 
Return/Destruction ......................................................................................................24 
8.6. 
Method of Assigning Subjects to Schedule A or Schedule B ....................................24 
8.7. 
Selection of Doses in the Study ..................................................................................24 
8.8. 
Selection and Timing of Dose for Each Subject .........................................................25 
8.9. 
Blinding ......................................................................................................................25 
9. 
PRIOR AND CONCOMITANT THERAPY .............................................................25 
10. 
TREATMENT COMPLIANCE .................................................................................25 
11. 
STUDY PROCEDURES ............................................................................................25 
11.1. 
Procedure Schedule ....................................................................................................26 
11.2. 
Detailed Description of Study Visits ..........................................................................27 
11.2.1. 
Screening Visit (Day -28 to Day -2) ...........................................................................27 
11.2.2. 
Day -1 (Period 1 and Period 2): Admission ................................................................27 
11.2.3. 
Day 1 (Period 1 and Period 2) ....................................................................................27 
11.2.4. 
Day 2 (Period 1 and Period 2) ....................................................................................28 
11.2.5. 
Days 6-8 post last dose: Post-Study Follow-up ..........................................................29 
12. 
DRUG CONCENTRATION MEASUREMENTS ....................................................29 
12.1. 
Pharmacokinetic Blood Sampling ..............................................................................29 
12.2. 
Pharmacokinetic Urine Collection ..............................................................................30 
12.3. 
Determination of Plasma and Urine Drug Concentrations .........................................30 
12.4. 
Incurred Samples Reanalysis ......................................................................................31 


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13. 
SAFETY ASSESSMENTS ........................................................................................31 
13.1. 
Subject’s Safety ..........................................................................................................31 
13.2. 
Adverse Events ...........................................................................................................32 
13.2.1. 
Definition of Adverse Event .......................................................................................32 
13.2.2. 
Recording of Adverse Events .....................................................................................33 
13.2.3. 
Grades of Adverse Event Severity ..............................................................................34 
13.2.4. 
Assessment of Causal Relationship ............................................................................34 
13.2.5. 
Reporting of Serious Adverse Events .........................................................................35 
13.2.6. 
Monitoring of Subjects with Adverse Events .............................................................36 
13.3. 
Pregnancy ...................................................................................................................36 
13.4. 
Laboratory ...................................................................................................................36 
13.4.1. 
Routine Laboratory Assessments ...............................................................................36 
13.4.2. 
Virology ......................................................................................................................37 
13.4.3. 
Drugs of Abuse and Alcohol ......................................................................................37 
13.5. 
Vital signs ...................................................................................................................37 
13.6. 
Physical Examination .................................................................................................38 
13.7. 
ECG Measurements ....................................................................................................38 
13.8. 
12-Lead ECG Holter ...................................................................................................38 
13.9. 
Concomitant Medication ............................................................................................38 
14. 
APPROPRIATENESS OF MEASUREMENTS ........................................................38 
15. 
STATISTICAL METHODS .......................................................................................39 
15.1. 
Primary Objectives and Analysis ................................................................................39 
15.2. 
Subject Population for Analyses .................................................................................39 
15.2.1. 
Safety Analysis Set .....................................................................................................39 
15.2.2. 
PK Analysis Set ..........................................................................................................39 
15.3. 
Subjects Characteristics ..............................................................................................40 
15.4. 
Sample Size Calculation .............................................................................................40 
15.5. 
Plasma and Urine Concentrations ...............................................................................40 
15.6. 
Estimation of pharmacokinetic parameters ................................................................41 
15.7. 
Pharmacokinetic Analysis ..........................................................................................42 
15.8. 
Safety Variables and Analyses ...................................................................................42 
15.8.1. 
ECGs ...........................................................................................................................42 
15.8.2. 
Vital Signs ..................................................................................................................43 


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15.8.3. 
Adverse Events ...........................................................................................................43 
15.8.4. 
Safety Laboratory tests ...............................................................................................43 
15.9. 
Procedures for Deviations from Planned Analyses ....................................................44 
16. 
DOCUMENTATION .................................................................................................44 
16.1. 
Study File and Site Documents...................................................................................44 
16.2. 
Study Documents Supplied by the Sponsor ................................................................44 
16.3. 
Maintenance and Retention of Records ......................................................................44 
17. 
ADMINISTRATIVE PROCEDURES .......................................................................45 
17.1. 
Sponsor Responsibilities .............................................................................................45 
17.2. 
Investigator Responsibilities .......................................................................................45 
18. 
REGULATORY COMPLIANCE ..............................................................................46 
19. 
ETHICAL CONDUCT OF THE TRIAL / GOOD CLINICAL PRACTICE .............46 
20. 
PROTOCOL MODIFICATION/PREMATURE TERMINATION ...........................47 
21. 
POLICY FOR PUBLICATION AND DATA PRESENTATION .............................47 
INVESTIGATOR’S AGREEMENT .............................................................................................48 
REFERENCES ..............................................................................................................................49 
APPENDIX 1. NORMAL RANGES FOR VITAL SIGNS AND ECG PARAMETERS .........50 
 


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LIST OF TABLES 
Table 1: 
CYT-BIO-02-07 PK Results .......................................................................................14 
Table 2: 
Single Dose PK Results ..............................................................................................14 
Table 3: 
Cytisine Product Information .....................................................................................23 
Table 4: 
Schedule of Study Procedures ....................................................................................26 
Table 5: 
Blood Sampling for Pharmacokinetic Assessments During each Period ...................29 
Table 6: 
Adverse Event Severity ..............................................................................................34 
Table 7: 
Assessment of Causal Relationship to Cytisine..........................................................34 
Table 8: 
Cytisine Pharmacokinetic Parameters ........................................................................41 
 
LIST OF FIGURES 
Figure 1: 
Study Design Overview ..............................................................................................16 
 


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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
ABBREVIATION/TERM DEFINITION 
ADR 
Adverse Drug Reaction 
AE 
Adverse Event 
ANOVA 
Analysis of Variance 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
AUC0-t 
AUC from Time Zero to Last Sampling Time with Quantifiable 
Concentrations 
AUC0-∞ 
AUC from Time Zero to Infinity 
%AUC 
Residual Area or Percentage of Extrapolated Part of AUC0-∞ 
BMI 
Body Mass Index 
Clast 
Last Quantifiable Concentration 
Cmax 
Maximum Observed Plasma Concentration 
CRF 
Case Report Form 
CV% 
Coefficient of Variation 
ECG 
Electrocardiogram 
EMA 
European Medicines Agency 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
GMR 
Geometric Means Ratio` 
ICH 
International Conference on Harmonization 
IMP 
Investigational Medicinal Product (for this protocol indicates 
cytisine 1.5 mg film coated tablet) 
LC/MS/MS 
Liquid Chromatography-tandem Mass Spectrometry 
LLOQ 
Lower Limit of Quantification 
MedDRA 
Medical Dictionary for Regulatory Activities 
PK 
Pharmacokinetics 
RBC 
Red Blood Cell Count 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SD 
Standard Deviation 
SOC 
MedDRA System Organ Class 
Tmax 
Time to Maximum Observed Concentration 
t1/2 
Apparent Terminal Elimination Half-life 
UADR 
Unexpected Adverse Drug Reaction 
UAE 
Unexpected Adverse Event 
ULOQ 
Upper Limit of Quantification 
WBC 
White Blood Cell Count 
λz 
Apparent Terminal Elimination Rate Constant 


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1. 
ETHICS 
1.1. 
Research Ethics Committee  
This study protocol will be submitted to the Research Ethics Committee (REC) for review and 
approval.  The approval of the REC must be obtained before commencement of any study 
procedures. 
The favourable opinion is conditional upon the Sponsor registering the clinical trial in a publicly 
accessible database, within 6 weeks of the first participant recruited.   
All substantial protocol amendments must be approved by the REC responsible for the study.  
Non-substantial amendments will not require prior approval by the REC. 
If the study is stopped due to adverse events (AEs), it will not be recommenced without 
reference to the REC responsible for the study. 
The outcome of the study (e.g. completed) will be reported to the REC responsible for the study 
within 90 days of completion of the last subject’s final study procedures.  In the event of the 
study being prematurely terminated, a summary safety report will be submitted to the REC 
responsible for the study within 15 days. 
A summary of the clinical study report will be submitted to the REC responsible for the study 
within 1 year of completion of the last subject’s final study procedures. 
The REC will be informed that 
 is a commercial organisation and that the study is funded 
by Achieve Life Science. The subjects who take part in the clinical study will be paid for their 
inconvenience and have been informed that there will be no benefits gained by their 
participation. All potential conflicts of interest will be declared by the Investigators.  
1.2. 
Subject Information and Consent 
Potential subjects who volunteer for participation in the study will be informed of the aims, 
methods, anticipated benefits and potential hazards of the study and any possible discomfort it 
may entail.  Information will be given in both oral and written form and in the manner deemed 
appropriate by the Clinical Unit standard operating procedures (SOPs). Each subject will also be 
informed of his/her right to withdraw from the study at any time, for any reason. 
A written explanation (participant information sheet) and informed consent form will be 
provided and the subject will be allowed sufficient time to consider the study information. Prior 
to signing the informed consent form, the subject will be given an opportunity to discuss any 
issues concerning the study with an Investigator who has suitable knowledge of the study and 
will have all questions answered openly and honestly. 
If the subject is willing to participate in the study, the informed consent form will be signed and 
personally dated by the subject and the person taking consent. The subject will receive a copy of 
the informed consent form together with the participant information sheet and the original signed 
informed consent form will be retained with the study records at the Investigator site.  In 
addition, the actions and completion of the consenting process will be recorded in the subject’s 
medical record (i.e., source document). 


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1.3. 
Indemnity Arrangements 
The Sponsor and 
 carry insurance to pay compensation for injury, accident, ill health or 
death caused by participation in this study without regard to proof of negligence in accordance 
with the insurance and compensation in the event of injury in phase I clinical trials 2012, 
guidance issued by the ABPI, the BioIndustry Association and the Clinical Contract Research 
Association in consultation with the Department of Health and the National Research Ethics 
Service. 
2. 
INTRODUCTION AND BACKGROUND 
2.1. 
Smoking and Health 
Tobacco smoking contributes to some 5 million premature deaths each year worldwide.1 
Smoking is highly addictive, with more than 95% of unaided attempts at cessation failing to last 
6 months.2  Every year that a smoker delays quitting beyond the mid-30s, it has been estimated 
that the person loses 3 months of life expectancy.3  The World Health Organization’s Framework 
Convention on Tobacco Control identifies evidence-based approaches to promote smoking 
cessation, which include mass-media campaigns, tax increases on tobacco, and help for smokers 
wanting to stop.4 
2.2. 
Cytisine 
 containing the active substance cytisine has 
been licensed and marketed in Central and Eastern Europe for several decades by 
 
), but remains relatively unknown outside Eastern Europe despite requests for 
market licensing worldwide.5-7 
Cytisine’s mechanism of action has assisted basic pharmacologists in understanding the complex 
pharmacology of the various subtypes of the nicotinic acetylcholine receptor.8  These studies 
have shown that both nicotine and cytisine bind strongly and preferentially to pre-synaptic α4β2 
receptors9,10 that mediate the release of dopamine in the shell of the nucleus accumbens.  This 
receptor subtype has been implicated in the development and maintenance of nicotine 
dependence11 and was the primary target for the drug varenicline, which has proved effective in 
aiding smoking cessation.12 
Two studies have evaluated the pharmacokinetics of cytisine in man. Study CYT–BIO-02-07 
was conducted according to the principles of Good Clinical Practice (GCP) and sponsored by 
  The pharmacokinetic profile of cytisine in plasma was determined following 
administration of 1.5 mg cytisine (in the form of a 
 tablet) in 36 male volunteers.  All drug 
administrations were given in the fasted state.  The main outcomes are summarised in the table 
below and are as follows: 
 


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Table 1: 
CYT-BIO-02-07 PK Results 
Cmax 
15.55 ng/mL 
AUC0-t 
59.80 ng.h/mL 
AUC0-∞ 
73.63 ng.h/mL 
Tmax 
0.92 h  
(≈ 55 min) 
T½ 
4.09 h 
 
• The mean Cmax (n=36) was 15.55 ± 6.55 ng/ml (95% CI: 13.33 - 17.76).  
• The concentration-time curves over 0-t period (AUC0-t) for cytisine was 59.80 
ng*hr/ml ± 16.77 (95% CI: 54.13 - 65.48).  
• The AUC0-∞ was 73.63 ng*hr/ml ± 21.27 (95% CI: 66.43 - 80.83).  
• Time to maximal plasma concentration (Tmax) ranged from 0.33 h to 1.75 h with the 
mean Tmax value at 0.92 h ± 0.40 (95% CI: 0.78 ÷ 1.06).  
• The plasma elimination half-life ranged from 2.74 h to 6.36 h with a mean of 4.09 h ± 
0.82 (95% CI: 3.81 - 4.37). 
• Of note: cumulated cytisine levels excreted in the urine during the 24 hr indicated that 
~64% of administered dose was excreted as parent compound (34.71% - 100.24%). 
A second study13 reported the pharmacokinetic profile of cytisine in seven healthy subjects (aged 
between 20 and 39 years) following administration of 3 mg cytisine (in the form of a 
 
tablet). Subjects were eligible if they were current cigarette smokers at the time of the study and 
on average, the subjects smoked 10.6 cigarettes per day. There was no restriction on diet or 
smoking during the study. The main outcomes are summarised in the table below and are as 
follows: 
Table 2: 
Single Dose PK Results 
Cmax 
27.76 ng/mL 
T½ 
4.8 h 
Tmax 
1-2 h 
VD 
115 L 
CL 
16.7 L/h 
 
• The peak concentrations (Cmax) of cytisine were between 23.37 and 32.04 ng/mL. The 
mean Cmax was 27.76 ng/mL.  


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• Plasma elimination half-life was calculated to be 4.8 h.  
• 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. 
• Of note: cytisine was still detectable in the urine collected at 24 h for all subjects 
(mean 24 h concentration was 428.15 ng/mL).  
• Data together in this study were modeled using NONMEM to estimate the volume of 
distribution (VD) at 115 L and clearance (CL) at 16.7 L/h. 
3. 
RATIONALE FOR THE STUDY 
No studies in man have been conducted to examine the pharmacokinetic profile of cytisine in the 
fed versus fasted state.  A recent bioavailability study in a limited number of dogs, where 
cytisine was given in the fed and fasted states, indicated a potential 28% increase in Cmax 
occurred in the presence of food.  Therefore, this study will compare the pharmacokinetics of 
cytisine in the fed and fasted state in 24 healthy volunteers to more clearly determine to what 
level a potential food effect (Fed state) might occur with cytisine administration and whether 
dietary restrictions need to be defined in a future Phase 3 trial design for cytisine. In addition, 
assessment for any potential effect of cytisine on QT/QTc interval prolongation or other cardiac 
safety measures will be explored and analyzed in relationship to the cytisine pharmacokinetic 
profile.  
4. 
STUDY OBJECTIVES 
4.1. 
Primary Objective 
1. To compare the bioavailability (Cmax and AUC0-∞) of cytisine in subjects under fed and 
fasted conditions, following administration of 3 mg cytisine (2 x 1.5 mg cytisine tablets). 
4.2. 
Secondary Objectives 
1. To compare the AUC0-t, T½ and Tmax of cytisine in subjects under fed and fasted 
conditions, following administration of 3 mg cytisine (2 x 1.5 mg cytisine tablets). 
2. To assess the safety and tolerability of cytisine at the 3 mg dose level under fed and 
fasted conditions. 
3. To assess the renal elimination of cytisine via measurement of urinary concentrations of 
cytisine. 
4. To explore possible effects of other study parameters on the bioavailability of cytisine 
(e.g. gender, BMI). 
5. To explore for cytisine effects on QT/QTc interval prolongation and cardiac safety. 


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5. 
INVESTIGATIONAL PLAN 
5.1. 
Study Design 
This will be an open-label, randomised, 2-period, single-dose crossover Phase I study conducted 
in healthy volunteers as shown below: 
Figure 1: 
Study Design Overview  
 
 
The study will be comprised of a pre-study screen, followed by 2 treatment periods (1 and 2) and 
a post-study follow-up.   
Screening (Day 28 to Day 2): Screening assessments will be carried out within 28 days before 
first administration of IMP. Eligible subjects will be asked to return for the treatment periods.  
Continued eligibility will be confirmed pre-dose during each treatment period. 
Treatment Periods (Day -1 to Day 2):  Eligible subjects will receive a single-dose of IMP 
under fed and fasted conditions over 2 treatment periods.  
• Each treatment period will be approximately 2 days’ duration and will require 2 
overnight stays in the Clinical Unit.  Each period will begin the afternoon before 
dosing on Day -1 until 24 hours (h) postdose (Day 2).   
• During each treatment period, subjects will arrive at the Clinical Unit on Day -1 and 
will undergo an overnight fast of at least 10 h prior to dosing on Day 1. Holter 
monitoring will also be initiated. Randomisation will occur on the morning of Day 1, 
Period 1, at least 1 hour  prior to dosing, and IMP will be administered under fasted 
(after an overnight fast of at least 10 h) or fed (after a high fat breakfast) conditions 
and subjects will be discharged 24 h postdose (Day 2).   
• Pharmacokinetic (PK) samples (plasma and urine) will be collected predose and up to 
24 h postdose (Day 2) during each period (17 plasma and 6 urine samples per period) 
for the measurement of plasma and urine cytisine concentrations. Safety will also be 
evaluated at specified times.   
 
R 
A 
N 
D 
O 
M 
I 
Z 
E 
Schedule B 
 
Initial dose of cytisine (3 mg) given after fasted state 
(Treatment Period 1) followed by second dose of cytisine (3 
mg) given after fed state (Treatment Period 2). 
Schedule A 
 
Initial dose of cytisine (3 mg) given after fed state 
(Treatment Period 1) followed by second dose of cytisine (3 
mg) given after fasted state (Treatment Period 2). 
 
S 
C 
R 
E 
E 
N 
 
 
F 
 
O 
 
L 
 
L 
 
O 
   W 
 
 
 
U 
 
P 


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• Continuous ECG measurements will be obtained via the Holter monitor initiated on 
Day -1 and continued until approximately 24 hours post dosing.   
• Final ECGs and blood draw for repeat haematology and chemistries will be obtained 
just prior to discharge on Day 2. 
• There will be at least 72 hours between dose administrations. 
Post Study: Post study telephone follow-up will be conducted 6 to 8 days after last dose of IMP. 
If any adverse events are recorded at post study telephone assessment, arrangements will be 
made with the subject, so that they are followed-up appropriately and the final outcome 
determined.  
The end of study is defined as completion of safety follow-up on all subjects. The maximum 
duration of this study is estimated to be around 6 weeks (screening to last subject’s last visit).   
5.2. 
Discussion of Study Design 
This study design minimizes the influence of inter-subject variability, which is assumed to be 
higher than the intra-subject variability, as each subject will be administered cytisine in both the 
fed and fasted states. As each subject serves as his own control, the precision of the comparison 
should be improved. 
The blood samples for the determination of cytisine plasma concentrations will be collected prior 
to and up to 24 hours after cytisine administration. Taking into account the known terminal 
elimination half-life of cytisine (about 4 hours), this sampling schedule is expected to provide a 
reliable estimate of the extent of exposure.  
The washout of at least 72 hours between dose administration will allow a complete elimination 
of cytisine between doses and is deemed sufficiently long to avoid any relevant carry-over effect. 
6. 
SELECTION OF STUDY POPULATION 
24 subjects (approximately 12 males and 12 females) will be required to complete the study.   
The study is to be conducted in healthy subjects and therefore participants are not expected to 
derive any therapeutic benefit from taking part.  A healthy subject population with carefully 
considered inclusion/exclusion criteria will avoid the potential for interaction of cytisine with 
any underlying disease state or concomitant medication that might be necessary to administer to 
patients, while ensuring that subjects are fit and well enough for participation in the study.   
The following eligibility criteria are designed to select subjects for whom protocol treatment and 
procedures are considered appropriate.  All relevant medical and non-medical conditions should 
be taken into consideration when deciding whether this protocol is suitable for a particular 
subject. 
6.1. 
Inclusion Criteria 
6.1.1. 
To be Confirmed at Screening 
1. Healthy males and females between 18 and 55 years of age. 


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a. If a female subject of child bearing potential, a negative pregnancy test at screening 
and admission and willing to use an effective method of contraception (unless of non-
childbearing potential or where abstaining from sexual intercourse is in line with the 
preferred and usual lifestyle of the subject) from first dose until 3 months after last 
dose of IMP.  
b. If a female subject of non-child bearing potential, a negative pregnancy test at 
screening and admission.  For the purposes of this study, this is defined as the subject 
being amenorrheic for at least 12 consecutive months or at least 4 months post-
surgical sterilisation (including bilateral fallopian tube ligation or bilateral 
oophorectomy with or without hysterectomy).  Menopausal status will be confirmed 
by demonstrating at screening that levels of follicle stimulating hormone (FSH) fall 
within the respective pathology reference range.  In the event a subject's menopause 
status has been clearly established (for example, the subject indicates she has been 
amenorrheic for 10 years), but FSH levels are not consistent with a post-menopausal 
condition, determination of subject eligibility will be at Investigator's discretion 
following consultation with the Sponsor. 
c. If a male subject, willing to use an effective method of contraception (unless 
anatomically sterile or where abstaining from sexual intercourse is in line with the 
preferred and usual lifestyle of the subject) from first dose until 3 months after last 
dose of IMP. 
2. Subject with a body mass index (BMI) of 18-32 kg/m2. BMI = body weight in kg / 
[height in m]2. 
3. Subject with no clinically significant abnormal serum biochemistry, haematology and 
urine examination values within 28 days before the first dose of IMP.   
4. Subject with negative urinary drugs of abuse screen, determined within 28 days before 
the first dose of IMP (a positive alcohol or cotinine result may be repeated at 
Investigator's discretion). 
5. Subject with negative human immunodeficiency virus (HIV), hepatitis B surface antigen 
(Hep B) and hepatitis C virus antibody (Hep C) results. 
6. Subject with no clinically significant abnormalities in 12-lead ECG determined after 
minimum of 5 minutes in supine position within 28 days before the first dose of IMP. 
7. Subject with no clinically significant abnormalities in vital signs (systolic blood pressure 
between 90-150 mmHg, diastolic blood pressure (DBP) between 50 and 90 mmHg, and 
pulse rate (PR) between 40-110 bpm, measured on the dominant arm after minimum of 5 
minutes in supine position) determined within 28 days before first dose of IMP. 
8. Subject must be available to complete the study (including post study follow-up) and 
comply with study restrictions. 
9. Subject must provide written informed consent to participate in the study. 
6.1.2. 
To be Re-Confirmed Prior to Dosing 
1. Subject continues to meet all screening inclusion criteria (before the first dose only). 
2. Subject has a negative urinary drugs of abuse screen (including alcohol & cotinine). 


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3. Female subject has a negative pregnancy test.  
6.2. 
Exclusion Criteria 
6.2.1. 
To be Confirmed at Screening 
1. Known hypersensitivity/allergy reaction to varenicline, other cytisine-derivatives or any 
of the excipients in the 
formulation 
). 
2. History of severe hypersensitivity reactions to any other drugs. 
3. History of any medical condition (e.g. gastrointestinal, renal or hepatic) or surgical 
condition (e.g. cholecystectomy, gastrectomy) that may affect drug pharmacokinetics 
(absorption, distribution, metabolism or excretion). 
4. Difficulty in donating blood on either arm or known history. 
5. History of alcoholism or drug abuse within last 2 years. 
6. Regular nicotine intake (e.g., smoking, nicotine patch, nicotine chewing gum or 
electronic cigarettes) within previous 3 months and inability to refrain from nicotine 
intake from Screening until end of study. 
7. Use of prescription or non-prescription drugs, including vitamins, herbal and dietary 
supplements within 14 days (or 5 half-lives, whichever is longer) prior to the first dose of 
IMP, unless in the opinion of the Investigator the medication will not interfere with the 
study procedures or compromise subject safety. 
8. Participated in any investigational drug clinical trial within the previous 3 months or a 
marketed drug trial within the previous 30 days prior to randomization on Day 1 of 
Period 1. 
9. Donation of 450 mL or more blood or had history of significant blood loss due to any 
reason or had plasmapheresis within 3 months before the first dose of IMP. 
10. Any special food restrictions that may hinder ability to consume the high fat breakfast 
provided during the study; such as lactose intolerance, vegan, low-fat, low sodium, etc.  
11. Any inability or difficulty in fasting. 
12. Inability to communicate well with Investigators (i.e., language problem, poor mental 
development or impaired cerebral function). 
13. Any other condition that the Principal Investigator considers making the subject 
unsuitable for this study. 
6.2.2. 
To be Re-Confirmed Prior to Dosing: 
1. Development of any exclusion criteria since screening (before the first dose only). 
2. Use of prescription or non-prescription drugs, including vitamins, herbal and dietary 
supplements since screening, unless in the opinion of the Investigator the medication will 
not interfere with the study procedures or compromise subject safety. 
3. Participation in a clinical study since the screening visit (before the first dose only). 


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4. Donation of 450 mL or more blood since the screening visit (before the first dose only). 
6.3. 
Removal of Subjects from Therapy or Assessment 
Each subject will be informed of their right to withdraw from the study at any time and for any 
reason. 
An Investigator will withdraw a subject from the study at any time for any of the following 
reasons: 
• If a subject experiences a serious or intolerable AE, that prevents them from 
continuing. 
• If a subject incurs a significant protocol violation which impacts on their safety or the 
scientific integrity of the study (this will be discussed on a case-by-case basis with the 
Sponsor). 
• At the request of the Sponsor. 
• If it is considered that the subject’s health is compromised by remaining in the study 
or the subject is not sufficiently cooperative. 
• If a subject is lost to follow-up. 
The reasons for any subject withdrawal will be recorded on the study completion form of the 
case report form (CRF). 
If a subject is withdrawn or chooses to withdraw from the study for any reason, every possible 
effort will be made to perform the evaluations described for the post-study follow-up.  The data 
collected from withdrawn subjects will be included in the study report. 
In the event of any abnormalities considered to be clinically significant, subjects will be followed 
up with appropriate medical management until values are considered to be clinically acceptable.  
Referral or collaborative care will be organised if considered necessary. 
Since 24 subjects are required to complete the study, subjects who withdraw from the study 
before receiving any study medication will be replaced.  Subjects who are withdrawn from the 
study due to significant drug-related AEs will not be replaced.  Replacement of all other subjects 
withdrawn from the study after receiving study medication will be decided on a -case-by-case 
basis by the Chief Investigator (or deputy) and Sponsor. 
7. 
RETRICTIONS PRIOR AND DURING THE STUDY 
To avoid possible negative effects on the measurement of plasma drug concentrations and/or on 
subject’s safety, several restrictions are to be adopted as outlined in the eligibility criteria and in 
the following sections.  
7.1. 
Confinement 
Each subject will complete 2 treatment periods (Period 1 and Period 2). Each period will require 
2 nights stay (arrive at the unit in the afternoon of Day -1 and discharged 24 hours post dose on 
Day 2). 


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7.2. 
Diet and Fluid Restrictions 
7.2.1. 
Meal Times 
Fasting: 
• Subjects will fast overnight for at least 10 hours before dosing. 
• Lunch will be served: approximately 4 h post-dose. 
• Dinner will be served: approximately 8 h post-dose. 
• Snack will be served: approximately 12 h post-dose. 
Fed: 
• Subjects will fast overnight for at least 10 hours. 
• A high fat breakfast will be served at 30 min pre-dose (breakfast needs to be finished 
at least 5 minutes before dosing).   
• Lunch will be served: approximately 4 h post-dose. 
• Dinner will be served: approximately 8 h post-dose. 
• Snack will be served: approximately 12 h post-dose. 
The example of a high fat (approximately 50% of total caloric content of the meal) and high-
calorie (approximately 800-1000 calories) breakfast provided in the US Food and Drug 
Administration guidance document for food-effect bioavailability and fed bioequivalence studies 
(Guidance for Industry, U.S. Department of Health and Human Services Food and Drug 
Administration Center for Drug Evaluation and Research (CDER), December 2002) is 
approximately 150, 250 and 500-600 calories from protein, carbohydrate and fat respectively. 
Suggested content to be:  2 eggs fried in butter, 2 strips of bacon, 2 slices of toast, 4 ounces of 
hash brown potatoes and 8 ounces of whole milk.  Consumption of the standardized breakfast 
will be documented. 
7.2.2. 
Fluid Intake 
No fluids (apart from water taken with dose) are allowed from 1 h prior to dosing until 1 h 
afterwards.  Water is then allowed ad libitum. Decaffeinated tea and coffee as well as 
squash/cordial are allowed from 4 h post-dose. 
7.2.3. 
Alcohol Intake 
The consumption of alcohol will be limited to a maximum of 2 units per day from 7 days prior to 
the first dose of IMP.  Alcohol will be avoided completely for at least 2 days prior to the first 
dose of IMP and throughout the study period.  Any deviation outside this alcohol intake 
restriction will be assessed on a case-by-case basis at Investigator’s discretion (provided the 
subject’s alcohol intake will not impact in the safety aspects and objectives of the study and the 
subject has a negative alcohol screen prior to dosing). 


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7.2.4. 
Caffeine 
Food or drink containing caffeine, including coffee, tea, cola, energy drinks or chocolates will be 
avoided completely for 2 days prior to dosing and whilst the subjects are resident in the Clinical 
Unit (during each dosing period). 
7.2.5. 
Poppy Seeds 
Subjects must not eat food containing poppy seeds for 3 days before each visit to the Clinical 
Unit, as consumption of poppy seeds can lead to a positive opiate result in the drugs of abuse 
test. 
7.2.6. 
Grapefruit Juice and Other Restrictions  
No food or drink containing grapefruit, cranberry, or Seville oranges (including marmalade and 
fruit juices), and/or food or drink, sweets, candies or other confectionary containing liquorice 
will be allowed from 7 days before the first dose of IMP until the final study visit. 
Each subject will be questioned on the specific points at admission to the clinical unit (Day -1 of 
each period). If a subject admits a non-compliance with these restrictions, the Principal 
Investigator or attending Research Physician will decide whether or not the subject will be 
permitted to remain in the study. Non-compliance with these restrictions will be noted. 
7.3. 
Other Life-Style Restrictions 
7.3.1. 
Strenuous Exercise 
Strenuous exercise must be avoided completely from 3 days before the first dose of IMP until the 
final study visit. 
7.3.2. 
Blood Donation 
Subjects will be advised that they should not donate blood for at least 3 months after the final 
study visit. 
7.3.3. 
Contraception 
To prevent pregnancy female subjects of childbearing potential and male subjects with female 
partners of child bearing potential must take adequate contraceptive precautions for the entire 
duration of study participation. Adequate contraceptive precautions include: 
1. Established use of oral, injected or implanted hormonal methods of contraception. 
2. Placement of an intrauterine device (IUD) or intrauterine system (IUS).  
3. Barrier methods of contraception: Condom or occlusive cap (diaphragm or cervical/vault 
caps) with spermicidal foam/gel/film/cream/suppository.  
4. Male sterilisation (with the appropriate post-vasectomy documentation of the absence of 
sperm in the ejaculate). [For female subjects on the study, the vasectomised male partner 
should be the sole partner for that subject]. 






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8.8. 
Selection and Timing of Dose for Each Subject 
Doses will be administered at approximately 9.00 a.m.  All subjects will have been fasting for at 
least 10 h overnight prior to dosing. Subjects assigned to be fasting prior to dosing will remain 
fasting until lunch and subjects assigned to be fed prior to dosing will start their high-fat 
breakfast 30 minutes prior to the dose.  Lunch will be provided 4 h after dosing for all subjects. 
8.9. 
Blinding 
The study is open-label.  However, as stated in the EMA guidelines on investigation of 
bioavailability and bioequivalence (CPMP/EWP/QWP/1401/98 Rev.1/Corr** London, 20 
January 2010), analysis of the samples should be conducted without information on treatment, 
therefore staff responsible for drug concentration and ECG analysis will be blinded to the 
treatment schedule. 
9. 
PRIOR AND CONCOMITANT THERAPY 
Prior Medication:  Prescription or non-prescription drugs, including vitamins, herbal and 
dietary supplements should not be taken within 14 days (or 5 half-lives, whichever is longer) 
prior to the first dose of IMP, unless in the opinion of the Investigator and Sponsor's Responsible 
Physician the medication will not interfere with the study procedures or would compromise 
subject safety.  Prescription or non-prescription drugs, including vitamins, herbal and dietary 
supplements taken during the 14 days before the first dose of IMP, and the reason for taking 
them, will be noted in the subject’s CRF.  
Inclusion of subjects who have taken prior medication will be reviewed on a case-by-case basis 
in relation to the safety aspects and objectives of this study.   
Concomitant Medication: Prescription or non-prescription drugs, including vitamins, herbal 
and dietary supplements should not be taken throughout the duration of the study, with the 
exception of paracetamol (which may be taken as an analgesic to a maximum of 2 g in 24 h (500 
mg 4 times a day) and ibuprofen (which may be taken as an analgesic to a maximum of 1.2 g in 
24 h (400 mg 3 times a day).  
If intake of ANY prior or concomitant medication is necessary during the study, the daily dosage, 
duration and reasons for administration will be recorded on the subject’s CRF. 
Chewing gum will not be allowed during confinement periods. 
10. 
TREATMENT COMPLIANCE 
Each dose of IMP will be taken under supervision and a hand and mouth check conducted.  The 
exact dosing time for each subject will be recorded on the subject’s CRF. 
11. 
STUDY PROCEDURES 
All subjects will be evaluated for inclusion in the study during the Screening Period. Subjects 
who are eligible for the study will be randomized according to the randomization schedule. 


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Subjects will receive their study medications on Day 1 of each period followed by the required 
blood and urine samples and ECG Holter monitoring.  The subjects will be released from the 
clinical research facility after the last PK sample is collected and safety assessments completed 
in each period (approximately 24 hours post-last dose, mornings of Day 2 of each period). All 
subjects are required to complete a telephone follow-up on Day 6-8 days post last dose. 
11.1. 
Procedure Schedule 
Table 4 provides a summary of required study evaluations. Screening evaluations are to occur 
within a 28-day interval from initiation of screening evaluations to randomization on Day 1 of 
Period 1, pre-dose. 
Table 4: 
Schedule of Study Procedures 
 
Days 
Screening 
Study Day (Period 1 and Period 2) 
Post-Study 
Telephone 
Call  
Days 
-28 to -2 
Day -1 
1  
2  
6-8 days 
after last 
dose 
Written informed consent 
X 
 
 
 
 
Demographic data/height/weight 
X 
 
 
 
 
Randomization 
 
 
X (Pre-first 
dose in  
Period 1) 
 
 
Vital signs 
X 
X 
X1 
 
 
Medical history 
X 
 
 
 
 
Medical history update 
 
X2 
 
 
 
Prior and concomitant medication 
X 
X 
X 
X 
 
Physical examination 
X 
 
 
 
 
12-lead ECG 
X 
 
 
X3 
 
Holter monitor for continuous ECG 
readings4 
 
X 
X 
X 
 
Pregnancy test for all females 
X 
(Serum) 
X 
(Urine) 
 
 
 
FSH (females only) 
X 
 
 
 
 
Haematology 
X 
 
 
X 
 
Biochemistry 
X 
 
 
X 
 
Urinalysis 
X 
 
 
X 
 
Drugs-of-abuse tests in urine 
X 
X 
 
 
 
Verification of eligibility criteria  
X 
X 
X 
 
 
Cytisine administration 
 
 
X 
 
 
Blood and urine collection for PK 
analysis 
 
 
X 
X 
 
Adverse events monitoring 
 
X5 
X5 
X5 
X5 
 
1 Vital signs (supine blood pressure, pulse rate and oral temperature) will be recorded at pre-dose and 
again approximately 2 hours after administration on Day 1 of each period. 
2 Clinically relevant changes will be reported as adverse events.  
3 Repeat 12-lead ECG and assess prior to discharge. 
4      Attach ECG Holter monitor and begin recording on Day -1 and continue until 24 hours post dose. 
5      Adverse event recording to begin upon admissions on Day -1 of Period 1 to post study. 


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11.2. 
Detailed Description of Study Visits 
11.2.1. 
Screening Visit (Day -28 to Day -2) 
All subjects will complete the following screening procedures within 28 days of Day 1 in order 
to verify required inclusion and exclusion criteria.  
• Written informed consent 
• Demographic data 
• Medical history data 
• Prior and concomitant medication 
• Physical examination 
• Vital signs 
• 12-lead ECGs (triplicate) 
• Clinical laboratory safety tests (biochemistry, haematology, urinalysis, virology, 
drugs of abuse and serum pregnancy test for females) 
• Verification of eligibility criteria 
11.2.2. 
Day -1 (Period 1 and Period 2): Admission 
The results of screening for each subject must be reviewed by the Principal Investigator prior to 
subject’s admission to assure eligibility criteria have been met.   
On Day -1 of each period, the subject will check into the research facility and upon arrival to the 
research facility, the following procedures are to be performed: 
1. Repeat urine testing for drugs of abuse and urine pregnancy test for female subjects.  
2. Verify that all subjects still satisfy the inclusion and exclusion criteria. 
3. Attach ECG Holter monitor, checking to ensure recording has started.  Note: Holter 
monitors to remain attached to subjects until 24 hours post-dosing. 
All subjects will fast for minimum of 10 hours prior to dosing on Day 1. 
All subjects will remain in clinic overnight. 
11.2.3. 
Day 1 (Period 1 and Period 2) 
Administration of cytisine will occur on Day 1 of each period for all subjects.  
The following study procedures are to be performed prior to dosing:  
1. Check to ensure ECG Holter monitor is functioning properly after overnight monitoring. 
2. Vital signs (supine blood pressure, pulse rate and oral temperature) will be recorded at 
pre-dose and again approximately 2 hours after administration. 
3. Any clinically relevant changes should be documented as adverse events. 
4. Prior and concomitant medication documented. 


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5. Pre-dose blood draw for PK to be taken within 60 minutes prior to dosing. 
For Period 1 only:  After Day 1 pre-dose study procedures have been successfully completed 
and all eligibility criteria has been verified, subjects will be randomized onto Schedule A or 
Schedule B. 
Schedule A (12 subjects):  
• Period 1: Cytisine (2 x 1.5 mg tablets) will be administered 30 minutes after the start 
of a high fat breakfast (fed state) with 240 mL (8 fluid ounces) water.  No additional 
fluids are to be taken until 1 hour post-dosing 
• Period 2: Cytisine (2 x 1.5 mg tablets) will be administered after the overnight fast of 
at least 10 hours and subject will continue fasting until lunch (fasting state). Dosing 
will be administered with 240 mL (8 fluid ounces) water.  Except for fluids taken for 
dosing, no fluids will be allowed from 1 hour before dosing until 1 hour post-dosing. 
Schedule B (12 subjects):  
• Period 1: Cytisine (2 x 1.5 mg tablets) will be administered after the overnight fast of 
at least 10 hours and subject will continue fasting until lunch (fasting state). Dosing 
will be administered with 240 mL (8 fluid ounces) water.  Except for fluids taken for 
dosing, no fluids will be allowed from 1 hour before dosing until 1 hour post-dosing. 
• Period 2: Cytisine (2 x 1.5 mg tablets) will be administered 30 minutes after the start 
of a high fat breakfast (fed state) with 240 mL (8 fluid ounces) water.  No additional 
fluids are to be taken until 1 hour post-dosing. 
Blood and urine samples will be collected and processed as outlined in (Section 12.1) and 
(Section 12.2) prior to, and after administration of cytisine (time zero).   
No food should be allowed for at least 4 hours post-dose for either arm until lunch is served for 
all subjects. 
All subjects will remain in the clinic overnight. 
11.2.4. 
Day 2 (Period 1 and Period 2) 
Remaining blood and urine samples for pharmacokinetics will be collected (Section 12.1). 
1. Check to ensure ECG Holter monitor is functioning properly after overnight monitoring. 
2. Document concomitant medications and any adverse events. 
3. At 24 hours post-dosing, collect blood for haematology and biochemistry and collect 
urine for urinalysis.  
4. Just prior to discharge after 24 hours post-dosing, remove ECG Holter monitor. 
5. Obtain 12-lead ECG (in triplicate) similar to what was done at screen and review prior to 
discharge. 
Upon final review by the study physician, the subject will be released from the clinical research 
facility and will be scheduled for return on Day 7.  


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11.2.5. 
Days 6-8 post last dose: Post-Study Follow-up 
A Post-Study Follow-up telephone call will occur 6-8 days after last dosing to document any 
changes in adverse event(s) observed at discharge as well as any new adverse events. If any new 
adverse events are recorded at this follow-up call, arrangements will be made with the subject to 
come into the clinic for assessment and then followed until outcome determined.  
12. 
DRUG CONCENTRATION MEASUREMENTS 
12.1. 
Pharmacokinetic Blood Sampling 
17 venous blood samples (volume of 7.5 mL each, total volume of approximately 128 mL) will 
be collected into pre-cooled lithium heparin tubes during each period, according to the following 
schedule: 
Table 5: 
Blood Sampling for Pharmacokinetic Assessments During each Period   
Blood Sample Number 
Time from Dosing 
1 
Pre-dose 
(within 60 minutes  
prior to dosing) 
2 
00:15 
3 
00:20 
4 
00:30 
5 
00:45 
6 
01:00 
7 
01:30 
8 
02:00 
9 
02:30 
10 
03:00 
11 
04:00 
12 
06:00 
13 
08:00 
14 
12:00 
15 
14:00 
16 
16:00 
17 
24:00 
 


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The pre-dose blood samples will be collected within 60 minutes prior to dosing.  The post-dose 
blood samples will be collected within +/-1 minute from the scheduled sampling time. The clock 
time will be recorded and reported for all blood draws and all subjects.  A deviation greater than 
+/- 2 minutes will be reported as a protocol deviation and its cause will be recorded.  
In case blood sampling for pharmacokinetics and other procedures coincide in time, blood draws 
will have priority unless other procedures are necessary for assuring subject’s safety.  
Immediately upon sampling, the blood sample for pharmacokinetic analysis will be identified 
with a bar-coded label bearing details of study number, subject number, study phase/period, 
sampling time point, sample type and a unique 9-digit identification number. The samples will be 
logged and then centrifuged at 1500 x g, 4°C for 10 minutes. Two equal aliquots of plasma will 
be transferred to polypropylene tubes labeled identically to the original blood sample (except 
sample type being plasma instead of whole blood) and logged when stored at approximately        
-80°C, pending analysis.   
12.2. 
Pharmacokinetic Urine Collection 
Six urine samples will be collected at the following time points during each period: 
• Pre-dose (The pre-dose urine sample will be collected within 30 minutes prior to 
dosing) 
• 0-2 hour 
• 2-4 hour 
• 4-8 hour 
• 8-12 hour 
• 12-24 hour 
The urine collection containers will be identified with a bar coded label bearing details of the 
study number, subject number, study period number, sampling time-point, sample type and a 
unique 9 digit identification number. 
The urine collection containers will be kept chilled (at approximately 4°C) during collections.  
After the termination of each fractional collection the volume excreted will be recorded and 2 x 
10mL aliquots retained for determination of cytisine concentrations.  The aliquots will be 
labelled identically to the original collection container and will be stored at -20°C pending 
analysis. 
12.3. 
Determination of Plasma and Urine Drug Concentrations 
Cytisine concentrations (ng/mL) in plasma and urine will be determined using a validated liquid 
chromatography tandem mass spectrometry method and will be conducted in accordance with 
Good Laboratory Practice regulations and guidelines. Methods validation must be completed 
prior to start of the analysis. 
The sample analysis will only begin after the last sample of a study subject is collected. All 
samples from each subject will be analyzed in the same analytical batch.  In each run, standard 


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and quality control samples will be distributed throughout the batch containing study samples. 
Samples with concentrations above the ULOQ will be diluted and re-assayed.  
Full details, including the lower (LLOQ) and upper (ULOQ) limits of quantification range,  will 
be provided in a Bioanalytical Phase Plan written by 
 
Plasma and urine samples of withdrawn subjects should also be analysed for drug concentrations 
and pharmacokinetic parameters (providing withdrawal was not due to withdrawal of consent).  
Unacceptable values due to analytical reasons will be handled in accordance with the 
Bioanalytical Phase Plan.   
Details of the methodologies used and the results obtained, including details of re-measured 
samples, will be given in the Analytical Study Report (ASR). 
12.4. 
Incurred Samples Reanalysis 
In order to establish the reproducibility of the assay, 10% of the first 1000 samples and 5% of the 
number of samples exceeding 1000 samples will be reanalyzed. 
Both the original and replicate values will be presented in the Analytical Study Report with the 
percent difference between the two values. The original value will be the one used for 
pharmacokinetic analysis. 
13. 
SAFETY ASSESSMENTS 
13.1. 
Subject’s Safety 
Only healthy subjects are eligible for the study.  The healthy status will be determined by 
meeting the inclusion and exclusion criteria. Subjects’ safety will be monitored during the study.  
Adverse events will be monitored and recorded throughout the study.  
Prior to the first IMP dosing only, the Principal Investigator or Research Physician will assess 
the clinical significance of results of laboratory investigations for haematology and serum 
chemistry values outside the defined normal ranges, as provided by the laboratory. All changes 
from screening that are observed on Day 2 during the study and meet clinically-significant 
definitions for laboratory adverse events will be recorded. 
In addition to the planned times, any safety procedures can be performed at any time considered 
necessary by the Principal Investigator or attending Research Physician.  
On each cytisine dosing day, at least one physician (Principal Investigator or Research 
Physician) will physically remain at the clinical site for investigational product administration 
and until 4 hours post-dose, and will remain available on call at all times during the entire period 
of the study.  
In case any clinically significant abnormality is observed at pre-dose, Principal Investigator or 
attending Research Physician will decide whether the subject will proceed to investigational 
product administration or will be withdrawn from the study. 
Safety will be assessed by consideration of all adverse events reported by or elicited from the 
subject and abnormalities detected on haematology, serum chemistry tests, urinalysis, and 12-


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lead ECG. Worsening of other preexisting medical conditions and any changes to concomitant 
medications/treatments will also be taken into account in this evaluation.  
All adverse events (serious and non-serious) beginning on Day 1 (prior to dosing) through post-
study follow-up telephone call will be recorded in the subject’s CRF.  
13.2. 
Adverse Events 
13.2.1. 
Definition of Adverse Event 
The definitions are adopted in accordance with the Directive 2001/20/EC, ICH-E2A and the 
European Commission Detailed Guidance 2011/C 172/01. 
An Adverse Event (AE) is defined as any untoward medical occurrence in a clinical trial subject 
administered a medicinal product and which does not necessarily have a causal relationship with 
the treatment. 
An AE can therefore be any unfavorable and unintended sign (including any clinical significant 
abnormal laboratory finding), symptom, or disease temporally associated with the use of an 
investigational medicinal product, whether or not considered related to the investigational 
medicinal product. 
An Adverse Drug Reaction (ADR) means all untoward and unintended responses to a 
medicinal product related to any dose administered.  The phrase ‘response to a medicinal 
product’ means that a causal relationship has at least a reasonable possibility, i.e. the relationship 
cannot be ruled out and is judged by the Investigator as at least possible (see definition below). 
An Unexpected Adverse Drug Reaction (UADR)/Unexpected Adverse Event (UAE) means 
an adverse reaction/event, the nature or severity of which is not consistent with the applicable 
product information, namely in the Investigator Brochure for an unauthorized investigational 
product or in the SmPC for an authorized product. 
The expected/unexpected status should be evaluated and assessed, by the Sponsor, based on the 
reference safety information available since expectedness in Pharmacovigilance refers strictly to 
the information listed or mentioned in the applicable reference safety information and not to 
events that might be anticipated from knowledge of the pharmacological properties of a 
substance or because it was foreseeable due to the health status (e.g., age, medical history) of the 
study subjects. 
A Serious Adverse Event (SAE) or Serious Adverse Reaction (SAR) is defined as an AE that 
results in any of the following: 
• Results in death  
• Is life-threatening  
• Requires hospitalization or prolongs existing inpatient’s hospitalization 
• Results in persistent or significant disability or incapacity 
• Results in a congenital abnormality or birth defect 
• Is an important medical event which requires medical intervention to prevent any of 
the above outcomes 


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SUSARs: AEs which meet all of the following criteria: 
• Serious. 
• Unexpected (i.e., is not consistent with the applicable product information e.g. 
Investigator's brochure for an unapproved IMP or SmPC for an authorised product). 
• There is at least a reasonable possibility that there is a causal relationship between the 
event and the medicinal product.  
will be classified as suspected unexpected serious adverse reactions (SUSARs). 
Important medical events are those which may not be immediately life-threatening, but may 
jeopardize the subject and may require intervention to prevent one of the other serious outcomes 
listed above. Examples of such events are intensive treatment in an emergency room or at home 
for allergic bronchospasm, or blood dyscrasias or convulsions that do not result in 
hospitalization. 
The term “life-threatening” in the definition of “serious” refers to an event in which the subject 
was at risk of death at the time of the event; it does not refer to an event which hypothetically 
might have caused death if it were more severe. For example, drug-induced hepatitis that 
resolves without evidence of hepatic failure would not be considered life threatening even 
though drug-induced hepatitis can be fatal. 
Inpatient hospitalization or prolongation of existing hospitalization means that hospital inpatient 
admission and/or prolongation of hospital stay were required for treatment of AE or occurred as 
a consequence of the event. It does not refer to pre-planned elective hospital admission for 
treatment of a pre-existing condition that has not significantly worsened, or to diagnostic 
procedures. 
13.2.2. 
Recording of Adverse Events 
All of the following details will be recorded in the subject’s CRF for each AE: 
• Full description of AE. 
• Date and time of onset. 
• Date and time of resolution. 
• Severity of event, to be assessed by an Investigator in accordance with the definitions 
below. 
• Relationship to IMP to be assessed by an Investigator in accordance with the 
definitions below. 
• Action taken (if any). 
• Outcome and details of any further follow-up. 
Adverse events documented in the CRF without a stop date should be reviewed at the end of 
each treatment period until final resolution or until it is medically justifiable to stop further 
follow up (e.g. a chronic condition has been reached.)  Documentation of adverse events should 
be updated as necessary. 


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13.2.3. 
Grades of Adverse Event Severity 
The following grades will be used by an Investigator to describe the severity of all AEs 
(including clinically-significant laboratory AEs) as shown in Table 6.  Only 1 severity grade will 
be used for each AE (e.g. mild - moderate is not acceptable). 
Table 6: 
Adverse Event Severity 
Severity of AE 
Definition 
Mild 
No interference with activity 
Moderate 
Some interference with activity not requiring medical 
intervention 
Severe 
Prevents daily activity and requires medical intervention 
If an adverse event has multiple aspects, the aspect in the highest intensity will be graded. It is 
emphasized that the term severe is a measure of intensity; thus a severe AE is not necessarily 
serious. For example, itching for several days may be rated as severe; however, may not be 
clinically serious.  
13.2.4. 
Assessment of Causal Relationship 
The causal relationship between an adverse event and cytisine will be determined and 
documented by the responsible Investigator, or designee, according to best medical judgment as 
shown in Table 7.  
Table 7: 
Assessment of Causal Relationship to Cytisine 
Category 
Description 
Not Related 
The event is definitely not associated with cytisine.  
Unlikely 
The event was most probably produced by other factors such as the subject’s clinical 
state, therapeutic intervention or concomitant therapy, and does not follow a known 
response pattern to cytisine 
Possible 
The event follows a reasonable temporal sequence from the time of cytisine 
administration, and/or follows a known response pattern to the investigational 
product, but could have been produced by other factors such as the subject’s clinical 
state, therapeutic intervention or concomitant therapy. 
Probable 
The event follows a reasonable temporal sequence from the time of cytisine 
administration, and/or follows a known response pattern to the product, and could not 
have been produced by other factors such as the subject’s clinical state, therapeutic 
intervention or concomitant therapy. 
Definite 
The event follows a reasonable temporal sequence from the time of cytisine 
administration, and/or follows a known response pattern to the product, and could not 
have been produced by other factors such as the subject’s clinical state, therapeutic 
intervention or concomitant therapy, and either occurs immediately following 
investigational product administration, or improves on stopping the investigational 
product. 


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13.2.5. 
Reporting of Serious Adverse Events 
Any new SAE that occurs during a treatment period should be recorded and reported 
immediately. All SAEs including those that are ongoing at the end of study treatment will be 
followed until each event resolves or is assessed as chronic.  
In order to satisfy regulatory requirements, any Serious Adverse Event, whether deemed study 
drug-related or not, must be reported to the Sponsor or designee as soon as possible after the 
Investigator or coordinator has become aware of its occurrence.  SAE form completion and 
reporting must not be delayed even if all of the information is not available at the time of the 
initial contact. 
SAEs must be reported within 24 h of knowledge of the event by submitting an initial SAE 
report via email or fax to 
 
  
 
 will notify the Sponsor and Sponsor's Responsible Physician of the 
SAE via email within 24 h of receipt of the initial SAE report. 
Additional information (follow-up) about any SAE unavailable at the initial reporting should be 
forwarded by the site within 24 hours of the information becoming available.  
The following information should be provided to accurately and completely record the event: 
• Investigator name and center number 
• Subject number 
• Subject initials, if permitted 
• Subject demographics 
• Clinical event 
− description 
− date of onset 
− severity 
− treatment 
− relationship to study drug (causality) 
− action taken regarding study drug 
• If the AE resulted in death: 
− cause of death (whether or not the death was related to study drug) 
− autopsy findings (if available) 
• Medical history case report form (copy) 
• Concomitant medication case report form (copy) 


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• Any relevant reports (laboratory, discharge, x-ray, etc.)  
Subjects who have had an SAE during the treatment period must be followed clinically until all 
parameters (including laboratory) have either resolved or been assessed as chronic.  
SUSARs should be reported to the REC, MHRA and Sponsor in accordance with applicable 
regulatory requirements for expedited reporting.  It is 
 responsibility to report SUSARs 
to the REC and MHRA.  It is the Sponsor’s responsibility to report any SUSAR to the FDA. 
13.2.6. 
Monitoring of Subjects with Adverse Events 
In the event of any abnormalities considered to be clinically significant by the investigating 
physician, subjects will be followed up with appropriate medical management until: 
• It has resolved/returned to normal or baseline. 
• The event has stabilised at a level acceptable to the Investigator and is not considered to 
be clinically significant. 
13.3. 
Pregnancy 
Serum pregnancy test will be performed for all females regardless of post-menopausal or 
sterilised status at screening.  Pregnancy tests will be performed by 
  
Serum pregnancy tests will be performed using the Roche Cobas c6000 analyser series 
comprising of c501 and e601 modules.   
Pregnancies must be reported immediately and followed up to determine outcome, including 
spontaneous or voluntary termination, details of the birth, and the presence or absence of any 
birth defects, congenital abnormalities, or maternal and/or new-born complications.  Pregnancy 
outcomes must be collected for females who took the IMP and the female partners of any males 
who took the IMP.  Consent to report information regarding these pregnancy outcomes should be 
obtained from the mother.   
13.4. 
Laboratory 
13.4.1. 
Routine Laboratory Assessments 
Routine laboratory safety  samples will be analysed by 
 at screening 
and again on Day 2 of treatment periods 1 and 2 for each subject. Printed laboratory reports will 
include normal reference ranges.  A decision regarding whether the result outside the reference 
range is of clinical significance or not shall be made by an Investigator and the report will be 
annotated accordingly.  Clinically significant abnormalities at screening or occurring during the 
study will be recorded on the AE page. The reference ranges for laboratory parameters will also 
be filed in the Investigator site file. 
Haematology: Haemoglobin (HGB), haematocrit (HCT), mean cell volume (MCV), mean cell 
haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), red blood cells (RBC), 
white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMP), monocytes (MONO), 
eosinophils (EOS), basophils (BASO) and platelets (PLT).  


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Samples will be collected into a 2.7 mL ethylenediaminetetraacetic acid (EDTA) Sarstedt 
Monovette® collection tube and analysed using the Siemens Advia 2120® or Siemens Advia 
120®. 
Biochemistry: Total protein (TP), albumin (ALB), total bilirubin (BIL-T), alanine transaminase 
(ALT), aspartate transaminase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase 
(ALKP), glucose (GLU), sodium (NA), potassium (K), calcium (CA), bicarbonate (HCO3), 
creatinine (CREA) and urea. 
At screening, biochemistry samples will be collected into a Sarstedt Monovette® 4.7 mL z-gel 
collection tube.  This volume allows sufficient serum for virology and pregnancy test screening.  
At all other visits, biochemistry safety samples will be collected into a 4.5 mL plain Sarstedt 
Monovette® collection tube.  Biochemistry samples will be analysed using the Roche Cobas 
c6000 analyser series comprising of c501 and e601 modules. 
Urinalysis: pH, specific gravity, protein, blood, glucose, leukocyte esterase, ketones, 
urobilinogen, bilirubin & nitrites.  In the event that the ‘dipstick’ is positive, RBCs, WBCs, 
epithelial cells, crystals, bacteria and casts will be examined microscopically.  The microscopy 
will be performed at 
 or referred to a local specialist hospital laboratory for analysis. 
A mid-stream urine sample will be collected into a 20 mL Sterilin tube. Urinalysis will be 
performed using the Siemens Clinitek 500 analyser. 
13.4.2. 
Virology 
Virology (Hep B, Hep C and HIV) will be performed at screening.  
Virology will be analysed from the Sarstedt Monovette® 4.7 mL z-gel biochemistry screen by 
 using the Roche Cobas c6000 analyser series comprising of c501 
and e601 modules. 
13.4.3. 
Drugs of Abuse and Alcohol 
Urine alcohol and drugs of abuse screen: Cannabinoids, amphetamines, cocaine, 
benzodiazepines, opiates and cotinine. 
A mid-stream urine sample will be collected into a 20 mL Sterilin tube.  At time-points when 
both urinalysis and drugs of abuse / alcohol screening is required, all testing will be performed 
from a single 20 mL sample. 
Drug of abuse and alcohol samples will be analysed by 
 using the 
Roche Cobas c6000 analyser series comprising of c501 and e601 modules.Vital signs  
13.5. 
Vital signs 
Systolic/diastolic blood pressure and pulse rate and oral temperature.   
Measurements will be recorded in the supine position after 5 min supine.  Blood pressure, pulse 
and temperature will be measured by the DINAMAP* Compact Vital Signs Monitor (Model TS) 
or equivalent.  Normal ranges for vital signs are presented in Appendix 1. 


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13.6. 
Physical Examination 
A physical examination will be performed by an Investigator.  The examination will include 
general appearance, head, ears, eyes, nose, throat, neck, skin, cardiovascular system, respiratory 
system, gastrointestinal system, central nervous system, lymph nodes and musculoskeletal. An 
Investigator can examine other body systems if required, at their discretion. 
13.7. 
ECG Measurements 
Screening and discharge ECGs will be conducted using a 12-lead ECG.  Heart rate, PR interval, 
QRS width, QT interval, and QT interval corrected using Fridericia’s formula (QTcF) will be 
documented.  
12-lead ECG recordings will be made using a MAC 5500 or equivalent.  Each ECG trace should 
be labelled with the study number, subject number, subject initials and date of birth. An 
Investigator will provide an interpretation of each tracing.  Clinically significant abnormalities 
will be recorded on the AE page. Normal ranges for 12-lead ECG parameters are presented in 
Appendix 1. 
13.8. 
12-Lead ECG Holter 
12-lead ECG Holter recording will begin on Day -1 and continue until approximately 24 hours 
post dosing during each study period. 
13.9. 
Concomitant Medication 
All prior and concomitant medications taken during the study will be recorded in the subject’s 
CRF. 
14. 
APPROPRIATENESS OF MEASUREMENTS 
All measurements performed in the study are standard measurements. 
The total volume of blood to be collected from each subject during the study (approximately 
280 mL) is considered acceptable.   
Table 8: 
Summary of Blood Volume 
Procedure 
Visit 
No. Samples 
Blood Volume 
per Sample 
(mL) 
No. Treatment 
Periods 
Blood Volume 
(mL) 
Biochemistry  
Screeninga 
Study Day 2  
1 
2 
4.7 
4.5 
2 
4.7 
9.0 
Haematology  
Screening 
Study Day 2 
1 
2 
2.7 
2.7 
2 
2.7 
5.4 
PK Drug Conc. 
Measurement 
Treatment 
Period 
17 
7.5 
2 
255 
Total Blood Volume 276.8 
a From the screening biochemistry blood samples, the serum pregnancy test and virology screen will be conducted.  


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15. 
STATISTICAL METHODS 
15.1. 
Primary Objectives and Analysis 
All data recorded during the course of the study or derived during the programming phase will be 
presented in individual data listings. 
Unless specified otherwise, continuous variables will be summarised with the following 
descriptive statistics: n (number of observations), arithmetic mean, standard deviation (SD), 
minimum value, median and maximum values. For PK summaries, the geometric mean and 
coefficient of variation (%CV) will also be used to summarise the data. Categorical data will be 
summarised with frequencies and percentages.  
Unless specified otherwise, the minimum and maximum statistics will be presented with the 
same precision as the original data.  The mean, median and geometric mean will be presented to 
one more decimal place than the original data. The SD will be presented to two more decimal 
places than the original data. Unscheduled measurements will be listed in the individual data 
listings.  With the exception of baseline, unscheduled measurements will be excluded from the 
descriptive statistical analysis.  In case an unscheduled measurement is performed immediately 
after the scheduled measurement due to an error in the previous measurement, the unscheduled 
measurement will be included in the analysis and the original erroneous measurement will be 
excluded. 
For subject characteristics and safety analyses, missing data will not be replaced; descriptive 
statistics and statistical analysis will be performed on the basis of the available data only.  
Subjects discontinued will be included in the descriptive statistics if they have received the 
investigational product and their examinations were performed at the same scheduled time as 
other subjects.  All data recorded on discontinued subjects will be listed. 
15.2. 
Subject Population for Analyses 
15.2.1. 
Safety Analysis Set 
All subjects who received at least one dose of cytisine will constitute the safety population.  
15.2.2. 
PK Analysis Set 
The PK Analysis Set will include all subjects who receive both doses of cytisine and comply 
with the following criteria: 
• Do not have an occurrence of vomiting or diarrhoea which renders the concentration 
profile unreliable;  
• Do not use a concomitant medication which renders the concentration profile 
unreliable;  
• Do not have a pre-dose concentration that is greater than 5% of the corresponding 
Cmax; 


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• Have at least one evaluable concentration that is preceded by a lower evaluable 
concentration and followed by a lower evaluable concentration for the calculation of 
Cmax, Tmax and AUCs (i.e. at least 3 evaluable concentrations in total); 
• Do not violate the protocol (major protocol violation) in a way that may invalidate or 
bias the results. 
15.3. 
Subjects Characteristics 
Separate summaries of  demographics,  will be provided for both safety and PK analysis sets if 
applicable. 
The protocol violations will be listed per subject, describing the nature of the violation. Subjects 
failing to complete the study (as well as the date and reasons for discontinuation) will be 
displayed. Time may be added if relevant to the nature of the violation. 
Medical history will be referred in accordance with the Medical Dictionary for Regulatory 
Activities (MedDRA), version 20.0 or higher. Medications will be mentioned according to the 
Anatomical Therapeutic Chemical (ATC) classification system using WHO Drug Dictionary. 
15.4. 
Sample Size Calculation 
A total of approximately 24 subjects will be randomized to the study, with approximately 12 
subjects per treatment schedule arm.  
Subject numbers are based on the following guidance:  No previous pharmacokinetic studies 
with cytisine have reported the effects of repeated administration, so no estimates of intra-subject 
variability of Cmax or AUC are available.  A recent bioavailability study in a limited number of 
dogs,14 where cytisine was given in the fed and fasted states, indicated an increased Cmax in the 
presence of food of 28%.   A previous study13 reported a total coefficient of variation (%CV) for 
Cmax of 42%.  Under the assumptions that a similar food effect will be observed in humans and 
that the intra-subject %CV will be less than the total %CV obtained from the previous study, 
based on an estimated geometric mean ratio for Cmax of 1.30 (30% increase) and an estimated 
%CV of 35%, a sample size of 24 should be sufficient to meet the objectives of the study. 
Subjects who withdraw from study at any time after randomization will not be automatically 
replaced. Replacement based on the number and reason of withdrawals will be at discretion of 
the Sponsor, following discussion with the Principal Investigator.   
15.5. 
Plasma and Urine Concentrations 
Descriptive statistics to summarize plasma and urine concentrations at each time point.  
Concentrations below the LLOQ will be replaced by zero for calculation of descriptive statistics.  
Individual drug plasma concentrations versus time profiles will be plotted per subject on both a 
linear and a semi-logarithmic scale.  For plotting of individual data in linear scale, concentrations 
below the LLOQ will be replaced by zero.  For plotting of individual data in semi-logarithmic 
scale, concentrations below the LLOQ will be set to missing.  Graphical presentation of 
individual data will be based on actual blood sampling times. 


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Arithmetic mean drug plasma concentration versus time profiles will be presented on both a 
linear and a semi-logarithmic scale.   
The post-dose blood samples will be collected within ±2 minutes from the scheduled sampling 
time. A deviation greater than ±2 minutes will be reported as a protocol deviation.   
15.6. 
Estimation of pharmacokinetic parameters 
Plasma: The following pharmacokinetic parameters for cytisine will be derived by standard non-
compartmental methods from the concentration versus time profiles: 
Table 8: 
Cytisine Pharmacokinetic Parameters 
Parameter 
Description 
Cmax 
Maximum observed plasma concentration post dose, directly obtained from the observed 
concentration versus time profile. 
Tmax 
Time of occurrence of maximum observed plasma concentration. 
AUC0-t 
Area under the plasma concentration versus time curve (AUC) from time zero to the last 
sampling time at which concentrations were at or above the LLOQ, calculated by the 
linear-up/log-down trapezoidal rule. 
AUC0-∞ 
Total AUC from time zero to infinity, calculated as AUC0-∞ = AUC0-t + (Clast/λz), where Clast 
is the last measurable plasma concentration and λz the apparent terminal elimination rate 
constant. 
%AUC 
Residual area or percentage of extrapolated part for the calculation of AUC0-∞, calculated as 
100*[1-(AUC0-t/AUC0-∞)] 
λz 
Apparent terminal elimination rate constant. 
t1/2 
Apparent terminal elimination half-life, calculated from ln 2/λz  
No values of λz, AUC0-∞, %AUC and t1/2 will be reported for cases where λz cannot be 
reliably determined. 
Pharmacokinetic parameters will be estimated from the plasma concentration versus time 
profiles for all subjects in the safety analysis set.  Actual sampling times will be used for the 
pharmacokinetic analysis. All calculations will be performed using raw data.  
The terminal elimination rate constant (λz) will be determined by plotting the concentration data 
versus time on a semi-logarithmic scale.  The parameter will be estimated by linear least square 
regression analysis, using the last three (or more) non-zero concentrations.  The upper and the 
lower timepoints, as well as the number of timepoints, used for λz estimation will be reported.  
AUC0-t will be calculated by the linear-up/log-down trapezoidal method. 
Individual pharmacokinetic parameters and descriptive statistics for the pharmacokinetic 
parameters obtained for each treatment will be presented by treatment and gender (males, 
females, overall) and by treatment and BMI.  If no reliable pharmacokinetic parameter could be 
determined for more than 1/3 of the subjects, only the minimum and maximum values will be 
presented for that parameter and all other descriptive statistics will be omitted.  Missing 
pharmacokinetic parameter data will not be imputed.  


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Urine: The urine PK parameters amount excreted in urine over time (Ae) and percent of drug 
excreted in urine (Ae%) will be derived from the urine concentration-time data for all subjects in 
the safety analysis set. Derived urine PK parameters will be listed and summarised by treatment 
and gender (males, females, overall).   
15.7. 
Pharmacokinetic Analysis 
For males, females and overall (data from both genders pooled) following logarithmic 
transformation of Cmax, AUC0-t and AUC0-inf values will be subjected to an analysis of variance 
(ANOVA) including fixed effects for sequence, period, treatment and subject nested within 
sequence.  Point estimates and 90% confidence intervals (CI) will be constructed for the 
contrasts between treatments using the residual mean square error obtained from the ANOVA. 
The point and interval estimates will be back-transformed to give estimates of the ratios of the 
geometric least squares means (LSmeans) and corresponding 90% CI. Estimated geometric 
means will also be presented for each treatment. 
In addition, for the purpose of exploring whether BMI may have an effect on bioavailability, the 
above analysis will be performed by BMI category (BMI of 18-22, 23-27 or 28-32 kg/m2). 
A comparison of Tmax between treatments will be performed using the Wilcoxon Signed-Rank 
test. In addition, the Hodges-Lehmann estimate of the median difference in Tmax and 
corresponding 95% CI will be presented. This analysis will be performed by gender and overall. 
15.8. 
Safety Variables and Analyses  
All safety data will be listed using the safety analysis set. 
15.8.1. 
ECGs 
The ECG extraction timepoints will be from extraction windows which precede the PK blood 
draws.  The following ECG parameters will be extracted in triplicate from Holter recordings at 
45, 30 and 15 minutes prior to pre-dose and at pre-dose, 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12 h and 
24h post dose (Period 1 and Period 2): 
• Heart Rate (HR) 
• PR intervals  
• QRS  
• QT  
• QTcF 
If a large change in heart rate is observed during the study, the individual QT correction method 
(QTcI) will be used as the primary QT correction method. 
The extracted ECG parameters will be listed with any out of normal range values flagged (“H” -
high or “L” -low).  The ECG results will assessed as either “normal” or “abnormal”. Any 
comments on abnormal results will also be provided. Descriptive statistics of absolute and 
change from baseline values at each time point will be tabulated.   
In addition, frequencies of QTcF data will be calculated according to the following categories: 


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For absolute values: 
• QTcF ≤ 450 mSec 
• 450 < QTcF ≤ 480 mSec 
• 480 < QTcF ≤ 500 mSec 
• QTcF > 500 mSec. 
For change from baseline: 
• QTcF increase ≤ 30 mSec  
• 30 < QTcF increase ≤ 60 mSec  
• QTcF increase > 60 mSec. 
Abnormalities in ECGs will be classified in terms of clinical significance. Clinically significant 
abnormalities will be reported as AEs 
In order to explore the effects of cytisine on QTc prolongation, plasma concentrations will be 
plotted against change from baseline QTcF values. 
15.8.2. 
Vital Signs 
Vital signs parameters will be listed with any out of normal range values flagged (“H” -high or 
“L” -low) as described in the Statistical Analysis Plan (SAP).   
Abnormalities in vital signs will be classified in terms of clinical significance. Clinically 
significant abnormalities will be reported as AEs. 
15.8.3. 
Adverse Events 
AEs will be tabulated and summarised according to the MedDRA version 20.0 or higher, and 
classified by system organ class (SOC) and preferred term (PT). The following information 
recorded or computed is used for the description of the adverse events: reported Term; SOC and 
PT by MedDRA coding; onset date, onset time, offset date and offset time; seriousness; severity 
(intensity); relationship (causality); action taken; outcome. 
Treatment-emergent adverse events (TEAEs) are defined as AEs not present prior to first 
administration of investigational product, or AEs present before first administration of 
investigational product that worsen after the subject receives the first dose of investigational 
product. TEAEs that occur after administration of investigational product in a given period are 
assigned to the treatment administered in that period. A separate listing of SAEs will be 
presented, if applicable. 
Frequencies of TEAEs will be presented by SOC and PT, severity and relationship, by 
Fed/Fasting regimens and overall. 
15.8.4. 
Safety Laboratory tests 
Biochemistry, haematology and urinalysis parameters will be listed with any out of normal/alert 
range values flagged.  Laboratory test results which are out of normal range will also be 
presented separately along with normal reference ranges. 


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Abnormalities in clinical laboratory tests will be classified in terms of clinical significance. 
Clinically significant abnormalities will be reported as AEs. 
15.9. 
Procedures for Deviations from Planned Analyses 
The planned statistical analyses for this study will be described in detail in a SAP. Modifications 
or additions to the analyses described above will be described in the SAP. Any decisions to 
deviate from the planned analyses described in the protocol and SAP will be documented in the 
clinical study report. The clinical study report will also provide a detailed explanation for any 
deviations from the planned analyses. 
16. 
DOCUMENTATION  
16.1. 
Study File and Site Documents 
Prior to the initiation of the study, the following items must be received by the Sponsor from the 
site: 
• Confidential Disclosure Agreement  
• Signed protocol, and amendment(s) page(s)  
• The Principal Investigator’s curriculum vitae and where required current medical 
license 
• Signed Clinical Study Agreement 
• Signed Financial Disclosure Form from the relevant site personnel 
• Signed Data Protection Form (for EU member states only) 
• Competent/Regulatory Authority written approval (if applicable) 
• EC written approval for the protocol, amendment(s), Informed Consent Form, Subject 
Information Sheet (if applicable), advertisements (if applicable) 
• EC Membership list or an official statement from the EC/IRB stating the EC/IRB is in 
compliance with the EU Directive on Good Clinical Practice (GCP) 
• Local laboratory certification and normal ranges 
16.2. 
Study Documents Supplied by the Sponsor 
The Sponsor will supply the Investigator with the following items: 
• Current version of the Investigator’s Brochure  
• Current version of study protocol 
16.3. 
Maintenance and Retention of Records 
It is the responsibility of the Investigator to maintain a comprehensive and centralized filing 
system of all relevant documentation. 


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• Investigators will be instructed to retain all study records required by the Sponsor and 
regulatory authorities in a secure and safe facility with limited access for one of the 
following time periods based on notification from the Sponsor: 
− For a period of at least 2 years from the last marketing approval worldwide or for 
at least 15 years, whichever is the greater 
− Or a period of at least two years after discontinuation of clinical development of 
the investigational product as confirmed by the Sponsor 
− For a longer period if required by local regulations 
The Investigator will be instructed to consult with the Sponsor before disposal of any study 
records and to provide written notification to the Sponsor of any change in the location, 
disposition, or custody of the study files. 
17. 
ADMINISTRATIVE PROCEDURES 
17.1. 
Sponsor Responsibilities 
The study will be monitored by representatives of the Sponsor and/or designated contract 
research organizations (CROs). Routine monitoring visits will be conducted to: 
a. Assure compliance with the study protocol. 
b. Verify that the research facilities, including laboratories and equipment, are adequate 
to safely and properly conduct the study. 
c. Verify that the investigational product is stored properly and under the proper 
conditions, is in sufficient supply, and that receipt, use, and return of investigational 
product at the study sites are controlled and documented adequately. 
d. Verify that written informed consent was obtained before any protocol-specific 
screening procedures are performed solely for the purpose of determining eligibility 
for the clinical study and/or prior to the provision of study medication. 
e. Review the subject CRFs and source documents to ensure that reported study data are 
accurate, complete, and verifiable from source documents. 
f. Ensure that adequate records of clinical trial supplies are maintained. 
g. Verify that the Investigator and study site personnel are adequately qualified 
throughout the study. 
h. Verify that the safety information and amendments are submitted to the 
IRBs/ECs/REBs. 
17.2. 
Investigator Responsibilities 
All requested study data must be entered on the CRFs for the study. An explanation should be 
provided for all missing data. Correction of data on a CRF will be made with identification of the 
individual making the correction and date of the correction. Only individuals who are identified 
on the Delegation of Responsibility Form(s) may correct data on the CRF. For those subjects 
who withdraw before completion of their specified treatment regimen, all available efficacy and 
safety data must be entered in the CRF. The reason for withdrawal must be specified. Incomplete 


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or inconsistent data on the CRFs will result in data queries that will be returned to the 
Investigator for resolution. 
The Investigator must maintain adequate and accurate source documents upon which CRFs for 
each subject are based. The source documents are to be separate and distinct from the CRFs, 
except for cases in which the Sponsor has predetermined that direct entry into specified pages of 
the subject’s CRF is appropriate. The documents to be maintained must include, but are not 
limited to, detailed notes on: 
a. The medical history prior to participation in the study 
b. The basic identifying information, such as demographics, that link the subject’s 
source documents with the CRFs 
c. The results of all diagnostic tests performed, diagnoses made, therapy provided, and 
any other data on the condition of the subject 
d. The subject’s exposure to study treatment 
e. All AEs 
f. The subject’s exposure to any concomitant therapy, including dates of administration 
g. All relevant observations and data on the condition of the subject throughout the 
study 
h. The oral and written communication with the subject regarding the study treatment, 
including the risks and benefits of the study. The date of informed consent must be 
recorded in the source documentation. 
18. 
REGULATORY COMPLIANCE 
Quality Assurance representatives from the Sponsor or their delegate, the MHRA, and all other 
regulatory agencies as required will be allowed to periodically visit the Investigators to discuss 
the conduct of the trial and, upon request, to inspect the records of the trial. These reviews are 
necessary to ensure that the study is conducted according to standards consistent with the ICH 
GCP Guideline. 
The Investigator agrees to discuss and correct, if necessary, any problems or deficiencies that are 
found during the course of these reviews. 
19. 
ETHICAL CONDUCT OF THE TRIAL / GOOD CLINICAL 
PRACTICE 
This trial will be conducted in accordance with the Declaration of Helsinki, as well as the ICH 
Guidelines on GCP, the US Code of Federal Regulations, the Food and Drugs Act (Health 
Canada), and local requirements regarding IRB/EC/REB committee review, informed consent, 
and other statutes or regulations regarding the protection of the rights and welfare of human 
subjects participating in biomedical research.  The Chief Investigator shall be responsible for 
ensuring that the clinical study is performed in accordance with the following: 
• Declaration of Helsinki (Brazil, 2013). 
• Association of the British Pharmaceutical Industry (ABPI) Guidelines for Phase 1 
Trials (2012). 


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• International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline 
for Good Clinical Practice (GCP). 
• The Medicines for Human Use (Clinical Trials) Regulations 2004 (Statutory 
Instrument 2004 No. 1031) and subsequent amendments. 
• Applicable local standard operating procedures (SOPs). 
As this clinical study will be conducted in the United Kingdom, it has been registered in the 
EudraCT database and a Clinical Trials Authorisation (CTA) will be obtained from the 
Medicines and Healthcare products Regulatory Agency (MHRA) prior to the start of the study in 
accordance with Part 3, Regulation 12 of the United Kingdom (UK) Statutory Instrument.  In 
addition, this study protocol will be included in an Investigational New Drug (IND) application 
with the US FDA for cytisine development. 
20. 
PROTOCOL MODIFICATION/PREMATURE TERMINATION 
All protocol amendments must be written and approved by the Sponsor. Each IRB, EC, or REB 
will review and approve amendments prior to their implementation in the study. IRB, EC, or 
REB approval need not be obtained prior to removal of an immediate hazard to subjects. 
The Sponsor may terminate the protocol early if safety or other issues occur. 
The study may be suspended or terminated by either the Principal Investigator or the Sponsor 
after mutual consultation at any time for scientific and safety reasons.  Furthermore, the study 
may also be terminated prematurely by the Sponsor for important corporate reasons, or due to 
instruction of the Regulatory Authorities due to safety reasons.  
Following a decision of temporary suspension or discontinuation, it is a responsibility of the 
Principal Investigator to inform the study subjects, Ethics Committee and Regulatory Agency, 
stating the reasons for premature termination. 
 shall be responsible for expedited 
reporting and/or notification to MHRA/EMA. The Sponsor shall be responsible for expediting 
reporting and/or notification to other regulatory authorities, as applicable.  
21. 
POLICY FOR PUBLICATION AND DATA PRESENTATION 
The Sponsor encourages the scientific publication of data from clinical research trials. However, 
Investigators may not present or publish partial or complete study results individually. The 
Principal Investigators and the Sponsor may propose appropriate scientific manuscripts or 
abstracts from the study data. Any manuscript or abstract proposed by the Investigators must be 
reviewed to ensure accuracy of data represented and commented upon in writing by the Sponsor 
prior to submission for publication. Investigators agree to consider the comments of the Sponsor 
in good faith and the Sponsor agrees in good faith not to impose limitations on access to the 
complete study data or unreasonable or inappropriate restrictions on publication of the study 
results. In case of publication, confidentiality of the study volunteers will be maintained. 


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INVESTIGATOR’S AGREEMENT  
Protocol No. ACH-CYT-01 
I have carefully read the foregoing protocol including all appendices and agree that it contains all 
the necessary information for conducting the study safely. 
I will conduct this study in strict accordance with this protocol and according to the current GCP 
guidelines and will attempt to complete the study within the time designated. 
I will provide copies of the protocol and all other information submitted by the Sponsor relating 
to pre-clinical and prior clinical experience to all personnel for whom I am responsible that 
participate in the study. I will discuss this information with them to assure that they are 
adequately informed regarding the drug and conduct of the study. 
I agree to keep records on all subject information (case report forms, shipment and drug return 
forms and all other information collected during the study) in accordance with the current GCP 
and local regulations. 
 
 
 
 
Site Principal Investigator’s name 
 
Sponsor’s Representative's name 
 
 
 
Signature 
 
Signature 
 
 
 
Date (ddMmmYYYY) 
 
Date (ddMmmYYYY) 
 
 
 
Institution 
 


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REFERENCES 
1. 
WHO. WHO Report on the Global Tobacco Epidemic, 2008 The MPOWER package. 
Geneva2008. 
2. 
Hughes JR, Keely J, Naud S. Shape of the relapse curve and long-term abstinence among 
untreated smokers. Addiction (Abingdon, England) 2004;99:29-38. 
3. 
Doll R, Peto R, Boreham J, Sutherland I. Mortality in relation to smoking: 50 years' 
observations on male British doctors. BMJ (Clinical research ed) 2004;328:1519. 
4. 
WHO. WHO Framework Convention on Tobacco Control2003. 
5. 
Aveyard P, West R. Cytisine and the failure to market and regulate for human health. 
Thorax 2013;68:989. 
6. 
Prochaska JJ, Das S, Benowitz NL. Cytisine, the world's oldest smoking cessation aid. 
BMJ (Clinical research ed) 2013;347:f5198. 
7. 
Stapleton JA. The case for licensing cytosine now for smoking cessation is 
overwhelming. BMJ (Clinical research ed) 2013;347:f5736. 
8. 
Gopalakrishnan M, Monteggia LM, Anderson DJ, et al. Stable expression, pharmacologic 
properties and regulation of the human neuronal nicotinic acetylcholine alpha 4 beta 2 receptor. 
The Journal of pharmacology and experimental therapeutics 1996;276:289-97. 
9. 
Tutka P, Zatonski W. Cytisine for the treatment of nicotine addiction: from a molecule to 
therapeutic efficacy. Pharmacological reports : PR 2006;58:777-98. 
10. 
Etter JF, Lukas RJ, Benowitz NL, West R, Dresler CM. Cytisine for smoking cessation: a 
research agenda. Drug and alcohol dependence 2008;92:3-8. 
11. 
Gotti C, Guiducci S, Tedesco V, et al. Nicotinic acetylcholine receptors in the 
mesolimbic pathway: primary role of ventral tegmental area alpha6beta2* receptors in mediating 
systemic nicotine effects on dopamine release, locomotion, and reinforcement. The Journal of 
neuroscience : the official journal of the Society for Neuroscience 2010;30:5311-25. 
12. 
Hughes JR, Stead LF, Hartmann-Boyce J, Cahill K, Lancaster T. Antidepressants for 
smoking cessation. The Cochrane database of systematic reviews 2014:Cd000031. 
13. 
Jeong SH, Newcombe D, Sheridan J, Tingle M. Pharmacokinetics of cytisine, an alpha4 
beta2 nicotinic receptor partial agonist, in healthy smokers following a single dose. Drug testing 
and analysis 2015;7:475-82. 
14. 
SRI Study No. M2161-16. 
 
 


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APPENDIX 1. NORMAL RANGES FOR VITAL SIGNS AND ECG 
PARAMETERS 
 
 



SAP_001.pdf: