Understanding Factors That Influence Electronic Cigarette Nicotine Delivery Through PET Imaging of Beta-2 Nicotinic Acetylcholine Receptors
Understanding Factors That Influence Electronic Cigarette Nicotine Delivery Through PET Imaging of Beta-2 Nicotinic Acetylcholine Receptors
The goal of this proposal is to test the overall hypothesis that e-cigarettes (ECs) provide
 efficient nicotine delivery to the brain and arterial blood in non-EC-naïve smokers and
 e-cigarette users and that EC liquid characteristics modulate this effect. This study will
 test the hypothesis through complementary methods that include [ [18F]NCFHEB (aka
 [18F]Flubatine) PET neuroimaging, arterial blood nicotine measurements, and subjective drug
 effects assessments.
C.2 Subject Inclusion and Assessments Overall study design: Non-EC-naïve smokers or
 e-cigarette users (n=20) will abstain from tobacco smoking/nicotine products for 5 days prior
 to each PET scan. In a randomized order (counterbalanced), after baseline quantification of
 receptor availability, subjects will be scanned while using an EC: (1) "JUUL" 5%, a common
 commercially available unmodified nicotine salt EC, and (2) JUUL 3%, a lower nicotine
 strength product. A third optional scan where subjects smoke a cigarette may be conducted but
 is not required. During scanning, subjects will undergo arterial blood nicotine level
 measurements following EC use and measures of nicotine withdrawal, craving, and drug effects
 will be measured. 5-7 days of smoking abstinence will be required prior to each PET scan to
 ensure no residual nicotine occupies receptors. Subjects will go back to smoking as usual
 after first scan for 2-3 weeks prior to subsequent smoking abstinence/PET scanning. Subjects
 will be provided with smoking cessation resources upon request.

 C.2.b Subjects: Twenty non-EC-naïve tobacco dependent smokers or e-cigarette users will be
 recruited for the study. All research subjects will be recruited under guidelines of the Yale
 University Institutional Review Board (Human Investigation Committee). Smokers will be
 recruited from the community via IRB-approved advertising. After completing informed consent,
 subjects will have a psychiatric, physical and neurological examination, laboratory tests and
 an electrocardiogram (EKG) (see C.2.c Clinical Assessments below). This is considered the
 screening phase. Subjects will be excluded for major medical, neurological or psychiatric
 illness, abnormal laboratory tests, or contraindication to PET or MR imaging. A urine drug
 screen and pregnancy test are performed at screening and prior to each imaging session and
 participants are excluded for a positive test. Detailed inclusion/exclusion criteria can be
 found in Protection of Human Subjects section. All women will be required to have a negative
 urine pregnancy test and all subjects are required to have a negative urine drug toxicology
 test prior to radiotracer administration.

 C.2.c Clinical Assessments: A standardized battery will be administered to all subjects at
 intake including Health Questionnaire, the SCID, assessments of demographics, measures of
 tobacco use, alcohol and drug use, family history of smoking, mood (PROMIS short forms for
 depression, anxiety, and anger), Center for Epidemiologic Studies Depression Scale), anxiety
 (State Trait Anxiety Inventory), impulsivity (Brief - Barratt Impulsivity Scale, smoking
 dependence (PROMIS nicotine dependence scale), smoking craving (Tiffany QSU) and withdrawal
 (Minnesota Nicotine Withdrawal Scale). Mood and smoking assessments will be repeated on each
 scan day before and after challenge. Product liking, craving and the Drug Effects
 Questionnaire (DEQ) will be assessed on a subjective scale (0-100, low-high) immediately
 following product use. The DEQ will measure acute effects consisting of seven items: drug
 strength, high, feeling stimulated, good effects, bad effects, wanting more drugs, and drug
 liking.

 Baseline and after each PET scan/e-cig challenge:

 -  Health Questionnaire

 -  PROMIS Dyspnea Severity Item Pool

 -  PROMIS Dyspnea Characteristics

 -  PROMIS Fatigue Short Form

 -  Functional Assessment of Chronic Illness Therapy (FACIT) Cough item

 -  The health questionnaire titled 'assessment of health problems' (and provided as an
 attachment via a comment in IRES) will be added to the protocol and will be completed at
 intake and before each exposure. Note that potential subjects with chronic symptoms that
 would interfere with monitoring of vaping complications, will not be enrolled.

 Positive findings of a change in symptom or single severe symptom would trigger clinical
 referral to a treating physician or the emergency room.

 C.2.d Contingency Management: All smokers/e-cigarette users will be helped to remain
 abstinent for 5- 7 days prior to each PET scan with highly successful contingency management
 techniques that have been used extensively in the laboratory before. Subjects will return to
 smoking use as usual after the first PET scan and repeat smoking abstinence prior to the next
 PET scan. This technique has been successful in the past to study nicotine occupancy of
 receptors after multiple challenges by recruiting smokers who are non-treatment seeking.
 Subjects will be met daily over the course of abstinence provide support and obtain CO and
 urine cotinine levels to ensure smoking abstinence. Subjects who are not able to abstain from
 tobacco smoking (2 consecutive days without a decrease in urine cotinine levels or a sudden
 increase in urine cotinine or CO levels) will be withdrawn from the study.

 C.2.e - MRI/cognitive testing Subjects will be asked to go to the MRRC at The Anlyan Center
 for Medical Research & Education (TAC, 300 Cedar Street) to have an MRI (Magnetic Resonance
 Imaging) scan of their brain. The purpose of the MRI is to help us identify the different
 regions of their brain on the PET scans.

 The MRI scan is a routine way to get pictures of the inside of the body. Subjects will be
 asked if they are carrying any metallic objects before they move toward the MRI system. These
 objects will be held for them in a locked cabinet in the MRI Center to avoid having these
 objects fly toward the magnet when they approach it. They will also be asked to walk through
 a metal detector. They will be asked to lie still in the MRI scanner for about 30 minutes.
 The scanner looks like a deep tunnel. They will be inside the tunnel from head to knees. They
 will not be able to see out of it, but they will be able to hear us and be heard if they wish
 to say anything. They will hear a drumming noise when the camera is taking pictures of their
 brain. If they feel uncomfortable during the scan, the scan will be ended at any time they
 wish. However, if they cannot complete the MRI scan, they will not be able to participate in
 any more of these studies.

 Two sessions of testing of their memory, attention, and concentration may take place. This
 will take approximately 1hr total. They will be given a break between these sessions. This
 may take place on the same day as the MRI scan, or on another day prior to PET scans if that
 works better with their and staff schedule.

 During this visit, they may play a computer game called the Face Game. The goal of the Face
 Game is to win as much money as possible. They may earn money by quickly and correctly
 pressing one of two keys on the keyboard, each time they see a face on the screen. They will
 press one key if they think that the mouth on the face is long, and the other if they think
 it is short.

 Cold Pressor Task Subjects may also be asked to participate in the cold pressor task during
 the study to test the body's physiological response to cold water. They will be asked to
 immerse your hand in a bucket of ice cold water maintained at 0-4°C. They will be told to
 raise their other hand when they begin to feel pain and to remove their hand from the water
 when they can no longer tolerate the pain. While their hand is immersed in water, they will
 also be asked to rate their pain on a scale from 0-100 and their heart rate and blood
 pressure will be monitored.

 Impulsivity Subjects may be asked to play a game in which they will use a computer mouse to
 click a balloon pump that inflates a balloon on the screen. The objective of the task is to
 get the largest amount of the money possible while avoiding balloon explosions. This
 assessment tests their impulsivity. They may be asked to play this game on the day of their
 PET scan too.

 C.3. PET Imaging Procedures PET procedures will be conducted at the Yale University PET
 Center. Female subjects will be given a urine pregnancy test prior to the initiation of any
 imaging procedures. If the test is positive, the scans will be canceled. Subjects will
 receive a minimum of 2 and maximum of 3 PET scans. The 3rd scan is optional.

 PET scans: Prior to the PET scans, a T1-weighted MRI will be acquired for all subjects on a
 3.0 Tesla Siemens Trio camera to rule out any brain abnormalities and for anatomical
 reference in the image analysis.

 PET scans are acquired as subjects lie supine on the scanner bed. Venous catheters will be
 used for intravenous administration of the radiotracer, and possibly for additional venous
 blood sampling. A radial artery catheter will be inserted by an experienced physician before
 the PET scan to draw arterial blood samples for metabolite analysis and for determination of
 the fraction of plasma radioactivity unbound to protein.

 Infusion of the radiotracer takes 210 minutes (3.5 hours). Subjects will be scanned for 2
 hours (starting 90 minutes after initiation of radiotracer infusion).

 [18F]NCFHEB will be synthesized and administered on PET scan days as a bolus plus constant
 infusion with a Kbol of 360 minutes over 3.5 hours. PET data will be acquired on the MCT. A
 6-min transmission scan will be acquired at the beginning or end of each PET scan to correct
 for attenuation. Arterial blood samples will be taken to obtain a metabolite-corrected
 [18F]NCFHEB input function. Baseline receptor availability will be measured 90-120mins after
 radiotracer infusion. Then, subjects will start a challenge (NIDA EC or JUUL EC,
 counterbalanced order) at 125min for 5 minutes in the scanner. Thereafter, post nicotine
 scanning will continue until end of 4 hrs of infusion. Vital signs (blood pressure, pulse,
 respiration) and ECG's will be obtained before and after radiotracer administration. The same
 procedures will follow for the 2nd PET scan.

 Use of EC: Subjects will receive 2 different scans and will puff one of two EC products (in
 random order, counterbalanced): "JUUL" 5% (50 mg/ml), a common commercially available
 unmodified nicotine salt EC, and (2) JUUL 3% (30 mg/ml), a lower nicotine strength product.
 Only tobacco flavor will be used. A training session to familiarize smokers with the EC
 product used in the study will be conducted at an appointment prior to the PET scans.
 Subjects will be advised to take longer and slower puffs from the EC (at least 3-4 seconds)
 as compared with puffing a cigarette. During each scan, subjects will be instructed to puff
 on the EC for up to 5 minutes. All users will be directly observed during EC use to ensure
 they do not have any problems operating the device. If available, a mouthpiece to measure
 puff topography may be used as previously described.

 Use of Regular Cigarette: A third PET scan using a regular cigarette may be offered for
 cigarette smokers but is not required for study completion.

 Blood Samples: On PET scan days, arterial blood samples will be collected prior to
 radiotracer administration and at multiple time points (t = 0, 0.3, 1, 3, 5, 10, 20, 30, 60,
 90 minutes post challenge) after EC/cigarette use to determine nicotine concentrations in the
 blood, which will be assayed using liquid chromatography tandem-mass spectrometry with
 deuterated internal standards.47 These data will be used to calculate pharmacokinetic
 patterns of nicotine delivery under each condition (Cmax, Tmax, and AUC for nicotine).

 Image Analysis and Outcome Measure: List-mode PET data will be reconstructed with OSEM with
 built-in corrections for attenuation, normalization, scatter, randoms, deadtime and subject
 motion.53,54 Following image reconstruction, a summed image will be registered to the
 subject's MRI. Regions of interest (ROIs) will be anatomically defined using a template
 [Anatomical Automatic Labeling (AAL)] coregistered to the subject's MRI image. ROIs used for
 calculation of receptor occupancy will be: cerebellum, frontal, parietal, temporal, and
 occipital cortices, caudate, putamen, hippocampus, and amygdala. These regions are chosen
 because they were previously examined in β2*-nAChR studies, have significant density in the
 human brain, and are relevant to the craving and withdrawal properties of nicotine. The
 thalamus will not be included because equilibrium is not reached in this region with the
 bolus-infusion paradigm. The volume of distribution VT will be estimated using the
 equilibrium ratio between total radioactivity concentration in tissue and parent
 concentration in arterial plasma. Baseline VT will be estimated from 90-120 min after
 infusion start, previously shown to establish good equilibrium in brain Post-ECig VT will be
 estimated from 180-210 minutes after infusion start (55-85 minutes post challenge). Prior
 preliminary data show that this is a sufficient amount of time for a new steady state to be
 achieved. Receptor occupancy and VND will be estimated using the Lassen plot approach.
Inclusion Criteria:

 -  Men and women, aged 21-55 years

 -  Able to read and write English

 -  Able to give voluntary, written informed consent

 -  Current cigarette smoking or e-cigarette use

 -  Non-treatment seeking

 -  Exhaled CO > 10 ppm or urine cotinine > 50 ng/ml

 -  Agreement to only use e-cigarette products provided in the study

 -  Not naïve to vaping (3 or more months and 12 or more times).

 Exclusion Criteria:

 -  Current medical condition such as neurological, pulmonary, cardiovascular, endocrine,
 renal, liver, or thyroid pathology

 -  History of or current neurological or psychiatric disorder including drug or alcohol
 dependence (as per SCID for DSM-5) except Nicotine Dependence

 -  Regular or current use of any prescription, herbal or illegal psychotropic medications
 in the past 1 year, with no current illegal drug use confirmed by urine toxicology
 (including cannabis)

 -  Drink more than 14 drinks per week for women or 21 drinks per week for men;

 -  Women who are pregnant or nursing

 -  Individuals who are currently taking medication that may affect cholinergic system or
 nicotine replacement therapy prescribed for smoking cessation;

 -  Contraindications to MRI such as claustrophobia or metal in their body, or to PET such
 as history of cancer Patient unlikely to be able to complete the study as determined
 by the PI or research assistants

 -  Blood donation within eight weeks of the start of the study

 -  History of a bleeding disorder or are currently taking anticoagulants (such as
 Coumadin, Heparin, Pradaxa, Xarelto)

 -  Known hypersensitivity to propylene glycol

 -  Planning to quit smoking with a set goal or time for quit attempt

 -  Untreated, unresolved acute pulmonary conditions (recurring bronchitis and Reactive
 airway disorder, as examples).

 -  Chronic symptoms on Health Questionnaire that would interfere with monitoring of
 vaping complication

 -  Naïve to vaping (vaping for less than 3 months and less than 12 times)