Clinical trial NCT04603391
Conditions: Drug Interaction
Sponsor: University of Florida
Start date: 2021-02-25; Completion date: 2021-12-31
Enrollment: 14 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The proposed study will assess the drug interaction potential between oral cannabidiol (Epidiolex®) and the carboxylesterase 1 (CES1) substrate methylphenidate (Ritalin®) in 12 healthy research subjects

Detailed description:
Cannabidiol (CBD) is a widely utilized nonpsychoactive cannabinoid available as an OTC supplement, a component of medical cannabis, and a prescriptive treatment of childhood epilepsies. In vitro studies suggest CBD may inhibit a number of drug-metabolizing enzymes, including carboxylesterase 1 (CES1). The aim of this study was to evaluate effect of CBD on the disposition of the CES1 substrate methylphenidate (MPH). This was a randomized, placebo-controlled, crossover study involving 12 healthy subjects. Each subject ingested 750 mg of CBD solution, or alternatively, a placebo solution twice daily for a 3-day run-in period followed by an additional CBD dose (or placebo) and a single 10 mg dose of MPH and completed serial blood sampling for pharmacokinetic analysis. MPH and CBD concentrations were measured by liquid chromatography with tandem mass spectrometry.

Arms: arm 1: Subjects received a three day run in of CBD 750 mg orally twice daily (administered as as 7.5 mL of Epidiolex® solution \[100 mg/ml\]). On day four, all subjects returned to the clinical research center to receive one oral dose of CBD 750 mg (administered as as 7.5 mL of Epidiolex® solution \[100 mg/ml\]) and one 10 mg tablet of dl-methylphenidate (Ritalin®) orally in a fed state (a 30 minute standardized breakfast).

After a minimum washout period of five days, subjects received a three day run in of 7.5 ml of Epidiolex® placebo solution (no CBD) orally twice daily. On day four, all subjects returned to the clinical research center to receive one oral dose of 7.5 mL of Epidiolex® placebo solution (no CBD) and one 10 mg tablet of dl-methylphenidate (Ritalin®) orally in a fed state (a 30 minute standardized breakfast). arm 2: Subjects received a three day run in of 7.5 ml of Epidiolex® placebo solution (no CBD) orally twice daily. On day four, all subjects returned to the clinical research center to receive one oral dose of 7.5 mL of Epidiolex® placebo solution (no CBD) and one 10 mg tablet of dl-methylphenidate (Ritalin®) orally in a fed state (a 30 minute standardized breakfast).

After a minimum washout period of five days, subjects received a three day run in of CBD 750 mg orally twice daily (administered as as 7.5 mL of Epidiolex® solution \[100 mg/ml\]). On day four, all subjects returned to the clinical research center to receive one oral dose of CBD 750 mg (administered as as 7.5 mL of Epidiolex® solution \[100 mg/ml\]) and one 10 mg tablet of dl-methylphenidate (Ritalin®) orally in a fed state (a 30 minute standardized breakfast).
Interventions: intervention 1: dl-Methylphenidate plus Cannabidiol intervention 2: dl-Methylphenidate plus Cannabidiol Placebo solution
Intervention descriptions: intervention 1: Subjects will be administered one 10 mg tablet of dl-methylphenidate (Ritalin®) plus 7.5 mL Epidiolex® solution intervention 2: Subjects will be administered one 10 mg tablet of dl-methylphenidate (Ritalin®) plus 7.5 mL of Epidiolex® placebo solution

Protocol documents:
Prot_SAP_001.pdf: 
1 
 
 
Title: Cannabidiol and CES1 Interactions in Healthy Subjects 
 
NCT Number: NCT04603391 
 
10/25/2022  


2 
 
College of Pharmacy, Department of Pharmacotherapy & Translational Research 
(PTR), College of Pharmacy 
Clinical Research Protocol 
An Assessment of the Drug Interaction Potential Between Oral Cannabidiol 
(Epidiolex®) and the CES1 Substrate Methylphenidate in Healthy Volunteers 
IRB Protocol No: 
 
202002547 
Principal Investigator:  
John S. Markowitz, Pharm.D. 
 
 
 
 
 
                           
 
Department of Pharmacotherapy & Translational Research 
College of Pharmacy 
Office Phone: 352-273-6262 
 
 
 
 
Cell Phone: 352-284-2248 
 
 
 
 
Email: jmarkowitz@cop.ufl.edu 
 
Co-Investigator: 
 
Reginald F. Frye, Pharm.D., Ph.D. 
 
 
 
 
Department of Pharmacotherapy & Translational Research 
 
 
 
 
College of Pharmacy 
 
Co-Investigator: 
 
Ludmila De Faria, MDAssociate 
Professor 
UF Department of Psychiatry 
 
Study Sponsor: 
State of Florida Consortium for Medical Marijuana Clinical 
Outcomes Research 
 


3 
 
PREFACE: COVID-19 CONSIDERATIONS 
Because of the COVID-19 viral pandemic, certain precautions for the personal safety of the 
subjects and the safety of UF study staff and personnel must be maintained. Although it is 
anticipated that the specific nature of these precautions will evolve over time (and the course of 
the study), certain basic precautions shared by UF and Shands Faculty, staff, students and 
visitors will be observed that may be subject to change per UF guidelines. 
Accordingly, all research subjects, will be required to wear a mask or cloth face covering when in 
UF and UF Health facilities.  Face coverings must be worn in all patient care/ research areas and 
when in public/common areas, including lobbies, elevators, stairwells, and bathrooms.  Further, 
the subject and one or more people are within 6 feet of each other outside of any UF building, 
they must also wear a mask or cloth face covering. This is explained explicitly in the Informed 
Consent document. There may be the need to show evidence of a negative COVID-19 test prior 
to participation in the study. 
While in the CRC research unit where subject evaluations and primary study day activities will 
take place, there may be further directions provided by CRC staff related to CRC-specific policy 
and procedures regarding these precautions. Such procedures may include a temperature 
checks and several questions about any recent travel or known contact with COVID-19 positive 
individuals by the research subject. Any such CRC unit specifics are not available to the PI at the 
time of this submission. Nevertheless, this possibility is also explained to subjects in the 
Informed Consent document. 
1. 
INTRODUCTION  
Introduction and Background 
Epidiolex® (cannabidiol [CBD]) solution is an FDA-approved medication derived from cannabis 
that is approved for the treatment of seizures associated with Lennox-Gastaut syndrome (LGS) 
or Dravet syndrome (DS) in patients 2 years of age and older. Additionally, CBD and CBD in 
combination with other cannabinoids is presently being studied for an expanding array of other 
pediatric clinical indications including Childhood Absence Seizures, Prader-Willi Syndrome, 
Autism Spectrum Disorder, Fragile X Syndrome, Attention Deficit Hyperactivity Disorder 
(ADHD), and others.1 Importantly, ADHD is the most common neuropsychiatric disorder of 
childhood, often persists into adulthood and the psychostimulant d,l-methylphenidate (Ritalin®) 
accounts for approximately 50% of all stimulant usage in the world. In the US alone there has 
been an approximately 10-fold increase in methylphenidate (MPH) prescribing since 1990, with 
18 million prescriptions dispensed in 2010, including 1.9 million new starts on MPH, making it 
the 5th most commonly prescribed medication to children ages 2 -11 and the single most 
frequently prescribed medication of any type in those aged 12-17 years.2 


4 
 
Thus, more likely than not, a very large number of pediatric patients who are receiving 
cannabidiol and/or other cannabinoids in clinical studies or in clinical care, will also be 
receiving MPH concomitantly. Any DDI between the two would be of clinical relevance. 
One of the most important aspects of safety considered in all drug therapies is the potential for 
the occurrence of drug-drug interactions (DDIs) of clinical significance. In the case of Epidiolex® 
(cannabidiol), the product sponsor, GW / Greenwich, has performed the requisite in vitro and in 
vivo DDI assessments as part of the FDA drug approval process.3 However, these studies were 
almost exclusively assessments of the potential influence of CBD on the Phase I cytochrome 
P450 (CYP) enzyme system. Recently, our group carried out the first in vitro assessment of the 
effects of CBD and other major cannabinoids on 
another Phase I drug metabolizing enzyme 
carboxylesterase 1 (CES1).4  
The results of our just published study indicate that 
CBD and other cannabinoids can significantly inhibit 
CES1. This finding is significant in that functional and 
unimpeded hepatic CES1 is responsible for both the 
detoxification/deactivation of numerous CES1 
substrates including methylphenidate (which relies almost exclusively on the CES1 pathway and 
no CYP 450 enzymes). Indeed in a reaction catalyzed by CES1, MPH is exclusively hydrolyzed to 
the inactive metabolite ritalinic acid.5 Additionally, CES1 is involved in the deactivation of 
numerous other substrates and also the activation of a number of medications formulated as 
prodrugs (e.g. oseltamivir). Table 1 contains numerous examples of known CES1 substrates 
including chemicals, toxins, and medications representing essentially every major therapeutic 
drug class.6 
Potential Drug-drug Interaction Implications 
Our recently published in vitro 
studies suggest that the use 
of CBD alone and or in 
combination with other 
cannabinoids, concurrently 
with MPH would be 
anticipated to impede the 
normal deactivation (de-
esterification) of MPH, and 
potentially lead to 
concentration associated 
adverse effects or toxicities.  
However, in vitro studies, 
ACE inhibitors 
CNS Agents 
Antihyperlipidemics 
benazepril        perindopril 
enalapril           quinapril 
Imidapril           ramipril 
moexipril          trandolapril 
 
methylphenidate 
cocaine 
heroin 
meperidine 
flumazenil 
simvastatin 
lovastatin 
clofibrate 
fenofibrate 
Antiplatelets/Anticoagulants 
Anticancer Agents 
Chemical warfare 
agents 
clopidogrel 
dabigatran etexilate 
capecitabine 
irinotecan 
sarin 
soman 
tabun 
Antiviral Agents 
Immunosuppressive 
Pesticides 
oseltamivir 
sofosbuvir 
mycophenolate mofetil 
ciclesonide 
trans-permethrin 
para-nitrophenyl valerate 
Endogenous compounds 
Miscellaneous 
Synthetic 
Cannabinoids 
cholesterol 
fatty acid ethyl esters 
oxybutynin 
AB-PINACA, AB-
FUBINACA 
PB-22, 5F-PB-22 
Table 1: Substrates of CES1 as established by in vitro assay 
COCH3
NH
O
H
H
COH
NH
O
dl-methylphenidate
Ritalinic Acid
Inactive Metabolite 
CES1
     


5 
 
particularly of natural products, have a number of recognized limitations and in vitro indications 
of DDI liability are not always borne out in clinical confirmatory studies.7 Accordingly, an 
assessment of whether these recent in vitro findings translate into a clinically relevant DDI 
requires confirmation through a formal interaction study in healthy individuals.  In 20088 we 
documented the first genetically deficient (CES1) metabolizer of MPH- the consequences of 
which are seen in the figure below (vs 19 normal  
 
subjects). As can be appreciated from this example, impaired CES1 activity can result in drastic 
elevations of MPH, even after a single dose of an immediate-release formulation. Finally, 
beyond the concerns of CES1 inhibition which may lead to the inability adequately to 
deactivate/detoxify and clear a given CES1 substrate (e.g. methylphenidate) is the inability to 
activate a number of prodrugs (e.g. oseltamivir) which require functional CES1 to be converted 
to their active moiety.9  
Clinical Evidence of CBD Inhibition of CES1 Activity 
Gaston and coworkers10 conducted a study which assessed the potential pharmacokinetic 
interactions between CBD (Epidiolex®) and commonly co-administered antiepileptic drugs 
(AEDs) in adults and children with treatment-resistant epilepsy. Notably, among other findings, 
CBD was found to dose-dependently, and significantly, increase concentrations of rufinamide 
(Banzel®), an AED almost exclusively metabolized via CES1. The investigators were unable to 
articulate a clear mechanism of the drug-drug interaction based upon the known in vitro 
activities of CBD and speculated that the sesame oil vehicle used in the Epidiolex® formulation 
might play a role. However, our recent findings lead us to believe that CBD inhibition of CES1, 
an unknown phenomenon at the time of the Gaston et al publication, is the likely explanation. 
The magnitude of the interaction is not well described in the paper and there are a number of 
study limitations that would limit such determinations. First, subjects were started on 5 
mg/kg/day (in divided doses) and potentially titrated to a maximum of 50 mg/kg/day but it is 
not reported what final CBD doses were. Secondly, only single blood samples for rufinamide 
were obtained and these were drawn during a 4-hour sampling window rather than a narrow 
range (i.e. 1 hour) more typical of rigorous drug-drug interaction assessment. A significant CBD-


6 
 
rufinamide interaction would not necessarily be an impediment to using the two together, but 
clinical awareness is important relative to dosing. Indeed, CBD might actually lessen the dosing 
requirements for rufinamide and decrease the cost of treatment.  
3. 
PURPOSE OF PRESENT STUDY 
To address the question of whether CBD can inhibit CES1 activity in a clinically relevant manner, 
we propose the conduct of a small (n=12) healthy volunteer study assessing the potential 
influence of Epidiolex® (cannabidiol) on the pharmacokinetic disposition of a single dose of the 
known CES1 substrate MPH in healthy volunteers. Such an assessment will directly answer the 
question of whether orally administered CBD, at clinically relevant doses, can significantly alter 
(impede) the metabolism of co-administered MPH. Furthermore, such an assessment, wherein 
MPH also functions as a CES1 probe drug substrate, will also provide some insight as to the 
possibility of CBD influencing a wide array of other CES1 substrates (Table 1). 
 
4. 
HYPOTHESIS  
Epidiolex® (CBD) will produce modest increases in the Cmax and area under the plasma 
concentration-time curve from 0 to 8 hours (AUC 0→8h) of a single dose of the recognized CES1 
substrate, dl-methylphenidate (Ritalin®) in healthy volunteers and confirm our recent in vitro 
findings. 
 
5. 
SPECIFIC AIMS  
 
The Specific Aims are as follows: 
1) To determine if the single dose pharmacokinetics of the psychostimulant and CES1 
substrate, methylphenidate (Ritalin® others), are altered by concomitant use of 
Epidiolex® (CBD).  
2) To determine if the single dose pharmacokinetics of the psychostimulant and CES1 
substrate, methylphenidate (Ritalin® others), are altered by concomitant exposure to 
the inactive components in the vehicle solution used for the Epidiolex® formulation 
 
6. 
METHODS 
A. 
Clinical Protocol Overview 
An open-label, randomized crossover design is proposed wherein healthy volunteer subjects 
(n=12) would receive a single dose of immediate-release dl-methylphenidate (Ritalin®) 
concomitantly with orally administered CBD (Epidiolex®) solution or an equal volume of 
Epidiolex® placebo solution (i.e vehicle with no CBD) which have been dosed to plasma steady-
state conditions. Serial blood samples will then be collected for methylphenidate analysis over 
the ensuing 8-hour period during both Epidiolex® exposure phases. Methylphenidate 
concentrations will be determined using a validated LC-MS/MS assay.11 Standard 
pharmacokinetic parameters will be calculated including methylphenidate peak concentration 


7 
 
(Cmax), the time to Cmax (tmax), the terminal elimination half-life (t1⁄2β) and the area under the 
plasma concentration-time curve (AUC). Comparisons between study phases would then reveal 
any significant influence of CBD. The study lasts about 6 weeks and involves 5 visits to the CRC 
although 2 of these 5 visits are only to pick up study medications (see Table 2)  
A general schematic of the proposed clinical study design is presented in the Figure Below: 
 
Research Subjects 
The study population will consist of normal healthy volunteers. The study population will 
consist of 12 healthy adult subjects (6 females, 6 males completing the study), aged 21-45 
years, who are healthy as assessed by medical history, physical examination, and routine 
laboratory tests including complete blood count, Comprehensive Metabolic Panel, urinalysis, 
and urine tetrahydrocannabinol (THC) screen. THC is a surrogate marker for cannabis use which 
would generally also result in exposure to CBD as well. Since outside use of CBD could confound 
the proposed study, THC positive subjects will be excluded. Participating females of child-
bearing potential must have a negative urine pregnancy test prior to enrollment and avoid 
pregnancy during study participation. Female subjects will be tested prior to each scheduled 
study day (urine pregnancy test) to provide additional assurance. A subject’s Body Mass Index 
(BMI) must be between 18.5 to 28 kg/m2 (inclusive). 
With the exception of oral contraceptives, no other prescription or OTC medications are 
permitted during study participation. These restrictions also extend to botanical/nutritional 
supplements, vitamins and “energy drinks”. All subjects must have no clinically significant 
diseases or clinically significant abnormal laboratory values as assessed during the screening 


8 
 
medical history, physical exam, and laboratory evaluations. Informed Consent forms must be 
signed by the eligible subject prior to the initiation of any study procedures.  
 
The specific Inclusion/Exclusion criteria are as follows: 
 
 
Inclusion Criteria 
* 
Signed Informed Consent 
* 
Age: 21-45 years  
* 
Gender: males and females (50:50) 
* 
Race or ethnicity: no restrictions 
* 
Body Mass Index (BMI) between 18.5 to 28 kg/m2 (inclusive) 
* 
Satisfactory completion of the screening medical history, physical exam, and laboratory 
evaluations.    
* 
Females of child-bearing potential must have a negative urine pregnancy test prior to 
enrollment and avoid pregnancy during study participation. 
* 
With the exception of oral contraceptives, subjects must not be taking prescription or 
OTC medication for the duration of study participation 
* 
Subjects must have no ongoing use of any botanical/nutritional supplement, vitamin, or 
energy drink for the duration of study participation 
* 
Negative Urine THC screen.  
Exclusion Criteria 
* 
The presence of a known allergy, hypersensitivity, or adverse reaction to CBD or 
cannabis, or sesame seed oil 
* 
The presence of a known allergy, hypersensitivity, or adverse reaction to 
methylphenidate or dexmethylphenidate (Focalin®) 
* 
A history (within the past year) or presence of clinically significant cardiovascular, 
cerebrovascular, renal, hepatic, gastrointestinal, pulmonary, immunological, 
hematological, endocrine, or neurologic disease will render subjects ineligible for the 
study.  
* 
The presence of any surgical or medical condition (active or chronic) that may interfere 
with drug absorption, distribution, metabolism, or excretion including; 
 
Gastric bezoar 
 
Swallowing disorders 
 
Strictures 
 
Fistulas  
 
GI obstruction 
 
Severe dsyphasgia 
 
Crohn’s disease  
 
Diverticulitis 


9 
 
 
* 
A positive urine pregnancy test. 
 
* 
A positive Urine THC Screen  
- we will not retain any information on any subjects with a positive urine THC 
screen, including the reason for excluding them from the study. 
 
* 
Any concomitant prescription medication, OTC medication, herbal or other 
dietary supplement or vitamins during the study period.
All subjects must be medication-free from 7 Days before initiation of the first active study day, 
through the duration of the study. This exclusion the use of vitamins, herbal preparations and 
OTC supplements.  
 
Informed Consent/Screening Process:  
All subjects will provide written informed consent approved by the University of Florida 
Institutional Review Board (IRB) prior to participating in the study. The Screening Visit will be 
conducted in the UF Shands CRC located in the Clinical Translational Research Building in 
Gainesville. It is expected to last approximately 1 hour.  
 
Up to 18 subjects may screened for the clinical protocol with a goal of 12 subjects completing 
the entire protocol.  All study protocols, informed consent forms, and Health Insurance 
Portability and Accountability Act (HIPAA) forms must be approved by the University of Florida 
(UF) Investigational Review Board (IRB) prior to initiating any study procedure or recruitment. 
At a screening/informed consent visit, the study design, purposes and inclusion/exclusion 
criteria will be explained to each prospective subject. Written informed consent will be 
obtained prior to commencing any study procedures.  
 
After obtaining written Informed Consent, study subjects will be interviewed about their 
medical history and the protocol’s Inclusion/Exclusion criteria will be discussed. All potential 
participants must be nonsmokers, not taking prescription or over-the-counter medications or 
botanical/nutritional supplements (inclusive of vitamins). Additionally, participants are 
requested to abstain from alcohol use 24 hours prior to the study health screen lab work or any 
scheduled study visit should they participate fully in the study.  
 
During the screening visit, subjects will have blood samples drawn for health screening 
purposes including baseline serum chemistries including liver enzymes, complete blood count 
(CBC), urinalysis, a urine THC screen, and a urine pregnancy test (women) which will have a 
history and physical exam performed by the study physician (See Appendix for details). Copies 
of the laboratory results will be made available to study subjects at their request, and this is 
conveyed in the Informed Consent.  


10 
 
 
An Assessment of the Drug Interaction Potential Between Oral Cannabidiol and Methylphenidate in Healthy 
Volunteers. Clinical Protocol Version # 2 12/01/20 
Page 10 of 20 
 
Study Visits 
This section describes the study procedures for the two major study visits at the University CRC 
following Informed Consent and a satisfactory history and physical and medical screening.  
 
Cannabidiol Pre-Exposure: In a previous normal volunteer study, subjects dosed with 750 mg of 
of Epidiolex® (cannabidiol) solution twice daily achieved steady-state conditions after 
approximately two (2) days.13  Accordingly, three (3) days prior to their first scheduled full 
pharmacokinetic study day at the CRC, and depending on assigned randomization sequence (to 
be assigned by the UF Investigational Pharmacy Services), subjects will have commenced self-
dosing with either A) CBD 750 mg (administered as 7.5 ml of Epidiolex® solution [100 mg/ml] of 
CBD) or B) 7.5 ml of Epidiolex® placebo solution containing no CBD. 
 
CRC Check in and Preparation 
Following an overnight fast (abstention from eating any foodstuffs after 9 pm the evening prior 
to the scheduled visit), subjects will arrive at the UF Clinical and Translational Science Institute 
CRC the morning of each of the two active Study Days where they will remain for approximately 
8 hours for each of two separate formulation administration and blood drawing phases (for 
pharmacokinetic assessments) of the study.  After checking in, and under medical supervision, 
skilled CRC staff will place an indwelling venous catheter in each subject’s arm to facilitate serial 
blood sampling. Female subjects will provide a urine sample for a pregnancy test, the results of 
which will be read and documented.  Baseline blood pressure, heart rate and temperature, will 
be obtained and vital signs (B.P. and H.R. only) will be repeated at approximately 12 noon, prior 
to lunch. 
 
Drug Administration 
At approximately 8:00 AM, on the day of the first full day dosing and pharmacokinetic 
assessment, subjects will be fed a standardized breakfast which they will have approximately 30 
min to consume. After breakfast, subjects will be administered one (1) 10 mg tablet of dl-
methylphenidate (Ritalin®) and depending on the previous randomization sequence, either A) 
CBD 750 mg (administered as 7.5 ml of Epidiolex® solution [100 mg/ml] of CBD) or B) 7.5 ml of 
Epidiolex® placebo solution containing no CBD. Subjects will then be given 240 ml of room 
temperature water and asked to drink it in its entirety.  A standard lunch will later be provided 
to all subjects approximately 4 hours post-dosing. The composition and amount of food eaten 
will be recorded.  Subjects will consume an identical meal on the subsequent second study visit. 
 
Blood sample collection and processing for methylphenidate analysis 
An indwelling venous catheter will be placed to facilitate serial blood sampling. A total of 9 
blood samples (~10 ml each) will be taken over an 8-hour period during each of the two 


11 
 
 
An Assessment of the Drug Interaction Potential Between Oral Cannabidiol and Methylphenidate in Healthy 
Volunteers. Clinical Protocol Version # 2 12/01/20 
Page 11 of 20 
primary study days. Blood samples will be collected in one 6 ml gray stoppered Vacutainers® 
(Becton Dickinson) which contain sodium oxalate to prevent clotting and sodium fluoride to 
prevent methylphenidate hydrolysis). 
Specific time points of blood collection will be immediately prior to the dose (0 time point), of 
methylphenidate (Ritalin®) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8 hrs. All samples will be drawn in 
heparinized tubes and stored on ice until centrifugation at 4°C.  Each ~10 ml of whole blood 
sample is anticipated to yield ~ 5 ml of plasma following centrifugation.  Plasma samples will be 
split into duplicate, approximately equal volumes (i.e. ~2.5 ml) labeled storage tubes, and 
frozen immediately at –70°C. CRC lab personnel will be asked to be split individual plasma 
samples and store them in duplicate as a measure of redundancy should the investigators 
encounter unanticipated difficulties in conducting the analysis. 
   
This single dose assessment of methylphenidate pharmacokinetics will be conducted both in 
the presence and absence of CBD as a single dose assessment in a randomized crossover 
fashion. Following the completion of the initially randomized Epidiolex® exposure (active vs 
placebo) administration and sample collection, a minimum 5-day wash-out period will transpire 
prior to a return for the second single-day assessment of the alternate assignment (under 
identical study conditions and collection times). Based upon the relatively short half-lives of 
methylphenidate and CBD, 5 days will be an adequate wash-out period.  
 
There will again be a 3-day self-dosing with either A) CBD 750 mg (administered as 7.5 ml of 
Epidiolex® solution [100 mg/ml] of CBD) or B) 7.5 ml of Epidiolex® PLACEBO solution prior to 
the second CRC Study Day.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


12 
 
 
An Assessment of the Drug Interaction Potential Between Oral Cannabidiol and Methylphenidate in Healthy 
Volunteers. Clinical Protocol Version # 2 12/01/20 
Page 12 of 20 
The General Schedule of Study Events is depicted below in Table 2: 
 
 
 
*A second LFT (after baseline screening) will be drawn only on the PK visit in which active 
Epidiolex is given (dependent on randomization) 
 
 
 
TABLE 2 
 
 
 
 
 
 
 
STUDY EVENT 
Study 
Consent 
Enrollment 
History & 
Physical 
with 
screening 
labs 
drawn 
Pick up 
“run-in” 
meds 
from CRC 
PK 
Sampling 
Day #1 
Wash-
out 5-
day min 
Pick up 
“run-in” 
meds 
from CRC 
PK 
Sampling 
Day #2 
VISIT # 
0 
1 
2 
3 
 
4 
5 
Study week  
1 
2 
 
3 
4 
 
5-6 
Informed Consent 
X 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
Physical Exam 
 
X 
 
 
 
 
 
Body Weight/BMI 
 
X 
 
 
 
 
 
Height 
 
X 
 
 
 
 
 
Vital Signs 
 
X 
 
X 
 
 
X 
Basic metabolic 
panel, LFTs, CBC, 
urinalysis, urine 
THC 
 
X 
 
LFTs* 
 
 
LFTs* 
Urine Pregnancy 
Test  
 
X 
 
X 
 
 
X 
Administer Study 
Drugs 
  
 
 
X 
 
 
X 
Intensive PK 
Sampling Day (CRC) 
 
 
 
X  
 
 
X  


13 
 
 
An Assessment of the Drug Interaction Potential Between Oral Cannabidiol and Methylphenidate in Healthy 
Volunteers. Clinical Protocol Version # 2 12/01/20 
Page 13 of 20 
Compliance with Outpatient CBD Dosing 
There is no way to absolutely confirm compliance in studies that involve self-administration of 
medications. However, as a measure to promote compliance, research subjects will be asked to 
save the empty oral syringes containing their CBD doses and bring them to the CRC at the time 
of check-in so that the investigators can examine and count them and confirm they were used. 
 
Analytical Methods 
Methylphenidate analysis 
All analytical methods for this project have been developed, validated, and published by the PI 
previously.10 This validated LC-MS/MS enantioselective method is established in the PI’s 
laboratory will be utilized to quantify d- and l-methylphenidate plasma concentrations. 
 
Pharmacokinetic Analysis 
The enantiospecific assay will permit calculation of separate pharmacokinetic values for d- l- as 
well as total methylphenidate concentrations. Estimates will be made using a non-
compartmental analysis using WinNonlin 5.3 (Pharsight, Mountain View, CA). The Cmax and time 
to maximum plasma concentration (Tmax) will be obtained directly from the plasma 
concentration-time data. The terminal elimination rate constant (λz) will be estimated by linear 
least-squares regression of the terminal portion of the plasma concentration-time curve, and 
the corresponding elimination half-live (t 1/2) was then calculated using the formula t1/2 = 
0.693/λz. The area under the plasma concentration-time curve from time 0 to infinity (AUC0→inf) 
for these single-dose assessments and AUC0→8h will be calculated according to the linear 
trapezoidal rule. The apparent clearance (CL/F) will be calculated using the formula 
dose/AUC0→inf. The apparent volume distribution (V/F) will be estimated by dividing CL/F by λz.  
 
Outcome Measures 
Differences in the geometric mean ratios (GMR) of the pharmacokinetic parameters will be 
compared between the two exposure conditions; i.e. methylphenidate + CBD vs 
methylphenidate + placebo. 
 
Statistical Analysis:  Results will be presented as median (range) for Tmax and mean ± standard 
deviation (S.D.) for other data. The differences are considered statistically significant when the 
P values are less than 0.05. 
 
 
 
 
 
 


14 
 
 
An Assessment of the Drug Interaction Potential Between Oral Cannabidiol and Methylphenidate in Healthy 
Volunteers. Clinical Protocol Version # 2 12/01/20 
Page 14 of 20 
 
7. 
PROTECTION OF HUMAN SUBJECTS 
 
1. 
Risks to Human Subjects 
a. 
Human Subjects Involvement and Characteristics:  
Twelve (12) healthy men and women (50:50) between the ages of 18-50 years will complete 
this study. The number of prospective subjects will also allow for dropouts and failed screening 
evaluations. We anticipate a dropout/screening failure rate of approximately 5% based on past 
experience. All subjects will give written informed consent using a consent form approved by 
the University of Florida Institutional Review Board. Subjects will have to pass a screening 
evaluation based on history, physical examination and biochemical and urinalysis tests 
described above. Specific Inclusion and Exclusion criteria are outlined above under the Study 
Population section.  
 
Abstinence from alcohol (in any form) for 24 hours prior to the screening visit as well as any of 
the scheduled study visits is required. No special populations, such as children less than 18 
years of age, pregnant women, or prisoners will be included in this study. The racial and age mix 
of the study will be representative of the population of Alachua County, FL. There will be no 
exclusion based on race, sex, or ethnicity.  
 
b. 
Sources of Materials: The following information will be collected from study 
participants for use in the research studies: demographic information (age, race/ethnicity, 
current medications, past medical history, and family history), body weight, height, history and 
physical exam, smoking status, and typical alcohol consumption. The laboratory data to be 
obtained from study participants include typical screening labs (i.e. complete blood count, liver 
function tests) and pregnancy tests for women with childbearing potential. Data will be kept in 
locked files that are only accessible to the investigators. During these studies, blood and urine 
samples will be collected during the health screening and the pharmacokinetic studies. The 
blood and urine will be obtained specifically for research purposes in order to meet our specific 
aims.  
 
c. 
Potential Risks:  
Venipuncture: The risks of drawing blood include temporary discomfort and inconvenience 
from the needle stick, and intravenous catheter placement. The procedures proposed have a 
potential for causing minor pain, bruising, bleeding, blood clots, and rarely, infection and 
swelling, and fainting could occur. 
Dietary modification:  During this study subjects will be asked to fast after 9 pm the evening 
prior to each of the two Full Study Day visits. Consumption of water during the fasting period is 
acceptable. 


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The most common side effects/adverse effects associated the two FDA-approved medications 
administered to the research subjects are as follows; 
 
Cannabidiol (Epidiolex®): In the proposed study subjects will be take CBD 750 mg (or placebo) 
twice daily on their own for 3-Days in a run-up to the active Study Day at the CRC, and one 750 
mg dose of CBD (or placebo) in the CRC. The 750 mg dose corresponds to a 10 mg/kg dose in a 
70 kg adult. In a recently published report of the pharmacokinetics of Epidiolex® solution in 
healthy adult volunteers receiving 1500 mg twice daily (i.e. 2 x the dose proposed here) for 6 
days, CBD was well-tolerated with the diarrhea, nausea, headache, somnolence and some 
instances of elevations in LFTs reported as the most common adverse events, all characterized 
as mild to moderate in severity.13  
Although Epidiolex® prescribing literature recommends a dose titration in patients (2.5mg/kg 
bid x 7 days, 5mg/kg bid x 7 days, and then 10 mg/kg bid), there is precedent in the literature 
for initiating Epidiolex® at 10mg/kg bid or higher (as proposed here) in healthy adult volunteer  
pharmacokinetic and drug interaction assessments without issues of tolerability.12.13 
Accordingly, we anticipate no untoward or serious adverse events associated with the short-
term exposure to 750 mg bid of CBD in this study. 
Liver Function Tests: Per Epidiolex® full prescribing literature, active treatment may cause dose-
related elevations of liver transaminases (alanine aminotransferase [ALT] and/or aspartate 
aminotransferase [AST]). In controlled studies for indicated seizure disorders, the incidence of 
ALT elevations above 3 times the upper limit of normal (ULN) was 13% in Epidiolex®-treated 
patients compared with 1% in patients on placebo. Importantly, most of these studies involved 
patients also treated with other anticonvulsants (e.g. valproate) and involved exposures far 
longer than the 7 doses subjects will receive in the present study. Nevertheless, as an added 
safety measure, we will obtain baseline LFTs as part of the subject screening process and will 
repeat the LFT assessment after their last dose is received on the CRC unit. 
 
Methylphenidate (Ritalin®):  In the proposed study subjects will be given a single 10 mg dose of 
immediate-release dl-methylphenidate (Ritalin®) on two separate occasions at least 5 days 
apart. Methylphenidate has been in continuous clinical use for over 60 years and is the most 
commonly employed stimulant used to treat ADHD) in children and adults with approximately 
18 million prescriptions dispensed annually in the US. Methylphenidate is well tolerated, has an 
excellent track record of safety, and serious adverse reactions are extremely rare. The most 
common side effects reported (with ongoing clinical treatment) are decreased appetite, 
stomachache, nervousness and insomnia. Insomnia associated with methylphenidate use is 
generally associated with taking late afternoon or evening doses. In the present study, a 
modest single 10 mg dose will be administered early in the morning. Clinically, immediate-
release methylphenidate is routinely administered to children in doses of 60 mg/day or greater.  


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In the present study, the potential inhibition of methylphenidate metabolism (via CBD 
inhibition of CES1) is being assessed. In theory, if significant inhibition does occur, subjects 
would effectively be receiving a higher “dose” of methylphenidate than the 10 mg 
administered. However, even a doubling or tripling of the methylphenidate concentrations 
would still produce levels well within the range of clinically used dosing parameters (i.e. 60 
mg/day and higher). 
Moreover, he principal investigator (JSM) has performed numerous healthy volunteer studies in 
which methylphenidate was administered to healthy adult subjects in doses ranging from 10 
mg immediate release to 40 mg sustained release with and without alcohol co-administration 
without any subject ever experiencing any serious adverse event. 14,15,16,17 
 
2. 
Adequacy of Protection Against Risks 
 
a. 
Recruitment and Informed Consent: Subjects will be recruited through advertisements 
that will be placed on bulletin boards (e.g. IRB-Approved Flyers). These sources of recruitment 
for healthy volunteers have been very successful in the past. Advertisements will be submitted 
to the University of Florida IRB for approval prior to use. Informed Consent will be obtained 
prior to any screening procedures by the PI or his designee after thorough discussion with the 
individual regarding the protocol, potential risks, and potential benefits of participation. 
 
b. 
Protections Against Risk: The risk of adverse experiences in this study will be minimized 
by utilizing only qualified individuals to conduct the study, the staff in the UF Shands CRC. 
Appropriate attention to detail in the experimental setting will be emphasized. Moreover, this 
study will consist of single doses of two marketed botanical and vitamin formulations (i.e. one 
exposure to each formulation). Thus, the likelihood of formulation-associated adverse events 
should be minimized. Immediate medical treatment will be provided for any illness or injury 
resulting from this study. Trained nursing staff is present in the UF Shands CRC at all times and 
the physician co-investigator (Rajesh Mohandas, MD) will also be available to evaluate the 
subjects. In the event that a subject experiences an intolerable side effect, the subject will be 
withdrawn from the study immediately and followed to a satisfactory resolution of the 
effect(s). A subject may also be removed from the study if in the opinion of the physician 
investigator it is in the subject’s best interest. Other risks of participation are minimal.  
 
c. 
Potential Benefits of the Proposed Research to Human Subjects and Others 
There is no direct benefit to the subjects as the aims of this research are to evaluate the 
potential drug interactions between CBD and methylphenidate with implications for other CES1 
substrate medications (Table 1). No disease or condition is present or being treated. All 
procedures necessary for the completion of these studies will be performed at no cost to the 


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Page 17 of 20 
study subjects (or their insurance providers). There is some intrinsic value to the 
comprehensive physical assessment and health screen the subjects will receive, and copies of 
their laboratory results will be provided to subjects at their request.  
 
All subjects will receive monetary compensation for study participation. Other than 
compensation for participation, there are no direct clinical benefits to the subjects for 
participating in this study.  
 
Payment for Participation: 
Subjects will receive a $500 at the completion of the entire research protocol.  Parking at UF 
CRC is free. If it is determined during the initial screening procedure that a subject cannot 
participate in the study, each will be paid $25.00 for completing the screening procedure.  If 
subjects are enrolled and elect not to complete the entire protocol for any reason, or are 
discontinued from the study by the investigators before protocol completion, each will receive 
partial compensation that will be pro-rated based upon the extent of their participation in the 
study to that point. For example, should a subject complete the first CRC study day for PK 
assessment and decide to discontinue the study, or be discontinued by the PI for any reason, 
they would be entitled to one half of the full study payment (i.e $250). 
 
If subjects are paid for taking part in this study, their name and social security number will be 
reported to the appropriate university employees for the purpose of making and recording the 
payment. Research subjects will be responsible for paying income taxes on any payments 
provided by the study that total $600 or more or if the patient is a nonresident alien.  Payment 
will be processed through the University of Florida Accounts Payable department and the 
University must report the amount received to the Internal Revenue Service (IRS). 
 
8. 
Data and Safety Monitoring Plan 
This study involves a small number of healthy volunteer subjects receiving single doses of a 
dietary supplement formulation who will be closely monitored by the Investigators and 
research personnel at the Shands CRC. All procedures will be performed in accordance with the 
Declaration of Helsinki on biomedical research involving human subjects as well as HIPPA. All 
biohazard materials such as blood samples will be handled and disposed in accordance with 
corresponding federal and state laws.  
 
 
Study Limitations 
Methylphenidate is an excellent representative probe substrate of CES1 and perturbations in its 
metabolism and disposition by CBD can reliably be attributed to this interaction mechanism 
under the described protocol. However, the results cannot necessarily be assumed to be 


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applicable to all CES1 substrates which might be co-administered with Epidiolex® solution. 
Nevertheless, it is accepted within the scientific and regulatory communities that clinical 
assessment utilizing probe drug methodology is a valid approach to provide general clinical 
insight into potential drug interaction liabilities beyond what in vitro studies can tell us.18  
 
Finally, although not a limitation of the present study design per se, study results with 
Epidiolex® solution cannot be assumed to be applicable to other CBD formulations or CBD 
dosing route. 
 
Conflict of Interest: The investigators DO NOT have a significant financial interest that would 
reasonably appear to be affected by the proposed research activities. 
 
 
9. 
REFERENCES 
 
1. https://clinicaltrials.gov/ct2/results?term=cannabidiol&Search=Search (Accessed 5/9/2019). 
2. Chai G, Governale L, McMahon AW, Trinidad JP, Staffa J, Murphy D. Trends of outpatient 
prescription drug utilization in US children, 2002-2010. Pediatrics. 2012;130(1):23-31. 
Epub 2012/06/20. doi: 10.1542/peds.2011-2879. PubMed PMID: 22711728. 
3. FDA Center for Drug Evaluation and Research. Epidiolex® (cannabidiol) Clinical 
Pharmacology and Biopharmaceutic(s) Review, Application #210365Orig1s000. Available 
at:https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/210365Orig1s000ClinPh
armR.pdf. Accessed August 23, 2018. 
4. Qian Y, Wang X, Markowitz JS. In vitro inhibition of carboxylesterase 1 by major 
cannabinoids and selected metabolites. Drug Metab Dispos 2019; dmd.118.086074; 
DOI: https://doi.org/10.1124/dmd.118.086074 
5. Zhu H-J, Patrick KS, Yuan H-J, Wang J-S, Donovan JL, DeVane, CL, Malcolm R, Johnson JA, 
Youngblood GL, Sweet DH, Langaee TY, Markowitz JS. Two CES1 gene mutations lead to 
dysfunctional carboxylesterase 1 activity in man: Clinical significance and molecular 
basis. Am J Hum Genetics 2008;82:1241-48. (PMID: 18485328) 
6. Laizure SC, Herring V, Hu Z, et al. The role of human carboxylesterases in drug 
metabolism: have we overlooked their importance? Pharmacotherapy. Feb 
2013;33(2):210-222. 
7. Markowitz JS, Zhu H-J. Limitations of in vitro assessments of the drug interaction 
potential of botanical supplements. Planta Med 2012;78(13): 1421-1427 
8. Zhu H-J, Patrick KS, Yuan H-J, Wang J-S, Donovan JL, DeVane, CL, Malcolm R, Johnson JA, 
Youngblood GL, Sweet DH, Langaee TY, Markowitz JS. Two CES1 gene mutations lead to 
dysfunctional carboxylesterase 1 activity in man: Clinical significance and molecular 
basis. Am J Hum Genetics 2008;82:1241-48. (PMID: 18485328) 
9. Zhu H-J, Markowitz JS. Activation of the antiviral prodrug oseltamivir is impaired by two 
newly identified carboxylesterase 1 variants. Drug Metab Dispos 2009;37:264-7. 


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10. Gaston TE, Bebin EM, Cutter GR, Liu Y3, Szaflarski JP. Interactions between cannabidiol 
and commonly used antiepileptic drugs. Epilepsia. 2017 Sep;58(9):1586-1592. 
11. Zhu HJ, Patrick KS,  Markowitz JS. Enantiospecific determination of dl-methylphenidate 
and dl-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: 
Application to human ethanol interactions. J Chromatogr B 2011;879:783-88. (PMID: 
21931492) 
12. Morrison G, Crockett J, Blakey G, Sommerville K. A Phase 1, open-label, pharmacokinetic 
trial to investigate possible drug-drug interactions between clobazam, stiripentol, or 
valproate and cannabidiol in healthy subjects. Clin Pharmacol Drug Dev. 2019 Feb 21. 
doi: 10.1002/cpdd.665. 
13. Taylor L, Gidal B, Blakey G, Tayo B, Morrison G. A phase I, randomized, double‑blind, 
placebo‑controlled, single ascending dose, multiple dose, and food effect trial of the 
safety, tolerability and pharmacokinetics of highly purified cannabidiol in healthy 
subjects. CNS Drugs 2018;32:1053–1067 
14. Markowitz JS, Logan BK, Diamond F, Patrick KS. Detection of the novel metabolite 
ethylphenidate following methylphenidate overdose with alcohol co-ingestion. J Clin 
Psychopharmacol 1999;19:362-6. 
14. DeVane CL, Markowitz JS, Carson SW, Boulton DW, Gill HS, Nahas Z, Risch SC. Single 
dose methylphenidate pharmacokinetics in CYP 2D6 extensive and poor metabolizers. J 
Clin Psychopharmacol 2000;20:347-9 
15. Markowitz JS, DeVane CL, Boulton DW, Nahas Z, Risch SC, Diamond F, Patrick KS. 
Ethylphenidate formation in human subjects after the administration of a single dose of 
methylphenidate and alcohol. Drug Metab Dispos 2000;28:620-24. 
16. Markowitz JS, Straughn AB, Patrick KS, DeVane CL, Pestreich L, Lee J, Wang Y, Muniz R. 
Pharmacokinetics of methylphenidate after oral administration of two modified-release 
formulations in healthy adults. Clin Pharmacokinet 2003;42:393-401. 
17. Zhu HJ, Patrick KS, Straughn AB, Reeves OT, Bernstein H, Shi, H, Johnson HJ, Knight, JM, 
Smith AT, Malcolm RJ, Markowitz JS. Ethanol interactions with dexmethylphenidate and 
dl-methylphenidate spheroidal oral drug absorption systems in healthy volunteers. J Clin 
Psychopharmacol 2017;37:419-428 
18. Gurley BJ, Markowitz JS, Williams DK, Barone GW. Practical considerations when 
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J Pharmacokinetics 2017;2(1):57-69 
 
 
 
 
 
 
 


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APPENDIX 
 
Laboratory Studies for Health Screening 
Comprehensive Metabolic Panel 
Glucose 
Creatinine 
BUN 
AST 
ALT 
Alkaline phosphatase 
Total bilirubin  
Sodium 
Potassium 
Chloride  
Calcium 
Total protein 
Albumin 
CBC with Differential 
 
Urine THC Screen