Quantification of Estradiol's Impact on Nucleotides in Different Cellular Populations of the Lower Gastrointestinal Tract
Phase: PHASE1
Conditions: HIV/AIDS
Sponsor: University of North Carolina, Chapel Hill

STUDY PROTOCOL 
 
Quantification of Estradiol’s Impact on Nucleotides in Cellular 
Populations of the Lower GI Tract 
 
 
NCT number NCT03917420 
Document Date 25 February 2019 
 

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Quantification of Estradiol's Impact on Nucleotides in Different Cellular 
Populations of the Lower Gastrointestinal Tract  
 
Protocol Number IRB # 19-0333 
 
Principal Investigator: 
Mackenzie Cottrell, PharmD, MS 
 
 
 
 
 
Funded by: 
National Institute of Allergy and Infectious Disease 
 

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Table of Contents 
1.  List of Abbreviations and Acronyms ............................................................................. 4 
2.  List of Figures and Tables.............................................................................................. 7 
3.  Terminology for Study Drug ......................................................................................... 8 
4.  Protocol Team Roster .................................................................................................... 9 
5.  Protocol Summary ....................................................................................................... 10 
6.  Overview of Study Design and Allocation Scheme .................................................... 11 
A) Allocation and Tissue Sampling Scheme: ............................................................... 11 
B) Study Procedure Time and Event: ........................................................................... 12 
7.  Key Roles ..................................................................................................................... 13 
7.1 
Protocol identification ........................................................................................ 13 
7.2 
Clinical Laboratories .......................................................................................... 13 
7.3 
Data Management Center ................................................................................... 14 
7.4 
Study Operations ................................................................................................ 16 
8.  Scientific Rationale ...................................................................................................... 16 
9.  Study Objectives .......................................................................................................... 18 
10.  Study Design .............................................................................................................. 18 
10.1 
Identification of Study Design ........................................................................ 18 
10.2 
Description of Study Population..................................................................... 19 
10.3 
Clinical Trial Experience ................................................................................ 19 
10.4 
Study Sampling............................................................................................... 19 
10.5 
Primary Endpoints: ......................................................................................... 20 
10.6 
Secondary Endpoints: ..................................................................................... 20 
10.7 
Time to Complete Accrual ............................................................................. 20 
10.8 
Expected Duration of Participation ................................................................ 21 
10.9 
Clinical Study Site .......................................................................................... 21 
11.  Study Population ........................................................................................................ 21 
11.1 
Recruitment .................................................................................................... 21 
11.2 
Subject Withdrawal ........................................................................................ 22 
11.3 
Inclusion Criteria ............................................................................................ 22 
11.4 
Exclusion Criteria ........................................................................................... 23 
11.5 
Co-enrollment Guidelines............................................................................... 24 
12.  Study Product ............................................................................................................. 25 
12.1 
Study Product Description .............................................................................. 25 
12.2 
Administration ................................................................................................ 25 
12.3 
Supply and Accountability ............................................................................. 25 
12.4 
Dietary Restrictions and Prohibited Medications and Practices ..................... 25 
12.5 
Toxicity Management ..................................................................................... 26 
12.6 
Adverse Effects Associated with Use for Treatment of HIV Infection .......... 26 
13. Study Procedures ........................................................................................................ 27 
13.1 
Pre-screening .................................................................................................. 27 
13.2 
Screening Visit ............................................................................................... 27 
13.3 
Enrollment Visit ............................................................................................. 28 
13.4 
Biopsy Visit 1 and 2 ....................................................................................... 29 
13.5 
Follow-up Visit ............................................................................................... 30 
13.6 
Behavioral Assessment ................................................................................... 31 

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13.7 
Clinical Evaluations and Procedures .............................................................. 31 
14.  Assessment of Safety ................................................................................................. 31 
14.1 
Safety Monitoring ........................................................................................... 31 
14.2 
Clinical Data and Safety Review .................................................................... 31 
14.3 
Adverse Events Definition and Reporting Requirements ............................... 32 
14.4 
Serious Adverse Events .................................................................................. 33 
14.5 
Adverse Event Relationships .......................................................................... 34 
14.6 
Grading Severity of Events............................................................................. 34 
14.7 
Pregnancy and Pregnancy Outcomes ............................................................. 34 
14.8 
Social Harms Reporting.................................................................................. 36 
15.  Clinical Management ................................................................................................. 36 
15.1 
Dose Modification Instructions ...................................................................... 38 
15.2 
Discontinuation of Study Product in the Presence of Toxicity ...................... 38 
15.3 
Pregnancy ....................................................................................................... 38 
15.4 
Criteria for Early Termination of Study Participation .................................... 39 
16.  Statistical Consideration ............................................................................................ 39 
16.1  
Statistical analysis........................................................................................... 39 
16.2 
Sample Size and Power Calculations ............................................................. 39 
16.3 
Participant Accrual, Follow-up and Retention ............................................... 40 
17.  Data and Safety Monitoring and Analysis ................................................................. 40 
17.1 
Clinical Data Monitoring ................................................................................ 40 
17.2 
Sample and Data Analysis .............................................................................. 40 
18.  Data Handling and Record Keeping .......................................................................... 41 
18.1 
Data Management Responsibilities ................................................................ 41 
18.2 
Quality Control and Quality Assurance .......................................................... 41 
18.3 
Study Coordination ......................................................................................... 41 
19.  Clinical Site Monitoring ............................................................................................ 42 
19.1 
Human Participants Protection ....................................................................... 42 
19.2 
Institutional Review Boards ........................................................................... 42 
19.3 
Risk Benefits Statement.................................................................................. 43 
19.4 
Risks ............................................................................................................... 43 
19.5 
Benefits ........................................................................................................... 45 
19.6 
Informed Consent Process .............................................................................. 45 
19.7 
Participation Confidentiality ........................................................................... 46 
19.8 
Special Populations......................................................................................... 47 
19.9 
Pregnant Women ............................................................................................ 47 
19.10 
Children .......................................................................................................... 47 
19.11 
Compensation ................................................................................................. 48 
19.12 
Communicable Disease Reporting ................................................................. 48 
19.13 
Access to HIV-related Care ............................................................................ 48 
19.14 
HIV Counseling and Testing .......................................................................... 48 
19.15 
Care for Participants Identified as HIV-infected ............................................ 49 
19.16 
Study Discontinuation .................................................................................... 49 
20.  Publication Policy ...................................................................................................... 49 
21.  References .................................................................................................................. 49 
 

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1.  List of Abbreviations and Acronyms 
ACTU 
AIDS Clinical Trials Unit 
AE 
adverse event 
AIDS 
Acquired Immunodeficiency Syndrome 
aPTT 
activated partial thromboplastin time 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
AUC 
area under the time vs. concentration curve 
BP 
blood pressure 
CBC 
complete blood count 
CPAC 
Clinical Pharmacology Analytical Chemistry 
CPT 
cell preparation tube 
CRF 
case report form 
CT 
Chlamydia (Chlamydia trachomatis) 
CTRC 
Clinical and Translational Research Center 
DAIDS 
Division of AIDS 
DBS 
dried blood spots 
DNA 
deoxyribonucleic acid 
EAE 
expedited adverse event (reporting)  
EC 
ethics committee 
eCcr 
estimated calculated creatinine clearance 
FDA 
(United States) Food and Drug Administration 
FHT 
feminizing hormone therapy 
FTC 
emtricitabine 
FTC-TP 
emtricitabine triphosphate 
GC 
gonorrhea (Neisseria Gonorrhoeae) 
GCP 
good clinical practices 
GI 
gastrointestinal 
HepBsAg 
hepatitis B surface antigen 
Hep C Ab 
hepatitis C antibody 
HIV 
Human Immunodeficiency Virus 

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HR 
heart rate 
ICH 
International Conference on Harmonisation 
IDS 
investigational drug service 
IRB 
institutional review board 
IUD 
intrauterine device 
LLN 
lower limit of normal  
LLOQ 
lower limit of quantification 
MRN 
medical record number 
MSM 
men who have sex with men 
NC TraCS 
North Carolina Translational and Clinical Sciences Institute  
NIAID 
(United States) National Institute of Allergy and Infectious 
Diseases 
NIH 
(United States) National Institutes of Health 
NRTI 
nucleos(t)ide reverse transcriptase inhibitor 
OHRP 
Office for Human Research Protections 
PD 
pharmacodynamics 
PI 
principal investigator 
PK 
pharmacokinetics 
PrEP 
pre-exposure prophylaxis 
PSRT 
Protocol Safety Review Team 
PT 
prothrombin test 
QA 
quality assurance 
QC 
quality control 
RPR 
rapid plasma reagin test for syphilis  
RR 
respiration rate 
SAE 
serious adverse event 
SOP 
standard operating procedures 
SST 
serum separator tube 
STI 
sexually transmitted infection 
TDF 
tenofovir disoproxil fumarate 

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TDF/FTC 
Truvada® (tenofovir disoproxil fumarate and emtricitabine in 
fixed dose combination)  
TFV 
tenofovir 
TFV-DP 
tenofovir diphosphate 
TGW 
transgender women 
TraCS 
The North Carolina Translational and Clinical Sciences Institute 
ULN 
upper limit of normal 
UNC 
University of North Carolina 

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2.  List of Figures and Tables  
 
Figure 1. Sampling Schema Pictorial................................................................................ 11 
Figure 2. Theoretical Framework ..................................................................................... 16 
Figure 3. Truvada® Identification ..................................................................................... 25 
Figure 4. Power Determination ......................................................................................... 39 
 
Table 1 Sample collection and bioanalytical measurement 
20 
 
 

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3.  Terminology for Study Drug 
 
Abbreviation 
Compound 
name 
Comments 
TDF 
Tenofovir 
disoproxil 
fumarate 
This is the inactive, oral formulation of tenofovir 
(trade name: Viread). The ester form enhances oral 
absorption and bioavailability. TDF is rapidly 
metabolized after dosing to the de-esterified pro-drug, 
tenofovir (TFV), which is also inactive. 
TFV 
Tenofovir 
This is the inactive, de-esterified form of TDF. This is 
the form of the drug that is measured in serum, blood, 
other body fluids, and tissue samples. 
TFV-DP 
Tenofovir 
diphosphate 
This is the active, phosphorylated form of tenofovir 
that is generated in cells. This is the form of the drug 
that is measured in cells (e.g., PBMCs). It is rapidly 
dephosphorylated to the inactive form outside of cells, 
and has a very short half-life outside of cells in tissue. 
FTC 
Emtricitabine 
This antiretroviral drug is co-formulated with TDF in 
Truvada® (TDF/FTC). FTC is an inactive pro-drug 
that is activated in cells by phosphorylation. This is the 
form of emtricitabine that is measured in serum, blood, 
other body fluids, and tissue samples. 
FTC-TP 
Emtricitabine 
triphosphate 
This is the active, phosphoylated form of FTC that is 
generated in cells. This is the form of the drug that is 
measured in cells (e.g., PBMCs). 
Truvada® 
Tenofovir plus 
Emtricitabine 
This is the co-formulated drug produced by Gilead 
Sciences, Inc. Each pill contains 300 mg of TDF and 
200 mg of FTC. 

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4.  Protocol Team Roster 
Principle investigator: 
Mackenzie Cottrell, PharmD., MS 
Division of Pharmacotherapy and Experimental Therapeutics  
UNC Eshelman School of Pharmacy 
1092 Genetic Medicine Building, CB# 7361 
120 Mason Farm Road 
Chapel Hill NC, 27599-7569, USA  
Email: mlcottre@email.unc.edu  
Phone: 919-843-0321 
Fax: 919-962-1020 
Study Medical Officer:  
Cynthia Gay, MD, MPH 
Division of Infectious Diseases  
CB# 7030, 2112 Bioinformatics Building  
130 Mason Farm Road, 2nd Floor  
Chapel Hill, NC, 27599-7030, USA  
Email: cynthia_gay@med.unc.edu 
Phone: 919-843-2726  
Fax: 919-966-8928  
Study Physician Assistant/Project Manager:  
Heather Asher Prince, MPA, PA-C, DFAAPA 
Division of Infectious Diseases  
1092 Genetic Medicine Building, CB 7361  
120 Mason Farm Road 
Chapel Hill, NC 27599, USA  
Email: princeh@med.unc.edu  
Phone: 919-962-5344 
Fax: 919-966-1020 

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5.  Protocol Summary  
Protocol Chair: 
Mackenzie Cottrell, PharmD, MS 
 
Sample Size: 
10 cisgender females  
Study Population:  
Premenopausal healthy volunteer cisgender women between 
18-49 years of age with an intact gastrointestinal tract 
Study Site(s): 
NC TraCS Clinical Translational Research Center at the 
University of North Carolina, Chapel Hill, NC USA 
Study Design: 
Single-center, open-label PK sampling study 
Study Duration: 
Women will be enrolled in the study within 42 days of 
screening, depending on the timing of their menstrual cycle, 
and then will be on active study for two weeks. A follow-up 
visit will occur within 14 days.   
Study Products: 
Truvada® 
Study Regimen: 
Five witnessed doses daily prior to each sampling visit 
 
Primary Objectives: 
To assess the impact of high and low in vivo estradiol 
exposure on PrEP nucleotide concentrations in different 
cellular populations of the lower GI tract  
Hypothesis:  
PrEP nucleotide concentrations in total rectal cells collected 
via cytobrush will significantly correlate with concentrations 
in CD4+ T cells isolated from rectal tissue biopsies regardless 
of in vivo estradiol exposure. 
Secondary Objectives:  
To quantify the relationship between estradiol, progesterone 
and testosterone on PrEP nucleotide concentrations in rectal 
and peripheral blood mononuclear cells 
 
To quantify the relationship between estradiol, progesterone 
and testosterone on PrEP concentrations in plasma 
 

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6.  Overview of Study Design and Allocation Scheme 
A) Allocation and Tissue Sampling Scheme: 
 
Figure 1. Sampling Schema Pictorial 
 

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B) Study Procedure Time and Event: 
 
  Time Period 
→  
Activity ↓  
Screen  
  
  
(-42 to  
0 Days)  
Enrollment 
/Dose 1 
  
  
Dosing 
Days  
  
  
  
  
Sampling 
Visit 1 
  
  
  
Dosing 
Days 
  
Sampling 
Visit 2  
  
Follow-up  
  
  
(Within 14 
days)  
Eligibility  
X  
X  
  
  
  
  
  
Informed Consent  
X  
  
  
  
  
  
  
Review of Medical History  
X  
X  
  
  
  
  
  
History of Menses  
X  
  
  
  
  
  
  
Vital Signs a  
X  
 
 
X  
 
X  
X  
Complete Physical  
Examination  
X  
  
  
  
  
  
  
Targeted Physical  
Examination  
  
  
  
X  
  
 X 
X  
Virology Tests b  
X  
  
  
  
  
  
 X 
Pregnancy Test c  
X  
X  
  
X  
 
X  
X  
STD Tests d  
X  
  
  
  
  
  
  
Urine Drug Test  
X  
  
  
  
  
  
  
Safety Labs e  
X  
X  
  
  
  
  
X  
Medication Dosing f 
  
X  
X  
  
X  
  
  
Blood Sampling   
  
 
 
X  
 
X  
  
Rectal Cell Collection by 
cytobrush  
 
 
 
X  
 
X  
  
Rectal Tissue Biopsy  
  
 
 
X  
 
X  
  
Adverse Event Assessment  
  
X  
  
X  
 
X  
X  
a. 
Vital signs include: blood pressure, pulse, respiratory rate, temperature, and weight.  
Height should be documented at the screening visit.  
b. 
Virology tests will include HIV Ag/Ab test, HbsAg, and Hepatitis C antibody at 
screening visit.  HIV testing will be repeated at follow up, as well as anytime 
infection is suspected. 
 

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c. 
Serum pregnancy test will be obtained at screening visit.  Urine pregnancy test will 
be obtained at enrollment and follow up.  Particpants will also have urine pregnancy 
test prior to sampling on assigned biopsy days.   
d. 
Participants will be tested for the following sexually transmitted diseases: syphilis, 
gonorrhea, and chlamydia.  
e. 
Safety labs include: CBC with differential, urine drug screen, and the following 
serum chemistries: Na, K, Cl, CO2, BUN, SCr, glucose, Alb, total protein, AST, 
ALT, Alkaline phosphatase, total bilirubin.  PT/INR and aPTT will be obtained at 
screening. CBC without differential at follow up. 
f. 
Member of study team will witness dose administration.    
 
7.  Key Roles 
7.1 
Protocol identification 
 
Protocol title: Quantification of Estradiol's Impact on Nucleotides in Different 
Cellular Populations of the Lower Gastrointestinal Tract 
 
 
7.2 
Clinical Laboratories 
 
McLendon Labs 
The following samples/tests will be processed at UNC Hospitals McLendon Core 
Lab: 
1. Urine drug screen 
2. Serum pregnancy test 
3. Syphilis (RPR) titer 
4. HIV diagnostic testing 
5. Hepatitis testing 
6. Safety labs 
a. CBC (with differential) 
b. Coagulation tests (PT/INR and aPTT) 
c. Serum Chemistries 
 
UNC CFAR Virology, Immunology and Microbiology (VIM) Core 

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The following samples/tests will be processed at UNC STD-CRC Core Lab: 
1. Chlamydia test 
2. Gonorrhea test 
 
Reproductive Endocrinology Laboratory 
The following analyses will be performed in a core lab directed by Dr. Frank 
Stanczyk at the Keck School of Medicine of the University of Sothern California: 
1. Estradiol, Progesterone, Testosterone serum concentrations 
 
UNC CFAR Clinical Pharmacology and Analytical Chemistry (CPAC) Core 
The following analyses will be performed at the CPAC lab: 
1. Plasma PrEP (TFV and FTC) concentrations 
2. PBMC PrEP nucleotide (TFVdp, FTCtp, dATP and dCTP) concentrations 
3. Rectal tissue and total rectal cells PrEP nucleotide (TFVdp, FTCtp, dATP and 
dCTP) concentrations 
This laboratory, directed by Angela Kashuba, was created to provide clinical 
pharmacology expertise and laboratory support to facilitate clinical and preclinical 
HIV/AIDS research. The Core has been CLIA certified since December 2004 and 
CAP certified since 2008 and participates in four proficiency testing activities per 
year. The Core has > 90% accuracy for all antiretrovirals tested, has been awarded 
certificates of excellence and is approved to perform TDM for all PIs and NNRTIs.  
Sample storage, processing, and HPLC methods are performed in approximately 6000 
square feet of space in in the UNC Eshelman School of Pharmacy Genetic Medicine 
Building.  This laboratory has validated analytical methods for all currently marketed 
antiretrovirals.   
 
7.3 
Data Management Center 
 
Data management will be maintained by the research coordinator with oversight from 
the principle investigator.  The study records will be stored in a locked filing cabinet 
in a locked office located at 1100 Genetic Medicine Building, 120 Mason Farm Road, 

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Chapel Hill, NC 27599.  Data will be transferred by the investigators to a password 
protected Microsoft Access database and stored on the UNC Secure Network 
Attached Storage (Secure Nas) server, which is administered and maintained by UNC 
IT staff, and has custom port configurations (Windows firewall) which block all 
unused ports and limit access to open ports based on IP address.  Security logging and 
auditing follow industry best practices.  Access to the server is restricted to key 
personnel including investigators, and computer support staff.  User access to server 
resources via network connection is limited via firewall configuration, IP address 
filtering, and encrypted user authentication protocols.  Access to the study data will 
be further restricted via user roles.  Application security for data entry will be 
protected using firewalling and IP address filtering.  User authentication will be 
managed by a single-sign-on model of log in. Passwords for access to server 
resources are managed in a Microsoft Active Directory repository.  Passwords for 
applications are managed via a single-sign-on (SSO) application which stores 
passwords using a one-way 128 bit hashing algorithm (MD5).  Password policies 
force users to employ “strong” passwords, which use numbers, letters and special 
characters.  Passwords must change at least every 90 days.  All data will be de-
identified and assigned a nonsense random unique identifier.   

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7.4 
Study Operations 
 
This study will be conducted in accordance with ICH/GCP, The Code of Federal 
Regulations, Office for Human Research Protections (OHRP) and FDA regulations 
and guidance, in addition to department Standard Operating Procedures   
 
The Principal Investigator (PI) accepts ultimate responsibility for the quality of the 
data, subject safety and protocol adherence for all research conducted through the 
UNC NC TraCS CTRC. She also accepts responsibility for the overall functioning of 
the site. 
 
The PI is responsible for oversight of activities and responsibilities delegated to other 
staff.  All staff are to be appropriately educated in their obligations as described 
above and trained in the areas for which they have delegated responsibility.  
 
8.  Scientific Rationale 
 
Female steroid sex hormones modulate enzymes involved in maintaining 
PrEP nucleotide balance. Currently only 
a fixed dose combination of 2 
nucleos(t)ide reverse transcriptase 
inhibitors (NRTIs), tenofovir disoproxil 
fumarate (TDF) and emtricitabine (FTC), 
is FDA approved for HIV pre-exposure 
prophylaxis (PrEP). TDF and FTC are 
intracellularly phosphorylated to their 
active forms (TFVdp and FTCtp), which 
compete with the host cells’ naturally 
occurring deoxynucleotides (dATP and 
dCTP) for incorporation into the proviral 
DNA strand to block reverse 
Figure 2 Theoretical Framework 
Tenofovir (TFV) is intracellularly phosphorylated to 
its active form (TFVdp), which is chemically similar 
to deoxyadenosine triphosphate (dATP). TFVdp 
competes for incorporation into proviral HIV DNA 
by reverse transcriptase (RT), which results in chain 
termination. Estradiol reduces TFVdp and increases 
dATP minimizing the likelihood that RT will 
incorporate TFVdp. 

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transcription. The female sex steroid hormone, estradiol, increases expression of 
5’nucleotidase enzymes involved in maintaining intracellular nucleotide balance.1 
Particularly, ecto-5’nucleotidase (located on the extracellular surface of the plasma 
membrane) catalyzes AMP to ADP, which is a precursor in the biochemical 
formation of dATP. Conversely, intracellularly expressed cytosolic 5’nucleotidase 
may dephosphorylate and deactivate PrEP’s active metabolites (TFVdp and FTCtp; 
Figure 2).  
 
Female sex steroid hormones may disproportionately impact PrEP 
pharmacology in different cellular populations. Previously, investigators have 
utilized several different sampling strategies from which to draw conclusions about 
PrEP pharmacology in HIV transmission sites. These surrogate measures include 1) 
collecting biopsies for measurement in isolated CD4+ T cells – generally considered 
gold standard; 2) tissue homogenization for measurement in mixed cell populations; 
3) collecting swabs and cytobrush for measurement in fluid or mixed cell populations. 
A previous clinical study noted a predictive relationship between parent TFV 
measured in rectal fluids versus tissue homogenates in individuals taking oral TDF.2 
Additionally, Yang et al. demonstrated a predictive correlation between TFVdp in 
mixed cell populations of homogenized rectal tissue vs CD4+ T cells isolated from 
rectal tissue following topical application of TFV gel.3 Yet the impact of estradiol on 
these surrogate relationships has not been well characterized. One in vitro study 
utilizing very high concentrations of TFV (1mg/ml) suggests that estradiol could 
disproportionately impact TFVdp formation in epithelial cells (enhanced formation) 
vs CD4+ T cells (diminished formation)4, which could confound the relationship 
between surrogate measures of mixed vs isolated cell populations. 
 
We aim to quantify estradiol’s impact on PrEP nucleotide concentrations 
in mixed cell populations obtained via cytobrush sampling vs CD4+ T cells 
isolated from rectal tissue biopsies in order to understand whether cytobrush is a 
valid PrEP pharmacology surrogate in people taking exogenous estradiol. 
 

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9.  Study Objectives  
9.1 
Primary Objective:  
To assess the impact of high and low in vivo estradiol exposure on PrEP 
nucleotide concentrations in different cellular populations of the lower GI 
tract 
9.2 
Secondary Objective(s):  
To quantify the relationship between estradiol, progesterone and testosterone 
on PrEP nucleotide concentrations in rectal and peripheral blood mononuclear 
cells 
To quantify the relationship between estradiol, progesterone and testosterone 
on PrEP concentrations in plasma 
 
10.  Study Design 
 
10.1 
Identification of Study Design 
 
This is a single center, open-label, multi-matrix sampling study of PrEP nucleotides 
in mucosal tissue concentrations measured after multiple doses of Truvada® 
(TDF/FTC). Participants will take a single daily dose of study drug for five days 
before each sampling visit. The visits will be scheduled during the early follicular 
phase of the menstrual cycle (approximately Days 2-5 after the first day of menses, 
Visit 1) when estradiol is predicted to be the lowest; and the late follicular phase 
(approximately Days 12-15 after the first day of the menses, Visit 2) when estradiol is 
predicted to be highest.  After participant education, informed consent, and screening 
for study eligibility, participants will be evaluated at baseline. 
 

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10.2 
Description of Study Population 
 
Healthy, HIV-uninfected premenopausal women (N=10) will be enrolled based on the 
following inclusion criteria: ≥18 years of age, no clinical evidence of sexually 
transmitted infections (STIs), unimpaired renal function (>60ml/min; as estimated by 
Cockcroft-Gault), a regular menstrual cycle (defined as at least 1 day of menses 
occurring every 21-35 days), being willing to refrain from inserting anything rectally 
for 3 days prior and 7 days post biopsy visits. We have carefully considered 
allowance of different forms of contraception in this study and their impact on 
endogenous hormonal fluctuation (See section 14.7 for details).  
 
10.3 
Clinical Trial Experience  
 
The UNC Chapel Hill Center for AIDS Research has considerable prior experience 
with the conduct of cervical, vaginal, and gastrointestinal biopsy studies in women.  
We recently enrolled 48 women in a phase I dose ranging study PK study 
(NCT01330199, clinicaltrials.gov) requiring all three types of biopsies to be 
performed.  Prior to this study, UNC enrolled the majority of women in HPTN 066 
(NCT01276600, clinicaltrials.gov) which required all three types of biopsies to be 
performed within 60 minutes of each other.  In both cases, these biopsies were 
performed successfully and UNC enrolled to completion.  Approximately 10 women 
who participated in the aforementioned study (NCT01330199), have expressed 
interest in participating in additional studies of the similar nature.  As part of the 
screening and informed consent process, every participant will receive a detailed 
description of the procedures involved and the intensity of the protocol. 
 
10.4 
Study Sampling  
 
At both sampling visits, the following samples will be collected in all participants: 
1. Blood plasma  
2. Serum 

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3. PBMCs  
4. Rectal tissue biopsies 
5. Rectal cell collection by cytobrush 
 
10.5 
Primary Endpoints:  
TFVdp, FTCtp, dATP and dCTP concentrations in mixed rectal cells collected 
via cytobrush vs CD4+ T cells isolated from rectal tissue biopsies during the 
early (low estradiol) and late (high estradiol) follicular phases of the menstrual 
cycle 
 
10.6 
Secondary Endpoints:  
1. Estradiol, progesterone and testosterone concentrations in serum 
2. TFVdp, FTCtp, dATP and dCTP concentrations in peripheral blood 
mononuclear cells 
3. TFV and FTC concentrations in plasma 
 
Table 1 Sample collection and bioanalytical measurement 
Matched Visit 1 and 2 Measurements 
Collection Matrix  
Bioanalytical assay 
PBMCs 
TFVdp FTCtp dATP dCTP 
Total rectal cells 
TFVdp FTCtp dATP dCTP 
Rectal CD4 cells 
TFVdp FTCtp dATP dCTP 
Plasma 
TFV FTC 
Serum 
Estradiol progesterone testosterone 
TFVdp=tenofovir diphosphate; FTCtp=emtricitabine triphosphate; dATP=deoxyadenosine 
triphosphate; dCTP=deoxycytidine triphosphate; TFV=tenofovir; FTC=emtricitabine 
 
10.7 
Time to Complete Accrual  
 
Based on our prior experience with several other pharmacokinetic studies that 
collected rectal tissue and samples from other matrices, we anticipate an accrual rate 
of two participants per month for a total recruitment time of 5 months. 
 

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10.8 
Expected Duration of Participation 
 
Once enrolled in the study, participants will participate in the study for approximately 
2 weeks and then have a follow up visit approximately 1-14 days after the last sample 
is collected.  No study data will be collected after the follow-up visit unless the 
participant has an adverse event (AE), which will be followed until resolution.   
 
10.9 
Clinical Study Site 
 
The NC TraCs Clinical Translational Research Center at the University of North 
Carolina, Chapel Hill, NC USA 
 
11.  Study Population 
 
11.1 
Recruitment 
 
Our study participants will be recruited from the Chapel Hill-Raleigh-Durham area 
(an area with a population of approximately 3 million).  We will use the following 
recruitment strategies:   
1. Listing our study with NC TraCS’s UNC Clinical Trials Searchable Database 
and on the Research Match Searchable Database 
2. Sending IRB approved emails to the UNC-CH campus listserv 
3. Posting IRB approved fliers across campus 
4. Contacting a list of subjects who have expressed interest in any of our 
previous healthy volunteer studies 
 
Potential participants will be given an email address or a phone number to call if they 
are interested, and the study coordinator conducts a brief initial IRB-approved 
screening interview over the phone in a private area to determine further eligibility.  
If the subject is potentially eligible, a screening visit is scheduled; if one or more of 

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the basic inclusion criteria are not met, the subject is informed that they are not 
eligible.   
 
11.2 
Subject Withdrawal 
 
There is a low chance of participant withdrawal from the study due to the relatively 
short study period.  If a participant does withdraw before completion of all biopsies 
and samples, it will be necessary to replace them in order to have a complete set of 
data.  If a subject completes all biopsies but misses more than 1 outpatient dose it will 
be necessary to replace the subject in order to have a complete set of data.  If a 
subject is unable to provide all of the serum samples because of poor vascular access 
or other issues it will be necessary to replace the subject in order to have a complete 
set of data.  
 
11.3 
Inclusion Criteria 
 
1. Healthy cisgender pre-menopausal female participants between the ages of 18 
and 49 years, inclusive on the date of screening (Healthy is defined as no 
irregular menstrual cycles or clinically relevant abnormalities identified by a 
detailed medical history, full physical examination, including blood pressure 
and pulse rate measurement, and clinical laboratory tests.  
2. Regular menstrual cycles defined as at least 1 day of menses occurring every 
21-35 days) 
3. Estimated calculated creatinine clearance (eCcr) of at least 60 mL/min by the 
Cockcroft-Gault formula where: eCcr (female) in mL/min = [(140 - age in 
years) x (weight in kg) x 0.85] / (72x serum creatinine in mg/dL). 
4. Negative serum pregnancy test at screening  
5. All participants should be using at least one of the following methods of 
contraception* from the screening visit through 72 hours prior to inpatient 
admission (at which time the women will be asked to remain abstinent until 
after their follow-up visit): 

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a. Non continuous systemic hormonal contraceptives that permit 
intermittent menstruation 
b. IUD (non-hormonal) placed at least 1 month prior to study enrollment 
c. Bilateral tubal ligation (Sterilization)  
d. Vasectomized male partners 
e. Condom + Spermicide 
f. *Unless engaged in sexual activity with female only sex partners or 
abstinent for at least 3 months prior with no intention of becoming 
sexually active during the study period.  Any history of recent or 
present concomitant male sex partners will be addressed and ruled out 
in the context of screening participants for eligibility for the protocol 
6. Evidence of a personally signed and dated informed consent document 
indicating that the subject has been informed of all pertinent aspects of the 
trial. 
7. Willing and able to comply with scheduled visits, treatment plan, laboratory 
tests, and other trial procedures. 
8. Subject must be willing to abstain from sexual intercourse, and all and 
intrarectal objects and products for at least 72 hours prior to Sampling #1 until 
study completion.  
9. Subject must be HIV-1 and Hepatitis B and C negative as documented on 
screening labs.  
10. Subject must not be actively involved in the conception process and must be 
non-lactating. 
11. Subject must be able to swallow pills and have no allergies to any component 
of the study product 
 
11.4 
Exclusion Criteria 
 
1. Evidence or history of clinically significant hematological, renal, endocrine, 
pulmonary, gastrointestinal, cardiovascular, hepatic, psychiatric, neurologic, 

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or allergic disease (including documented drug allergies, but excluding 
untreated, asymptomatic, seasonal allergies at time of dosing). 
2. Participants with a history of hysterectomy 
3. Participants who are pregnant, possibly pregnant or lactating 
4. History of febrile illness within five days prior to first dose. 
5. Any condition possibly affecting drug absorption (eg, gastrectomy or other 
significant alterations of the gastrointestinal tract) 
6. A positive urine drug screen. 
7. An untreated-positive test for syphilis, gonorrhea, or Chlamydia at screening. 
8. Any clinically relevant laboratory chemistry or hematology result Grade 2 or 
greater according to the DAIDS Laboratory Grading Tables  
9. Treatment with an investigational drug within 4 months preceding the first 
dose of study product.   
10. History of regular alcohol consumption exceeding 14 drinks (1 drink = 5 
ounces (150 mL) of wine or 12 ounces (360 mL) of beer or 1.5 ounces (45 
mL) of spirits) per week. 
11. Participation in a clinical trial involving rectal biopsies within 6 months 
preceding the first dose of trial medication. 
12. Blood donation of approximately 1 pint (500 mL) within 56 days prior to 
dosing.  
13. Any condition which, in the opinion of the investigator, is likely to interfere 
with follow-up or ability to take the study medication appropriately. 
14. Unwilling or unable to comply with the dietary and concomitant drug 
restrictions in regard to study drug administration as outlined in the study 
procedures and prohibited medications sections. 
15. Women utilizing continuous hormonal contraception options such as 
Seasonique, injectables, implants, and hormonal IUDs  
 
11.5 
Co-enrollment Guidelines 
  

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Participants cannot have participated in a clinical trial in which they received 
treatment with an investigational drug within 4 months preceding the first dose of 
trial medication or have participated in a clinical trial involving rectal biopsies within 
6 months preceding the first dose of trial medication. 
 
12.  Study Product  
 
12.1 
Study Product Description  
 
Truvada® (Figure 3) is an FDA-approved pill that contains two different HIV 
medicines combined in one pill: emtricitabine and tenofovir disoproxil fumarate.  
Both medicines belong to the group of HIV drugs called nucleoside reverse 
transcriptase inhibitors (NRTIs) made by Gilead, Inc. 
 
 
12.2 
Administration 
 
The participant will take a single oral daily dose of Truvada for the five days before 
Sampling Visit #1 and Sampling Visit #2 as scheduled.  Doses can be given in 
person, or through video conferencing programs, such as SKYPE. A study team 
member will witness all doses and record the times of administration. The drug name 
and dose will be confirmed with visual inspection during a time-out procedure prior 
to dose administration.   
 
 
12.3 
Supply and Accountability 
 
The medications will be purchased and dispensed by the Investigational Drug Service 
at UNC Hospitals.   
 
12.4 
Dietary Restrictions and Prohibited Medications and Practices 
Figure 3 Truvada® Identification 

25February2019 v1.0 
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1. Participants must comply with a low fiber diet for 3 days prior to all rectal 
biopsies.  
2. Participants must consume only clear liquids 12 hours prior to all rectal 
biopsies, refraining from anything containing red or purple dyes.  
 
12.5 
Toxicity Management 
 
Laboratory tests will be taken before study drug administration to look for baseline 
abnormalities and these will be compared to the laboratory tests to be taken at the 
follow-up visit after study drug exposure.  Any side effects will be managed 
symptomatically on a case by case basis.  Participants will be advised to contact study 
staff if any adverse effects occur at any time during the study period and to seek 
medical attention as they see appropriate.  A follow up call will be made by the 
research coordinator to assess for adverse events the day following any visits where a 
biopsy was performed.  
 
12.6 
Adverse Effects Associated with Use for Treatment of HIV Infection 
 
Emtricitabine 
Single dose emtricitabine safety data has not been published.  For multiple doses, 
greater than 10% of patients have shown the following adverse effects: headache, 
dizziness, tiredness, inability to sleep, unusual dreams, loose or watery stools, upset 
stomach (nausea) or vomiting, abdominal pain, rash, itching, skin darkening of the 
palms and/or soles, increased cough, runny nose. Less than 10 % of patients 
experienced abnormal liver function tests, increases in pancreatic enzyme, increased 
triglycerides, and increased creatinine phosphokinase. 
 
Tenofovir disoproxil fumarate 
A phase I single-dose study with tenofovir 300 mg showed it was generally well 
tolerated. The most frequently reported adverse events were headache (19%) and 

25February2019 v1.0 
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dizziness (13%). Flatulence (7%), hot flush (3%), and an increase in alanine 
aminotransferase (3%) were also reported.  
 
13. Study Procedures 
 
Study specific procedures are presented in table format in the Schedule of Events.  
All patients will attend an outpatient screening visit, an enrollment visit, two 
outpatient biopsy visits, and a follow-up visit. Dosing visits will occur in person or 
through videoconferencing. All on-site visits will be conducted at the UNC NC 
TraCS Clinical Translational Research Center.  During the outpatient biopsy visits 
tissue will be collected by insertion of an anoscope for the rectal biopsies and rectal 
cells.  We currently have a list of approximately 10 women who have expressed 
interest in participating in this study after having participated in a previous 
pharmacokinetic study where biopsies of this nature were collected. As part of the 
screening and informed consent process, every participant will receive a detailed 
description of the procedures involved and the intensity of the protocol. 
 
13.1 
Pre-screening 
 
Persons interested in participating will be pre-screened by telephone interview using a 
standardized IRB-approved questionnaire to assess general eligibility prior to 
scheduling a screening visit.  Participants may be provided a copy of the informed 
consent prior to the screening visit, if requested. 
 
13.2 
Screening Visit 
 
After providing written informed consent, potential participants may be screened for 
eligibility.  
The screening visit will consist of the following activities: 
1. Review study overview and obtain written informed consent 

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2. Review approved contraception options to be used during the entire study 
period 
3. Assign Participant ID 
4. Collect demographic information 
5. Collect medical/menstrual history and review contraception history  
6. Perform physical exam 
7. Vitals (blood pressure, heart rate, respiratory rate, height, weight (Safety vitals 
obtained on arrival to research clinic by nursing staff may be used) 
8. Obtain safety labs: 
a. CBC with differential (includes hemoglobin, hematocrit, WBC and 
differential count, platelets) 
b. Coagulation studies (PT/INR and aPTT) 
c. Serum Chemistry Panel (Sodium, potassium, chloride, bicarbonate, 
blood urea nitrogen, creatinine, serum glucose, total protein, albumin, 
aspartate aminotransferase, alanine aminotransferase, alkaline 
phosphatase, total bilirubin) 
9. Serum Pregnancy Test  
10. Urine Drug Test (amphetamine, barbiturate, benzodiazepine, cannabinoid 
cocaine, methadone, opiates)  
11. Virology tests (HIV antibody combo, HBsAg, Hep C Ab) 
12. STI tests (syphilis, gonorrhea, chlamydia)  
 
13.3 
Enrollment Visit 
 
Within 42 days of the screening visit, all participants will present on their scheduled 
date for their enrollment visit.  A repeat urine pregnancy test will be performed.  If 
participant still meets eligibility criteria, the first dose of study drug will be 
administered by study staff, and they will be provided with 4 more doses to take 
home. 
 

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13.4 
Biopsy Visit 1 and 2 
 
Patients will be admitted to the Center for Translational and Clinical Research 
(CTRC) at University of North Carolina Chapel Hill Hospital at approximately Days 
2-5 and 12-15 of their menstrual cycle, after 5 days of dosing study product. 
 
This visit will consist of the following activities: 
1. Targeted physical examination, as indicated 
2. Vitals (BP, HR, RR, weight) 
3. Urine Pregnancy test 
4. Blood draw  
a. Blood to be collected in one 3 mL K2 EDTA (purple top tube) and 
stored on ice for no more than 1 hour prior to processing blood plasma 
by centrifuging at 1700rcf (converts to 2800rpm) for 10 minutes at 
4°C. Blood plasma will be aliquoted into an appropriately labeled 
cryovial and temporarily stored at -20°C until transferred to permanent 
storage at -80°C in the CPAC laboratory.   
b. Blood to be collected in one 8 mL Cell Preparation tube (CPT) with 
Sodium Citrate and stored upright at room temperature for no more 
than 4 hours before transferring to the CPAC laboratory and initiating 
processing procedures for PBMCs as according to CPAC SOP0345.  
c. Blood to be collected into one 6mL Serum Separator Tube (SST) and 
processed to serum by allowing to coagulate for 30 minutes at room 
temperature then centrifuging at 1700rcf (converts to 2800rpm) for 10 
minutes at 4° C. Serum will be aliquoted into an appropriately labeled 
cryovial and temporarily stored at -20°C until transferred to permanent 
storage at -80°C in the CPAC laboratory.   
5. Tissue samples will be obtained: 
a. Digital exam will be performed prior to insertion of the anoscope.  
KY-jelly lubricant will be used for digital exam and anoscope 
insertion, but no anesthetic will be applied. Rectal cells will first be 

25February2019 v1.0 
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collected with a cytobrush, placed immediately into a Falcon tube, and 
transported fresh back to the CPAC lab on ice. 10 biopsies will be 
collected from the rectal mucosa using 2.4mm radial jaw, single use, 
biopsy forceps by a board-certified gastroenterologist.  Tissue will be 
transferred to two appropriately labeled, cryovials with 5 pieces of 
tissue in each cryovial.  The samples will be transported fresh back to 
the CPAC lab on ice. 
6. Adverse event assessment using a standard adverse events questionnaire will 
be conducted after the last sample is collected, prior to discharge to home.  
7. Discharge to home 
 
The patient will be offered a meal prior to discharge. 
During Visit 1, the participant will be given a pill bottle containing the 5 doses of 
study product that they will take for the five days prior to Biopsy Visit 2, and dosing 
appointments will be scheduled. 
 
The day following biopsies, each subject will be contacted in person/phone by a study 
team member to assess for adverse events.  Additional study visits will be scheduled 
for any subject experiencing an adverse event. 
 
13.5 
Follow-up Visit 
 
The follow up visit will consist of the following activities: 
1. Targeted physical examination, as indicated 
2. Vitals (BP, HR, RR, Temp and weight) 
3. Safety labs: 
a. CBC  
b. Serum Chemistry Panel (Sodium, potassium, chloride, bicarbonate, 
blood urea nitrogen, creatinine, glucose, total protein, albumin, 
aspartate aminotransferase, alanine aminotransferase, alkaline 
phosphatase, total bilirubin) 

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c. HIV test 
4. Urine Pregnancy test 
 
13.6 
Behavioral Assessment 
 
Adherence to behavior guidelines, approved contraception and medications taken will 
be assessed prior to the biopsies by a series of questions to be asked prior to 
proceeding.  Women who do not have sex that could result in pregnancy, will only be 
tested at screening for pregnancy.   
 
13.7 
Clinical Evaluations and Procedures 
 
The extent of examination for the brief physical exam will be determined by clinical 
judgment based on the interim medical history. 
 
 
14.  Assessment of Safety 
 
14.1 
Safety Monitoring 
 
Adverse events will be monitored throughout the study.  Adverse events will be 
assessed formally before discharge to home at the inpatient visit, after each outpatient 
visit and at the follow-up visit during which participants will answer standardized 
questions regarding to prompt reporting of adverse events associated with the study 
medication and procedures.  The research coordinator will contact participants the 
day following all biopsy procedures. 
 
14.2 
Clinical Data and Safety Review 
 
A multi-tiered safety review process will be followed for the duration of this study.  
The study site investigators are the first layer of this tiered system and are responsible 

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for the initial evaluation and reporting of safety information at the participant level, 
and for alerting the Protocol Safety Review Team (PSRT) if unexpected concerns 
arise.  The PSRT will consist of the following study site investigators: Cynthia Gay, 
MD (Study Physician), Heather Prince, PA-C (Study Physician Assistant), Mackenzie 
Cottrell, PharmD (Principal Investigator). 
During the trial, the PSRT will review safety reports (all AEs included, independent 
of determination of relatedness to study products).  In addition to these routine safety 
data reviews, the PSRT will convene on an ad hoc basis to make decisions regarding 
the handling of any significant safety concerns.  If necessary, experts external to the 
protocol team, representing expertise in the fields of microbicides, biostatistics, and 
medical ethics may be invited to join the PSRT safety review.  
14.3 
Adverse Events Definition and Reporting Requirements 
 
An adverse event (AE) is defined as any untoward medical occurrence in a clinical 
research participant administered an investigational product and which does not 
necessarily have a causal relationship with the investigational product.  As such, an 
AE can be an unfavorable or unintended sign (including an abnormal laboratory 
finding, for example), symptom or disease temporally associated with the use of an 
investigational product, whether or not considered related to the product.  This 
definition is applied to all the study groups and is applied to all groups beginning 
from the time of consent. The term “investigational product” for this study refers to 
all study products. Study participants will be instructed to contact the study site staff 
to report any AEs they may experience at any time between consent and follow-up.  
In the case of a life-threatening event, they will be instructed to seek immediate 
emergency care. Where feasible and medically appropriate, participants will be 
encouraged to seek evaluation where the study clinician is based, and to request that 
the clinician be contacted upon their arrival. Study staff will obtain written 
permission from the participant to obtain and use records from non-study medical 
providers to complete any missing data element on a CRF related to an adverse event.  
All participants reporting an untoward medical occurrence will be followed clinically, 

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until the occurrence resolves (returns to baseline) or stabilizes.  
The study physician will determine AE resolution or stabilization in their best clinical 
judgment.  Study site staff will document in source documents all AEs reported by or 
observed in enrolled study participants regardless of severity and presumed 
relationship to study product. Study staff will record all AEs on case report forms.  
 
14.4 
Serious Adverse Events 
 
Serious adverse events (SAEs) will be defined by the Manual for Expedited 
Reporting of Adverse Events to DAIDS (Version 2.0, dated January 2010) as AEs 
occurring at any dose that:  
1. Results in death  
2. Is life-threatening  
3. Results in persistent or significant disability/incapacity  
4. Is a congenital anomaly/birth defect    
5. Requires inpatient hospitalization or prolongation of existing hospitalization 
6. The following are examples of hospitalization that will not be considered to be 
AEs: 
a. Protocol-specified admission (e.g. for procedure required by study 
protocol)  
b. Admission for treatment of target disease of the study, or for pre-
existing condition (unless it is a worsening or increase in frequency of 
hospital admissions as judged by the clinical investigator)  
c. Diagnostic admission (e.g. for a work-up of an existing condition such 
as persistent pretreatment lab abnormality)  
d. Administrative admission (e.g. for annual physical)  
e. Social admission (e.g. placement for lack of place to sleep)  
f. Elective admission (e.g. for elective surgery)  
 
Important medical events that may not result in death, be life-threatening, or require 

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hospitalization may be considered a serious adverse drug experience when, based 
upon appropriate medical judgment, they may jeopardize the patient or subject and 
may require medical or surgical intervention to prevent one of the outcomes listed 
above.   
Study staff will report information on all AEs and SAEs to the IRB in accordance 
with all applicable regulations and requirements.   
 
14.5 
Adverse Event Relationships 
 
The relationship of all AEs to study product will be assessed per the Manual for 
Expedited Reporting of Adverse Events to DAIDS (Version 2.0, dated January 2010). 
Per the Manual for Expedited Reporting of Adverse Events to DAIDS (Version 2.0, 
dated January 2010), the relationship categories that will be used for this study are: 
1. Related: AE and administration of study product are related in time, and a 
direct association can be demonstrated 
2. Possibly related: There is a reasonable possibility that the adverse event, 
incident, experience or outcome may have been caused by the procedures 
involved in the research.  
3. Not related: There is not a reasonable possibility that the AE is related to the 
study agent(s)  
 
14.6 
Grading Severity of Events  
 
The Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse 
Events, Version 2.1, March 2017 will be the primary tools for grading adverse events 
for this protocol.   
14.7 
Pregnancy and Pregnancy Outcomes 
 

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Participation in this study requires taking a Pregnancy Category B medication outside 
of routine clinical care; therefore only women not intending to become pregnant will 
be enrolled into this study. The duration of medication dosing is short (5 days before 
each sampling visit) and scheduled during the early phases of the menstrual cycle 
(pre-ovulation); therefore the risk of unintentional in utero drug exposure is low. 
Women will be tested at screening using a serum pregnancy test (human chorionic 
gonadotropin; hCG) and immediately prior to enrollment and each sampling visit 
using a urine pregnancy test. They will be required to use at least 1 pregnancy 
prevention strategy during their participation in the study. These may include 
abstinence for 3 months preceding; monogamy with a vasectomized cisgender male 
or a cisgender female partner; condoms with spermicide; a non-hormonal IUD; and 
hormonal contraception. Because the goal of this study is to characterize nucleotide 
concentrations within an individual during high and low estradiol exposure, only 
hormonal contraception options that do not impede cycle fluctuation will be allowed. 
Estradiol has been well characterized in women taking traditional 21/7 hormonal 
contraception regimens that allow for monthly menses. While peak estradiol 
concentrations are slightly diminished (1.6-1.8 fold) after initiating this contraceptive 
regimen, estradiol’s overall concentration vs time profile follows a predictable pattern 
similar to the pre-contraception pattern.5,6 Furthermore, peak estradiol concentrations 
are still 6-9 fold greater than trough concentrations during these contraceptive cycles. 
Therefore, we will include women utilizing a traditional 21/7 hormonal contraception 
regimen in this study. Since estradiol fluctuations over the contraceptive cycle are 
dramatically reduced by shortening the on/off interval to 23/5 days5, women utilizing 
continuous hormonal contraception options such as Seasonique, injectables, implants, 
and hormonal IUDs will be excluded from this study.  
 
Pregnancy-related data will be collected using the pregnancy CRFs for all 
pregnancies detected during the study. After the participant’s follow-up study visit, 
the study team will make every attempt to follow the participant until an outcome of 
the pregnancy can be ascertained to allow reporting of these outcomes meeting EAE 
criteria.  Pregnancy outcomes that meet criteria for EAE reporting as described above 

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(e.g., maternal complications, congenital anomalies) occurring among participants 
known to be pregnant at the Final Study Visit will continue to be expeditiously 
reported.  
 
14.8 
Social Harms Reporting 
 
Although study sites make every effort to protect participant privacy and 
confidentiality, it is possible that participants' involvement in the study could become 
known to others, and that social harms may result (i.e., because participants could 
become known as HIV-infected or at "high risk" for HIV infection).  For example, 
participants could be treated unfairly or discriminated against, or could have problems 
being accepted by their families and/or communities.  Social harms that are judged by 
the Investigator of Record to be serious or unexpected will be reported to the IRB at 
least annually, or according to their individual requirements. In the event that a 
participant reports social harm, every effort will be made by study staff to provide 
appropriate care and counseling to the participant, and/or referral to appropriate 
resources for the safety of the participant as needed.  While maintaining participant 
confidentiality, study sites may engage their CABs in exploring the social context 
surrounding instances of social harm. 
15.  Clinical Management 
 
In general, the site investigator has the discretion to discontinue study product 
use/participation at any time if they feel that continued product use would be harmful 
to the participant or interfere with treatment deemed clinically necessary.  Unless 
otherwise specified below, the investigator should immediately consult the PSRT for 
further guidance regarding permanent discontinuation.  
The site investigator or designee will document all discontinuations on applicable 
case report forms.  

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15.1 
Dose Modification Instructions 
 
Study dose will not be modified. 
 
15.2 
Discontinuation of Study Product in the Presence of Toxicity 
 
Stopping rules have been created to protect the health and safety of study participants. 
If a single subject experiences a Serious Adverse Event, enrollment will be held while 
the protocol undergoes review.  If 2 or more participants experience Grade 3 or Grade 
4 adverse events, enrollment will be held while the protocol undergoes review.   
 
Any Grade 3 or 4 or serious adverse event related to a study intervention (blood draw, 
biopsy) or the study drug will result in subject discontinuation.  Participants who are 
withdrawn for safety reasons will complete a follow-up visit within 14 days the last 
sample collection visit. 
 
15.3 
Pregnancy 
 
Pregnant and lactating women are excluded from this study.  Routine serum 
pregnancy testing is being performed at the screening visit and routine urine testing is 
being performed at other study visits as noted. If a participant becomes pregnant at 
any time during the course of the study, study treatment and interventions would be 
terminated, but she will continue with follow-up visits and safety lab assessments. 
Pregnancy-related data will be collected using the Antiretroviral Pregnancy Registry 
forms for all pregnancies detected during the study. The study team will make a very 
strong attempt to maintain contact with the pregnant participant until the outcome of 
the pregnancy is determined or it becomes clear that it will be impossible to obtain 
outcome information.  

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15.4 
Criteria for Early Termination of Study Participation 
 
Participants may voluntarily withdraw from the study for any reason at any time.  The 
site investigator also may withdraw participants from the study to protect their safety 
and/or if they are unwilling or unable to comply with required study procedures, after 
consultation with the PSRT.  Participants also may be withdrawn if the study 
sponsors, government or regulatory authorities (including the Office of Human 
Research Protections), or site IRBs terminate the study prior to its planned end date.  
Every reasonable effort is made to complete a final evaluation of participants who 
withdraw or are withdrawn from the study prior to completing follow-up.  Study staff 
members will record the reason(s) for all withdrawals in participants’ study records.  
 
16.  Statistical Consideration 
16.1  Statistical analysis.  
 
We hypothesize that PrEP nucleotide concentrations in total rectal cells obtained via 
cytobrush will significantly correlate with isolated CD4+ T cells regardless of 
estradiol exposure. To test this hypothesis TFVdp and dATP concentrations in total 
rectal cells collected by cytobrush at each visit will be compared to CD4+ T cells 
isolated from rectal biopsies using Pearson’s correlation. We expect strong 
correlations (>0.8) to exist during both visits. Linear regression will be performed for 
log10 concentrations of total rectal cells (X) vs isolated CD4+ T cells (Y) to describe 
the relationship between these sampling matrices within 
each visit. These analyses will also be performed for 
FTCtp and dCTP. 
 
16.2 
Sample Size and Power Calculations 
 
Power determination for the correlation analysis was 
carried out using the R-package pwr.7 This package 
Figure 4 Power Determination 
Sample size required to detect a 
positive correlation given various 
scenarios of r and beta values. 

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implements the power calculation methods described in Cohen, 1988.8 Assuming r is 
>0.78, a sample size of 10 women achieves 80% power (Figure 2). This assumed r 
value is more conservative than our previous observation of TFV concentration in 
rectal fluid versus tissue homogenate where r was 0.944. 
 
16.3 
Participant Accrual, Follow-up and Retention 
 
A total of 10 evaluable women will be enrolled over a period of approximately 5 
months.  Participants who complete all visits but the follow-up visit will still be 
considered evaluable.  All unevaluable participants will be replaced. 
 
 17.  Data and Safety Monitoring and Analysis 
 
17.1 
Clinical Data Monitoring 
 
The study physician will meet with the PA for screen and entry reviews.  The key 
indicators that will be assessed include informed consents, eligibility criteria, drug 
dosing, protocol required evaluations, review of subject responsibilities, and 
contraception counseling.  Additionally, laboratory results, concomitant medications, 
proper error documentation, adherence to unit specific SOPs, and CRF completion 
will be assessed.  When meeting for “on-study” reviews, the key indicators that will 
be assessed are SAE reporting, adverse event monitoring, and implementation of new 
protocol versions and amendments.  Additionally, missed visits, proper symptom 
grading, laboratory results, concomitant medications, proper error documentation, 
adherence to unit specific SOPs, and CRF completion will be reassessed.  Additional 
training on source documentation and good clinical practices will be addressed as 
needed.  
 
17.2 
Sample and Data Analysis 
 

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All blood, cells, and rectal tissue samples will be analyzed by the Clinical 
Pharmacology and Analytical Chemistry Laboratory (CPAC) at the UNC School of 
Pharmacy, which is directed by Angela Kashuba, PharmD. TFV and FTC will be 
quantified in plasma by validated LC-MS/MS methods (lower limit of quantification; 
LLOQ=1ng/ml). TFVdp, FTCtp, dATP and dCTP will be quantified in PBMCs and 
rectal cell lysates by validated LC-MS/MS methods (LLOQ=0.02ng/ml). These 
analyses will be performed at the UNC CFAR Clinical Pharmacology and Analytical 
Chemistry Core (see Kashuba letter).  
 
18.  Data Handling and Record Keeping 
 
18.1 
Data Management Responsibilities 
 
The investigator will maintain, and store securely, complete, accurate and current 
study records throughout the study.  The investigator will retain all study records for 
at least three years following the completion of the study.  Study records must be 
maintained on site for the entire period of study implementation.   
18.2 
Quality Control and Quality Assurance 
 
The quality control and assurance of clinical study data will be the responsibility of 
the study physician assistant, Heather Prince.  All clinical charts prepared by study 
staff will be reviewed by Heather Prince for quality assurance within 7 days of the 
subject visit.  Quality assurance for analytical methods and drug concentration data 
will be performed by the quality control officer Hannah Bryan.      
 
18.3 
Study Coordination 
 
The coordination of subject visits will be the responsibility of the clinical team, led by 
the study Physician Assistant.   
 

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19.  Clinical Site Monitoring 
 
All clinical and experimental activities related to this study will be conducted at the 
University of North Carolina at Chapel Hill.  There will be no enrollment outside this 
institution.  The investigators listed in the team roster have affiliations with UNC 
Eshelman School of Pharmacy and UNC School of Medicine/Center of Infectious 
Disease.  Site quality management will be the responsibility of the principal 
investigator and co-investigators, and this process will follow the guidelines and 
operating policies already established by the UNC ACTU. The Clinical 
Pharmacology and Analytical Chemistry Core (CPAC) and its Director, will provide 
oversight for laboratory personnel who are not governed by the ACTU policies.  
 
The principal investigator will hold weekly meetings to discuss clinical research and 
laboratory activities. Concerns in these matters will be addressed during these 
meetings. 
 
19.1 
Human Participants Protection 
 
The investigators will make efforts to minimize risks to participants.  Volunteers and 
study staff members will take part in a thorough informed consent process.  Before 
beginning the study, the investigators will have obtained IRB approval and the 
protocol will have been submitted to the FDA.  The investigators will permit audits 
by the FDA or any of their appointed agents.  
19.2 
Institutional Review Boards 
 
The final protocol (version 1.0) will be submitted along with the informed consent to 
the UNC IRB.  Modifications will be made to version 1.0 after initial review for final 
approval by the UNC IRB.  Subsequent to initial review and approval, the IRB will 
review the protocol at least annually.  The Investigator will make safety and progress 
reports to the IRB at least annually. These reports will include the total number of 
participants enrolled in the study, the number of participants who completed the 

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study, all changes in the research activity, and all unanticipated problems involving 
risks to human participants or others. 
 
19.3 
Risk Benefits Statement  
 
This research study is designed to gain new knowledge for society. Participants 
participating in this research will not receive any direct benefit from their 
participation. Certain risks are associated with the study procedures and are listed 
below.    
 
19.4 
Risks 
1. Blood Draws:   
Risks associated with blood draws include bleeding, discomfort, feelings of 
dizziness or faintness, and/or bruising, swelling and/or infection.   
 
2. STI Screening/Reporting:   
Disclosure of STI status may cause sadness or depression in volunteers.  
Partner notification of STI status may cause problems in their relationships 
with their sexual partners. Additionally, participants could misunderstand the 
current experimental status of the study medication and as a result increase 
their HIV risk behaviors while in the study. The following STIs are required 
by law to be reported to the NC State Department of Health if positive: HIV, 
Syphilis, gonorrhea, Chlamydia, Hepatitis B and C. 
 
3. Anoscopy with rectal biopsy and cells:  
This is a commonly practiced medical procedure and the procedures done in 
this trial will not involve any unusual risks or discomforts.  The risks 
associated with these procedures include mild discomfort and the feeling of 
having a “bloated stomach.”  Anoscopic biopsies are painless and heal quickly 
(usually within three days).  On extremely rare occasions, the procedure or 
biopsies may lead to pain, infection (sepsis), bleeding or perforation of the 

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gastrointestinal tract.  Perforation occurs approximately once out of every 
100,000 procedures.  If this extremely rare complication occurs, antibiotics 
and surgery to repair the perforation may be necessary.  
 
4. Confidentiality:   
Participation in clinical research includes the risks of loss of confidentiality 
and discomfort with personal nature of questions. Although the study site 
makes every effort to protect participant privacy and confidentiality, it is 
possible that participants’ involvement in the study could become known to 
others, and that social harms may result.  
 
5. Study Drug:  
 
Truvada® tablets are fixed-dose combination tablets containing emtricitabine 
(FTC) 200mg and tenofovir disoproxil fumarate (TDF) 300mg in each table.  
There is some experience in healthy volunteers taking Truvada®.  No new or 
unexpected side effects are observed with the fixed-dose tablet than those 
observed when each drug is given separately.  The dosing regimens will be 
consistent with package insert recommendations.  Individual daily doses of 
this magnitude were studied during development of both TDF and FTC and 
these were well tolerated.  In our previous studies using the fixed-dose tablet 
in healthy volunteer research projects, there were no serious adverse advents 
attributed to study drug. 
 
FTC.  The following side effects have been associated with the use of FTC: 
Headache, dizziness, tiredness, inability to sleep, unusual dreams, loose or 
watery stools, upset stomach (nausea) or vomiting, abdominal pain, rash, 
itching, skin darkening of the palms/soles, increased cough, runny nose, and 
abnormal liver function tests. 
 

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TDF.  The most common side effects associated with oral TDF in patients 
with HIV infection are nausea, headache, diarrhea, vomiting, flatulence, 
asthenia, abdominal distension and anorexia.  Less common side effects of 
TDF include kidney toxicities and low blood phosphate.  Other side effects 
reported in the post-marketing period include weakness, pancreatitis, 
dizziness, shortness of breath, and rash.  TDF has been associated with 
decreased bone mineral density in HIV-infected patients taking TDF tables for 
24-48 weeks.   
 
19.5 
Benefits 
 
Participants in this study will experience no direct benefit.  Participants and others 
may benefit in the future from information learned from this study.  Specifically, 
information learned in this study may lead to the development of safe and effective 
interventions to prevent HIV transmission. Additionally, participants will be referred 
for treatment for any incidental findings detected during screening and other 
examinations.    
19.6 
Informed Consent Process 
 
The investigators will obtain informed consent from each subject before starting any 
study procedures according to the standards set forth in the ICH Good Clinical 
Practice guidelines and per unit SOPs.  The process will include reviewing consent 
forms with potential participants in a confidential setting and explaining all risks and 
benefits associated with participation of the study.  This involves reading over the 
IRB-approved consent form with the subject in a private space, soliciting questions 
from the subject, allowing the subject ample time alone to review the form, soliciting 
questions again, and then offering the subject the opportunity to sign the consent 
form.  To ensure understanding, study staff may ask questions of the participants 
regarding study procedures.  The consent forms will use language that is sufficiently 
simple for lay persons to comprehend.  Participants will not be coerced into 
participating. Children under the age of 18 years, decisionally impaired adults and 

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non-English speakers will not be enrolled in this study.  Each subject will be provided 
with a photocopy of all documents that she signs.  The informed consent process will 
cover all elements of informed consent required by research regulations.  In addition, 
the process specifically will address the following topics of importance to this study: 
1. The unknown safety and unproven efficacy of the study products 
2. The need to practice safer sex behaviors regardless of study treatment group 
3. The importance of adherence to the study visit and procedures schedule 
4. The potential medical risks of study participation (and what to do if such risks 
are experienced) 
5. The potential social harms associated with study participation (and what to do 
if such harms are experienced) 
6. The real yet limited benefits of study participation  
7. The distinction between research and clinical care  
8. The right to withdraw from the study at any time  
The informed consent process will include an assessment of each potential 
participant’s understanding prior to enrollment and sequential assignment of concepts 
identified by the protocol team as essential to the informed consent decision.  
Participants who are not able to demonstrate adequate understanding of key concepts 
after exhaustive educational efforts will not be enrolled in the study.    
19.7 
Participation Confidentiality 
 
Confidentiality will be maintained by storing all specimens for current and future use 
with a unique identifying number, which will be linked to the subject’s name, social 
security number, address, telephone number, and hospital medical record (MR) 
number.  The principal investigators and study staff will be the only people with 

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access to the identifying information.  Any information provided to other people 
working on this study will be given with the study ID number, not other identifying 
information.  The records will be secured in a locked file cabinet in the locked office 
specifically designated for study record storage purposes. 
 
All electronic data for this study will be stored on a dedicated University server which 
contains extensive protections and securities. The server is housed and administered 
at the server farm at the Manning Data Center, located at 211 Manning Drive, Chapel 
Hill, NC 27599-3420.  
 
19.8 
Special Populations 
 
Children under the age of 18 years, decisionally impaired adults and non-English 
speakers will not be enrolled in this study.   
 
19.9 
Pregnant Women 
 
Women who test positive for pregnancy at the screening visit or prior to study drug 
administration at the inpatient pharmacokinetic visit, will not be eligible to participate 
in this study.  A serum pregnancy test will be performed at the screening visit and 
urine pregnancy tests at all study visits, and additionally if clinically indicated.  
During the informed consent process, women will be informed that none of the study 
products are methods of contraception and about the current knowledge of effects of 
these products on a developing human fetus.    
19.10 Children  
 
The NIH has mandated that children be included in research trials when appropriate. 
This study meets “Justifications for Exclusion” criteria for younger children as set 
forth by the NIH.  Specifically, “insufficient data are available in adults to judge 
potential risk in children” and “children should not be the initial group to be involved 
in research studies.”  This study does not plan to enroll children under 18 years old.  

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19.11 Compensation 
 
Participants will be compensated for their time and effort in this study.  Participants 
will be provided with parking voucher to cover the cost of parking in the UNC 
Memorial Hospital’s designated visitor parking deck.  Meals will be provided when 
having a biopsy.  Participants will be given $40 for the screening, and $25 for the 
follow-up visit. They will be compensated $5 for each dose, for $50 total.  They will 
receive $225 for each biopsy day ($200 for biopsy and $25 for blood). A participant 
may be compensated $565 for completing all aspects of the study.  Stipends will be 
provided at screening and follow-up. 
 
Pro-rated amounts will be provided to participants who are unable or unwilling to 
complete the protocol-specified visits.    
 
19.12 Communicable Disease Reporting 
 
Study staff will comply with all applicable local requirements to report 
communicable diseases including HIV identified among study participants to local 
health authorities. Participants will be made aware of all reporting requirements 
during the study informed consent process.  
19.13 Access to HIV-related Care 
 
Participants found to be HIV-infected will be referred to available sources of medical 
and psychosocial care and support, and local research studies for HIV-infected adults. 
 
 
19.14 HIV Counseling and Testing 
 
HIV test-related counseling will be provided to all potential study participants who 
consent to undergo HIV screening to determine their eligibility for this study, and to 
all enrolled participants at each follow-up HIV testing time point.  Testing will be 

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performed in accordance with the algorithm in Appendix 3.  Counseling will be 
provided in accordance with standard HIV counseling policies and methods at the site 
and additionally will emphasize the unknown efficacy of the study products in 
preventing HIV infection.  In accordance with the policies of the US NIH, 
participants must receive their HIV test results to take part in this study.   
 
19.15 Care for Participants Identified as HIV-infected 
 
Participants will be provided with their HIV test results in the context of post-test 
counseling.  Participants found to be HIV-infected will be referred to available 
sources of medical and psychosocial care and support, and local research studies for 
HIV-infected adults. Our medical officer, Dr. Cynthia Gay, is an attending physician 
in the UNC Infectious Disease clinic and can provide ready access to multi-
disciplinary care for any participant who tests positive. 
19.16 Study Discontinuation 
 
This study may be discontinued at any time by the US FDA, the OHRP, or the site 
IRB. 
20.  Publication Policy 
 
Any presentation, abstract, or manuscript will be made available to Gilead Sciences 
prior to publication, with the final decision remaining with the PI. 
 
21.  References 
 
1.  
Shen Z, Fahey J V, Bodwell JE, et al. Estradiol regulation of nucleotidases in 
female reproductive tract epithelial cells and fibroblasts. PLoS One. 
2013;8(7):e69854. doi:10.1371/journal.pone.0069854 [doi]. 
2.  
Cottrell ML, Prince HM, Allmon A, et al. Cervicovaginal and Rectal Fluid as a 
Surrogate Marker of Antiretroviral Tissue Concentration: Implications for Clinical 

25February2019 v1.0 
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Trial Design. J Acquir Immune Defic Syndr. 2016;72(5):498-506. 
doi:10.1097/QAI.0000000000000996 [doi]. 
3.  
Yang KH, Hendrix C, Bumpus N, et al. A multi-compartment single and multiple 
dose pharmacokinetic comparison of rectally applied tenofovir 1% gel and oral 
tenofovir disoproxil fumarate. Abstract O_09. Presented at 15th Intl. Workshop on 
Clin Pharm of HIV and Hepatitis Therapy. 
4.  
Shen Z, Fahey J V, Bodwell JE, Rodriguez-Garcia M, Kashuba ADM, Wira CR. 
Sex hormones regulate tenofovir-diphosphate in female reproductive tract cells in 
culture. PLoS One. 2014;9(6):e100863. doi:10.1371/journal.pone.0100863. 
5.  
Spona J, Elstein M, Feichtinger W, et al. Shorter pill-free interval in combined oral 
contraceptives decreases follicular development. Contraception. 1996;54(2):71-77. 
6.  
Sullivan H, Furniss H, Spona J, Elstein M. Effect of 21-day and 24-day oral 
contraceptive regimens containing gestodene (60  microg) and ethinyl estradiol (15 
microg) on ovarian activity. Fertil Steril. 1999;72(1):115-120. 
7.  
Champley S. pwr: Basic Functions for Power Analysis. 2018. https://cran.r-
project.org/package=pwr. 
8.  
Cohen J. Statistical Power Analysis for the Behavioral Sciences. Vol 2nd ed. 
(Erlbaum L, ed.). Hillsdale, NJ; 1988.