A Phase I Trial for the Evaluation of the Two-way Pharmacokinetic-pharmacodynamic (PD) Interaction of Gender Affirming Exogenous Estrogen (With Testosterone Suppression) on TDF/FTC PrEP in Transgender Women (TGW)
Phase: PHASE1
Conditions: HIV, Prophylaxis, Transgender
Sponsor: Johns Hopkins University

TGW GAHT Interaction Study 
 
 
 
 
1 
 
 
 
26th April 2023 
 
A Phase I trial for the evaluation of the two-way pharmacokinetic-
pharmacodynamic interaction of gender affirming exogenous 
estrogen (with testosterone suppression) on TDF/FTC PrEP in 
transgender women (TGW) 
 
Funded by: 
Division of AIDS, US National Institute of Allergy and Infectious Diseases 
US National Institutes of Health 
 
 
Principal Investigator: Mark A. Marzinke 
 
DAIDS Program Officer: Roberta Black 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Program Grant #: 1R01AI45675-01 
 
 
 
Version 3.0 
26th, April 2023 
 
 
NCT04760691

TGW GAHT Interaction Study 
 
 
 
 
2 
 
 
 
26th, April 2023 
TABLE OF CONTENTS 
TABLE OF CONTENTS ................................................................................................................................. 2 
LIST OF ABBREVIATIONS AND ACRONYMS ............................................................................................. 5 
PROTOCOL TEAM ROSTER ........................................................................................................................ 9 
IMPROVING PREP PROTECTION OF TRANSGENDER WOMEN THROUGH MECHANISTIC 
PHARMACOKINETIC UNDERSTANDING .................................................................................................. 11 
INVESTIGATOR SIGNATURE FORM ......................................................................................................... 11 
PROTOCOL SUMMARY .............................................................................................................................. 12 
1 
KEY ROLES ......................................................................................................................................... 16 
1.1 
PROTOCOL IDENTIFICATION ............................................................................................................. 16 
1.2 
FUNDING AGENCY, SPONSOR, AND MONITOR IDENTIFICATION ........................................................... 16 
1.3 
DAIDS MEDICAL OFFICER ............................................................................................................... 16 
1.4 
STUDY SITE INVESTIGATORS ........................................................................................................... 16 
1.5 
STUDY SITE PHARMACIES ............................................................................................................... 17 
1.6 
CLINICAL LABORATORIES ................................................................................................................. 17 
1.7 
RESEARCH LABORATORIES AND ANALYTICAL UNITS .......................................................................... 17 
1.8 
DATA CENTER ................................................................................................................................ 18 
1.9 
STUDY OPERATIONS ....................................................................................................................... 18 
2 
INTRODUCTION .................................................................................................................................. 19 
2.1 
BACKGROUND ON GENDER AFFIRMING ESTROGEN-BASED THERAPY IN TGW ...................................... 20 
2.2 
RATIONALE FOR GONADOTROPIN RELEASING HORMONE AGONISTS FOR ANDROGEN SUPPRESSION .... 21 
2.3 
PREP CLINICAL TRIALS AND PHARMACOLOGY IN TGW ..................................................................... 22 
2.4 
THE CONTRIBUTION OF HORMONAL THERAPIES ON RENAL FUNCTION ................................................ 24 
2.5 
BACKGROUND AND RATIONALE OF PLANNED PREP-GAHT INTERACTIONS IN TGW ASSESSMENTS ..... 25 
2.5.1 
Safety Assessments .................................................................................................................. 25 
2.5.2 
Drug Analysis ............................................................................................................................ 25 
2.5.3 
Ex vivo colon tissue explant HIV challenge ............................................................................... 25 
2.5.4 
Direct Measurement of Glomerular Filtration ............................................................................. 25 
2.5.5 
Hormone Analysis ..................................................................................................................... 26 
2.5.6 
Additional Biomarkers of Renal Function ................................................................................... 26 
2.5.7 
Assessment of Participant Adherence with Study Product/Intervention(s) ................................. 26 
2.6 
DESCRIPTION OF STUDY PRODUCTS ................................................................................................ 26 
3 
OBJECTIVES AND ENDPOINTS ......................................................................................................... 27 
3.1 
OBJECTIVES ................................................................................................................................... 27 
3.1.1 
Objectives & Endpoints ............................................................................................................. 28 
4 
STUDY DESIGN ................................................................................................................................... 28 
4.1 
IDENTIFICATION OF STUDY DESIGN .................................................................................................. 28 
4.2 
DESCRIPTION OF STUDY POPULATION .............................................................................................. 30 
4.3 
TIME TO COMPLETE ACCRUAL ......................................................................................................... 30 
4.4 
EXPECTED DURATION OF PARTICIPATION ......................................................................................... 30 
5 
STUDY POPULATION ......................................................................................................................... 30 
5.1 
SELECTION OF STUDY POPULATION ................................................................................................. 30 
5.2 
RECRUITMENT ................................................................................................................................ 30 
5.3 
RETENTION .................................................................................................................................... 31 
5.4 
INCLUSION CRITERIA ....................................................................................................................... 31 
5.5 
EXCLUSION CRITERIA ...................................................................................................................... 32 

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6 
STUDY PRODUCTS ............................................................................................................................. 33 
6.1 
REGIMEN ........................................................................................................................................ 33 
6.2 
ADMINISTRATION ............................................................................................................................ 34 
6.3 
STUDY PRODUCT ............................................................................................................................ 34 
6.3.1 Tenofovir/Emtricitabine ................................................................................................................ 35 
6.3.2 
Leuprolide acetate ..................................................................................................................... 35 
6.3.3 
Estrace® .................................................................................................................................... 35 
6.3.4 
Iohexol (OmnipaqueTM 300) ....................................................................................................... 35 
6.4 
STUDY PRODUCT SUPPLY AND ACCOUNTABILITY .............................................................................. 36 
6.5 
ACCOUNTABILITY ............................................................................................................................ 36 
6.6 
DISPENSING ................................................................................................................................... 36 
6.7 
RETRIEVAL OF UNUSED PRODUCT ................................................................................................... 36 
6.8 
CONCOMITANT MEDICATIONS AND PROCEDURES .............................................................................. 36 
6.9 
PROHIBITED MEDICATIONS AND PROCEDURES .................................................................................. 36 
6.5 
RECOMMENDED PROCEDURES ........................................................................................................ 37 
7 
STUDY PROCEDURES ....................................................................................................................... 37 
7.1 
PROCEDURE OVERVIEW .................................................................................................................. 37 
7.2 
VISIT 1 - SCREENING VISIT .............................................................................................................. 37 
7.3 Visit 2 – Baseline Visit (Day 0) ........................................................................................................ 38 
7.4 
DIRECTLY OBSERVED VISITS (CONDUCTED FOR 7 DAYS PRIOR TO INTENSIVE PK SAMPLING VISITS 1, 3, 
4, 5, 7) 40 
7.5 
Visits 1, 3, 4, 5, 7 –Day 1 (Dosing Visits) ................................................................................... 40 
7.7 
Visits 2, 6, 8 - Safety/Adherence Visits ...................................................................................... 42 
* SAFETY LABS WILL ONLY BE COLLECTED AT VISIT 8 .................................................................................... 43 
7.8 
EXIT FOLLOW-UP PHONE CALL ........................................................................................................ 43 
7.9 
INTERIM VISITS ............................................................................................................................... 43 
7.5 
PARTICIPANTS WHO WITHDRAW OR ARE WITHDRAWN FROM THE STUDY ........................................... 44 
7.6 
PARTICIPANTS WHO BECOME INFECTED WITH HIV ............................................................................ 44 
7.10 
CLINICAL EVALUATIONS AND PROCEDURES ...................................................................................... 45 
7.11 
LABORATORY EVALUATIONS ............................................................................................................ 46 
7.11.1 
Clinical Laboratory Testing .................................................................................................... 46 
7.11.2 
Research Laboratory Testing ................................................................................................ 46 
7.12 
SPECIMEN COLLECTION, HANDLING, AND PROCESSING ..................................................................... 46 
7.13 
STORAGE OF SPECIMENS FOR FUTURE USE ..................................................................................... 47 
7.14 
BIOHAZARD CONTAINMENT .............................................................................................................. 47 
8 
ASSESSMENT OF SAFETY ................................................................................................................ 47 
8.1 
SAFETY MONITORING ...................................................................................................................... 47 
8.2 
CLINICAL DATA SAFETY REVIEW ...................................................................................................... 47 
8.3 
ADVERSE EVENTS DEFINITIONS AND REPORTING REQUIREMENTS ..................................................... 48 
8.3.1 
Adverse Events ......................................................................................................................... 48 
8.3.2 
Serious Adverse Events ............................................................................................................ 48 
8.3.3 
Adverse Event Relationship to Study Product ........................................................................... 49 
8.4 
EXPEDITED ADVERSE EVENT (EAE) DEFINITIONS AND REPORTING REQUIREMENTS ........................... 49 
8.5 
REPORTING OF ADVERSE REACTIONS TO THE RESPONSIBLE IRBS .................................................... 50 
8.6 
SOCIAL HARMS REPORTING............................................................................................................. 50 
8.7 
WITHDRAWAL OF SUBJECTS DUE TO ADVERSE EVENTS .................................................................... 51 
9 
CLINICAL MANAGEMENT .................................................................................................................. 51 
9.1 
GRADING SYSTEM ........................................................................................................................... 51 
9.2 
DOSE MODIFICATION ....................................................................................................................... 51 
9.3 
DISCONTINUATION OF STUDY PRODUCT(S) IN THE PRESENCE OF TOXICITY ........................................ 52 
9.4 
GENERAL CRITERIA FOR DISCONTINUATION OF STUDY PRODUCT(S) .................................................. 52 

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9.5 
CRITERIA FOR PROGRESSION TO THE NEXT DOSING PHASE .............................................................. 52 
9.6 
CRITERIA FOR EARLY TERMINATION OF STUDY PARTICIPATION .......................................................... 52 
9.7 
STUDY PARTICIPANT REPLACEMENT ................................................................................................ 52 
10 
STATISTICAL CONSIDERATIONS ..................................................................................................... 53 
10.1 
OVERVIEW AND SUMMARY OF DESIGN .............................................................................................. 53 
10.2 
STUDY ENDPOINTS ......................................................................................................................... 53 
10.3 
STUDY HYPOTHESES ...................................................................................................................... 53 
10.4 
ACCRUAL AND SAMPLE SIZE ............................................................................................................ 53 
10.5 
RANDOMIZATION AND BLINDING ....................................................................................................... 54 
10.6 
DATA ANALYSIS .............................................................................................................................. 54 
10.6.1 
Descriptive Statistics and Graphics ....................................................................................... 54 
10.6.2 
Safety .................................................................................................................................... 54 
10.6.3 
Pharmacokinetics .................................................................................................................. 55 
10.6.4 
Explant Studies ..................................................................................................................... 55 
10.6.5 
Missing Data.......................................................................................................................... 55 
11 
DATA HANDLING AND RECORDKEEPING ....................................................................................... 56 
11.1 
DATA MANAGEMENT RESPONSIBILITIES ............................................................................................ 56 
11.2 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS .................................................. 56 
11.3 
QUALITY CONTROL AND QUALITY ASSURANCE .................................................................................. 56 
11.4 
STUDY COORDINATION .................................................................................................................... 57 
12 
CLINICAL SITE MONITORING ............................................................................................................ 57 
13 
HUMAN SUBJECTS PROTECTIONS .................................................................................................. 57 
13.1 
INSTITUTIONAL REVIEW BOARDS ...................................................................................................... 58 
13.2 
PROTOCOL REGISTRATION .............................................................................................................. 58 
13.3 
RISK-BENEFIT STATEMENT .............................................................................................................. 58 
13.3.1 
Risks ..................................................................................................................................... 58 
13.3.2 
Benefits ................................................................................................................................. 61 
13.4 
INFORMED CONSENT PROCESS ....................................................................................................... 61 
13.5 
PARTICIPANT CONFIDENTIALITY ....................................................................................................... 62 
13.6 
SPECIAL POPULATIONS ................................................................................................................... 63 
13.6.1 
Women .................................................................................................................................. 63 
13.6.2 
Children ................................................................................................................................. 63 
13.7 
COMPENSATION .............................................................................................................................. 63 
13.8 
COMMUNICABLE DISEASE REPORTING ............................................................................................. 63 
13.9 
ACCESS TO HIV-RELATED CARE ...................................................................................................... 64 
13.9.1 
HIV Counseling ..................................................................................................................... 64 
13.9.2 
Care for Participants Identified as HIV-infected ..................................................................... 64 
13.10 
STUDY DISCONTINUATION............................................................................................................ 64 
14 
LIST OF APPENDICES ........................................................................................................................ 64 
15 
REFERENCES ..................................................................................................................................... 65 
APPENDIX I: SCHEDULE OF STUDY VISITS AND EVALUATIONS .......................................................... 70 
APPENDIX II: HIV TESTING ALGORITHM ................................................................................................. 73 
APPENDIX III:  RECTAL TISSUE BIOPSY ALLOCATION ......................................................................... 75 
APPENDIX IV: TOXICITY TABLES ............................................................................................................. 76 
APPENDIX V: SAMPLE INFORMED CONSENT FORM ............................................................................. 77 
 

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LIST OF ABBREVIATIONS AND ACRONYMS 
 
AE   
 
Adverse Event 
AIDS  
 
Acquired Immunodeficiency Syndrome 
ALT 
 
 
Alanine Transaminase 
ANCOVA 
 
Analysis of Covariance 
ANOVA 
 
Analysis of Variance 
API 
 
 
Active Pharmaceutical Ingredient 
ART  
 
Antiretroviral Therapy 
AST  
 
Aspartate Aminotransferase 
AUC  
 
Area Under the Curve 
BLQ  
 
Below the Limit of Quantitation 
BMD  
 
Bone Mineral Density 
BP 
 
 
Blood Pressure 
BUN  
 
Blood Urea Nitrogen 
°C 
 
 
Celsius  
CAPRISA 
 
Centre for the AIDS Programme of Research in South Africa 
CBC  
 
Complete Blood Count 
CCR5  
 
Chemokine Receptor 5 
CD4   
 
Cluster of Differentiation 4 
CDC  
 
Centers for Disease Control and Prevention 
CFR  
 
Code of Federal Regulations 
cGMP  
 
Current Good Manufacturing Practices 
CGM  
 
Cisgender men 
CGW  
 
Cisgender women 
CT 
 
 
Chlamydia  
 
Cl 
 
 
Chloride 
CLIA  
 
Clinical Laboratory Improvement Amendments 
Cmax 
 
 
Maximum Serum Concentration 
CMP  
 
Comprehensive Metabolic Panel 
CO2  
 
Carbon Dioxide 
C-PMPA 
 
Radiolabeled Tenofovir 
Cr 
 
 
Creatinine 
CRF  
 
Case Report Form 
CRS  
 
Clinical Research Site 
CT  
 
 
Chlamydia trachomatis 
CVL  
 
Cervicovaginal Lavage 
CXCR4 
 
CXC Chemokine Receptor 4 
d4T 
 
 
Stavudine 
DAIDS 
 
Division of AIDS 
DNA  
 
Deoxyribonucleic Acid 
DP 
 
 
Diphosphate 
DSMB  
 
Data and Safety Monitoring Board 
ELISA  
 
Enzyme-Linked Immunosorbent Assay 
EAE  
 
Expedited Adverse Event 
ES 
 
 
Endocrine Society 
ET 
 
 
Eastern Time 
FDA  
 
Food and Drug Administration 
FTC  
 
Emtricitabine 

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FSH  
 
Follicle Stimulating Hormone 
g 
 
 
Gram 
GAHT  
 
Gender-affirming hormonal therapy 
GC 
 
 
Neisseria gonorrhoeae 
GCP  
 
Good Clinical Practices 
GLP  
 
Good Laboratory Practices 
HBsAg 
 
Hepatitis B Surface Antigen 
HHS  
 
Health and Human Services 
HIV 
 
 
Human Immunodeficiency Virus 
HPTN  
 
HIV Prevention Trials Network 
HR 
 
 
Heart Rate 
hr 
 
 
Hour 
HSV  
 
Herpes Simplex Virus 
IATA 
 
 
International Air Transport Association 
IB 
 
 
Investigator’s Brochure 
IDS 
 
 
Investigational Drug Service 
IoR 
 
 
Investigator of Record  
IPCP  
 
Integrated Pre-Clinical/Clinical Program 
iPrEx  
 
Pre-Exposure Prophylaxis Initiative 
IRB 
 
 
Institutional Review Board 
IGF 
 
 
Insulin-like Growth Factor 
IV 
 
 
Intravenous 
JHU  
 
Johns Hopkins University 
K 
 
 
Potassium 
kg 
 
 
Kilogram 
LC-MS 
 
Liquid Chromatography-Mass Spectroscopy                 
LC-UV 
 
Liquid Chromatography-Ultraviolet 
LH 
 
 
Leutinizing Hormone 
LL 
 
 
Local Laboratory 
LLOQ  
 
Lower Limit of Quantification 
mg 
 
 
Milligram 
μg 
 
 
Micrograms 
min 
 
 
Minute 
mITT  
 
Modified Intent To Treat Analysis 
mL 
 
 
Milliliter  
MMC  
 
Mucosal Mononuclear Cells 
mOsmol 
 
MilliOsmole, or one-thousandth of an osmole 
MO 
 
 
Medical Officer  
MSM  
 
Men who have Sex with Men 
MTN  
 
Microbicide Trials Network 
MTT  
 
[1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] 
N-9 
 
 
Nonoxynol-9 
Na 
 
 
Sodium 
NAAT  
 
Nucleic acid amplification test 
NIAID  
 
National Institute of Allergy and Infectious Diseases 
NIH 
 
 
(United States) National Institutes of Health 
NL 
 
 
Network Laboratory 
NOAEL 
 
No Observed Adverse Effect Level 
NNRTI 
 
Non-Nucleoside Reverse Transcriptase Inhibitor 

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NRTI  
 
Nucleoside Reverse Transcriptase Inhibitor 
O.D.  
 
Optical density 
OHRP  
 
Office for Human Research Protections 
Osmol  
Osmole; one osmole is defined as a unit of osmotic pressure equivalent to the 
amount of solute that dissociates in solution to form one mole (Avogadro's 
number) of particles (molecules and ions) 
PBMC  
 
Peripheral Blood Mononuclear Cells 
PCR  
 
Polymerase Chain Reaction 
PD 
 
 
Pharmacodynamic(s) 
PEP  
 
Post-Exposure Prophylaxis 
PIV 
 
 
Peripheral IV 
PK 
 
 
Pharmacokinetic(s) 
PMPA  
 
9-[(R)-2-(phosphonomethoxy)propyl] adenine monohydrate 
PO4 
 
 
Phosphate 
PoR  
 
Pharmacist of Record 
PrEP  
 
Pre-Exposure Prophylaxis 
PRO  
 
Protocol Registration Office 
PSRT  
 
Protocol Safety Review Team 
PTID  
 
Participant Identification Number 
PRN  
 
As needed 
(qc)-RT PCR  
Quantitative Competitive 
QD 
 
 
Daily 
RAI                 
Receptive Anal Intercourse (refers to coitus only, does not include manual 
stimulation or the use of sex toys or purgatives) 
RMP  
 
Rectal Microbicide Program 
RNA 
 Ribonucleic Acid 
RPR  
 
Rapid Plasma Reagin 
RRI 
 
 
Rabbit Rectal Irritation 
RSC  
 
Regulatory Support Center  
RRR  
 
Relative Risk Reduction 
RT 
 
 
Reverse Transcriptase 
RU 
 
 
Research Unit 
SAE  
 
Serious Adverse Experience 
SADR  
 
Suspected Adverse Drug Reaction 
SHIV  
 
Simian/Human Immunodeficiency Virus 
SIV 
 
 
Simian Immunodeficiency Virus 
SMC  
 
Safety Monitoring Committee 
SMS  
 
Short Message Service 
SOP  
 
Standard Operating Procedure 
sRAI  
 
Simulated RAI 
SSP  
 
Study Specific Procedures 
SPECT/CT  
Single Photon Computed Tomography/Computed Tomography 
STD/ STI 
 
Sexually Transmitted Disease/Infection 
SUSAR 
 
Suspected and Unexpected Serious Adverse Reactions 
99mTc-DTPA  
Technetium-99m-DTPA 
Tmax 
 
 
The time at which maximal concentration is reached 
TAF 
 
 
Tenofovir Alafenamide Fumarate (formerly GS7340) 
TDF  
 
Tenofovir Disoproxil Fumarate (oral tenofovir) 
Temp  
 
Temperature  

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TFV 
 
 
Tenofovir 
TFV-DP 
 
Tenofovir Diphosphate 
TER  
 
Transepithelial Resistance 
TGW  
 
Transgender Women 
UA 
 
 
Urinalysis 
ULN  
 
Upper Limit of Normal 
ULOQ  
 
Upper Limit of Quantification 
URAI  
 
Unprotected Receptive Anal Intercourse 
USP  
 
United States Pharmacopeia 
UTI 
 
 
Urinary tract infection 
VI 
 
 
Virus Isolation 
vRNA  
 
Viral Ribonucleic Acid 
WPATH 
 
World Professional Association for Transgender Health 

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PROTOCOL TEAM ROSTER 
Rahul Bakshi, PH.D 
The Johns Hopkins University School of Medicine 
301 Biophysics, 
725 N Wolfe Street 
Baltimore, MD 21205 
 
Phone: 410-955-1889 
Email:  rbakshi@jhmi.edu 
 
 
Adrian S Dobs, MD, MHS 
Co-Investigator 
The Johns Hopkins University School of Medicine 
1830 E Monument St 328 
Baltimore, MD 21205 
 
Phone: 443-287-4000  
Fax:  (410) 367-2042 
Email:  adobs@jhmi.edu 
Craig W. Hendrix, M.D. 
Co-Investigator 
Director,  
Division of Clinical Pharmacology 
The Johns Hopkins University School of Medicine 
600 North Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
Phone: 410-955-9707 
Fax: 410-955-9708 
Email: cwhendrix@jhmi.edu 
Claire Knezevic, PhD 
Co-investigator 
Assistant Director, Clinical Pharmacology Analytical 
Laboratory 
The Johns Hopkins University School of Medicine 
1800 Orleans Street, Sheikh Zayed Tower B1020-G 
Baltimore, MD 21287 
 
Phone: 443-287-2018 
Fax: 410-955-0767 
Email: cknezev1@jhmi.edu 
Mark Marzinke, PhD 
Principal Investigator 
The Johns Hopkins University School of Medicine 
1800 Orleans Street, Sheikh Zayed Tower B1020-F 
Baltimore, MD 21287 
 
Phone: 443-287-7516 
Fax: 410-955-0767 
Email: mmarzin@jhmi.edu 
Michael A Rosenblum, PHD 
Co-Investigator 
The Johns Hopkins University School of Public Health 
615 N Wolfe St E3616 
Baltimore, MD 21205-2103 
 
Phone:  (410) 614-9400 
Fax:  410-955-0958 
Email:  mrosen@jhu.edu 
 
Sasha Bessleman, PharmD 
Investigational Drug Service, Osler 100 
Johns Hopkins Hospital 
600 N. Wolfe St. 
Baltimore, MD  21287 
 
Phone: 410-955-6337 
Email: abeselm1@jhmi.edu 
 
Roberta Black, PhD 
Program Officer  
Clinical Microbicide Research Program 
DAIDS, NIAID, NIH  
5601 Fishers Lane Room 8B62 
Rockville, MD 20852 
 
Phone: 301-496-8199 
Email: rblack@niaid.nih.gov 

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Kate Marych, BSN, RN 
Senior Research Nurse 
The Johns Hopkins Hospital 
600 North Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
Phone: 410-955-1218Email: kdepros1@jhmi.edu 
Santiago Alvarez Arango, MD 
Co-Investigator 
The Johns Hopkins Hospital 
600 North Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
Phone: 410-614-2724 
Email: salvarez@jhmi.edu 
 
Jennifer Hoffman, CRNP 
Research Nurse Manager 
The Johns Hopkins Hospital 
600 North Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
Phone: 410-955-1318 
Email: jhoffm45@jhmi.edu 
 
 
 
 
 

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Improving PrEP protection of transgender women through mechanistic 
pharmacokinetic understanding  
 
INVESTIGATOR SIGNATURE FORM 
 
Version 3.0 
 26 April 2023 
 
Funded by: 
Division of AIDS, US National Institute of Allergy and Infectious Diseases 
US National Institutes of Health 
 
 
 
I will conduct the study in accordance with the provisions of this protocol and all applicable protocol-
related documents. I agree to conduct this study in compliance with United States (US) Health and 
Human Service regulations (45 CFR 46); applicable U.S. Food and Drug Administration regulations; 
standards of the International Conference on Harmonization Guideline for Good Clinical Practice (E6); 
Institutional Review Board/Ethics Committee determinations; all applicable in-country, state, and local 
laws and regulations; and other applicable requirements (e.g., US National Institutes of Health, 
Division of AIDS) and institutional policies. 
 
 
I have read and understand the information in the Investigator's Brochure(s), including the potential 
risks and side effects of the products under investigation, and will ensure that all associates, 
colleagues, and employees assisting in the conduct of the study are informed about the obligations 
incurred by their contribution to the study. 
 
 
____________________________ 
Name of Investigator of Record 
 
 
____________________________ 
______________________________ 
Signature of Investigator of Record  
Date 

TGW GAHT Interaction Study 
 
 
 
 
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26th, April 2023 
A Phase I trial for the evaluation of the two-way pharmacokinetic-
pharmacodynamic 
interaction 
of 
gender 
affirming 
exogenous 
estrogen (with testosterone suppression) on TDF/FTC PrEP in 
transgender women (TGW). 
PROTOCOL SUMMARY 
 
Short Title: PrEP-GAHT Interactions in TGW 
 
Clinical Phase: Phase 1 
 
Sponsor: NIH/Division of AIDS 
 
Protocol Chair:  Mark A. Marzinke (JHU)  
 
Study Site Investigators:  Craig Hendrix, MD  
Adrian Dobs, MD 
 
                                            Santiago Alvarez Arango, MD          
                                            Jennifer Hoffman, CRNP                                    
 
Sample Size:  20 evaluable, self-identifying transgender women (TGW)   
 
Study Population:  
HIV-negative, self-identifying TGW who are not currently on any form of 
GAHT (including   estrogen-based therapies, spironolactone, and 
progesterone), or who are willing to abstain from feminizing therapies until 
serum total testosterone concentrations exceed 2 ng/mL (cisgender male 
reference interval). 
 
Participating Clinical Research Site (CRS): Johns Hopkins University (JHU) 
 
Study Rationale and Objectives:  TGW frequently use estrogen-based gender affirming hormonal 
therapies (GAHT) to promote feminization and decrease gender dysphoria. A highly vulnerable 
population, TGW are 49-times more likely to acquire HIV as compared to cisgender men and women; 
therefore, pre-exposure prophylaxis (PrEP, tenofovir disoproxil fumarate/emtricitabine), which 
prevents HIV acquisition in high-risk populations, is greatly needed by TGW. Because the interaction 
between GAHT and PrEP is poorly understood, an investigation on the impact of exogenous hormonal 
therapies on PrEP effectiveness (and vice versa) is required to optimize dosing regimens for HIV 
protection, while also not impacting the effects of GAHT on reducing gender dysphoria.  
 
Study Objectives: This is a Non-IND study designed to more closely evaluate the two-way pharmacokinetic-
pharmacodynamic (PK-PD) interaction of GAHT (estrogen with testosterone suppression) on TDF/FTC PrEP in 
TGW through the completion of a five-phase PK-PD study, in which TGW take daily PrEP, followed by a 
testosterone suppressing agent alone and in conjunction with low- and high-dose estrogen therapy (Table 1).  To 
reduce variability, observed PrEP administration will be conducted for 7 days prior to each intensive PK visit 
while on PrEP.  PrEP and hormone doses will be directly observed by study personnel either in person, by live 
video streaming, or with a timestamped video. In the high-dose estrogen phase of the clinical trial, estrogen 
dosing may be split to a morning and an evening dose; only one dose per day will be monitored using one of the 
aforementioned strategies.   Study objectives including safety, pharmacokinetics (PK) and pharmacodynamics 
(PD), are essential in understanding the impact of co-administration of PrEP and GAHT on downstream drug and 
hormone concentrations (Table 2). 
 
Table 1: Drug administration during each phase of the PK-PD clinical study 

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PK Phase 
PrEP 
GnRH Agonist 
Low-Dose Estrogen 
High-Dose Estrogen 
1 
X 
 
 
 
2 
X 
X 
 
 
3 
X 
X 
X 
 
4 
X 
X 
 
X 
5 
 
X 
 
X 
 
Study Design: The PrEP-GAHT Interactions in TGW protocol is a phase 1, open label study to 
compare the safety, PK and PD of five sequential phases of PrEP administration in the presence or 
absence of testosterone-reducing therapies or dose-escalated estrogen therapy.  Each participant 
will undergo a Screening Visit to evaluate eligibility. Following Baseline evaluation, eligible 
participants will receive 300 mg TDF/200 mg FTC (Truvada®, Gilead Sciences) once daily for seven 
days, using the aforementioned direct observation approaches, to achieve steady state drug PrEP 
concentrations.  After one week of therapy, participants will undergo intensive PK analysis as well as 
collection of colorectal biopsies for PD testing (PK1).  During the PK-intensive day, iohexol will be 
administered intravenously for the empirical determination of renal function and measured glomerular 
filtration rate (mGFR).  While concurrently on PrEP, participants will then be intramuscularly 
administered depot leuprolide acetate (11.25 mg Lupron®).  Two weeks post-injection, when 
testosterone concentrations are far below the lower limit of normal of total testosterone in cisgender 
men (typically < 200 ng/dL, or < 2 ng/mL), sampling for PK, PD, and renal function will be performed 
(PK2).  Participants will then immediately begin low-dose oral estrogen therapy (1 mg 17β-estradiol) 
in conjunction with PrEP for one week, at which time samples will be collected for the analyses 
described above (PK3).  While still on PrEP, participants will then transition to high-dose estrogen 
therapy (6 mg 17β-estradiol) for the remainder of the study.  One-week post-high dose estrogen 
therapy in the presence of PrEP, pharmacologic and renal samples will be collected for analysis 
(PK4).  PrEP will then be discontinued, and two weeks later, samples will be collected to assess renal 
function and hormonal concentrations, and evaluate the presence of any remaining PrEP in plasma, 
PBMC, or colorectal tissue (though it should be near undetectable levels for most analytes according 
to our prior data) (PK5).  The schema is summarized in Figure 1.  Safety assessments, including 
history/physical, chemistry/hematology labs at screening and interim history will be performed at each 
study-intensive visit.  Additionally, periodic assessments of gender dysphoria will be conducted at 
baseline and a convenient time during PK visits throughout the study. To ensure compliance, 
participants will undergo direct observation of dosing each day of the week prior to PK visits, using 
the aforementioned strategies.  
 
 

TGW GAHT Interaction Study 
 
 
 
 
14 
 
 
 
26th, April 2023 
 
 
Table 2: Study Objectives and Endpoints 
*as defined by the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, Corrected 
Version 2.1 - July 2017, and Addendum 3 (Rectal Grading Tables for Use in Microbicide Studies) 
 
Study Schema: 
STUDY 
DRUGS 
& 
STUDY 
WEEK 
WK 0 
WK 1 
WK 2 
WK 3 
WK 4 
WK 5 
WK 6 
WK 7 
TDF/FTC 300mg/200mg po qd 
  
  
  
  
  
  
  
Leuprolide acetate 11.25 mg sc 
  
  
  
  
  
  
  
Estradiol 1 mg po qd 
  
  
Estradiol 6 mg po qd 
  
  
  
  
PK 
VISIT 
# 
& 
PK 
ASSESSMENTS 
  
PK1 
  
PK2 
PK3 
PK4 
  
PK5  
TDF/FTC 
x 
x 
x 
x 
  
Low/No Testosterone 
x 
x 
x 
x 
Estradiol 
  
  
  
  
Low 
High 
  
High 
PD ASSESSMENTS 
 
 
 
 
 
 
 
 
FSH/LH 
x 
x 
 
x 
x 
x 
 
x 
Colorectal HIV infectivity 
x 
x 
 
x 
x 
x 
 
x 
Figure 1. Dosing and PK sampling schema to assess the two drug-hormone interaction between PrEP and GAHT. 
 
 
Study Products:  
Tenofovir disoproxil fumarate (TDF) 300 mg/emtricitabine (FTC) 200 mg study product tablets are 
manufactured and marketed by Gilead Sciences, Inc. under the trade name Truvada®. 
Study Product Acquisition.  17β-estradiol (1 x 1 mg or 3 x 2 mg) is manufactured and marketed by 
Allergan, Inc.  Leuprolide acetate is formulated as an 11.25 mg depot, and is manufactured and 
Objectives 
Endpoints & Data Analysis 
• Describe the impact of GAHT 
on TDF/FTC PrEP PK-PD 
• Describe the bi-directional 
impact of TDF/FTC & GAHT 
combinations 
• Non-compartmental analysis of TFV/FTC in plasma and urine 
• Concentrations of all analytes in blood, PBMC, & colorectal 
tissue  
• Compare PK1 to PK2, PK3, PK4 for GAHT impact on PrEP 
• Compare PK5 to PK2-4 for PrEP impact on GAHT 
• Describe the impact of GAHT 
on PrEP suppressed HIV 
susceptibility  
• Describe changes in explant p24 antigen under all PK 
conditions 
• Correlation and Emax model of drug concentration-p24 
relationship  
• Explain the mechanism of 
estrogen impact on PrEP PK 
• Mechanistic modeling of PK change  
• Renal changes with addition of GAHT 
• Describe adverse effects 
associated with PrEP & GAHT 
• Grade 2 or higher AEs* 
• Assess gender dysphoria will 
participants are on study 
product 
• Gender dysphoria evaluations at baseline and during PK1 
through PK5 

TGW GAHT Interaction Study 
 
 
 
 
15 
 
 
 
26th, April 2023 
marketed by AbbVie, Inc.  The study product will be purchased and dispensed per investigator 
prescription by the Investigational Drug Service (IDS) Pharmacy at The Johns Hopkins Hospital. 
 
Study Duration:  
Participant accrual will take approximately 3 years and each participant will be on study for approximately 
4 months (up to 2 months in screening and 56 days under observationon study drugs). Total study 
duration is about 4 years. 

TGW GAHT Interaction Study 
 
 
 
 
16 
 
 
 
26th, April 2023 
1 
KEY ROLES 
 
1.1 
Protocol Identification 
 
Protocol Title: 
A Phase I trial for the evaluation of the two-way pharmacokinetic-
pharmacodynamic interaction of gender affirming exogenous 
estrogen (with testosterone suppression) on TDF/FTC PrEP in 
transgender women (TGW). 
 
Short Title: 
 
PrEP-GAHT Interactions in TGW  
 
Date:  
 
26 April 2023 
 
1.2 
Funding Agency, Sponsor, and Monitor Identification 
 
Funding Agency: 
Division of AIDS 
National Institute of Allergy and Infectious Diseases (NIAID), NIH 
5601 Fishers Lane 
Rockville, MD 20852 USA 
 
Monitor: 
 
Drug Development Unit  
Johns Hopkins University 
 
 
Division of Clinical Pharmacology 
 
 
600 N. Wolfe Street, Blalock 569 
 
 
Baltimore, MD 21287 
 
1.3 
DAIDS Officers 
 
Program Officer: 
Roberta Black, PhD 
Chief, Clinical Microbicide Research Branch 
DAIDS, NIAID, NIH 
5601 Fishers Lane, Room 8B62 
Rockville, MD 20852 
 
 
1.4 
Study Site Investigators 
 
Principal 
Investigator:  
Mark Marzinke, PhD, DABCC 
Departments of Pathology and Medicine 
Johns Hopkins University School of Medicine  
1800 Orleans Street, Sheikh Zayed Tower, Room B1-1020G 
Baltimore, MD 21287  
 
Co-Investigators: 
Craig Hendrix, MD 
Division of Clinical Pharmacology 
Johns Hopkins University 
600 N. Wolfe Street, Blalock 569 
Baltimore, MD 21287 

TGW GAHT Interaction Study 
 
 
 
 
17 
 
 
 
26th, April 2023 
 
Adrian Dobs, MD 
Division of Endocrinology 
Johns Hopkins University 
1830 E. Monument St 328 
Baltimore, MD 21205Santiago Alvarez Arango, MD 
Co-Investigator 
The Johns Hopkins Hospital 
600 North Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
 
Jennifer Hoffman, CRNP 
Research Nurse Manager 
The Johns Hopkins Hospital 
600 North Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
 
 
 
1.5 
Study Site Pharmacies 
 
Johns Hopkins University: Johns Hopkins Investigational Drug Service 
Johns Hopkins Hospital 
600 N. Wolfe Street, Osler 100 
Baltimore, MD 21287 
Phone: 410-955-6337  
Fax: 410-614-8074 
 
1.6 
Clinical Laboratories 
 
JHU Laboratories:  
Johns Hopkins Medical Laboratories 
401 N. Broadway, Weinberg Bldg. Rm 2335 
Baltimore, MD 21231 
 
Johns Hopkins Medical Laboratories 
600 N. Wolfe Street, Carnegie 417 
Baltimore, MD 21287 
 
 
1.7 
Research Laboratories and Analytical Units 
 
JHU Laboratories:  
Clinical Pharmacology Analytical Laboratory 
Johns Hopkins University 
600 N. Wolfe Street, Osler 500 
Baltimore, MD 21287 USA 
 
5200 Eastern Ave 
MFL Center Tower, Suite 6000 

TGW GAHT Interaction Study 
 
 
 
 
18 
 
 
 
26th, April 2023 
Baltimore, MD 21224 USA 
 
1.8 
Data Center 
Johns Hopkins University, School of Medicine 
Division of Clinical Pharmacology 
Drug Development Unit  
600 N. Wolfe Street, Blalock 569 
Baltimore, MD 21287 
 
1.9 
Study Operations 
 
Johns Hopkins University, School of Medicine 
Division of Clinical Pharmacology 
Drug Development Unit 
600 N. Wolfe Street, Blalock 569 
Baltimore, MD 21287 

TGW GAHT Interaction Study 
 
 
 
 
19 
 
 
 
26th, April 2023 
2 
INTRODUCTION  
 
More than 1.3 million adults in the U.S. identify as transgender, with the highest percentage (2.78%) 
of transgender individuals living in the Washington DC area1. The term transgender describes 
persons whose gender identity is not congruent with the sex they were assigned at birth and those 
associated sexual characteristics2. Due to this gender dysphoria, many transgender individuals 
experience distress and face significant physiological, social, and psychological challenges. All of 
these factors greatly impact the quality of life within the community3. Further, transgender individuals 
face significant barriers in terms of employment and healthcare, with up to 28% and 48% of 
transgender persons postponing healthcare due to discrimination or financial constraints, 
respectively4,5. Organizations such as the World Professional Association for Transgender Health 
(WPATH), a multidisciplinary society with expertise in multiple facets of transgender health, provide 
guidelines in the culturally competent care to transgender persons, including gender affirming 
hormonal therapy (GAHT)6. Despite these guidelines, significant knowledge gaps still exist in 
transgender care, especially where it overlaps with other health needs of this vulnerable population. 
For example, with increased risk of HIV acquisition, the transgender community needs access to 
effective HIV pre-exposure prophylaxis (PrEP) with the confidence that drugs for PrEP and GAHT do 
not interfere with each other’s efficacy7. The focus of this proposal is to better characterize the 
pharmacologic interactions between estrogen-based GAHT and PrEP drugs. 
 
HIV infection remains a public health concern. Current estimates indicate that there are nearly 37 
million individuals worldwide living with the disease, with nearly 2 million new infections occurring 
annually8. The primary modality for the prevention of HIV in high-risk populations is the administration 
of antiretroviral agents for PrEP. Currently, two fixed-dose formulations containing both tenofovir 
(TFV) and emtricitabine (FTC) - 300 mg TFV disoproxil fumarate (TDF)/200 mg FTC (Truvada®, 
Gilead Sciences) and 25 mg TFV alafenamide fumarate (TAF)/200 mg FTC (Descovy®, Gilead 
Sciences) are the only FDA-approved regimens to prevent HIV acquisition. TFV and FTC are 
nucleotide and nucleoside reverse transcriptase inhibitors (NRTIs), respectively, that achieve their 
antiviral effects as the intracellular phosphorylated derivatives, TFV-diphosphate (TFV-DP) and FTC 
triphosphate (FTC-TP). With excellent adherence to the daily TDF/FTC regimen, protection from HIV 
acquisition can be as high as 100%9. However, small reductions in adherence can result in significant 
reductions of protective efficacy, indicating that HIV protection is sensitive to drug concentration10-13. 
 
Among those most affected by the disease, transgender communities, in particular transgender 
women (TGW), are disproportionately burdened by HIV infection. A recent meta-analysis reported an 
estimated HIV prevalence of 19.1% in TGW, with a 49-times greater risk for HIV acquisition when 
compared to adult cisgender men (CGM) and women (CGW) of reproductive age14. Based on clinical 
trial data, HIV prevalence amongst TGW was highest in the United States, South America, Indonesia, 
and India14. Further, compared to their cisgender counterparts, TGW sex workers are four times more 
likely to be living with HIV. Due to the disproportionate risk for HIV acquisition in TGW, PrEP uptake 
and efficacy is critical. However, the impact of GAHT on PrEP pharmacology, and vice versa, has not 
been extensively characterized. Therefore, a thorough pharmacological evaluation of the relationship 
between GAHT and PrEP is needed to ensure sufficient protection against HIV acquisition and to 
understand the potential impact of PrEP on hormonal therapies.  
 
The PrEP-GAHT Interactions in TGW protocol will allow for the closer evaluation of the two-way 
pharmacokinetic-pharmacodynamic (PK-PD) interaction of GAHT (estrogen with testosterone 
suppression) on TDF/FTC PrEP in TGW through the completion of a five-phase PK-PD study, in 

TGW GAHT Interaction Study 
 
 
 
 
20 
 
 
 
26th, April 2023 
which TGW take daily PrEP, followed by a testosterone suppressing agent alone and in conjunction 
with low- and high-dose estrogen therapy. 
 
2.1 
 Background on gender affirming estrogen-based therapy in TGW 
 
Based on a survey of more than 7,000 transgender and gender non-conforming individuals in the 
U.S., 72% of TGW reported accessing GAHT, with higher percentages of hormonal use in 
transgender individuals undertaking transition-related surgeries4. The WPATH and the Endocrine 
Society have published transgender-focused guidelines that include estrogen therapy as the 
foundation for achieving feminization in TGW6,15. Via a negative feedback mechanism, elevated 
concentrations of estrogens can inhibit the hypothalamic release of gonadotropin releasing hormone 
(GnRH), thereby disrupting the hypothalamic-pituitary-testis axis, resulting in the reduction of 
testosterone concentrations16-20. Estrogen therapy, through interactions with the estrogen receptor, 
can stimulate feminizing characteristics, including changes in fat distribution, reduction of male-
pattern hair growth, and dermatological changes, as well as decreased frequencies of erections and 
sexual desire21. However, estrogen therapy alone is typically insufficient to achieve maximal 
androgen suppression, and consequently, adjunctive therapies, including anti-androgenic 
medications or GnRH agonists, are co-administered as part of gender affirming feminizing therapeutic 
regimens. Physical changes may occur in the first 3-12 months post-initiation of hormonal 
replacement therapy. 
 
Estrogen-based regimens used for feminization in TGW are administered at dosages higher than 
those used in the treatment of CGW for pre- and post-menopausal symptoms. Oral estrogen doses 
range from 1 mg/daily to 8 mg/daily for transition management15,22,23.  Transdermal estradiol patches 
release from 0.025 to 0.2 mg/day; intramuscular injectable doses range from 2-10 mg of estrogen 
conjugates weekly24. Estrogens can be administered as the “bioidentical” human 17β-estradiol (orally 
or transdermally), as synthetic or semisynthetic hormone conjugates (such as estradiol valerate and 
estradiol cypionate), or as equine estrogens. Supraphysiological doses of estradiol are associated 
with significant adverse effects, including venous thromboembolic disease (VTE); van Kesteren and 
colleagues observed a 20-fold increase in VTE in TGW using the semisynthetic ethinyl estradiol24-28. 
Ethinyl estradiol, consequently, is not recommended by the Endocrine Society in the treatment and 
management of TGW15. However, VTE is likely a risk with all formulations. Estrogens are also 
associated 
with 
a 
moderate 
risk 
for 
macroprolactinoma, 
coronary 
artery 
disease, 
hypertriglyceridemia, cholelithiasis, and hypertension15.  A summary of recommended estrogen-
based hormone preparations and dosing has been published by the Center of Excellence for 
Transgender Health (Table 3). 
 
Table 3. Estrogen preparations and dosing recommendations by the Center of Excellence for 
Transgender Health29 (modified). 
 
Estrogen 
Initial-low 
Initial 
Maximum 
Estradiol 
oral/sublingual 
1 mg/day 
2-4 mg/day 
8 mg/day 
Estradiol 
transdermal 
50 mcg 
100 mcg 
100-400 mcg 
Estradiul valerate 
intramuscular 
<20 mg IM 
Q2W 
20 mg IM 
Q2W 
40 mg IM 
Q2W 

TGW GAHT Interaction Study 
 
 
 
 
21 
 
 
 
26th, April 2023 
Estradiol 
cypionate 
intramuscular 
<2 mg IM 
Q2W 
2 mg IM 
Q2W 
5 mg IM 
Q2W 
 
 
 
2.2 
 Rationale for Gonadotropin Releasing Hormone Agonists for Androgen Suppression 
 
Adjunctive medications are typically required to reduce testosterone concentrations to reflect levels 
observed in CGW30. Commonly used in feminizing GAHT, the mineralcorticoid receptor antagonist 
spironolactone has a weak affinity for the androgen receptor, resulting in reduced testosterone binding 
through receptor competition31. However, the efficacy of spironolactone in decreasing the effects of 
testosterone in TGW is suboptimal32,33. Therefore, other drugs have been used to further promote 
feminization in TGW. GnRH agonists disrupt the hypothalamus-pituitary-testis axis via initial 
stimulation and subsequent desensitization of the hypothalamic GnRH receptor; suppression of 
GnRH results in decreased production of luteinizing hormone (LH) and follicle stimulating hormone 
(FSH) from the pituitary gland34,35. The terminal effect of chronic administration of GnRH agonists is 
decreased testosterone production, and they are commonly used in the treatment of prostate cancer. 
While these agonists have been used to suppress pubertal development, they have also shown 
efficacy in demasculinization in TGW22,36–38. In a cohort of 60 TGW treated with monthly doses of the 
GnRH agonist goserelin acetate (3.8 mg) and 6 mg/daily 17β-estradiol valerate, serum total and free 
testosterone concentrations decreased from 18.75 to 0.53 nmol/L and 0.396 to 0.004 nmol/L, 
respectively22. There was also an 8.9-fold increase in serum estradiol concentrations, with no 
significant adverse effects observed. Retrospective studies have also demonstrated that the GnRH 
agonists cyproterone acetate and leuprolide acetate are also effective in the reduction of LH, FSH, 
and testosterone concentrations39. Notably, adverse effects were not observed when these agents 
were used in conjunction with low- to medium-dose estrogen therapies. Also, while cyproterone 
acetate is not approved by the FDA for use in the United States, long-acting leuprolide acetate is, and 
is marketed as a monthly 3.75 mg or three-month 11.25 mg intramuscular depot LupronTM.  Previous 
studies in a prostate cancer cohort demonstrated that one injection of LupronTM resulted in an initial 
increase of testosterone concentrations 3 days post-injection, but by 21 days post-administration, 
78.7% of CGM achieved medical castration (testosterone concentrations <0.2 ng/mL; Figure 1)40.  
 
 
Figure 2. Reduction in testosterone concentrations in cisgender men post-leuprolide acetate administration 
from a clinical study.  Mean plasma testosterone concentrations reached Cmax of 606 ng/mL 3 days post-injection.  By 

TGW GAHT Interaction Study 
 
 
 
 
22 
 
 
 
26th, April 2023 
14 days post-injection, mean concentrations were reduced by 4-fold.  By Day 21, 78.7% of study participants reached 
total testosterone concentrations <0.2 ng/mL.  Adapted from Marberger et al.40. 
 
The use of GnRH agonists, in conjunction with low-dose estrogen, may mitigate some of the side 
effects associated with high-dose estrogen-containing regimens, and is more potent in its inhibition 
of testosterone production as compared to anti-androgenic agents (which do not directly inhibit the 
hypothalamic production of GnRH), resulting in increased demasculinization. 
 
2.3 
PrEP Clinical Trials and Pharmacology in TGW 
 
While TGW have been enrolled in HIV clinical trials evaluating PrEP uptake and efficacy, their 
numbers remain small, understudied, and overall findings may not be applicable to them5,41,42. Within 
the iPrEx trial and the subsequent open label extension cohort, 14% of participants who received 
PrEP self-identified as TGW. While incidence of seroconversion did not differ between cisgender men 
who have sex with men (MSM) and TGW, TGW exhibited lower PrEP metabolite concentrations41. In 
PrEP clinical trials, while non-adherence is likely the dominant cause of suboptimal drug 
concentrations, numerous other factors could modify drug concentrations in addition to adherence. 
For example, in CGW, vaginal dosing of TFV in the presence of vaginal dysbiosis is associated with 
lower systemic concentrations of TFV, and is attributed to decreased intravaginal microbiome-
mediated TFV metabolism, sufficient enough to reduce the protective efficacy of a TFV gel43,44. We 
and another group have recently observed reduced TFV and FTC concentrations in TGW taking 
estrogens, possibly reducing the HIV protection effects of PrEP. 
 
TDF has been evaluated for interactions with hormonal contraceptives. Pharmacokinetic (PK) findings 
demonstrated mean TFV Cmax and area under the curve (AUC) of 340 ng/mL and 2970 ng-hr/mL, 
respectively, in CGW taking a norgestimate-ethinyl estradiol oral contraceptive agent; these TFV 
findings were consistent with historical data in CGW not on oral contraceptives45. At the cellular level, 
however, in vitro studies from the female genital tract have demonstrated decreased NRTI uptake in 
the presence of ethinyl estradiol and etonogestrel in several cell types, including dendritic and CD8+ 
cells46. Further, CD4+ cells from the female genital tract showed decreased TFV-DP concentrations 
in the presence of progesterone +/- estradiol47. It has also been reported that progesterone and 
testosterone impact NRTI phosphorylation in peripheral blood mononuclear cells (PBMC), indicating 
another endocrine effect on TFV metabolism48. Nicol, et al., reported differential expression of drug 
transporters between colorectal and vaginal tissue, including a 2.9-fold increase in the ABCC2 gene 
in vaginal tissue as compared to colorectal tissue49. ABCC2 encodes the multidrug resistance protein 
2, which is involved in estradiol glucuronide clearance50. In a recent review, Anderson and colleagues 
discussed the research gaps in understanding PrEP efficacy in TGW, and highlighted the need for 
well-controlled clinical trials to more comprehensively describe the impact of hormonal therapies in 
TGW on PrEP7. 
 
To begin to address this knowledge gap regarding the potential impact of GAHT on PrEP 
pharmacology, our group conducted a Center for AIDS Research (CFAR)-funded pilot study to 
characterize the potential differences in PrEP PK between CGM and TGW51. The transgender 
community, in Baltimore and more broadly, voiced concerns that, despite the high risk of HIV infection 
in TGW and the promise of PrEP to reduce that risk, there remains substantial concern that PrEP 
drugs might adversely impact GAHT5. This concern, for which there was no evidence to the contrary, 
creates a reluctance to initiate PrEP among TGW. To begin to address these concerns, we enrolled 
8 CGM not on exogenous estrogen and 8 TGW on exogenous estrogen for gender affirmation. Study 
participants were administered 300 mg TDF/200 mg FTC under direct observation for seven days, 
enough to establish steady state concentrations based on our prior experience52. Unable to fund the 

TGW GAHT Interaction Study 
 
 
 
 
23 
 
 
 
26th, April 2023 
enrollment of larger numbers or to afford the time to recruit estrogen-naïve TGW, we enrolled TGW 
on a variety of estrogen regimens (Table 4). Within the transgender cohort, serum estradiol 
concentrations needed to exceed 100 pg/mL, which is within the normal reference limits for 
premenopausal CGW and consistent with the upper limit of normal for postmenopausal CGW on 
HRT50. In addition, 75% of TGW participants also used the anti-androgenic compound spironolactone.   
 
Table 4. GAHT used by TGW enrolled in CFAR pilot51. 
 
 
 
 
 
 
 
 
 
 
 
On the 8th day of daily observed oral TDF/FTC PrEP dosing, we collected blood for PrEP drug PK 
testing as well as estradiol, LH, FSH, and total testosterone. Colorectal biopsies were also collected 
for localized PK and ex vivo explant challenge by HIV to estimate HIV susceptibility. Plasma TFV 
trough concentrations (C24) in TGW were lower by 32% (p = 0.010) when compared to CGM; AUC0-
24 trended 27% lower (p = 0.065) and clearance (CLss/F) trended 38% higher (p = 0.065) in TGW 
compared to CGM.  Plasma TFV Cmax and V/F were not different between gender cohorts. In TGW, 
plasma FTC trough (C24) and AUC0-24 were lower by 32% (p = 0.038) and 24% (p = 0.028) 
respectively, while CLss/F was higher by 31% (p = 0.028) when compared to CGM; V/F trended higher 
in TGW by 26% (p = 0.065) when compared to CGM. FTC Cmax was not different between cohorts. 
There were no statistically significant PK differences between TGW and CGM in PBMC or colon tissue 
for any analytes. However, our pilot was only powered to detect several-fold differences in 
phosphorylated metabolites. As noted, we did not control for estrogen regimens or use of 
spironolactone.   
 
 
Figure 3. AUC0-24 of (a) plasma TFV (left) and (b) FTC (right) in CGM (solid line) and TGW (dashed line). 
 
Despite 100% adherence observed throughout our study, 38% and 13% of TGW in our study 
appeared to have TFV plasma and PBMC TFV-DP, respectively, which fell below daily adherence 
PID 
Estrogen 
Spironolactone 
(mg) 
1010 
Premarin 1.25 mg daily 
0 
1011 
Estradiol 6 mg po daily 
200 
1012 
Premarin 2.5 mg daily 
50 
1017 
Estradiol 0.5 mg IM every 2 wks 
50 
1018 
Estradiol valerate IM 20 mg every 2 wks 
200 
1019 
Estradiol valerate IM 40 mg every 2 wks 
+ Premarin 6.25 mg daily 
0 
1020 
Estradiol 20 mg IM every 2 weeks 
100 
1021 
Estradiol 1.5 mg IM every week 
200 

TGW GAHT Interaction Study 
 
 
 
 
24 
 
 
 
26th, April 2023 
benchmarks from HPTN 066 – used to identify persons with less than daily dosing and potentially 
reduced PrEP protection.   
 
To further corroborate these findings, the Thai Red Cross reported that estradiol valerate 2 mg and 
cyproterone acetate 25 mg reduced TFV concentrations by 17% (AUC0-24), while TDF/FTC did not 
affect plasma estrogen or testosterone concentrations53. In contrast to the Hopkins pilot study, the 
Thai Red Cross study did not assess FTC concentrations or any intracellular concentrations of TFV-
DP and FTC-TP; similarly, both studies used estrogen-experienced research participants. In contrast 
with U.S. practices, the Thai study also used cyproterone for testosterone suppression, which is not 
licensed for marketing in the U.S., as well as relatively low estrogen doses compared to typical U.S. 
doses. 
 
There are a number of studies that have aimed to understand the interplay between antiretrovirals 
and GAHT; however, each differs in their study design and population.  A summary of ongoing or 
completed PrEP/ART trials in TGW are summarized below (Table 5): 
 
Table 5. Planned, completed, and ongoing PrEP/ART trials in TGW.  
Study 
PI 
N 
GAHT 
Naïve 
Estrogen  
Regimen 
Androgen  
Regimen 
Adherence 
Measure 
Renal  
Function 
Status 
Hopkins R01a 
Marzinke 
20 
yes 
estradiol 
 (1 mg, 6 mg) 
leuprolide 
 acetate 
DOT 
MEMS 
mGFR, eGFR 
cystatin C 
Planned 
Hopkins CFARb 
Shieh 
8 
no 
various 
spironolactone 
DOT 
eGFR 
Completed 
Thai Red Crossb 
Hiransuthikul 
20 
no 
estradiol 
valerate 
cyproterone  
acetate 
unclear 
- 
Completed 
Nebraskac 
Cirrincione 
15 
no 
estradiol  
(po, sl, td) 
spironolactone 
unclear 
- 
Ongoing 
UNC Chapel Hillc 
Cottrell 
4 
no 
varied 
unclear 
unclear 
- 
Ongoing 
TG Youth & 
PrEPc 
Hosek 
24 
no 
estradiol 
spironolactone 
video DOT 
none 
Ongoing 
 
2.4 
The Contribution of Hormonal Therapies on Renal Function  
 
Recently, two observational studies demonstrated short-term changes in creatinine concentrations in 
transgender individuals in a GAHT-dependent fashion54,55. Decreased creatinine concentrations 
(implying improved renal clearance) was observed in TGW on a variety of estrogen-based GAHT. 
In our pilot, 75% of both CGM and TGW were African American. BMI was higher in TGW (median 
30.1) as compared to CGM (22.7), p=0.05. The most striking difference between the two cohorts was 
in regards to renal function. eGFR using the CKD-EPI equation was estimated as 133 mL/min/BSA 
in TGW and 111 mL/min/BSA in CGM (p=0.002). This difference was also statistically significant using 
the eGFR MDRD equation (p=0.005), as well as estimated creatinine clearance using the Cockcroft-
Gault equation (p=0.01). These data suggest differential renal function in the presence of exogenous 
estrogens; however, neither the duration of time on therapy nor direct evaluation of filtration capacity 
were evaluated in this cohort.   
 
Conversely, exogenous testosterone in transgender men (TGM) resulted in increased creatinine 
concentrations over time55. Previous studies have also demonstrated a similar, short-term impact of 
hormonal therapies on renal function. Acutely, diabetic CGW with a history of hypertension have 
shown improved creatinine clearance rates and decreased proteinuria when placed on estrogen-
based hormone replacement therapy (HRT) consisting of estradiol and norgestrel56. Potential 
explanations for the impact of exogenous estrogens on improved renal function include hormonal 
effects on proximal tubule secretion, changes in muscle mass, or changes in renal vasculature.   

TGW GAHT Interaction Study 
 
 
 
 
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2.5 
Background and Rationale of Planned PrEP-GAHT Interactions in TGW Assessments 
 
Because the interaction between GAHT and PrEP is poorly understood, an investigation on the impact 
of exogenous hormonal therapies on PrEP effectiveness (and vice versa) is required to optimize 
dosing regimens for HIV protection, while also not impacting the effects of GAHT on reducing gender 
dysphoria. Thus, safety, PK, and PD are highly relevant readout in the evaluation of the relationship 
between GAHT and PrEP.  
2.5.1 Safety Assessments 
Safety assessments includes history/physical, chemistry/hematology labs at screening and 
asssement of adverse events, collection of safety labs, review on concomitant medications, and 
interim history and targeted exams, as needed, during study visits and throughout the study. 
2.5.2 Drug Analysis  
TFV, FTC, and their metabolites will be measured using validated, liquid chromatographic- mass 
spectrometric (LC-MS) assays in the CPAL (Dr. Marzinke’s laboratory)52,57.  Post PBMC-lysis and 
tissue homogenization, drugs will be isolated from specimen sources using either protein precipitation 
(PrEP in plasma, tissue) or anion exchange chromatography and solid phase extraction (PrEP 
metabolites in PBMC and tissue) approaches52,58,59.  Analytes will undergo chromatographic 
separation and drug-specific ion detection via mass spectrometry.  Assay lower limits of quantification 
are as follows:  plasma TFV and FTC: 0.31 ng/mL; tissue TFV and FTC: 0.05 and 0.25 ng/sample, 
respectively; tissue and PBMC TFV-DP and FTC-TP: 5 fmol/sample and 50 fmol/sample, 
respectively.  All assays are validated in accordance with the FDA, Guidance for Industry: 
Bioanalytical Method Validation recommendations60.  The NIH-funded Clinical Pharmacology Quality 
Assurance (CPQA) Program peer-reviews all bioanalytical methods. Further, the CPAL participates 
in CPQA proficiency testing for plasma TFV and FTC assays semi-annually61. Dr. Marzinke’s 
laboratory is also CLIA-certified and CAP-accredited. 
2.5.3 Ex vivo colon tissue explant HIV challenge 
Endoscopic colonic biopsies, acquired during PK-intensive visits, can be challenged ex vivo with HIV 
(R5 HIVBal in our studies) to assess the alterations of susceptibility to HIV infection. With parallel PK 
testing, this readout can provide evidence of changing protection from HIV over time. Ex vivo 
challenges were previously conducted in our pilot TGW study, and we observed no statistically 
significant difference in HIV acquisition between TGW and CGM at PrEP steady state. These data 
suggest that while there may be an impact of hormonal therapies on systemic TFV and FTC 
concentrations, susceptibility of HIV acquisition in colorectal tissue was not impacted by feminizing 
therapies. As with phosphorylated analyte concentrations, however, we only had power to detect 
several-fold changes in explant tissue HIV susceptibility drug regimens.  Further, there were a variety 
of feminizing GAHT used by study participants.  The designed PrEP-GAHT Interactions in TGW study 
will definitively evaluate testosterone ablation or dose-escalated estrogen administration on HIV 
susceptibility in colonic tissue. 
2.5.4 Direct Measurement of Glomerular Filtration 
Creatinine clearance and eGFR are indirect estimates of renal filtration.  Therefore, GFR will be 
empirically measured (mGFR) through the administration and evaluation of the exogenous molecule 
iohexol, which is cleared solely by glomerular filtration without tubular secretion or reabsorption62-64.  

TGW GAHT Interaction Study 
 
 
 
 
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Post-intravenous administration, blood samples will be collected over a 4-hour period and analyzed 
via by liquid chromatography-UV (LC-UV) at the University of Minnesota.   
2.5.5  Hormone Analysis 
Safety hormone concentrations, including serum testosterone concentrations, will be measured by 
the CLIA-certified, CAP-accredited Johns Hopkins Hospital Core Laboratories.  To formally evaluate 
the impact of Truvada® on hormone concentrations, batched analysis of hormone concentrations will 
be conducted.  Serum total and free testosterone concentrations, as well as estradiol measurements, 
will be conducted via LC-MS analysis.  LH and FSH measurements will be analyzed via 
immunoassay.  Samples will be tested at the Brigham and Women’s Brigham Research Assay Core, 
which is a CLIA- certified laboratory accredited by the Joint Commission.   
2.5.6  Additional Biomarkers of Renal Function      
Additional renal markers will also be measured, including cystatin C, which is a 13 kDa lysosomal 
cysteine proteinase inhibitor that is freely filtered at the glomerulus, and is completely resorbed and 
catabolized at the proximal tubules65.  Cystatin C has also been used as a marker of renal function, 
and its production and clearance is independent of changes in muscle mass66,67.  Samples collected 
for cystatin C will be sent to the Johns Hopkins Hospital Core Laboratories for analysis. 
2.5.7 Assessment of Participant Adherence with Study Product/Intervention(s)  
To maximize participant safety of and adherence to Truvada® and estrogen-based hormonal 
therapies, adherence will be monitored using several complementary tools to evaluate Truvada® and 
estrogen adherence while on study.  Adherence will be primarily evaluated via direct observed therapy 
(DOT).  PrEP and hormone doses will be directly observed by study personnel either in person or by 
live video streaming (e.g., FaceTime, WhatsApp). In the high-dose estrogen phase of the clinical trial, 
estrogen dosing may be split to a morning and an evening dose; only one dose per day will be 
monitored using one of the aforementioned strategies. An additional measure to evaluate adherence 
will be the participant’s provision of their pill containers to study visits.  IDS will perform a manual pill 
count.  Subjects who demonstrate poor adherence, as determined by study staff, will be required to 
take the remaining medication under in-person DOT.  If the subject misses more than one dose across 
two of the PK periods, they will be removed from the study. A participant may be replaced at the PI’s 
discretion 
2.5.8 Assessment of Gender Dysphoria 
Periodic assessments of gender dysphoria will be conducted at baseline and a convenient time during 
PK visits throughout the study.  This will be achieved by asking research participants to rate how they 
feel about their gender throughout the study. 
 
2.6 
Description of Study Products 
 
Tenofovir disoproxil fumarate/emtricitabine (TDF/FTC; Truvada®) 
TFV is licensed as the prodrug form, TFV disoproxil fumarate (TDF) which is available in a 300 mg 
dosage form as well as in fixed dose combination with emtricitabine (200 mg), also an HIV nucleoside 
reverse transcriptase inhibitor (NRTI), marketed as Truvada® and indicated for prevention of HIV 
infection in persons at risk of HIV infection as well as for treatment in combination with another NRTI 
and a more potent antiretroviral drug.  TFV is also marketed in other prodrug forms, including TFV 
alafenamide fumarate (TAF); however, the TDF prodrug form will be evaluated in this phase 1 study. 
 

TGW GAHT Interaction Study 
 
 
 
 
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26th, April 2023 
Tenofovir disoproxil fumarate, an acyclic nucleoside phosphonate diester analog of adenosine 
monophosphate, is initially converted to tenofovir by diester hydrolysis and further undergoes 
phosphorylation by cellular enzymes to form tenofovir diphosphate. In turn, tenofovir diphosphate 
competes with deoxyadenosine 5'-triphosphate, which is then incorporated into DNA resulting in chain 
termination, thus, preventing the activity of HIV-1 reverse transcriptase. It is also a weak inhibitor of 
mammalian DNA polymerase alpha, beta, and mitochondrial DNA polymerase gamma. 
 
Emtricitabine is a synthetic nucleoside analog of cytidine. The active metabolite, emtricitabine 5'-
triphosphate, inhibits the activity of HIV-1 reverse transcriptase by competing with the natural 
substrate deoxycytidine 5'-triphosphate, which is then incorporated into nascent viral DNA resulting 
in chain termination. It is also a weak inhibitor of mammalian DNA polymerase alpha, beta, epsilon 
and mitochondrial DNA polymerase gamma. 
 
Leuprolide Acetate (Lupron Depot®) 
Leuprolide acetate is a synthetic nonapeptide gonadotropin-releasing hormone or luteinizing 
hormone-releasing hormone (GnRH or LH-RH) analog that exhibits a potent reversible inhibition of 
gonadotropin secretion, through suppression of testicular and ovarian steroidogenesis. Initial 
administration of the drug results in an increased luteinizing hormone (LH) and follicle stimulating 
hormone (FSH) which leads to a transient rise in gonadal steroids, however, its continuous 
administration results in reduced LH and FSH concentrations. 
 
Estradiol 
Estrogens regulate the transcription of a limited number of genes and act through binding to nuclear 
receptors in estrogen-responsive tissues. Estrogens also modulate the pituitary secretion of the 
gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), through a negative 
feedback mechanism reducing the levels of these hormones seen in postmenopausal women. 
Estradiol is the principal intracellular human estrogen and is substantially more potent than its 
metabolites at the receptor level. 
 
Future Application of PrEP-GAHT Interactions in TGW.  
Taken together, the PrEP-GAHT Interactions in TGW study will provide criticial information on the 
two-way impact between gender affirming femininizing therapies and PrEP.  Using a traditional drug-
drug interaction study design, these five dosing phases will evaluate the influence of PrEP on GAHT 
pharmacology as well as the roles that testosterone ablation and dose-escalated estrogen have on 
systemic and localized PrEP concentrations.  The findings from this work may result in personalized 
PrEP dosing regimens for TGW on GAHT to maximize PrEP efficacy.   
 
3 
OBJECTIVES AND ENDPOINTS 
 
3.1 
Objectives 
 
The global objective of this study is to evaluate the two-way pharmacokinetic-pharmacodynamic (PK-
PD) interaction of GAHT (estrogen with testosterone suppression) on TDF/FTC PrEP in TGW through 
the completion of a five-phase PK-PD study, in which TGW take daily PrEP, followed by a 
testosterone suppressing agent alone and in conjunction with low- and high-dose estrogen therapy  
Study objectives including safety, pharmacokinetics (PK) and pharmacodynamics (PD) are essential 
in understanding the impact of co-administration of PrEP and GAHT on downstream drug and 
hormone concentrations. 

TGW GAHT Interaction Study 
 
 
 
 
28 
 
 
 
26th, April 2023 
 
3.1.1 Objectives & Endpoints 
 
Table 2: Study Objectives and Endpoints 
*as defined by the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, 
Corrected Version 2.1 - July 2017, and Addendum 3 (Rectal Grading Tables for Use in Microbicide Studies) 
 
4 
STUDY DESIGN 
 
4.1 
Identification of Study Design 
 
This is a Non-IND phase I, open label study of a 5 period drug-drug interaction study to better define 
the magnitude of PrEP-GAHT drug-drug interactions in order to assure protective dosing in TGW for 
whom both PrEP and GAHT are indicated and desired. 
 
After completing a screening evaluation, up to 40 individuals will be enrolled to complete 20 evaluable 
participants at the Johns Hopkins University (JHU).  The 20 HIV-negative, self-identifying TGW should 
be naïve GAHT (including estrogen-based therapies, spironolactone, and progesterone) or willing to 
abstain from feminizing therapies until serum total testosterone concentrations exceed 200 ng/dL 
(cisgender male reference interval).  Baseline testosterone concentrations will be evaluated at 
screening; if participants have testosterone concentrations below 200 ng/dL, testosterone levels will 
be re-evaluated every 2-4 weeks for up to 6 weeks until they exceed 200 ng/dL.  If testosterone levels 
remain <200 ng/dL after 6 weeks, the participant will be considered a screen failure. 
 
Once enrolled and when testosterone concentrations are ≥ 200 ng/dL, eligible participants will receive 
300 mg TDF/200 mg FTC (Truvada®, Gilead Sciences), once daily for seven days under 
aforementioned direct observation (in person observation, live video streaming, or with a time-
stamped video) to achieve steady state drug PrEP concentrations.  After one week of therapy, 
participants will undergo intensive PK analysis as well as collection of colorectal biopsies for PD 
testing (PK1).  During the PK-intensive day, iohexol will be administered intravenously for the 
Objectives 
Endpoints & Data Analysis 
• Describe the impact of GAHT 
on TDF/FTC PrEP PK-PD 
• Describe the bi-directional 
impact of TDF/FTC & GAHT 
combinations 
• Non-compartmental analysis of TFV/FTC in plasma and 
urine 
• Concentrations of all analytes in blood, PBMC, & colorectal 
tissue Compare PK1 to PK2, PK3, PK4 for GAHT impact on 
PrEP 
• Compare PK5 to PK2-4 for PrEP impact on GAHT 
• Describe the impact of GAHT 
on PrEP suppressed HIV 
susceptibility  
• Describe changes in explant p24 antigen under all PK 
conditions 
• Correlation and Emax model of drug concentration-p24 
relationship  
• Explain the mechanism of 
estrogen impact on PrEP PK 
• Mechanistic modeling of PK change  
• Renal changes with addition of GAHT 
• Describe adverse effects 
associated with PrEP & GAHT 
• Grade 2 or higher AEs* 
• Assess gender dysphoria will 
participants are on study 
product 
• Gender dysphoria evaluations at baseline and during PK1 
through PK5 

TGW GAHT Interaction Study 
 
 
 
 
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26th, April 2023 
empirical determination of renal function and mGFR.  While concurrently on PrEP, participants will 
then be intramuscularly administered depot leuprolide acetate (11.25 mg Lupron®).  Two weeks post-
injection, when testosterone concentrations are below the lower limit of normal of total testosterone 
in cisgender men (typically < 200 ng/dL), sampling for PK, PD, and renal function will be performed 
(PK2).  Participants will then immediately begin low-dose oral estrogen therapy (1 mg 17β-estradiol) 
in conjunction with PrEP for one week, at which time samples will be collected for analyses described 
above (PK3).  While still on PrEP, participants will then transition to high-dose estrogen therapy (6 
mg 17β-estradiol) for the remainder of the study.  One-week post-high dose estrogen therapy in the 
presence of PrEP, pharmacological and renal samples will be collected for analysis (PK4).  PrEP will 
then be discontinued, and two weeks later, samples will be collected to assess renal function and 
hormonal concentrations, and evaluate the presence of any remaining PrEP in plasma, PBMC, or 
colorectal tissue (though it should be near undetectable levels for most analytes according to our prior 
data) (PK5).  Safety assessments, including history/physical, chemistry/hematology labs at screening 
and interim history will be performed at each study-intensive visit, as needed.  To ensure compliance, 
participants will have PrEP and hormone dosing monitored via direct observation by study personnel 
either in person, by live video streaming, or with a timestamped video.  In the high-dose estrogen 
phase of the clinical trial, estrogen dosing may be split to a morning and an evening dose; only one 
dose per day will be monitored using one of the aforementioned strategies. 
 
Overview of study visits and detailed study events are shown in the Study Schema and outlined in 
Schedule of Events in Appendix I.  
 
STUDY DRUGS & STUDY 
WEEK 
WK 0 
WK 1 
WK 2 
WK 3 
WK 4 
WK 5 
WK 6 
WK 7 
TDF/FTC 300mg/200mg po qd 
  
  
  
  
  
  
  
Leuprolide acetate 11.25 mg sc 
  
  
  
  
  
  
  
Estradiol 1 mg po qd 
  
  
Estradiol 6 mg po qd 
  
  
  
  
PK VISIT # & PK 
ASSESSMENTS 
  
PK1 
  
PK2 
PK3 
PK4 
  
PK5  
TDF/FTC 
x 
x 
x 
x 
  
Low/No Testosterone 
x 
x 
x 
x 
Estradiol 
  
  
  
  
Low 
High 
  
High 
PD ASSESSMENTS 
 
 
 
 
 
 
 
 
FSH/LH 
x 
x 
 
x 
x 
x 
 
x 
Colorectal HIV infectivity 
x 
x 
 
x 
x 
x 
 
x 
Figure 1.  Study Schema for PrEP-GAHT Interactions in TGW Protocol. Dosing and PK sampling phases indicates 
TDF/FTC PrEP alone (PK1), followed by testosterone suppressing GnRH agonist leuprolide acetate (PK2), then two levels 
of estradiol (low-dose [PK3] and high dose-estrogen [PK4]), before cessation of PrEP while on high-dose estrogen (PK5). 
The iterative dosing phases allow assessment of two-way drug-drug interactions: PrEP on GAHT and vice versa. 
Additionally, the effect of full suppression of testosterone on PrEP, distinct from the estrogen effect, is enabled. 
 
During each PK-intensive day, blood will be collected prior to administration to a PrEP dose, followed 
by collection at 1, 2, 3, 4, , and 24 hours for TFV ad FTC quantification; blood will also be collected 
for PBMC isolation at pre-dose and 24 hour time points. Urine will be collected and pooled 0-4 h and 
4-24 h post-dose for TFV, FTC and creatinine.  Serum will be collected pre-dose on each PK-intensive 
day for the quantification of estradiol, testosterone, LH, FSH, and cystatin C.  For further PK/PD 
analysis, flexible sigmoidoscopy with rectal tissue biopsies will be collected to measure systemic drug 
concentrations in colorectal tissue.  Biopsies will also be used for ex vivo infectivity assays.  The 
sampling schedules and laboratory processing procedures will be described in the Study Specific 
Procedures (SSP) manual.  At each study visit, particpants will have two peripheral intravenous (PIV) 

TGW GAHT Interaction Study 
 
 
 
 
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26th, April 2023 
access lines inserted (if applicable and the participant’s vascular access allows), one will be used for 
frequent blood sampling and the second PIV will be used for the administration of iohexol for the direct 
assessment of renal function.  Following iohexol administration, blood GFR sampling will be obtained 
pre-dose and at 2 and 4 hours  hours post-injection.   This will occur at baseline and at each PK phase 
of the study.  
Periodic assessments of gender dysphoria will be also be conducted at baseline and at a convenient 
time during PK visits throughout the study (which coincide with steady-state on each sequential drug 
regimen). This assessment will involve  asking research participants to rate  their response to the 
following statements, based on a 1-5 scale (1 meaning ‘completely disagree’ and , 5 meaning 
‘completely agree’.) 
• I feel good about my gender today. 
• I feel bad about my gender today. 
 
4.2 
Description of Study Population 
 
This study will include HIV-negative TGW who meet the criteria outlined in Sections 5.4 and 5.5. 
 
4.3 
Time to Complete Accrual 
 
Participant accrual will take approximately 3 years.   
 
4.4 
Expected Duration of Participation 
 
Each participant will be on study for approximately 4 months (up to 2 months in screening and 56 days 
under observationon study drugs). Total study duration is about 4 years. 
 
5 
STUDY POPULATION 
 
5.1 
Selection of Study Population 
 
The inclusion and exclusion criteria outlined in Sections 5.4 and 5.5 will be utilized to ensure the 
appropriate selection of study participants. 
 
5.2 
Recruitment 
 
Participants will be recruited from a variety of sources, using the following key strategies: 
 
• Listings of prior research participants who have given informed consent to be reached for future 
studies for which they may be eligible 
• Participant referrals (participants refer their friends or partners who may meet eligibility criteria) 
• Passive self-referral: interested individuals see an IRB-approved study poster, flyer, brochure, 
website, or social media posting advertising the study and call the study site directly 
• Recruitment services that will use social media-based recruitment strategies involving the posting 
of IRB-approved recruitment materials 
 
Referrals from other study coordinators and providers, including referrals from care providers of the 
Chase-Brexton Clinic, and TGW patients under the clinical care of Dr. Adrian Dobs. In order to assist 

TGW GAHT Interaction Study 
 
 
 
 
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with medical management of GAHT following study protocol completion, and to ensure appropriate 
management of any gender dysphoric symptoms that occur while on study, all participants will be 
required to be under the care of a health care provider for transgender health management. Tha name 
and contact information of the health care provider will be recorded in the study record. 
 
Study staff will meet as needed to discuss current recruitment status, targets, and strategies.  Included 
in the plans will be measures to specifically target recruitment of TGW, including community outreach 
to encourage referrals by word of mouth, and use of IRB-approved recruitment materials designed to 
specifically target recruitment among these populations. Staff also will follow-up with all persons who 
express an interest in the study to ensure that screening appointments are scheduled and carried out 
in a timely manner. 
 
5.3 
Retention 
 
Once participants enroll in this study, the study site will make every effort to retain them for the 
duration of follow-up in order to minimize possible bias associated with loss-to-follow-up. Beginning 
with the informed consent process, components of such procedures include: 
 
• Thorough explanation of the study visit schedule and procedural requirements during the consent 
process with re-emphasis at each study visit.   
• Thorough explanation of the importance and inter-relatedness of all three product doses and 
sampling phases to the overall success of the study.  
• Use of appropriate and timely visit reminder mechanisms (via email and/or telephone/text). 
• Immediate and multifaceted follow-up (i.e. phone/text, email, and/or any other communication 
methods available at the disposal of site’s study staff) on missed visits. 
• The protocol principal investigator in consultation with the site investigator will determine if research 
participants who fail to complete all intensive PK visits should be replaced. 
 
5.4 
Inclusion Criteria 
 
Individuals who meet the following criteria are eligible for inclusion in this study: 
 
1. 
18 years of age or older 
2. 
Self-identifying as a transgender woman 
3. 
Not currently taking any gender affirming hormonal therapy (GAHT) with a total testosterone 
concentration of ≥ 200 ng/dL, or willing to abstain from feminizing therapies (including 
estradiol, spironolactone, progesterone, etc.) until total total testosterone concentrations are 
≥ 200 ng/dL. Note: Testosterone may be retested every 2-4 weeks during screening to 
determine eligibility up to 6 weeks. 
4. 
HIV-1 uninfected at screening as documented by Combo Ag/Ab HIV-1/HIV-2 immunoassay 
5. 
Understand and agree to local STI reporting requirements 
6. 
Able and willing to communicate in English 
7. 
Able and willing to provide written informed consent to take part in the study 
8. 
Able and willing to provide adequate information for locator purposes 
9. 
Able and willing to participate in a directly observed study, which may occur in person, using 
livestreaming or a timestamped video? 

TGW GAHT Interaction Study 
 
 
 
 
32 
 
 
 
26th, April 2023 
10. 
Availability to return for all study visits, barring unforeseen circumstances 
11. 
Willing to abstain from insertion of anything (drug, enema, penis, or sex toy) in rectum for 72 
hours before and 72 hours after each flexible sigmoidoscopy  
12. 
Willing to refrain from aspirin and NSAID use for one week before and after each study 
biopsy visit 
13. 
Willing and able to use condoms for all RAI for the duration of participation 
14. 
Willing and able to participate in a directly observed study, which may occur in person, using 
livestreaming or a timestamped video 
15. 
Has an identified healthcare provider for transgender health management 
16. 
Agree not to participate in other research studies involving drugs and/or medical devices for 
the duration of the study 
 
5.5 
Exclusion Criteria 
 
Individuals who meet any of the following criteria at screening will be excluded from the study: 
 
1. 
Not currently on any PrEP regimen (e.g., Truvada®, Descovy®) 
2. 
History of chronic Hepatitis B infection, as documented by positive HBsAg at screening 
3. 
≥ Grade 2 laboratory abnormality at baseline as defined by Division of AIDS Table for 
Grading the Severity of Adult and Pediatric Adverse Events, Corrected Version 2.1 - July 
2017, and Addendum 3 (Rectal Grading Tables for Use in Microbicide Studies) 
4. 
Significant colorectal symptom(s) as determined by medical history or by participant self-
report (including but not limited to presence of any unresolved injury, infectious or 
inflammatory condition of the local mucosa, history of inflammatory bowel disease, 
presence of symptomatic hemorrhoids, and presence of any painful anorectal conditions 
that would be tender to manipulation) 
5. 
At screening or within the past 2 months: participant-reported symptoms and/or clinical or 
laboratory diagnosis of active rectal infection requiring treatment per current CDC 
guidelines or symptomatic urinary tract infection (UTI).  Infections requiring treatment 
include Chlamydia (CT), gonorrhea (GC), syphilis, active HSV lesions, chancroid, genital 
sores or ulcers, and, if clinically indicated, genital warts. Note that HSV seropositivity with 
no active genital lesions is not an exclusion criterion. (Note: if an STI apart from HIV is 
detected, the participant will be referred for treatment and can be retested in 30 days and 
rescreened once.) 
6. 
History of an underlying clinically significant cardiac arrhythmia or renal disease (including 
creatinine clearance < 60 mL/min using Cockcroft-Gault equation)  
7. 
Serum phosphate < 2.3 mg/dL 
8. 
History of severe or recent cardiac or pulmonary event 
9. 
History of significant gastrointestinal bleeding 
10. 
Current use of warfarin or heparin or other anticoagulant medications associated with 
increased risk for bleeding following mucosal biopsy (e.g., daily high dose aspirin [>81 mg], 
NSAIDs, or Pradaxa®) 
11. 
Use of systemic or anorectal immunomodulatory medications within 4 weeks of enrollment 
or planned use at any time during study participation 

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33 
 
 
 
26th, April 2023 
12. 
Per participant report, use of any rectally administered products containing N-9 (including 
condoms) or investigational products within 4 weeks of enrollment, or planned use of either 
at any time during study participation 
13. 
Known allergic reaction to TFV, FTC, leuprolide acetate, or estradiol or other components 
of the test articles 
14. 
Current known HIV-infected partner(s) 
15. 
Symptoms suggestive of acute HIV seroconversion at screening and enrollment 
16. 
Any other condition or prior therapy that, in the opinion of the investigator, would preclude 
informed consent, make study participation unsafe, make the individual unsuitable for the 
study or unable to comply with the study requirements. Such conditions may include, but 
are not limited to, current or recent history of severe, progressive, or uncontrolled 
substance abuse, or renal, hepatic, hematological, gastrointestinal, endocrine, pulmonary, 
neurological, or cerebral disease. 
6 
STUDY PRODUCTS 
 
6.1 
Regimen 
 
After completion of written informed consent and all screening procedures, eligible participants will 
be asked to come to the DDU research clinic for baseline evaluations, including laboratory 
assessment of hormonal status and biopsies for baseline HIV explant challenge PD assessment 
before any study drugs are administered. At this baseline visit, participants will be dispensed one-
week supply of 300 mg TDF/200 mg FTC (Truvada®, Gilead Sciences) PrEP.  Participants will take 
one pill daily for seven days under direct observation, starting the day after the baseline study visit, 
to achieve steady state PrEP drug concentrations.   
 
After 6 days of Truvada® PrEP dosing via DOT, participants will return to the DDU research clinic to 
undergo the first intensive 24-hour PK sampling visit (Visit # 9, PK1 D1 Visit), including collection of 
colorectal biopsies for PK and PD testingand administration of intravenous iohexol for the empirical 
determination of renal function and GFR.  At completion of the 24-hour PK sampling visit (Visit #10, 
PK1 D2 Visit), participants will start another 7 days of Truvada® PrEP via DOT and receive an 
intramuscular injection of depot leuprolide acetate (11.25 mg Lupron Depot®).  
 
Participants will continue to take Truvada® PrEP via DOT for 5 days, after which, they will return to 
the DDU research clinic for their 7th dose of Truvada® PrEP via in-person DOT, a 1 week refill of 
Truvada® PrEP and a safety assessment (Visit #16, Day 14).  
 
After 6 days of Truvada® PrEP dosing via DOT, when testosterone concentrations are projected to 
be below the lower limit of normal of total testosterone in cisgender men (typically < 200 ng/dL), 
participants will return to the DDU research clinic to undergo the second intensive 24-hour PK 
sampling visit (Visit #23, PK2 D1 Visit) including collection of colorectal biopsies for PK and PD testing 
and administration of intravenous iohexol for the empirical determination of renal function and GFR.  
At completion of the 24-hour PK sampling (Visit #24, PK2 D2 Visit), participants will start another 7 
days of Truvada® PrEP via DOT and begin daily low-dose oral estrogen therapy (1 mg 17β-estradiol), 
taken concomitantly with Truvada® PrEP via DOT. 
 
 
After 5 days of low-dose estradiol in combination with Truvada® PrEP dosing via DOT, participants 
will return to the DDU research clinic to undergo the third intensive 24-hour PK sampling visit (Visit 

TGW GAHT Interaction Study 
 
 
 
 
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#30, PK3 D1 Visit) including collection of colorectal biopsies for PK and PD testing and administration 
of intravenous iohexol for the empirical determination of renal function and GFR.  At completion of 
the 24-hour PK sampling (Visit #31, PK3 D2 Visit), participants will start another 7 days of Truvada® 
PrEP via DOT and transition to high-dose estrogen therapy (6 mg 17β-estradiol), which they will 
continue on for 2 weeks.  The high-dose estrogen therapy may be split into two administration periods, 
one taken in the AM, and one taken in the PM, andonly the estrogen dose taken concomitantly with 
the Truvada® PrEP dose will be monitored via DOT  
 
After 5 days of high-dose estradiol in combination with Truvada® PrEP dosing via DOT, participants 
will return to the DDU research clinic to undergo the fourth intensive 24-hour PK sampling visit (Visit 
#37, PK4 D1 Visit) including collection of colorectal biopsies for PK and PD testing and administration 
of intravenous iohexol for the empirical determination of renal function and GFR.  Participants will 
complete Truvada® PrEP dosing at this visit. At completion of the 24-hour PK sampling (Visit #38, 
PK4 D2 Visit), participants will start another 7 days of high-dose estrodiol via DOT without Truvada® 
PrEP.  Participants will continue to take the high-dose estrodial via DOT without Truvada® PrEP, for 
5 days, after which, they will return to the DDU research clinic for their 7th dose of high-dose estrogen 
therapy via in-person DOT, a one-week refill of high-dose estrodiol and a safety assessment (Visit 
#44, Day 42).  
 
After 6 days of high-dose estradiol dosing via DOT without Truvada® PrEP, participants will return to 
the DDU research clinic to undergo the fifth and final intensive 24-hour PK sampling visit (Visit #51, 
PK5 D1 Visit) including collection of colorectal biopsies for PK and PD testing and administration of 
intravenous iohexol for the empirical determination of renal function and GFR. Participants will 
complete the high-dose estradiol at this visit, may elect to stay on estrogen after completion of the 
study, and this will be coordinated with their primary care providers.  Participants will return the next 
day for the final 24-hour PK sampling visit (Visit #52, PK5 D2 Visit).   Participants will complete the 
high-dose estradiol at this visit, may elect to stay on estrogen after completion of the study, and this 
will be coordinated with their primary care providers. 
 
After 6 days, participants will return to the DDU research clinic for the final safety visit (Visit # 53). 
 
6.2 Administration 
 
Figure 1. Schema of PK Dosing Phases 
STUDY DRUGS & STUDY 
WEEK 
WK 0 
WK 1 
WK 2 
WK 3 
WK 4 
WK 5 
WK 6 
WK 7 
TDF/FTC 300mg/200mg po qd 
  
  
  
  
  
  
  
Leuprolide acetate 11.25 mg sc 
  
  
  
  
  
  
  
Estradiol 1 mg po qd 
  
  
Estradiol 6mg po qd 
  
  
  
  
PK VISIT # & PK 
ASSESSMENTS 
  
PK1 
  
PK2 
PK3 
PK4 
  
PK5  
TDF/FTC 
x 
x 
x 
x 
  
Low/No Testosterone 
x 
x 
x 
x 
Estradiol 
  
  
  
  
Low 
High 
  
High 
PD ASSESSMENTS 
 
 
 
 
 
 
 
 
FSH/LH 
x 
x 
 
x 
x 
x 
 
x 
Colorectal HIV infectivity 
x 
x 
 
x 
x 
x 
 
x 
 
6.3 Study Product 
 

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26th, April 2023 
Table 6.  Study products to be administered during this study. 
Trade Name  Study Drug 
Formulation 
Dose Period 
Regimen  
Truvada®   
tenofovir disoproxil fumarate  
emtricitabine 
(fixed dose combination) 
300 mg 
200 mg 
Baseline 
through Visit 4 
1 x 300mg/200mg tablet 
daily with food 
EstraceTM 
17β-estradiol  
17β-estradiol 
1 mg 
2 mg 
Visit 3-4 
Visit 4-7 
1 x 1 mg tablet daily (1 mg) 
3 x 2 mg tablet daily (6 mg) 
Lupron 
Depot® 
leuprolide acetate  
11.25 mg 
Visit 1 
prefilled syringe injection  
6.3.1 Tenofovir/Emtricitabine 
Tenofovir/Emtricitabine: A daily oral dose of TDF/FTC 300 mg/200mg (Truvada®) will be prescribed 
and dispensed at baseline visit (V 2, day 0) and continued over 5 weeks.  
The dose selected is the FDA-recommended oral PrEP dose.   
6.3.2 Leuprolide acetate 
Lupron Depot® injection 11.25 mg will be administered at Visit #10 (Day 8). The syringe contains 
sterile lyophilized microspheres, which is leuprolide incorporated in a biodegradable copolymer of 
lactic and glycolic acids. The product is available in a prefilled dual-chamber syringe containing 
sterile lyophilized microspheres which, when mixed with diluent, become a suspension. The product 
is supplied as a kit, containing: 
• 
one prefilled dual-chamber syringe 
• 
one plunger 
• 
alcohol swabs 
• 
a complete prescribing information enclosure 
 
Kits should be stored at 25ºC, with excursions permitted to 15-30 ºC. The suspension will be prepared 
in the Investigational Drug Service Pharmacy and the injection will be administered by a qualified 
DDU clinician. The entire contents of the suspension is injected intramuscularly in the ventrogluteal 
muscle with the needle inserted completely at a 90 degree angle.  The syringe should be discarded 
if not used within 2 hours. 
6.3.3 Estrace® 
Estradiol: A daily oral low-dose of estradiol (Estrace®) 1mg, will be prescribed and dispensed at Visit 
#24 (PK2 D2, Day 22) and continued for 7 days.  At Visit #31 (PK3 D2, Day 29), the total daily dose 
of Estrace® will be increased to 6 mg, taken as three 2-mg tablets, daily through Visit #51 (PK5 D1, 
Day 49). Estrace® tablets will be dispensed by the Johns Hopkins IDS and provided to the participants 
by the DDU staff. The drug product should be stored at 20-25ºC (USP Controlled Room Temperature). 
6.3.4 Iohexol  
Iohexol (Omnipaque™ 240; Omnipaque™ 300; Omnipaque™ 350): Iohexol will be prepared and 
administered in accordance with practices outlined in the Omnipaque™ prescribing information. 
Specifically, participants will be encouraged to maintain good hydration 24 hours prior to and 24 hours 
following iohexol administration. A single volume dose of Omnipaque™ will be administered 
intravenously during the intensive sampling visits so that a total dose of 1500 mg of iohexol is 
delivered over two minutes. This is the usual adult dosage for measurement of GFR. The 
concentration of Omnipaque™ used in any individual participant will be kept consistent for all iohexol 
dose administrations during their participation in the protocol. The iohexol injection will be prepared 
and dispensed by the Johns Hopkins IDS Pharmacy and administered by a qualified clinician in the 

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Johns Hopkins University Institute for Clinical Research (ICTR) unit. Omnipaque™ should be stored 
at controlled room temperature, 20°-25°C (68°- 77°F); excursions permitted to 15°-30°C (59°-86°F) 
[see USP Controlled Room Temperature]. Samples will be analyzed at the University of Minneosta.   
 
6.4 
Study Product Supply and Accountability 
 
The Hopkins Investigational Drug Service (IDS) Pharmacy will be used to support the study, which 
includes labeling, drug storage, dispensing and obtaining drug supplies.  Inventory management such 
as recordkeeping requirements, tracking of drug supply, and accounting for all doses dispensed will 
also be done through IDS. 
 
6.5 
Accountability 
 
All study products will be available to the study staff through the designated site pharmacy or 
compounding pharmacy, once they have been labeled appropriately by the PoR. The PoR is required 
to maintain complete records of all study products received and dispensed.  
 
6.6 
Dispensing 
 
Study products will be dispensed from the pharmacy to the designated study staff for an enrolled 
participant, upon receipt of a written prescription from an authorized prescriber.  
 
6.7 
Retrieval of Unused Product 
 
Any dispensed, unused study product will be returned to the investigational drug pharmacy.   
 
6.8 
Concomitant Medications and Procedures 
 
With the exception of medications listed as prohibited, enrolled study participants may use 
concomitant medications during study participation.  All concomitant medications, over-the-counter 
preparations, vitamins and nutritional supplements, recreational drugs, and herbal preparations 
reported throughout the course of the study will be recorded in the source documents and on case 
report forms designated for that purpose. 
 
6.9 
Prohibited Medications and Procedures 
 
Participants will be advised to refrain from RAI or any practices which include rectal insertion of any 
product, including those used during sexual intercourse (drug/medication, penis, other objects, sex 
toy, or douche) for 72 hours before and after each flexible sigmoidoscopy with biopsy collection. 
 
Study participants will be prohibited from using the following medications throughout the study period: 
heparin (including Lovenox®), warfarin, Plavix® (clopidogrel bisulfate), immunomodulatory 
medications, rectally administered products containing N-9 (including over-the-counter products), or 
investigational products. Other drugs, such as aspirin or NSAIDS that are associated with increased 
likelihood of bleeding following mucosal biopsy, should be restricted for use prior to flexible 
sigmoidoscopy as required by clinical guidelines. 
 

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Based on the Truvada® U.S. prescribing information, concomitant use of TFV with medications that 
decrease renal function or compete for active tubular secretion may increase concentrations of TFV 
or other renally eliminated medications. 
 
If participants report using any of these medications or products while on study or within the specified 
time frame noted, their study product administration will be put on hold, pending discussions with 
study site investigator and/or DAIDS MO. Study staff will actively review medication use with each 
research participant during screening and throughout study participation. Participants will also be 
queried as to their adherence regarding insertion of rectal products 72 hours before and after each 
biopsy collection visit. 
 
6.5 
Recommended Procedures 
 
Condoms will be provided for use by all participants enrolled in this study.  Using condoms will be 
recommended for all sexual encounters where the participant will be engaging in receptive anal 
intercourse (RAI), for the duration of study participation. In addition to instructions regarding 
abstinence prior to and following flexible sigmoidoscopy with biopsy, participant education about risk 
reduction will be provided. These will be included in instructions provided as Pre-Post visit reminders 
and STI risk reduction. In the event that a participant needs additional supply of condoms between 
visits, she may request these from study staff at any time.   
 
7 
STUDY PROCEDURES 
 
7.1 
Procedure overview 
 
This section includes information on visit-specific study procedures. An overview of the study visits 
and evaluations is available in protocol Appendix I:  Schedule of Events. Unless otherwise specified, 
the laboratory procedures listed in this section are performed at the local study site laboratories.  
 
7.2 
Visit 1 - Screening Visit  
 
Prescreening via a phone call or email will take place with potential participants. Participants 
interested in the study can request that the informed consent documents be mailed out or emailed to 
them prior to the Screening Visit. 
 
Written informed consent will be obtained at the screening before any procedures are initiated.  For 
participants who do not meet the eligibility criteria, screening will be discontinued once ineligibility is 
determined. Participants who do not meet the eligibility criteria may be rescreened at a later date, if 
appropriate, as in the case of a concomitant medication washout period. In such cases, the length of 
the washout period will be determined by the PI based on safety considerations and the elimination 
half-life of the concomitant medication.  Screening labs may be repeated once to determine eligibility. 
 
The following activities will take place at the screening visit: 
 

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Table 10:  Visit #1 - Screening Visit 
Component 
Procedure/Analysis 
Administrative 
 
• Obtain written informed consent 
• Assign participant ID (PTID) 
• Assess eligibility 
• Obtain demographic and locator information 
• Provide counseling: 
o HIV/STI risk reduction counseling 
o HIV pre/post test counseling 
o Participant education: flexible sigmoidoscopy procedure with rectal 
tissue biopsy 
Clinical 
• Obtain complete medical history, documenting pre-existing conditions 
• Review and record concomitant medications 
• Perform general physical exam 
• Obtain vital signs – blood pressure (BP), heart rate (HR), temperature 
(temp), height (inches) and weight (kg) 
Safety and 
Screening Labs*   
• Collect blood specimens for: 
• Complete Blood Count (CBC) with differential 
• Comprehensive Metabolic Panel (CMP) 
• Prothrombin Time (PT)/Partial Thromboplastin Time (PTT) 
• PO4 
• Lipid Panel 
• Estradiol 
• Testosterone, total only 
• HBsAg  
• HIV testing  
• Syphilis testing (confirmatory test as needed) 
• Collect rectal swab for: 
• Rectal NAAT for GC/CT 
 
* Screening laboratory values are valid up to 60 days.  Additional tests can be ordered for abnormal POCT urine dipstick results. 
 
7.3 Visit #2 – Baseline Visit (Day 0) 
 
The Baseline visit serves to establish pre-dose conditions for safety assessments, hormone levels, 
kidney function, and PD assessment of HIV challenge of colon explant tissue.  
 
If deemed eligible based on the results from the screening visit, participants will be contacted to 
schedule a baseline visit and asked to refrain from RAI and the insertion of anything in their anorectum 
for 72 hours prior to the visit.  
 
The Baseline Visit must take place ≤60 days after the Screening Visit. If >60 days pass before the 
Baseline Visit, participants will need to be re-screened and re-consented. 
 
The following activities will take place at the baseline visit:   
 

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Table 11: Visit #2 – Baseline Visit (Day 0) 
Component 
Procedure/Analysis 
Administrative 
• Confirm participant understood and signed informed consent 
• Review/update locator information 
• Confirm eligibility 
• Ensure participant complied with pre-visit instructions  
• Provide counseling and education: 
o HIV/STI risk reduction counseling 
o Pre/Post visit reminders 
o Flexible sigmoidoscopy with rectal tissue biopsy information sheet 
 
Clinical 
• Review/update medical history 
• Review/update concomitant medications 
• Review any available test results 
•  Conduct gender dysphoria assessment 
• Perform symptom-directed physical exam, as needed 
• Obtain vital signs (HR, BP, temp, weight) 
• Insert PIV x2 (if applicable) 
Safety Labs:  
• Collect blood specimens for: 
• Complete Blood Count (CBC) with differential 
• Comprehensive Metabolic Panel (CMP) 
• PO4 
• Estradiol 
• Testosterone, total only 
• HIV testing   
Study Product 
• Adminster Iohexol 
• Dispense 7 day supply of TDF/FTC 
Study Labs:  
• Collect blood specimens for: 
• 
Estradiol 
• 
Testosterone, free and total 
• 
LH/FSH 
• 
Cystatin C  
Study Labs: 
GFR 
• Collect blood specimens for GFR calculations: 
• 
Pre-Dose 
• 2hrs 
• 4hrs 
 
Cleansing 
Douche 
• Cleansing douche will be administered at least one hour prior to flexible 
sigmoidoscopy  
Rectal 
Specimens 
• Perform flexible sigmoidoscopy with rectal tissue biopsy collection (5) 
 
 

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7.4 
Directly Observed Therapy (DOT) Visits (Conducted prior to Intensive PK Sampling 
Visits 
 
The following activities will take place at the DOT visits: 
 
Table 12: DOT Visits 
 
Component 
Procedure/Analysis 
Clinical 
• Assess/Update AEs 
• Review/update medical history 
• Review/update concomitant medications 
• Perform symptom-directed physical exam as needed 
Study Product 
• Participant will take TDF/FTC or estradiol dose under direct observation 
by study personnel. 
 
 
7.5 
Visits # #23, #30, #37 and #51 - Intensive PK Sampling Visits Day 1 (PK D1 Visits) 
 
The following activities will take place at the PK Day 1 Visits: 

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Table 13: Intensive PK Sampling Visits Day 1 (PK D1 Visits) 
Component 
Procedure/Analysis 
Administrative 
• Review/update locator information 
Ensure participant complied with pre-visit instructions  
• 
Adherence monitoring 
Clinical 
• Assess/Update AEs 
• Review/update medical history 
• Review/update concomitant medications 
• Review any available test results 
• Conduct gender dysphoria assessment 
• Perform symptom-directed physical exam, as needed 
• Collect vital signs (HR, BP, temp, weight) 
• Insert PIV x2 (if applicable) 
Safety Labs 
• Collect blood specimens for: 
• Serum Cr 
• Testosterone, only total (Visit #23, PK2 D1 only) 
• HIV Ag/Ab  
Study Labs 
• Collect blood specimens for: 
• Estradiol 
• Testosterone, free and total 
• LH/FSH 
• Cystatin C 
Study Labs: 
GFR  
• Collect blood specimens for GFR calculations: 
Pre-Dose 
• 2hrs 
• 4hrs 
 
Study Labs: PK 
and PBMC 
• Collect blood specimens for PK: 
• Pre-Dose 
• 1hr 
• 2hrs 
• 
3hrs4hr 
•  Collect blood specimens for PBMC 
• Pre-Dose 
Study Labs: 
Urine 
• Collect urine specimens for TFV/FTC and Creatinine 
• 0-4hrs 
• 4-24 hrs (collected at home by participant) 
 
 
 
7.6 
Visits #10, #24, #31, #38, #52  - Intensive PK Sampling Visits Day 2 (PK D2 Visits) 
 

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The following activities will take place at the PK Day 2 Visits: 
 
Table 14: Intensive PK Sampling Visits Day 2 (PK 24hr Visits) 
Component 
Procedure/Analysis 
Administrative 
• Provide counseling and education: 
o HIV/STI risk reduction counseling 
o Pre/Post visit reminders 
Flexible sigmoidoscopy with rectal tissue biopsy information sheet 
Clinical 
• Assess/Update AEs 
• Review/update medical history 
• Review/update concomitant medications 
• Review any available test results 
• Perform symptom-directed physical exam, as needed 
• Collect vital signs (HR, BP, temp) 
Study Labs:  PK 
and PBMC 
• Collect blood specimens for PK: 
• 24hrs 
• Collect blood specimens for PBMC: 
• 
24 hrs 
• Collect 4-24hrs Urine  
 
Cleansing 
Douche 
• 
Cleansing douche will be administered at least one hour prior to flexible 
sigmoidoscopy  
Rectal 
Specimens 
• Perform flexible sigmoidoscopy with rectal tissue biopsy collection (5) 
Study Product 
• Dispense TDF/FTC (Visits #10, 24, 31) 
• Inject Lupron Depot (Visit #10)  
• Dispense Estradiol, 1 mg (Visit #24) 
• Dispense Estradiol, 6 mg (Visits #31, 38) 
 
 
7.7 
Visits #16, #44 and #53 - Safety Visits 
 
The following activities will take place at the Safety Visits: 
 
 
 
 
 
 
 
 

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Table 15: Safety Visits 
Component 
Procedure/Analysis 
Administrative 
• Review/update locator information 
• Provide counseling and education: 
o HIV/STI risk reduction counseling 
o Pre/Post visit reminders 
 
Clinical 
• Assess/update AEs 
• Review/update medical history 
• Review/update concomitant medications 
• Review any available test results 
• Perform symptom-directed physical exam, as needed 
• Collect vital signs (HR, BP, temp) 
Safety Labs 
• Collect blood specimens for: 
• CBC with differential1 
• CMP1 
• PO41 
• Lipid Panel2 
• Testosterone2, total only 
• Syphilis2 
• HIV testing1 
• Collect rectal swab for: 
• Rectal NAAT for GC/CT2 
Study Product  
• Dispense TDF/FTC (Visit #16) 
• Dispense Estradiol 6 mg (Visit #44) 
1 Safety labs collected at Visit #16 and #53 
2 Safety labs collected only at Visit #53 
 
 
 
7.8 
Exit Follow-up Phone Call 
 
After the final study visit, follow-up call or early termination visit, a final contact may be required to 
provide laboratory test results and post-test counseling.  All final contacts will be documented in 
participant study records.   
 
7.9 
Interim Visits 
 
Interim visits may be performed at any time during the study.  All interim contacts and visits will be 
documented in the source documentation and on applicable case report forms.   
 
Some Interim Visits may occur for administrative reasons.  For example, the participant may have 
questions for study staff.  Other interim visits may occur for clinical purpsoes, such as to measure 
testosterone levels or in response to AEs experienced by study participants.  When interim visits are 
completed in response to participant reports of AEs, study staff will assess the reported event clinically 
and provide or refer the participant to appropriate medical care, if clinically indicated.   

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The following activities will take place at Interim Visits: 
 
Table 16: Interim Visit 
Component 
Procedure/Analysis 
Administrative 
• Review/update locator information 
•  
Clinical 
• Assess/update AEs 
• Review/update medical history 
• Review/update concomitant medications 
• Review any available test results 
• Perform symptom-directed physical exam, as needed 
• Obtain vital signs (HR, BP, Temp), as needed 
Specimens 
• Collect specimens as appropriate/indicated 
 
7.5 
Participants Who Withdraw or Are Withdrawn from the Study 
 
If the participant withdraws or is withdrawn from the study after receiving study product, an Early 
Termination Visit will be conducted, if possible. 
 
The following activities will take place at the Early Termination Visit: 
 
Table 17: Early Termination Visit 
Component 
Procedure/Analysis 
Administrative 
• Review/update demographic and locator information 
• Review STI risk reduction counseling and provide condoms 
Clinical 
• Assess/update AEs 
• Review/update medical history 
• Review/update concomitant medications 
• Review any available test results 
• Perform symptom-directed physical exam, as needed 
• Obtain vital signs (HR, BP, Temp) 
Safety Labs and 
STI Screening 
• Collect blood specimens for: 
• CBC with differential 
• CMP 
• Lipid Panel 
• Estradiol 
• Testosterone, total only 
• HIV Testing  
• Syphilis 
• Collect rectal swab for: 
• Rectal NAAT for GC/CT 
 
 
7.6 
Participants Who Become Infected with HIV 
 

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During the study protocol, subjects will be regularly tested for HIV exposure and queried for high-risk 
behaviors. If there is a concern of possible HIV exposure, HIV and STI screening will be offered at 
that time. If a participant self-reports seroconversion while on study, or if one of the HIV tests 
performed during the study protocol is confirmed positive, study staff will provide counseling and direct 
the participant to the appropriate HIV care.   
 
Study staff will also request documentation of lab results or, failing availability of test results, will 
perform HIV Testing, with confirmatory testing as needed. These results will be kept in the subject’s 
study chart and recorded on study case report forms (CRFs).   
 
Participants will be withdrawn from the study if they test positive for HIV antibody or RNA. Any 
participant who tests positive for HIV while enrolled in the study will be immediately removed from the 
study and referred to the appropriate care provider for additional counseling and treatment as 
necessary. HIV 4th generation EIA and RNA test results will be available prior to any subsequent 
study intervention. No additional doses of study product will be dispensed. Participants will be 
compensated for their study participation up until the time of removal. 
 
7.10 Clinical Evaluations and Procedures 
 
The following history and physical exam components will be conducted at select visits. 
 
Medical History: 
• Each participant will have a complete review of their medical history at screening and will be asked 
about any changes to their medical history at each visit. 
• Concomitant medications will be reviewed and recorded at screening and updated as needed at 
every study visit. 
 
Physical Exam: 
• A complete physical examination will be completed at screening and a targeted symptom-directed 
physical exam will be done during subsequent study visits as needed. 
• Height (at screening only) and Weight (at screening and PK iohexol visits only) 
• Vital signs 
o Heart Rate/Pulse 
o Blood Pressure 
o Temperature 
 
Rectal Exam, Cleansing Douche, and Rectal Specimen Collection: 
The participant will be positioned in the left lateral decubitus position for the following procedures:  
• Rectal exam: The examiner will conduct a visual examination of the anus and surrounding area 
and note any abnormality.  The examiner will then insert a lubricated (Good Clean Love, Good 
Clean Love, Inc. Eugene, OR) gloved finger into the anal canal and sweep around the internal 
anal circumference. 
• Rectal specimen collection:  
o Swabs for GC/CT will be inserted into the rectum and placed in contact with the rectal 
wall, turned 360 degrees and then removed.  
• A pre-procedural cleansing douche will be self-administered by the participant prior to all flexible 
sigmoidoscopies.  

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• Rectal tissue biopsy collection via flexible sigmoidoscopy: Up to 5 biopsies will be taken via a 
flexible sigmoidoscope at approximately 15-20 cm using large-cup biopsy forceps.  
 
 
 
 
7.11 Laboratory Evaluations 
 
7.11.1 Clinical Laboratory Testing 
 
The local clinical laboratory or designee will run the following, as indicated: 
• Blood specimens: 
• CBC with differential 
• CMP, which includes blood urea nitrogen (BUN), creatinine (Cr), aspartate 
aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase test 
(ALP), sodium (Na), potassium (K), calcium (Ca), carbon dioxide (CO2), chloride (Cl), 
glucose, albumin, total bilirubin, total protein, and calculated eCRF (calculated by the JH 
laboratories using CKD-EPI) 
• Phosphate (PO4) 
• HBsAg 
• HIV Testing 
• Syphilis Testing 
• Testosterone, total only 
• Estradiol 
• Rectal swabs: 
• Rectal GC/CT (NAAT) 
 
7.11.2 
Research Laboratory Testing 
 
Johns Hopkins University: 
• Plasma and PBMC cell lysate for PK (Marzinke) 
• Colonic tissue homogenate and extracted cell lysates for PK/PD (Marzinke) 
• Urine for PK and creatinine 
• Cystatin-C 
 
University of Minnesota:  
• Iohexol  
 
Brigham and Women’s Hospital: 
• Testosterone, total and free 
• Estradiol 
• LH 
• FSH 
 
 
7.12 Specimen Collection, Handling, and Processing 
 

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Study sites will adhere to the standards of good clinical laboratory practice, DAIDS Laboratory 
Requirements and site standard operating procedures for proper collection, processing, labeling, 
transport, and storage of specimens at the local laboratories.   
(http://www.niaid.nih.gov/LabsAndResources/resources/DAIDSClinRsrch/Documents/gclp.pdf)  
 
Specimen collection, testing, and storage at the site laboratories will be documented per standard 
site practice. In cases where laboratory results are not available due to administrative or laboratory 
error, sites are permitted to re-draw specimens that are intended for use in the screening as well as 
ongoing safety assessments process. Specimens will be handled in accordance with Requirements 
for DAIDS Sponsored and/or Funded Laboratories in Clinical Trials.   
 
7.13 Storage of Specimens for Future Use 
 
Study staff will store all specimens collected according to site SOPs. Specimens will not be labeled 
with any personal identifiers.  Storage of all study samples will follow local standard operating 
procedure to ensure the anonymity and confidentiality of the trial research participants. Specimens 
remaining at the end of the study will be transferred to a designated central bio-repository after all 
protocol-required and quality assurance testing has been completed. Study participants’ permission 
to do so will have been documented by signing informed consent which will include appropriate IRB-
approved language. All screening samples for individuals who do not eventually enroll in the study 
will be destroyed. 
 
7.14 Biohazard Containment 
 
As the transmission of blood-borne pathogens can occur through contact with contaminated needles, 
blood, and blood products, appropriate blood and secretion precautions will be employed by all 
personnel in the drawing of blood and shipping and handling of all specimens for this study, as 
currently recommended by the United States Centers for Disease Control and Prevention. All 
infectious specimens will be transported in accordance with US regulations 42 Code of Federal 
Regulations (CFR) 72. 
 
8 
ASSESSMENT OF SAFETY 
 
8.1 
Safety Monitoring 
 
The study site investigator is responsible for continuous close safety monitoring of all study 
participants and for promptly alerting the PI and the protocol team if unexpected concerns arise. A 
sub-group of the protocol team, including the grant PI, co-PI, and study site investigatorswill serve as 
the Protocol Safety Review Team (PSRT). Study status reports will be submitted to the PSRT every 
month for review.  
 
These reports will include all adverse events (AEs) reported for the study, regardless of their 
relationship to the study products.  
 
The study team will meet, as needed, throughout the period of study implementation to review the 
safety data, discuss product use management, and address any potential safety concerns. 
 
8.2 
Clinical Data Safety Review 
 

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A multi-tiered safety review process will be followed for the duration of this study.  The study site 
investigators are the first layer and are responsible for the initial evaluation and reporting of safety 
information at the participant level, and for promptly alerting the PSRT if unexpected concerns arise.  
In the event of any study participant experiencing an AE ≥ Grade 3, the study site investigator will 
review and discuss the appropriate study management with the PSRT.   
 
During the trial, the PSRT will review status reports described above and conduct calls to clarify the 
data as appropriate.  The content, format and frequency of the safety reports will be agreed upon in 
advance of study implementation.  If necessary, external experts representing expertise in the fields 
of HIV drug pharmacology, biostatistics, and medical ethics may be invited to review the events.  
 
8.3 
Adverse Events Definitions and Reporting Requirements 
8.3.1 Adverse Events 
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 a study product whether or not considered related to the product. The term 
“investigational product” for this study refers to TDF/FTC, leuprolide acetate, and estradiol. 
 
Study participants will be instructed to contact the study site staff to report any AEs they may 
experience at any time between enrollment and completion of their participation.  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 site 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, until the occurrence resolves (returns to 
baseline) or stabilizes.  
 
The study site investigators will determine AE resolution or stabilization in their best clinical judgment, 
but may seek medical consultation regarding follow-up or additional evaluations of an AE from the 
PSRT. Study site staff will document in source documents and CRFs all AEs reported by or observed 
in enrolled study participants, regardless of severity and presumed relationship to study products. 
AEs will be graded by the standard Division of AIDS Table for Grading the Severity of Adult and 
Pediatric Adverse Events, Corrected Version 2.1 - July 2017.  
 
8.3.2 Serious Adverse Events 
Serious adverse events (SAEs) will be defined as AEs occurring at any dose that: 
 
• Results in death 
• Is life-threatening 
• Results in persistent or significant disability/incapacity 
• Is a congenital anomaly/birth defect 
• Requires inpatient hospitalization or prolongations of existing hospitalization 
 

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Note: Per ICH SAE definition, hospitalization itself is not an adverse event, but is an outcome of the 
event. Thus, hospitalization in the absence of an adverse event is not regarded as an AE, and is not 
subject to expedited reporting. The following are examples of hospitalization that are not considered 
to be AEs:  
o Protocol-specified admission (e.g. for procedure required by study protocol)  
o 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)  
o Diagnostic admission (e.g. for a work-up of an existing condition such as persistent 
pretreatment lab abnormality)  
o Administrative admission (e.g. for annual physical)  
o Social admission (e.g. placement for lack of place to sleep)  
o Elective admission (e.g. for elective surgery)  
 
Important medical events that may not result in death, be life-threatening, or require hospitalization 
may be considered a serious adverse drug experience when, based upon appropriate medical 
judgment, they may jeopardize the research subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above. 
8.3.3 Adverse Event Relationship to Study Product 
The relationship of all AEs to study product will be assessed by the study investigators. The 
relationship categories that will be used for this study are: 
 
• Related:  There is a reasonable possibility that the AE may be related to the study agent(s) 
• Not related: There is not a reasonable possibility that the AE is related to the study agent(s) 
 
8.4 
Expedited Adverse Event (EAE) Definitions and Reporting Requirements  
 
Expedited Adverse Event Reporting  
Events that meet SAE criteria and are unexpected (not previously reported to the agency or included 
in the IB) must be reported in an expedited manner. The study Principal Investigator will assume 
responsibility for the reporting of Serious Adverse Events to the Johns Hopkins Medicine Institutional 
Review Board (JHM IRB).  Copies of this final report will be forwarded to the DAIDS PO for this 
protocol.   
 
Unexpectedly Fatal or Life-threatening Event  
Unexpectedly fatal or life-threatening events, will be reported to the JHM IRBvia telephone and fax 
as soon as possible, but no more than seven calendar days after receipt of the information. Any 
additional information received will be sent to the IRB as soon as possible, but no more than 15 days 
after receipt of the information. 
 
Serious and Unexpected Event  
Serious and unexpected events must be reported to the JHM IRB within 15 days.  Also required to 
be reported to the agency within 15 days are any laboratory findings suggesting significant risk for 
human subjects including: 
• Carcinogenicity 
• Mutagenicity 

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26th, April 2023 
• Teratogenicity 
 
Follow-Up Information 
Additional relevant information received about an event must be reported as soon as possible to the 
JHM IRB with a copy to the DAIDS PO, in no more than 15 days.  
 
Report Format 
Safety reports will be drafted by the study team, in consultation with the Principal Investigator and 
DAIDS PO using the JHM IRB Protocol Event Reporting format.  
 
For all EAEs submitted, the study site must file an update to the DAIDS PO with the final or stable 
outcome unless the initial EAE submitted had a final or stable outcome noted already.  
 
 
Grading Severity of Events 
The Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, Corrected 
Version 2.1 dated July 2017 and Addendum 3 (Rectal Grading Tables for Use in Microbicide Studies) 
will be the primary tools for grading adverse events for this protocol.    In cases where an AE is 
covered in multiple tables, Addendum 3 (Rectal Grading Table for Use in Microbicide Studies) will be 
the grading scale utilized.   
 
The DAIDS AE Grading Tables are available on the RSC website at  
https://rsc.niaid.nih.gov/clinical-research-sites/daids-adverse-event-grading-tables 
 
 
EAE Reporting Period 
• 
The expedited AE reporting period for this study is defined as the entire study duration for an 
individual participant (from enrollment until the participant’s final study contact).   
• 
After the protocol-defined AE reporting period, unless otherwise noted, only suspected 
unexpected serious adverse reactions will be reported to the DAIDS PO if the study staff becomes 
aware of the events on a passive basis (from publicly available information). 
 
8.5 
Reporting of Adverse Reactions to the Responsible IRBs 
 
The study site investigator will report adverse reactions to the responsible IRBs in accordance with 
respective IRB policies and procedures. 
 
Follow-up information to a reported adverse event will be submitted to the IRB as soon as the relevant 
information is available.  If the results of the study site investigator’s follow-up investigation show that 
an adverse event that was initially determined to not require reporting to the IRB does, in fact, meet 
the requirements for reporting, the study site investigator will report it as soon as possible in 
accordance with respective IRB policies and procedures. 
 
8.6 
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 

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26th, April 2023 
infection). For example, participants could be treated unfairly or discriminated against, or could have 
problems being accepted by their families and/or communities and/or employer. 
 
Social harms that are judged by the study site investigator to be serious or unexpected will be reported 
to the DAIDS Program Officer and the responsible site IRB (JHU) at least annually or according to 
individual IRB 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. 
 
8.7 
Withdrawal of Subjects Due to Adverse Events 
 
Clinical Management Section 9 below includes guidelines for withdrawal of research participants from 
this study. 
 
9 CLINICAL MANAGEMENT 
 
Guidelines for clinical management and product hold/discontinuation are outlined in this section.  In 
general, the study site investigator has the discretion to hold study product at any time if he or she 
feels that continued product use would be harmful to the participant, or interfere with participant’s 
acute medical condition (such as e.g. viral gastroenteritis), or with treatment deemed clinically 
necessary according to the judgment of the investigator. 
 
9.1 
Grading System 
 
The primary grading system for this study is Rectal Grading Table for Use in Microbicide Studies, 
which is Addendum 3 to the DAIDS AE Grading Table Version 2.1, dated July 2017; it can be found 
at Regulatory Support Center (DAIDS RSC) website:  
 
 
https://rsc.niaid.nih.gov/sites/default/files/rectal-tox-table-updates-05-11-2012.pdf 
 
9.2 
Dose Modification 
 
This study involves five different test conditions composed of five different drug combinations. If any 
of the study products are not well tolerated according to either the research participant who requests 
withdrawal, the clinical judgement of a study clinician, or if a participant experiences a Grade 3 AE 
attributable to the study product, then the subject will be discontinued form the study, followed until 
AE resolution or stabilization, and replaced. The participant will be included in the safety analysis. 
Only research participants who successfully complete all five test conditions and PK assessments 
will be considered evaluable for PK and PD endpoints.  
 
All Grade 3 or greater AEs will be reviewed with the PSRT, or designees, as soon as they occur. A 
Grade 4 AE also requires immediate PSRT, or designee, involvement, an automatic pause in further 
dosing, and review by PSRT. Study participants who experience the following will be permanently 
discontinued from product use, but may continue with study sampling if deemed medically appropriate 
by the PSRT:  
• 
Any Grade 3 event deemed related to study products 
• 
Any Grade 4 event 

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26th, April 2023 
 
The PSRT will consider replacement of any subject who fails to complete all five intensive PK 
sampling visits. 
 
9.3 
Discontinuation of Study Product(s) in the Presence of Toxicity 
 
Study participants will be permanently discontinued from product use and discontinued from the study 
following appropriate safety evaluation by the study site investigator or a designated sub-investigator 
in the event of the following: 
• 
HIV seroconversion 
• 
Any Grade 3 event deemed related to study products 
• 
Any Grade 4 event 
• 
Clinical reasons determined by the investigator or PSRT 
 
9.4 General Criteria for Discontinuation of Study Product(s) 
 
Study participants will be permanently discontinued from product use by the study site investigator or 
a designated sub-investigator in the event of the following: 
• 
HIV seroconversion 
• 
Any Grade 3 event deemed related to study products 
• 
Any Grade 4 event 
• 
Clinical reasons determined by the investigator or PSRT 
 
9.5 
Criteria for Progression to the Next Dosing Phase 
 
In the event of a Grade 2 event deemed related to study product, the PSRT will consider participant 
safety in determining if the participant may remain on protocol with close follow-up. 
 
9.6 
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. Participants also may be withdrawn if the study 
sponsor, government or regulatory authorities (including the Office of Human Research Protections), 
DAIDS 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.   
 
9.7 Study Participant Replacement 
 
Individuals that miss or incorrectly complete a PK intensive visit may be replaced at the discretion of 
the protocol chair. Accordingly, the protocol chair will determine if research participants who fail to 
complete all biopsy collection sessions should be replaced. Replacement subjects would be assigned 
the same sampling schedule as the subject they are replacing. 
 

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10 
STATISTICAL CONSIDERATIONS 
 
10.1 Overview and Summary of Design 
 
Eligible participants will receive 300 mg TDF/200 mg FTC (Truvada®, Gilead Sciences), once daily 
for seven days under direct observation to achieve steady state drug PrEP concentrations. After one 
week of therapy, participants will undergo intensive PK analysis as well as collection of colorectal 
biopsies for PD testing (PK1). During the PK-intensive day, iohexol will be administered intravenously 
for the empirical determination of renal function and glomerular filtration rate (GFR). While 
concurrently on PrEP, participants will then be intramuscularly administered depot leuprolide acetate 
(11.25 mg LupronTM). Two weeks post-injection, sampling for PK, PD, and renal function will be 
performed (PK2). Participants will then immediately begin low-dose oral estrogen therapy (1 mg 17β-
estradiol) in conjunction with PrEP for one week, at which time samples will be collected for the 
analyses described above (PK3). While on PrEP, participants will then transition to high-dose 
estrogen therapy (6 mg 17β-estradiol) for the remainder of the study. One week post-high dose 
estrogen therapy in the presence of PrEP, pharmacological and renal samples will be collected for 
analysis (PK4). PrEP will then be discontinued, and two weeks later, samples will be collected to 
assess renal function and hormonal concentrations (PK5). The presence of any remaining PrEP in 
plasma, PBMC or colorectal tissue will also be evaluated. Truvada® PrEP or hormone drug dosing 
will be administered under direct observation (DOT). Safety assessments include history/physical, 
chemistry/hematology labs at screening and interim history before each phase of the study.  
 
10.2 Study Endpoints 
 
Study endpoints to be assessed are each paired with specific objectives as listed in Section 3, 
Objectives & Endpoints.  
 
10.3 Study Hypotheses 
 
This protocol and derived data will provide information to test the following study hypotheses: 
 
• GAHT decreases TDF/FTC PrEP concentrations in an estradiol dosage-dependent manner; but 
there is no effect of TDF/FTC PrEP on feminizing GAHT (as assessed by testosterone 
measurements). 
 
10.4 Accrual and Sample Size 
 
Up to a total of 40 individuals will be enrolled at at JHU, in order to complete 20 evaluable participants.  
Participants will be HIV-1 seronegative, self-identify as a TGW, generally healthy, and at least 18 
years of age.  Healthy participants must also not be on any hormonal therapies, or be willing to abstain 
from estrogen or anti-androgenic therapies, until testosterone concentrations reach > 2 ng/mL. 
 
Based on prior studies with similar eligibility requirements, is the expected enrollment rate would be 
1 participant per month. Therefore, accrual is expected to take approximately 20 months.  
 
For paired analyses of change within participants between the 5 pharmacologic conditions, 20 
research participants provides 80% power to detect a 37% relative difference in PBMC TDF-DP 
concentration, assuming a standard deviation of 0.3 (in terms of log10 concentration), which is the 
observed standard deviation of PBMC TFV-DP measurements in the HPTN 066 trial data set. This 

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will provide sufficient power to detect a similar change in active drug concentrations in PBMC as we 
saw with plasma TFV in the TGW Pilot study. 
 
10.5 Randomization and Blinding 
 
Enrolled participants will not be randomized, as this is by necessity an open-label study. This will be 
an unblinded study. As described in Section 9, in the event that a study site investigator is concerned 
that a participant might be put at undue risk by continuing product use, the product use may be 
discontinued. 
 
10.6 Data Analysis 
 
10.6.1 Descriptive Statistics and Graphics 
 
Descriptive statistics and graphics will be used to summarize the characteristics of endpoints among 
the five dosing phases. Continuous measures will be summarized by means and standard deviations 
or median and interquartile range depending on data distribution.  For binomial endpoints, 95% exact 
confidence intervals will be calculated.  Given the small sample size and possible non-normality of 
the data, non-parametric tests will be used to test for differences among (Kruskal-Wallis) and between 
(Wilcoxon rank sum test) test formulations. 
 
10.6.2 Safety 
For the safety analysis, the number and the frequency of all adverse events (AEs) will be tabulated 
for each of the five dosing phases.  To be able to determine whether excessive AEs are occurring, 
the proportion of subjects that experience an AE will be calculated for each phase.  A single summary 
outcome of this type (yes/no) can be reasonable assumed to follow a Bernoulli distribution.   
 
Table shows for selected true underlying rates between .01 and .20 the probability of one or more 
participants experiencing AE’s in a sample of 20.  From this table we can see that we have at least a 
80% chance of one or more participants experiencing an AE when the true rate is at least 8% and a 
probability of approximately 18% when the true rate is no more than 1%. Unless the true rate is at 
least 2%, the probability of two or more subjects experiencing AEs will be quite small – 5%.           
 
Table 18: Probability of Events for Selected True Rates of AEs 
Event Rate 
Probability of 
missing 
toxicity              
(1-event rate)n 
Power to detect 
one or more 
events                        
1-(1-p)n 
Power to detect 
two or more 
events         
Power to detect 
three or more 
events         
0.01 
0.818 
0.182 
0.017 
0.001 
0.02 
0.668 
0.332 
0.060 
0.007 
0.03 
0.544 
0.456 
0.120 
0.021 
0.04 
0.442 
0.558 
0.190 
0.044 
0.05 
0.358 
0.642 
0.264 
0.075 
0.06 
0.290 
0.710 
0.340 
0.115 
0.07 
0.234 
0.766 
0.413 
0.161 
0.08 
0.189 
0.811 
0.483 
0.212 
0.09 
0.152 
0.848 
0.548 
0.267 

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0.10 
0.122 
0.878 
0.608 
0.323 
0.11 
0.097 
0.903 
0.662 
0.380 
0.12 
0.078 
0.922 
0.711 
0.437 
0.13 
0.062 
0.938 
0.754 
0.492 
0.14 
0.049 
0.951 
0.792 
0.545 
0.15 
0.039 
0.961 
0.824 
0.595 
0.16 
0.031 
0.969 
0.853 
0.642 
0.17 
0.024 
0.976 
0.877 
0.685 
0.18 
0.019 
0.981 
0.898 
0.725 
0.19 
0.015 
0.985 
0.916 
0.761 
0.20 
0.012 
0.988 
0.931 
0.794 
 
Additional safety analyses will also tabulate the number and type of AEs observed overall, and by 
severity, site, and study product.  AEs that lead to discontinuation of study participation will be 
tabulated separately. 
 
10.6.3 Pharmacokinetics 
Pharmacokinetic parameters in all matrices will be estimated using non-compartmental analysis, 
including the maximum concentration (Cmax), time to maximum concentration (Tmax), and area 
under the concentration versus time curve (from 0-24 hours, AUC0-24h) for plasma TFV and FTC. 
These will be summarized using descriptive statistics by PK period. Colorectal tissue concentrations 
of TFV, FTC, TFV-DP, and FTC-TP will be summarized by time interval and PK period using 
descriptive statistics. Sample size is conditioned primarily on plasma TFV and FTC as well as PBMC 
TFV-DP and FTC-TP.  
10.6.4 
Explant Studies 
Pharmacodynamic antiviral effects will be explored by assessing HIV-1 p24 antigen production after 
ex vivo HIV challenge of colorectal biopsy explants from participants sampled at each PK phase of 
the study. Paired PK-PD data pairs will be pooled across all study participants within each condition 
and fit to variations of the traditional Emax model to establish the concentration-response relationship 
for each condition. Comparisons across conditions will also be compared. Sample size is not 
conditioned on this readout.  
10.6.5 
Missing Data 
All reasonable efforts will be made to obtain complete data for all participants; however, missing 
observations will occur due to missed visits, participants lost to follow-up, or noncompliance. 
Research participants who fail to complete dosing and evaluations relevant to all study product doses 
for any reason may be replaced at discretion of the study site investigator after consultation with the 
Sponsor.  Data for PK, distribution, and permeability will be required of all periods of the study or the 
research participant will be replaced.   
 
Plasma and PBMC samples obtained outside the specified window due to schedule conflicts with 
anoscopy and biopsy collection will not substantially affect our ability to capture data needed for study 
endpoints and will not adversely affect achieving related study objectives. 
 

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As previously noted, all available data will be used for safety purposes. All available data will also be 
used for descriptive purposes. Formal statistical comparisons between study phases, however, must 
necessarily be limited to subjects who complete use of all dosing phases.  
 
The familywise error rate corrections will be used to adjust for multiple comparisons. 
 
Missing data adjustment methods will be investigated using multiple imputation and sensitivity 
analyses in the event of possible informative missingness. 
 
11 
DATA HANDLING AND RECORDKEEPING 
 
11.1 Data Management Responsibilities 
 
Study staff will provide case report forms and site Standard Operating Procedures as part of the study 
activation process. All case report forms that will be used as source documents will be identified.  
 
Close coordination between protocol team members will be necessary to track study progress, 
respond to queries about proper study implementation, and address other issues in a timely manner.  
Rates of accrual, adherence, and follow-up will be monitored closely by the team.  
 
11.2 Source Documents and Access to Source Data/Documents 
 
All study sites will maintain source documents and data in accordance with Requirements for Source 
Documentation: 
 
 
https://www.niaid.nih.gov/sites/default/files/daids-sourcedocpolicy.pdf 
 
Each investigator will maintain and securely store complete, accurate, and current study records 
throughout the study.  This described study is a non-IND trial and there are no investigational products 
involved in the study.  There will be no marketing application based on the data or findings from this 
work.  
 
Study records must be maintained on site for the entire period of study implementation.  Thereafter, 
instructions for record storage will adhere to the DAIDS Storage and Retention of Clinical Research 
Records Policy.  No study records may be moved to an off-site location or destroyed prior to receiving 
approval from both DAIDS and Sponsor. 
 
11.3 Quality Control and Quality Assurance 
 
Quality control and quality assurance procedures will be performed by all study sites as specified in 
Requirements for Clinical Quality Management Plans: 
 
 
https://www.niaid.nih.gov/sites/default/files/qmppolicy.pdf 
 
 
 

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11.4 Study Coordination 
 
Study implementation will also be guided by a common study-specific procedures manual that 
provides further instructions and operational guidance on conducting study visits; data and forms 
processing; specimen collection, processing, and shipping; AE assessment, management and 
reporting; dispensing study products and documenting product accountability; and other study 
operations. 
 
Close coordination between protocol team members is necessary to track study progress, respond to 
queries about proper study implementation, and address other issues in a timely manner. Rates of 
accrual, retention, follow-up, and AE incidence will be monitored closely by the team. 
 
12 
CLINICAL SITE MONITORING 
 
Non-network study monitoring will be carried out with prior approval of the study monitoring plan by 
DAIDS. Site monitoring visits will be conducted to assess overall study compliance, as required per 
Requirements for On-Site Monitoring: 
 
https://www.niaid.nih.gov/sites/default/files/onsitemonitor_reqs.pdf 
 
Study monitors will visit the site to complete the following: 
• 
Assess compliance with the study protocol, Good Clinical Practices (GCP) guidelines, and 
applicable regulatory requirements, including US CFR Title 45 Part 46 and Title 21 Parts 50, 
56, and 312 
• 
Review informed consent forms, procedures, and documentation 
• 
Perform source document verification to ensure the accuracy and completeness of study data 
• 
Verify proper collection and storage of biological specimens 
• 
Verify proper storage, dispensing, and accountability of investigational study products 
• 
Assess implementation and documentation of internal site quality management procedures 
• 
Assess site staff training needs 
 
Johns Hopkins University Clinical Pharmacology will provide site study monitoring. Site investigators 
will allow study monitors to inspect study facilities and documentation (e.g., informed consent forms, 
clinic and laboratory records, other source documents, CRFs), as well as observe the performance 
of study procedures. Investigators also will allow inspection of all study-related documentation by 
authorized representatives of DAIDS and U.S. regulatory authorities. A site visit log will be maintained 
at the study site to document all visits. The outcomes of the monitoring visits and the subsequent 
reports of resolutions of any identified problems will be provided to DAIDS. 
 
13 
HUMAN SUBJECTS PROTECTIONS 
 
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 
appropriate regulatory agencies involved in this trial, including amendment of IND# 126136.  The 
investigators will permit audits by the NIH, Sponsor, and Office for Human Research Protections 
(OHRP), the FDA or any of their appointed agents. 

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13.1 Institutional Review Boards 
 
The participating study site is responsible for assuring that this protocol, the associated site-specific 
informed consent documents, and study-related materials are reviewed by an IRB responsible for 
oversight of research conducted at the study sites. Any amendments to the protocol, informed consent 
documents, and other study-related materials must be approved by the study site IRB prior to 
implementation. 
 
Subsequent to the initial review and approval, the study site IRB must review the study at least 
annually. These reports will include the total number of participants enrolled in the study, the number 
of participants who completed the study, all changes in the research activity, and all unanticipated 
problems involving risks to human subjects or others.  
 
13.2 Protocol Registration 
 
Prior to implementation of this protocol, and any subsequent full version amendments, the study site 
must have the protocol and the protocol consent form(s) approved, as appropriate, by their local 
institutional review board (IRB)/ethics committee (EC) and any other applicable regulatory entity (RE). 
Upon receiving final approval, the site will submit all required protocol registration documents to the 
DAIDS PRO at the Regulatory Support Center (RSC).  The DAIDS PRO will review the submitted 
protocol registration packet to ensure that all of the required documents have been received. 
 
Site-specific informed consent forms (ICFs) WILL NOT be reviewed or approved by the DAIDS PRO, 
and sites will receive an Initial Registration Notification when the DAIDS PRO receives a complete 
registration packet. Receipt of an Initial Registration Notification indicates successful completion of 
the protocol registration process. The site will not receive any additional notifications from the DAIDS 
PRO for the initial protocol registration. A copy of the Initial Registration Notification should be retained 
in the site's regulatory files. 
 
Upon receiving final IRB/EC and any other applicable RE approval(s) for an amendment, the study 
site should implement the amendment immediately. The site is required to submit an amendment 
registration packet to the DAIDS PRO at the RSC. The DAIDS PRO will review the submitted protocol 
registration packet to ensure that all the required documents have been received. Site-specific ICF(s) 
WILL NOT be reviewed and approved by the DAIDS PRO and sites will receive an Amendment 
Registration Notification when the DAIDS PRO receives a complete registration packet. A copy of the 
Amendment Registration Notification should be retained in the site's regulatory files. 
 
For additional information on the protocol registration process and specific documents required for 
initial and amendment registrations, refer to the current version of the DAIDS Protocol Registration 
Manual, available at  
https://www.niaid.nih.gov/sites/default/files/prmanual.pdf 
 
13.3 Risk-Benefit Statement 
13.3.1 Risks 
 
General 
Disclosure of STI status may cause sadness or depression in volunteers.  Participation in clinical 
research includes the risks of loss of confidentiality and discomfort with the personal nature of 

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questions. Participants may experience slight embarrassment from receiving a douche administered 
by the clinical staff. Although all efforts will be made to protect participant privacy and confidentiality, 
it is possible that participant's involvement in the study could become known to others, and that social 
harms may result (i.e. 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, employer, and/or communities. 
 
Participants in sites requiring partner notification in response to diagnosed STIs or HIV infection could 
have problems in their relationships with their sexual partners.  Participants also could have problems 
in their partner relationships associated with use or attempted use of study products.  
 
Risk of blood draws and PIVs 
Phlebotomy and insertion and use of peripheral intravenous catheters (PIVs) are generally safe 
procedures, but there are some risks. Common risks include discomfort, site pain, tenderness, 
bleeding, bruising, redness, or swelling due to vein irritation from the needle or catheter. Less 
common risks include local site infection, infiltration or the leakage of fluid into the surrounding 
area/tissue, or clotting of the vein. In rare cases, it may result in fainting. Very rarely PIVs can be 
associated with a venous air embolism where air enters the vein and can potentially travel to your 
heart, brain, or lungs and cause damage. Study staff will adhere to universal precautions and infection 
control guidelines to reduce risks. 
 
Rectal Swabs 
There is the risk of mild discomfort from this procedure, in addition to a slight risk of bleeding.  
 
General Risks of Cleansing Douche 
The main risk from having a douche is temporary discomfort. The douche bottle nozzle consists of 
hollow plastic tube, tapered slightly toward the opening, and coated with paraffin as lubricant. It is 
about the thickness of a pencil and will be passed through the anal canal to administer approximately 
120-125 mL of fluid into the rectum. This may cause a “bloated” or “cramping” feeling.  Some air may 
be pumped into the rectum as well, causing flatulence.  The tube is small, but it might cause some 
anal or rectal discomfort if the subject has any hemorrhoids or other painful conditions. There is a 
remote possibility that the douche tip could cause perforation. 
 
TDF/FTC PrEP 
In PrEP trials of TDF/FTC, more than 2% of participants receiving TDF/FTC reported adverse 
reactions of headache, abdominal pain and decreased weight more frequently than those receiving 
placebo. Other side effects associated with TDF/FTC are less frequent. Side effects associated with 
use of TDF or FTC are listed below: 
 
The following side effects have been associated with the use of TDF: 
• Upset stomach, vomiting, gas, loose or watery stools 
• Generalized weakness 
• Dizziness 
• Depression 
• Headache 
• Abdominal pain 
• Worsening or new kidney damage or failure 
• Inflammation or swelling and possible damage to the pancreas and liver 
• Shortness of breath 

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• Rash 
• Allergic reaction: symptoms may include fever, rash, upset stomach, vomiting, loose or watery 
stools, abdominal pain, achiness, shortness of breath, a general feeling of illness or a potentially 
serious swelling of the face, lips, and/or tongue 
• Bone pain and bone changes such as thinning and softening which may increase the risk of 
breakage 
• Muscle pain and muscle weakness 
 
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, which sometimes can be a sign of an allergic reaction 
• Skin darkening of the palms and/or soles 
• Increased cough 
• Runny nose 
• Abnormal liver function tests, which could mean liver damage 
• Increases in pancreatic enzyme 
• Increased triglycerides 
• Increased creatine phosphokinase, which could mean muscle damage 
 
Leuprolide acetate 
Commonly reported side effects of leuprolide include: arthropathy, asthenia, decreased testicular 
size, dermatological reaction, edema, gastrointestinal disease, headache, hot flash, increased lactate 
dehydrogenase, pain, signs and symptoms of injection site, urination disorder, and diaphoresis.  
Other side effects include: respiratory system disorder, abnormal hepatic function tests, dehydration, 
dizziness, dyspnea, impotence, increased serum alkaline phosphatase, increased serum aspartate 
aminotransferase, insomnia, myalgia, nausea and vomiting, neuromuscular disease, paresthesia, 
prolonged partial thromboplastin time, sleep disorder, and vertigo. 
 
17β-estradiol 
Commonly reported side effects of estradiol include: cerebrovascular accident, infection, malignant 
neoplasm of breast (in women, though theoretical in TGW), headache, and mastalgia.  
Other side effects include: abdominal pain, limb pain, pruritus, sinusitis, nausea, and skin rash. 
Venous thromboembolism risk associated with oral estradiol varies in the literature ranging from no 
additional risk to 4-times greater than background risk, which is itself low, ranging from 1 in 1,000 to 
1 in 10,000 population. Several cases of prolactin elevations and growth of pituitary prolactinoma 
have been reported.  
 
Iohexol Infusions 
Iohexol is approved for use as an intravenous contrast agent, but is very commonly used in clinical 
research settings for direct GFR measurements in healthy research participants. Side effects of 
iohexol associated with intravenous use are generally either (1) chemotoxic reactions resulting from 
the physicochemical properties of the contrast media, as well as the dose, speed of injection, and 
hemodynamic disturbances and/or injuries to organs or vessels perfused by the contrast medium; or, 

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(2) idiosyncratic reactions including all other reactions, which occur more frequently in patients 20 to 
40 years old. Idiosyncratic reactions may or may not be dependent on the amount of dose injected, 
the speed of injection, and the radiographic procedure. Idiosyncratic reactions are subdivided into 
minor, intermediate, and severe. The minor reactions are self-limited and of short duration; the severe 
reactions are life-threatening and treatment is urgent and mandatory. As in our prior use of iohexol 
(JHMIRB Protocol #IRB00033945), we will obtain an IND exemption for an FDA approved drug being 
used in a non-approved population. This is regularly approved under 21 CFR 312.2(b) given that there 
is no intent to modify the label, no intent for commercial purposes, performed under IRB approval, 
and the dose, route, frequency, and intended population does not pose an increased risk with iohexol 
use. 
 
Flexible Sigmoidoscopy with Rectal Tissue Biopsies 
Flexible sigmoidoscopy is a commonly practiced medical procedure and the endoscopic 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”.  Endoscopic 
biopsies are painless and heal quickly, usually within 3 days. On extremely rare occasions, the 
endoscopic procedure or biopsies may lead to pain, infection (sepsis), bleeding or perforation of the 
gastrointestinal tract. Perforation occurs approximately once out of every 1 in 1,000 to 10,000 
procedures. If this extremely rare complication occurs, antibiotics and surgery to repair the tear may 
be necessary.  
 
Prep instructions for sigmoidoscopy will include no solid food eight hours prior to the procedure. A 
cleansing douche will be administered as enema prep for all flexible sigmoidoscopy procedures 
 
13.3.2 
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. Participants 
also may appreciate the benefit of earlier diagnosis of STIs; additionally, participants will be referred 
for treatment for any incidental findings detected during screening and other study-related 
examinations. 
 
13.4 Informed Consent Process 
 
It is the responsibility of the study site investigator to ensure that the Elements of Informed Consent 
(21 CFR 50.25 and ICH GCP 4.8.10) and Health Insurance Portability Accountability Act (HIPAA) 
guidelines are followed and documented in the source document file.  The process for obtaining 
informed consent from potential research participants should be clearly documented and 
appropriately filed within the site’s standard operating procedures. 
 
Written informed consent will be obtained from each study participant prior to initiation of any study 
procedures.  Written informed consent also will be obtained for long-term specimen storage and 
possible future testing. In obtaining and documenting informed consent, the investigators and their 
sub-investigators will comply with applicable local and US regulatory requirements and will adhere to 
GCP and to the ethical principles that have their origin in the Declaration of Helsinki.  Study staff must 
document the informed consent process in accordance with the Requirements for Source 
Documentation at: 
 

TGW GAHT Interaction Study 
 
 
 
 
62 
 
 
 
26th, April 2023 
 
 
https://www.niaid.nih.gov/sites/default/files/daids-sourcedocpolicy.pdf 
 
Participants will be provided with copies of the informed consent forms.  The study site is responsible 
for developing study informed consent forms for local use, based on the templates in the Appendices 
that describe the purpose of the study, the procedures to be followed, and the risks and benefits of 
participation, in accordance with all applicable regulations. 
 
The informed consent process will cover all elements of informed consent required by research 
regulations.  In addition, the process will specifically address the following topics of importance to this 
study: 
 
• The unknown safety and unproven efficacy of the study products 
• The need to practice safer sex behaviors regardless of study treatment group 
• The importance of adherence to the study visit and procedures schedule 
• The potential medical risks of study participation (and what to do if such risks are experienced) 
• The potential social harms associated with study participation (and what to do if such harms 
are experienced) 
• The limited benefits of study participation 
• The distinction between research and clinical care 
• The right to withdraw from the study at any time 
 
The informed consent process will include an assessment of each potential participant’s 
understanding of concepts identified by the protocol team as essential to the informed consent 
decision. Study sites will follow the IRB guidance regarding this assessment.  
 
If during the trial, a consent revision is being presented, including new information that might affect 
the research participant’s willingness to participate, participants will be informed of the revisions. If a 
research participant terminates the study and consent form revision occurs after their participation 
has ended, they do not need to sign the revised consent form. 
 
13.5 Participant Confidentiality 
 
All study procedures will be conducted in private and every effort will be made to protect participant 
privacy and confidentiality to the extent possible. The study site will implement confidentiality 
protections that reflect the local study implementation plan and the input of study staff and community 
representatives to identify potential confidentiality issues and strategies to address them.  In addition 
to local considerations, the protections described below will be implemented at all sites. 
 
All study-related information will be stored securely at the study site.  All participant information will 
be stored in locked file cabinets in areas with access limited to study staff.  Data collection, process, 
and administrative forms, laboratory specimens and other reports will be identified by a coded number 
only, to maintain participant confidentiality. Forms, lists, logbooks, appointment books and any other 
listings that link participant ID numbers to other identifying information will be stored in a separate, 
locked file in an area with limited access. All local databases will be secured with password-protected 
access systems.  Participants’ study information will not be released without their written permission, 
except as necessary for review, monitoring, and/or auditing. 
 

TGW GAHT Interaction Study 
 
 
 
 
63 
 
 
 
26th, April 2023 
The site investigator will make study documents (e.g., consent forms, drug distribution forms, eCRFs) 
and pertinent hospital or clinic records readily available for inspection by the local IRB, the site 
monitors, the FDA, the NIAID, the OHRP, JHU legal representatives (attorneys that may review 
records in the case where there is concern about a possible research-related injury or violation of 
confidentiality) and other JHU staff and other local, US, or international regulatory entities for 
confirmation of the study data.  
 
13.6 Special Populations 
 
This section outlines considerations made for the inclusion or exclusion of special populations in this 
study. 
13.6.1  Women 
All minorities are eligible for the proposed studies, and no individual will be excluded on the basis of 
racial or ethnic background.  We expect the racial and ethnic composition of our studies to be similar 
to the demographics of the community served by the Johns Hopkins Hospital, Chase-Brexton Health 
Services and Whitman-Walker Health (referral bases). Approximately 70% of the transgender 
population are racial and ethnic minorities at both our primary patient referral partner, Chase-Brexton 
Clinic, as well as in our past transgender women (TGW) clinical studies in our Drug Development Unit 
(DDU) at The Johns Hopkins Hospital. Focused ongoing efforts to encourage clinical trial participation 
by minorities within the Johns Hopkins program have resulted in enhanced participation. This high 
percentage of racial and ethnic minorities also reflects the demographics of the larger HIV epidemic 
in Baltimore and Washington, DC.  
 
This is a phase I pharmacokinetic study targeted to self-identifying TGW. We will exclude cisgender 
men and cisgender women from participation in the study based on study design. Inclusion criteria 
include self-identifying TGW who are naïve to gender affirming hormonal therapy or are willing to 
abstain from feminizing therapies until total testosterone concentrations reach a cisgender male 
reference interval. As the proposed study is aimed to evaluate drug-drug interactions between PrEP 
and exogenous gender affirming estrogen-based hormonal therapies, we are therefore limiting 
enrollment to self-identifying TGW to increase the efficiency of successful recruiting based on 
behavioral grounds.   
13.6.2 
 Children 
Pediatric TGW aged 18-21 are eligible to participate and provide clinical materials for laboratory 
studies. Children less than 18 years of age will be excluded.  
 
13.7 Compensation 
 
Pending IRB approval, participants will be compensated for their time and effort in this study, and/or 
be reimbursed for travel to study visits, child care, and time away from work according to site’s 
practice.  Reimbursement amounts will be specified in the site’s informed consent form. 
 
13.8 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. 
 

TGW GAHT Interaction Study 
 
 
 
 
64 
 
 
 
26th, April 2023 
13.9 Access to HIV-related Care 
13.9.1 HIV Counseling 
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.  Testing will be performed in accordance with 
the algorithm in Appendix II. Counseling will be provided in accordance with standard HIV counseling 
policies and methods at the participating study site and additionally will emphasize the unknown 
efficacy of the study products in preventing HIV infection.  In accordance with NIH policies, 
participants must receive their HIV test results to take part in this study. Referral for additional 
counseling related to testing or diagnosis will occur if needed or requested by the participant. 
13.9.2 Care for Participants Identified as HIV-infected 
Participants will be provided with their HIV test results in the context of post-test counseling. Per site 
SOPs, 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. 
 
13.10 Study Discontinuation 
 
This study may be discontinued at any time by DAIDS/NIAID, Sponsor, , the OHRP, or site IRB. 
 
 
 
 
14 
LIST OF APPENDICES 
 
APPENDIX I: SCHEDULE OF STUDY VISITS AND EVALUATIONS 
APPENDIX II: HIV TESTING ALGORITHM 
APPENDIX III: RECTAL TISSUE BIOPSY ALLOCATION 
APPENDIX IV: TOXICITY TABLES 
APPENDIX V: SAMPLE INFORMED CONSENT 

TGW GAHT Interaction Study 
 
 
 
 
65 
 
 
 
26th, April 2023 
15 
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67. Rule AD, Bergstralh EJ, Slezak JM, Bergert J, Larson TS. Glomerular filtration rate estimated 
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APPENDIX I: SCHEDULE OF STUDY VISITS AND EVALUATIONS 
 
 
 
STUDY VISIT 
 
Screen 
Base 
line 
DO
T 
PK  SERIES 1 
D1           D2 
DOT 
Safety 
DOT 
PK SERIES 
2 
D1          D2 
DOT 
PK SERIES 
3 
D1          D2 
DOT 
 PK SERIES 
4 
D1          D2 
DOT 
Safety 
DOT 
PK SERIES 
5 
D1          D2 
 
Safety 
(+ 2 
weeks
) 
Visit Number 
1 
2 
3-8 
9 
10 
11-15 
16 
17-22 
23 
24 
25-29 
30 
31 
32-36 
37 
38 
39-43 
44 
45-50 
51 
52 
53 
Study Day 
-60 
0 
1-6 
7 
8 
9-13 
14 
15-20 
21 
22 
23-27 
28 
29 
30-34 
35 
36 
37-41 
42 
43-48 
49 
50 
56 
ADMINISTRATIVE 
Informed Consent 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility assessment 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Obtain/Update 
Locator 
Information 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Obtain/Update 
Medical 
History/Concomitant 
Meds 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Assess/Update AEs 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Adherence Monitoring 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Gender 
Dysphoria 
Assessment 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
CLINICAL 
Physical Exam1 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
 
x 
x 
x 
Vital Signs 
 (BP HR, Temp, Ht2, Wt2) 
x 
x 
 
x 
x 
 
x 
 
x 
x 
 
x 
x 
 
x 
x 
 
x 
 
x 
x 
x 
Insert 
PIV 
x 
2 
(if 
applicable) 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Hormone 
Testing 
Estradiol 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
Testosteron
e 
(total only) 
x 
x 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
x 
Safety 
Labs 
CBC w/ Diff 
x 
x 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
CMP (eGFR) 
x 
x 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
x 
PO4 
x 
x 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
PT/PTT 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum 
Cr 
(eGFR) 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Lipid panel5 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
STD 
Testing: 
Blood 
HBsAg 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Syphilis 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
HIV 
x 
x 
 
x 
 
 
x 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
x 

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STUDY VISIT 
Screen 
Base 
line 
DO
T 
PK SERIES 1 
D1           D2 
DOT 
Safety 
DOT 
PK SERIES 
2 
D1          D2 
DOT 
PK SERIES 
3 
D1          D2 
DOT 
PK SERIES 
4 
D1          D2 
DOT 
Safety 
DOT 
PK SERIES 
5 
D1          D2 
Safety 
(+ 2 
weeks
) 
Visit Number 
1 
2 
3-8 
9 
10 
11-15 
16 
17-22 
23 
24 
25-29 
30 
31 
32-36 
37 
38 
39-43 
44 
45-50 
51 
52 
53 
Study Day 
-60 
0 
1-6 
7 
8 
9-13 
14 
15-20 
21 
22 
23-27 
28 
29 
30-34 
35 
36 
37-41 
42 
43-48 
49 
50 
56 
CLINICAL CONTINUED 
STD 
Testing: 
Rectal 
Swab 
GC/CT NAAT 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
Cleansing Douche 
 
x 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
DOSING/STUDY PRODUCT  
Iohexol Administration 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Dispense TDF/FTC 
 
x 
 
 
x 
 
x 
 
 
x 
 
 
x 
 
 
 
 
 
 
 
 
 
DOT Dose of TDF/FTC 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
Lupron Injection® 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense Estradiol 1 mg 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
DOT Dose of Estradiol 
1mg 
 
 
 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
Dispense Estradiol 6 mg 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
x 
 
x 
 
 
 
 
DOT Dose of Estradiol 
6mg 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
STUDY SPECIMEN COLLECTION 
Serum Estradiol 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Serum Testosterone  
(free and total) 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Serum LH, FSH 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Serum Cystatin 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
Plasma 
PK TFV, 
FTC 
Pre-Dose 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
 
 
 
x 
 
 
1hr 
 
 
 
2hr 
 
 
 
3hr 
 
 
 
4hr 
 
 
 
24hr 
 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
PBMC 
TFV-DP, 
FTC-TP 
Pre-Dose 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
24hr 
 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 

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26th, April 2023 
 
1 Full physical exam at screening only, targeted PE at all other visits as needed 
2 Height at screening only, weight at screening and iohexol visits only 
3 Additional test may be ordered for abnormal results 
4 DOT may consist of in person observation, live video streaming, or with use of a time-stamped video 
5 Fasting, total cholesterol, triglycerides, HDL, LDL 
6 Colonic tissue collection for PK/PD - 5 total biopsies collected, 2 for PD (explant), 3 for PK studies. 
7If a participant has difficult vein access and is not a candidate for 2 PIVs, a single PIV is acceptable; a saline flush will be performed after iohexol administration, and a waste tube will be collected prior to 
collection of samples for iohexol measurement of drug quantification.  
 
 
 
STUDY VISIT 
Screen 
Bas
e 
line 
DO
T 
PK  SERIES 
1         D1       
D2 
DOT 
Safety 
DOT 
 
PK  SERIES 
2 
   D1          
D2 
DOT 
 
PK  SERIES 
3 
  D1          D2 
DOT 
 
PK  SERIES 
4 
D1          D2 
DOT 
Safety 
DOT 
 PK  SERIES 
5 
   D1          D2 
Safety 
 (+ 2 
weeks) 
Visit Number 
1 
2 
3-8 
9 
10 
11-15 
16 
17-22 
23 
24 
25-29 
30 
31 
32-36 
37 
38 
39-43 
44 
45-50 
51 
52 
53 
Study Day 
-60 
0 
1-6 
7 
8 
9-13 
14 
15-20 
21 
22 
23-27 
28 
29 
30-34 
35 
36 
37-41 
42 
43-48 
49 
50 
56 
STUDY SPECIMEN COLLECTION CONTINUED 
Iohexo
l GFR 
Pre-
Dose 
 
x 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
2hrs 
 
 
 
4hrs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine 
TFV, 
FTC, Cr 
0-4hr 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
4-24hr 
 
 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
x 
 
Colon 
Biopsies 
TFV, FTC, TFV-DP, 
FTC-TP, 
Explant 
HIV Challenge6 
 
x 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
x 
 
 
 
 
 
x 
 

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APPENDIX II: HIV TESTING ALGORITHM 
In accordance with CDC guidelines for HIV serology testing, The Johns Hopkins Hospital HIV Laboratory has moved to a 4th 
generation assay which is a more sensitive and specific assay. The 4th generation assay is a combined antigen and antibody assay 
for HIV1 and HIV2 serological detection. All reactive samples will be confirmed with a rapid multi-spot assay for HIV-1 and HIV-2. 
The Western blot confirmatory assay is no longer performed. Any discrepant results will go on to HIV RNA viral load testing. As 
with the previous HIV serology, no reactive results will be released prior to confirmatory testing.  
 

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Combo Ag/AB  
HIV-1/HIV-2 
Antibody 
Immunoassay
Reactive 
Nonreactive
 HIV-1/HIV-2 Multispot 
Ab Confirmation
Testing Stops and 
Report
JHH HIV Lab HIV Screening Testing Algorithm Overview
If Multispot Negative/
Indeterminate
HIV-1 Viral Load 
Testing to resolve 
discrepancy
Testing Stops and 
Report
If Multispot 
Positive

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APPENDIX III:  Rectal Tissue Biopsy Allocation 
Table 19. Number of Samples Taken at Each Biopsy Visit & Their Allocation for Testing 
 
 
Baseline 
24 hr Post-PK 
Intensive Visits 
PK (tissue 
homogenates) 
3 
3 
Explant 
2 
2 
 
Of note, the overall total number of biopsies for the entire study, given one baseline visit and the sampling visits, will be 30 
biopsies per participant. 

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APPENDIX IV: TOXICITY TABLES 
 
The Division of AIDS Rectal Grading Table for Use in Microbicide Studies (DAIDS AE Grading 
Table Addendum 3, Clarification dated May 2012) will be the primary tool for grading adverse 
events for this protocol.   
 
Adverse events not included in the Rectal Grading Table will be graded by the most current 
Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse 
Events, Corrected Version 2.1. (July 2017). In cases where an AE is covered in both tables, 
DAIDS AE Grading Table Addendum 3 will be the grading scale utilized.   
 
All Division of AIDS grading tables are available online at: http://rsc.tech-
res.com/safetyandpharmacovigilance/gradingtables.aspx  
 
 
 

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APPENDIX V: SAMPLE INFORMED CONSENT FORM 
 
This is a sample consent form document only. Each participating site is responsible for 
developing its own informed consent documents. Institutions may require specific informed 
consent language (e.g., HIPAA language), have different policies on compensation, financial 
obligation, costs and injury, etc.